Publications
2021
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(2021) Frontiers in Plant Science. 11, 604349. Abstract
Aromatic amino acids (AAAs) synthesized in plants via the shikimate pathway can serve as precursors for a wide range of secondary metabolites that are important for plant defense. The goals of the current study were to test the effect of increased AAAs on primary and secondary metabolic profiles and to reveal whether these plants are more tolerant to abiotic stresses (oxidative, drought and salt) and to Phelipanche egyptiaca (Egyptian broomrape), an obligate parasitic plant. To this end, tobacco (Nicotiana tabacum) plants were transformed with a bacterial gene (AroG) encode to feedback-insensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase, the first enzyme of the shikimate pathway. Two sets of transgenic plants were obtained: the first had low expression of the AroG protein, a normal phenotype and minor metabolic changes; the second had high accumulation of the AroG protein with normal, or deleterious morphological changes having a dramatic shift in plant metabolism. Metabolic profiling analysis revealed that the leaves of the transgenic plants had increased levels of phenylalanine (up to 43-fold), tyrosine (up to 24-fold) and tryptophan (up to 10-fold) compared to control plants having an empty vector (EV) and wild type (WT) plants. The significant increase in phenylalanine was accompanied by higher levels of metabolites that belong to the phenylpropanoid pathway. AroG plants showed improved tolerance to salt stress but not to oxidative or drought stress. The most significant improved tolerance was to P. aegyptiaca. Unlike WT/EV plants that were heavily infected by the parasite, the transgenic AroG plants strongly inhibited P. aegyptiaca development, and only a few stems of the parasite appeared above the soil. This delayed development of P. aegyptiaca could be the result of higher accumulation of several phenylpropanoids in the transgenic AroG plants and in P. aegyptiaca, that apparently affected its growth. These findings indicate that high levels of AAAs and their related metabolites have the potential of controlling the development of parasitic plants.
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(2021) Autophagy. 17, 11, p. 1-14 Abstract
Reticulophagy, the selective autophagy of endoplasmic reticulum (ER) components, is known to operate in eukaryotes from yeast and unicellular algae to animals and plants. Thus far, only ER-stress induced reticulophagy was reported and analyzed in plants. In this study we characterize a reticulophagy pathway in Arabidopsis thaliana that is triggered by dark-induced starvation but not by ER stress. This pathway is defined by the previously reported ATG8-interacting proteins, ATI1 and ATI2. We further identified the ER-localized MSBP1 (Membrane Steroid Binding Protein 1) as an ATI1- and ATI2-interacting protein and an autophagy cargo, and show that ATI1 and ATI2 serve as its cargo receptors. Together, these findings expand our knowledge on plant responses during energy deprivation and highlight the role of this special type of reticulophagy in this process.
2020
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(2020) New Phytologist. 228, 1, p. 151-162 Abstract
Methylation of internal adenosine at nitrogen-6 position (m(6)A) is the most abundant post-transcriptional modification in eukaryotic RNAs. These modifications are recognized by m(6)A-binding proteins ('readers') that affect downstream functions. In plants, the scope of gene expression regulation by reader proteins is not clear. Here, overexpression and loss-of-function mutants were used to characterize the role of the Arabidopsis m(6)A reader ECT2 in proteasome regulation. ECT2 regulates the mRNA levels of the proteasome regulator PTRE1 and of several 20S proteasome subunits, resulting in enhanced 26S proteasome activity. This regulation is dependent on ECT2 m(6)A binding function. Interestingly, though ECT2 positively regulates proteasome activity in both young and mature plants, PTRE1 has different regulatory effects in different developmental stages. In mature plants, PTRE1 inhibits 26S proteasome activity, while in seedlings PTRE1 knockout mutants have reduced 26S proteasome activity. Taken together, our results suggest a novel epitranscriptomic mechanism of proteasome regulation by ECT2 that is used to fine tune proteasome activity by affecting the expression of PTRE1 and 20S proteasome subunits.
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(2020) Plant Science. 290, 110289. Abstract
Botrytis cinerea is a major plant pathogen, causing losses in crops during growth and storage. Here we show that increased accumulation of phenylalanine (Phe) and Phe-derived metabolites in plant leaves significantly reduces their susceptibility to B. cinerea. Arabidopsis, petunia and tomato plants were enriched with Phe by either overexpressing a feedback-insensitive E.coli DAHP synthase (AroG*), or by spraying or drenching detached leaves or whole plants with external Phe, prior to infection with B. cinerea. Metabolic analysis of Arabidopsis and petunia plants overexpressing AroG* as well as wt petunia plants treated externally with Phe, revealed an increase in Phe-derived phenylpropanoids accumulated in their leaves, and specifically in those inhibiting B. cinerea germination and growth, suggesting that different compounds reduce susceptibility to B. cinerea in different plants. Phe itself had no inhibitory effect on germination or growth of B. cinerea, and inhibition of Phe metabolism in petunia plants treated with external Phe prevented decreased susceptibility to the fungus. Thus, Phe metabolism into an array of metabolites, unique to each plant and plant organ, is the most probable cause for increased resistance to Botrytis. This mechanism may provide a basis for ecologically friendly control of a wide range of plant pathogens.
2019
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(2019) Proceedings of the National Academy of Sciences of the United States of America. 116, 45, p. 22872-22883 Abstract
RNA silencing is a major antiviral defense mechanism in plants and invertebrates. Plant ARGONAUTE1 (AGO1) is pivotal in RNA silencing, and hence is a major target for counteracting viral suppressors of RNA-silencing proteins (VSRs). P0 from Turnip yellows virus (TuYV) is a VSR that was previously shown to trigger AGO1 degradation via an autophagy-like process. However, the identity of host proteins involved and the cellular site at which AGO1 and P0 interact were unknown. Here we report that P0 and AGO1 associate on the endoplasmic reticulum (ER), resulting in their loading into ER-associated vesicles that are mobilized to the vacuole in an ATG5- and ATG7-dependent manner. We further identified ATG8-Interacting proteins 1 and 2 (ATI1 and ATI2) as proteins that associate with P0 and interact with AGO1 on the ER up to the vacuole. Notably, ATI1 and ATI2 belong to an endogenous degradation pathway of ER-associated AGO1 that is significantly induced following P0 expression. Accordingly, ATI1 and ATI2 deficiency causes a significant increase in posttranscriptional gene silencing (PTGS) activity. Collectively, we identify ATI1 and ATI2 as components of an ER-associated AGO1 turnover and proper PTGS maintenance and further show how the VSR P0 manipulates this pathway.
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(2019) Trends in Plant Science. 24, 3, p. 189-191 Abstract
miRNAs act as negative modulators of target genes and play key roles in post-transcriptional gene regulation through sequence-specific mRNA cleavage and translational inhibition. Two recent reports highlight the orchestrated role of miRNA2111 and miRNA172b in plant innate immunity [1,2].
2018
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(2018) Journal of Experimental Botany. 69, 22, p. 5489-5506 Abstract
Lysine (Lys) connects the mitochondria! electron transport chain to amino acid catabolism and the tricarboxylic acid cycle. However, our understanding of how a deficiency in Lys biosynthesis impacts plant metabolism and growth remains limited. Here, we used a previously characterized Arabidopsis mutant (dapat) with reduced activity of the Lys biosynthesis enzyme L,L-diaminopimelate aminotransferase to investigate the physiological and metabolic impacts of impaired Lys biosynthesis. Despite displaying similar stomata! conductance and internal CO2 concentration, we observed reduced photosynthesis and growth in the dapat mutant. Surprisingly, whilst we did not find differences in dark respiration between genotypes, a lower storage and consumption of starch and sugars was observed in dapat plants. We found higher protein turnover but no differences in total amino acids during a diurnal cycle in dapat plants. Transcriptional and two-dimensional (isoelectric focalization/SDS-PAGE) proteome analyses revealed alterations in the abundance of several transcripts and proteins associated with photosynthesis and photorespiration coupled with a high glycine/serine ratio and increased levels of stress-responsive amino acids. Taken together, our findings demonstrate that biochemical alterations rather than stomatal limitations are responsible for the decreased photosynthesis and growth of the dapat mutant, which we hypothesize mimics stress conditions associated with impairments in the Lys biosynthesis pathway.
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(2018) Plant Science. 275, p. 11-18 Abstract
Amino acids play vital roles in the central metabolism of seeds. They are primarily utilized for the synthesis of seed-storage proteins, but also serve as precursors for the biosynthesis of secondary metabolites and as a source of energy. Here, we aimed at describing the knowledge accumulated in recent years describing the changes occurring in the contents of free amino acids (FAAs) during seed development. Since several essential amino acids are found in low levels in seeds (e.g., Lys, Met, Thr, Val, Leu, Ile and His), or play unique functional roles in seed development (e.g., Pro and the non-proteinogenic γ-aminobutyrate [GABA]), we also briefly describe studies carried out in order to alter their levels in seeds and determine the effects of the manipulation on seed biology. The lion share of these studies highlights strong positive correlations between the biosynthetic pathways of FAAs, meaning that when the levels of a certain amino acid change in seeds, the contents of other FAAs tend to elevate as well. These observations infer a tight regulatory network operating in the biosynthesis of FAAs during seed development.
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(2018) The Plant Endoplasmic Reticulum. p. 239-249 Abstract
Macroautophagy (hereafter referred to as autophagy) is a conserved mechanism in eukaryotic cells that delivers unneeded cellular components for degradation in the lytic organelle. In plants, as in other eukaryotes, autophagy begins in the formation of cup-shaped double membranes that engulf cytosolic material. The double membrane closes to form autophagosomes that are then transported to the vacuole for degradation. Autophagy can function as a bulk nonselective process or as a selective process targeting specific proteins, protein aggregates, organelles, or other cellular components for degradation. The endoplasmic reticulum (ER) is linked to autophagy-related processes in multiple ways. The ER was suggested as a possible site for the nucleation of autophagosomes, and as a source for autophagosomal membranes. Furthermore, autophagy has an important role in ER homeostasis, and the ER is a target for a selective type of autophagy, ER-phagy, in response to ER stress. However, the detailed molecular mechanisms, especially in plants, are only now starting to be revealed. In this chapter, we describe the use of confocal imaging to follow the delivery of fluorescently tagged ER-associated proteins to the vacuole. We also describe the utilization of fluorescent protein fusions to look at the co-localization of a protein of interest with the autophagosome marker protein ATG8, a core autophagy machinery protein that is essential for selective autophagy processes.
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(2018) Plant Reproduction. 31, p. 203-211 Abstract
Aspartate (Asp)-family pathway, via several metabolic branches, leads to four key essential amino acids: Lys, Met, Thr, and Ile. Among these, Lys and Met have received the most attention, as they are the most limiting amino acid in cereals and legumes crops, respectively. The metabolic pathways of these four essential amino acids and their interactions with regulatory networks have been well characterized. Using this knowledge, extensive efforts have been devoted to augmenting the levels of these amino acids in various plant organs, especially seeds, which serve as the main source of human food and livestock feed. Seeds store a number of storage proteins, which are utilized as nutrient and energy resources. Storage proteins are composed of amino acids, to guarantee the continuation of plant progeny. Thus, understanding the seed metabolism, especially with respect to the accumulation of aspartate-derived amino acids Lys and Met, is a crucial factor for sustainable agriculture. In this review, we summarized the Asp-family pathway, with some new examples of accumulated Asp-family amino acids, particularly Lys and Met, in plant seeds. We also discuss the recent advances in understanding the roles of Asp-family amino acids during seed development.
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(2018) Journal of Experimental Botany. 69, 6, p. 1335-1353 Abstract
Autophagy is a eukaryotic catabolic pathway essential for growth and development. In plants, it is activated in response to environmental cues or developmental stimuli. However, in contrast to other eukaryotic systems, we know relatively little regarding the molecular players involved in autophagy and the regulation of this complex pathway. In the framework of the COST (European Cooperation in Science and Technology) action TRANSAUTOPHAGY (2016-2020), we decided to review our current knowledge of autophagy responses in higher plants, with emphasis on knowledge gaps. We also assess here the potential of translating the acquired knowledge to improve crop plant growth and development in a context of growing social and environmental challenges for agriculture in the near future.
2017
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(2017) Seed Development and Germination. p. 811-831 Abstract
Humans and monogastric animals cannot synthesize ten out of the twenty protein amino acids and therefore should obtain them in the diet. Among the essential amino acids, lysine and threonine are considered exceedingly important, inasmuch as they are among the most limiting essential amino acids in cereal grains, which represent the largest source of food worldwide. Cereal based diets for livestock are routinely supplemented with lysine plus threonine, and such supplements were proven to be of high beneficial value to livestock growth (Bright and Shewry, 1983; Cuaron et aL, 1984; Fuller et aL, 1979).
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(2017) International Journal of Molecular Sciences. 18, 6, 1306. Abstract
To feed the worlds growing population, increasing the yield of crops is not the only important factor, improving crop quality is also important, and it presents a significant challenge. Among the important crops, horticultural crops (particularly fruits and vegetables) provide numerous health compounds, such as vitamins, antioxidants, and amino acids. Essential amino acids are those that cannot be produced by the organism and, therefore, must be obtained from diet, particularly from meat, eggs, and milk, as well as a variety of plants. Extensive efforts have been devoted to increasing the levels of essential amino acids in plants. Yet, these efforts have been met with very little success due to the limited genetic resources for plant breeding and because high essential amino acid content is generally accompanied by limited plant growth. With a deep understanding of the biosynthetic pathways of essential amino acids and their interactions with the regulatory networks in plants, it should be possible to use genetic engineering to improve the essential amino acid content of horticultural plants, rendering these plants more nutritionally favorable crops. In the present report, we describe the recent advances in the enhancement of essential amino acids in horticultural plants and possible future directions towards their bio-fortification.
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(2017) Frontiers in Plant Science. 8, 769. Abstract
Phenylalanine (Phe) is a precursor for a large group of plant specialized metabolites, including the fragrant volatile benzenoidphenylpropanoids (BPs). In plants, the main pathway leading to production of Phe is via arogenate, while the pathway via phenylpyruvate (PPY) is considered merely an alternative route. Unlike plants, in most microorganisms the only pathway leading to the synthesis of Phe is via PPY. Here we studied the effect of increased PPY production in petunia on the formation of BPs volatiles and other specialized metabolites originating from Phe both in flowers and leaves. Stimulation of the pathway via PPY was achieved by transforming petunia with PheA∗, a gene encoding a bacterial feedback insensitive bi-functional chorismate mutase/prephenate dehydratase enzyme. PheA∗ overexpression caused dramatic increase in the levels of flower BP volatiles such as phenylacetaldehyde, benzaldehyde, benzyl acetate, vanillin, and eugenol. All three BP pathways characterized in petunia flowers were stimulated in PheA∗ flowers. In contrast, PheA∗ overexpression had only a minor effect on the levels of amino acids and non-volatile metabolites both in the leaves and flowers. The one exception is a dramatic increase in the level of rosmarinate, a conjugate between Phe-derived caffeate and Tyr-derived 3,4-dihydroxyphenylacetate, in PheA∗ leaves. PheA∗ petunia flowers may serve as an excellent system for revealing the role of PPY in the production of BPs, including possible routes directly converting PPY to the fragrant volatiles. This study emphasizes the potential of the PPY route in achieving fragrance enhancement in flowering plants.
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(2017) Frontiers in Plant Science. 7, 2030. Abstract
Plants are sessile organisms that cannot escape from stressful environments, such as drought, high salinity, high temperature, and shortage of essential minerals in the soil. Hence, plants have evolved processes that protect them from these harmful conditions. One of these major processes is autophagy (which means, \u201cself-eating\u201d), a mechanism that destroys specific compounds that participate in efficient growth and requires extensive energy input and on the other hand stimulates biological processes that protects from the stress. Autophagy can be either a bulk process, turning over bulk amounts of various components in response to major stresses, such as serious accumulation of damaging compounds in the soil, or a selective process turning over specific components in response to specific and/or relatively minor environmental cues, such as minor shortage of rain and/or non-significant shortage of minerals in the soil.
2016
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(2016) Plant Biotechnology Journal. 14, 12, p. 2300-2309 Abstract
Targeted manipulation of phenylalanine (Phe) synthesis is a potentially powerful strategy to boost biologically and economically important metabolites, including phenylpropanoids, aromatic volatiles and other specialized plant metabolites. Here, we use two transgenes to significantly increase the levels of aromatic amino acids, tomato flavour-associated volatiles and antioxidant phenylpropanoids. Overexpression of the petunia MYB transcript factor, ODORANT1 (ODO1), combined with expression of a feedback-insensitive E.coli 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (AroG), altered the levels of multiple primary and secondary metabolites in tomato fruit, boosting levels of multiple secondary metabolites. Our results indicate that coexpression of AroG and ODO1 has a dual effect on Phe and related biosynthetic pathways: (i) positively impacting tyrosine (Tyr) and antioxidant related metabolites, including ones derived from coumaric acid and ferulic acid; (ii) negatively impacting other downstream secondary metabolites of the Phe pathway, including kaempferol-, naringenin- and quercetin-derived metabolites, as well as aromatic volatiles. The metabolite profiles were distinct from those obtained with either single transgene. In addition to providing fruits that are increased in flavour and nutritional chemicals, coexpression of the two genes provides insights into regulation of branches of phenylpropanoid metabolic pathways.
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(2016) Journal of Experimental Botany. 67, 14, p. 4009-4011 Abstract
Lysine is considered an essential amino acid required at sufficient levels to prevent malnutrition and serious diseases, particularly in developing countries. It is mostly obtained from various plant foods. In the current issue (pages 42854296) Yang and colleagues report on successful stimulation of lysine biosynthesis and suppression of its catabolism in transgenic rice plants without changing the plant phenotype. This approach led to the production of high-lysine rice plants, rendering them as nutritionally favourable crops.
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(2016) Autophagy. 12, 5, p. 876-887 Abstract
ABSTRACT: Most of the proteins that are specifically turned over by selective autophagy are recognized by the presence of short Atg8 interacting motifs (AIMs) that facilitate their association with the autophagy apparatus. Such AIMs can be identified by bioinformatics methods based on their defined degenerate consensus F/W/Y-X-X-L/I/V sequences in which X represents any amino acid. Achieving reliability and/or fidelity of the prediction of such AIMs on a genome-wide scale represents a major challenge. Here, we present a bioinformatics approach, high fidelity AIM (hfAIM), which uses additional sequence requirementsthe presence of acidic amino acids and the absence of positively charged amino acids in certain positionsto reliably identify AIMs in proteins. We demonstrate that the use of the hfAIM method allows for in silico high fidelity prediction of AIMs in AIM-containing proteins (ACPs) on a genome-wide scale in various organisms. Furthermore, by using hfAIM to identify putative AIMs in the Arabidopsis proteome, we illustrate a potential contribution of selective autophagy to various biological processes. More specifically, we identified 9 peroxisomal PEX proteins that contain hfAIM motifs, among which AtPEX1, AtPEX6 and AtPEX10 possess evolutionary-conserved AIMs. Bimolecular fluorescence complementation (BiFC) results verified that AtPEX6 and AtPEX10 indeed interact with Atg8 in planta. In addition, we show that mutations occurring within or nearby hfAIMs in PEX1, PEX6 and PEX10 caused defects in the growth and development of various organisms. Taken together, the above results suggest that the hfAIM tool can be used to effectively perform genome-wide in silico screens of proteins that are potentially regulated by selective autophagy. The hfAIM system is a web tool that can be accessed at link: http://bioinformatics.psb.ugent.be/hfAIM/.
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(2016) Annual Review of Plant Biology. 67, p. 153-178 Abstract
Although amino acids are critical for all forms of life, only proteogenic amino acids that humans and animals cannot synthesize de novo and therefore must acquire in their diets are classified as essential. Nine amino acids-lysine, methionine, threonine, phenylalanine, tryptophan, valine, isoleucine, leucine, and histidine-fit this definition. Despite their nutritional importance, several of these amino acids are present in limiting quantities in many of the world's major crops. In recent years, a combination of reverse genetic and biochemical approaches has been used to define the genes encoding the enzymes responsible for synthesizing, degrading, and regulating these amino acids. In this review, we describe recent advances in our understanding of the metabolism of the essential amino acids, discuss approaches for enhancing their levels in plants, and appraise efforts toward their biofortification in crop plants.
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(2016) Trends in Plant Science. 21, 2, p. 134-144 Abstract
Autophagy is a major cellular degradation pathway in eukaryotes. Recent studies have revealed the importance of autophagy in many aspects of plant life, including seedling establishment, plant development, stress resistance, metabolism, and reproduction. This is manifested by the dual ability of autophagy to execute bulk degradation under severe environmental conditions, while simultaneously to be highly selective in targeting specific compartments and protein complexes to regulate key cellular processes, even during favorable growth conditions. Delivery of cellular components to the vacuole enables their recycling, affecting the plant metabolome, especially under stress. Recent research in Arabidopsis has further unveiled fundamental mechanistic aspects in autophagy which may have relevance in non-plant systems. We review the most recent discoveries concerning autophagy in plants, touching upon all these aspects. Autophagy is involved in almost every aspect of plant life, including germination, seedling establishment, development, reproduction, metabolism, and stress tolerance.Proteins that are involved in fundamental processes of autophagy, such as autophagosome biogenesis, were recently characterized in plants.Autophagy is intimately associated with other intracellular trafficking pathways.Several selective autophagy pathways were recently identified in Arabidopsis; most are common to all eukaryotes. Nevertheless, some pathways were initially discovered in plants and others are plant-specific.As an intracellular recycling system, autophagy is highly important for proper plant metabolism and nutrient allocation, both during stress and favorable growth conditions.
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2015
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(2015) Frontiers in Plant Science. 6, SEPTEMBER, 785. Abstract
Glutamate derived γ-aminobutyric acid (GABA) is synthetized in the cytosol prior to delivery to the mitochondria where it is catabolized via the TCA cycle. GABA accumulates under various environmental conditions, but an increasing number of studies show its involvement at the crossroad between C and N metabolism. To assess the role of GABA in modulating cellular metabolism, we exposed seedlings of A. thaliana GABA transporter gat1 mutant to full nutrition medium and media deficient in C and N combined with feeding of different concentrations (0.5 and 1 mM) of exogenous GABA. GC-MS based metabolite profiling showed an expected effect of medium composition on the seedlings metabolism of mutant and wild type alike. That being said, a significant interaction between GAT1 deficiency and medium composition was determined with respect to magnitude of change in relative amino acid levels. The effect of exogenous GABA treatment on metabolism was contingent on both the medium and the genotype, leading for instance to a drop in asparagine under full nutrition and low C conditions and glucose under all tested media, but not to changes in GABA content. We additionally assessed the effect of GAT1 deficiency on the expression of glutamate metabolism related genes and genes involved in abiotic stress responses. These results suggest a role for GAT1 in GABA-mediated metabolic alterations in the context of the C-N equilibrium of plant cells.
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(2015) Frontiers in Plant Science. 6, JULY, 538. Abstract
Environmental stresses such as high light intensity and temperature cause induction of the shikimate pathway, aromatic amino acids (AAA) pathways, and of pathways downstream from AAAs. The induction leads to production of specialized metabolites that protect the cells from oxidative damage. The regulation of the diverse AAA derived pathways is still not well understood. To gain insight on that regulation, we increased AAA production in red grape Vitis vinifera cv. Gamay Red cell suspension, without inducing external stress on the cells, and characterized the metabolic effect of this induction. Increased AAA production was achieved by expressing a feedback-insensitive bacterial form of 3-deoxy- D-arabino-heptulosonate 7-phosphate synthase enzyme (AroG*) of the shikimate pathway under a constitutive promoter. The presence of AroG* protein led to elevated levels of primary metabolites in the shikimate and AAA pathways including phenylalanine and tyrosine, and to a dramatic increase in phenylpropanoids. The AroG* transformed lines accumulated up to 20 and 150 fold higher levels of resveratrol and dihydroquercetin, respectively. Quercetin, formed from dihydroquercetin, and resveratrol, are health promoting metabolites that are induced due to environmental stresses. Testing the expression level of key genes along the stilbenoids, benzenoids, and phenylpropanoid pathways showed that transcription was not affected by AroG*. This suggests that concentrations of AAAs, and of phenylalanine in particular, are rate-limiting in production of these metabolites. In contrast, increased phenylalanine production did not lead to elevated concentrations of anthocyanins, even though they are also phenylpropanoid metabolites. This suggests a control mechanism of this pathway that is independent of AAA concentration. Interestingly, total anthocyanin concentrations were slightly lower in AroG* cells, and the relative frequencies of the different anthocyanins changed as well.
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(2015) AIMS bioengineering. 2, 2, p. 75-92 Abstract
The tomato (Solanum lycopersicum) fruit is an excellent source of antioxidants, dietary fibers, minerals and vitamins and therefore has been referred to as a "functional food". Ripe tomato fruits produce a large number of specialized metabolites including volatile organic compounds. These volatiles serve as key components of the tomato fruit flavor, participate in plant pathogen and herbivore defense, and are used to attract seed dispersers. A major class of specialized metabolites is derived from the shikimate pathway followed by aromatic amino acid biosynthesis of phenylalanine, tyrosine and tryptophan. We attempted to modify tomato fruit flavor by overexpressing key regulatory genes in the shikimate pathway. Bacterial genes encoding feedback-insensitive variants of 3-Deoxy-D-Arabino-Heptulosonate 7-Phosphate Synthase (DAHPS; AroG(209-9)) and bi-functional Chorismate Mutase/Prephenate Dehydratase (CM/PDT; PheA(12)) were expressed under the control of a fruit-specific promoter. We crossed these transgenes to generate tomato plants expressing both the AroG(209) and PheA(12) genes. Overexpression of the AroG(209-9) gene had a dramatic effect on the overall metabolic profile of the fruit, including enhanced levels of multiple volatile and non-volatile metabolites. In contrast, the PheA(12) overexpression line exhibited minor metabolic effects compared to the wild type fruit. Co-expression of both the AroG(209-9) and PheA(12) genes in tomato resulted overall in a similar metabolic effect to that of expressing only the AroG(209-9) gene. However, the aroma ranking attributes of the tomato fruits from PheA(12)//AroG(209-9) were unique and different from those of the lines expressing a single gene, suggesting a contribution of the PheA(12) gene to the overall metabolic profile. We suggest that expression of bacterial genes encoding feedback-insensitive enzymes of the shikimate pathway in tomato fruits provides a useful metabolic engineering tool for the modification of fruits aroma and the generation of new combinations of tomato flavors.
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(2015) Trends in Plant Science. 20, 5, p. 264-265 Abstract
Degradation of chloroplasts is a hallmark of both natural and stress-induced plant senescence. Autophagy and senescence-associated vacuoles are two established cellular pathways for chloroplast degradation. Recently, a third independent pathway for chloroplast degradation was reported. Here we will discuss this new discovery in relation to the other known pathways.
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(2015) Plant Biotechnology Journal. 13, 1, p. 125-136 Abstract
Purple Petunia × hybrida V26 plants accumulate fragrant benzenoid-phenylpropanoid molecules and anthocyanin pigments in their petals. These specialized metabolites are synthesized mainly from the aromatic amino acids phenylalanine. Here, we studied the profile of secondary metabolites of petunia plants, expressing a feedback-insensitive bacterial form of 3-deoxy-di-arabino-heptulosonate 7-phosphate synthase enzyme (AroG*) of the shikimate pathway, as a tool to stimulate the conversion of primary to secondary metabolism via the aromatic amino acids. We focused on specialized metabolites contributing to flower showy traits. The presence of AroG* protein led to increased aromatic amino acid levels in the leaves and high phenylalanine levels in the petals. In addition, the AroG* petals accumulated significantly higher levels of fragrant benzenoid-phenylpropanoid volatiles, without affecting the flowers' lifetime. In contrast, AroG* abundance had no effect on flavonoids and anthocyanins levels. The metabolic profile of all five AroG* lines was comparable, even though two lines produced the transgene in the leaves, but not in the petals. This implies that phenylalanine produced in leaves can be transported through the stem to the flowers and serve as a precursor for formation of fragrant metabolites. Dipping cut petunia stems in labelled phenylalanine solution resulted in production of labelled fragrant volatiles in the flowers. This study emphasizes further the potential of this metabolic engineering approach to stimulate the production of specialized metabolites and enhance the quality of various plant organs. Furthermore, transformation of vegetative tissues with AroG* is sufficient for induced production of specialized metabolites in organs such as the flowers.
2014
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(2014) Plant Cell. 26, 10, p. 4084-4101 Abstract
Selective autophagy has been extensively studied in various organisms, but knowledge regarding its functions in plants, particularly in organelle turnover, is limited. We have recently discovered ATG8-INTERACTING PROTEIN1 (ATI1) from Arabidopsis thaliana and showed that following carbon starvation it is localized on endoplasmic reticulum (ER)-associated bodies that are subsequently transported to the vacuole. Here, we show that following carbon starvation ATI1 is also located on bodies associating with plastids, which are distinct from the ER ATI bodies and are detected mainly in senescing cells that exhibit plastid degradation. Additionally, these plastid-localized bodies contain a stroma protein marker as cargo and were observed budding and detaching from plastids. ATI1 interacts with plastid-localized proteins and was further shown to be required for the turnover of one of them, as a representative. ATI1 on the plastid bodies also interacts with ATG8f, which apparently leads to the targeting of the plastid bodies to the vacuole by a process that requires functional autophagy. Finally, we show that ATI1 is involved in Arabidopsis salt stress tolerance. Taken together, our results implicate ATI1 in autophagic plastid-to-vacuole trafficking through its ability to interact with both plastid proteins and ATG8 of the core autophagy machinery.
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(2014) Frontiers in Plant Science. 5, 447. Abstract
Seeds are the major organs responsible for the evolutionary upkeep of angiosperm plants. Seeds accumulate significant amounts of storage compounds used as nutrients and energy reserves during the initial stages of seed germination. The accumulation of storage compounds requires significant amounts of energy, the generation of which can be limited due to reduced penetration of oxygen and light particularly into the inner parts of seeds. In this review, we discuss the adjustment of seed metabolism to limited energy production resulting from the suboptimal penetration of oxygen into the seed tissues. We also discuss the role of photosynthesis during seed development and its contribution to the energy status of developing seeds. Finally, we describe the contribution of amino acid metabolism to the seed energy status, focusing on the Asp-family pathway that leads to the synthesis and catabolism of Lys, Thr, Met, and Ile.
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(2014) International Journal of Molecular Sciences. 15, 5, p. 7624-7638 Abstract
Macroautophagy (hereafter referred to as autophagy) is a cellular mechanism dedicated to the degradation and recycling of unnecessary cytosolic components by their removal to the lytic compartment of the cell (the vacuole in plants). Autophagy is generally induced by stresses causing energy deprivation and its operation occurs by special vesicles, termed autophagosomes. Autophagy also operates in a selective manner, recycling specific components, such as organelles, protein aggregates or even specific proteins, and selective autophagy is implicated in both cellular housekeeping and response to stresses. In plants, selective autophagy has recently been shown to degrade mitochondria, plastids and peroxisomes, or organelle components such as the endoplasmic-reticulum (ER) membrane and chloroplast-derived proteins such as Rubisco. This ability places selective-autophagy as a major factor in cellular steady-state maintenance, both under stress and favorable environmental conditions. Here we review the recent advances documented in plants for this cellular process and further discuss its impact on plant physiology.
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(2014) Frontiers in Plant Science. 5, APR, 134. Abstract
Trafficking of proteins from the endoplasmic reticulum (ER) to the vacuole is a fundamental process in plants, being involved both in vacuole biogenesis as well as with plant growth and response to environmental stresses. Although the canonical transport of cellular components from the ER to the vacuole includes the Golgi apparatus as an intermediate compartment, there are multiple lines of evidence that support the existence of a direct ER-to-vacuole, Golgi-independent, trafficking route in plants that uses the autophagy machinery. Plant autophagy was initially described by electron microscopy, visualizing cellular structures that are morphologically reminiscent of autophagosomes. In some of these reports these structures were shown to transport vacuole residing proteins, particularly seed storage proteins, directly from the ER to the vacuole. More recently, following the discovery of the proteins of the core autophagy machinery, molecular tools were implemented in deciphering the involvement of autophagy in this special trafficking route. Here we review the relatively older and more recent scientific observations, supporting the involvement of autophagy in the special cellular trafficking pathways of plants.
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(2014) Omics Technologies and Crop Improvement. p. 343-366 Abstract
Although amino acids primarily serve as the building blocks of proteins, in plants amino acids also serve as important alternative energy substrates, particularly when plants are exposed to stresses that cause energy deprivation. The contribution of amino acids to energy homeostasis in response to stress occurs through their catabolism, which funnels their carbon backbones into the tricarboxylic acid (TCA) cycle, providing an alternative source of electrons for the mitochondrial electron transport chain. As such, amino acid catabolism, which also includes the association of the amino acid Glu and the γ-aminobutyric acid (GABA) shunt, is becoming recognized as a critical element of energy metabolism in stress and carbon starvation. In the first part of the present review, we focus on recent findings concerning modes of action of amino acid catabolism as an alternative source of energy in stress conditions. Second, we reveal how systems biology approaches, including gene coexpression analysis and transcript and metabolic profiling, led to the discovery of new biological processes associating plant amino-acid (AA) metabolism with response and adaptation to stress.
2013
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(2013) Journal of Experimental Botany. 64, 14, p. 4441-4452 Abstract
Tomato (Solanum lycopersicum) fruit contains significant amounts of bioactive compounds, particularly multiple classes of specialized metabolites. Enhancing the synthesis and accumulation of these substances, specifically in fruits, are central for improving tomato fruit quality (e.g. favour and aroma) and could aid in elucidate pathways of specialized metabolism. To promote the production of specialized metabolites in tomato fruit, this work expressed under a fruit ripening-specific promoter, E8, a bacterial AroG gene encoding a 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAHPS), which is feedback-insensitive to phenylalanine inhibition. DAHPS, the first enzyme of the shikimate pathway, links between the primary and specialized metabolism derived from aromatic amino acids. AroG expression influenced the levels of number of primary metabolites, such as shikimic acid and aromatic amino acids, as well as multiple volatile and non-volatile phenylpropanoids specialized metabolites and carotenoids. An organoleptic test, performed by trained panellists, suggested that the ripe AroG-expressing tomato fruits had a preferred floral aroma compare with fruits of the wild-type line. These results imply that fruit-specific manipulation of the conversion of primary to specialized metabolism is an attractive approach for improving fruit aroma and favour qualities as well as discovering novel fruit-specialized metabolites.
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(2013) PLANT SIGNALING & BEHAVIOR. 8, 10, p. doi: 10.4161/psb.26732 Abstract
The exocyst complex is a multi-subunits evolutionary conserved complex, which was originally shown to be primarily associated with vesicular transport to the plasma membrane. A recent report (Kulich et al., 2013 Traffic; In Press) revealed that AtEXO70B1, one of the multiple subunits of the exocyst complex of Arabidopsis thaliana plants, is co-transported with the autophagy-associated Atg8f protein to the vacuole. This pathway does not involve the Golgi apparatus. The co-localization of AtEXO70B1 and Atg8f suggests either that both of these proteins are co-transported together to the vacuole or, alternatively, that Atg8 binds to a putative Atg8 interacting motif (AIM) located within the AtEXO70B1 polypeptide, apparently forming a tethering complex for an autophagic complex that is transported to the vacuole. In the present addendum, by tooling a bioinformatics approach, we show that AtEXO70B1 as well as the additional 20 paralogs of Arabidopsis EXO70 exocyst subunits each possess one or more AIMs whose consensus sequence implies their high fidelity binding to Atg8. This indicates that the autophagy machinery is strongly involved in the assembly, transport, and apparently also the function of AtEXO70B1 as well as the exocyst sub complex.
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(2013) Acta Crystallographica Section F-Structural Biology And Crystallization Communications. 69, 2, p. 84-89 Abstract
Diaminopimelate aminotransferase (DAP-AT) is an enzyme in the lysine-biosynthesis pathway. Conversely, ALD1, a close homologue of DAP-AT in plants, uses lysine as a substrate in vitro. Both proteins require pyridoxal-5-phosphate (PLP) for their activity. The structure of ALD1 from the flowering plant Arabidopsis thaliana (AtALD1) was solved at a resolution of 2.3 Å. Comparison of AtALD1 with the previously solved structure of A. thaliana DAP-AT (AtDAP-AT) revealed similar interactions with PLP despite sequence differences within the PLP-binding site. However, sequence differences between the binding site of AtDAP-AT for malate, a purported mimic of substrate binding, and the corresponding site in AtALD1 led to different interactions. This suggests that either the substrate itself, or the substrate-binding mode, differs in the two proteins, supporting the known in vitro findings.
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(2013) Plant Biotechnology Journal. 11, 2, p. 211-222 Abstract
Humans, as well as farm animals, cannot synthesize a number of essential amino acids, which are critical for their survival. Hence, these organisms must obtain these essential amino acids from their diets. Cereal and legume crops, which represent the major food and feed sources for humans and livestock worldwide, possess limiting levels of some of these essential amino acids, particularly Lys and Met. Extensive efforts were made to fortify crop plants with these essential amino acids using traditional breeding and mutagenesis. However, aside from some results obtained with maize, none of these approaches was successful. Therefore, additional efforts using genetic engineering approaches concentrated on increasing the synthesis and reducing the catabolism of these essential amino acids and also on the expression of recombinant proteins enriched in them. In the present review, we discuss the basic biological aspects associated with the synthesis and accumulation of these amino acids in plants and also describe recent developments associated with the fortification of crop plants with essential amino acids by genetic engineering approaches.
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(2013) Plant Physiology. 161, 2, p. 628-643 Abstract
The aim of this work was to investigate the effect of decreased cytosolic phosphoenolpyruvate carboxykinase (PEPCK) and plastidic NADP-dependent malic enzyme (ME) on tomato (Solanum lycopersicum) ripening. Transgenic tomato plants with strongly reduced levels of PEPCK and plastidic NADP-ME were generated by RNA interference gene silencing under the control of a ripening-specific E8 promoter. While these genetic modifications had relatively little effect on the total fruit yield and size, they had strong effects on fruit metabolism. Both transformants were characterized by lower levels of starch at breaker stage. Analysis of the activation state of ADP-glucose pyrophosphorylase correlated with the decrease of starch in both transformants, which suggests that it is due to an altered cellular redox status. Moreover, metabolic profiling and feeding experiments involving positionally labeled glucoses of fruits lacking in plastidic NADP-ME and cytosolic PEPCK activities revealed differential changes in overall respiration rates and tricarboxylic acid (TCA) cycle flux. Inactivation of cytosolic PEPCK affected the respiration rate, which suggests that an excess of oxaloacetate is converted to aspartate and reintroduced in the TCA cycle via 2-oxoglutarate/glutamate. On the other hand, the plastidic NADP-ME antisense lines were characterized by no changes in respiration rates and TCA cycle flux, which together with increases of pyruvate kinase and phosphoenolpyruvate carboxylase activities indicate that pyruvate is supplied through these enzymes to the TCA cycle. These results are discussed in the context of current models of the importance of malate during tomato fruit ripening.
2012
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(2012) Journal of Experimental Botany. 63, 14, p. 4995-5001 Abstract
Plants represent the major sources of human foods and livestock feeds, worldwide. However, the limited content of the essential amino acid lysine in cereal grains represents a major nutritional problem for human and for livestock feeding in developed countries. Optimizing the level of lysine in cereal grains requires extensive knowledge on the biological processes regulating the homeostasis of this essential amino acid as well as the biological consequences of this homeostasis. Manipulating biosynthetic and catabolic enzymes of lysine metabolism enabled an enhanced accumulation of this essential amino acid in seeds. However, this approach had a major effect on the levels of various metabolites of the tricarboxylic acid (TCA) cycle, revealing a strong interaction between lysine metabolism and cellular energy metabolism. Recent studies discussed here have shed new light on the metabolic processes responsible for the catabolism of lysine, as well as isoleucine, another amino acid of the aspartate-family pathway, into the TCA cycle. Here we discuss progress being made to understand biological processes associated with the catabolism of amino acids of the aspartate-family pathway and its importance for optimal improvement of the nutritional quality of plants.
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(2012) Plant Journal. 70, 6, p. 954-966 Abstract
Plants need to continuously adjust their transcriptome in response to various stresses that lead to inhibition of photosynthesis and the deprivation of cellular energy. This adjustment is triggered in part by a coordinated re-programming of the energy-associated transcriptome to slow down photosynthesis and activate other energy-promoting gene networks. Therefore, understanding the stress-related transcriptional networks of genes belonging to energy-associated pathways is of major importance for engineering stress tolerance. In a bioinformatics approach developed by our group, termed 'gene coordination', we previously divided genes encoding for enzymes and transcription factors in Arabidopsis thaliana into three clusters, displaying altered coordinated transcriptional behaviors in response to multiple biotic and abiotic stresses (Plant Cell, 23, 2011, 1264). Enrichment analysis indicated further that genes controlling energy-associated metabolism operate as a compound network in response to stress. In the present paper, we describe in detail the network association of genes belonging to six central energy-associated pathways in each of these three clusters described in our previous paper. Our results expose extensive stress-associated intra- and inter-pathway interactions between genes from these pathways, indicating that genes encoding proteins involved in energy-associated metabolism are expressed in a highly coordinated manner. We also provide examples showing that this approach can be further utilized to elucidate candidate genes for stress tolerance and functions of isozymes.
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(2012) Autophagy. 8, 5, p. 838-839 Abstract
Selective autophagy, mediated by Atg8 binding proteins, has not been extensively studied in plants. Plants possess a large gene family encoding multiple isoforms of the Atg8 protein. We have recently reported the identification of two new, closely homologous Arabidopsis thaliana plant proteins that bind the Arabidopsis Atg8f protein isoform. These two proteins are specific to plants and have no homologs in nonplant organisms. The expression levels of the genes encoding these proteins are elevated during carbon starvation and also during late stages of seed development. Exposure of young seedlings to carbon starvation induces the production of a newly identified compartment decorated by these Atg8-binding proteins. This compartment dynamically moves along the endoplasmic reticulum membrane and is also finally transported into the vacuole. Enhanced or suppressed expression of these Atg8-binding proteins respectively enhances or suppresses seed germination under suboptimal germination conditions, indicating that they contribute to seed germination vigor.
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(2012) Plant Signaling and Behavior. 7, 6, p. 685-687 Abstract
Autophagy is a mechanism used for the transport of macromolecules to the vacuole for degradation. It can be either non-selective or selective, resulting from the specific binding of target proteins to Atg8, an essential autophagyrelated protein. Nine Atg8 homologs exist in the model plant Arabidopsis thaliana, suggesting possible different roles for different homologs. In a previous report published in the Plant Cell, our group identified two plant-specific proteins, termed ATI1 and ATI2, which bind Atg8f, as a representative of the nine Atg8 homologs. The proteins were shown to associate with novel starvationinduced bodies that move on the ER network and reach the lytic vacuole. Altered expression level of the proteins was also shown to affect the ability of seeds to germinate in the presence of the germination inhibiting hormone ABA. In the present addendum article, we demonstrate that, in addition to Atg8f, ATI1 binds Atg8h, an Atg8 homolog from a different sub-family, indicating that ATI1 is not a specific target of Atg8f.
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(2012) NEW PHYTOLOGIST. 194, 2, p. 430-439 Abstract
The shikimate pathway of plants mediates the conversion of primary carbon metabolites via chorismate into the three aromatic amino acids and to numerous secondary metabolites derived from them. However, the regulation of the shikimate pathway is still far from being understood. We hypothesized that 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAHPS) is a key enzyme regulating flux through the shikimate pathway. To test this hypothesis, we expressed a mutant bacterial AroG gene encoding a feedback-insensitive DAHPS in transgenic Arabidopsis plants. The plants were subjected to detailed analysis of primary metabolism, using GC-MS, as well as secondary metabolism, using LC-MS. Our results exposed a major effect of bacterial AroG expression on the levels of shikimate intermediate metabolites, phenylalanine, tryptophan and broad classes of secondary metabolite, such as phenylpropanoids, glucosinolates, auxin and other hormone conjugates. We propose that DAHPS is a key regulatory enzyme of the shikimate pathway. Moreover, our results shed light on additional potential metabolic bottlenecks bridging plant primary and secondary metabolism.
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(2012) Protoplasma. 249, 2, p. 285-299 Abstract
Autophagy is an evolutionary conserved process of bulk degradation and nutrient sequestration that occurs in all eukaryotic cells. Yet, in recent years, autophagy has also been shown to play a role in the specific degradation of individual proteins or protein aggregates as well as of damaged organelles. The process was initially discovered in yeast and has also been very well studied in mammals and, to a lesser extent, in plants. In this review, we summarize what is known regarding the various functions of autopahgy in plants but also attempt to address some specific issues concerning plant autophagy, such as the insufficient knowledge regarding autophagy in various plant species other than Arabidopsis, the fact that some genes belonging to the core autophagy machinery in various organisms are still missing in plants, the existence of autophagy multigene families in plants and the possible operation of selective autophagy in plants, a study that is still in its infancy. In addition, we point to plant-specific autophagy processes, such as the participation of autophagy during development and germination of the seed, a unique plant organ. Throughout this review, we demonstrate that the use of innovative bioinformatic resources, together with recent biological discoveries (such as the ATG8-interacting motif), should pave the way to a more comprehensive understanding of the multiple functions of plant autophagy.
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(2012) Plant Cell. 24, 1, p. 288-303 Abstract
Atg8 is a central protein in bulk starvation-induced autophagy, but it is also specifically associated with multiple protein targets under various physiological conditions to regulate their selective turnover by the autophagy machinery. Here, we describe two new closely related Arabidopsis thaliana Atg8-interacting proteins (ATI1 and ATI2) that are unique to plants. We show that under favorable growth conditions, ATI1 and ATI2 are partially associated with the endoplasmic reticulum (ER) membrane network, whereas upon exposure to carbon starvation, they become mainly associated with newly identified spherical compartments that dynamically move along the ER network. These compartments are morphologically distinct from previously reported spindle-shaped ER bodies and, in contrast to them, do not contain ER-lumenal markers possessing a C-terminal HDEL sequence. Organelle and autophagosome-specific markers show that the bodies containing ATI1 are distinct from Golgi, mitochondria, peroxisomes, and classical autophagosomes. The final destination of the ATI1 bodies is the central vacuole, indicating that they may operate in selective turnover of specific proteins. ATI1 and ATI2 gene expression is elevated during late seed maturation and desiccation. We further demonstrate that ATI1 overexpression or suppression of both ATI1 and ATI2, respectively, stimulate or inhibit seed germination in the presence of the germinationinhibiting hormone abscisic acid.
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2011
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(2011) Plant Physiology. 157, 3, p. 1026-1042 Abstract
In seeds, glutamate decarboxylase (GAD) operates at the metabolic nexus between carbon and nitrogen metabolism by catalyzing the unidirectional decarboxylation of glutamate to form γ-aminobutyric acid (GABA). To elucidate the regulatory role of GAD in seed development, we generated Arabidopsis (Arabidopsis thaliana) transgenic plants expressing a truncated GAD from Petunia hybrida missing the carboxyl-terminal regulatory Ca2+-calmodulin-binding domain under the transcriptional regulation of the seed maturation-specific phaseolin promoter. Dry seeds of the transgenic plants accumulated considerable amounts of GABA, and during desiccation the content of several amino acids increased, although not glutamate or proline. Dry transgenic seeds had higher protein content than wild-type seeds but lower amounts of the intermediates of glycolysis, glycerol and malate. The total fatty acid content of the transgenic seeds was 50% lower than in the wild type, while acyl-coenzyme A accumulated in the transgenic seeds. Labeling experiments revealed altered levels of respiration in the transgenic seeds, and fractionation studies indicated reduced incorporation of label in the sugar and lipid fractions extracted from transgenic seeds. Comparative transcript profiling of the dry seeds supported the metabolic data. Cellular processes up-regulated at the transcript level included the tricarboxylic acid cycle, fatty acid elongation, the shikimate pathway, tryptophan metabolism, nitrogen-carbon remobilization, and programmed cell death. Genes involved in the regulation of germination were similarly up-regulated. Taken together, these results indicate that the GAD-mediated conversion of glutamate to GABA during seed development plays an important role in balancing carbon and nitrogen metabolism and in storage reserve accumulation.
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(2011) Plant Signaling and Behavior. 6, 9, p. 1294-1296 Abstract
The response of plants to environmental cues, particularly stresses, involves the coordinated induction or repression of gene expression. In a previous study, we developed a bioinformatics approach to analyze the mutual expression pattern of genes encoding transcription factors and metabolic enzymes upon exposure of Arabidopsis plants to abiotic and biotic stresses. The analysis resulted in three gene clusters, each displaying a unique expression pattern. In the present addendum, we address the composition of each of these three clusters in regard to the functional identity of their encoded proteins as enzymes or transcription factors.
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(2011) Planta. 233, 5, p. 1025-1040 Abstract
Initial steps of aspartate-derived biosynthesis pathway (Asp pathway) producing Lys, Thr, Met and Ile are catalyzed by bifunctional (AK/HSD) and monofunctional (AK-lys) aspartate kinase (AK) enzymes. Here, we show that transcription of all AK genes is negatively regulated under darkness and low sugar conditions. By using yeast one-hybrid assays and complementary chromatin immunoprecipitation analyses in Arabidopsis cells, the bZIP transcription factors ABI5 and DPBF4 were identified, capable of interacting with the G-box-containing enhancer of AK/HSD1 promoter. Elevated transcript levels of DPBF4 and ABI5 under darkness and low sugar conditions coincide with the repression of AK gene expression. Overexpression of ABI5, but not DPBF4, further increases this AK transcription suppression. Concomitantly, it also increases the expression of asparagines synthetase 1 (ASN1) that shifts aspartate utilization towards asparagine formation. However, in abi5 or dpbf4 mutant and abi5, dpbf4 double mutant the repression of AK expression is maintained, indicating a functional redundancy with other bZIP-TFs. A dominant-negative version of DPBF4 fused to the SRDX repressor domain of SUPERMAN could counteract the repression and stimulate AK expression under low sugar and darkness in planta. This effect was verified by showing that DPBF4-SRDX fails to recognize the AK/HSD1 enhancer sequence in yeast one-hybrid assays, but increases heterodimmer formation with DPBF4 and ABI5, as estimated by yeast two-hybrid assays. Hence it is likely that heterodimerization with DPBF4-SRDX inhibits the binding of redundantly functioning bZIP-TFs to the promoters of AK genes and thereby releases the repressing effect. These data highlight a novel transcription control of the chloroplast aspartate pathway that operates under energy limiting conditions.
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(2011) Plant Cell. 23, 4, p. 1264-1271 Abstract
The expression pattern of any pair of genes may be negatively correlated, positively correlated, or not correlated at all in response to different stresses and even different progression stages of the stress. This makes it difficult to identify such relationships by classical statistical tools such as the Pearson correlation coefficient. Hence, dedicated bioinformatics approaches that are able to identify groups of cues in which there is a positive or negative expression correlation between pairs or groups of genes are called for. We herein introduce and discuss a bioinformatics approach, termed Gene Coordination, that is devoted to the identification of specific or multiple cues in which there is a positive or negative coordination between pairs of genes and can further incorporate additional coordinated genes to form large coordinated gene networks. We demonstrate the utility of this approach by providing a case study in which we were able to discover distinct expression behavior of the energy-associated gene network in response to distinct biotic and abiotic stresses. This bioinformatics approach is suitable to a broad range of studies that compare treatments versus controls, such as effects of various cues, or expression changes between a mutant and the control wild-type genotype.
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(2011) Current Opinion in Biotechnology. 22, 2, p. 239-244 Abstract
Plants synthesize a myriad of secondary metabolites (SMs) that are derived from central or primary metabolism. While these so-called natural products have been targets for plant metabolic engineering attempts for many years, the immense value of manipulating the interface between committed steps in secondary metabolism pathways and those in primary metabolism pathways has only recently emerged. In this review we discuss a few of the major issues that should be taken into consideration in attempts to engineer the primary to secondary metabolism interface. The availability of carbon, nitrogen and sulfur resources will have a major impact on the production of specific classes of primary metabolites (PMs) and consequently on the levels and composition of SMs derived from these PMs. Recent studies have shown that transcription factors associated with the synthesis of a given class of SMs coactivate the expression of genes encoding metabolic enzymes associated with primary pathways that supply precursors to these SMs. In addition, metabolic engineering approaches, which alter post-transcriptional feedback and feedforward regulatory mechanisms of the primary-secondary metabolism interface, have been highly fruitful in Taylormade enhancements of the content of specific beneficial SMs. Lastly, the evolution of pathways of secondary metabolism from pathways of primary metabolism highlights the need to consider cases in which common enzymatic reactions and pathways take place between the two. Taken together, the available information indicates a supercoordinated gene expression networks connecting primary and secondary metabolism in plants, which should be taken into consideration in future attempts to metabolically engineer the various classes of plant SMs.
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(2011) NEW PHYTOLOGIST. 189, 1, p. 148-159 Abstract
Lysine is a nutritionally important essential amino acid, but significant elevation of its levels in Arabidopsis seeds, by enhancing its synthesis and blocking its catabolism, causes a retardation of germination. Here, we hypothesized that this negative effect is associated with changes in primary metabolism and gene expression programs that are essential for early germination. Seeds at different stages of germination sensu stricto of the seed-high-lysine genotype were subjected to detailed analysis of primary metabolism, using GC-MS, as well as microarray analysis and two-dimensional, isoelectric focusing, sodium dodecylsulfate polyacrylamide gel electrophoresis, to detect storage protein mobilization. Our results exposed a major negative effect of the seed-specific increased lysine synthesis and knockout of its catabolism on the levels of a number of TCA cycle metabolites. This metabolic alteration also influences significantly the transcriptome, primarily attenuating the boost of specific transcriptional programs that are essential for seedling establishment, such as the onset of photosynthesis, as well as the turnover of specific transcriptional programs associated with seed embryonic traits. Our results indicate that catabolism of the aspartic acid family of amino acids is an important contributor to the energy status of plants, and hence to the onset of autotrophic growth-associated processes during germination.
2010
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(2010) Molecular Plant. 3, 6, p. 956-972 Abstract
The aromatic amino acids phenylalanine, tyrosine, and tryptophan in plants are not only essential components of protein synthesis, but also serve as precursors for a wide range of secondary metabolites that are important for plant growth as well as for human nutrition and health. The aromatic amino acids are synthesized via the shikimate pathway followed by the branched aromatic amino acids biosynthesis pathway, with chorismate serving as a major intermediate branch point metabolite. Yet, the regulation and coordination of synthesis of these amino acids are still far from being understood. Recent studies on these pathways identified a number of alternative cross-regulated biosynthesis routes with unique evolutionary origins. Although the major route of Phe and Tyr biosynthesis in plants occurs via the intermediate metabolite arogenate, recent studies suggest that plants can also synthesize phenylalanine via the intermediate metabolite phenylpyruvate (PPY), similarly to many microorganisms. Recent studies also identified a number of transcription factors regulating the expression of genes encoding enzymes of the shikimate and aromatic amino acids pathways as well as of multiple secondary metabolites derived from them in Arabidopsis and in other plant species.
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(2010) Amino Acids. 39, 4, p. 1023-1028 Abstract
Amino acid metabolism is among the most important and best recognized networks within biological systems. In plants, amino acids serve multiple functions associated with growth. Besides their function in protein synthesis, the amino acids are also catabolized into energyassociated metabolites as well we into numerous secondary metabolites, which are essential for plant growth and response to various stresses. Despite the central importance of amino acids in plants growth, elucidation of the regulation of amino acid metabolism within the context of the entire system, particularly transcriptional regulation, is still in its infancy. The different amino acids are synthesized by a number of distinct metabolic networks, which are expected to possess regulatory cross interactions between them for proper coordination of their interactive functions, such as incorporation into proteins. Yet, individual amino acid metabolic networks are also expected to differentially cross interact with various genome-wide gene expression programs and metabolic networks, in respect to their functions as precursors for various metabolites with distinct functions. In the present review, we discuss our recent genomics, metabolic and bioinformatics studies, which were aimed at addressing these questions, focusing mainly on the Asp-family metabolic network as the main example and also comparing it to the aromatic amino acids metabolic network as a second example (Angelovici et al. in Plant Physiol 151:2058-2072, 2009; Less and Galili in BMC Syst Biol 3:14, 2009; Tzin et al. in Plant J 60:156- 167, 2009). Our focus on these two networks is because of the followings: (i) both networks are central to plant metabolism and growth and are also precursors for a wide range of primary and secondary metabolites that are indispensable to plant growth; (ii) the amino acids produced by these two networks are also essential to the nutrition and health of human and farm animals; and (iii) both networks contain branched pathways requiring extensive regulation of fluxes between the different branches. Additional views on the biochemistry, regulation and functional significance of the Asp-family and aromatic amino acid networks and some of their associated metabolites that are discussed in the present report, as well as the nutritional importance of Lys and Trp to human and farm animals, and attempts to improve Lys level in crop plants, can be obtained from the following reviews as examples (Radwanski and Last in Plant Cell 7:921-934, 1995; Halkier and Gershenzon in Annu Rev Plant Biol 57:303-333, 2006; Ufaz and Galili in Plant Physiol 147:954-961, 2008; Jander and Joshi in Mol Plant 3:54-65, 2010).
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(2010) Trends in Plant Science. 15, 4, p. 211-218 Abstract
The development of orthodox seeds concludes by a desiccation phase. The dry seeds then enter a phase of dormancy, also called the after-ripening phase, and become competent for germination. We discuss physiological processes as well as gene expression and metabolic programs occurring during the desiccation phase in respect to their contribution to the desiccation tolerance, dormancy competence and successful germination of the dry seeds. The transition of developing seeds from the phase of reserve accumulation to desiccation is associated with distinct gene expression and metabolic switches. Interestingly, a significant proportion of the gene expression and metabolic signatures of seed desiccation resemble those characterizing seed germination, implying that the preparation of the seeds for germination begins already during seed desiccation.
2009
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(2009) Plant Physiology. 151, 4, p. 2058-2072 Abstract
In order to elucidate transcriptional and metabolic networks associated with lysine (Lys) metabolism, we utilized developing Arabidopsis (Arabidopsis thaliana) seeds as a system in which Lys synthesis could be stimulated developmentally without application of chemicals and coupled this to a T-DNA insertion knockout mutation impaired in Lys catabolism. This seed-specific metabolic perturbation stimulated Lys accumulation starting from the initiation of storage reserve accumulation. Our results revealed that the response of seed metabolism to the inducible alteration of Lys metabolism was relatively minor; however, that which was observable operated in a modular manner. They also demonstrated that Lys metabolism is strongly associated with the operation of the tricarboxylic acid cycle while largely disconnected from other metabolic networks. In contrast, the inducible alteration of Lys metabolism was strongly associated with gene networks, stimulating the expression of hundreds of genes controlling anabolic processes that are associated with plant performance and vigor while suppressing a small number of genes associated with plant stress interactions. The most pronounced effect of the developmentally inducible alteration of Lys metabolism was an induction of expression of a large set of genes encoding ribosomal proteins as well as genes encoding translation initiation and elongation factors, all of which are associated with protein synthesis. With respect to metabolic regulation, the inducible alteration of Lys metabolism was primarily associated with altered expression of genes belonging to networks of amino acids and sugar metabolism. The combined data are discussed within the context of network interactions both between and within metabolic and transcriptional control systems.
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(2009) Plant Science. 177, 5, p. 450-459 Abstract
The creation of a water-stress environment usually starts with a reduction in air relative humidity (RH) while soil water potential still reflects favorable conditions. Arabidopsis plants subjected to low RH (17%) exhibited a significant increase in leaf specific hydraulic conductance, reducing the water potential. However, no detectable effects on stomatal performance or osmotic leaf adjustment were noted relative to plants exposed to high RH. In the present study, we profiled gene expression in roots 2.5 and 5 h after shoot exposure to low RH. Multiple genes with various putative biological roles were identified as differentially expressed under these conditions; among them were aquaporins, some of whose expression was induced under low RH. Transcription of two aquaporin was localized to the roots, especially to cells around the vascular system and to cells of the differentiation zone, and to leaf trichomes. Our results suggest that plant roots perceive the low RH stimulus from shoots through a sensing mechanism(s), leading to distinct plant transcriptional responses, potentially reflecting activation of various biological processes. This activation might be a prelude to the expected forthcoming drought conditions, providing the plant with enhanced resistance to future water-stress situations.
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(2009) Plant Journal. 60, 1, p. 156-167 Abstract
Plants can synthesize the aromatic amino acid Phe via arogenate, but it is still not known whether they also use an alternative route for Phe biosynthesis via phenylpyruvate, like many micro-organisms. To examine this possibility, we expressed a bacterial bi-functional PheA (chorismate mutase/prephenate dehydratase) gene in Arabidopsis thaliana that converts chorismate via prephenate into phenylpyruvate. The PheA-expressing plants showed a large increase in the level of Phe, implying that they can convert phenylpyruvate into Phe. In addition, PheA expression rendered the plants more sensitive than wild-type plants to the Trp biosynthesis inhibitor 5-methyl-Trp, implying that Phe biosynthesis competes with Trp biosynthesis from their common precursor chorismate. Surprisingly, GC-MS, LC-MS and microarray analyses showed that this increase in Phe accumulation only had a very minor effect on the levels of other primary metabolites as well as on the transcriptome profile, implying little regulatory cross-interaction between the aromatic amino acid biosynthesis network and the bulk of the Arabidopsis transcriptome and primary metabolism. However, the levels of a number of secondary metabolites derived from all three aromatic amino acids (Phe, Trp and Tyr) were altered in the PheA plants, implying regulatory cross-interactions between the flux of aromatic amino acid biosynthesis from chorismate and their further metabolism into various secondary metabolites. Taken together, our results provide insights into the regulatory mechanisms of aromatic amino acid biosynthesis and their interaction with central primary metabolism, as well as the regulatory interface between primary and secondary metabolism.
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(2009) Molecular Genetics and Metabolism. 96, 2, p. S13-S14 Abstract
Keywords: Biochemistry & Molecular Biology; Genetics & Heredity; Medicine, Research & Experimental
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(2009) BMC Systems Biology. 3, 14. Abstract
Background: Being sessile organisms, plants should adjust their metabolism to dynamic changes in their environment. Such adjustments need particular coordination in branched metabolic networks in which a given metabolite can be converted into multiple other metabolites via different enzymatic chains. In the present report, we developed a novel "Gene Coordination" bioinformatics approach and use it to elucidate adjustable transcriptional interactions of two branched amino acid metabolic networks in plants in response to environmental stresses, using publicly available microarray results. Results: Using our "Gene Coordination" approach, we have identified in Arabidopsis plants two oppositely regulated groups of "highly coordinated" genes within the branched Asp-family network of Arabidopsis plants, which metabolizes the amino acids Lys, Met, Thr, Ile and Gly, as well as a single group of "highly coordinated" genes within the branched aromatic amino acid metabolic network, which metabolizes the amino acids Trp, Phe and Tyr. These genes possess highly coordinated adjustable negative and positive expression responses to various stress cues, which apparently regulate adjustable metabolic shifts between competing branches of these networks. We also provide evidence implying that these highly coordinated genes are central to impose intra- and inter-network interactions between the Asp-family and aromatic amino acid metabolic networks as well as differential system interactions with other growth promoting and stress-associated genome-wide genes. Conclusion: Our novel Gene Coordination elucidates that branched amino acid metabolic networks in plants are regulated by specific groups of highly coordinated genes that possess adjustable intra-network, inter-network and genome-wide transcriptional interactions. We also hypothesize that such transcriptional interactions enable regulatory metabolic adjustments needed for adaptation to the stresses.
2008
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(2008) Journal of Experimental Botany. 59, 14, p. 4029-4043 Abstract
Eukaryotes contain a ubiquitous family of autophagy-associated Atg8 proteins. In animal cells, these proteins have multiple functions associated with growth, cancer, and degenerative diseases, but their functions in plants are still largely unknown. To search for novel functions of Atg8 in plants, the present report tested the effect of expression of a recombinant AtAtg8 protein, fused at its N-terminus to green fluorescent protein (GFP) and at its C-terminus to the haemagglutinin epitope tag, on the response of Arabidopsis thaliana plants to the hormones cytokinin and auxin as well as to salt and osmotic stresses. Expression of this AtAtg8 fusion protein modulates the effect of cytokinin on root architecture. Moreover, expression of this fusion protein also reduces shoot anthocyanin accumulation in response to cytokinin feeding to the roots, implying the participation of AtAtg8 in cytokinin-regulated root-shoot communication. External application of cytokinin leads to the formation of novel GFP -AtAtg8-containing structures in cells located in the vicinity of the root vascular system, which are clearly distinct in size and dynamic movement from the GFP -AtAtg8-containing autophagosome-resembling structures that were observed in root epidermis cells. Expression of the AtAtg8 fusion construct also renders the plants more sensitive to a mild salt stress and to a lesser extent to a mild osmotic stress. This sensitivity is also associated with various changes in the root architecture, which are morphologically distinct from those observed in response to cytokinin. The results imply multiple functions for AtAtg8 in different root tissues that may also be regulated by different mechanisms.
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(2008) Plant Physiology. 147, 1, p. 316-330 Abstract
Using a bioinformatics analysis of public Arabidopsis (Arabidopsis thaliana) microarray data, we propose here a novel regulatory program, combining transcriptional and posttranslational controls, which participate in modulating fluxes of amino acid metabolism in response to abiotic stresses. The program includes the following two components: (1) the terminal enzyme of the module, responsible for the first catabolic step of the amino acid, whose level is stimulated or repressed in response to stress cues, just-in-time when the cues arrive, principally via transcriptional regulation of its gene; and (2) the initiator enzyme of the module, whose activity is principally modulated via posttranslational allosteric feedback inhibition in response to changes in the level of the amino acid, just-in-case when it occurs in response to alteration in its catabolism or sequestration into different intracellular compartments. Our proposed regulatory program is based on bioinformatics dissection of the response of all biosynthetic and catabolic genes of seven different pathways, involved in the metabolism of 11 amino acids, to eight different abiotic stresses, as judged from modulations of their mRNA levels. Our results imply that the transcription of the catabolic genes is principally more sensitive than that of the biosynthetic genes to fluctuations in stress-associated signals. Notably, the only exception to this program is the metabolic pathway of Pro, an amino acid that distinctively accumulates to significantly high levels under abiotic stresses. Examples of the biological significance of our proposed regulatory program are discussed.
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(2008) Molecular Genetics and Metabolism. 93, 2, p. S15-S15 Abstract
Keywords: Biochemistry & Molecular Biology; Genetics & Heredity; Medicine, Research & Experimental
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(2008) Trends in Plant Science. 13, 1, p. 14-19 Abstract
Much of the recent work on the γ-aminobutyrate (GABA) shunt in plants has concentrated on stress/pest-associated and signalling roles. However, fifty years after the structural elucidation of the pathway, aspects of its regulation and even of its biological significance remain largely obscure. Here, we assess the importance of GABA metabolism in plants, reviewing relevant biological circumstances and taking advantage of high-throughput data accessibility and computational approaches. We discuss the premise that GABA metabolism plays a major role in carbon and nitrogen primary metabolism. We further evaluate technological developments that will likely allow us to address the quantitative importance of this shunt within the biological processes to which it contributes.
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(2008) Advances in Plant Biochemistry and Molecular Biology. C ed. p. 49-80 Abstract
Amino acids are not only building blocks of proteins but also participate in many metabolic networks that control growth and adaptation to the environment. In young plants, amino acid biosynthesis is regulated by a compound metabolic network that links nitrogen assimilation with carbon metabolism. This network is strongly regulated by the metabolism of four central amino acids, namely glutamine, glutamate, aspartate, and asparagine (Gln, Glu, Asp, and Asn), which are then converted into all other amino acids by various biochemical processes. Amino acids also serve as major transport molecules of nitrogen between source and sink tissues, including transport of nitrogen from vegetative to reproductive tissues. Amino acid metabolism is subject to a concerted regulation by physiological, developmental, and hormonal signals. This regulation also appears to be different between source and sink tissues. The importance of amino acids in plants does not only stem from being central regulators of plant growth and responses to environmental signals, but amino acids are also effectors of the nutritional quality of human foods and animal feeds. Since mammals cannot synthesize about half of the 20-amino acid building blocks of proteins, they rely on obtaining them from foods and feeds. Yet, the major crop plants contain limited amounts of some of these so-called "essential amino acids," which decreases nutritional value. Recent genetic engineering and more recently genomic approaches have significantly boosted our understanding of the regulation of amino acid metabolism in plants and their participation in growth, stress response, and reproduction. In addition, genetic engineering approaches have improved the content of essential amino acids in plants, particularly the contents of lysine and methionine, which are often most limiting.
2007
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(2007) Plant Biotechnology Journal. 5, 5, p. 579-590 Abstract
Gaucher's disease, a lysosomal storage disorder caused by mutations in the gene encoding glucocerebrosidase (GCD), is currently treated by enzyme replacement therapy using recombinant GCD (Cerezyme®) expressed in Chinese hamster ovary (CHO) cells. As complex glycans in mammalian cells do not terminate in mannose residues, which are essential for the biological uptake of GCD via macrophage mannose receptors in human patients with Gaucher's disease, an in vitro glycan modification is required in order to expose the mannose residues on the glycans of Cerezyme®. In this report, the production of a recombinant human GCD in a carrot cell suspension culture is described. The recombinant plant-derived GCD (prGCD) is targeted to the storage vacuoles, using a plant-specific C-terminal sorting signal. Notably, the recombinant human GCD expressed in the carrot cells naturally contains terminal mannose residues on its complex glycans, apparently as a result of the activity of a special vacuolar enzyme that modifies complex glycans. Hence, the plant-produced recombinant human GCD does not require exposure of mannose residues in vitro, which is a requirement for the production of Cerezyme®. prGCD also displays a level of biological activity similar to that of Cerezyme® produced in CHO cells, as well as a highly homologous high-resolution three-dimensional structure, determined by X-ray crystallography. A single-dose toxicity study with prGCD in mice demonstrated the absence of treatment-related adverse reactions or clinical findings, indicating the potential safety of prGCD. prGCD is currently undergoing clinical studies, and may offer a new and alternative therapeutic option for Gaucher's disease.
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(2007) Metabolomics. 3, 3, p. 357-369 Abstract
This article raises the complex issue of improving plant nutritional value through metabolic engineering and the potential of using RNAi and micro RNA technologies to overcome this complexity, focusing on a few key examples. It also highlights current knowledge of RNAi and microRNA functions and discusses recent progress in the development of new RNAi vectors and their applications. RNA interference (RNAi) and microRNA (miRNA) are recent breakthrough discoveries in the life sciences recognized by the 2006 Nobel Prize in Physiology or Medicine. The importance of these discoveries relates not only to elucidating the fundamental regulatory aspects of gene expression, but also to the tremendous potential of their applications in plants and animals. Here, we review recent applications of RNAi and microRNA for improving the nutritional value of plants, discuss applications of metabolomics technologies in genetic engineering, and provide an update on the related RNAi and microRNA technologies.
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(2007) Journal of Experimental Botany. 58, 10, p. 2653-2660 Abstract
A quantitative trait locus has previously been identified in maize (Zea mays L.) that influences the level of free amino acids in the endosperm, especially those from the aspartate pathway: lysine, threonine, methionine, leucine, and isoleucine. Because this locus occurs in a region of the genome containing ask2, a monofunctional aspartate kinase, the nature of the monofunctional aspartate kinase genes in the parental inbreds, Oh545o2 and Oh51Ao2, was investigated. Two genes, Ask1 and Ask2 were isolated, and Ask2 was mapped to the ask2 locus. Nucleotide sequence analysis of the Ask2 alleles from Oh545o2 and Oh51Ao2 showed they differ by one amino acid. Both alleles complemented a yeast aspartate kinase mutant, hom3, and based on the growth of the yeast mutant it appeared that Ask2-Oh545o2 produces an enzyme with greater total activity than that encoded by the Oh51Ao2 allele. The results suggest that the higher level of free amino acids derived from the aspartate pathway in Oh545o2 endosperm results from a single amino acid change in the ASK2 enzyme that has pleiotropic effects on its activity.
2006
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(2006) Plant Physiology. 142, 3, p. 839-854 Abstract
While the metabolic networks in developing seeds during the period of reserve accumulation have been extensively characterized, much less is known about those present during seed desiccation and subsequent germination. Here we utilized metabolite profiling, in conjunction with selective mRNA and physiological profiling to characterize Arabidopsis (Arabidopsis thaliana) seeds throughout development and germination. Seed maturation was associated with a significant reduction of most sugars, organic acids, and amino acids, suggesting their efficient incorporation into storage reserves. The transition from reserve accumulation to seed desiccation was associated with a major metabolic switch, resulting in the accumulation of distinct sugars, organic acids, nitrogen-rich amino acids, and shikimate-derived metabolites. In contrast, seed vernalization was associated with a decrease in the content of several of the metabolic intermediates accumulated during seed desiccation, implying that these intermediates might support the metabolic reorganization needed for seed germination. Concomitantly, the levels of other metabolites significantly increased during vernalization and were boosted further during germination sensu stricto, implying their importance for germination and seedling establishment. The metabolic switches during seed maturation and germination were also associated with distinct patterns of expression of genes encoding metabolism-associated gene products, as determined by semiquantitative reverse transcription-polymerase chain reaction and analysis of publicly available microarray data. When taken together our results provide a comprehensive picture of the coordinated changes in primary metabolism that underlie seed development and germination in Arabidopsis. They furthermore imply that the metabolic preparation for germination and efficient seedling establishment initiates already during seed desiccation and continues by additional distinct metabolic switches during vernalization and early germination.
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(2006) Plant Molecular Biology. 61, 1-2, p. 255-268 Abstract
The essential amino acid methionine is a substrate for the synthesis of S-adenosyl-methionine (SAM), that donates its methyl group to numerous methylation reactions, and from which polyamines and ethylene are generated. To study the regulatory role of methionine synthesis in tomato fruit ripening, which requires a sharp increase in ethylene production, we cloned a cDNA encoding cystathionine γ-synthase (CGS) from tomato and analysed its mRNA and protein levels during tomato fruit ripening. CGS mRNA and protein levels peaked at the "turning" stage and declined as the fruit ripened. Notably, the tomato CGS mRNA level in both leaves and fruit was negatively affected by methionine feeding, a regulation that Arabidopsis, but not potato CGS mRNA is subject to. A positive correlation was found between elevated ethylene production and increased CGS mRNA levels during the ethylene burst of the climacteric ripening of tomato fruit. In addition, wounding of pericarp from tomato fruit at the mature green stage stimulated both ethylene production and CGS mRNA level. Application of exogenous methionine to pericarp of mature green fruit increased ethylene evolution, suggesting that soluble methionine may be a rate limiting metabolite for ethylene synthesis. Moreover, treatment of mature green tomato fruit with the ethylene-releasing reagent Ethephon caused an induction of CGS mRNA level, indicating that CGS gene expression is regulated by ethylene. Taken together, these results imply that in addition to recycling of the methionine moieties via the Yang pathway, operating during synthesis of ethylene, de novo synthesis of methionine may be required when high rates of ethylene production are induced.
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(2006) Amino Acids. 30, 2, p. 121-125 Abstract
Lysine is a nutritionally important essential amino acid, whose synthesis in plants is strongly regulated by the rate of its synthesis. Yet, lysine level in plants is also finely controlled by a super-regulated catabolic pathway that catabolizes lysine into glutamate and acetyl Co-A. The first two enzymes of lysine catabolism are synthesized from a single LKR/SDH gene. Expression of this gene is subject to compound developmental, hormonal and stress-associated regulation. Moreover, the LKR/SDH gene of different plant species encodes up to three distinct polypeptides: (i) a bifunctional enzyme containing the linked lysine-ketoglutarate (LKR) and saccharopine dehydrogenase (SDH) whose LKR activity is regulated by its linked SDH enzyme; (ii) a monofunctional SDH encoded by an internal promoter, which is a part of the coding DNA region of the LKR/SDH gene; and (iii) a monofunctional, highly potent LKR that is formed by polyadenylation within an intron. LKR activity in the bifunctional LKR/SDH polypeptide is also post-translationally regulated by phosphorylation by casein kinase-2 (CK2), but the consequence of this regulation is still unknown. Why is lysine metabolism super-regulated by synthesis and catabolism? A hypothesis addressing this important question is presented, suggesting that lysine may serve as a regulator of plant growth and interaction with the environment.
2005
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(2005) Journal of Experimental Botany. 56, 421, p. 2839-2849 Abstract
Arabidopsis plants possess a family of nine AtAtg8 gene homologues of the yeast autophagy-associated Apg8/Aut7 gene. To gain insight into how these genes function in plants, first, the expression patterns of five AtAtg8 homologues were analysed in young Arabidopsis plants grown under favourable growth conditions or following exposure to prolonged darkness or sugar starvation. Promoters, plus the entire coding regions (exons and introns) of the AtAtg8 genes, were fused to the p-glucuronidase reporter gene and transformed into Arabidopsis plants. In all plants, grown under favourable growth conditions, β-glucuronidase staining was much more significant in roots than in shoots. Different genes showed distinct spatial and temporal expression patterns in roots. In some transgenic plants, p-glucuronidase staining in leaves was induced by prolonged darkness or sugar starvation. Next, Arabidopsis plants were transformed with chimeric gene-encoding Atg8f protein fused to N-terminal green fluorescent protein and C-terminal haemagglutinin epitope tags. Analysis of these plants showed that, under favourable growth conditions, the Atg8f protein is efficiently processed and is localized to autophagosome-resembling structures, both in the cytosol and in the central vacuole, in a similar manner to its processing and localization under starvation stresses. Moreover, treatment with a cocktail of proteasome inhibitors did not prevent the turnover of this protein, implying that its turnover takes place in the vacuoles, as occurs in yeasts. The results suggest that, in plants, the cellular processes involving the Atg8 genes function efficiently in young, non-senescing tissues, both under favourable growth conditions and under starvation stresses.
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(2005) Journal of Experimental Botany. 56, 412, p. 525-536 Abstract
Lysine catabolism in plants is initiated by a bifunctional LKR/SDH (lysine-ketoglutarate reductase/saccharopine dehydrogenase) enzyme encoded by a single LKR/SDH gene. Yet, the AtLKR/SDH gene of Arabidopsis also encodes a second gene product, namely a monofunctional SDH. To elucidate the regulation of lysine catabolism in Arabidopsis through these two gene products of the AtLKR/SDH gene, an analysis was carried out on the effects of the hormones, abscisic acid and jasmonate, as well as various metabolic and stress signals, including lysine itself, on their mRNA and protein levels. The response of the two gene products to the various treatments was only partially co-ordinated, but the levels of the monofunctional SDH mRNA and protein were always in excess over their bifunctional LKR/SDH counterparts. These results suggest that lysine catabolism is regulated primarily by the first enzyme LKR, while the excess level of SDH enables efficient flux of lysine catabolism following the LKR step. Analysis of transgenic plants expressing β-glucoronidase fusion constructs with the AtLKR/SDH and monofunctional AtSDH promoters demonstrated that transcriptional regulation contributes to the modulation of expression of the bifunctional LKR/SDH and monofunctional SDH gene products in response to hormonal and metabolic signals. To test whether the enhanced expression of the LKR/SDH gene under various hormonal and metabolic signals is correlated with enhanced lysine catabolism, wild-type Arabidopsis and a knockout mutant lacking lysine catabolism were exposed to abscisic acid and sugar starvation. Free lysine accumulated to significantly higher levels in this knockout mutant than in the wild-type plants.
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(2005) Biological Chemistry. 386, 9, p. 817-831 Abstract
Plants represent the major source of food for humans, either directly or indirectly through their use as livestock feeds. Plant foods are not nutritionally balanced because they contain low proportions of a number of essential metabolites, such as vitamins and amino acids, which humans and a significant proportion of their livestock cannot produce on their own. Among the essential amino acids needed in human diets, Lys, Met, Thr and Trp are considered as the most important because they are present in only low levels in plant foods. In the present review, we discuss approaches to improve the levels of the essential amino acids Lys and Met, as well as of sulfur metabolites, in plants using metabolic engineering approaches. We also focus on specific examples for which a deeper understanding of the regulation of metabolic networks in plants is needed for tailor-made improvements of amino acid metabolism with minimal interference in plant growth and productivity.
2004
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(2004) Trends in Biotechnology. 22, 9, p. 463-469 Abstract
RNA interference (RNAi) is an ancient mechanism of gene suppression, whose machinery and biological functions are only partially understood. Intensive studies have focused on developing RNAi technologies for treating human diseases and for improving plant traits. Yet application of RNAi to improving the nutritional value of plants for human and animal nutrition, and development of the related RNAi technologies are still in their infancy. Here we discuss current knowledge of plant RNAi function, as well as concepts and strategies for the improvement of plant nutritional value through the development of plant RNAi technologies.
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(2004) Plant Physiology. 135, 1, p. 129-136 Abstract
The functional role of Lys catabolism in balancing Lys levels in plants has only been directly demonstrated in developing seeds. Seed-specific expression of a bacterial feedback-insensitive dihydrodipicolinate synthase (DHPS) in an Arabidopsis knockout mutant of the AtLKR/SDH gene that regulates Lys catabolism synergistically boosted Lys accumulation in mature seeds, but it also severely reduced the growth of seedlings derived from them. Here we further tested whether the inhibition of seedling growth was due to a negative physiological effect of excess Lys on seed maturation or to defective postgermination catabolism of Lys, which accumulated in the mature seeds. To address these questions, we coexpressed a bacterial DHPS gene with an RNAi construct of AtLKR/SDH, both under control of the same seed-specific promoter, to restrict Lys synthesis and catabolism to the developing seeds. Coexpression of these genes boosted seed Lys content and caused a significant, metabolically unanticipated increase in Met content, similarly to our previous report using plants expressing the bacterial DHPS on an AtLKR/SDH knockout background. However, postgermination seedling growth was significantly improved when the reduction of Lys catabolism was restricted to seed development, suggesting that defective postgermination Lys catabolism was responsible for inhibition of seedling growth in the AtLKR/SDH knockout plants expressing the bacterial DHPS gene in a seed-specific manner. Constitutive expression of the bacterial DHPS in the AtLKR/SDH knockout mutant boosted Lys levels in vegetative tissues in a similar manner to that observed in seeds, further demonstrating that Lys catabolism plays an important regulatory role in balancing Lys levels.
2003
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(2003) Plant Physiology. 133, 3, p. 1407-1415 Abstract
In plants, excess cellular lysine (Lys) is catabolized into glutamic acid and acetyl-coenzyme A; yet, it is still not clear whether this pathway has other functions in addition to balancing Lys levels. To address this issue, we examined the effects of stress-related hormones, abscisic acid (ABA), and jasmonate, as well as various metabolic signals on the production of the mRNA and polypeptide of the bifunctional Lys-ketoglutarate reductase (LKR)/saccharopine dehydrogenase (SDH) enzyme, which contains the first two linked enzymes of Lys catabolism. The level of LKR/SDH was strongly enhanced by ABA, jasmonate, and sugar starvation, whereas excess sugars and nitrogen starvation reduced its level; thus this pathway appears to fulfill multiple functions in stress-related and carbon/nitrogen metabolism. Treatments with combination of hormones and/or metabolites, as well as use of ABA mutants in conjunction with the tester sugars mannose and 3-O-methyl-glucose further supported the idea that the hormonal and metabolic signals apparently operate through different signal transduction cascades. The stimulation of LKR/SDH protein expression by ABA is regulated by a signal transduction cascade that contains the ABI1-1 and ABI2-1 protein phosphatases. By contrast, the stimulation of LKR/SDH protein expression by sugar starvation is regulated by the hexokinase-signaling cascade in a similar manner to the repression of many photosynthetic genes by sugars. These findings suggest a metabolic and mechanistic link between Lys catabolism and photosynthesis-related metabolism in the regulation of carbon/nitrogen partitioning.
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(2003) Molecular Plant-Microbe Interactions. 16, 5, p. 382-388 Abstract
Arbuscular mycorrhizae (AM) represent an ancient symbiosis between mycorrhizal fungi and plant roots which co-evolved to exhibit a finely tuned, multistage interaction that assists plant growth. Direct screening efforts for Myc- plant mutants resulted in the identification of a tomato (Lycopersicon esculentum L. cv. Micro-Tom) mutant, M20, which was impaired in its ability to support the premycorrhizal infection (pmi) stages. The Myc- phenotype of the M20 mutant was a single Mendelian recessive trait, stable for nine generations, and nonallelic to a previously identified M161 pmi mutant. The M20 mutant was resistant to infection by isolated AM spores and colonized roots. Formation of Glomus intraradices appressoria on M20 roots was normal, as on wild-type (WT) plants, but in significantly reduced numbers. A significant reduction in spore germination was observed in vitro in the presence of M20 exudates relative to WT. Our results indicate that this new mutant shares similar physiological characteristics with the M161 pmi mutant, but has a more suppressive Myc- phenotype response.
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(2003) Molecular Breeding. 11, 3, p. 187-201 Abstract
So far two different strategies for engineering high methionine (Met) grain legumes were followed separately in several laboratories: a) The transfer of foreign genes encoding Met-rich proteins, and b) the engineering of Met biosynthesis pathways. In some cases a down regulation of the formation of endogenous sulfur-containing compounds was observed due to the expression of Met-rich foreign proteins. Since this might result from competition of the foreign protein with endogenous compounds for limited Met supply both strategies were combined in the present work. Double transformants of narbon bean (Vicia narbonensis L.) were generated which express seed-specifically the Met-rich Brazil nut 2S albumin (BNA) as well as a feed-back insensitive bacterial aspartate kinase (AK) known to stimulate Met biosynthesis in transgenic tobacco seeds. In order to produce double transformants a homozygous transgenic BNA line of narbon bean was either retransformed with the AK gene or crossed with an AK line. For the first time the influence of a deregulated AK on amino acids of the aspartate pathway was studied in seeds of a transgenic legume. Effects of expressing the foreign genes on inorganic sulphate, free and protein-bound Met and other amino acids of the aspartate pathway as well as on free sulphhydryl compounds of mature seeds were analysed. AK lines had 10 to 12 percent and the BNA line 80 percent increased Met in mature seeds. Double transformants showed additive but not synergistic effects of the expression of AK and BNA gene on seed Met. In their mature seeds protein-bound Met reached levels 2.0 to 2.4 times higher than in the wildtype. The Met level of best line corresponds approximately to the FAO standard for Met in a nutritionally balanced protein for human food or for feeding monogastric animals.
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(2003) Plant Cell. 15, 4, p. 845-853 Abstract
To elucidate the relative significance of Lys synthesis and catabolism in determining Lys level in plant seeds, we expressed a bacterial feedback-insensitive dihydrodipicolinate synthase (DHPS) in a seed-specific manner in wild-type Arabidopsis as well as in an Arabidopsis knockout mutant in the Lys catabolism pathway. Transgenic plants expressing the bacterial DHPS, or the knockout mutant, contained ∼12-fold or ∼5-fold higher levels, respectively, of seed free Lys than wild-type plants. However, the combination of these two traits caused a synergistic ∼80-fold increase in seed free Lys level. The dramatic increase in free Lys in the knockout mutant expressing the bacterial DHPS was associated with a significant reduction in the levels of Glu and Asp but also with an unexpected increase in the levels of Gln and Asn. This finding suggested a special regulatory interaction between Lys metabolism and amide amino acid metabolism in seeds. Notably, the level of free Met, which competes with Lys for Asp and Glu as precursors, was increased unexpectedly by up to ∼38-fold in the various transgenic and knockout plants. Together, our results show that Lys catabolism plays a major regulatory role in Lys accumulation in Arabidopsis seeds and reveal novel regulatory networks of seed amino acid metabolism.
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(2003) Plant Cell. 15, 2, p. 439-447 Abstract
Most of the symplastic water transport in plants occurs via aquaporins, but the extent to which aquaporins contribute to plant water status under favorable growth conditions and abiotic stress is not clear. To address this issue, we constitutively overexpressed the Arabidopsis plasma membrane aquaporin, PIP1b, in transgenic tobacco plants. Under favorable growth conditions, PIP1b overexpression significantly increased plant growth rate, transpiration rate, stomatal density, and photosynthetic efficiency. By contrast, PIP1b overexpression had no beneficial effect under salt stress, whereas during drought stress it had a negative effect, causing faster wilting. Our results suggest that symplastic water transport via plasma membrane aquaporins represents a limiting factor for plant growth and vigor under favorable conditions and that even fully irrigated plants face limited water transportation. By contrast, enhanced symplastic water transport via plasma membrane aquaporins may not have any beneficial effect under salt stress, and it has a deleterious effect during drought stress.
2002
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(2002) Journal of Biological Chemistry. 277, 51, p. 49655-49661 Abstract
Lysine-ketoglutarate reductase/saccharopine dehydrogenase (LKR/SDH) is a bifunctional enzyme catalyzing the first two steps of lysine catabolism in animals and plants. To elucidate the biochemical signification of the linkage between the two enzymes of LKR/SDH, namely lysine ketoglutarate and saccharopine dehydrogenase, we employed various truncated and mutated Arabidopsis LKR/SDH polypeptides expressed in yeast. Activity analyses of the different recombinant polypeptides under conditions of varying NaCl levels implied that LKR, but not SDH activity, is regulated by functional interaction between the LKR and SDH domains, which is mediated by the structural conformation of the linker region connecting them. Because LKR activity of plant LKR/SDH enzymes is also regulated by casein kinase 2 phosphorylation, we searched for such potential regulatory phosphorylation sites using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and site-directed mutagenesis. This analysis identified Ser-458 as a candidate for this function. We also tested a hypothesis suggesting that an EF-hand-like sequence at the C-terminal part of the LKR domain functions in a calcium-dependent assembly of LKR/SDH into a homodimer. We found that this region is essential for LKR activity but that it does not control a calcium-dependent assembly of LKR/SDH. The relevance of our results to the in vivo function of LKR/SDH in lysine catabolism in plants is discussed. In addition, because the linker region between LKR and SDH exists only in plants but not in animal LKR/SDH enzymes, our results suggest that the regulatory properties of LKR/SDH and, hence, the regulation of lysine catabolism are different between plants and animals.
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(2002) Journal of Experimental Botany. 53, 378, p. 2277-2278 Abstract
The 3 cleavage and polyadenylation of mRNAs has been studied in detail in animals and yeast, but not in plants. Aimed at elucidating the regulation of mRNA 3 end formation in plants, three Arabidopsis cDNAs encoding homologues of the animal proteins CstF-64, CstF-77 and CstF-50 that form the cleavage stimulating factor of the polyadenylation machinery have been cloned. It is shown experimentally that the N-terminal domain of the Arabidopsis CstF-64 homologue binds the mRNA 3 non-coding region in an analogous manner to the animal protein. It is also shown that the Arabidopsis CstF-64 and CstF-77 homologues strongly interact with each other in a similar way to their animal counterparts. These results imply that these Arabidopsis homologues belong to the polyadenylation machinery of nuclear mRNAs.
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(2002) Plant Physiology. 130, 1, p. 147-154 Abstract
Both plants and animals catabolize lysine (Lys) via two consecutive enzymes, Lys-ketoglutarate reductase (LKR) and saccharopine dehydrogenase (SDH), which are linked on a single polypeptide encoded by a single LKR/SDH gene. We have previously shown that the Arabidopsis LKR/SDH gene also encodes a monofunctional SDH that is transcribed from an internal promoter. In the present report, we have identified two cDNAs derived from cotton (Gossypium hirsutum) boll abscission zone that encode a novel enzymatic form of Lys catabolism, i.e. a catabolic monofunctional LKR. The monofunctional LKR mRNA is also encoded by the LKR/SDH gene, using two weak polyadenylation sites located within an intron. In situ mRNA hybridization and quantitative reverse transcriptase-polymerase chain reaction analyses also suggest that the cotton monofunctional LKR is relatively abundantly expressed in parenchyma cells of the abscission zone. DNA sequence analysis of the LKR/SDH genes of Arabidopsis, maize (Zea mays), and tomato (Lycopersicon esculentum) suggests that these genes can also encode a monofunctional LKR mRNA by a similar mechanism. To test whether the LKR/SDH and monofunctional LKR enzymes possess different biochemical properties, we used recombinant Arabidopsis LKR/SDH and monofunctional LKR enzymes expressed in yeast (Saccharomyces cerevisiae) cells. The Km of the monofunctional LKR to Lys was nearly 10-fold lower than its counterpart that is linked to SDH. Taken together, our results suggest that the LKR/SDH locus of plants is a super-composite locus that can encode three related but distinct enzymes of Lys catabolism. These three enzymes apparently operate in concert to finely regulate Lys catabolism during plant development.
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(2002) Trends in Plant Science. 7, 4, p. 153-156 Abstract
The sulfur-containing amino acid methionine is a nutritionally important essential amino acid and is the precursor of several metabolites that regulate plant growth and responses to the environment. Methionine production is largely regulated by cystathionine γ-synthase, the first specific enzyme for its synthesis. This enzyme competes in a complex manner with threonine synthase, the last enzyme in threonine biosynthesis, for their common substrate O-phosphohomoserine. New genetic and molecular data suggest that methionine synthesis and catabolism are coordinately regulated by novel post-transcriptional and post-translational mechanisms that are associated with a regulatory part within the N-terminal part of cystathionine γ-synthase.
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(2002) Annual Review of Plant Biology. 53, p. 27-43 Abstract
Lysine is one of the most limiting essential amino acids in vegetative foods consumed by humans and livestock. In addition to serving as a building block of proteins, lysine is also a precursor for glutamate, an important signaling amino acid that regulates plant growth and responses to the environment. Recent genetic, molecular, and biochemical evidence suggests that lysine synthesis and catabolism are regulated by novel concerted mechanisms. These include intracellular compartmentalization of enzymes and metabolites, complex transcriptional and posttranscriptional controls of genes encoding enzymes in lysine metabolism during plant growth and development, as well as interactions between different metabolic fluxes. The recent advances in our understanding of the regulation of lysine metabolism in plants may also prove valuable for future production of high-lysine crops.
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(2002) Critical Reviews in Plant Sciences. 21, 3, p. 167-204 Abstract
Recent advances in gene isolation, plant transformation, and genetic engineering are being used extensively to alter metabolic pathways in plants by tailormade modifications to single or multiple genes. Many of these modifications are directed toward increasing the nutritional value of plant-derived foods and feeds. These approaches are based on rapidly growing basic knowledge, understanding, and predictions of metabolic fluxes and networks. Some of the predictions appear to be accurate, while others are not, reflecting the fact that plant metabolism is more complex than we presently understand. Tailor-made modifications of plant metabolism has so far been directed into improving the levels of primary metabolites that are essential for growth and development of humans and their livestock. Yet, the list of improved metabolites is expected to grow tremendously after new discoveries in nutritional, medical, and health sciences. Despite our extensive knowledge of metabolic networks, many of the genes encoding enzymes, particularly those involved in secondary metabolism, are still unknown. These genes are being discovered at an accelerated rate by recent advances in genetic and genomics approaches. In the present review, we discuss examples in which the nutritional and health values of plant-derived foods and feeds were improved by metabolic engineering. These include modifications of the levels of several essential amino acids, lipids, fatty acids, minerals, nutraceuticals, antinutritional compounds, and aromas.
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(2002) Metabolic Engineering. 4, 1, p. 3-11 Abstract
Amino acid pathways are important targets for plant metabolic engineering. Since plants represent the major global food supply, large efforts are devoted to increasing the content of "essential" amino acids, which are absolutely required in human foods and animal feeds. Engineering of amino acids is also undertaken to improve plant growth and stress tolerance. Many of the pathways of amino acid metabolism in plants have been elucidated, and genes encoding most of the enzymes are now available. The expression of recombinant genes in transgenic plants, coupled with genetic and biochemical approaches, has contributed significantly to the understanding of regulatory networks of the metabolism of amino acids and their incorporation into proteins. This knowledge is now being extensively applied to metabolic engineering of crops, and this is reflected by a large patent literature. The problems of engineering plant amino acid metabolism, and ways to solve them, are discussed using the essential amino acids lysine and methionine as examples.
2001
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(2001) Journal of Experimental Botany. 52, 365, p. 2387-2388 Abstract
Wheat storage proteins are deposited in the vacuole of maturing endosperm cells by a novel pathway that is the result of protein body formation by the endoplasmic reticulum followed by autophagy into the central vacuole, bypassing the Golgi apparatus. This model predicts a reduced role of the Golgi in storage protein accumulation, which has been supported by electron microscopy observations. To study this issue further, wheat cDNAs encoding three distinct proteins of the endomembrane system were cloned and characterized. The proteins encoded were homologues (i) of the ER translocon component Sec61α, (ii) the vacuolar sorting receptor BP-80 which is located in the Golgi and clathrin-coated prevacuole vesicles (CCV), and (iii) the Golgi COPI coatomer component COPα. During endosperm development, the levels of all three mRNAs were highest in young stages, before the onset of storage protein synthesis, and declined with seed maturation. However, the relative mRNA levels of BP-80,/Sec61α and the COPα/Sec61α were lower during the onset of storage protein synthesis than at earlier stages of endosperm development. These results support previous studies, suggesting a reduced function of the Golgi apparatus in wheat storage protein transport and deposition.
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(2001) Plant Physiology. 126, 4, p. 1539-1545 Abstract
Plants possess both anabolic and catabolic pathways for the essential amino acid lysine (Lys). However, although the biosynthetic pathway was clearly shown to regulate Lys accumulation in plants, the functional significance of Lys catabolism has not been experimentally elucidated. To address this issue, we have isolated an Arabidopsis knockout mutant with a T-DNA inserted into exon 13 of the gene encoding Lys ketoglutarate reductase/saccharopine dehydrogenase. This bifunctional enzyme controls the first two steps of Lys catabolism. The phenotype of the LKR/SDH knockout was indistinguishable from wild-type plants under normal growth conditions, suggesting that Lys catabolism is not an essential pathway under standard growth conditions. However, mature seeds of the knockout mutant over-accumulated Lys compared with wild-type plants. This report provides the first direct evidence for the functional significance of Lys catabolism in regulating Lys accumulation in seeds. Such a knockout mutant may also provide new perspectives to improve the level of the essential amino acid Lys in plant seeds.
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(2001) Plant Journal. 27, 6, p. 561-569 Abstract
Vesicular arbuscular mycorrhizal fungi infect plants by means of both spores and vegetative hyphae at early stages of symbiosis. Using 2500 M2 fast-neutron-mutagenized seeds of the miniature tomato (Lycopersicon esculentum) cultivar, Micro-Tom, we isolated a mutant, M161, that is able to resist colonization in the presence of Glomus intraradices spores. The myc- phenotype of the mutant was stable for nine generations, and found to segregate as a single Mendelian recessive locus. The mutant exhibited morphological and growth-pattern characteristics similar to those of wild-type plants. Alterations of light intensity and day/night temperatures did not eliminate the myc- characteristic. Resistance to mycorrhizal fungal infection and colonization was also evident following inoculation with the fungi Glomus mosseae and Gigaspora margarita. Normal colonization of M161 was evident when mutant plants were grown together with arbuscular mycorrhizal-inoculated wild-type plants in the same growth medium. During evaluation of the pre-infection stages in the mutant rhizosphere, spore germination and appressoria formation of G. intraradices were lower by 45 and 70%, respectively, than the rates obtained with wild-type plants. These results reveal a novel, genetically controlled step in the arbuscular mycorrhizal colonization process, governed by at least one gene, which significantly reduces key steps in pre-mycorrhizal infection stages.
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(2001) Current Opinion in Plant Biology. 4, 3, p. 261-266 Abstract
Lysine is a nutritionally important essential amino acid whose level in plants is largely regulated by the rate of its synthesis. In some plant tissues and under some stress conditions, however, lysine is also efficiently catabolized into glutamate and several other stress-related metabolites by novel mechanisms of metabolic regulation. Lysine catabolism is important for mammalian brain function; it is possible that the generation of glutamate regulates nerve transmission signals via glutamate receptors. Plants also possess homologues of animal glutamate receptors. It is thus likely that lysine catabolism also regulates various plant processes via these receptors.
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(2001) Journal of Plant Physiology. 158, 4, p. 515-520 Abstract
Lysine is among the most important essential amino acids in the diet of human and livestock because it is presented in severely limiting amounts in cereals and other important crops. Attempts to increase lysine levels in plants were made by reducing the sensitivity of the key enzyme in lysine biosynthesis, namely dihydrodipicolinate synthase, to feedback inhibition by lysine. However, these studies showed that in plant seeds, lysine accumulation is determined not only by the rate of its synthesis, but also by the rate of its catabolism via the α-amino adipic acid pathway. Our laboratory is currently studying the regulation of lysine catabolism in plants in order to explore potentials of reducing lysine catabolic fluxes in transgenic plants. In plants, like animals, lysine is catabolized via saccharopine by two consecutive enzymes, lysine-ketoglutarate reductase (LKR) and saccharopine dehydrogenase (SDH), which are linked on a single bifunctional polypeptide. Yet SDH activity of the LKR/SDH polypeptide may be limiting in vivo because of its non-physiological pH optimum of activity. In some plants, like Arabidopsis and canola, this is overcome by the presence of an additional monofunctional SDH enzyme, which is encoded by the same locus that encodes the bifunctional LKR/SDH enzyme. Results from our, and other laboratories imply that attempts to generate high-lysine crop plants should take into account a seed-specific reduction of lysine catabolism.
2000
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(2000) Biochemical Journal. 351, 1, p. 215-220 Abstract
Whereas plants and animals use the α-aminoadipic acid pathway to catabolize lysine, yeast and fungi use the very same pathway to synthesize lysine. These two groups of organisms also possess structurally distinct forms of two enzymes in this pathway, namely lysine-oxoglutarate reductase (lysine-ketoglutarate reductase; LKR) and saccharopine dehydrogenase (SDH): in plants and animals these enzymes are linked on to a single bifunctional polypeptide, while in yeast and fungi they exist as separate entities. In addition, yeast LKR and SDH possess bidirectional activities, and their anabolic function is regulated by complex transcriptional and post-transcriptional controls, which apparently ascertain differential accumulation of intermediate metabolites; in plants, the regulation of the catabolic function of these two enzymes is not known. To elucidate the regulation of the catabolic function of plant bifunctional LKR/SDH enzymes, we have used yeast as an expression system to test whether a plant LKR/SDH also possesses bi-directional LKR and SDH activities, similar to the yeast enzymes. The Arabidopsis enzyme complemented a yeast SDH, but not LKR, null mutant. Identical results were obtained when deletion mutants encoding only the LKR or SDH domains of this bifunctional polypeptide were expressed individually in the yeast cells. Moreover, activity assays showed that the Arabidopsis LKR possessed catabolic, but not anabolic, activity, and its uni-directional activity stems from its structure rather than its linkage to SDH. Our results suggest that the uni-directional activity of LKR plays an important role in regulating the catabolic function of the α-amino adipic acid pathway in plants.
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(2000) Plant Journal. 23, 2, p. 195-203 Abstract
Both plants and animals catabolise lysine via saccharopine by two consecutive enzymes, lysine-ketoglutarate reductase (LKR) and saccharopine dehydrogenase (SDH), which are linked on a single polypeptide. We recently demonstrated that Arabidopsis plants possess not only a bifunctional LKR/SDH but in addition a monofunctional SDH enzyme. We also speculated that these two enzymes may be controlled by a single gane (G. Tang et al., Plant Cell, 1997, 9, 1305-1318). By expressing several epitope-tagged and GUS reporter constructs, we demonstrate in the present study that the Arabidopsis monofunctional SDH is encoded by a distinct gene, which is, however, nested entirely within the coding and 3' non-coding regions of the larger bifunctional LKR/SDH gene. The entire open reading frame of the monofunctional SDH gene, as well as some components of its promoter, are also parts of the translated coding sequence of the bifunctional LKR/SDH gene. These special structural characteristics, combined with the fact that the two genes encode simultaneously two metabolically related but distinct enzymes, render the LKR/SDH locus a novel type of a composite locus. Not all plant species possess an active monofunctional SDH gene and the production of this enzyme is correlated with an increased flux of lysine catabolism. Taken together, our results suggest that the composite LKR/SDH locus serves to control an efficient, highly regulated flux of lysine catabolism.
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(2000) Israel Journal of Plant Sciences. 48, 3, p. 181-187 Abstract
Metabolic processes are regulated by complex networks of interacting mechanisms that utilize various cellular machineries. Such complex networks may be well exemplified by the synthesis, accumulation, and degradation of storage proteins in developing and germinating seeds. Our laboratories are using plant seeds as a model system for studying the regulation of production of the essential amino acid lysine, the control of synthesis of storage proteins and their transport to the storage vacuoles, and the de novo formation of new forms of lytic vacuoles in germinating seeds which fuse with the storage vacuoles to enable degradation of the storage protein sand their mobilization into the germinating embryo. We show that: (i) production of lysine in developing seeds is regulated by complex pathways of synthesis and catabolism that involve the sensing of free lysine levels in the seeds, and (ii) analysis of the deposition of storage proteins in seed storage vacuoles and their subsequent degradation during germination provide novel insights into the biogenesis and function of vacuoles in plants.
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(2000) Plant Physiology. 123, 2, p. 655-663 Abstract
Both in mammals and plants, excess lysine (Lys) is catabolized via saccharopine into α-amino adipic semialdehyde and glutamate by two consecutive enzymes, Lys-ketoglutarate reductase (LKR) and saccharopine dehydrogenase (SDH), which are linked on a single bifunctional polypeptide. To study the control of metabolite flux via this bifunctional enzyme, we have purified it from developing soybean (Glycine max) seeds. LKR activity of the bifunctional LKR/SDH possessed relatively high K(m) for its substrates, Lys and α-ketoglutarate, suggesting that this activity may serve as a rate-limiting step in Lys catabolism. Despite their linkage, the LKR and SDH enzymes possessed significantly different pH optima, suggesting that SDH activity of the bifunctional enzyme may also be rate-limiting in vivo. We have previously shown that Arabidopsis plants contain both a bifunctional LKR/SDH and a monofunctional SDH enzymes (G. Tang, D. Miron, J.X. Zhu-Shimoni, G. Galili [1997] Plant Cell 9: 1-13). In the present study, we found no evidence for the presence of such a monofunctional SDH enzyme in soybean seeds. These results may provide a plausible regulatory explanation as to why various plant species accumulate different catabolic products of Lys.
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(2000) Plant Physiology. 124, 3, p. 1363-1371 Abstract
Arabidopsis plants possess a composite AtLKR/SDH locus encoding two different polypeptides involved in lysine catabolism: A bifunctional lysine-ketoglutarate reductase/saccharopine dehydrogenase (LKR/SDH) enzyme and a monofunctional SDH enzyme. To unravel the physiological significance of these two enzymes, we analyzed their subcellular localization and detailed biochemical properties. Sucrose gradient analysis showed that the two enzymes are localized in the cytosol and therefore may operate at relatively neutral pH values in vivo. Yet while the physiological pH may provide an optimum environment for LKR activity, the pH optima for the activities of both the linked and non-linked SDH enzymes were above pH 9, suggesting that these two enzymes may operate under suboptimal conditions in vivo. The basic biochemical properties of the monofunctional SDH, including its pH optimum as well as the apparent Michaelis constant (K(m)) values for its substrates saccharopine and nicotinamide adenine dinucleotide at neutral and basic pH values, were similar to those of its SDH counterpart that is linked to LKR. Taken together, our results suggest that production of the monofunctional SDH provides Arabidopsis plants with enhanced levels of SDH activity (maximum initial velocity), rather than with an SDH isozyme with significantly altered kinetic parameters. Excess levels of this enzyme might enable efficient flux of lysine catabolism via the SDH reaction in the unfavorable physiological pH of the cytosol.
1999
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(1999) EMBO Journal. 18, 14, p. 3973-3980 Abstract
Cellular functions require adequate homeostasis of several divalent metal cations, including Mg2+ and Zn2+. Mg2+ the most abundant free divalent cytoplasmic cation, is essential for many enzymatic reactions, while Zn2+ is a structural constituent of various enzymes. Multicellular organisms have to balance not only the intake of Mg2+ and Zn2+, but also the distribution of these ions to various organs. To date, genes encoding Mg2+ transport proteins have not been cloned from any multicellular organism. We report here the cloning and characterization of an Arabidopsis thaliana transporter, designated AtMHX, which is localized in the vacuolar membrane and functions as an electrogenic exchanger of protons with Mg2+ and Zn2+ ions. Functional homologs of AtMHX have not been cloned from any organism. Ectopic overexpression of AtMHX in transgenic tobacco plants render them sensitive to growth on media containing elevated levels of Mg2+ or Zn2+, but does not affect the total amounts of these minerals in shoots of the transgenic plants. AtMHX mRNA is mainly found at the vascular cylinder, and a large proportion of the mRNA is localized in close association with the xylem tracheary elements. This localization suggests that AtMHX may control the partitioning of Mg2+ and Zn2+ between the various plant organs.
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Regulation of lysine catabolism in plants(1999) Plant Biotechnology And In Vitro Biology In The 21St Century. 36, p. 311-314 Abstract
Seeds of many crop plants are considered as a poor nutritional value food source, due to very low levels of the essential amino acid lysine. Attempts to increase lysine content in seeds are hampered by the presence of an active catabolic process that degrades lysine via saccharopine into other metabolites. The first enzyme in this catabolic process is lysine ketoglutarate reductase (LKR). We have recently obtained evidence that strongly suggest that lysine finely controls its own level in seeds by maintaining LKR activity balanced using two separate complementary mechanisms. I) LKR activity is post-translationally stimulated by lysine via a Ca+2 dependent intracellular signaling cascade that ends in the phosphorylation of the enzyme, thus, preventing lysine to accumulate to high levels. LI) subsequent binding of lysine to the active site of the enzyme may expose phosphate residues on the surface of LKR rendering them more accessible to dephosphorylation, thus allowing lysine to accumulate to a sufficient level needed for protein synthesis.
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T-DNA and "gain of function" tobacco mutants with altered threonine metabolism(1999) Plant Biotechnology And In Vitro Biology In The 21St Century. 36, p. 273-276 Abstract
A transferred DNA (T-DNA) tagging vector, containing four enhancers of the 35S gene promoter (Walden, 94), was used to obtain "gain of function" tobacco mutants with altered threonine metabolism. From 150 million transferred protoplasts, 17 plants were regenerated whose growth was resistant to a high level of threonine and to its toxic analog hydroxynorvaline. The majority of these plants contained a single T-DNA insert, genetically co-segregating with the threonine resistance. The mutants consisted of two categories: threonine overproducers and threonine non-overproducers. The overproducer mutants were probably connected with regulation of threonine biosynthesis while the non-overproducers mutants may be results of altered threonine sequestration.
1998
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(1998) Molecular Plant-Microbe Interactions. 11, 6, p. 489-497 Abstract
A differentially displayed cDNA clone (MD17) was isolated from tobacco roots (Nicotiana tabacum cv. Xanthi-nc) infected with the arbuscular mycorrhizal (AM) fungus Glomus intraradices. The isolated DNA fragment exhibited a reduced level of expression in response to AM establishment and 90% identity with the 3' noncoding sequence of two basic chitinases (EC 3.2.1.14) from N. tabacum. Northern (RNA) blots and Western blots (immunoblots), probed with tobacco basic chitinase gene-specific probe and polyclonal antibodies raised against the chitinase enzyme, yielded hybridization patterns similar to those of MD17. Moreover, the up-regulation of the 32-kDa basic chitinase gene expression in tobacco roots by (1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH) was less effective in mycorrhizal roots than in nonmycorrhizal controls. Suppression of endogenous basic chitinase (32-kDa) expression was also observed in transgenic mycorrhizal plants that constitutively express the 34-kDa basic chitinase A isoform. When plants were grown with an increased phosphate supply, no suppression of the 32-kDa basic chitinase was obtained. These findings indicate that during the colonization and establishment of G. intraradices in tobacco roots, expression of the basic chitinase gene is down-regulated at the mRNA level.
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(1998) Plant Physiology. 116, 3, p. 1023-1028 Abstract
Although the control of carbon fixation and nitrogen assimilation has been studied in detail, relatively little is known about the regulation of carbon and nitrogen flow into amino acids. In this paper we report our study of the metabolic regulation of expression of an Arabidopsis aspartate kinase/homoserine dehydrogenase (AK/ HSD) gene, which encodes two linked key enzymes in the biosynthetic pathway of aspartate family amino acids. Northern blot analyses, as well as expression of chimeric AK/HSD-β-glucuronidase constructs, have shown that the expression of this gene is regulated by the photosynthesis-related metabolites sucrose and phosphate but not by nitrogenous compounds. In addition, analysis of AK/HSD promoter deletions suggested that a CTTGACTCTA sequence, resembling the binding site for the yeast GCN4 transcription factor, is likely to play a functional role in the expression of this gene. Nevertheless, longer promoter fragments, lacking the GCN4-like element, were still able to confer sugar inducibility, implying that the metabolic regulation of this gene is apparently obtained by multiple and redundant promoter sequences. The present and previous studies suggest that the conversion of aspartate into either the storage amino acid asparagine or aspartate family amino acids is subject to a coordinated, reciprocal metabolic control, and this biochemical branch point is a part of a larger, coordinated regulatory mechanism of nitrogen and carbon storage and utilization.
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(1998) Plant Molecular Biology. 38, 1-2, p. 1-29 Abstract
The endoplasmic reticulum (ER) is the port of entry of proteins into the endomembrane system, and it is also involved in lipid biosynthesis and storage. This organelle contains a number of soluble and membrane-associated enzymes and molecular chaperones, which assist the folding and maturation of proteins and the deposition of lipid storage compounds. The regulation of translocation of proteins into the ER and their subsequent maturation within the organelle have been studied in detail in mammalian and yeast cells, and more recently also in plants. These studies showed that in general the functions of the ER in protein synthesis and maturation have been highly conserved between the different organisms. Yet, the ER of plants possesses some additional functions not found in mammalian and yeast cells. This compartment is involved in cell to cell communication via the plasmodesmata, and, in specialized cells, it serves as a storage site for proteins. The plant ER is also equipped with enzymes and structural proteins which are involved in the process of oil body biogenesis and lipid storage. In this review we discuss the components of the plant ER and their function in protein maturation and biogenesis of oil bodies. Due to the large number of cited papers, we were not able to cite all individual references and in many cases we refer the readers to reviews and references therein. We apologize to the authors whose references are not cited.
1997
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(1997) Plant Cell. 9, 8, p. 1305-1316 Abstract
In plant and mammalian cells, excess lysine is catabolized by a pathway that is initiated by two enzymes, namely, lysine-ketoglutarate reductase and saccharopine dehydrogenase. In this study, we report the cloning of an Arabidopsis cDNA encoding a bifunctional polypeptide that contains both of these enzyme activities linked to each other. RNA gel blot analysis identified two mRNA bands- a large mRNA containing both lysine-ketoglutarate reductase and saccharopine dehydrogenase sequences and a smaller mRNA containing only the saccharopine dehydrogenase sequence. However, DNA gel blot hybridization using either the lysine-ketoglutarate reductase or the saccharopine dehydrogenase cDNA sequence as a probe suggested that the two mRNA populations apparently are encoded by the same gene. To test whether these two mRNAs are functional, protein extracts from Arabidopsis cells were fractionated by anion exchange chromatography. This fractionation revealed two separate peaks- one containing both coeluted lysine-ketoglutarate reductase and saccharopine dehydrogenase activities and the second containing only saccharopine dehydrogenase activity. RNA gel blot analysis and in situ hybridization showed that the gene encoding lysine-ketoglutarate reductase and saccharopine dehydrogenase is significantly upregulated in floral organs and in embryonic tissues of developing seeds. Our results suggest that lysine catabolism is subject to complex developmental and physiological regulation, which may operate at gene expression as well as post-translational levels.
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(1997) Journal of Biological Chemistry. 272, 24, p. 15488-15495 Abstract
Wheat high molecular weight glutenin subunits (HMW-GS) are the most important determinants of its superiority for making leavened bread. Following synthesis, these proteins are sequestered into the endoplasmic reticulum and assemble into extremely large elastic polymers, linked by noncovalent and intermolecular disulfide bonds. To study the structural requirements for the assembly of HMW-GS, we have expressed in transgenic wheat a recombinant protein between two cognate x- and y-type subunits. In contrast to the natural polymerized x- and y-type HMW-GS, a significant amount of the recombinant subunit remained monomeric. Nonreducing SDS- polyacrylamide gel electrophoresis, coupled with limited proteolysis, showed that the monomeric form of the recombinant subunit contained an unusual intramolecular disulfide bond, linking an N-terminal cysteine to the single C-terminal cysteine residue. In addition, sucrose gradient analysis revealed that this intramolecular disulfide bond impeded the ability of the recombinant subunit to assemble into polymers. Despite of its altered assembly, a notable amount of the overexpressed recombinant subunit was also present in glutenin polymers. Moreover, its presence significantly altered the subunit composition of the polymer. Our results show that it is possible to modify gluten assembly and properties by expressing recombinant HMW-GS in transgenic wheat, and have a major implication for the improvement of wheat breadmaking quality.
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(1997) Plant Molecular Biology. 34, 2, p. 287-294 Abstract
As in many bacterial species, the first enzymatic reaction of the aspartate-family pathway in plants is mediated by several isozymes of aspartate kinase (AK) that are subject to feedback inhibition by the end-product amino acids lysine or threonine. So far, only cDNAs and genes encoding threonine-sensitive AKs have been cloned from plants. These were all shown to encode polypeptides containing two linked activities, namely AK and homoserine dehydrogenase (HSD), similar to the Escherichia coli thrA gene encoding a threonine-sensitive bifunctional AK/HSD isozyme. In the present report, we describe the cloning of a new Arabidopsis thaliana cDNA that is relatively highly homologous to the E. coli lysC gene encoding the lysine-sensitive AK isozyme. Moreover, similar to the bacterial lysine-sensitive AK, the polypeptide encoded by the present cDNA is monofunctional and does not contain an HSD domain. These observations imply that our cloned cDNA encodes a lysine-sensitive AK. Southern blot hybridization detected a single gene highly homologous to the present cDNA, plus an additional much less homologous gene. This was confirmed by the independent cloning of an additional Arabidopsis cDNA encoding a lysine-sensitive AK (see accompanying paper). Northern blot analysis suggested that the gene encoding this monofunctional AK cDNA is abundantly expressed in most if not all tissues of Arabidopsis.
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(1997) Plant Physiology. 113, 3, p. 695-706 Abstract
Although the regulation of amino acid synthesis has been studied extensively at the biochemical level, it is still not known how genes encoding amino acid biosynthesis enzymes ate regulated during plant development. In the present report, we have used the β-glucuronidase (GUS) reporter gene to study the regulation of expression of an Arabidopsis thaliana aspartate kinase-homoserine dehydrogenase (AK/HSD) gene in transgenic tobacco plants. The polypeptide encoded by the AK/HSD gene comprises two linked key enzymes in the biosynthesis of aspartate-family amino acids. AK/HSD-GUS gene expression was highly stimulated in apical and lateral meristems, lateral buds, young leaves, trichomes, vascular and cortical tissues of growing stems, tapetum and other tissues of anthers, pollen grains, various parts of the developing gynoecium, developing seeds, and, in some transgenic plants, also in stem and leaf epidermal trichomes. AK/HSD-GUS gene expression gradually diminished upon maturation of leaves, stems, floral tissues, and embryos. GUS expression was relatively low in roots. During seed development, expression of the AK/HSD gene in the embryo was coordinated with the initiation and onset of storage protein synthesis, whereas in the endosperm it was coordinated with the onset of seed desiccation. Upon germination, AK/HSD-GUS gene expression in the hypocotyl and the cotyledons was significantly affected by light. The expression pattern of the A. thaliana AK/HSD-GUS reporter gene positively correlated with the levels of aspartate-family amino acids and was also very similar to the expression pattern of the endogenous tobacco AK/HSD mRNA as determined by in situ hybridization.
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(1997) Advances In Botanical Research Incorporating Advances In Plant Pathology, Vol 25: The Plant Vacuole. 25, C, p. 113-140 Abstract
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(1997) Plant Journal. 12, 6, p. 1453-1458 Abstract
In plant seeds, the essential amino acid lysine autoregulates its own level by modulating the activity of its catabolic enzyme lysine-ketoglutarate reductase via an intracellular signaling cascade, mediated by Ca2+ and protein phosphorylation/dephosphorylation. In the present report, it has been further tested whether the activity of soybean lysine-ketoglutarate reductase, as well as that of saccharopine dehydrogenase, the second enzyme in the pathway of lysine catabolism, are modulated by direct phosphorylation of the bifunctional polypeptide containing both of these linked activities. Incubation of purified lysine-ketoglutarate reductase/saccharopine dehydrogenase with casein kinase II resulted in a significant phosphorylation of the bifunctional enzyme. Moreover, in vibrio dephosphorylation of the bifunctional polypeptide with alkaline phosphatase significantly inhibited the activity of lysine-ketoglutarate reductase, but not of its linked enzyme saccharopine dehydrogenase. The inhibitory effect of alkaline phosphatase on lysine-ketoglutarate reductase activity was dramatically stimulated by binding of lysine to the enzyme. Our results suggest that in plant seeds, active lysine-ketoglutarate reductase is a phospho-protein, and that its activity is modulated by opposing actions of protein kinases and phosphatases. Moreover, this modulation is subject to a compound regulation by lysine.
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Expression of bacterial dihydrodipicolinate synthase in transgenic plants: Potentials to improve lysine content in forage, grain and tuber crops(1997) Horticultural Biotechnology In Vitro Culture And Breeding. 447, p. 551-559 Abstract
Plants generally contain low proportions of several essential amino acids, particularly lysine. The potential of increasing lysine content in vegetative tissues and storage organs of plants was studied by expression in transgenic tobacco plants of a chimeric gene encoding a bacterial dihydrodipicolinate synthase (DHPS), a key enzyme in lysine biosynthesis. The bacterial enzyme is much less sensitive to feedback inhibition by lysine than its plant counterparts. Constitutive expression of the bacterial enzyme caused a dramatic elevation of free lysine in vegetative tissues, which in some cases amounted up to 40 mol % of total free amino acids. In addition, constitutive lysine overproduction caused an alteration in plant phenotype, including loss of apical dominance, reduced plant height, bushy appearance, altered leaf structure and delayed flowering. In order to study the potential of the bacterial DHPS to increase lysine content in storage organs, we have expressed this bacterial enzyme in a seed-specific manner in transgenic tobacco plants. Although lysine synthesis was enhanced during seed development in the transgenic plants, this amino acid failed to over accumulate in mature seeds. The low lysine levels in mature seeds of the transgenic plants was correlated with a lysine-dependent stimulation of another enzyme, lysine-ketoglutarate reductase, which catabolizes lysine into saccharopine. This stimulation was shown to operate via an intracellular signaling cascade, mediated by Ca2+ and protein phosphorylation. The potential of the bacterial DHPS, either alone or in combination with additional genes, to improve the nutritional quality of forage, grain and tuber crops is discussed.
1996
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(1996) Journal of Biological Chemistry. 271, 31, p. 18869-18874 Abstract
Following synthesis, wheat gliadin storage proteins are deposited into protein bodies inside the endomembrane system in a way that enables not only their efficient accumulation and dehydration during seed maturation, but also their rapid rehydration and degradation during germination. In the present report, we studied the mechanism of gliadin deposition and whether it was controlled by the conformation of these proteins. Although gliadins are generally known to be insoluble in aqueous solutions, sucrose gradient analysis showed that a considerable amount of these proteins appeared as relatively soluble monomers in developing grains. In vitro reduction of the intramolecular disulfide bonds that are present in natural monomeric gliadins caused their precipitation into insoluble aggregates. In addition, pulse- chase experiments in the absence or presence of reducing agents showed that formation of intramolecular disulfide bonds also played a major role in folding and deposition of the gliadins in vivo. Our results imply that following sequestration into the endoplasmic reticulum, the gliadins fold into relatively soluble monomers, which are incompetent for rapid aggregation and gradually assemble into protein bodies. This pattern of deposition apparently depends on the conformation of the gliadins, which is stabilized by intramolecular disulfide bonds formed between the conserved cysteines. The contribution of this study to the understanding of the evolution and function of gliadins is discussed.
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(1996) Plant Molecular Biology. 32, 4, p. 611-620 Abstract
In prokaryotes and plants the synthesis of the essential amino acids lysine and threonine is predominantly regulated by feed-back inhibition of aspartate kinase (AK) and dihydrodipicolinate synthase (DHPS). In order to modify the flux through the aspartate family pathway in barley and enhance the accumulation of the corresponding amino acids, we have generated transgenic barley plants that constitutively express mutant Escherichia coli genes encoding lysine feed back insensitive forms of AK and DHPS. As a result, leaves of primary transformants (T0) exhibited a 14-fold increase of free lysine and an 8-fold increase in free methionine. In mature seeds of the DHPS transgenics, there was a 2-fold increase in free lysine, arginine and asparagine and a 50% reduction in free proline, while no changes were observed in the seeds of the two AK transgenic lines analysed. When compared to that of control seeds, no differences were observed in the composition of total amino acids. The introduced genes were inherited in the T1 generation where enzymic activities revealed a 2.3-fold increase of AK activity and a 4.0-9.5-fold increase for DHPS. T1 seeds of DHPS transformants showed the same changes in free amino acids as observed in T0 seeds. It is concluded that the aspartate family pathway may be genetically engineered by the introduction of genes coding for feed-back insensitive enzymes, preferentially giving elevated levels of lysine and methionine.
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Wheat storage proteins: Assembly, transport and deposition in protein bodies(1996) Plant Physiology and Biochemistry. 34, 2, p. 245-252 Abstract
Wheat storage proteins are co-translationally inserted into the endoplasmic reticulum (ER) and then accumulate in protein bodies inside vacuoles. It appears that a significant amount of these storage proteins assemble into protein bodies within the ER and that these protein bodies are subsequently internalized into vacuoles by a process that is analogous to autophagy and does not utilize the Golgi complex. Folding and assembly of the storage proteins within the ER is not a spontaneous process, but is apparently assisted by ER-resident proteins. These include molecular chaperones as well as enzymes that catalyze the formation, isomerization and perhaps also dissociation of disulfide bonds.
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(1996) Plant Molecular Biology. 32, 4, p. 727-734 Abstract
To study the regulation of lysine and threonine metabolism in plants, we have transformed Arabidopsis thaliana with chimeric genes encoding the two bacterial enzymes dihydrodipicolinate synthase (DHPS) and aspartate kinase (AK). These bacterial enzymes are much less sensitive to feedback inhibition by lysine and threonine than their plant counterparts. Transgenic plants expressing the bacterial DHPS overproduced lysine, but lysine levels were quite variable within and between transgenic genotypes and there was no direct correlation between the levels of free lysine and the activity of DHPS. The most lysine-overproducing plants also exhibited abnormal phenotypes. However, these phenotypes were detected only at early stages of plant growth, while at later stages, new buds emerged that looked completely normal and set seeds. Wild-type plants exhibited relatively high levels of free threonine, suggesting that in Arabidopsis AK regulation may be more relaxed than in other plants. This was also supported by the fact that expression of the bacterial AK did not cause any dramatic elevation in this amino acid. Yet, the relaxed regulation of threonine synthesis in Arabidopsis was not simply due to a reduced sensitivity of the endogenous AK to feedback inhibition by lysine and threonine because growth of wild-type plants, but not of transgenic plants expressing the bacterial AK, was arrested in media containing these two amino acids. The present results, combined with previous studies from our laboratory, suggest that the regulation of lysine and threonine metabolism is highly variable among plant species and is subject to complex biochemical, physiological and environmental controls. The suitability of these transgenic Arabidopsis plants for molecular and genetic dissection of lysine and threonine metabolism is also discussed.
1995
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The lysine-dependent stimulation of lysine catabolism in tobacco seed requires calcium and protein phosphorylation(1995) Plant Cell. 7, 11, p. 1963-1970 Abstract
The accumulation of free lysine in tobacco seed triggers the stimulation of lysine-ketoglutarate reductase, an enzyme that acts in lysine catabolism. The mechanism of lysine-ketoglutarate reductase stimulation was studied in two different systems: (1) developing seeds of wild-type plants in which the low basal lysine-ketoglutarate reductase activity can be stimulated by the exogenous addition of lysine; and (2) developing seeds of transgenic tobacco plants expressing a bacterial dihydrodipicolinate synthase in which lysine-ketoglutarate reductase activity is stimulated by endogenous lysine overproduction. In both systems, the stimulation of lysine-ketoglutarate reductase activity was significantly reduced when treated with the Ca2+ chelator EGTA. Moreover, the inhibitory effect of EGTA was overcome by the addition of Ca2+ but not Mg2+, suggesting that the lysine-dependent activation of lysine-ketoglutarate reductase requires Ca2+. This was further confirmed by a significant stimulation of lysine-ketoglutarate reductase activity following the treatment of wild-type seeds with ionomycin (an ionophore that increases Ca2+ flow into the cytoplasm). In addition, treatment of wild-type seeds with the protein phosphatase inhibitor okadaic acid triggered a significant induction in lysine-ketoglutarate reductase activity, whereas treatment of the transgenic seeds with the protein kinase inhibitor K-252a caused a significant reduction in its activity. Thus, we conclude that the stimulation of lysine-ketoglutarate reductase activity by lysine in tobacco seed operates through an intracellular signaling cascade mediated by Ca2+ and protein phosphorylation.
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Purification, characterization, and intracellular localization of glycosylated protein disulfide isomerase from wheat grains(1995) Plant Physiology. 108, 1, p. 327-335 Abstract
Wheat (Triticum aestivum) storage proteins fold and assemble into complexes that are linked by intra- and intermolecular disulfide bonds, but it is not yet clear whether these processes are spontaneous or require the assistance of endoplasmic reticulum (ER)-resident enzymes and molecular chaperones. Aiming to unravel these processes, we have purified and characterized the enzyme protein disulfide isomerase (PDI) from wheat endosperm, as well as studied its developmental expression and intracellular localization. This ER-resident enzyme was previously shown to be involved in the formation of disulfide bonds in secretory proteins. Wheat PDI appears as a 60-kD glycoprotein and is among the most abundant proteins within the ER of developing grains. PDI is notably upregulated in developing endosperm in comparison to embryos, leaves, and roots. In addition, the increase in PDI expression in grains appears at relatively early stages of development, preceding the onset of storage protein accumulation by several days. Subcellular localization analysis and immunogold labeling of electron micrographs showed that PDI is not only present in the lumen of the ER but is also co-localized with the storage proteins in the dense protein bodies. These observations are consistent with the hypothesis that PDI is involved in the assembly of wheat storage proteins within the ER.
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(1995) Methods In Cell Biology. Vol. 50. p. 497-517 Abstract
This chapter discusses the synthesis of plant proteins in heterologous systems. The recent advent in recombinant DNA and gene transfer technologieswhich enables the construction of mutant genes and their subsequent expression in plant and animal cellshas provided an impetus for a wide array of studies on the synthesis, assembly, and transport of plant proteins. An important outcome of these studies is that many processes, though not all, are independent of the eukaryotic cell type in which the protein of interest is synthesized. It appears, for instance, that plant secretory proteins can fold, assemble, and acquire transport competence when expressed in animal cells. The major advantages of such heterologous systems are the ease of analysis and fast results, compared to the use of transgenic plants. Moreover, in specific cases, heterologous systems can be used for specific experiments that are either difficult or impossible to perform in plant cells.
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(1995) Journal of Plant Physiology. 145, 5-6, p. 626-631 Abstract
Following sequestration into the endoplasmic reticulum (ER), the storage proteins of wheat (Triticum aestivum L.) may either be retained and packaged into protein bodies (PB) inside the organelle or be exported to the Golgi complex. To unravel the signals and mechanisms regulating the assembly and sorting of these proteins within the ER, we expressed wild type and mutant forms of a γ type gliadin in Xenopus oocytes. A considerable amount of the wild type γ-gliadin was secreted via the Golgi into the medium, while still a significant proportion was retained within the ER of the oocytes where it assembled into dense PB. A deletion mutant of the γ-gliadin, encoding only the N-terminal region, which is composed of tandem repeats of a consensus PQQPFPQ sequence, was entirely retained within the oocytes, while another deletion mutant encoding only the C-terminal unique-sequence region of this protein was entirely secreted. Retention of the γ-gliadin within the ER could not be explained by rapid precipitation or assembly into insoluble deposits inasmuch as protein could diffuse rather efficiently within the organelle for several hours. In contrast, mutants of the γ-gliadin, lacking specific conserved cysteines in the C-terminal region, were entirely retained within the oocytes and were unable to diffuse within the ER. We thus hypothesize that the assembly and sorting of wheat gliadins within the ER are determined by concerted interactions between the Nand C-terminal regions of these proteins with ER resident proteins.
1994
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(1994) Applied Biochemistry and Biotechnology. 48, 3, p. 149-171 Abstract
The garlic plant (Allium sativum) alliinase (EC 4.4.1.4), which catalyzes the synthesis of allicin, was purified to homogeneity from bulbs using various steps, including hydrophobic chromatography. Molecular and biochemical studies showed that the enzyme is a dimer of two subunits of MW 51.5 kDa each. Its K m using synthetic S-allylcysteine sulfoxide (+isomer) as substrate was 1.1 mM, its pH optimum 6.5, and its isoelectric point 6.35. The enzyme is a glycoprotein containing 6% carbohydrate. N-terminal sequences of the intact polypeptide chain as well as of a number of peptides obtained after cyanogen bromide cleavage were obtained. Cloning of the cDNAs encoding alliinase was performed by a two-step strategy. In the first, a cDNA fragment (pAli-1-450 bp) was obtained by PCR using a mixed oligonucleotide primer synthesized according to a 6-amino acid segment near the N- terminal of the intact polypeptide. The second step involved screening of garlic λgt11 and λZAPII cDNA libraries with pAli-1, which yielded two clones; one was nearly full length and the second was full length. These clones exhibited some degree of DNA sequence divergence, especially in their 3 noncoding regions, suggesting that they were encoded by separate genes. The nearly full length cDNA was fused in frame to a DNA encoding a signal peptide from a wheat gliadin, and expressed in Xenopus oocytes. This yielded a 50 kDa protein that interacted with the antibodies against natural bulb alliinase. Northern and Western blot analyses showed that the bulb alliinase was highly expressed in bulbs, whereas a lower expression level was found in leaves, and no expression was detected in roots. Strikingly, the roots exhibited an abundant alliinase activity, suggesting that this tissue expressed a distinct alliinase isozyme with very low homology to the bulb enzyme.
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Role of conserved cysteines of a wheat gliadin in its transport and assembly into protein bodies in Xenopus oocytes(1994) Journal of Biological Chemistry. 269, 9, p. 6677-6682 Abstract
Following sequestration into the endoplasmic reticulum, wheat gliadin storage proteins may either be retained and packaged into protein bodies inside the organelle or be transported via the Golgi apparatus to vacuoles and condense into protein bodies at a post-endoplasmic reticulum location. To unravel the mechanism of this complex process of deposition, we expressed wild-type and mutant forms of two closely related γ and aggregated gliadins in Xenopus oocytes. Although a considerable amount of the γ-gliadin was secreted to the medium, its closely related aggregated gliadin was entirely retained within the oocytes. This differential secretion was largely due to structural variations in the C-terminal regions of the proteins. Retention of the wild-type aggregated and γ-gliadins within the endoplasmic reticulum could not be explained by rapid assembly into insoluble deposits inasmuch as both proteins could diffuse rather efficiently within the organelle for several hours. To address more closely the role of the C-terminal region in the transport and assembly of the γ-gliadin within the endoplasmic reticulum, 3 cysteine codons in this region were mutated, one at a time, to serine codons. The cysteine-replacement mutants improperly aggregated within the endoplasmic reticulum forming denser deposits compared with the wild- type protein.
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Mechanisms of assembly of wheat high molecular weight glutenins inferred from expression of wild-type and mutant subunits in transgenic tobacco(1994) Journal of Biological Chemistry. 269, 12, p. 8924-8930 Abstract
Following sequestration into the endoplasmic reticulum, wheat high molecular weight glutenin subunits (HMW-GS) assemble into polymers through intermolecular disulfide bond formation. These polymers, which also include low molecular weight glutenin subunits (LMW-GS), have a broad distribution of molecular mass reaching up to several million daltons. To study the mechanism of assembly of the HMW-GS, we have expressed x- and y-type HMW-GS in transgenic tobacco plants. Both types, when expressed individually or in combination, were incorporated into polymers. Partial reduction of polymers formed by different subunits resulted in different patterns of release of homodimers, heterodimers, and monomers. This suggested different arrangements of intermolecular disulfide bonds or different peptide conformations in the vicinity of the disulfide bonds linking x-x, y-y, and x-y type HMW-GS. A mutant of the x-type subunit, lacking a conserved cysteine in the C-terminal domain, assembled into oligomers linked by intermolecular disulfide bonds, but not into large polymers. This mutant was deposited, however, in dense protein bodies, similar to those formed by the native HMW-GS, suggesting that polymer formation and packaging into protein bodies may be the result of different types of interactions. Pulse-chase labeling of proteins in wheat endosperm showed that the assembly of the HMW-GS into insoluble polymers occurs by a slow process which apparently continues after the initiation of protein body formation.
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(1994) Proceedings of the National Academy of Sciences of the United States of America. 91, 7, p. 2577-2581 Abstract
The regulation of synthesis and accumulation of the essential amino acid lysine was studied in seeds of transgenic tobacco plants expressing, in a seed-specific manner, two feedback-insensitive bacterial enzymes: dihydrodipicolinate synthase (EC 4.2.1.52) and aspartate kinase (EC 2.7.2.4). High-level expression of the two bacterial enzymes resulted in only a slight increase in free lysine accumulation at intermediate stages of seed development, while free lysine declined to the low level of control plants toward maturity. To test whether enhanced catabolism may have contributed to the failure of free lysine to accumulate in seeds of transgenic plants, we analyzed the activity of lysine-ketoglutarate reductase (EC 1.5.1.7), an enzyme that catabolizes lysine into saccharopine. In both the control and the transgenic plants, the timing of appearance of lysine-ketoglutarate reductase activity correlated very closely with that of dihydrodipicolinate synthase activity, suggesting that lysine synthesis and catabolism were coordinately regulated during seed development. Notably, the activity of lysine- ketoglutarate reductase was significantly higher in seeds of the transgenic plants than in the controls. Coexpression of both bacterial enzymes in the same plant resulted in a significant increase in the proportions of lysine and threonine in seed albumins. Apparently, the normal low steady-state levels of free lysine and threonine in tobacco seeds may be rate limiting for the synthesis of seed proteins, which are relatively rich in these amino acids.
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(1994) Biochemical Society Transactions. 22, 4, p. 921-925 Abstract
Keywords: BACTERIAL DIHYDRODIPICOLINATE SYNTHASE; DESENSITIZED ASPARTATE KINASE; TOBACCO PLANTS; KETOGLUTARATE REDUCTASE; NICOTIANA-SYLVESTRIS; EXPRESSION; MUTANT; MAIZE; ACCUMULATION; CHLOROPLASTS
1993
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(1993) FEBS Letters. 336, 3, p. 403-407 Abstract
Certain endogenous Xenopus mRNAs, carrying a destabilizing 3' AU-rich sequence, are unusually very stable in oocytes and become unstable only after fertilization. In addition, heterologous short lived mRNA, containing 3' AU-rich sequences, appear to be very stable when injected into Xenopus oocytes. In the present study, a human interferon β (hu-IFNβ) mRNA, carrying the destabilizing 3' AU-rich element, was used as a probe to identify Xenopus proteins that specifically bind to the 3' AU-rich element as well as to study their relative levels during early embryonic development. While three major proteins that specifically bind to the 3' AU-rich element were detected in human SV80 cells, that naturally express hu-IFNβ (proteins termed AU-F1, F2 and F3), only two proteins, migrating similarly to the SV80 AU-F1 and AU-F3, were detected in cytoplasmic extracts from Xenopus oocytes or eggs. Following fertilization, the intensity of the Xenopus AU-F1 and AU-F3 proteins increased considerably and a new protein, corresponding to SV80 AU-F2, was also detected. Cyclohexamide applied either at the morula or at the early blastula stages reduced the intensity of the AU-binding factors, while actinomycin D did not, indicating that the levels of these factors during these stages are regulated posttranscriptionally. In contrast, application of each of these metabolic inhibitors at the late blastula stage increased the intensity of the AU-binding proteins. The possible function of these AU-binding factors in regulating the expression and half life of AU-rich mRNAs is discussed.
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(1993) Plant Molecular Biology. 23, 4, p. 759-768 Abstract
The essential amino acids lysine and threonine are synthesized in higher plants by two separate branches of a common pathway. This pathway is primarily regulated by three key enzymes, namely aspartate kinase (AK), dihydrodipicolinate synthase (DHPS) and homoserine dehydrogenase (HSD), but how these enzymes operate in concert is as yet unknown. Addressing this issue, we have expressed in transgenic tobacco plants high levels of bacterial AK and DHPS, which are much less sensitive to feedback inhibition by lysine and threonine than their plant counterparts. Such expression of the bacterial DHPS by itself resulted in a substantial overproduction of lysine, whereas plants expressing only the bacterial AK overproduced threonine. When both bacterial enzymes were expressed in the same plant, the level of free lysine exceeded by far the level obtained by the bacterial DHPS alone. This increase, however, was accompanied by a significant reduction in threonine accumulation compared to plants expressing the bacterial AK alone. Our results suggested that in tobacco plants the synthesis of both lysine and threonine is under a concerted regulation exerted by AK, DHPS, and possibly also by HSD. We propose that the balance between lysine and threonine synthesis is determined by competition between DHPS and HSD on limiting amounts of their common substrate 3-aspartic semialdehyde, whose level, in turn, is determined primarily by the activity of AK. The potential of this molecular approach to increase the nutritional quality of plants is discussed.
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(1993) Molecular and Cellular Biology. 13, 6, p. 3487-3493 Abstract
The 3 AU-rich region of human beta-1 interferon (hu-IFNβ) mRNA was found to act as a translational inhibitory element. The translational regulation of this 3 AU-rich sequence and the effect of its association with the poly(A) tail were studied in cell-free rabbit reticulocyte lysate. A poly(A)-rich hu-IFNβ mRNA (110 A residues) served as an inefficient template for protein synthesis. However, translational efficiency was considerably improved when the poly(A) tract was shortened (11 A residues) or when the 3 AU-rich sequence was deleted, indicating that interaction between these two regions was responsible for the reduced translation of the poly(A)-rich hu-IFNβ mRNA. Differences in translational efficiency of the various hu-IFNβ mRNAs correlated well with their polysomal distribution. The poly(A)-rich hu-IFNβ mRNA failed to form large polysemes, while its counterpart bearing a short poly(A) tail was recruited more efficiently into large polysemes. The AU-rich sequence-binding activity was reduced when the RNA probe contained both the 3 AU-rich sequence and long poly(A) tail, supporting a physical association between these two regions. Further evidence for this interaction was achieved by RNase H protection assay. We suggest that the 3 AU-rich sequence may regulate the translation of hu-IFNβ mRNA by interacting with the poly(A) tail.
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SEED-SPECIFIC EXPRESSION OF A BACTERIAL DESENSITIZED ASPARTATE KINASE INCREASES THE PRODUCTION OF SEED THREONINE AND METHIONINE IN TRANSGENIC TOBACCO(1993) Plant Journal. 3, 5, p. 721-727 Abstract
In order to study the regulation of threonine and methionine synthesis in plant seeds, tobacco plants were transformed with a chimeric gene containing the coding DNA sequence of a mutant lysC gene from Escherichia coli fused to a promoter from a phaseolin seed storage protein gene. The bacterial mutant lysC gene codes for aspartate kinase (AK) which is desensitized to feedback inhibition by lysine and threonine. Increased AK activity, compared with control non-transformed plants, was detected in seeds but not in leaves, roots and flowers of the transgenic plants. This expression was accompanied by a significant increase in the levels of free threonine and methionine in the seed. The level of these amino acids also correlated positively with the levels of the bacterial enzyme. No alteration in plant phenotype and 'average seed weight' was observed in any of the transgenic plants, indicating that plant growth and seed development were normal. This study demonstrates, for the first time, that the threonine and methionine biosynthetic pathways are active in plant seeds. Thus, targeting of the production of favorable biosynthetic enzymes to plant seeds may represent a desirable molecular approach for production of crop plants with a more balanced nutritional quality.
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(1993) Plant Physiology. 102, 1, p. 61-69 Abstract
Following their sequestration into the endoplasmic reticulum (ER), wheat storage proteins may either be retained and packaged into protein bodies within this organelle or transported via the Colgi to vacuoles. We attempted to study the processes of transport and packaging of wheat storage proteins using the heterologous expression system of yeast. A wild-type wheat γ-gliadin, expressed in the yeast cells, accumulated mostly within the ER and was deposited in protein bodies with similar density to natural protein bodies from wheat endosperm. This suggested that wheat storage proteins contain sufficient information to initiate the formation of protein bodies in the ER of a heterologous system. Only a small amount of the γ-gliadin was transported to the yeast vacuoles. When a deletion mutant of the γ-gliadin, lacking the entire N-terminal repetitive region, was expressed in the yeast cells, the mutant was unable to initiate the formation of protein bodies within the ER and was completely transported to the yeast vacuole. This strongly indicated that the information for packaging into dense protein bodies within the ER resides in the N-terminal repetitive region of the γ-gliadin. The advantage of using yeast to identify the signals and mechanisms controlling the transport of wheat storage proteins and their deposition in protein bodies is discussed.
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(1993) Plant Cell. 5, 4, p. 443-450 Abstract
Following sequestration into the endoplasmic reticulum (ER), wheat storage proteins are naturally either retained and packaged into protein bodies within this organelle or exported to the Golgi apparatus. To identify protein domains that control the sorting of wheat storage proteins within the ER, a wild-type γ-gliadin storage protein as well as two of its deletion mutants, each bearing either of the two autonomous N- and C-terminal regions, were expressed in Xenopus oocytes. Our results demonstrated that the N-terminal region of the gliadin, which is composed of several tandem repeats of the consensus sequence PQQPFPQ, was entirely retained within the ER and accumulated in dense protein bodies. In contrast, the C-terminal autonomous region was efficiently secreted to the medium. The wild-type γ-gliadin, containing both regions, was secreted at a lower rate and less efficiently than its C-terminal region. These results suggest that sorting of the wheat γ-gliadin within the ER may be determined by a balance between two opposing signals: one functions in the retention and packaging of the storage protein within the ER, while the second renders the protein competent for export from this organelle to the Golgi apparatus.
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(1993) Nature biotechnology. 11, 6, p. 715-718 Abstract
Potato plants transformed with a chimeric gene encoding a bacterial desensitized aspartate kinase were selected for resistance to the presence of lysine plus threonine in the regeneration and rooting media. Similarly, plants transformed with a chimeric gene encoding a bacterial dihydrodipicolinate synthase were selected for resistance to the toxic lysine analog S-aminoethyl L-cysteine. In both cases, resistant plants were regenerated, and all were apparently transgenic based on their content of dihydrodipicolinate synthase or aspartate kinase activities that were significantly higher, and much less sensitive to lysine and threonine inhibition, than the endogenous activities in control untransformed plants. Our data suggest that these novel selectable markers may be useful for the isolation of transgenic plants expressing relatively high levels of the gene of interest.
1992
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Evidence for a novel route of wheat storage proteins to vacuoles(1992) Journal of Cell Biology. 119, 5, p. 1117-1128 Abstract
Wheat seed storage proteins are deposited in protein bodies (PB) inside vacuoles, but their subcellular site of aggregation and their route to vacuoles are still controversial. In the present work, an ultra structural analysis of developing wheat endosperm at early to mid maturation was performed to address these issues. Golgi complexes were rarely detected, indicating that their role in wheat storage protein transport is limited. In contrast, a considerable amount of PB was detected in the cytoplasm. Many of these PB were surrounded by RER membranes and were enlarged by fusion of smaller PB. Small, electron lucent vesicles were detected around the surfaces of the PB in the cytoplasm, or attached to them, suggesting that such attachments and subsequent fusion of the vesicles with each other lead to the formation of small vacuoles containing PB inclusions. Immunogold labeling with serum raised against yeast-BiP, an ER-localized protein, demonstrated that the wheat BiP homolog was present within the PB in the cytoplasm as well as inside vacuoles. This confirmed that the PB were formed within the RER and that the Golgi complex was not involved in their transport to vacuoles. It is concluded that a considerable part of the wheat storage proteins aggregate into PB within the RER and are then transported as intact PB to the vacuoles by a novel route that does not utilize the Golgi complex.
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(1992) Molecular Genetics and Genomics. 235, 2-3, p. 279-284 Abstract
Genetic transformation of cereals by direct DNA delivery via microprojectile bombardment has become an established procedure in recent years. But the derivation of functional transgenic plants, especially in wheat, is still problematic, mainly due to low efficiency of DNA delivery and the reduced regeneration capability of microprojectile-bombarded tissue. We focussed on these two aspects and found that the regeneration of scutellar calli of wheat can be rendered highly efficient and considerably accelerated by a liquid culture phase in screen rafts. We also found that the expression of a reporter gene following DNA delivery by microprojectile can be improved by maintaining the scutellar calli in 0.25 M mannitol before and after bombardment, by bombardment in the presence of silver thiosulfate and Ca(NO3)2 (rather than CaCl2) and by the elimination of spermidine from the DNA/microprojectile mixture. A protocol that includes all these features leads to several-fold higher transient expression of the reporter gene than have previously published procedures.
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(1992) Plant Physiology. 100, 3, p. 1157-1163 Abstract
In higher plants, the synthesis of the essential amino acid threonine is regulated primarily by the sensitivity of the first enzyme in its biosynthetic pathway, aspartate kinase, to feedback inhibition by threonine and lysine. We aimed to study the potential of increasing threonine accumulation in plants by means of genetic engineering. This was addressed by the expression of a mutant, desensitized aspartate kinase derived from Escherichia coli either in the cytoplasm or in the chloroplasts of transgenic tobacco (Nicotiana Tabacum cv Samsun NN) plants. Both types of transgenic plants exhibited a significant overproduction of free threonine. However, threonine accumulation was higher in plants expressing the bacterial enzyme in the chloroplast, indicating that compartmentalization of aspartate kinase within this organelle was important, although not essential. Threonine overproduction in leaves was positively correlated with the level of the desensitized enzyme. Transgenic plants expressing the highest leaf aspartate kinase activity also exhibited a slight increase in the levels of free lysine and isoleucine, both of which share a common biosynthetic pathway with threonine, but showed no significant change in the level of other free amino acids. The present study proposes a new molecular biological approach to increase the limiting content of threonine in higher plants.
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(1992) Plant Molecular Biology. 19, 5, p. 815-823 Abstract
The essential amino acid lysine is synthesized in higher plants by a complex pathway that is predominantly regulated by feedback inhibition of two enzymes, namely aspartate kinase (AK) and dihydrodipicolinate synthase (DHPS). Although DHPS is thought to play a major role in this regulation, the relative importance of AK is not known. In order to study this regulation, we have expressed in the chloroplasts of transgenic potato plants a DHPS derived from Escherichia coli at a level 50-fold above the endogenous DHPS. The bacterial enzyme is much less sensitive to lysine inhibition than its potato counterpart. DHPS activity in leaves, roots and tubers of the transgenic plants was considerably higher and more resistant to lysine inhibition than in control untransformed plants. Furthermore, this activity was accompanied by a significant increase in level of free lysine in all three tissues. Yet, the extent of lysine overproduction in potato leaves was significantly lower than that previously reported in leaves of transgenic plants expressing the same bacterial enzyme, suggesting that in potato, AK may also play a major regulatory role in lysine biosynthesis. Indeed, the elevated level of free lysine in the transgenic potato plants was shown to inhibit the lysine-sensitive AK activity in vivo. Our results support previous reports showing that DHPS is the major rate-limiting enzyme for lysine synthesis in higher plants, but they suggest that additional plant-specific regulatory factors are also involved.
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(1992) FEBS Letters. 307, 2, p. 185-189 Abstract
The cDNA coding for pre-peanut agglutinin (PNA) was isolated from a bacterial expression library. It codes for a polypeptide of 273 amino acids composed of a hydrophobic signal peptide of 23 amino acids and a mature protein of 250 amino acids. The sequence of the latter is identical to that of native PNA, determined very recently by conventional methods, except that it contains 14 additional amino acids at the C-terminus. Bacterial cells harboring a plasmid with the prePNA-cDNA, produced two PNA cross-reacting proteins; one migrated on SDS-PAGE identically with the native lectin (apparent mol. wt. 31 kDa); the other, at 35 kDa, was a β-galactosidase pre-PNA fusion protein. The former protein possessed an N-terminal sequence identical to that of the mature, native PNA, suggesting that it was processed from the 35 kDa prePNA precursor. Only the 31 kDa protein was exported into the bacterial periplasmic space, and had the ability to bind to galactose-Sepharose. The isolated processed protein had the same hemagglutinating activity as the native lectin, when assayed with sialidase-treated human erythrocytes. Like the native lectin, it did not agglutinate the untreated cells, was not inhibited by N-acetylgalactosamine, and was inhibited by GAlβl→3GalNAc 30-times more strongly than by galactose.
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(1992) Plant Journal. 2, 2, p. 203-209 Abstract
A major nutritional drawback of many crop plants is their low content of several essential amino acids, particularly lysine. The biosynthesis of lysine in plants is regulated by several feedback loops. Dihydrodipicolinate synthase (DHPS) from Escherichia coli, a key enzyme in lysine biosynthesis, which is considerably less sensitive to lysine accumulation than the endogenous plant enzyme has been expressed in chloroplasts of tobacco leaves. Expression of the bacterial enzyme was accompanied by a significant increase in the level of free lysine. No increase in proteinbound lysine was evident. Free lysine accumulation was positively correlated with the level of DHPS activity in various transgenic plants. Compartmentalization of DHPS in the chloroplast was essential for its participation in lysine biosynthesis as no lysine overproduction was obtained in transgenic plants that expressed the bacterial enzyme in the cytoplasm. The elevated level of free lysine in the transgenic plants was sufficient to inhibit, in vivo, a second key enzyme in lysine biosynthesis, namely, aspartate kinase, with no apparent influence on lysine accumulation. The present report not only provides a better understanding of the regulation of lysine biosynthesis in higher plants but also offers a new strategy to improve the production of this essential amino acid.
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(1992) Theoretical And Applied Genetics. 83, 3, p. 385-391 Abstract
Three different 3 noncoding sequences of wheat rubisco small subunit (SSU) genes (RbcS) were used as probes to identify the gene members of different RbcS subfamilies in the common wheat cultivar Chinese Spring (CS). All genes of the wheat RbcS multigene family were previously assigned to the long arm of homoeologous group 5 and to the short arm of homoeologous group 2 chromosomes of cv CS. Extracted DNA from various aneuploids of these homoeologous groups was digested with four restriction enzymes and hybridized with three different 3 noncoding sequences of wheat SSU clones. All RbcS genes located on the long arm of homoeologous group 5 chromosomes were found to comprise a single subfamily, while those located on the short arm of group 2 comprised three subfamilies. Each of the ancestral diploid genomes A, B, and D has at least one representative gene in each subfamily, suggesting that the divergence into subfamilies preceded the differentiation into species. This divergence of the RbcS genes, which is presumably accompanied by a similar divergence in the 5 region, may lead to differential expression of various subfamilies in different tissues and in different developmental stages, in response to different environmental conditions. Moreover, members of one subfamily that belong to different genomes may have diverged also in the coding sequence and, consequently, code for distinguishable SSU. It is assumed that such utilization of the RbcS multigene family increases the adaptability and phenotypic plasticity of common wheat over its diploid progenitors.
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(1992) Plant Physiology. 98, 2, p. 433-441 Abstract
The high molecular weight glutenin subunits are considered one of the most important components of wheat (Triticum aestivum) gluten, but their structure and interactions with other gluten proteins are still unknown. Understanding the role of these proteins in gluten formation may be aided by analyses of the conformation and interactions of individual wild-type and modified subunits expressed in heterologous systems. In the present report, the bacterium Escherichia coli was used to synthesize four naturally occurring X- and Y-type wheat high molecular weight glutenin subunits of the Glu-1D locus, as well as four bipartite chimeras of these proteins. Naturally occurring subunits synthesized in the bacteria exhibited sodium dodecyl sulfate-polyacrylamide gel electrophoresis migration properties identical to those of high molecular weight glutenin subunits extracted from wheat grains. Wild-type and chimeric subunits migrated in sodium dodecyl sulfate gels differently than expected based on their molecular weights due to conformational properties of their N- and C-terminal regions. Results from cycles of reductive cleavage and oxidative reformation were consistent with the formation of both inter- and intramolecular disulfide bonds in patterns and proportions that differed among specific high molecular weight glutenin species. Comparison of the chimeric and wild-type proteins indicated that the two C-terminal cysteines of the Y-type subunits are linked by intramolecular disulfide bonds, suggesting that the role of these cysteines in glutenin polymerization may be limited.
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(1992) Plant Physiology. 99, 2, p. 718-724 Abstract
Storage proteins of wheat grains (Triticum L. em Thell) are deposited in protein bodies inside vacuoles. However, the subcellular sites and mechanisms of their aggregation into protein bodies are not clear. In the present report, we provide evidence for two different types of protein bodies, low- and high-density types that accumulate concurrently and independently in developing wheat endosperm cells. Gliadins were present in both types of protein bodies, whereas the high molecular weight glutenins were localized mainly in the dense ones. Pulse-chase experiments verified that the dense protein bodies were not formed by a gradual increase in density but, presumably, by a distinct, quick process of storage protein aggregation. Subcellular fractionation and electron microscopy studies revealed that the wheat homolog of immunoglobulin heavy-chain-binding protein, an endoplasmic reticulum-resident protein, was present within the dense protein bodies, implying that these were formed by aggregation of storage proteins within the endoplasmic reticulum. The present results suggest that a large part of wheat storage proteins aggregate into protein bodies within the rough endoplasmic reticulum. Because these protein bodies are too large to enter the Golgi, they are likely to be transported directly to vacuoles. This route may operate in concert with the known Golgi-mediated transport to vacuoles in which the storage proteins apparently condense into protein bodies at a postendoplasmic reticulum location. Our results further suggest that although gliadins are transported by either one of these routes, the high molecular weight glutenins use only the Golgi bypass route.
1991
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(1991) Theoretical And Applied Genetics. 82, 5, p. 615-620 Abstract
Animal and plant cells contain a family of constitutively expressed HSP-70 cognate proteins that are localized in different subcellular locations and are presumed to play a role in protein folding and transport. Utilizing antibodies raised against the yeast endoplasmicreticulum-localized HSP-70 cognate termed BiP/GRP-78, as well as antibodies raised against the Escherichia coli HSP-70 protein DnaK, we have identified and characterized a large family of closely related proteins in wheat. One protein band of 78 kDa that is apparently closely related to yeast BiP was localized in the endoplasmic reticulum. This band cross-reacted with the yeast BiP but not with the DnaK-specific antibodies. The yeast BiP antibodies also recognized a cytoplasmic protein of 70 kDa that is probably related to the HSC-70 cognate proteins. These two proteins were further confirmed as HSP-70 cognates by their ability to bind to an ATP-agarose column. Probing of proteins from purified wheat mitochondrial preparations with the yeast BiP and DnaK-specific antibodies showed that this organelle contained a family of HSP-70-related proteins. The yeast BiP antibodies recognized two mitochondrial proteins of 60 and 58 kDa, but failed to detect any protein in the size rang of 70 to 80 kDa. However, the presence of immunologically distinct proteins of 90 and 78 kDa, as well as of lower molecular weight from this family in the mitochondria, was shown by probing with the DnaK-specific antibodies. A new protein of 30 kDa, cross-reacting with anti-yeast BiP antibodies, was detected only in developing seeds, close to their maturity. The evolution of HSP-70 cognate proteins in wheat as shown in this study is discussed.
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POLYMORPHISM OF HIGH-MOLECULAR-WEIGHT GLUTENINS IN WILD TETRAPLOID WHEAT - SPATIAL AND TEMPORAL VARIATION IN A NATIVE SITE(1991) Israel Journal of Plant Sciences. 40, 6-May, p. 451-479 Abstract
Variation in the electrophoretic pattern of the high-molecular-weight (HMW) glutenin subunits was studied in the Ammiad population of wild tetraploid wheat, Triticum turgidum var. dicoccoides (genome AABB), during a 5-year period (1983-4 to 1987-8). These storage proteins were analysed following one-dimensional sodium-dodecyl-sulphate polyacrylamide-gel electrophoresis (SDS PAGE), using seeds collected annually from individual plants at 249 defined sampling sites distributed in 11 habitats. Since plants did mt grow at all the sampling points each year, 1108 accessions were analysed altogether. The population was found to be highly polymorphic: the HMW glutenin loci of genome A, Glu-Al-1 and Glu-Al-2, had four and two alleles. respectively, and those of genome B. Glu-Bl-1 and Glu-Bl-2, had five and seven alleles, respectively. The A-genome alleles appeared in 4 combinations, and the B-genome alleles appeared in 12 combinations. There were 18 intergenomic combinations (A and B genotypes), some of which were very rare while others were abundant and distributed along transects in clusters. The spatial distribution of these genotypes was nonrandom, with each of the 11 habitats characterized by different genotype frequencies. Yearly changes in genotypes, mostly occurring in the last 2 years of the study, had little effect on the total frequencies of die various genotypes. A high affinity was found between specific HMW glutenin genotypes and certain habitats. This affinity may have resulted from a random fixation of specific genotypes in different habitats (founder effect) or, alternatively, from natural selection, thus indicating either linkage between HMW glutenin alleles and adaptive genes (hitchhiking effect) or fitness of some of these allele combinations to specific micro-environments.
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(1991) Theoretical And Applied Genetics. 81, 1, p. 98-104 Abstract
The genes coding for the Rubisco small subunit (SSU) and for the α-subunit of the Rubisco-binding protein were located to chromosome arms of common wheat. HindIII-digested total DNA from the hexaploid cultivar Chinese Spring and from ditelosomic and nullisomic-tetrasomic lines was probed with these two genes, whose chromosomal location was deduced from the disappearance of or from changes in the relative intensity of the relevant band(s). The Rubisco SSU pattern consisted of 14 bands, containing at least 21 different types of DNA fragments, which were allocated to two homoeologous groups: 15 to the short arm of group 2 chromosomes (4 to 2AS, 7 to 2BS, and 4 to 2DS) and 6 to the long arm of group 5 chromosomes (2 on each of arms 5AL, 5BL, and 5DL). The pattern of the Rubisco-binding protein consisted of three bands, each containing one type of fragment. These fragments were located to be on the short arm of group 2 chromosomes. The restriction fragment length polymorphism (RFLP) patterns of several hexaploid and tetraploid lines were highly conserved, whereas the patterns of several of their diploid progenitors were more variable. The variations found in the polyploid species were mainly confined to the B genome. The patterns of the diploids T. monococcum var. urartu and Ae. squarrosa were similar to those of the A and D genome, respectively, in polyploid wheats. The pattern of T. monococcum var. boeoticum was different from the patterns of the A genome, and the patterns of the diploids Ae. speltoides, Ae. longissima, and Ae. Searsii differed from that of the B genome.
1990
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(1990) Plant Cell. 2, 9, p. 941-950 Abstract
[alpha]-Gliadins and [gamma]-gliadins are two closely related wheat storage proteins that evolved from a common ancestral gene. However, synthesis of [alpha]-gliadins and [gamma]-gliadins in Xenopus laevis oocytes revealed striking differences in their subcellular routing. The major portion of [alpha]-gliadin accumulated inside the oocyte, whereas most of the [gamma]-gliadin was secreted. Disruption of the Golgi apparatus by monensin revealed that the major part of secretion of [gamma]-gliadin is Golgi mediated. The difference in the subcellular route between [alpha]-gliadin and [gamma]-gliadin may be attributed to differential transport from the endoplasmic reticulum to the Golgi apparatus, a process that is generally the rate-limiting step in protein secretion. Coinjection of the two mRNAs had no effect on their routing, indicating no interaction between them. Our results support the hypothesis that subcellular transport of gliadins in wheat endosperm occurs in two separate routes; one is Golgi mediated, and the other is not. We also show that the subcellular transport may be markedly affected by small structural variations within closely related storage proteins.
1989
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(1989) Proceedings of the National Academy of Sciences of the United States of America. 86, 20, p. 7756-7760 Abstract
An engineered DNA fragment containing a DNA sequence encoding a wheat high molecular weight glutenin subunit was cloned into a bacterial expression vector that is based on bacteriophage T7 RNA polymerase. The resulting plasmid directed the synthesis of large amounts of the mature form of the wheat high molecular weight glutenin subunit in Escherichia coli. This protein comigrated in SDS/PAGE with a native high molecular weight glutenin subunit extracted from wheat endosperm and cross-reacted with antibodies raised against purified wheat high molecular weight glutenins. The wheat subunit synthesized in E. coli also exhibited pI and solubility characteristics identical to those of native wheat subunits. Moreover, the wheat glutenin subunit produced in E. coli cells self-assembled into oligomers linked by intermolecular disulfide bonds, a process that plays an important role in the assembly of native glutenins during gluten formation. The large amounts of a purified wheat subunit obtained from E. coli will enable investigators to analyze the three-dimensional structure of that protein and to identify protein sequences that affect the bread-making quality of wheat dough.
1988
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(1988) Heredity. 61, 1, p. 63-72 Abstract
Polymorphism of the high molecular weight glutenin subunits was studied in 456 accessions of the wild wheat Triticum turgidum var. dicoccoides (2n = 4x = 28; genomes AABB), originating from 21 populations in Israel. A total of 50 different SDS PAGE migration patterns were observed, resulting from the combinations of 15 subunit patterns of the A genome and 24 subunit patterns of the B genome. Most migration patterns consisted of five subunits, varying between three and six. The migration patterns of the A genome had zero to three subunitstwo being most common. The apparent molecular weights (MWs) of the slowest migrating subunit (114, 000 to 103, 500) and of the next in rate of migration (106, 000 to 96, 000) were highly correlated (r = 0·97). Also, both subunits were either present (in most accessions) or absent. In 82.3 per cent of the accessions, the third subunit (MW 76, 000 to 71, 500) was absent, while in 16.9 per cent of the accessions all three subunits of the A genome were absent. The migration patterns of the B genome had one to three subunitsthree being most common. The slowest migrating subunit (99, 500 to 93, 000) was present in almost all cases (99·3 per cent). The MWs of the next two subunits (90, 500 to 82, 000 and 86, 000 to 78, 000, respectively) were highly correlated (r = 0·95). Also, either both subunits were present, as in most cases (94·4 per cent), or absent (5·6 per cent). A nomenclature for the genes encoding for the HMW glutenins is proposed based on the following model: The three subunit groups controlled by each genome are encoded by two genes. In genome A, one gene (Glu-A1-1), with 12 alleles, encodes for the two correlated subunit groups 1Ax and 1Ax; the other gene (Glu-A1-2), with 3 alleles, encodes for the fast-migrating subunit group (1Ay). In genome B, one gene (Glu-B1-1), with 8 alleles, encodes for the slow-migrating subunit group (1Bx), and the other gene (Glu-B1-2), with 10 alleles, encodes for the two correlated subunit groups, 1By and 1By. The polymorphism of the HMW glutenin genes found in var. dicoccoides is much higher than that of cultivated wheats as well as of genes coding for enzymes in var. dicoccoides.
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(1988) Science. 240, 4852, p. 662-664 Abstract
Zeins, the storage proteins of maize, are totally lacking in the essential amino acids lysine and tryptophan. Lysine codons and lysine- and typtophan-encoding oligonucleotides were introduced at several positions into a 19-kilodalton zein complementary DNA by oligonucleotide-mediated mutagenesis. A 450-base pair open reading frame from a simian virus 40 (SV40) coat protein was also engineered into the zein coding region. Messenger RNAs for the modified zeins were synthesized in vitro with an SP6 RNA polymerase system and injected into Xenopus laevis oocytes. The modifications did not affect the translation, signal peptide cleavage, or stability of the zeins. The ability of the modified zeins to assemble into structures similar to maize protein bodies was assayed by two criteria: assembly into membrane-bound vesicles resistant to exogenously added protease, and ability to self-aggregate into dense structures. All of the modified zeins were membrane-bound; only the one containing a 17-kilodalton SV40 protein fragment was unable to aggregate. These findings suggest that it may be possible to create high-lysine corn by genetic engineering.
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Inactivity of high-molecular-weight glutenin genes in wild diploid and tetraploid wheats(1988) Proceedings of the seventh international wheat genetics symposium, held at Cambridge, UK, 13-19 July 1988. Vol. 1. p. 81-86 Abstract
1987
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Genetic studies on storage proteins in wheat(1987) Israel Agresearch, Journal of the Agricultural Research Organization. 1, p. 87-99 Abstract
1986
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(1986) Proceedings of the National Academy of Sciences of the United States of America. 83, 17, p. 6524-6528 Abstract
Several aneuploid lines and one intervarietal substitution line of the hexaploid wheat Triticum aestivum (2n = 6χ = 42; genomes AABBDD) cv. Chinese Spring were used to study the effects of different doses of chromosomes 1B, 1D, or 1A on the amount of the high molecular weight ('HMW') glutenins and gliadins of endosperm. These homeologous chromosomes carry HMW glutenin and gliadin gene clusters on their long and short arms, respectively. Increasing the dosage of chromosome 1B of Chinese Spring in plants having in their 3n endosperm zero or the normal three doses of the homeologue 1D, as well as in plants carrying in their endosperm one dose of 1B of the cultivar Timstein, had a dual effect: on one hand, a nonlinear increase in the amount of each subunit encoded by the chromosome whose dosage was elevated and, on the other hand, a compensating nonspecific decrease in the amount of other HMW glutenin and gliadin subunits encoded either by the homeoalleles on 1D or by the homoalleles on 1B of Timstein, respectively. Deletion of chromosome arm 1BL, which carries only a few HMW glutenin genes, had no significant effect on the amount of HMW glutenins encoded by 1DL and HMW gliadins encoded by 1DS and 1BS. However, deletion of 1BS or 1DS, each carrying many gliadin genes, caused a significant but nonspecific increase in the HMW glutenins and gliadins encoded by the remaining arms of 1B and 1D. The possible mechanism and evolutionary implications of gene-dosage compensation in polyploid wheat are discussed.
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(1986) The Origin and domestication of cultivated plants : symposium / organized by Centro linceo interdisciplinare di scienze matematiche e loro applicazioni, Accademia nazionale dei Lincei, Rome, 25-27 November 1985. p. 83-100 Abstract
The polyploid species of the weat (Triticum and Aegilops) group constitute a classical example of evolutionary success through allopolyploidy. The evolutionary advantage of these polyploids over their diploid progenitors reflects a successful genètic system based on allopolyploidy, diploid-like cytological behavior and predominant self-pollination. The different genomes of the newly formed allopolyploids, derived recurrently from diverging diploid species, differ by numerous allelic variations of homoeologous loci. The permanent heterozygosity of the different homoeoalleles facilitated enzyme multiplicity and thereby, wider and greater adaptability. Whereas this genetic multiplication has an evolutionary advantage for loci coding for functional proteins, it may be redundant for others, e.g., multigene families such as rRNA genes or storage protein genes. Activity of all loci in such genes might result in over-production and inefficiency. One should expect, therefore, to find in polyploid wheat regulatory processes involved, on one hand, in the preservation of the activity of favorable gene loci and, on the other hand, in the reduction of the number and activity of the redundant ones. The latter include diploidization (inactivation) and gene dosage compensation (reduced gene expression) processes. The presented evidence indicates that diploidization is a non-random process achieved through mutations or intergenomic suppression. In contrast, gene dosage compensation is a non-specific process determined by several post-transcriptional rate-limiting factors. The evolutionary significance of these regulatory processes is discussed.
1985
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(1985) Theoretical And Applied Genetics. 69, 4, p. 429-435 Abstract
The effect of various chromosomes of Aegilops longissima when added to the common wheat cultivar 'Chinese Spring' was evaluated at two levels of nitrogen fertilization for absolute and relative amount of protein in the grain. All the added chromosomes of Ae. longissima increased protein percentage: protein increase by chromosomes D, C and A averaged 3.8% while that by chromosomes F, E, G and B averaged 1.7%. Addition lines F, D and C had a significantly higher protein weight per grain. On the other hand, lines A, E and G had reduced grain protein weight per grain as compared with that of 'Chinese Spring'. Line C carries the HMW glutenin and some of the gliadin subunits of Ae. longissima. The effect of this line, however, and obviously that of the other lines on protein content was through genes controlling the level of storage protein rather than through genes that code directly for these proteins. Nitrogen fertilization affected protein content and the relative amount of the various protein fractions in a similar manner in every addition line. When high levels of nitrogen fertilization were compared to low ones, the relative amount of the HMW glutenins remained constant while that of HMW gliadins increased and that of the LMW subunits decreased. In contrast to the nitrogen effect, increase in protein content by the addition of longissima chromosomes did not change the relative amounts of the various protein fractions.
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(1985) Theoretical And Applied Genetics. 69, 5-6, p. 583-589 Abstract
Total endosperm proteins extracted from both several common wheat cultivars and some intervarietal substitution lines derived from them were fractionated according to their molecular weight in a high resolution one-dimensional gel electrophoresis. The four donor cultivars and the recipient one - 'Chinese Spring', possessed differentially migrating protein bands in the fractions of high molecular weight (HMW) glutenins and gliadins. Several of these bands were identified for the first time in this study. By utilizing intervarietal substitution lines the control of the HMW glutenins and gliadins by chromosomes of homoeologous group 1 was either reaffirmed or, for the new bands, established. Several HMW gliadin subunits showed a considerable variation in their staining intensity in the intervarietal substitution lines indicating that their expression was dependent on the genetic background.
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1984
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(1984) Molecular Genetics and Genomics. 193, 2, p. 293-298 Abstract
The inheritance of the high molecular weight (HMW) glutenins and of several gliadins controlled, respectively, by the long and short arms of chromosome 1B of common wheat was studied. Analysis was carried out on the progeny of two inter-varietal crosses in which the parental lines possessed differentially migrating subunits as revealed by sodium dodecyl sulphate polyacrylamide gel electrophoresis. No recombination event was detected either within the fraction of the HMW glutenins or among most of the gliadin subunits studied indicating that they are controlled by tightly linked gene clusters. One gliadin subunit (B30) showed 25.5% recombination frequency with the rest of the gliadin subunits and 23.5% recombination frequency with the fraction of the HMW glutenin subunits. It has been concluded that this subunit is controlled by a separate locus (Gld-B6), proximal to the major gliadin gene cluster on the short arm of chromosome 1B. Consequently, the recombination percentage between the glutenin loci and most of the gliadin loci was calculated as 49.0 and the distance in centi-Morgans (cM) as 53.6. The estimated distance in cM is very close to the observed recombination percentage. A genetic map of these storage protein genes is presented.
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A DEFICIENCY OF THE RAPIDLY MIGRATING HIGH MOLECULAR-WEIGHT GLUTENIN SUBUNIT-D5 IN COMMON WHEAT(1984) Cereal Research Communications. 12, 4-Mar, p. 259-261 Abstract
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INTERGENOMIC SUPPRESSION OF ENDOSPERM PROTEIN GENES IN COMMON WHEAT(1984) Genome. 26, 6, p. 651-656 Abstract
1983
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Changes in chromatin structure at the replication fork. DNase I and trypsin-micrococcal nuclease effects on approximately 300- and 150-base pair nascent DNAs(1983) Journal of Biological Chemistry. 258, 18, p. 1274-1279 Abstract
DNase I, trypsin, and micrococcal nuclease are used to further probe the structure of nascent deoxyribonucleoprotein (DNP) fractions which appear after in vivo 20-s pulse labeling of sea urchin embryos with [3H]thymidine. We present evidence that the large nascent DNP which protects the approximately 300-base pair large nascent DNA consists of at least one nucleosome core. This is based on fractionation in denaturing polyacrylamide gels of DNA extracted from large nascent DNP fractions of a micrococcal nuclease + DNase I digest of nuclei. The data also suggest the existence of a DNase I-hypersensitive site(s) within the large nascent DNP; this is consistent with the hypothesis that the latter consists of closely packed dinucleosome cores. Histone H1 and non-histone proteins do not account for the previously reported unusual hyperresistance of the large nascent DNA against micrococcal nuclease. The protection offered this approximately 300-base pair nascent DNA was not eliminated by an 0.2-microgram/ml trypsin pretreatment which removes the above proteins from the chromatin. However, 5-10 micrograms/ml of trypsin, which remove a portion of the NH2 termini of the four core histones of nucleosomes, eliminate the hyperresistance of the large nascent DNA to subsequent micrococcal nuclease digestion, while nascent and bulk monomer DNAs remain unaffected. This indicates histone-histone and/or histone-DNA interactions within the large nascent DNP which differ from those of nascent and bulk mononucleosome cores.
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(1983) Theoretical And Applied Genetics. 66, 1, p. 77-86 Abstract
Endosperm protein subunits of 109 primitive and modern lines of hexaploid wheat, Triticum aestivum L. em. Thell., were fractionated by one-dimensional, high resolution, sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis (PAGE). A wide range of both qualitative and quantitative variation was observed in the fractions of the high molecular weight (HMW) glutenin and gliadin subunits of the different lines. The qualitative variation was expressed in the number of subunits per fraction and in their molecular weight, as determined by the differential rate of migration. The quantitative variation was expressed in the differential staining intensity of several subunits. The widest variation was detected in the HMW glutenin and gliadin subunits controlled by chromosome 1B while a much smaller variation was observed in those subunits controlled by chromosome 1A and further smaller variation in the subunits controlled by 1D. Only a small number of subunits in both fractions was found to be controlled by chromosome 1A indicating that diploidization of endosperm protein genes in common wheat has been non-random. The genetic and evolutionary implications of these findings are discussed.
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Diploidization of endosperm protein genes in polyploid wheats(1983) Proceedings of the Sixth International Wheat Genetics Symposium: held at Kyoto, Japan : November 28-December 3, 1983. p. 1119-1123 Abstract
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(1983) Theoretical And Applied Genetics. 64, 2, p. 97-101 Abstract
Total endosperm protein subunits, extracted from the common wheat cultivar Chinese Spring and from some of its aneuploid lines, were fractionated according to their molecular weight (MW) in an improved high resolution one-dimensional sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis (PAGE). The resolution obtained by this method and, in particular, that of the high molecular weight (HMW) glutenin and gliadin subunits approached that of a previous report in which two-dimensional fractionation system based on charge and MW was used. In the cultivar Chinese Spring, 21 discrete protein bands were resolved and the chromosomes controlling many of them were either reconfirmed, or, in some cases, established. The advantages of this high resolution SDS PAGE technique are discussed.
1981
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(1981) Nucleic Acids Research. 9, 16, p. 3991-4005 Abstract
Discrete deoxyribonucleoproteins (DNPs) containing nascent and/or bulk DNA, were obtained by fractionating micrococcal nuclease digests of nuclei from 3H-thymidine pulse (15-20 sec) and 14C-thymidine long (16 h) labeled sea urchin embryos in polyacrylamide gels. One of these DNPs was shown to contain the micrococcal nuclease resistant 300 bp "large nascent DNA" described in Cell 14, 259-267, 1978. The bulk and nascent mononucleosome fractions provided evidence for the preferential digestion by micrococcal nuclease of nascent over bulk linker regions to yield mononucleosome cores with nascent DNA.DNAase I was used to probe whether any nascent DNA is in nucleosomes. Nascent as well as bulk single-stranded DNA fragments occurred in multiples of 10.4 bases with higher than random frequencies of certain fragment sizes (for instance 83 bases) as expected from a nucleosome structure. However, a striking background of nascent DNA between nascent DNA peaks was observed. This was eliminated by a pulse-chase treatment or by digestion of pulse-labeled nuclei with micrococcal nuclease together with DNAase I. One of several possible interpretations of these results suggests that a transient change in nucleosome structure may have created additional sites for the nicking of nascent DNA by DNAase I; the micrococcal nuclease sensitivity of the interpeak radioactivity suggest its origin from the linker region.Endogenous nuclease of sea urchin embryos cleaves chromatin DNA in a manner similar to that of DNAase I.