Publications

In this study, a chilli pepper (Capsicum annuum) panel for post-harvest carotenoid retention was studied to elucidate underlying mechanisms associated with this commercial trait of interest. Following drying and storage, some lines within the panel had an increase in carotenoids approaching 50% compared with the initial content at the fresh fruit stage. Other lines displayed a 25% loss of carotenoids. The quantitative determination of carotenoid pigments with concurrent cellular analysis indicated that in most cases, pepper fruit with thicker (up to 4-fold) lipid exocarp layers and smooth surfaces exhibit improved carotenoid retention properties. Total cutin monomer content increased in medium/high carotenoid retention fruits and subepidermal cutin deposits were responsible for the difference in exocarp thickness. Cutin biosynthesis and cuticle precursor transport genes were differentially expressed between medium/high and low carotenoid retention genotypes, and this supports the hypothesis that the fruit cuticle can contribute to carotenoid retention. Enzymatic degradation of the cuticle and cell wall suggests that in Capsicum the carotenoids (capsanthin and its esters) are embedded in the lipidic exocarp layer. This was not the case in tomato. Collectively, the data suggest that the fruit cuticle could provide an exploitable resource for the enhancement of fruit quality.

Plants developed sophisticated mechanisms to receive environmental stimuli and generate appropriate signals to maintain optimal growth and stress responses. A fascinating strategy employed by plants is the use of long-distance mobile signals which can trigger local and distant responses across the entire plant. Some metabolites play a central role as long-distance mobile signals allowing plants to communicate across tissues and mount robust stress responses. In the review, we summarize the current state of knowledge regarding the various long-distance mobile metabolites and their function in stress response and signaling pathways. We also raise questions with respect to how can we identify new mobile metabolites and engineer them to improve plant health and resilience.

The plant hormone gibberellin (GA) regulates multiple developmental processes. It accumulates in the root elongating endodermis, but how it moves into this cell file and the significance of this accumulation are unclear. Here we identify three NITRATE TRANSPORTER1/PEPTIDE TRANSPORTER (NPF) transporters required for GA and abscisic acid (ABA) translocation. We demonstrate that NPF2.14 is a subcellular GA/ABA transporter, presumably the first to be identified in plants, facilitating GA and ABA accumulation in the root endodermis to regulate suberization. Further, NPF2.12 and NPF2.13, closely related proteins, are plasma membrane-localized GA and ABA importers that facilitate shoot-to-root GA₁₂ translocation, regulating endodermal hormone accumulation. This work reveals that GA is required for root suberization and that GA and ABA can act non-antagonistically. We demonstrate how the clade of transporters mediates hormone flow with cell-file-specific vacuolar storage at the phloem unloading zone, and slow release of hormone to induce suberin formation in the maturation zone.

This year marks the 25th anniversary of the coinage of the term metabolome [S. G. Oliver et al., Trends Biotech. 16, 373–378 (1998)]. As the field rapidly advances, it is important to take stock of the progress which has been made to best inform the disciplines future. While a medical-centric perspective on metabolomics has recently been published [M. Giera et al., Cell Metab. 34, 21–34 (2022)], this largely ignores the pioneering contributions made by the plant and microbial science communities. In this perspective, we provide a contemporary overview of all fields in which metabolomics is employed with particular emphasis on both methodological and application breakthroughs made in plant and microbial sciences that have shaped this evolving research discipline from the very early days of its establishment. This will not cover all types of metabolomics assays currently employed but will focus mainly on those utilizing mass spectrometry–based measurements since they are currently by far the most prominent. Having established the historical context of metabolomics, we will address the key challenges currently facing metabolomics and offer potential approaches by which these can be faced. Most salient among these is the fact that the vast majority of mass features are as yet not annotated with high confidence; what we may refer to as definitive identification. We discuss the potential of both standard compound libraries and artificial intelligence technologies to address this challenge and the use of natural variance–based approaches such as genome-wide association studies in attempt to assign specific functions to the myriad of structurally similar and complex specialized metabolites. We conclude by stating our contention that as these challenges are epic and that they will need far greater cooperative efforts from biologists, chemists, and computer scientists with an interest in all kingdoms of life than have been made to date. Ultimately, a better linkage of metabolome and genome data will likely also be needed particularly considering the Earth BioGenome Project.

Brassica rapa (B. rapa) and its subspecies contain many bioactive metabolites that are important for plant defense and human health. This study aimed at investigating the metabolite composition and variation among a large collection of B. rapa genotypes, including subspecies and their accessions. Metabolite profiling of leaves of 102 B. rapa genotypes was performed using ultra-performance liquid chromatography coupled with a photodiode array detector and quadrupole time-of-flight mass spectrometry (UPLC-PDA-QTOF-MS/MS). In total, 346 metabolites belonging to different chemical classes were tentatively identified; 36 out of them were assigned with high confidence using authentic standards and 184 were those reported in B. rapa leaves for the first time. The accumulation and variation of metabolites among genotypes were characterized and compared to their phylogenetic distance. We found 47 metabolites, mostly representing anthocyanins, flavonols, and hydroxycinnamic acid derivatives that displayed a significant correlation to the phylogenetic relatedness and determined four major phylometabolic branches; 1) Chinese cabbage, 2) yellow sarson and rapid cycling, 3) the mizuna-komatsuna-turnip-caitai; and 4) a mixed cluster. These metabolites denote the selective pressure on the metabolic network during B. rapa breeding. We present a unique study that combines metabolite profiling data with phylogenetic analysis in a large collection of B. rapa subspecies. We showed how selective breeding utilizes the biochemical potential of wild B. rapa leading to highly diverse metabolic phenotypes. Our work provides the basis for further studies on B. rapa metabolism and nutritional traits improvement.

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Chemical exchange often serves as the first step in plant-microbe interactions and exchanges of various signals, nutrients, and metabolites continue throughout the interaction. Here, we highlight the role of metabolite exchanges and metabolic crosstalk in the microbiome-root-shoot-environment nexus. Roots secret a diverse set of metabolites; this assortment of root exudates, including secondary metabolites such as benzoxazinoids, coumarins, flavonoids, indolic compounds, and terpenes, shapes the rhizosphere microbiome. In turn, the rhizosphere microbiome affects plant growth and defense. These inter-kingdom chemical interactions are based on a metabolic circular economy, a seemingly wasteless system in which rhizosphere members exchange (i.e. consume, reuse, and redesign) metabolites. This review also describes the recently discovered phenomenon 'Systemically Induced Root Exudation of Metabolites' in which the rhizosphere microbiome governs plant metabolism by inducing systemic responses that shift the metabolic profiles of root exudates. Metabolic exchange in the rhizosphere is based on chemical gradients that form specific microhabitats for microbial colonization and we describe recently developed high-resolution methods to study chemical interactions in the rhizosphere. Finally, we propose an action plan to advance the metabolic circular economy in the rhizosphere for sustainable solutions to the cumulative degradation of soil health in agricultural lands.

Solanum steroidal glycoalkaloids (SGAs) are renowned defense metabolites exhibiting spectacular structural diversity. Genes and enzymes generating SGAs precursor pathway, SGAs scaffold and glycosylated forms have been largely identified. Yet, majority of downstream metabolic steps creating the vast repertoire of SGAs remain untapped. Here, we discovered that members of the 2-OXOGLUTARATE-DEPENDENT DIOXYGENASE (2-ODD) family play a prominent role in SGAs metabolism, carrying out three distinct backbone-modifying oxidative steps in addition to the three formerly reported pathway reactions. The GLYCOALKALOID METABOLISM34 (GAME34) enzyme catalyzes the conversion of core SGAs to habrochaitosides in wild tomato S. habrochaites. Cultivated tomato plants overexpressing GAME34 ectopically accumulates habrochaitosides. These habrochaitosides enriched plants extracts potently inhibits Puccinia spp. spore germination, a significant Solanaceae crops fungal pathogen. Another 2-ODD enzyme, GAME33, acts as a desaturase (via hydroxylation and E/F ring rearrangement) forming unique, yet unreported SGAs. Conversion of bitter α-tomatine to ripe fruit, non-bitter SGAs (e.g. esculeoside A) requires two hydroxylations; while the known GAME31 2-ODD enzyme catalyzes hydroxytomatine formation, we find that GAME40 catalyzes penultimate step in pathway and generates acetoxy-hydroxytomatine towards esculeosides accumulation. Our results highlight the significant contribution of 2-ODD enzymes to the remarkable structural diversity found in plant steroidal specialized metabolism.

Nitrogen (N) fertilization is one of the main inputs to increase crop yield and food production. However, crops utilize only 30–40% of N applied; the remainder is leached into the soil, causing environmental and health damage. In this scenario, the improvement of nitrogen-use efficiency (NUE) will be an essential strategy for sustainable agriculture. Here, we compared two pairs of NUE-contrasting eggplant (Solanum melongena L.) genotypes, employing GC-MS and UPLC-qTOF-MS-based technologies to determine the differential profiles of primary and secondary metabolites in root and shoot tissues, under N starvation as well as at short- and long-term N-limiting resupply. Firstly, differences in the primary metabolism pathways of shoots related to alanine, aspartate and glutamate; starch, sucrose and glycine; serine and threonine; and in secondary metabolites biosynthesis were detected. An integrated analysis between differentially accumulated metabolites and expressed transcripts highlighted a key role of glycine accumulation and the related glyA transcript in the N-use-efficient genotypes to cope with N-limiting stress. Interestingly, a correlation between both sucrose synthase (SUS)- and fructokinase (scrK)-transcript abundances, as well as D-glucose and D-fructose accumulation, appeared useful to distinguish the N-use-efficient genotypes. Furthermore, increased levels of L-aspartate and L-asparagine in the N-use-efficient genotypes at short-term low-N exposure were detected. Granule-bound starch synthase (WAXY) and endoglucanase (E3.2.1.4) downregulation at long-term N stress was observed. Therefore, genes and metabolites related to these pathways could be exploited to improve NUE in eggplant.(This article belongs to the Special Issue Improving Nitrogen Use Efficiency in Model and Crop Plants: From Lab to Field)

Steroidal glycoalkaloids (SGAs) are protective metabolites constitutively produced by Solanaceae species. Genes and enzymes generating the vast structural diversity of SGAs have been largely identified. Yet, mechanisms of hormone pathways coordinating defence (jasmonate; JA) and growth (gibberellin; GA) controlling SGAs metabolism remain unclear.
We used tomato to decipher the hormonal regulation of SGAs metabolism during growth vs defence tradeoff. This was performed by genetic and biochemical characterisation of different JA and GA pathways components, coupled with in vitro experiments to elucidate the crosstalk between these hormone pathways mediating SGAs metabolism.
We discovered that reduced active JA results in decreased SGA production, while low levels of GA or its receptor led to elevated SGA accumulation. We showed that MYC1 and MYC2 transcription factors mediate the JA/GA crosstalk by transcriptional activation of SGA biosynthesis and GA catabolism genes. Furthermore, MYC1 and MYC2 transcriptionally regulate the GA signalling suppressor DELLA that by itself interferes in JA-mediated SGA control by modulating MYC activity through protein–protein interaction. Chemical and fungal pathogen treatments reinforced the concept of JA/GA crosstalk during SGA metabolism.
These findings revealed the mechanism of JA/GA interplay in SGA biosynthesis to balance the cost of chemical defence with growth.

The effects of abscisic acid (ABA) on plant growth, development, and response to the environment depend on local ABA concentrations. Here, we show that in Arabidopsis, ABA homeostasis is regulated by two previously unknown ABA transporters. Adenosine triphosphate–binding cassette subfamily G member 17 (ABCG17) and ABCG18 are localized to the plasma membranes of leaf mesophyll and cortex cells to redundantly promote ABA import, leading to conjugated inactive ABA sinks, thus restricting stomatal closure. ABCG17 and ABCG18 double knockdown revealed that the transporters encoded by these genes not only limit stomatal aperture size, conductance, and transpiration while increasing water use efficiency but also control ABA translocation from the shoot to the root to regulate lateral root emergence. Under abiotic stress conditions, ABCG17 and ABCG18 are transcriptionally repressed, promoting active ABA movement and response. The transport mechanism mediated by ABCG17 and ABCG18 allows plants to maintain ABA homeostasis under normal growth conditions.

This Perspective, from a large group of metabolomics experts, provides best practices and simplified reporting guidelines for practitioners of liquid chromatography- and gas chromatography-mass spectrometry-based metabolomics. Mass spectrometry-based metabolomics approaches can enable detection and quantification of many thousands of metabolite features simultaneously. However, compound identification and reliable quantification are greatly complicated owing to the chemical complexity and dynamic range of the metabolome. Simultaneous quantification of many metabolites within complex mixtures can additionally be complicated by ion suppression, fragmentation and the presence of isomers. Here we present guidelines covering sample preparation, replication and randomization, quantification, recovery and recombination, ion suppression and peak misidentification, as a means to enable high-quality reporting of liquid chromatography- and gas chromatography-mass spectrometry-based metabolomics-derived data.

Background: Polyphenols are secondary metabolites produced by plants to defend themselves from environmental stressors. We explored the effect of Wolffia globosa ‘Mankai’, a novel cultivated strain of a polyphenol-rich aquatic plant, on the metabolomic-gut clinical axis in vitro, in-vivo and in a clinical trial. Methods: We used mass-spectrometry-based metabolomics methods from three laboratories to detect Mankai phenolic metabolites and examined predicted functional pathways in a Mankai artificial-gut bioreactor. Plasma and urine polyphenols were assessed among the 294 DIRECT-PLUS 18-month trial participants, comparing the effect of a polyphenol-rich green-Mediterranean diet (+1240 mg/polyphenols/day, provided by Mankai, green tea and walnuts) to a walnuts-enriched (+440 mg/polyphenols/day) Mediterranean diet and a healthy controlled diet. Results: Approximately 200 different phenolic compounds were specifically detected in the Mankai plant. The Mankai-supplemented bioreactor artificial gut displayed a significantly higher relative-abundance of 16S-rRNA bacterial gene sequences encoding for enzymes involved in phenolic compound degradation. In humans, several Mankai-related plasma and urine polyphenols were differentially elevated in the green Mediterranean group compared with the other groups (p

Fruits of nonastringent persimmon cultivars, as compared to astringent ones, were more resistant to infection despite having lower polyphenol content. Metabolic analysis from the pulp of nonastringent "Shinshu", as compared to the astringent "Triumph", revealed a higher concentration of salicylic, coumaric, quinic, 5--feruloyl quinic, ferulic acids, β-glucogallin, gallocatechin, catechin, and procyanidins. Selected compounds like salicylic, ferulic, and ρ-coumaric acids inhibited growth, and higher activity was demonstrated for methyl ferulic and methyl ρ-coumaric acids. These compounds also reduced growth and the black spot disease in stored fruits. On the other hand, methyl gallic acid was a predominant compound in the "Triumph" pulp, as compared to the "Shinshu" pulp, and it augmented growth and . Our results might explain the high sensitivity of the cultivar "Triumph" to . It also emphasizes that specific phenolic compounds, and not the total phenol, affect susceptibility to fungal infection.

Fruit taste is determined by sugars, acids and in some species, bitter chemicals. Attraction of seed-dispersing organisms in nature and breeding for consumer preferences requires reduced fruit bitterness. A key metabolic shift during ripening prevents tomato fruit bitterness by eliminating α-tomatine, a renowned defence-associated Solanum alkaloid. Here, we combined fine mapping with information from 150 resequenced genomes and genotyping a 650-tomato core collection to identify nine bitter-tasting accessions including the 'high tomatine' Peruvian landraces reported in the literature. These 'bitter' accessions contain a deletion in GORKY, a nitrate/peptide family transporter mediating α-tomatine subcellular localization during fruit ripening. GORKY exports α-tomatine and its derivatives from the vacuole to the cytosol and this facilitates the conversion of the entire α-tomatine pool to non-bitter forms, rendering the fruit palatable. Hence, GORKY activity was a notable innovation in the process of tomato fruit domestication and breeding.

N-hydroxy-pipecolic acid (NHP) activates plant systemic acquired resistance (SAR). Enhanced defense responses are typically accompanied by deficiency in plant development and reproduction. Despite of extensive studies on SAR induction, the effects of NHP metabolism on plant growth remain largely unclear. In this study, we discovered that NHP glycosylation is a critical factor that fine-tunes the tradeoff between SAR defense and plant growth. We demonstrated that a UDP-glycosyltransferase (UGT76B1) forming NHP glycoside (NHPG) controls the NHP to NHPG ratio. Consistently, the ugt76b1 mutant exhibits enhanced SAR response and an inhibitory effect on plant growth, while UGT76B1 overexpression attenuates SAR response, promotes growth, and delays senescence, indicating that NHP levels are dependent on UGT76B1 function in the course of SAR. Furthermore, our results suggested that, upon pathogen attack, UGT76B1-mediated NHP glycosylation forms a “hand brake” on NHP accumulation by attenuating the positive regulation of NHP biosynthetic pathway genes, highlighting the complexity of SAR-associated networks. In addition, we showed that UGT76B1-mediated NHP glycosylation in the local site is important for fine-tuning SAR response. Our results implicate that engineering plant immunity through manipulating the NHP/NHPG ratio is a promising method to balance growth and defense response in crops.

Suberin lamellae, which provide a hydrophobic protective barrier against biotic and abiotic stresses, are widely deposited in various cell types during plant development and in response to stress. However, it remains unclear how developmental programs interact with stress responses to direct the precise spatiotemporal pattern of suberin deposition. In this study, we found that SHORT-ROOT (SHR), together with its downstream factor MYB36, guided suberization specifically in the root endodermis. Despite a partial dependence on abscisic acid (ABA), the suberization mediated by SHR and MYB36 appeared to derive from a slow readout of developmental programs, which was in contrast to the rapid but transient suberization induced by ABA. Furthermore, we found the MYB39 transcription factor functioned as a common downstream hub of the SHR/MYB36 pathway and the ABA-triggered response. MYB39 could directly bind to theFAR5(alcohol-forming fatty acyl-coenzyme A reductase) promoter to activate its expression. In addition, overexpression of MYB39 dramatically increased the amount of suberization in Arabidopsis roots. Our results provide important insights into the interaction between developmental programs and environmental stimuli in root suberization in Arabidopsis.

The development of genetic transformation methods is critical for enabling the thorough characterization of an organism and is a key step in exploiting any species as a platform for synthetic biology and metabolic engineering approaches. In this work we describe the development of an Agrobacterium rhizogenes-mediated hairy root transformation protocol for the crop and medicinal legume fenugreek (Trigonella foenum-graecum). Fenugreek has a rich and diverse content in bioactive specialised metabolites, notably diosgenin, which is a common precursor for synthetic human hormone production. This makes fenugreek a prime target for identification and engineering of specific biosynthetic pathways for the production of triterpene and steroidal saponins, phenolics, and galactomanans. Through this transformation protocol, we identified a suitable promoter for robust transgene expression in fenugreek. Finally, we establish the proof of principle for the utility of the fenugreek system for metabolic engineering programs, by heterologous expression of known triterpene saponin biosynthesis regulators from the related legume Medicago truncatula in fenugreek hairy roots.

Steroidal glycoalkaloids (SGAs) are defense specialized metabolites produced by thousands of Solanum species. These metabolites are remarkable in structural diversity formed following modifications in their core scaffold. In recent years, it became clear that a large portion of this chemical repertoire was acquired through various molecular mechanisms involving ‘hijacking’ of core metabolism enzymes. This was typically accompanied by gene duplication and divergence and further neofunctionalization as well as modified subcellular localization and evolution of new substrate preferences. In this review, we highlight recent findings in the SGAs biosynthetic pathway and elaborate on similar occurrences in other chemical classes that enabled evolution of specialized metabolic pathways and its underlying structural diversity.

Glycosylation is one of the most prevalent molecular modifications in nature. Single or multiple sugars can decorate a wide range of acceptors from proteins to lipids, cell wall glycans and small molecules, dramatically affecting their activity. Here, we discovered that by 'hijacking' an enzyme of the cellulose synthesis machinery involved in cell wall assembly, plants evolved cellulose synthase-like enzymes (Csls) and acquired the capacity to glucuronidate specialized metabolites, that is, triterpenoid saponins. Apparently, endoplasmic reticulum-membrane localization of Csls and of other pathway proteins was part of evolving a new glycosyltransferase function, as plant metabolite glycosyltransferases typically act in the cytosol. Discovery of glucuronic acid transferases across several plant orders uncovered the long-pursued enzymatic reaction in the production of a low-calorie sweetener from licorice roots. Our work opens the way for engineering potent saponins through microbial fermentation and plant-based systems.Evolution of a group of plant cellulose synthase-like enzymes into specialized glycosyltransferases in the endoplasmic reticulum membrane confers the ability to glucuronidate triterpenoid saponins and other specialized metabolites.

The broad variability of Cucumis melo (melon, Cucurbitaceae) presents a challenge to conventional classification and organization within the species. To shed further light on the infraspecific relationships within C. melo, we compared genotypic and metabolomic similarities among 44 accessions representative of most of the cultivar-groups. Genotyping-by-sequencing (GBS) provided over 20,000 single-nucleotide polymorphisms (SNPs). Metabolomics data of the mature fruit flesh and rind provided over 80,000 metabolomic and elemental features via an orchestra of six complementary metabolomic platforms. These technologies probed polar, semi-polar, and non-polar metabolite fractions as well as a set of mineral elements and included both flavor- and taste-relevant volatile and non-volatile metabolites. Together these results enabled an estimate of "metabolomic/elemental distance" and its correlation with the genetic GBS distance of melon accessions. This study indicates that extensive and non-targeted metabolomics/elemental characterization produced classifications that strongly, but not completely, reflect the current and extensive genetic classification. Certain melon Groups, such as Inodorous, clustered in parallel with the genetic classifications while other genome to metabolome/element associations proved less clear. We suggest that the combined genomic, metabolic, and element data reflect the extensive sexual compatibility among melon accessions and the breeding history that has, for example, targeted metabolic quality traits, such as taste and flavor.

Several months into the ongoing novel coronavirus disease 2019 (COVID-19) pandemic, this work provides a simple and direct projection of the outbreak spreading potential and the pandemic cessation dates in Chinese mainland, Iran, the Philippines and Chinese Taiwan, using the generalized logistic model (GLM). The short-term predicted number of cumulative COVID-19 cases matched the confirmed reports of those who were infected across the four countries and regions, and the long-term forecasts were capable to accurately evaluate the spread of the pandemic in Chinese mainland and Chinese Taiwan, where control measures such as social distancing were fully implemented and sustained, suggesting GLM as a valuable tool for characterizing the transmission dynamics process and the trajectory of COVID-19 pandemic along with the impact of interventions.

A recent study by Gao et al., (Nat. Genet., 2019) presents a tomato pan-genome that was constructed using genome sequences of 725 phylogenetically and geographically representative accessions. The study revealed 4873 genes that are absent from the reference genome, including important genes associated with both disease resistance and flavor, thereby providing an important breeding resource.

Crops are negatively affected by abiotic and biotic stresses, however, plant-microbe cooperation allows prompt buffering of these environmental changes. Microorganisms exhibit an extensive metabolic capability to assist plants in reducing these burdens. Interestingly, beneficial microbes may also trigger, at the host side, a sequence of events from signal perception to metabolic responses leading to stress tolerance or protection against biotic threats. Although plants are well known for their vast chemical diversity, plant-microbial interactions often stimulate the production of a rich and different repertoire of metabolites in plants. The targeted microbial-plant interactions reprogramming plant metabolism represent potential means to foster various pest managements. However, the molecular mechanisms of microbial modulation of plant metabolic plasticity are still poorly understood. Here, we review an increasing amount of reports providing evidence for alterations to plant metabolism caused by beneficial microbial colonization. In addition, we highlight the vital importance of these metabolic reprograms for plants under stress erratic conditions.

Biofilms formed by bacteria on plant roots play an important role in maintaining an optimal rhizosphere environment that supports plant growth and fitness. Bacillus subtilis is a potent plant growth promoter, forming biofilms that play a key role in protecting the host from fungal and bacterial infections. In this work, we demonstrate that the development of B. subtilis biofilms is antagonized by specific indole derivatives that accumulate during symbiotic interactions with plant hosts. Indole derivatives are more potent signals when the plant polysaccharide xylan serves as a carbon source, a mechanism to sustain beneficial biofilms at a biomass that can be supported by the plant. Moreover, B. subtilis biofilms formed by mutants resistant to indole derivatives become deleterious to the plants due to their capacity to consume and recycle plant polysaccharides. These results demonstrate how a dynamic metabolite - based dialogue can promote homeostasis between plant hosts and their beneficial biofilm communities.

The pollen wall is a complex, durable structure essential for plant reproduction. A substantial portion of phenylpropanoids (e.g. flavonols) produced by pollen grain tapetal cells are deposited in the pollen wall. Transcriptional regulation of pollen wall formation has been studied extensively, and a specific regulatory mechanism for Arabidopsis (Arabidopsis thaliana) pollen flavonol biosynthesis has been postulated. Here, metabolome and transcriptome analyses of anthers from mutant and overexpression genotypes revealed that Arabidopsis MYB99, a putative ortholog of the petunia (Petunia hybrida) floral scent regulator ODORANT1 (ODO1), controls the exclusive production of tapetum diglycosylated flavonols and hydroxycinnamic acid amides. We discovered that MYB99 acts in a regulatory triad with MYB21 and MYB24, orthologs of emission of benzenoids I and II, which together with ODO1 coregulate petunia scent biosynthesis genes. Furthermore, promoter-activation assays showed that MYB99 directs precursor supply from the Calvin cycle and oxidative pentose-phosphate pathway in primary metabolism to phenylpropanoid biosynthesis by controlling TRANSKETOLASE2 expression. We provide a model depicting the relationship between the Arabidopsis MYB triad and structural genes from primary and phenylpropanoid metabolism and compare this mechanism with petunia scent control. The discovery of orthologous protein triads producing related secondary metabolites suggests that analogous regulatory modules exist in other plants and act to regulate various branches of the intricate phenylpropanoid pathway.

The skin of fleshy fruit is typically covered by a thick cuticle. Some fruit species develop different forms of layers directly above their skin. Reticulation, for example, is a specialized suberin-based coating that ornaments some commercially important melon (Cucumis melo) fruit and is an important quality trait. Despite its importance, the structural, molecular, and biochemical features associated with reticulation are not fully understood. Here, we performed a multilevel investigation of structural attributes, chemical composition, and gene expression profiles on a set of reticulated and smooth skin melons. High-resolution microscopy, surface profiling, and histochemical staining assays show that reticulation comprises cells with heavily suberized walls accumulating large amounts of typical suberin monomers, as well as lignified cells localized underneath the specialized suberized cell layer. Reticulated skin was characterized by induced expression of biosynthetic genes acting in the core phenylpropanoid, suberin, lignin, and lignan pathways. Transcripts of genes associated with lipid polymer assembly, cell wall organization, and loosening were highly enriched in reticulated skin tissue. These signatures were exclusive to reticulated structures and absent in both the smooth surfaces observed in between reticulated regions and in the skin of smooth fruit. Our data provide important insights into the molecular and metabolic bases of reticulation and its tight association with skin ligno-suberization during melon fruit development. Moreover, these insights are likely to contribute to melon breeding programs aimed at improving postharvest qualities associated with fleshy fruit surface layer.

Soil organisms are diverse taxonomically and functionally. This ecosystem experiences highly complex networks of interactions, but may also present functionally independent entities. Plant roots, a metabolically active hotspot in the soil, take an essential part in shaping the rhizosphere. Tracking the dynamics of root-microbe interactions at high spatial resolution is currently limited due to methodological intricacy. In this study, we developed a novel microfluidics-based device enabling direct imaging of root-bacteria interactions in real time.

High-throughput RNA sequencing has proven invaluable not only to explore gene expression but also for both gene prediction and genome annotation. However, RNA sequencing, carried out on tens or even hundreds of samples, requires easy and cost-effective sample preparation methods using minute RNA amounts. Here, we present TranSeq, a high-throughput 3'-end sequencing procedure that requires 10- to 20-fold fewer sequence reads than the current transcriptomics procedures. TranSeq significantly reduces costs and allows a greater increase in size of sample sets analyzed in a single experiment. Moreover, in comparison with other 3'-end sequencing methods reported to date, we demonstrate here the reliability and immediate applicability of TranSeq and show that it not only provides accurate transcriptome profiles but also produces precise expression measurements of specific gene family members possessing high sequence similarity. This is difficult to achieve in standard RNA-seq methods, in which sequence reads cover the entire transcript. Furthermore, mapping TranSeq reads to the reference tomato genome facilitated the annotation of new transcripts improving >45% of the existing gene models. Hence, we anticipate that using TranSeq will boost large-scale transcriptome assays and increase the spatial and temporal resolution of gene expression data, in both model and non-model plant species. Moreover, as already performed for tomato (ITAG3.0; www.solgenomics.net), we strongly advocate its integration into current and future genome annotations.

Early blight (EB), caused by Alternaria solani, is a major threat to global tomato production. In comparison with cultivated tomato (Solanum lycopersicum), a wild relative, S. arcanum exhibits strong resistance against EB. However, molecular cascades operating during EB resistance in wild or cultivated tomato plants are largely obscure. Here, we provide novel insight into spatio-temporal molecular events in S. arcanum against A. solani. Transcriptome and co-expression analysis presented 33-WRKYs as promising candidates of which 12 SaWRKYs displayed differential expression patterns in resistant and susceptible accessions during EB disease progression. Among these, SaWRKY1 exhibited induced expression with significant modulation in xyloglucan endotrans hydrolase 5 (XTH5) and MYB2 expressions that correlated with the disease phenotypes. Electro-mobility shift assay confirmed physical interaction of recombinant SaWRKY1 to SaXTH5 and SaMYB2 promoters. Comparative WRKY1 promoter analysis between resistant and susceptible plants revealed the presence of crucial motifs for defence mechanism exclusively in resistant accession. Additionally, many defence-related genes displayed significant expression variations in both the accessions. Further, WRKY1 overexpressing transgenic plants exhibited higher levels of EB resistance while RNAi silencing lines had increased susceptibility to A. solani with altered expression of XTH5 and MYB2. Overall, these findings demonstrate the positive influence of WRKY1 in improving EB resistance in wild tomato and this could be further utilized as a potential target through genetic engineering to augment protection against A. solani in crop plants.

The awn in stork's bill (Erodium gruinum) seed dispersal units coils as it dries. This hygroscopic movement promotes the dissemination and sowing of the seeds. Here we aimed to understand the movement rate, by correlating water dynamics within the awn to the spatial variation in the chemical composition of the awn's cell walls. We followed the hygroscopic movement visually and measured the kinetics of water adsorption-desorption in segments along the awn. We integrated data from white light, fluorescence, and Raman microscopy, and Matrix Assisted Laser Desorption Ionization imaging to characterize the micro chemical makeup of the awn. We hydrolyzed awns and followed the change in the cell walls' composition and the effect on the movement. We found that the coil's top segment is more sensitive to humidity changes than the coil's base. At the top part of the coil, we found high concentration of modified lignin. In comparison, the base part of the awn contained lower concentration of mostly unmodified lignin. Ferulic acid concentration increased along the awn, apparently cross-linking hemicellulose and strengthening cell-to-cell adhesion. We propose that the high concentration of modified lignin at the coil's top increased the hydrophobicity of the cell walls, allowed faster water molecules dynamics; thus inducing fast reaction to ambient humidity. Strong cell-to-cell adhesion in this region created a durable tissue required for the awn's repeated movement that is induced by the diurnal humidity cycles.

Oxygenic photosynthetic organisms perform solar energy conversion of water and CO2 to O-2 and sugar at a broad range of wavelengths and light intensities. These cells also metabolize sugars using a respiratory system that functionally overlaps the photosynthetic apparatus. In this study, we describe the harvesting of photocurrent used for hydrogen production from live cyanobacteria. A non-harmful gentle physical treatment of the cyanobacterial cells enables light-driven electron transfer by an endogenous mediator to a graphite electrode in a bio-photoelectrochemical cell, without the addition of sacrificial electron donors or acceptors. We show that the photocurrent is derived from photosystem I and that the electrons originate from carbohydrates digested by the respiratory system. Finally, the current is utilized for hydrogen evolution on the cathode at a bias of 0.65 V. Taken together, we present a bio-photoelectrochemical system where live cyanobacteria produce stable photocurrent that can generate hydrogen.

Databases containing mass spectrometry (MS) spectral data (i.e., MS reference libraries) are currently the most reliable and widely accepted approach to annotate unknown features in MS-based metabolomics. While for gas chromatography (GC)-MS data, a strategy for collecting, storing, and comparing to raw data has been established, this is not the case for liquid chromatography (LC)-MS data. Here, we present our approach for high-throughput data collection and automated MS reference library generation, as applied recently in the WEIZMASS library of plant metabolites. Methodologies to experimentally generate pools of chemical standards and computationally convert them into a unique source of reference data are detailed.

The bacterial plant pathogen Agrobacterium tumefaciens uses quorum sensing (QS) in order to regulate the transfer of DNA into the host plant genome, and this results in the induction of crown gall tumors. The deleterious results of these infections are widespread and affect many species of fruit and crops. In order to further our understanding, of this process and to provide potential solutions, we evaluated a library of 3800 natural products from plant sources and identified potent compounds that are able to strongly modulate plant-bacterial interactions.

Key message: Exploration with high throughput leaf metabolomics along with functional genomics in wild tomato unreveal potential role of steroidal glyco-alkaloids and phenylpropanoids during early blight resistance. Abstract: Alternaria solani severely affects tomato (Solanum lycopersicum L.) yield causing early blight (EB) disease in tropical environment. Wild relative, Solanum arcanum Peralta could be a potential source of EB resistance; however, its underlying molecular mechanism largely remains unexplored. Hence, non-targeted metabolomics was applied on resistant and susceptible S. arcanum accessions upon A. solani inoculation to unravel metabolic dynamics during different stages of disease progression. Total 2047 potential metabolite peaks (mass signals) were detected of which 681 and 684 metabolites revealed significant modulation and clear differentiation in resistant and susceptible accessions, respectively. Majority of the EB-triggered metabolic changes were active from steroidal glycol-alkaloid (SGA), lignin and flavonoid biosynthetic pathways. Further, biochemical and gene expression analyses of key enzymes from these pathways positively correlated with phenotypic variation in the S. arcanum accessions indicating their potential role in EB. Additionally, transcription factors regulating lignin biosynthesis were also up-regulated in resistant plants and electrophoretic mobility shift assay revealed sequence-specific binding of rSaWRKY1 with MYB20 promoter. Moreover, transcript accumulation of key genes from phenylpropanoid and SGA pathways along with WRKY and MYB in WRKY1 transgenic tomato lines supported above findings. Overall, this study highlights vital roles of SGAs as phytoalexins and phenylpropanoids along with lignin accumulation unrevealing possible mechanistic basis of EB resistance in wild tomato.

The assembly of the lipophilic cuticle layer and suberin lamellae, approximately 450 million years ago, was a major evolutionary development that enabled plants to colonize terrestrial habitats. The cuticle layer is composed of cutin polyester and embedded cuticular waxes, whereas the suberin lamellae consist of very long chain fatty acid derivatives, glycerol, and phenolics cross-linked with alkyl ferulate-embedded waxes. Due to their substantial biological roles in plant life, the mechanisms underlying the assembly of these structures have been extensively investigated. In the last decade, the introduction of `omics' approaches, including genomics, transcriptomics, proteomics, and metabolomics, have been key in the identification of novel genetic and chemical elements involved in the formation and function of the cuticle layer and suberin lamellae. This review summarizes contemporary studies that utilized various large-scale, `omics' strategies in combination with novel technologies to unravel how building blocks and polymers of these lipophilic barriers are made, and moreover linking structure to function along developmental programs and stress responses. We anticipate that the studies discussed here will inspire scientists studying lipophilic barriers to integrate complementary `omics' approaches in their efforts to tackle as yet unresolved questions and engage the main challenges of the field to date.

The factors rate-limiting growth of photosynthetic organisms under optimal conditions are controversial [1–8]. Adaptation to extreme environments is usually accompanied by reduced performance under optimal conditions [9, 10]. However, the green alga Chlorella ohadii, isolated from a harsh desert biological soil crust [11–17], does not obey this rule. In addition to resistance to photodamage [17, 18], it performs the fastest growth ever reported for photosynthetic eukaryotes. A multiphasic growth pattern (very fast growth [phase I], followed by growth retardation [phase II] and additional fast growth [phase III]) observed under constant illumination and temperature indicates synchronization of the algal population. Large physiological changes at transitions between growth phases suggest metabolic shifts. Indeed, metabolome analyses at points along the growth phases revealed large changes in the levels of many metabolites during growth with an overall rise during phase I and decline in phase II. Multivariate analysis of the metabolome data highlighted growth phase as the main factor contributing to observed metabolite variance. The analyses identified putrescine as the strongest predictive metabolite for growth phase and a putative growth regulator. Indeed, extracellular additions of polyamines strongly affected the growth rate in phase I and the growth arrest in phase II, with a marked effect on O₂ exchange. Our data implicate polyamines as the signals harmonizing metabolic shifts and suggest that metabolic flexibility enables the immense growth capabilities of C. ohadii. The data provide a new dimension to current models focusing on growth-limiting processes in photosynthetic organisms where the anabolic and catabolic metabolisms must be strictly regulated.

S-Methylmethionine (SMM) was suggested previously to participate in the metabolism of methionine (Met) in seeds. To further reveal its roles, we had previously produced transgenic Arabidopsis (Arabidopsis thaliana) RNA interference (RNAi) seeds with lower transcript expression of CYSTATHIONINE g-SYNTHASE (AtCGS), Met’s main regulatory enzyme. Unexpectedly, these seeds accumulated significantly higher levels of Met compared with control seeds through an as yet unknown mechanism. Here, transcript and metabolic analyses coupled with isotope-labeled [¹³ C]SMM and [¹³ C]Met feeding experiments enabled us to reveal that SMM that was synthesized in rosette leaves of RNAi plants significantly contributed to the accumulation of Met in their seeds at late stages of development. Seed-specific repression of AtCGS in RNAi seeds triggered the induction of genes operating in the SMM cycle of rosette leaves, leading to elevated transport of SMM toward the seeds, where higher reconversion rates of SMM to Met were detected. The metabolic rearrangements in RNAi seeds resulted in an altered sulfur-associated metabolism, such as lower amounts of Cys and glutathione, as well as a differential composition of glucosinolates. Together, the data propose a novel cross talk existing between seeds and rosette leaves along with mutual effects between the Asp family and SMM pathways operating in these tissues. They also shed light on the effects of higher Met levels on seed physiology and behavior.

Silicate minerals are dominant soil components. Thus, plant roots are constantly exposed to silicic acid. High silicon intake, enabled by root silicon transporters, correlates with increased tolerance to many biotic and abiotic stresses. However, the underlying protection mechanisms are largely unknown. Here, we tested the hypothesis that silicon interacts with the plant hormones, and specifically, that silicic acid intake increases cytokinin biosynthesis. The reaction of sorghum (Sorghum bicolor) and Arabidopsis plants, modified to absorb high versus low amounts of silicon, to dark-induced senescence was monitored, by quantifying expression levels of genes along the senescence pathway and measuring tissue cytokinin levels. In both species, detached leaves with high silicon content senesced more slowly than leaves that were not exposed to silicic acid. Expression levels of genes along the senescence pathway suggested increased cytokinin biosynthesis with silicon exposure. Mass spectrometry measurements of cytokinin suggested a positive correlation between silicon exposure and active cytokinin concentrations. Our results indicate a similar reaction to silicon treatment in distantly related plants, proposing a general function of silicon as a stress reliever, acting via increased cytokinin biosynthesis.

Soil communities are diverse taxonomically and functionally. This ecosystem experiences highly complex networks of interactions, but may also present functionally independent entities. Plant roots, a metabolically active hotspot in the soil, take an essential part in below-ground interactions. While plants are known to release an extremely high portion of the fixated carbon to the soil, less information is known about the composition and role of C-containing compounds in the rhizosphere, in particular those involved in chemical communication. Specialized metabolites (or secondary metabolites) produced by plants and their associated microbes have a critical role in various biological activities that modulate the behavior of neighboring organisms. Thus, elucidating the chemical composition and function of specialized metabolites in the rhizosphere is a key element in understanding interactions in this below-ground environment. Here, we review key classes of specialized metabolites that occur as mostly non-volatile compounds in root exudates or are emitted as volatile organic compounds (VOCs). The role of these metabolites in below-ground interactions and response to nutrient deficiency, as well as their tissue and cell type-specific biosynthesis and release are discussed in detail.

Plant roots play a dominant role in shaping the rhizosphere, the environment in which interaction with diverse microorganisms occurs. Tracking the dynamics of root-microbe interactions at high spatial resolution is currently limited because of methodological intricacy. Here, we describe a microfluidics-based approach enabling direct imaging of root-bacteria interactions in real time. The microfluidic device, which we termed tracking root interactions system (TRIS), consists of nine independent chambers that can be monitored in parallel. The principal assay reported here monitors behavior of fluorescently labeled Bacillus subtilis as it colonizes the root of Arabidopsis thaliana within the TRIS device. Our results show a distinct chemotactic behavior of B. subtilis toward a particular root segment, which we identify as the root elongation zone, followed by rapid colonization of that same segment over the first 6 h of root-bacteria interaction. Using dual inoculation experiments, we further show active exclusion of Escherichia coli cells from the root surface after B. subtilis colonization, suggesting a possible protection mechanism against root pathogens. Furthermore, we assembled a double-channel TRIS device that allows simultaneous tracking of two root systems in one chamber and performed real-time monitoring of bacterial preference between WT and mutant root genotypes. Thus, the TRIS microfluidics device provides unique insights into the microscale microbial ecology of the complex root microenvironment and is, therefore, likely to enhance the current rate of discoveries in this momentous field of research.

Triterpenoids are widespread bioactive plant defence compounds with potential use as pharmaceuticals, pesticides and other high-value products. Enzymes belonging to the cytochrome P450 family have an essential role in creating the immense structural diversity of triterpenoids across the plant kingdom. However, for many triterpenoid oxidation reactions, the corresponding enzyme remains unknown. Here we characterize CYP716 enzymes from different medicinal plant species by heterologous expression in engineered yeasts and report ten hitherto unreported triterpenoid oxidation activities, including a cyclization reaction, leading to a triterpenoid lactone. Kingdom-wide phylogenetic analysis of over 400 CYP716s from over 200 plant species reveals details of their evolution and suggests that in eudicots the CYP716s evolved specifically towards triterpenoid biosynthesis. Our findings underscore the great potential of CYP716s as a source for generating triterpenoid structural diversity and expand the toolbox available for synthetic biology programmes for sustainable production of bioactive plant triterpenoids.

Betalains are tyrosine-derived red-violet and yellow pigments found in plants only of the Caryophyllales order, which hold both scientific and economic values. Their pH in-dependence and high stability make them a natural pigment of choice for food industries. Their strong antioxidant activities have prompted research into their potential health-promoting properties and led to commercialization of a variety of betalain-based dietary supplements. While the biosynthetic process of many natural colorants is well understood, many questions remain open with regards to biosynthesis of betalains. Transcriptome analysis of the betalain-producing plants red beet (Beta vulgaris) and four o'clocks (Mirabilis jalapa) led us to the identification of a novel betalain-related cytochrome P450-type gene, CYP76AD6, which catalyzes the first step in the betalain biosynthetic pathway, namely the 3-hydroxylation of tyrosine to form l-3,4-dihydroxyphenylalanine (L-DOPA) [1]. L-DOPA formation in red beet was found to be redundantly catalyzed by CYP76AD6 together with a known betalain-related enzyme, CYP76AD1. Gene silencing assays and recombinant expression in Nicotiana benthamiana and yeast cells revealed that while CYP76AD1 catalyzes both L-DOPA formation and its subsequent conversion to cyclo-DOPA, CYP76AD6 uniquely exhibits only tyrosine hydroxylase activity. The new findings enabled us to engineer stable betalain production through heterologous expression of three genes taking part in the fully decoded betalain biosynthetic pathway, namely CYP76AD1 and DOPA 4, 5-dioxygenase (BvDODA1) from red beet, and cyclo-DOPA-5-O-glucosyltransferase from four o'clocks (cDOPA5GT). High-quantity betalain production was achieved in a number of plant species, including tobacco (135 mg/kg fresh weight, leaf tissue) tomato (200 mg/kg, fruit) and eggplant (120 mg/kg, fruit). These betalain-producing transgenic plants offer an exceptional opportunity to study for example the health-promoting properties of betalains.

The work herein presents comprehensive analyses of the cuticular wax mixtures covering the flag leaf blade and peduncle of bread wheat (Triticum aestivum) cv. Bethlehem. Overall, Gas Chromatography Mass Spectrometry and Flame Ionization Detection revealed a wax coverage of flag leaf blades (16 mu g/cm(2)) a third that of peduncles (49 mu g/cm(2)). Flag leaf blade wax was dominated by 1-alkanols, while peduncle wax contained primarily beta-diketone and hydroxy-beta-diketones, thus suggesting differential regulation of the acyl reduction and beta-diketone biosynthetic pathways in the two analyzed organs. The characteristic chain length distributions of the various wax compound classes are discussed in light of their individual biosynthetic pathways and biosynthetic relationships between classes. Along with previously reported wheat wax compound classes (fatty acids, 1-alkanols, 1-alkanol esters, aldehydes, alkanes, beta-diketone, hydroxy-beta-diketones, alkylresorcinols and methyl alkylresorcinols), esters of 2-alkanols and three types of aromatic esters (benzyl, phenethyl and p-hydroxyphenethyl) are also reported. In particular, 2-heptanol esters were identified. Detailed analyses of the isomer distributions within 1-alkanol and 2-alkanol ester homologs revealed distinct patterns of esterified acids and alcohols, suggesting several wax ester synthases with very different substrate preferences in both wheat organs. Terpenoids, including two terpenoid esters, were present only in peduncle wax.

Attaining high-resolution spatial information is a recurrent challenge in biological research, particularly in the case of small-molecule distribution. Mass spectrometry imaging (MSI) is an innovative molecular histology technique that could provide such information. It allows in situ and label-free measurement of both the abundance and distribution of a variety of molecules at the tissue or single cell level. The application of MSI in plant research has received considerable attention; thus, in this review, we describe the current state of MSI in plants. In particular, we present an overview of MSI approaches, highlight the recent technical and methodological developments, and discuss a range of applications contributing to the field of plant science.

We present a resource for fine mapping of traits derived from the wild tomato species Solanum pennellii (LA0716). The population of backcross inbred lines (BILs) is composed of 446 lines derived after a few generations of backcrosses of the wild species with cultivated tomato (cultivar M82; LA3475), followed by more than seven generations of self-pollination. The BILs were genotyped using the 10K SOL-CAP single nucleotide polymorphism (SNP) -Chip, and 3700 polymorphic markers were used to map recombination break points relative to the physical map of Solanum lycopersicum. The BILs carry, on average, 2.7 introgressions per line, with a mean introgression length of 11.7 Mbp. Whereas the classic 76 introgression lines (ILs) partitioned the genome into 106 mapping bins, the BILs generated 633 bins, thereby enhancing the mapping resolution of traits derived from the wild species. We demonstrate the power of the BILs for rapid fine mapping of simple and complex traits derived from the wild tomato species.

The glaucous appearance of wheat (Triticum aestivum) and barley (Hordeum vulgare) plants, that is the light bluish-gray look of flag leaf, stem, and spike surfaces, results from deposition of cuticular β-diketone wax on their surfaces; this phenotype is associated with high yield, especially under drought conditions. Despite extensive genetic and biochemical characterization, the molecular genetic basis underlying the biosynthesis of β-diketones remains unclear. Here, we discovered that the wheat W1 locus contains a metabolic gene cluster mediating β-diketone biosynthesis. The cluster comprises genes encoding proteins of several families including type-III polyketide synthases, hydrolases, and cytochrome P450s related to known fatty acid hydroxylases. The cluster region was identified in both genetic and physical maps of glaucous and glossy tetraploid wheat, demonstrating entirely different haplotypes in these accessions. Complementary evidence obtained through gene silencing in planta and heterologous expression in bacteria supports a model for a β-diketone biosynthesis pathway involving members of these three protein families. Mutations in homologous genes were identified in the barley eceriferum mutants defective in β-diketone biosynthesis, demonstrating a gene cluster also in the β-diketone biosynthesis Cer-cqu locus in barley. Hence, our findings open new opportunities to breed major cereal crops for surface features that impact yield and stress response.

Betalains are tyrosine-derived red-violet and yellow pigments, found in plants only of the Caryophyllales order. Although much progress has been made in recent years in the understanding of the betalain biosynthetic process, many questions remain open with regards to several of the proposed steps in the pathway. Most conspicuous by its absence is the characterization of the first committed step in the pathway, namely the 3-hydroxylation of tyrosine to form l-3,4-dihydroxyphenylalanine (l-DOPA). We used transcriptome analysis of the betalain-producing plants red beet (Beta vulgaris) and four o'clocks (Mirabilis jalapa) to identify a novel, betalain-related cytochrome P450-type gene, CYP76AD6, and carried out gene silencing and recombinant expression assays in Nicotiana benthamiana and yeast cells to examine its functionality. l-DOPA formation in red beet was found to be redundantly catalyzed by CYP76AD6 together with a known betalain-related enzyme, CYP76AD1, which was previously thought to only catalyze a succeeding step in the pathway. While CYP76AD1 catalyzes both l-DOPA formation and its subsequent conversion to cyclo-DOPA, CYP76AD6 uniquely exhibits only tyrosine hydroxylase activity. The new findings enabled us to metabolically engineer entirely red-pigmented tobacco plants through heterologous expression of three genes taking part in the fully decoded betalain biosynthetic pathway.

Marine viruses are the most abundant biological entities in the oceans shaping community structure and nutrient cycling. The interaction between the bloom-forming alga Emiliania huxleyi and its specific large dsDNA virus (EhV) is a major factor determining the fate of carbon in the ocean, thus serving as a key host-pathogen model system. The EhV genome encodes for a set of genes involved in the de novo sphingolipid biosynthesis, not reported in any viral genome to date. We combined detailed lipidomic and biochemical analyses to characterize the functional role of this virus-encoded pathway during lytic viral infection. We identified a major metabolic shift, mediated by differential substrate specificity of virus-encoded serine palmitoyltransferase, a key enzyme of sphingolipid biosynthesis. Consequently, unique viral glycosphingolipids, composed of unusual hydroxylated C17 sphingoid bases (t17:0) were highly enriched in the infected cells, and their synthesis was found to be essential for viral assembly. These findings uncover the biochemical bases of the virus-induced metabolic rewiring of the host sphingolipid biosynthesis during the chemical "arms race" in the ocean.

Mass spectrometry imaging (MSI) is a mass spectrometry based molecular ion imaging technique. It provides the means for ascertaining the spatial distribution of a large variety of analytes directly on tissue sample surfaces without any labeling or staining agents. These advantages make it an attractive molecular histology tool in medical, pharmaceutical, and biological research. Likewise, MSI has started gaining popularity in plant sciences; yet, information regarding sample preparation methods for plant tissues is still limited. Sample preparation is a crucial step that is directly associated with the quality and authenticity of the imaging results, it therefore demands in-depth studies based on the characteristics of plant samples. In this review, a sample preparation pipeline is discussed in detail and illustrated through selected practical examples. In particular, special concerns regarding sample preparation for plant imaging are critically evaluated. Finally, the applications of MSI techniques in plants are reviewed according to different classes of plant metabolites.

Ethylene is the main regulator of climacteric fruit ripening, by contrast the putative role of other phytohormones in this process remains poorly understood. The present study brings auxin signaling components into the mechanism regulating tomato fruit ripening through the functional characterization of Auxin Response Factor2 (SlARF2) which encodes a downstream component of auxin signaling. Two paralogs, SlARF2A and SlARF2B, are found in the tomato genome, both displaying a marked ripening-associated expression but distinct responsiveness to ethylene and auxin. Down-regulation of either SlARF2A or SlARF2B resulted in ripening defects while simultaneous silencing of both genes led to severe ripening inhibition suggesting a functional redundancy among the two ARFs. Tomato fruits under-expressing SlARF2 produced less climacteric ethylene and exhibited a dramatic down-regulation of the key ripening regulators RIN, CNR, NOR and TAGL1. Ethylene treatment failed to reverse the non-ripening phenotype and the expression of ethylene signaling and biosynthesis genes was strongly altered in SlARF2 down-regulated fruits. Although both SlARF proteins are transcriptional repressors the data indicate they work as positive regulators of tomato fruit ripening. Altogether, the study defines SlARF2 as a new component of the regulatory network controlling the ripening process in tomato.

Polyamines are essential polycations present in all living cells. Polyamine levels are maintained from the diet and de novo synthesis, and their decline with age is associated with various pathologies. Here we show that polyamine levels oscillate in a daily manner. Both clock- and feeding-dependent mechanisms regulate the daily accumulation of key enzymes in polyamine biosynthesis through rhythmic binding of BMAL1:CLOCK to conserved DNA elements. In turn, polyamines control the circadian period in cultured cells and animals by regulating the interaction between the core clock repressors PER2 and CRY1. Importantly, we found that the decline in polyamine levels with age in mice is associated with a longer circadian period that can be reversed upon polyamine supplementation in the diet. Our findings suggest a crosstalk between circadian clocks and polyamine biosynthesis and open new possibilities for nutritional interventions against the decay in clock's function with age.

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.

Lipid remodeling is crucial for hypoxic tolerance in animals, whilst little is known about the hypoxia-induced lipid dynamics in plants. Here we performed a mass spectrometry-based analysis to survey the lipid profiles of Arabidopsis rosettes under various hypoxic conditions. We observed that hypoxia caused a significant increase in total amounts of phosphatidylserine, phosphatidic acid and oxidized lipids, but a decrease in phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Particularly, significant gains in the polyunsaturated species of PC, PE and phosphatidylinositol, and losses in their saturated and mono-unsaturated species were evident during hypoxia. Moreover, hypoxia led to a remarkable elevation of ceramides and hydroxyceramides. Disruption of ceramide synthases LOH1, LOH2 and LOH3 enhanced plant sensitivity to dark submergence, but displayed more resistance to submergence under light than wild type. Consistently, levels of unsaturated very-long-chain (VLC) ceramide species (22:1, 24:1 and 26:1) predominantly declined in the loh1, loh2 and loh3 mutants under dark submergence. In contrast, significant reduction of VLC ceramides in the loh1-1 loh3-1 knockdown double mutant and lacking of VLC unsaturated ceramides in the ads2 mutants impaired plant tolerance to both dark and light submergences. Evidence that C24:1-ceramide interacted with recombinant CTR1 protein and inhibited its kinase activity in vitro, enhanced ER-to-nucleus translocation of EIN2-GFP and stabilization of EIN3-GFP in vivo, suggests a role of ceramides in modulating CTR1-mediated ethylene signaling. The dark submergence-sensitive phenotypes of loh mutants were rescued by a ctr1-1 mutation. Thus, our findings demonstrate that unsaturation of VLC ceramides is a protective strategy for hypoxic tolerance in Arabidopsis.

Anthocyanins contribute to the appearance of fruit by conferring to them a red, blue or purple colour. In a food context, they have also been suggested to promote consumer health. In purple tomato tissues, such as hypocotyls, stems and purple fruits, various anthocyanins accumulate. These molecules have characteristic patterns of modification, including hydroxylations, methylations, glycosylations and acylations. The genetic basis for many of these modifications has not been fully elucidated, and nor has their role in the functioning of anthocyanins. In this paper, AnthOMT, an O-methyltransferase (OMT) mediating the methylation of anthocyanins, has been identified and functionally characterized using a combined metabolomics and transcriptomics approach. Gene candidates were selected from the draft tomato genome, and their expression was subsequently monitored in a tomato seedling system comprising three tissues and involving several time points. In addition, we also followed gene expression in wild-type red and purple transgenic tomato fruits expressing Rosea 1 and Delila transcription factors. Of the 57 candidates identified, only a single OMT gene showed patterns strongly correlating with both accumulation of anthocyanins and expression of anthocyanin biosynthesis genes. This candidate (AnthOMT) was compared to a closely related caffeoyl CoA OMT by recombinant expression in Escherichia coli, and then tested for substrate specificity. AnthOMT showed a strong affinity for glycosylated anthocyanins, while other flavonoid glycosides and aglycones were much less preferred. Gene silencing experiments with AnthOMT resulted in reduced levels of the predominant methylated anthocyanins. This confirms the role of this enzyme in the diversification of tomato anthocyanins.

Marine viruses are major ecological and evolutionary drivers of microbial food webs regulating the fate of carbon in the ocean. We combined transcriptomic and metabolomic analyses to explore the cellular pathways mediating the interaction between the bloom-forming coccolithophore Emiliania huxleyi and its specific coccolithoviruses (E. huxleyi virus [EhV]). We show that EhV induces profound transcriptome remodeling targeted toward fatty acid synthesis to support viral assembly. A metabolic shift toward production of viral-derived sphingolipids was detected during infection and coincided with downregulation of host de novo sphingolipid genes and induction of the viral-encoded homologous pathway. The depletion of host-specific sterols during lytic infection and their detection in purified virions revealed their novel role in viral life cycle. We identify an essential function of the mevalonate-isoprenoid branch of sterol biosynthesis during infection and propose its downregulation as an antiviral mechanism. We demonstrate how viral replication depends on the hijacking of host lipid metabolism during the chemical "arms race" in the ocean.

Taste has been the subject of human selection in the evolution of agricultural crops, and acidity is one of the three major components of fleshy fruit taste, together with sugars and volatile flavour compounds. We identify a family of plant-specific genes with a major effect on fruit acidity by map-based cloning of C. melo PH gene (CmPH) from melon, Cucumis melo taking advantage of the novel natural genetic variation for both high and low fruit acidity in this species. Functional silencing of orthologous PH genes in two distantly related plant families, cucumber and tomato, produced low-acid, bland tasting fruit, showing that PH genes control fruit acidity across plant families. A four amino-acid duplication in CmPH distinguishes between primitive acidic varieties and modern dessert melons. This fortuitous mutation served as a preadaptive antecedent to the development of sweet melon cultigens in Central Asia over 1,000 years ago.

Diatoms are ubiquitous marine photosynthetic eukaryotes responsible for approximately 20% of global photosynthesis. Little is known about the redox-based mechanisms that mediate diatom sensing and acclimation to environmental stress. Here we used a quantitative mass spectrometry-based approach to elucidate the redox-sensitive signaling network (redoxome) mediating the response of diatoms to oxidative stress. We quantified the degree of oxidation of 3,845 cysteines in the Phaeodactylum tricornutum proteome and identified approximately 300 redox-sensitive proteins. Intriguingly, we found redox-sensitive thiols in numerous enzymes composing the nitrogen assimilation pathway and the recently discovered diatom urea cycle. In agreement with this finding, the flux from nitrate into glutamine and glutamate, measured by the incorporation of 15N, was strongly inhibited under oxidative stress conditions. Furthermore, by targeting the redox-sensitive GFP sensor to various subcellular localizations, we mapped organellespecific oxidation patterns in response to variations in nitrogen quota and quality. We propose that redox regulation of nitrogen metabolism allows rapid metabolic plasticity to ensure cellular homeostasis, and thus is essential for the ecological success of diatoms in the marine ecosystem.

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.

The complex metabolic networks in plants are highly compartmentalized and biochemical steps of a single pathway can take place in multiple subcellular locations. Our knowledge regarding reactions and precursor compounds in the various cellular compartments has increased in recent years due to innovations in tracking the spatial distribution of proteins and metabolites. Nevertheless, to date only few studies have integrated subcellular localization criteria in metabolic engineering attempts. Here, we highlight the crucial factors for subcellular-localization-based strategies in plant metabolic engineering including substrate availability, enzyme targeting, the role of transporters, and multigene transfer approaches. The availability of compartmentalized metabolic network models for plants in the near future will greatly advance the integration of localization constraints in metabolic engineering experiments and aid in predicting their outcomes.

Steroidal glycoalkaloids (SGAs) such as a-solanine found in solanaceous food plants - as, for example, potato - are antinutritional factors for humans. Comparative coexpression analysis between tomato and potato coupled with chemical profiling revealed an array of 10 genes that partake in SGA biosynthesis. We discovered that six of them exist as a cluster on chromosome 7, whereas an additional two are adjacent in a duplicated genomic region on chromosome 12. Following systematic functional analysis, we suggest a revised SGA biosynthetic pathway starting from cholesterol up to the tetrasaccharide moiety linked to the tomato SGA aglycone. Silencing GLYCOALKALOID METABOLISM 4 prevented accumulation of SGAs in potato tubers and tomato fruit. This may provide a means for removal of unsafe, antinutritional substances present in these widely used food crops.

The agronomic and physiological characteristics of fruit were compared between two lines of 'Micro Tom' tomato which differed in their fruit cuticle chemistry. A wild-type (WT) and a mutant line (lecer6) deficient in -ketoacyl-CoenzymeA synthase (LeCER6), an enzyme involved in cuticular wax synthesis, were studied.The lecer6 line showed significantly lower levels of wax aliphatics and higher levels of terpenoids. Lecer6 fruit also had lower levels of cutin monomers than WT fruit. The agronomic characteristics of fruit from the mutant line were generally different from WT fruit. Osmotic values and dry weight percentages (% DW) were higher than in WT fruit. These findings can be explained by the higher water permeance of the lecer6 fruit cuticle, resulting in a higher rate of cuticular transpiration. Fruit from lecer6 plants were not as firm as WT fruit, which may also have resulted from excessive water loss through the cuticle. Higher rates of water loss in lecer6 fruit did not seem to be related to changes in cuticle thickness. The biomechanical properties of the cuticle also differed between the two tomato lines. Transient creep tests of isolated cuticles were used to generate stress-strain curves for the fruit cuticles of each line. At the immature (green) stage of development, cuticles of WT fruit were more elastic than those of lecer6 fruit, as shown by a lower elastic modulus. However, at the mature (red) stage of development,WT fruit cuticles had become more rigid, and no differences were apparent between the two lines. These data show that a single gene mutation for fruit cuticular wax synthesis has a pleiotropic effect on many important agronomic and physiological attributes.

Sweet melon cultivars contain a low level of organic acids and, therefore, the quality and flavor of sweet melon fruit is determined almost exclusively by fruit sugar content. However, genetic variability for fruit acid levels in the Cucumis melo species exists and sour fruit accessions are characterized by acidic fruit pH of 6. In this paper, we report results from a mapping population based on recombinant inbred lines (RILs) derived from the cross between the non-sour 'Dulce' variety and the sour PI 414323 accession. Results show that a single major QTL for pH co-localizes with major QTLs for the two predominant organic acids in melon fruit, citric and malic, together with an additional metabolite which we identified as uridine. While the acidic recombinants were characterized by higher citric and malic acid levels, the non-acidic recombinants had a higher uridine content than did the acidic recombinants. Additional minor QTLs for pH, citric acid and malic acid were also identified and for these the increased acidity was unexpectedly contributed by the non-sour parent. To test for co-localization of these QTLs with genes encoding organic acid metabolism and transport, we mapped the genes encoding structural enzymes and proteins involved in organic acid metabolism, transport and vacuolar H+ pumps. None of these genes co-localized with the major pH QTL, indicating that the gene determining melon fruit pH is not one of the candidate genes encoding this primary metabolic pathway. Linked markers were tested in two additional inter-varietal populations and shown to be linked to the pH trait. The presence of the same QTL in such diverse segregating populations suggests that the trait is determined throughout the species by variability in the same gene and is indicative of a major role of the evolution of this gene in determining the important domestication trait of fruit acidity within the species.

The human gut hosts a microbial community which actively contributes to the host metabolism and has thus remarkable effect on our health. Intestinal microbiota is known to interact remarkably with the dietary constituents entering the colon, causing major metabolic conversions prior to absorption. To investigate the effect of microbial metabolism on the phytochemical pool of rye bran, we applied an in vitro simulated colonic fermentation where samples were collected with intervals and analyzed by LC-MS based non-targeted metabolite profiling. The analyses revealed extensive metabolic turnover on the phytochemical composition of the bran samples, and showed effects on all the metabolite classes detected. Furthermore, the majority of the metabolites, both the precursors and the conversion products, remained unidentified indicating that there are numerous yet unknown phytochemicals, which can potentially affect on our health. This underlines the importance of comprehensive profiling assays and subsequent detailed molecular investigations in order to clarify the effect of microbiota on phytochemicals present in our everyday diet.

Recent advances in the performance of hyphenated technologies based on ultrapressure chromatography and high-sensitivity mass spectrometry have set the stage for a myriad of metabolomics studies in plants and other organisms. In this chapter, we describe the use of a UPLC (Ultraperformance Liquid Chromatography)-qTOF (quadrupole time-of-flight) system for profiling semipolar metabolites in the model fruit plant tomato. An optimized extraction method, instrument parameters and data treatment procedures are provided. The value of UPLC instruments, which use small particle size chromatographic columns, in terms of resolution, separation, and short injection times are presented. When coupled to a TOF mass spectrometer with high resolution and mass accuracy, good dynamic range, and a fast spectral acquisition capacity, this system is most suitable for the extensive profiling of hundreds of plant metabolites.

Steroidal alkaloids (SAs) are triterpene-derived specialized metabolites found in members of the Solanaceae family that provide plants with a chemical barrier against a broad range of pathogens. Their biosynthesis involves the action of glycosyltransferases to form steroidal glycoalkaloids (SGAs). To elucidate the metabolism of SGAs in the Solanaceae family, we examined the tomato (Solanum lycopersicum) GLYCOALKALOID METABOLISM1 (GAME1) gene. Our findings imply that GAME1 is a galactosyltransferase, largely performing glycosylation of the aglycone tomatidine, resulting in SGA production in green tissues. Downregulation of GAME1 resulted in an almost 50% reduction in a-tomatine levels (the major SGA in tomato) and a large increase in its precursors (i.e., tomatidenol and tomatidine). Surprisingly, GAME1-silenced plants displayed growth retardation and severe morphological phenotypes that we suggest occur as a result of altered membrane sterol levels caused by the accumulation of the aglycone tomatidine. Together, these findings highlight the role of GAME1 in the glycosylation of SAs and in reducing the toxicity of SA metabolites to the plant cell.

Floral organs display tremendous variation in their exterior that is essential for organogenesis and the interaction with the environment. This diversity in surface characteristics is largely dependent on the composition and structure of their coating cuticular layer. To date, mechanisms of flower organ initiation and identity have been studied extensively, while little is known regarding the regulation of flower organs surface formation, cuticle composition, and its developmental significance. Using a synthetic microRNA approach to simultaneously silence the three SHINE (SHN) clade members, we revealed that these transcription factors act redundantly to shape the surface and morphology of Arabidopsis flowers. It appears that SHNs regulate floral organs' epidermal cell elongation and decoration with nanoridges, particularly in petals. Reduced activity of SHN transcription factors results in floral organs' fusion and earlier abscission that is accompanied by a decrease in cutin load and modified cell wall properties. SHN transcription factors possess target genes within four cutin- and suberin-associated protein families including, CYP86A cytochrome P450s, fatty acyl-CoA reductases, GSDL-motif lipases, and BODYGUARD1-like proteins. The results suggest that alongside controlling cuticular lipids metabolism, SHNs act to modify the epidermis cell wall through altering pectin metabolism and structural proteins. We also provide evidence that surface formation in petals and other floral organs during their growth and elongation or in abscission and dehiscence through SHNs is partially mediated by gibberellin and the DELLA signaling cascade. This study therefore demonstrates the need for a defined composition and structure of the cuticle and cell wall in order to form the archetypal features of floral organs surfaces and control their cell-to-cell separation processes. Furthermore, it will promote future investigation into the relation between the regulation of organ surface patterning and the broader control of flower development and biological functions.

Previous studies showed that ABCG11 and ABCG12, two ATP-binding-cassette (ABC) transporters, are required for cuticular lipids extracellular secretion. Here, we characterized ABCG13, a third clade member, to widen our limited knowledge regarding assembly of the plant's cuticle. We isolated an abcg13 knockout mutant and used RNAi and artificial microRNA approaches to study the effect of ABCG13 loss-of-function. These plants were subsequently used to conduct a detailed analysis of cuticular lipids composition and cytological observations. ABCG13 loss-of-function resulted in cuticle-related phenotypes that were restricted to flowers, including inter-organ post-genital fusions. Apart from a significant reduction in flower cutin monomers, the macromorphology and micromorphology of abcg13 petal epidermis was strongly affected. We also found that ABCG13 is highly expressed in flowers, predominantly in petals and carpels. The results suggest that ABCG13 is required for the transport of flower cuticular lipids. This work introduces a new component to the recently emerging genetic network that makes the archetypal exterior of Arabidopsis flowers. While the question regarding the substrate specificity of the ABCG12-clade members remains open, these findings will facilitate future investigations regarding the interaction between the half-size ABCG-type transporters that likely take part in cuticle assembly.

Sortin1 is a chemical genetic-hit molecule that causes specific mislocalization of plant and yeast-soluble and membrane vacuolar markers. To better understand its mode of action, we designed a Sortin1-hypersensitive screen and identified several Sortin1-hypersensitive and flavonoid-defective mutants. Mechanistically, Sortin1 mimics the effect of the glutathione inhibitor buthionine sulfoximine and alters the vacuolar accumulation of flavonoids, likely blocking their transport through vacuole-localized ABC transporters. Structure-activity relationship studies conducted in Arabidopsis revealed the structural requirements for Sortin1 bioactivity and demonstrated that overlapping Sortin1 substructures can be used to discriminate between vacuolar-flavonoid accumulations and vacuolar-biogenesis defects. We conclude that Sortin1 is a valuable probe for dissecting novel links among flavonoid transport, vacuolar integrity, and the trafficking of vacuolar targeted cargoes in Arabidopsis.

External color has profound effects on acceptability of agricultural products by consumers. Carotenoids and chlorophylls are known to be the major pigments of melon (Cucumis melo L.) rinds. Flavonoids (especially chalcones and anthocyanins) are also prominent in other fruits but have not been reported to occur in melons fruit. We analyzed the pigments accumulating in rinds of different melon genotypes during fruit development. We found that melon rind color is based on different combinations of chlorophyll, carotenoids, and flavonoids according to the cultivar tested and their ratios changed during fruit maturation. Moreover, in "canary yellow" type melons, naringenin chalcone, a yellow flavonoid pigment previously unknown to occur in melons, has been identified as the major fruit colorant in mature rinds. Naringenin chalcone is also prominent in other melon types, occurring together with carotenoids (mainly β-carotene) and chlorophyll. Both chlorophyll and carotenoid pigments segregate jointly in an F₂ population originating from a cross between a yellow canary line and a line with green rind. In contrast, the content of naringenin chalcone segregates as a monogenic trait independently to carotenoids and chlorophyll. Transcription patterns of key structural phenylpropanoid and flavonoid biosynthetic pathway genes were monitored in attempts to explain naringenin chalcone accumulation in melon rinds. The transcript levels of CHI were low in both parental lines, but C4H, C4L, and CHS transcripts were upregulated in "Noy Amid", the parental line that accumulates naringenin chalcone. Our results indicate that naringenin chalcone accumulates independently from carotenoids and chlorophyll pigments in melon rinds and gives an insight into the molecular mechanism for the accumulation of naringenin chalcone in melon rinds.

Apart from its significance in the protection against stress conditions, the cuticular cover is essential for proper development of the diverse surface structures formed on aerial plant organs. This layer mainly consists of a cutin matrix, embedded and overlaid with cuticular waxes. Following their biosynthesis in epidermal cells, cutin and waxes were suggested to be exported across the plasma membrane by ABCG-type transporters such as DSO/ABCG11 to the cell wall and further to extracellular matrix. Here, additional aspects of DSO/ABCG11 function were investigated, predominantly in reproductive organs, which were not revealed in the previous reports. This was facilitated by the generation of a transgenic DSO/ABCG11 silenced line (dso-4) that displayed relatively subtle morphological and chemical phenotypes. These included altered petal and silique morphology, fusion of seeds, and changes in levels of cutin monomers in flowers and siliques. The dso-4 phenotypes corresponded to the strong DSO/ABCG11 gene expression in the embryo epidermis as well as in the endosperm tissues of the developing seeds. Moreover, the DSO/ABCG11 protein displayed polar localization in the embryo protoderm. Transcriptome analysis of the dso-4 mutant leaves and stems showed that reduced DSO/ABCG11 activity suppressed the expression of a large number of cuticle-associated genes, implying that export of cuticular lipids from the plasma membrane is a rate-limiting step in cuticle metabolism. Surprisingly, root suberin composition of dso-4 was altered, as well as root expression of two suberin biosynthetic genes. Taken together, this study provides new insights into cutin and suberin metabolism and their role in reproductive organs and roots development.

The cuticle covering plants' aerial surfaces is a unique structure that plays a key role in organ development and protection against diverse stress conditions. A detailed analysis of the tomato colorless-peel y mutant was carried out in the framework of studying the outer surface of reproductive organs. The y mutant peel lacks the yellow flavonoid pigment naringenin chalcone, which has been suggested to influence the characteristics and function of the cuticular layer. Large-scale metabolic and transcript profiling revealed broad effects on both primary and secondary metabolism, related mostly to the biosynthesis of phenylpropanoids, particularly flavonoids. These were not restricted to the fruit or to a specific stage of its development and indicated that the y mutant phenotype is due to a mutation in a regulatory gene. Indeed, expression analyses specified three R2R3-MYB-type transcription factors that were significantly down-regulated in the y mutant fruit peel. One of these, SlMYB12, was mapped to the genomic region on tomato chromosome 1 previously shown to harbor the y mutation. Identification of an additional mutant allele that co-segregates with the colorless-peel trait, specific downregulation of SlMYB12 and rescue of the y phenotype by overexpression of SlMYB12 on the mutant background, confirmed that a lesion in this regulator underlies the y phenotype. Hence, this work provides novel insight to the study of fleshy fruit cuticular structure and paves the way for the elucidation of the regulatory network that controls flavonoid accumulation in tomato fruit cuticle.

Advanced metabolomics technologies are anticipated to permit the identification and quantification of metabolites at the whole-metabolome scale. Yet, most of the metabolites either remain unknown or cannot be identified unambiguously. Moreover, the present approaches suffer from inaccuracies in relative quantification because of sample preparation and matrix effects. Here we present Dual Labeling of Metabolites for Metabolome Analysis (DLEMMA) as a valuable tool, which with analogy to DNA array assays enables the identification and relative quantification of differential metabolites in a single sample. DLEMMA was demonstrated as an efficient method for reducing the number of possible chemical structures assigned that exhibit the same elemental composition. Its strength was exemplified by the discovery of 10 novel Tryptophan derivatives. Furthermore, employing DLEMMA by feeding two Phenylalanine-labeled precursors, we could detect differential metabolites between trans-genic and control plants. The accuracy of relative quantification is also enhanced since DLEMMA provides identical matrixes for both samples, thus avoiding the effects of different complex biological matrixes on electrospray ionization. Hence, DLEMMA will complement and contribute to the advancement of metabolomics technologies and boost metabolic pathway discovery in diverse organisms.

The gene encoding stilbene synthase is frequently used to modify plant secondary metabolism with the aim of producing the self-defence phytoalexin resveratrol. In this study, strawberry (Fragaria×ananassa) was transformed with the NS-Vitis3 gene encoding stilbene synthase from frost grape (Vitis riparia) under the control of the cauliflower mosaic virus 35S and the floral filament-specific fil1 promoters. Changes in leaf metabolites were investigated with UPLC-qTOF-MS (ultra performance liquid chromatography-quadrupole time of flight mass spectrometry) profiling, and increased accumulation of cinnamate, coumarate, and ferulate derivatives concomitantly with a decrease in the levels of flavonols was observed, while the anticipated resveratrol or its derivatives were not detected. The changed metabolite profile suggested that chalcone synthase was down-regulated by the genetic modification; this was verified by decreased chalcone synthase transcript levels. Changes in the levels of phenolic compounds led to increased susceptibility of the transgenic strawberry to grey mould fungus.

Starches extracted from most plant species are phosphorylated. α-Glucan water dikinase (GWD) is a key enzyme that controls the phosphate content of starch. In the absence of its activity starch degradation is impaired, leading to a starch excess phenotype in Arabidopsis and in potato leaves, and to reduced cold sweetening in potato tubers. Here, we characterized a transposon insertion (legwd::Ds) in the tomato GWD (LeGWD) gene that caused male gametophytic lethality. The mutant pollen had a starch excess phenotype that was associated with a reduction in pollen germination. SEM and TEM analyses indicated mild shrinking of the pollen grains and the accumulation of large starch granules inside the plastids. The level of soluble sugars was reduced by 1.8-fold in mutant pollen grains. Overall, the transmission of the mutant allele was only 0.4% in the male, whereas it was normal in the female. Additional mutant alleles, obtained through transposon excision, showed the same phenotypes as legwd::Ds. Moreover, pollen germination could be restored, and the starch excess phenotype could be abolished in lines expressing the potato GWD homolog (StGWD) under a pollen-specific promoter. In these lines, where fertility was restored, homozygous plants for legwd::Ds were isolated, and showed the starch excess phenotype in the leaves. Overall, our results demonstrate the importance of starch phosphorylation and breakdown for pollen germination, and open up the prospect for analyzing the role of starch metabolism in leaves and fruits.

Plants produce a myriad of secondary metabolites (SMs), which constantly contribute to plants' interaction with the surroundings. Since ancient times and up to this day mankind has been using SMs as sources for medicines, spices, fragrances, pesticides, poisons, hallucinogens, stimulants, dyes, perfumery and countless more purposes. The shared value for both humans and plants makes SMs important targets for bioengineering. The formation of certain SM compounds may be restricted to single plant species, specific plant organs, cells or even particular cell compartments. Bioengineering can modulate the levels, time and site of production of natural products in plants. In this chapter we review the state of the art in the bioengineering of natural products at the whole plant level. In the first part of this review, we summarize the current and emerging bioengineering strategies and methods, including the use of the riboswitches, immunomodulation, synthetic microRNAs and Zinc-finger nucleases. The second and major part of this chapter provides examples from different fields of bioengineering in plants including: (a) the production of nutraceuticals, (b) modifying volatiles and pigments (in fruit and flowers), (c) production of medicinal agents and (d) aiding plants in the fight against biotic stresses. The experiments described here were conducted either in target plants, usually crop species, or as a form of a "proof of concept" in model plant species (e.g. Arabidopsis). Future challenges for SM bioengineering include reducing unwanted effects on plant fitness, transfer of knowledge from models to crops, the reduction of genomic position effects and the capacity to predict the outcome of bioengineering. These aspects are also discussed. The large and rapid advances made during the last decade in our understanding of the molecular genetic control of SM production and biological function provide an excellent foundation for successful bioengineering of these small molec

The anatomy of strawberry (Fragaria x ananassa) fruit, in which the achene is found on the outer part of the fruit, makes it an excellent species for studying the regulation of fruit development. It can provide a model for the cross talk between primary and secondary metabolism, whose role is of pivotal importance in the process. By combining gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry with the aim of addressing the metabolic regulation underlying fruit seed development, we simultaneously analyzed the composition of primary and secondary metabolites, separately, in achene and receptacle during fruit ripening of strawberry cultivar Herut. The results from these analyses suggest that changes in primary and secondary metabolism reflect organ and developmental specificities. For instance, the receptacle was characterized by increases in sugars and their direct derivatives, while the achene was characterized by a major decrease in the levels of carbon- and nitrogen-rich compounds, with the exception of storage-related metabolites (e.g. raffinose). Furthermore, the receptacle, and to a lesser extent the achene, exhibited dynamic fluctuations in the levels and nature of secondary metabolites across the ripening process. In the receptacle, proanthocyanidins and flavonol derivatives characterized mainly early developmental stages, while anthocyanins were abundant in the mature red stage; in the achene, ellagitannin and flavonoids were abundant during early and late development, respectively. Correlation-based network analysis suggested that metabolism is substantially coordinated during early development in either organ. Nonetheless, a higher degree of connectivity within and between metabolic pathways was measured in the achenes. The data are discussed within the context of current models both of the interaction of primary and secondary metabolism and of the metabolic interaction between the different plant organs.

Three-way junctions in folded RNAs have been investigated both experimentally and computationally. The interest in their analysis stems from the fact that they have significantly been found to possess a functional role. In recent work, three-way junctions have been categorized into families depending on the relative lengths of the segments linking the three helices. Here, based on ideas originating from computational geometry, an algorithm is proposed for detecting three-way junctions in data sets of genes that are related to a metabolic pathway of interest. In its current implementation, the algorithm relies on a moving window that performs energy minimization folding predictions, and is demonstrated on a set of genes that are involved in purine metabolism in plants. The pattern matching algorithm can be extended to other organisms and other metabolic cycles of interest in which three-way junctions have been or will be discovered to play an important role. In the test case presented here with, the computational prediction of a three-way junction in Arabidopsis that was speculated to have an interesting functional role is verified experimentally.

The cuticle fulfills multiple roles in the plant life cycle, including protection from environmental stresses and the regulation of organ fusion. It is largely composed of cutin, which consists of C_16-18 fatty acids. While cutin composition and biosynthesis have been studied, the export of cutin monomers out of the epidermis has remained elusive. Here, we show that DESPERADO (AtWBC11) (abbreviated DSO), encoding a plasma membrane-localized ATP-binding cassette transporter, is required for cutin transport to the extracellular matrix. The dso mutant exhibits an array of surface defects suggesting an abnormally functioning cuticle. This was accompanied by dramatic alterations in the levels of cutin monomers. Moreover, electron microscopy revealed unusual lipidic cytoplasmatic inclusions in epidermal cells, disappearance of the cuticle in postgenital fusion areas, and altered morphology of trichomes and pavement cells. We also found that DSO is induced by salt, abscisic acid, and wounding stresses and its loss of function results in plants that are highly susceptible to salt and display reduced root branching. Thus, DSO is not only essential for developmental plasticity but also plays a vital role in stress responses.

Freshwater is a limited and dwindling global resource; therefore, efficient water use is required for food crops that have high water demands, such as rice, or for the production of sustainable energy biomass. We show here that expression of the Arabidopsis HARDY (HRD) gene in rice improves water use efficiency, the ratio of biomass produced to the water used, by enhancing photosynthetic assimilation and reducing transpiration. These drought-tolerant, low-water-consuming rice plants exhibit increased shoot biomass under well irrigated conditions and an adaptive increase in root biomass under drought stress. The HRD gene, an AP2/ERF-like transcription factor, identified by a gain-of-function Arabidopsis mutant hrd-D having roots with enhanced strength, branching, and cortical cells, exhibits drought resistance and salt tolerance, accompanied by an enhancement in the expression of abiotic stress associated genes. HRD overexpression in Arabidopsis produces thicker leaves with more chloroplast-bearing mesophyll cells, and in rice, there is an increase in leaf biomass and bundle sheath cells that probably contributes to the enhanced photosynthesis assimilation and efficiency. The results exemplify application of a gene identified from the model plant Arabidopsis for the improvement of water use efficiency coincident with drought resistance in the crop plant rice.

The interface between plants and the environment plays a dual role as a protective barrier as well as a medium for the exchange of gases, water, and nutrients. The primary aerial plant surfaces are covered by a cuticle, acting as the essential permeability barrier toward the atmosphere. It is a heterogeneous layer composed mainly of lipids, namely cutin and intracuticular wax with epicuticular waxes deposited on the surface. We identified an Arabidopsis thaliana activation tag gain-of-function mutant shine (shn) that displayed a brilliant, shiny green leaf surface with increased cuticular wax compared with the leaves of wild-type plants. The gene responsible for the phenotype encodes one member of a clade of three proteins of undisclosed function, belonging to the plant-specific family of AP2/EREBP transcription factors. Overexpression of all three SHN clade genes conferred a phenotype similar to that of the original shn mutant. Biochemically, such plants were altered in wax composition (very long fatty acid derivatives). Total cuticular wax levels were increased sixfold in shn compared with the wild type, mainly because of a ninefold increase in alkanes that comprised approximately half of the total waxes in the mutant. Chlorophyll leaching assays and fresh weight loss experiments indicated that overexpression of the SHN genes increased cuticle permeability, probably because of changes in its ultrastructure. Likewise, SHN gene overexpression altered leaf and petal epidermal cell structure, trichome number, and branching as well as the stomatal index. Interestingly, SHN overexpressors displayed significant drought tolerance and recovery, probably related to the reduced stomatal density. Expression analysis using promoter-β-glucuronidase fusions of the SHN genes provides evidence for the role of the SHN clade in plant protective layers, such as those formed during abscission, dehiscence, wounding, tissue strengthening, and the cuticle. We propose that these diverse functions are mediated by regulating metabolism of lipid and/or cell wall components.

MADS box genes function to regulate vegetative, floral, and fruit development in plants. Here we characterize the expression pattern of a MADS box gene from strawberry (Fragaria x ananassa), designated STAG1. Sequence analysis revealed that STAG1 shared 68-91% amino acid sequence identity to AGAMOUS homologs from a variety of plant species. STAG1 transcripts were detected in stamens, carpels, and developing fruit. In situ hybridization revealed that STAG1 mRNA expression was restricted to the endothelium and the vascular bundles connecting the achenes to the inner part of the receptacle and was not evident in the receptacle of the fruit. Analysis of the expression of a GUS marker gene driven by the STAG1 promoter showed that during floral development, STAG1 was active in stamens, the base of the receptacle and the petals, and in the central pith and vascular tissue. During the ripening stage of fruit development, STAG1 activity was detected in achenes, pith cells, and cortical cells. Sequence analysis and expression patterns indicate that STAG1 is an AGAMOUS homolog of strawberry.

DNA microarray technology is a key element in today's functional genomics toolbox. The power of the method lies in miniaturization, automation and parallelism permitting large-scale and genome-wide acquisition of quantitative biological information from multiple samples. DNA microarrays are currently fabricated and assayed by two main approaches involving either in situ synthesis of oligonucleotides ('oligonucleotide microarrays') or deposition of pre-synthesized DNA fragments ('cDNA microarrays') on solid surfaces. To date, the main applications of microarrays are in comprehensive, simultaneous gene expression monitoring and in DNA variation analyses for the identification and genotyping of mutations and polymorphisms. Already at a relatively early stage of its application in plant science, microarrays are being utilized to examine a range of biological issues including the circadian clock, plant defence, environmental stress responses, fruit ripening, phytochrome A signalling, seed development and nitrate assimilation. Novel insights are obtained into the molecular mechanisms co-ordinating metabolic pathways, regulatory and signalling networks. Exciting new information will be gained in the years to come not only from genome-wide expression analyses on a few model plant species, but also from extensive studies of less thoroughly studied species on a more limited scale. The value of microarray technology to our understanding of living processes will depend both on the amount of data to be generated and on its clever exploration and integration with other biological knowledge arising from complementary functional genomics tools for 'profiling' the genome, proteome, metabolome and phenome.

An efficient adventitious shoot regeneration procedure was developed for Gypsophila paniculata L. Using cultivar Arbel, shoot regeneration from the three upper internodes of the stem was monitored on MS media supplemented with different cytokinins (thidiazuron, benzyladenine, kinetin or zeatin) and an auxin (naphthaleneacetic acid). Thidiazuron was found to be the most efficient cytokinin, with up to 100% of the explants forming shoots, at an average of up to 19 shoots per explant being regenerated. The highest percentage of shoot formation was observed in the stem explants originating from the first internode, with all cytokinins tested. The adventitious origin of shoots regenerated from stem explants was confirmed by scanning electron microscopy. The regeneration procedure was found to be applicable to five other gypsophila cultivars (Perfecta, Golan, Gilboa, Flamingo and Tavor). Regenerating plants were successfully transferred to soil, and did not differ in flower color, size or shape from standard vegetatively propagated plants derived from cuttings.