Publications

Stable isotope labeling (SIL) is a powerful tool for probing cellular metabolism and has been increasingly employed for metabolite annotation in mass spectrometry-based metabolomics. Herein, we outline the principles and methods of designing and performing SIL-based experiments in metabolomics studies. Further, we summarize different SIL approaches and highlight recent examples of their use for metabolite annotation, including biological feature recognition, degenerate feature annotation, elemental composition determination and metabolite structure elucidation, across diverse biological systems. Throughout, the opportunities, open challenges, and potential pitfalls of each SIL workflow are discussed. Finally, we summarize and compare the recently developed software tools for the analysis of SIL-based metabolomics data.

Steroidal glycoalkaloids (SGAs) are specialized metabolites produced by hundreds of Solanum species including food crops, such as tomato, potato and eggplant. Unlike true alkaloids, nitrogen is introduced at a late stage of SGA biosynthesis through an unknown transamination reaction. Here, we reveal the mechanism by which GLYCOALKALOID METABOLISM12 (GAME12) directs the biosynthesis of nitrogen-containing steroidal alkaloid aglycone in Solanum. We report that GAME12, a neofunctionalized γ-aminobutyric acid (GABA) transaminase, undergoes changes in both active site specificity and subcellular localization to switch from its renown and generic activity in core metabolism to function in a specialized metabolic pathway. Moreover, overexpression of GAME12 alone in engineered S. nigrum leaves is sufficient for de novo production of nitrogen-containing SGAs. Our results highlight how hijacking a core metabolism GABA shunt enzyme is crucial in numerous Solanum species for incorporating a nitrogen to a steroidal-specialized metabolite backbone and form defensive alkaloids. (Figure presented.)

Mescaline, among the earliest identified natural hallucinogens, holds great potential in psychotherapy treatment. Nonetheless, despite the existence of a postulated biosynthetic pathway for more than half a century, the specific enzymes involved in this process are yet to be identified. In this study, we investigated the cactus Lophophora williamsii (Peyote), the largest known natural producer of the phenethylamine mescaline. We employed a multi-faceted approach, combining de novo whole-genome and transcriptome sequencing with comprehensive chemical profiling, enzymatic assays, molecular modeling, and pathway engineering for pathway elucidation. We identified four groups of enzymes responsible for the six catalytic steps in the mescaline biosynthetic pathway, and an N-methyltransferase enzyme that N-methylates all phenethylamine intermediates, likely modulating mescaline levels in Peyote. Finally, we reconstructed the mescaline biosynthetic pathway in both Nicotiana benthamiana plants and yeast cells, providing novel insights into several challenges hindering complete heterologous mescaline production. Taken together, our study opens up avenues for exploration of sustainable production approaches and responsible utilization of mescaline, safeguarding this valuable natural resource for future generations.

Tomato is the highest value fruit and vegetable crop worldwide, yet produces α-tomatine, a renowned toxic and bitter-tasting anti-nutritional steroidal glycoalkaloid (SGA) involved in plant defense. A suite of modifications during tomato fruit maturation and ripening converts α-tomatine to the non-bitter and less toxic Esculeoside A. This important metabolic shift prevents bitterness and toxicity in ripe tomato fruit. While the enzymes catalyzing glycosylation and hydroxylation reactions in the Esculeoside A pathway have been resolved, the proposed acetylating step remains, to date, elusive. Here, we discovered that GAME36 (GLYCOALKALOID METABOLISM36), a BAHD-type acyltransferase catalyzes SGA-acetylation in cultivated and wild tomatoes. This finding completes the elucidation of the core Esculeoside A biosynthetic pathway in ripe tomato, allowing reconstitution of Esculeoside A production in heterologous microbial and plant hosts. The involvement of GAME36 in bitter SGA detoxification pathway points to a key role in the evolution of sweet-tasting tomato as well as in the domestication and breeding of modern cultivated tomato fruit.

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, 373378 (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, 2134 (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 spectrometrybased 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 variancebased 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.

In a recent paper in Nature Edith Heard from the European Molecular Biology Laboratory (EMBL) suggested that molecular biologists should \u201creconnect with nature\u201d by diversifying sampling locations. Although this approach has its own benefits, we suggest that advanced methods should rather be used to take hypothesis-based experiments to nature, thereby supplying a much-needed context for experimentation under controlled conditions. Following the CRISPR revolution, this approach has become accessible to many research groups. For the past years we developed the groundwork and initiated such experimentation. This included assembly of a mobile lab on a 4-wheel drive truck and examining genome edited metabolic mutants in wild, tobacco grown in nature. Our findings included both targeted answers to focused questions, but also surprising results that could only be reached while working in natural settings.

Plant response to pathogens attacks generally comes at the expense of growth. Defense priming is widely accepted as an efficient strategy used for augmenting resistance with reduced fitness in terms of growth and yield. Plant-derived small molecules, both primary as well as secondary metabolites, can function as activators to prime plant defense. Amino acids and their derivatives regulate numerous aspects of plant growth and development, and biotic and abiotic stress responses. In this review, we discuss the recent progress in understanding the roles of amino acids and related molecules in defense priming and their link with plant growth. We also highlight some of the outstanding questions and provide an outlook on the prospects of engineering the tradeoff between defense and growth in plants.

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.

R2R3MYB transcription factors play important roles in plant development; yet, the exact role of each of them remains to be resolved. Here we report that the Arabidopsis AtMYB31 is required for wax biosynthesis in epidermis of reproductive tissues, and is involved in seed development. AtMYB31 was ubiquitously expressed in both vegetative and reproductive tissues with higher expression levels in siliques and seeds, while AtMYB31 was localized to the nucleus and cytoplasm. Loss of function of AtMYB31 reduced wax accumulation in the epidermis of silique and flower tissues, disrupted seed coat epidermal wall development and mucilage production, altered seed proanthocyanidin and polyester content. AtMYB31 could direct activate expressions of several wax biosynthetic target genes. Altogether, AtMYB31, a R2R3MYB transcription factor, regulates seed development in Arabidopsis.

Grass pea (Lathyrus sativus L.) is a grain legume commonly grown in Asia and Africa for food and forage. It is a highly nutritious and robust crop, capable of surviving both droughts and floods. However, it produces a neurotoxic compound, β-N-oxalyl-L-α,β-diaminopropionic acid (β-ODAP), which can cause a severe neurological disorder when consumed as a primary diet component. While the catalytic activity associated with β-ODAP formation was demonstrated more than 50 years ago, the enzyme responsible for this activity has not been identified. Here, we report on the identity, activity, 3D structure, and phylogenesis of this enzyme β-ODAP synthase (BOS). We show that BOS belongs to the BAHD superfamily of acyltransferases and is structurally similar to hydroxycinnamoyl transferases. Employing molecular docking, we propose a mechanism for its catalytic activity, and using heterologous expression in tobacco leaves (N. benthamiana), we demonstrate that expression of BOS in the presence of its substrates is sufficient for β-ODAP production in vivo. The identification of BOS may pave the way towards engineering β-ODAP-free grass pea cultivars, which are safe for human and animal consumption.

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 proteinprotein 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.

Abstract Plant MICRORNA164 (miR164) plays diverse regulatory functions by post-transcriptional repression of certain NAM/ATAF/CUC-domain transcription factors. However, the involvement of miR164 in fleshy fruit development and ripening remains poorly understood. Here, de novo prediction of tomato (Solanum lycopersicum) MIR164 genes identified four genes (SlMIR164a-d), of which SlMIR164d has an atypically long pre-miRNA. The roles of the fruit expressed SlMIR164a, b, and d were studied by analysis of their Clustered Regularly Interspaced Short Palindromic Repeats mutants. The slmir164bCR mutant plants exhibited shoot and flower abnormalities characteristic of ectopic boundary specification, whereas the shoot and flower development of slmir164aCR and slmir164dCR mutants were indistinguishable from wild-type. Strikingly, the knockout of SlMIR164a practically eliminated sly-miR164 from the developing and ripening fruit pericarp. The sly-miR164-deficient slmir164aCR fruits were smaller than the wild-type, due to reduced pericarp cell division and expansion, and displayed intense red color and matte, instead of glossy appearance, upon ripening. We found that the fruit skin phenotypes were associated with morphologically abnormal outer epidermis and thicker cuticle. Quantitation of sly-miR164 target transcripts in slmir164aCR ripening fruits demonstrated the upregulation of SlNAM3 and SlNAM2. Specific expression of their miR164-resistant versions in the pericarp resulted in the formation of extremely small fruits with abnormal epidermis, highlighting the importance of their negative regulation by sly-miR164a. Taken together, our results demonstrate that SlMIR164a and SlMIR164b play specialized roles in development: SlMIR164b is required for shoot and flower boundary specification, and SlMIR164a is required for fruit growth including the expansion of its outer epidermis, which determines the properties of the fruit skin.

Plants are unsurpassed biochemists that synthesize a plethora of molecules in response to an ever-changing environment. The majority of these molecules, considered as specialized metabolites, effectively protect the plant against pathogens and herbivores. However, this defense most probably comes at a great expense, leading to reduction of growth (known as the growthdefense trade-off). Plants employ several strategies to reduce the high metabolic costs associated with chemical defense. Production of specialized metabolites is tightly regulated by a network of transcription factors facilitating its fine-tuning in time and space. Multifunctionality of specialized metabolitestheir effective recycling system by re-using carbon, nitrogen, and sulfur, thus re-introducing them back to the primary metabolite poolallows further cost reduction. Spatial separation of biosynthetic enzymes and their substrates, and sequestration of potentially toxic substances and conversion to less toxic metabolite forms are the plants solutions to avoid the detrimental effects of metabolites they produce as well as to reduce production costs. Constant fitness pressure from herbivores, pathogens, and abiotic stressors leads to honing of specialized metabolite biosynthesis reactions to be timely, efficient, and metabolically cost-effective. In this review, we assess the costs of production of specialized metabolites for chemical defense and the different plant mechanisms to reduce the cost of such metabolic activity in terms of self-toxicity and growth.

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. Re-sults: 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 com-pound degradation. In humans, several Mankai-related plasma and urine polyphenols were differ-entially elevated in the green Mediterranean group compared with the other groups (p

The aerial surfaces of plants are covered by a protective barrier formed by the cutin polyester and waxes, collectively referred to as the cuticle. Plant cuticles prevent the loss of water, regulate transpiration, and facilitate the transport of gases and solutes. As the cuticle covers the outermost epidermal cell layer, it also acts as the first line of defense against environmental cues and biotic stresses triggered by a large array of pathogens and pests, such as fungi, bacteria, and insects. Numerous studies highlight the cuticle interface as the site of complex molecular interactions between plants and pathogens. Here, we outline the multidimensional roles of cuticle-derived components, namely, epicuticular waxes and cutin monomers, during plant interactions with pathogenic fungi. We describe how certain wax components affect various pre-penetration and infection processes of fungi with different lifestyles, and then shift our focus to the roles played by the cutin monomers that are released from the cuticle owing to the activity of fungal cutinases during the early stages of infection. We discuss how cutin monomers can activate fungal cutinases and initiate the formation of infection organs, the significant impacts of cuticle defects on the nature of plantfungal interactions, along with the possible mechanisms raised thus far in the debate on how host plants perceive cutin monomers and/or cuticle defects to elicit defense responses.

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.

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 \u201chand brake\u201d 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.

The physiology of fruit ripening is defined as either climacteric or non-climacteric. In climacteric fruit respiration during ripening increases until it reaches a peak, which is accompanied by an increase in autocatalytic ethylene production, whereas the respiration of non-climacteric fruit does not increase and they have no requirement for ethylene to complete their ripening. In an attempt to gain further insight into the involvement of autocatalytic ethylene production with the climacteric rise in respiration, tomato fruit were harvested at three defined stages of maturity prior to the climacteric peak (mature green, breaker, and early orange) and immediately exposed to the gaseous molecule 1-methylcyclopropene (1-MCP). The gene expression profile at each of these stages was monitored after 24 h, using an Affymetrix tomato microarray chip. This approach enabled us to identify ethylene responsive genes that are commonly regulated at early stages of ripening, as well as new candidate genes. In addition, 1-MCP treatment affected the levels of metabolites related to methionine biosynthesis. Methionine feeds climacteric ethylene production and we found that promotors of the genes of enzymes that catalyze the production of homoserine and homocysteine (aspartokinase/homoserine dehydrogenases and cystathionine beta lyase, respectively), precursors in the methionine pathway, contain the AtSR1 binding motif. This binding motif is recognized by ethylene activated transcription factors, hence indicating a role for ethylene in methionine synthesis during early ripening, explaining the autocatalytic ethylene production during subsequent ripening stages.

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.

Climacteric ripening is related to a respiratory climax accompanied by an exponential increase in ethylene production. This association is not fully understood. Tomato fruit were exposed to the ethylene antagonist 1-methylcyclopropene (1-MCP) at different ripening stages and their transcriptome was monitored. ISOCITRATE DEHYDROGENASE 1 (ICDH, Solyc01g005560.2.1) was the only respiratory process related gene that was down regulated at all ripening stages including the mature green. Silencing SlICDH1using VIGS (virus indused gene silencing), to about 50 % of its native expression, in Micro-Tom tomatoes, reduced both respiration and ethylene production of the harvested fruit. Silenced SlICDH1 tomatoes also exhibited prolonged ripening and reduced susceptibility to the pathogen Botrytis cinerea. In addition, ethylene response factors binding sites were identified in the promotor and in the first intron of SlICDH1. These results suggest that regulation of SlICDH1 is a key step in the initiation of the climacteric rise of respiration via ethylene regulation.

Gene-editing techniques are currently revolutionizing biology, allowing far greater precision than previous mutagenic and transgenic approaches. They are becoming applicable to a wide range of plant species and biological processes. Gene editing can rapidly improve a range of crop traits, including disease resistance, abiotic stress tolerance, yield, nutritional quality and additional consumer traits. Unlike transgenic approaches, however, it is not facile to forensically detect gene-editing events at the molecular level, as no foreign DNA exists in the elite line. These limitations in molecular detection approaches are likely to focus more attention on the products generated from the technology than on the process in itself. Rapid advances in sequencing and genome assembly increasingly facilitate genome sequencing as a means of characterizing new varieties generated by gene-editing techniques. Nevertheless, subtle edits such as single base changes or small deletions may be difficult to distinguish from normal variation within a genotype. Given these emerging scenarios, downstream 'omics' technologies reflective of edited affects, such as metabolomics, need to be used in a more prominent manner to fully assess compositional changes in novel foodstuffs. To achieve this goal, metabolomics or 'non-targeted metabolite analysis' needs to make significant advances to deliver greater representation across the metabolome. With the emergence of new edited crop varieties, we advocate: (i) concerted efforts in the advancement of 'omics' technologies, such as metabolomics, and (ii) an effort to redress the use of the technology in the regulatory assessment for metabolically engineered biotech crops.

Root endodermis, the innermost cortical layer surrounding the root vasculature, serves as the foremost barrier to water, solutes, and nutrients taken up from soil. Endodermis barrier functionality is achieved via its hydrophobic coating of lignified Casparian strips and the suberin lamellae; nonetheless the regulatory mechanisms underlying endodermis suberization are still elusive. Here, we discovered that the Arabidopsis SUBERMAN (SUB) transcription factor controls the establishment of the root suberin lamellae. Transient expression of SUB in Nicotiana benthamiana leaves resulted in the induction of heterologous suberin genes, the accumulation of suberin-type monomers, and consequent deposition of suberin-like lamellae. We demonstrate that SUB exerts its regulatory roles by transactivating promoters of suberin genes. In Arabidopsis, SUB is expressed in patchy and continuous suberization root endodermal cells, and thus roots with higher or lower expression of SUB display altered suberin polymer deposition patterns and modified composition. While these changes did not interfere with Casparian strip formation they had a substantial effect on root uptake capacity, resulting in varied root and leaf ionomic phenotypes. Gene expression profiling revealed that SUB function impacts transcriptional networks associated with suberin, phenylpropanoids, lignin, and cuticular lipid biosynthesis, as well as root transport activities, hormone signalling, and cell wall modification. Our findings highlight SUB as a regulator of root endodermis suberization during normal development, and its characterization is thus a key step towards dissecting the molecular mechanisms partaking in root endodermal barrier functionalities.

Microbial communities associated with roots confer specific functions to their hosts, thereby modulating plant growth, health, and productivity. Yet, seminal questions remain largely unaddressed including whether and how the rhizosphere microbiome modulates root metabolism and exudation and, consequently, how plants fine tune this complex belowground web of interactions. Here we show that, through a process termed systemically induced root exudation of metabolites (SIREM), different microbial communities induce specific systemic changes in tomato root exudation. For instance, systemic exudation of acylsugars secondary metabolites is triggered by local colonization of bacteria affiliated with the genus Bacillus. Moreover, both leaf and systemic root metabolomes and transcriptomes change according to the rhizosphere microbial community structure. Analysis of the systemic root metabolome points to glycosylated azelaic acid as a potential microbiome-induced signaling molecule that is subsequently exuded as free azelaic acid. Our results demonstrate that rhizosphere microbiome assembly drives the SIREM process at the molecular and chemical levels. It highlights a thus-far unexplored long-distance signaling phenomenon that may regulate soil conditioning.

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.

Motivation: The use of stable isotope labeling is highly advantageous for structure elucidation in metabolomics studies. However, computational tools dealing with multiple-precursor-based labeling studies are still missing. Hence, we developed Miso, an R package providing automated and efficient data analysis workflow to detect the complete repertoire of labeled molecules from multiple-precursor- based labeling experiments.Results: The capability of Miso is demonstrated by the analysis of liquid chromatography-mass spectrometry data obtained from duckweed plants fed with one unlabeled and two differently labeled tyrosine (unlabeled tyrosine, tyrosine-H-2(4) and tyrosine-(C9N1)-C-13-N-15). The resulting data matrix generated by Miso contains sets of unlabeled and labeled ions with their retention time, m/z values and number of labeled atoms that can be directly utilized for database query and biological studies.

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.

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.

Sepals play important roles in protecting inner floral organs from various stresses and in guaranteeing timely flower opening. However, the exact role of sepals in coordinating interior and exterior signals remains elusive. In this study, we functionally characterized a heat shock protein gene, Arabidopsis HSP70-16, in flower opening and mild heat stress response, using combined genetics with anatomic, physiological, chemical, and molecular analyses. We showed that HSP70-16 is required for flower opening and mild heat response. Mutation of HSP70-16 led to a significant reduction in seed setting rate under 22 degrees C, which was more severe at 27 degrees C. Mutation of HSP70-16 also caused postgenital fusion at overlapping tips of two lateral sepals, leading to failed flower opening, abnormal floral organ formation, and impaired fertilization and seed setting. Chemical and anatomic analyses confirmed specific chemical and morphological changes of cuticle property in mutant lateral sepals, and qRT-PCR data indicated that expression levels of different sets of cuticle regulatory and biosynthetic genes were altered in mutants grown at both 22 degrees C and 27 degrees C temperatures. This study provides a link between thermal and developmental perception signals and expands the understanding of the roles of sepal in plant development and heat response.

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.

Current breeding relies mostly on random mutagenesis and recombination to generate novel genetic variation. However, targeted genome editing is becoming an increasingly important tool for precise plant breeding. Using the CRISPR-Cas system combined with the bean yellow dwarf virus rolling circle replicon, we optimized a method for targeted mutagenesis and gene replacement in tomato. The carotenoid isomerase (CRTISO) and phytoene synthase 1 (PSY1) genes from the carotenoid biosynthesis pathway were chosen as targets due to their easily detectable change of phenotype. We took advantage of the geminiviral replicon amplification as a means to provide a large amount of donor template for the repair of a CRISPR-Cas-induced DNA double-strand break (DSB) in the target gene, via homologous recombination (HR). Mutagenesis experiments, performed in the Micro-Tom variety, achieved precise modification of the CRTISO and PSY1 loci at an efficiency of up to 90%. In the gene targeting (GT) experiments, our target was a fast-neutron-induced crtiso allele that contained a 281-bp deletion. This deletion was repaired with the wild-type sequence through HR between the CRISPR-Cas-induced DSB in the crtiso target and the amplified donor in 25% of the plants transformed. This shows that efficient GT can be achieved in the absence of selection markers or reporters using a single and modular construct that is adaptable to other tomato targets and other crops.

Thousands of specialized, steroidal metabolites are found in a wide spectrum of plants. These include the steroidal glycoalkaloids (SGAs), produced primarily by most species of the genus Solanum, and metabolites belonging to the steroidal saponins class that are widespread throughout the plant kingdom. SGAs play a protective role in plants and have potent activity in mammals, including antinutritional effects in humans. The presence or absence of the double bond at the C-5,6 position (unsaturated and saturated, respectively) creates vast structural diversity within this metabolite class and determines the degree of SGA toxicity. For many years, the elimination of the double bond from unsaturated SGAs was presumed to occur through a single hydrogenation step. In contrast to this prior assumption, here, we show that the tomato GLYCOALKALOID METABOLISM25 (GAME25), a short-chain dehydrogenase/reductase, catalyzes the first of three prospective reactions required to reduce the C-5,6 double bond in dehydrotomatidine to form tomatidine. The recombinant GAME25 enzyme displayed 3β-hydroxysteroid dehydrogenase/ Δ
^5,4 isomerase activity not only on diverse steroidal alkaloid aglycone substrates but also on steroidal saponin aglycones. Notably, GAME25 down-regulation rerouted the entire tomato SGA repertoire toward the dehydro-SGAs branch rather than forming the typically abundant saturated α-tomatine derivatives. Overexpress-ing the tomato GAME25 in the tomato plant resulted in significant accumulation of α-tomatine in ripe fruit, while heterologous expression in cultivated eggplant generated saturated SGAs and atypical saturated steroidal saponin glycosides. This study demonstrates how a single scaffold modification of steroidal metabolites in plants results in extensive structural diversity and modulation of product toxicity.

In the last decade, plant genome-scale modeling has developed rapidly and modeling efforts have advanced from representing metabolic behavior of plant heterotrophic cell suspensions to studying the complex interplay of cell types, tissues, and organs. A crucial driving force for such developments is the availability and integration of \u201comics\u201d data (e.g., transcriptomics, proteomics, and metabolomics) which enable the reconstruction, extraction, and application of context-specific metabolic networks. In this chapter, we demonstrate a workflow to integrate gas chromatography coupled to mass spectrometry (GC-MS)-based metabolomics data of tomato fruit pericarp (flesh) tissue, at five developmental stages, with a genome-scale reconstruction of tomato metabolism. This method allows for the extraction of context-specific networks reflecting changing activities of metabolic pathways throughout fruit development and maturation.

Betalains are tyrosine-derived red-violet and yellow pigments found exclusively in plants of the Caryophyllales order, which have drawn both scientific and economic interest. Nevertheless, research into betalain chemistry, biochemistry, and function has been limited as comparison with other major classes of plant pigments such as anthocyanins and carotenoids. The core biosynthetic pathway of this pigment class has only been fully elucidated in the past few years, opening up the possibility for betalain pigment engineering in plants and microbes. In this review, we discuss betalain metabolism in light of recent advances in the field, with a current survey of characterized genes and enzymes that take part in betalain biosynthesis, catabolism, and transcriptional regulation, and an outlook of what is yet to be discovered. A broad view of currently used and potential new sources for betalains, including utilization of natural sources or metabolic engineering, is provided together with a summary of potential applications of betalains in research and commercial use.

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.

Steroidal glycoalkaloids (SGAs) are major secondary metabolites constitutively produced in cultivated potato Solanum tuberosum, and alpha-solanine and alpha-chaconine are the most abundant SGAs. SGAs are toxic to humans at high levels but their role in plant protection against pests and pathogens is yet to be established. In this study, levels of SGAs in potato were reduced by RNA interference (RNAi)-mediated silencing of GLYCOALKALOID METABOLISM 4 (GAME4)-a gene encoding cytochrome P450, involved in an oxidation step in the conversion of cholesterol to SGA aglycones. Two GAME4 RNAi lines, T8 and T9, were used to investigate the effects of manipulation of the SGA biosynthetic pathway in potato. Growth and development of an insect pest, Colorado potato beetle (CPB), were affected in these lines. While no effect on CPB leaf consumption or weight gain was observed, early instar larval death and accelerated development of the insect was found while feeding on leaves of GAME4 RNAi lines. Modulation of SGA biosynthetic pathway in GAME4 RNAi plants was associated with a larger alteration to the metabolite profile, including increased levels of one or both the steroidal saponins or phytoecdysteroids, which could affect insect mortality as well as development time. Colonization by Verticillium dahliae on GAME4 RNAi plants was also tested. There were increased pathogen levels in the T8 GAME4 RNAi line but not in the T9. Metabolite differences between T8 and T9 were found and may have contributed to differences in V. dahliae infection. Drought responses created by osmotic stress were not affected by modulation of SGA biosynthetic pathway in potato.

Betalains are tyrosine-derived red-violet and yellow plant pigments known for their antioxidant activity, health-promoting properties, and wide use as food colorants and dietary supplements. By coexpressing three genes of the recently elucidated betalain biosynthetic pathway, we demonstrate the heterologous production of these pigments in a variety of plants, including three major food crops: tomato, potato, and eggplant, and the economically important ornamental petunia. Combinatorial expression of betalain-related genes also allowed the engineering of tobacco plants and cell cultures to produce a palette of unique colors. Furthermore, betalain-producing tobacco plants exhibited significantly increased resistance toward gray mold (Botrytis cinerea), a pathogen responsible for major losses in agricultural produce. Heterologous production of betalains is thus anticipated to enable biofortification of essential foods, development of new ornamental varieties, and innovative sources for commercial betalain production, as well as utilization of these pigments in crop protection.

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), Mets 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.

The cuticle is a specialized cell wall layer that covers the outermost surface of the epidermal cells and has important implications for fruit permeability and pathogen susceptibility. In order to decipher the genetic control of tomato fruit cuticle composition, an introgression line (IL) population derived from a biparental cross between Solanum pennellii (LA0716) and the Solanum lycopersicum cultivar M82 was used to build a first map of associated quantitative trait loci (QTLs). A total of 24 cuticular waxes and 26 cutin monomers were determined. They showed changes associated with 18 genomic regions distributed in nine chromosomes affecting 19 ILs. Out of the five main fruit cuticular components described for the wild species S. pennellii, three of them were associated with IL3.4, IL12.1, and IL7.4.1, causing an increase in n-alkanes (≥C 30), a decrease in amyrin content, and a decrease in cuticle thickness of ∼50%, respectively. Moreover, we also found a QTL associated with increased levels of amyrins in IL3.4. In addition, we propose some candidate genes on the basis of their differential gene expression and single nucleotide polymorphism variability between the introgressed and the recurrent alleles, which will be the subjects of further investigation.

Current innovations in mass-spectrometry-based technologies allow deep coverage of protein expression. Despite its immense value and in contrast to transcriptomics, only a handful of studies in crop plants engaged with global proteome assays. Here, we present large-scale shotgun proteomics profiling of tomato fruit across two key tissues and five developmental stages. A total of 7738 individual protein groups were identified and reliably measured at least in one of the analyzed tissues or stages. The depth of our assay enabled identification of 61 differentially expressed transcription factors, including renowned ripening-related regulators and elements of ethylene signaling. Significantly, we measured proteins involved in 83% of all predicted enzymatic reactions in the tomato metabolic network. Hence, proteins representing almost the complete set of reactions in major metabolic pathways were identified, including the cytosolic and plastidic isoprenoid and the phenylpropanoid pathways. Furthermore, the data allowed us to discern between protein isoforms according to expression patterns, which is most significant in light of the weak transcript-protein expression correspondence. Finally, visualization of changes in protein abundance associated with a particular process provided us with a unique view of skin and flesh tissues in developing fruit. This study adds a new dimension to the existing genomic, transcriptomic and metabolomic resources. It is therefore likely to promote translational and post-translational research in tomato and additional species, which is presently focused on transcription.

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.

Annotation of metabolites is an essential, yet problematic, aspect of mass spectrometry (MS)-based metabolomics assays. The current repertoire of definitive annotations of metabolite spectra in public MS databases is limited and suffers from lack of chemical and taxonomic diversity. Furthermore, the heterogeneity of the data prevents the development of universally applicable metabolite annotation tools. Here we present a combined experimental and computational platform to advance this key issue in metabolomics. WEIZMASS is a unique reference metabolite spectral library developed from high-resolution MS data acquired from a structurally diverse set of 3,540 plant metabolites. We also present MatchWeiz, a multi-module strategy using a probabilistic approach to match library and experimental data. This strategy allows efficient and high-confidence identification of dozens of metabolites in model and exotic plants, including metabolites not previously reported in plants or found in few plant species to date.

The identification and characterization of new tomato (Solanum lycopersicum) mutants affected in fruit pigmentation and nutritional content can provide valuable insights into the underlying biology, as well as a source of new alleles for breeding programs. To date, all characterized pink-pigmented tomato fruit mutants appear to result from low SlMYB12 transcript levels in the fruit skin. Two new mutant lines displaying a pink fruit phenotype (pf1 and pf2) were characterized in this study. In the pf mutants, SlMYB12 transcripts accumulated to wild-type levels but exhibited the same truncation, which resulted in the absence of the essential MYB activation domain coding region. Allelism and complementation tests revealed that both pf mutants were allelic to the y locus and showed the same recessive null allele in homozygosis: Δy. A set of molecular and metabolic effects, reminiscent of those observed in the Arabidopsis (Arabidopsis thaliana) myb11 myb12 myb111 triple mutant, were found in the tomato Δy mutants. To our knowledge, these have not been described previously, and our data support the idea of their being null mutants, in contrast to previously described transcriptional hypomorphic pink fruit lines. We detected a reduction in the expression of several flavonol glycosides and some associated glycosyl transferases. Transcriptome analysis further revealed that the effects of the pf mutations extended beyond the flavonoid pathway into the interface between primary and secondary metabolism. Finally, screening for Myb-binding sites in the candidate gene promoter sequences revealed that 141 of the 152 co-down-regulated genes may be direct targets of SlMYB12 regulation.

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.

Steroidal glycoalkaloids (SGAs) are cholesterol-derived molecules produced by solanaceous species. They contribute to pathogen defence but are toxic to humans and considered as anti-nutritional compounds. Here we show that GLYCOALKALOID METABOLISM 9 (GAME9), an APETALA2/Ethylene Response Factor, related to regulators of alkaloid production in tobacco and Catharanthus roseus, controls SGA biosynthesis. GAME9 knockdown and overexpression in tomato and potato alters expression of SGAs and upstream mevalonate pathway genes including the cholesterol biosynthesis gene STEROL SIDE CHAIN REDUCTASE 2 (SSR2). Levels of SGAs, C24-alkylsterols and the upstream mevalonate and cholesterol pathways intermediates are modified in these plants. Δ(7)-STEROL-C5(6)-DESATURASE (C5-SD) in the hitherto unresolved cholesterol pathway is a direct target of GAME9. Transactivation and promoter-binding assays show that GAME9 exerts its activity either directly or cooperatively with the SlMYC2 transcription factor as in the case of the C5-SD gene promoter. Our findings provide insight into the regulation of SGA biosynthesis and means for manipulating these metabolites in crops.

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.

The outer epidermal layer of apple fruit is covered by a protective cuticle. Composed of a polymerized cutin matrix embedded with waxes, the cuticle is a natural waterproof barrier and protects against several abiotic and biotic stresses. In terms of apple production, the cuticle is essential to maintain long post-harvest storage, while severe failure of the cuticle can result in the formation of a disorder known as russet. Apple russet results from micro-cracking of the cuticle and the formation of a corky suberized layer. This is typically an undesirable consumer trait, and negatively impacts the post-harvest storage of apples. In order to identify genetic factors controlling cuticle biosynthesis (and thus preventing russet) in apple, a quantitative trait locus (QTL) mapping survey was performed on a full-sib population. Two genomic regions located on chromosomes 2 and 15 that could be associated with russeting were identified. Apples with compromised cuticles were identified through a novel and high-throughput tensile analysis of the skin, while histological analysis confirmed cuticle failure in a subset of the progeny. Additional genomic investigation of the determined QTL regions identified a set of underlying genes involved in cuticle biosynthesis. Candidate gene expression profiling by quantitative real-time PCR on a subset of the progeny highlighted the specific expression pattern of a SHN1/WIN1 transcription factor gene (termed MdSHN3) on chromosome 15. Orthologues of SHN1/WIN1 have been previously shown to regulate cuticle formation in Arabidopsis, tomato, and barley. The MdSHN3 transcription factor gene displayed extremely low expression in lines with improper cuticle formation, suggesting it to be a fundamental regulator of cuticle biosynthesis in apple fruit.

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.

Steroidal alkaloids (SAs) and their glycosylated forms (SGAs) are toxic compounds largely produced by members of the Solanaceae and Liliaceae plant families. This class of specialized metabolites serves as a chemical barrier against a broad range of pest and pathogens. In humans and animals, SAs are considered anti-nutritional factors because they affect the digestion and absorption of nutrients from food and might even cause poisoning. In spite of the first report on SAs nearly 200 years ago, much of the molecular basis of their biosynthesis and regulation remains unknown. Aspects concerning chemical structures and biological activities of SAs have been reviewed extensively elsewhere; therefore, in this review the latest insights to the elucidation of the SAs biosynthetic pathway are highlighted. Recently, co-expression analysis combined with metabolic profiling revealed metabolic gene clusters in tomato and potato that contain core genes required for production of the prominent SGAs in these two species. Elaborating the knowledge regarding the SAs biosynthetic pathway, the subcellular transport of these molecules, as well as the identification of regulatory and signaling factors associated with SA metabolism will likely advance understanding of chemical defense mechanisms in Solanaceae and Liliaceae plants. It will also provide the means to develop, through classical breeding or genetic engineering, crops with modified levels of anti-nutritional SAs.

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.

Riboswitches are RNA elements that bind small molecules and in turn regulate gene expression. This mechanism allows the cell to sense the intracellular concentration of these small molecules. A particular riboswitch typically regulates its adjacent gene by altering the transcription, the translation or the splicing of this gene. Recently, a riboswitch that binds thiamin pyrophosphate (TPP) was characterized and found to regulate thiamin biosynthesis in plants and algae. Furthermore, it appears that this element is an essential regulator of primary metabolism in plants. Manipulation of endogenous riboswitch activity resulted in metabolic phenotypes that underlined the role of these elements and their ligands in preserving metabolic homeostasis. This situation supports the hypothesis that riboswitches could be remnants of the most ancient metabolic regulators. Here, we review the mode of action of the plant and algal TPP riboswitch and its relevance to the metabolic network. We also discuss the potential engineering of riboswitches as metabolite sensors in plants and platforms for gene control. Whether additional such RNA-based mechanisms exist in plants and in algae is still an open question, yet, the importance of these elements to metabolic regulation is beyond doubt.

This chapter describes the structure and assembly of the cuticle, focusing on the genes and enzymes implicated in constructing the cuticle of fleshy fruit and its role in this organ. It is worth noting that, in the majority of studies, the 'peel' or 'skin' of the fruit is discussed. This is not a scientific term and describes in general the fruit outer layer including the pericarp and epidermal cells as well as the cuticle.

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.

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.

Plant development and survival is centered on complex regulatory networks composed of genes, proteins, hormone pathways, metabolites and signaling pathways. The recent advancements in whole genome biology have furthered our understanding of the interactions between these networks. As a result, numerous cell type-specific transcriptome profiles have been generated that have elucidated complex gene regulatory networks occurring at the cellular level, many of which were masked during whole-organ analysis. Modern technologies have also allowed researchers to generate multiple whole-organ metabolite profiles; however, only a limited number have been generated at the level of individual cells. Recent advancements in the isolation of individual cell populations have made cell type-specific metabolite profiles possible, enabling the enhanced detection and quantification of metabolites that were formerly unavailable when considering the whole organ. The comparison of metabolite and transcriptome profiles from the same cells has been a valuable resource to generate predictions regarding specific metabolite activity and function. In this review, we focus on recent studies that demonstrate the value of cell type-specific transcriptional profiles and their comparison with profiles generated from whole organs. Advancements in the isolation of single-cell populations will be highlighted, and the potential application towards generating detailed metabolic profiles will be discussed.

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.

Jasmonate signalling plays a central role in activating the plethora of responses that are elicited by herbivory. Solanum nigrum plants silenced in the expression of genes involved in jasmonic acid biosynthesis (irlox3), conjugation (irjar4) and perception (ircoi1) were used to study the function of these genes in the field and in the regulation of transcriptional and metabolic responses. In the field, damage from Noctuidea larvae was four- to fivefold higher on irlox3 and ircoi1 than on wild-type (WT) plants, whereas damage to irjar4 plants was similar to WT levels. Damage rates reflected plant survival rates; fewer irlox3 (78%) and ircoi1 (22%) plants survived compared with irjar4 and WT plants of which all plants survived. Gene expression profiling in leaves 3h after simulated herbivory revealed differential regulation of ∼700 genes in irlox3 and ircoi1 plants but of only six genes in irjar4 compared with WT plants. Surprisingly, transcriptional responses were not reflected in metabolomic responses; 48h after simulated herbivory, irjar4 plants showed a 50% overlap in their metabolic profile with ircoi1 plants. Together, these results reveal that SnJAR4 does not play a direct role in herbivore defence, but suggests that SnJAR4 is involved in responses other than those to herbivory. We assessed the role of jasmonyl-isoleucine (JA-Ile) in the non-model Solanum nigrum. Using transgenic plants silenced for genes in JA production (SnLOX3), conjugation of JA to JA-Ile (SnJAR4) and JA perception (SnCOI1), we show that JA-Ile signaling is not responsible for defense against Noctuidea herbivores in a natural environment. Transcriptional profiling shows that only a few genes are regulated by SnJAR4, in contrast to large-scale gene regulation by SnLOX3 and SnCOI1. However, an analysis of the metabolome over time following simulated herbivory, shows that a substantial number of ions are regulated by JA-Ile. Taken together, these results show that in contrast to other plant species, JA-Ile does not play a central role in activating S. nigrum's defenses, indicating that the fine-tuning of the JA-Ile-elicited herbivore response is a plant-specific process.

Variations in tissue development and spatial composition have a major impact on the nutritional and organoleptic qualities of ripe fleshy fruit, including melon (Cucumis melo). To gain a deeper insight into the mechanisms involved in these changes, we identified key metabolites for rational food quality design. The metabolome, volatiles and mineral elements were profiled employing an unprecedented range of complementary analytical technologies. Fruits were followed at a number of time points during the final ripening process and tissues were collected across the fruit flesh from rind to seed cavity. Approximately 2000 metabolite signatures and 15 mineral elements were determined in an assessment of temporal and spatial melon fruit development. This study design enabled the identification of: coregulated hubs (including aspartic acid, 2-isopropylmalic acid, β-carotene, phytoene and dihydropseudoionone) in metabolic association networks; global patterns of coordinated compositional changes; and links of primary and secondary metabolism to key mineral and volatile fruit complements. The results reveal the extent of metabolic interactions relevant to ripe fruit quality and thus have enabled the identification of essential candidate metabolites for the high-throughput screening of melon breeding populations for targeted breeding programmes aimed at nutrition and flavour improvement.

Plants synthesize a myriad of secondary metabolites (SMs) that are derived from central or primary metabolism. While these so-called natural products have been targets for plant metabolic engineering attempts for many years, the immense value of manipulating the interface between committed steps in secondary metabolism pathways and those in primary metabolism pathways has only recently emerged. In this review we discuss a few of the major issues that should be taken into consideration in attempts to engineer the primary to secondary metabolism interface. The availability of carbon, nitrogen and sulfur resources will have a major impact on the production of specific classes of primary metabolites (PMs) and consequently on the levels and composition of SMs derived from these PMs. Recent studies have shown that transcription factors associated with the synthesis of a given class of SMs coactivate the expression of genes encoding metabolic enzymes associated with primary pathways that supply precursors to these SMs. In addition, metabolic engineering approaches, which alter post-transcriptional feedback and feedforward regulatory mechanisms of the primary-secondary metabolism interface, have been highly fruitful in Taylormade enhancements of the content of specific beneficial SMs. Lastly, the evolution of pathways of secondary metabolism from pathways of primary metabolism highlights the need to consider cases in which common enzymatic reactions and pathways take place between the two. Taken together, the available information indicates a supercoordinated gene expression networks connecting primary and secondary metabolism in plants, which should be taken into consideration in future attempts to metabolically engineer the various classes of plant SMs.

Benzoxazinoids are metabolites occurring in a restricted group of plant species including crops such as rye, wheat, and maize. Focus on the analysis of benzoxazinoid metabolites has typically been due to their importance to plant biochemistry and physiology as highly bioactive molecules that plants use as alleochemicals to defend themselves against predators and infections. However, the potential dietary contribution of these compounds has not been addressed. This study conducted a detailed qualitative characterization of benzoxazinoid metabolites present in the whole grain rye and processed fractions of rye bran, and their presence was also detected in whole grain wheat samples. Several novel benzoxazinoid metabolites of the hydroxamic acids (2,4-dihydroxy-1,4- benzoxazin-3-one, DIBOA; 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one, DIMBOA), lactams (2-hydroxy-1,4-benzoxazin-3-one, HBOA), and benzoxazolinones (1,3-benzoxazol-2-one, BOA) were identified, including double-hexose derivatives of DIBOA, DIMBOA, and HBOA. This paper presents an important addition to the information on the phytochemical composition of rye and wheat grains, which deserves attention in the discussion of the potential health-promoting effects of these grains.

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.

Brunfelsia calycina flowers change colour from purple to white due to anthocyanin degradation, parallel to an increase in fragrance and petal size. Here it was tested whether the production of the fragrant benzenoids is dependent on induction of the shikimate pathway, or if they are formed from the anthocyanin degradation products. An extensive characterization of the events taking place in Brunfelsia flowers is presented. Anthocyanin characterization was performed using ultraperfomance liquid chromatography-quadrupole time of flight-tandem mass specrometry (UPLC-QTOF-MS/MS). Volatiles emitted were identified by headspace solid phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS). Accumulated proteins were identified by 2D gel electrophoresis. Transcription profiles were characterized by cross-species hybridization of Brunfelsia cDNAs to potato cDNA microarrays. Identification of accumulated metabolites was performed by UPLC-QTOF-MS non-targeted metabolite analysis. The results include characterization of the nine main anthocyanins in Brunfelsia flowers. In addition, 146 up-regulated genes, 19 volatiles, seven proteins, and 17 metabolites that increased during anthocyanin degradation were identified. A multilevel analysis suggests induction of the shikimate pathway. This pathway is the most probable source of the phenolic acids, which in turn are precursors of both the benzenoid and lignin production pathways. The knowledge obtained is valuable for future studies on degradation of anthocyanins, formation of volatiles, and the network of secondary metabolism in Brunfelsia and related species.

After fertilization, the expanding carpel of fleshy fruit goes through a phase change to ripening. Although the role of ethylene signalling in mediating climacteric ripening has been established, knowledge regarding the regulation of ethylene biosynthesis and its association with fruit developmental programs is still lacking. A functional screen of tomato transcription factors showed that silencing of the TOMATO AGAMOUS-LIKE 1 (TAGL1) MADS box gene results in altered fruit pigmentation. Over-expressing TAGL1 as a chimeric repressor suggested a role in controlling ripening, as transgenic tomato fruit showed reduced carotenoid and ethylene levels, suppressed chlorophyll breakdown, and down-regulation of ripening-associated genes. Moreover, fruits over-expressing TAGL1 accumulated more lycopene, and their sepals were swollen, accumulated high levels of the yellow flavonoid naringenin chalcone and contained lycopene. Transient promoter-binding assays indicated that part of the TAGL1 activity in ripening is executed through direct activation of ACS2, an ethylene biosynthesis gene that has recently been reported to be a target of the RIN MADS box factor. Examination of the TAGL1 transcript and its over-expression in the rin mutant background suggested that RIN does not regulate TAGL1 or vice versa. The results also indicated RIN-dependent and-independent processes that are regulated by TAGL1. We also noted that fruit of TAGL1 loss-of-function lines had a thin pericarp layer, indicating an additional role for TAGL1 in carpel expansion prior to ripening. The results add a new component to the current model of the regulatory network that controls fleshy fruit ripening and its association with the ethylene biosynthesis pathway.

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.

Metabolomics aims at the efficient determination of multiple chemical constituents present in a tissue, a cell layer or ideally a single cell. Metabolomics is currently applied in a large number of life science disciplines. Nuclear Magnetic Resonance (NMR) or Gas- and Liquid-Chromatography coupled to Mass Spectrometry (GC-MS and LC-MS) are the most widespread technologies employed in metabolomics assays. Soft fruit, one of the most metabolite-rich plant organs, was from the first to be subjected to metabolomics investigation. The interest in metabolite profiling of soft fruit could be explained by the large repertoire of metabolites belonging to diverse chemical classes that are formed and catabolized during fruit development, starting from the fertilized ovary up to the ripe, mature fruit. Moreover, fruit constitute an essential part of our diet and the breeding to achieve nutrient-rich varieties entails a comprehensive analysis of their metabolite content. Presently, hundreds of substances, including primary and secondary (or specialized) metabolites have been detected in fruit. Metabolomics has been employed also for following metabolism in transgenic plants, mutants and introgression lines populations. The latter experiments allowed the identification of genomic regions associated with metabolic quality traits. So far, most metabolomics assays in fruit have been focused on two species, namely, tomato and strawberry. It is expected that in the following years the use of metabolomics will be expanded to the investigation of numerous other fruit species.

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

Riboswitches are natural RNA sensors that affect post-transcriptional processes via their capacity to bind small molecules. To date, these mRNA structures have been shown to regulate the biosynthesis of essential metabolites, including vitamins and amino acids. Although bacterial riboswitches are widespread and characterized, only a single eukaryotic, thiamin-pyrophosphate-binding riboswitch has recently been discovered to direct gene expression by regulating mRNA splicing in fungi, green algae and land plants. It is unclear how widespread riboswitches are and what additional roles they have in eukaryotes. When engineered in plants, riboswitches can function autonomously to modulate gene expression. These discoveries not only trigger novel findings regarding RNA switches in plants, but also spur the exploitation of riboswitches for monitoring metabolite concentrations in planta.

Formation of flower organs and the subsequent pollination process require a coordinated spatial and temporal regulation of particular metabolic pathways. In this study a comparison has been made between the metabolite composition of individual flower organs of strawberry (Fragaria × ananassa) including the petal, sepal, stamen, pistil and the receptacle that gives rise to the strawberry fruit. Non-targeted metabolomics analysis of the semi-polar secondary metabolites by the use of UPLC-qTOF-MS was utilized in order to localize metabolites belonging to various chemical classes (e.g. ellagitannins, proanthocyanidins, flavonols, terpenoids, and spermidine derivatives) to the different flower organs. The vast majority of the tentatively identified metabolites were ellagitannins that accumulated in all five parts of the flower. Several metabolite classes were detected predominantly in certain flower organs, as for example spermidine derivatives were present uniquely in the stamen and pistil, and the proanthocyanidins were almost exclusively detected in the receptacle and sepals. The latter organ was also rich in terpenoids (i.e. triterpenoid and sesquiterpenoid derivatives) whereas phenolic acids and flavonols were the predominant classes of compounds detected in the petals. Furthermore, we observed extensive variation in the accumulation of metabolites from the same class in a single organ, particularly in the case of ellagitannins, and the flavonols quercetin, kaempferol and isorhamnetin. These results allude to spatially-restricted production of secondary metabolite classes and specialized derivatives in flowers that take part in implementing the unique program of individual organs in the floral life cycle.

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.

Metabolic engineering of terpenoids in plants is a fascinating research topic from two main perspectives. On the one hand, the various biological activities of these compounds make their engineering a new tool for improving a considerable number of traits in crops. These include for example enhanced disease resistance, weed control by producing allelopathic compounds, better pest management, production of medicinal compounds, increased value of ornamentals and fruit and improved pollination. On the other hand, the same plants altered in the profile of terpenoids and their precursor pools make a most important contribution to fundamental studies on terpenoid biosynthesis and its regulation. In this review we describe our recent results with terpenoid engineering, focusing on two terpenoid classes the monoterpenoids and sesquiterpenoids. The emerging picture is that engineering of these compounds and their derivatives in plant cells is feasible, although with some requirements and limitations. For example, in terpenoid engineering experiments crucial factors are the subcellular localisation of both the precursor pool and the introduced enzymes, the activity of endogenous plant enzymes which modify the introduced terpenoid skeleton, the costs of engineering in terms of effects on other pathways sharing the same precursor pool and the phytotoxicity of the introduced terpenoids. Finally, we will show that transgenic plants altered in their terpenoid profile exert novel biological activities on their environment, for example influencing insect behaviour.

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.