Vered Tzin

New Insights into the Shikimate and Aromatic Amino Acids Biosynthesis Pathways in Plants

The aromatic amino acids phenylalanine, tyrosine, and tryptophan in plants are not only essential components of protein synthesis, but also serve as precursors for a wide range of secondary metabolites that are important for plant growth as well as for human nutrition and health. The aromatic amino acids are synthesized via the shikimate pathway followed by the branched aromatic amino acids biosynthesis pathway, with chorismate serving as a major intermediate branch point metabolite. Yet, the regulation and coordination of synthesis of these amino acids are still far from being understood. Recent studies on these pathways identified a number of alternative cross-regulated biosynthesis routes with unique evolutionary origins. Although the major route of Phe and Tyr biosynthesis in plants occurs via the intermediate metabolite arogenate, recent studies suggest that plants can also synthesize phenylalanine via the intermediate metabolite phenylpyruvate (PPY), similarly to many microorganisms. Recent studies also identified a number of transcription factors regulating the expression of genes encoding enzymes of the shikimate and aromatic amino acids pathways as well as of multiple secondary metabolites derived from them in Arabidopsis and in other plant species.

Regulation of primary plant metabolism during plant-pathogen interactions and its contribution to plant defense

Plants are constantly exposed to microorganisms in the environment and, as a result, have evolved intricate mechanisms to recognize and defend themselves against potential pathogens. One of these responses is the downregulation of photosynthesis and other processes associated with primary metabolism that are essential for plant growth. It has been suggested that the energy saved by downregulation of primary metabolism is diverted and used for defense responses. However, several studies have shown that upregulation of primary metabolism also occurs during plant-pathogen interactions. We propose that upregulation of primary metabolism modulates signal transduction cascades that lead to plant defense responses. In support of this thought, we here compile evidence from the literature to show that upon exposure to pathogens or elicitors, plants induce several genes associated with primary metabolic pathways, such as those involved in the synthesis or degradation of carbohydrates, amino acids and lipids. In addition, genetic studies have confirmed the involvement of these metabolic pathways in plant defense responses. This review provides a new perspective highlighting the relevance of primary metabolism in regulating plant defense against pathogens with the hope to stimulate further research in this area.

The Biosynthetic Pathways for Shikimate and Aromatic Amino Acids in Arabidopsis thaliana

The aromatic amino acids phenylalanine, tyrosine and tryptophan in plants are not only essential components of protein synthesis, but also serve as precursors for a wide range of secondary metabolites that are important for plant growth as well as for human nutrition and health. The aromatic amino acids are synthesized via the shikimate pathway followed by the branched aromatic amino acid metabolic pathway, with chorismate serving as a major branch point intermediate metabolite. Yet, the regulation of their synthesis is still far from being understood. So far, only three enzymes in this pathway, namely, chorismate mutase of phenylalanine and tyrosine synthesis, tryptophan synthase of tryptophan biosynthesis and arogenate dehydratase of phenylalanine biosynthesis, proved experimentally to be allosterically regulated. The major biosynthesis route of phenylalanine in plants occurs via arogenate. Yet, recent studies suggest that an alternative route of phynylalanine biosynthesis via phenylpyruvate may also exist in plants, similarly to many microorganisms. Several transcription factors regulating the expression of genes encoding enzymes of both the shikimate pathway and aromatic amino acid metabolism have also been recently identified in Arabidopsis and other plant species.

Coordinated Gene Networks Regulating Arabidopsis Plant Metabolism in Response to Various Stresses and Nutritional Cues

This work introduces a bioinformatics approach that identifies positive and negative coordination of gene expression between sets of genes, or entire gene networks, in response to environmental or developmental cues. The approach is illustrated by a case study that identifies distinct expression behavior of the energy-associated gene network in response to different biotic and abiotic stresses. The expression pattern of any pair of genes may be negatively correlated, positively correlated, or not correlated at all in response to different stresses and even different progression stages of the stress. This makes it difficult to identify such relationships by classical statistical tools such as the Pearson correlation coefficient. Hence, dedicated bioinformatics approaches that are able to identify groups of cues in which there is a positive or negative expression correlation between pairs or groups of genes are called for. We herein introduce and discuss a bioinformatics approach, termed Gene Coordination, that is devoted to the identification of specific or multiple cues in which there is a positive or negative coordination between pairs of genes and can further incorporate additional coordinated genes to form large coordinated gene networks. We demonstrate the utility of this approach by providing a case study in which we were able to discover distinct expression behavior of the energy-associated gene network in response to distinct biotic and abiotic stresses. This bioinformatics approach is suitable to a broad range of studies that compare treatments versus controls, such as effects of various cues, or expression changes between a mutant and the control wild-type genotype.

Expression of a bacterial bi‐functional chorismate mutase/prephenate dehydratase modulates primary and secondary metabolism associated with aromatic amino acids in Arabidopsis

Plants can synthesize the aromatic amino acid Phe via arogenate, but it is still not known whether they also use an alternative route for Phe biosynthesis via phenylpyruvate, like many micro-organisms. To examine this possibility, we expressed a bacterial bi-functional PheA (chorismate mutase/prephenate dehydratase) gene in Arabidopsis thaliana that converts chorismate via prephenate into phenylpyruvate. The PheA-expressing plants showed a large increase in the level of Phe, implying that they can convert phenylpyruvate into Phe. In addition, PheA expression rendered the plants more sensitive than wild-type plants to the Trp biosynthesis inhibitor 5-methyl-Trp, implying that Phe biosynthesis competes with Trp biosynthesis from their common precursor chorismate. Surprisingly, GC-MS, LC-MS and microarray analyses showed that this increase in Phe accumulation only had a very minor effect on the levels of other primary metabolites as well as on the transcriptome profile, implying little regulatory cross-interaction between the aromatic amino acid biosynthesis network and the bulk of the Arabidopsis transcriptome and primary metabolism. However, the levels of a number of secondary metabolites derived from all three aromatic amino acids (Phe, Trp and Tyr) were altered in the PheA plants, implying regulatory cross-interactions between the flux of aromatic amino acid biosynthesis from chorismate and their further metabolism into various secondary metabolites. Taken together, our results provide insights into the regulatory mechanisms of aromatic amino acid biosynthesis and their interaction with central primary metabolism, as well as the regulatory interface between primary and secondary metabolism.

Alteration of Plant Primary Metabolism in Response to Insect Herbivory

The species and feeding habits of insect herbivores influence the changes in photosynthesis, carbon allocation, and nitrogen utilization that are observed in host plants. Plants in nature, which are continuously challenged by diverse insect herbivores, produce constitutive and inducible defenses to reduce insect damage and preserve their own fitness. In addition to inducing pathways that are directly responsible for the production of toxic and deterrent compounds, insect herbivory causes numerous changes in plant primary metabolism. Whereas the functions of defensive metabolites such as alkaloids, terpenes, and glucosinolates have been studied extensively, the fitness benefits of changes in photosynthesis, carbon transport, and nitrogen allocation remain less well understood. Adding to the complexity of the observed responses, the feeding habits of different insect herbivores can significantly influence the induced changes in plant primary metabolism. In this review, we summarize experimental data addressing the significance of insect feeding habits, as related to herbivore-induced changes in plant primary metabolism. Where possible, we link these physiological changes with current understanding of their underlying molecular mechanisms. Finally, we discuss the potential fitness benefits that host plants receive from altering their primary metabolism in response to insect herbivory.

Dynamic Maize Responses to Aphid Feeding Are Revealed by a Time Series of Transcriptomic and Metabolomic Assays

A transcriptomic and metabolomic profiling time course of maize foliar responses to aphid feeding identifies genes for the synthesis of benzoxazinoids, terpenes, and other induced defense metabolites. As a response to insect attack, maize (Zea mays) has inducible defenses that involve large changes in gene expression and metabolism. Piercing/sucking insects such as corn leaf aphid (Rhopalosiphum maidis) cause direct damage by acquiring phloem nutrients as well as indirect damage through the transmission of plant viruses. To elucidate the metabolic processes and gene expression changes involved in maize responses to aphid attack, leaves of inbred line B73 were infested with corn leaf aphids for 2 to 96 h. Analysis of infested maize leaves showed two distinct response phases, with the most significant transcriptional and metabolic changes occurring in the first few hours after the initiation of aphid feeding. After 4 d, both gene expression and metabolite profiles of aphid-infested maize reverted to being more similar to those of control plants. Although there was a predominant effect of salicylic acid regulation, gene expression changes also indicated prolonged induction of oxylipins, although not necessarily jasmonic acid, in aphid-infested maize. The role of specific metabolic pathways was confirmed using Dissociator transposon insertions in maize inbred line W22. Mutations in three benzoxazinoid biosynthesis genes, Bx1, Bx2, and Bx6, increased aphid reproduction. In contrast, progeny production was greatly decreased by a transposon insertion in the single W22 homolog of the previously uncharacterized B73 terpene synthases TPS2 and TPS3. Together, these results show that maize leaves shift to implementation of physical and chemical defenses within hours after the initiation of aphid feeding and that the production of specific metabolites can have major effects in maize-aphid interactions.

Expression of a bacterial feedback‐insensitive 3‐deoxy‐d‐arabino‐heptulosonate 7‐phosphate synthase of the shikimate pathway in Arabidopsis elucidates potential metabolic bottlenecks between primary and secondary metabolism

The shikimate pathway of plants mediates the conversion of primary carbon metabolites via chorismate into the three aromatic amino acids and to numerous secondary metabolites derived from them. However, the regulation of the shikimate pathway is still far from being understood. We hypothesized that 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAHPS) is a key enzyme regulating flux through the shikimate pathway. To test this hypothesis, we expressed a mutant bacterial AroG gene encoding a feedback-insensitive DAHPS in transgenic Arabidopsis plants. The plants were subjected to detailed analysis of primary metabolism, using GC-MS, as well as secondary metabolism, using LC-MS. Our results exposed a major effect of bacterial AroG expression on the levels of shikimate intermediate metabolites, phenylalanine, tryptophan and broad classes of secondary metabolite, such as phenylpropanoids, glucosinolates, auxin and other hormone conjugates. We propose that DAHPS is a key regulatory enzyme of the shikimate pathway. Moreover, our results shed light on additional potential metabolic bottlenecks bridging plant primary and secondary metabolism.

Alteration of the Interconversion of Pyruvate and Malate in the Plastid or Cytosol of Ripening Tomato Fruit Invokes Diverse Consequences on Sugar But Similar Effects on Cellular Organic Acid, Metabolism, and Transitory Starch Accumulation

Summary: Normal tomato ripening is influenced by alterations on both cytosolic phosphoenolpyruvate carboxykinase and plastidic NADP-malic enzyme. This study provides compelling evidence of their roles in starch biosynthesis, respiration rates, and tricarboxylic acid cycle flux. The aim of this work was to investigate the effect of decreased cytosolic phosphoenolpyruvate carboxykinase (PEPCK) and plastidic NADP-dependent malic enzyme (ME) on tomato (Solanum lycopersicum) ripening. Transgenic tomato plants with strongly reduced levels of PEPCK and plastidic NADP-ME were generated by RNA interference gene silencing under the control of a ripening-specific E8 promoter. While these genetic modifications had relatively little effect on the total fruit yield and size, they had strong effects on fruit metabolism. Both transformants were characterized by lower levels of starch at breaker stage. Analysis of the activation state of ADP-glucose pyrophosphorylase correlated with the decrease of starch in both transformants, which suggests that it is due to an altered cellular redox status. Moreover, metabolic profiling and feeding experiments involving positionally labeled glucoses of fruits lacking in plastidic NADP-ME and cytosolic PEPCK activities revealed differential changes in overall respiration rates and tricarboxylic acid (TCA) cycle flux. Inactivation of cytosolic PEPCK affected the respiration rate, which suggests that an excess of oxaloacetate is converted to aspartate and reintroduced in the TCA cycle via 2-oxoglutarate/glutamate. On the other hand, the plastidic NADP-ME antisense lines were characterized by no changes in respiration rates and TCA cycle flux, which together with increases of pyruvate kinase and phosphoenolpyruvate carboxylase activities indicate that pyruvate is supplied through these enzymes to the TCA cycle. These results are discussed in the context of current models of the importance of malate during tomato fruit ripening.

A Global Coexpression Network Approach for Connecting Genes to Specialized Metabolic Pathways in Plants

Global gene coexpression network analysis is a powerful approach for identifying known and novel plant-specialized metabolic pathways, irrespective of the genomic location of the constitutive genes. Plants produce diverse specialized metabolites (SMs), but the genes responsible for their production and regulation remain largely unknown, hindering efforts to tap plant pharmacopeia. Given that genes comprising SM pathways exhibit environmentally dependent coregulation, we hypothesized that genes within a SM pathway would form tight associations (modules) with each other in coexpression networks, facilitating their identification. To evaluate this hypothesis, we used 10 global coexpression data sets, each a meta-analysis of hundreds to thousands of experiments, across eight plant species to identify hundreds of coexpressed gene modules per data set. In support of our hypothesis, 15.3 to 52.6% of modules contained two or more known SM biosynthetic genes, and module genes were enriched in SM functions. Moreover, modules recovered many experimentally validated SM pathways, including all six known to form biosynthetic gene clusters (BGCs). In contrast, bioinformatically predicted BGCs (i.e., those lacking an associated metabolite) were no more coexpressed than the null distribution for neighboring genes. These results suggest that most predicted plant BGCs are not genuine SM pathways and argue that BGCs are not a hallmark of plant specialized metabolism. We submit that global gene coexpression is a rich, largely untapped resource for discovering the genetic basis and architecture of plant natural products.