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

The aim of this work was to investigate the effect of decreased cytosolic phosphoenolpyruvate carboxykinase (PEPCK) and plastidic NADP-dependent malic enzyme (NADP-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 in 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 transformats, which suggest that is due to an altered cellular redox status. Moreover, metabolic profiling and feeding experiments involving positional labelled glucoses of fruits lacking in plastidic NADP-malic enzyme 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 excess of oxaloacetate OAA is converted to aspartate and reintroduced in the TCA via 2-oxoglutarate/glutamate. On the other hand, the plastidic NADP-malic enzyme antisense lines were characterized by no changes in respiration rates and TCA cycle flux and together with an increase of pyruvate kinase and phosphoenolpyruvate carboxylase activities indicates that pyruvate is supply 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

Integrated metabolomics identifies CYP72A67 and CYP72A68 oxidases in the biosynthesis of Medicago truncatula oleanate sapogenins

Introduction: Triterpene saponins are important bioactive plant natural products found in many plant families including the Leguminosae. Objectives: We characterize two Medicago truncatula cytochrome P450 enzymes, MtCYP72A67 and MtCYP72A68, involved in saponin biosynthesis including both in vitro and in planta evidence. Methods: UHPLC-(-)ESI-QToF-MS was used to profile saponin accumulation across a collection of 106 M. truncatula ecotypes. The profiling results identified numerous ecotypes with high and low saponin accumulation in root and aerial tissues. Four ecotypes with significant differential saponin content in the root and/or aerial tissues were selected, and correlated gene expression profiling was performed. Results: Correlation analyses between gene expression and saponin accumulation revealed high correlations between saponin content with gene expression of -amyrin synthase, MtCYP716A12, and two cytochromes P450 genes, MtCYP72A67 and MtCYP72A68. In vivo and in vitro biochemical assays using yeast microsomes containing MtCYP72A67 revealed hydroxylase activity for carbon 2 of oleanolic acid and hederagenin. This finding was supported by functional characterization of MtCYP72A67 using RNAi-mediated gene silencing in M. truncatula hairy roots, which revealed a significant reduction of 2-hydroxylated sapogenins. In vivo and in vitro assays with MtCYP72A68 produced in yeast showed multifunctional oxidase activity for carbon 23 of oleanolic acid and hederagenin. These findings were supported by overexpression of MtCYP72A68 in M. truncatula hairy roots, which revealed significant increases of oleanolic acid, 2-hydroxyoleanolic acid, hederagenin and total saponin levels. Conclusions: The cumulative data support that MtCYP72A68 is a multisubstrate, multifunctional oxidase and MtCYP72A67 is a 2-hydroxylase, both of which function during the early steps of triterpene-oleanate sapogenin biosynthesis

Multi-level engineering facilitates the production of phenylpropanoid compounds in tomato

Phenylpropanoids comprise an important class of plant secondary metabolites. A number of transcription factors have been used to upregulate-specific branches of phenylpropanoid metabolism, but by far the most effective has been the fruit-specific expression of AtMYB12 in tomato, which resulted in as much as 10% of fruit dry weight accumulating as flavonols and hydroxycinnamates. We show that AtMYB12 not only increases the demand of flavonoid biosynthesis but also increases the supply of carbon from primary metabolism, energy and reducing power, which may fuel the shikimate and phenylalanine biosynthetic pathways to supply more aromatic amino acids for secondary metabolism. AtMYB12 directly binds promoters of genes encoding enzymes of primary metabolism. The enhanced supply of precursors, energy and reducing power achieved by AtMYB12 expression can be harnessed to engineer high levels of novel phenylpropanoids in tomato fruit, offering an effective production system for bioactives and other high value ingredients

Brevicoryne brassicae aphids interfere with transcriptome responses of Arabidopsis thaliana to feeding by Plutella xylostella caterpillars in a density‑dependent manner

Plants are commonly attacked by multiple herbivorous species. Yet, little is known about transcriptional patterns underlying plant responses to multiple insect attackers feeding simultaneously. Here, we assessed= transcriptomic responses of Arabidopsis thaliana plants to simultaneous feeding by Plutella xylostella caterpillars and Brevicoryne brassicae aphids in comparison to plants infested by P. xylostella caterpillars alone, using microarray analysis. We particularly investigated how aphid feeding interferes with the transcriptomic response to P. xylostella caterpillars and whether this interference is dependent on aphid density and time since aphid attack. Various JA-responsive genes were up-regulated in response to feeding by P. xylostella caterpillars. The additional presence of aphids, both at low and high densities, clearly affected the transcriptional plant response to caterpillars. Interestingly, some important modulators of plant defense signalling, including WRKY transcription factor genes and ABA-dependent genes, were differentially induced in response to simultaneous aphid feeding at low or high density compared with responses to P. xylostella caterpillars feeding alone. Furthermore, aphids affected the P. xylostella-induced transcriptomic response in a density dependent manner, which caused an acceleration in plant response against dual insect attack at high aphid density compared to dual insect attack at low aphid density. In conclusion, our study provides evidence that aphids influence the caterpillar-induced transcriptional response of A. thaliana in a density-dependent manner. It highlights the importance of addressing insect density to understand how plant responses to single attackers interfere with responses to other attackers and thus underlines the importance of the dynamics of transcriptional plant responses to multiple herbivory