Carbon balance and source‐sink metabolic changes in winter wheat exposed to high night‐time temperature

Carbon loss under high night‐time temperature (HNT) leads to significant reduction in wheat yield. Growth chamber studies were carried out using six winter wheat genotypes, to unravel postheading HNT (23°C)–induced alterations in carbon balance, source‐sink metabolic changes, yield, and yield‐related traits compared with control (15°C) conditions. Four of the six tested genotypes recorded a significant increase in night respiration after 4 days of HNT exposure, with all the cultivars regulating carbon loss and demonstrating different degree of acclimation to extended HNT exposure. Metabolite profiling indicated carbohydrate metabolism in spikes and activation of the TriCarboxylic Acid (TCA) cycle in leaves as important pathways operating under HNT exposure. A significant increase in sugars, sugar‐alcohols, and phosphate in spikes of the tolerant genotype (Tascosa) indicated osmolytes and membrane protective mechanisms acting against HNT damage. Enhanced night respiration under HNT resulted in higher accumulation of TCA cycle intermediates like isocitrate and fumarate in leaves of the susceptible genotype (TX86A5606). Lower grain number due to lesser productive spikes and reduced grain weight due to shorter grain‐filling duration determined HNT‐induced yield loss in winter wheat. Traits and mechanisms identified will help catalyze the development of physiological and metabolic markers for breeding HNT‐tolerant wheat.     

Using the knowns to discover the unknowns: MS‐based dereplication uncovers structural diversity in 17‐hydroxygeranyllinalool diterpene glycoside production in the Solanaceae

Exploring the diversity of plant secondary metabolism requires efficient methods to obtain sufficient structural insights to discriminate previously known from unknown metabolites. De novo structure elucidation and confirmation of known metabolites (dereplication) remain a major bottleneck for mass spectrometry‐based metabolomic workflows, and few systematic dereplication strategies have been developed for the analysis of entire compound classes across plant families, partly due to the complexity of plant metabolic profiles that complicates cross‐species comparisons. 17‐hydroxygeranyllinalool diterpene glycosides (HGL‐DTGs) are abundant defensive secondary metabolites whose malonyl and glycosyl decorations are induced by jasmonate signaling in the ecological model plant Nicotiana attenuata. The multiple labile glycosidic bonds of HGL‐DTGs result in extensive in‐source fragmentation (IS‐CID) during ionization. To reconstruct these IS‐CID clusters from profiling data and identify precursor ions, we applied a deconvolution algorithm and created an MS/MS library from positive‐ion spectra of purified HGL‐DTGs. From this library, 251 non‐redundant fragments were annotated, and a workflow to characterize leaf, flower and fruit extracts of 35 solanaceous species was established. These analyses predicted 105 novel HGL‐DTGs that were restricted to Nicotiana, Capsicum and Lycium species. Interestingly, malonylation is a highly conserved step in HGL‐DTG metabolism, but is differentially affected by jasmonate signaling among Nicotiana species. This MS‐based workflow is readily applicable for cross‐species re‐identification/annotation of other compound classes with sufficient fragmentation knowledge, and therefore has the potential to support hypotheses regarding secondary metabolism diversification.     

The effects of season and water availability on chemical composition,secondary metabolites and biological activity in plants

Plants react towards changes in their environment, which can be a result of biotic or abiotic activities. Numerous studies have investigated the effects of abiotic stress on plants, and how it affects the primary as well as secondary metabolism. Generally it is accepted that plants react to environmental stress by increasing secondary metabolites. This is however a very broad and simplified explanation and often inaccurate. Various examples are provided where plants react positively, and often negatively towards seasonal variation and water availability, resulting in a lowering of certain secondary metabolites concentration, while others are increased. Furthermore species differences, cultivars and interaction of other environmental factors such as temperature complicates a simple conclusion from the effect of stress on plants. The differential expression of genes in different species and in different metabolic pathways ensures a complex and very specific reaction of a plant to environmental stress. Overall the paper provides support for a complex and intricate response system which differs for each plant species, and could be explained by understanding and studying the different metabolic pathways responsible for secondary metabolite production.     

Targeted metabolite and transcript profiling for elucidating enzyme function: isolation of novel N‐methyltransferases from three benzylisoquinoline alkaloid‐producing species

An integrated approach using targeted metabolite profiles and modest EST libraries each containing approximately 3500 unigenes was developed in order to discover and functionally characterize novel genes involved in plant‐specialized metabolism. EST databases have been established for benzylisoquinoline alkaloid‐producing cell cultures of Eschscholzia californica, Papaver bracteatum and Thalictrum flavum, and are a rich repository of alkaloid biosynthetic genes. ESI‐FTICR‐MS and ESI‐MS/MS analyses facilitated unambiguous identification and relative quantification of the alkaloids in each system. Manual integration of known and candidate biosynthetic genes in each EST library with benzylisoquinoline alkaloid biosynthetic networks assembled from empirical metabolite profiles allowed identification and functional characterization of four N‐methyltransferases (NMTs). One cDNA from T. flavum encoded pavine N‐methyltransferase (TfPavNMT), which showed a unique preference for (±)‐pavine and represents the first isolated enzyme involved in the pavine alkaloid branch pathway. Correlation of the occurrence of specific alkaloids, the complement of ESTs encoding known benzylisoquinoline alkaloid biosynthetic genes and the differential substrate range of characterized NMTs demonstrated the feasibility of bilaterally predicting enzyme function and species‐dependent specialized metabolite profiles.     

Post‐flowering night respiration and altered sink activity account for high night temperature‐induced grain yield and quality loss in rice (Oryza sativa L.)

High night temperature (HNT) is a major constraint to sustaining global rice production under future climate. Physiological and biochemical mechanisms were elucidated for HNT‐induced grain yield and quality loss in rice. Contrasting rice cultivars (N22, tolerant; Gharib, susceptible; IR64, high yielding with superior grain quality) were tested under control (23°C) and HNT (29°C) using unique field‐based tents from panicle initiation till physiological maturity. HNT affected 1000 grain weight, grain yield, grain chalk and amylose content in Gharib and IR64. HNT increased night respiration (Rn) accounted for higher carbon losses during post‐flowering phase. Gharib and IR64 recorded 16 and 9% yield reduction with a 63 and 35% increase in average post‐flowering Rn under HNT, respectively. HNT altered sugar accumulation in the rachis and spikelets across the cultivars with Gharib and IR64 recording higher sugar accumulation in the rachis. HNT reduced panicle starch content in Gharib (22%) and IR64 (11%) at physiological maturity, but not in the tolerant N22. At the enzymatic level, HNT reduced sink strength with lower cell wall invertase and sucrose synthase activity in Gharib and IR64, which affected starch accumulation in the developing grain, thereby reducing grain weight and quality. Interestingly, N22 recorded lower Rn‐mediated carbon losses and minimum impact on sink strength under HNT. Mechanistic responses identified will facilitate crop models to precisely estimate HNT‐induced damage under future warming scenarios.     

Expression profiling of metabolic genes in response to methyl jasmonate reveals regulation of genes of primary and secondary sulfur-related pathways in Arabidopsis thaliana

The treatment of Arabidopsis thaliana with methyl jasmonate was used to investigate the reaction of 2467 selected genes of primary and secondary metabolism by macroarray hybridization. Hierarchical cluster analysis allowed distinctions to be made between diurnally and methyl jasmonate regulated genes in a time course from 30 min to 24 h. 97 and 64 genes were identified that were up- or down-regulated more than 2–fold by methyl jasmonate, respectively. These genes belong to 18 functional categories of which sulfur-related genes were by far strongest affected. Gene expression and metabolite patterns of sulfur metabolism were analysed in detail, since numerous defense compounds contain oxidized or reduced sulfur. Genes encoding key reactions of sulfate reduction as well as of cysteine, methionine and glutathione synthesis were rapidly up-regulated, but none of the known sulfur-deficiency induced sulfate transporter genes. In addition, increased expression of genes of sulfur-rich defense proteins and of enzymes involved in glucosinolate metabolism was observed. In contrast, profiling of primary and secondary sulfur metabolites revealed only an increase in the indole glucosinolate glucobrassicin upon methyl jasmonate treatment. The observed rapid mRNA changes were thus regulated by a signal independent of the known sulfur deficiency response. These results document for the first time how comprehensively the regulation of sulfur-related genes and plant defense are connected. This interaction is discussed as a new approach to differentiate between supply- and demand-driven regulation of the sulfate assimilation pathway.     

A bell pepper cultivar tolerant to chilling enhanced nitrogen allocation and stress‐related metabolite accumulation in the roots in response to low root‐zone temperature

Two bell pepper (Capsicum annuum) cultivars, differing in their response to chilling, were exposed to three levels of root‐zone temperatures. Gas exchange, shoot and root phenology, and the pattern of change of the central metabolites and secondary metabolites caffeate and benzoate in the leaves and roots were profiled. Low root‐zone temperature significantly inhibited gaseous exchange, with a greater effect on the sensitive commercial pepper hybrid (Canon) than on the new hybrid bred to enhance abiotic stress tolerance (S103). The latter was less affected by the treatment with respect to plant height, shoot dry mass, root maximum length, root projected area, number of root tips and root dry mass. More carbon was allocated to the leaves of S103 than nitrogen at 17°C, while in the roots at 17°C, more nitrogen was allocated and the ratio between C/N decreased. Metabolite profiling showed greater increase in the root than in the leaves. Leaf response between the two cultivars differed significantly. The roots accumulated stress‐related metabolites including γ‐aminobutyric acid (GABA), proline, galactinol and raffinose and at chilling (7°C) resulted in an increase of sugars in both cultivars. Our results suggest that the enhanced tolerance of S103 to root cold stress, reflected in the relative maintenance of shoot and root growth, is likely linked to a more effective regulation of photosynthesis facilitated by the induction of stress‐related metabolism.     

Transcript profiling of jasmonate‐elicited Taxus cells reveals a β‐phenylalanine‐CoA ligase

Plant cell cultures constitute eco‐friendly biotechnological platforms for the production of plant secondary metabolites with pharmacological activities, as well as a suitable system for extending our knowledge of secondary metabolism. Despite the high added value of taxol and the importance of taxanes as anticancer compounds, several aspects of their biosynthesis remain unknown. In this work, a genomewide expression analysis of jasmonate‐elicited Taxus baccata cell cultures by complementary DNA‐amplified fragment length polymorphism (cDNA‐AFLP) indicated a correlation between an extensive elicitor‐induced genetic reprogramming and increased taxane production in the targeted cultures. Subsequent in silico analysis allowed us to identify 15 genes with a jasmonate‐induced differential expression as putative candidates for genes encoding enzymes involved in five unknown steps of taxane biosynthesis. Among them, the TB768 gene showed a strong homology, including a very similar predicted 3D structure, with other genes previously reported to encode acyl‐CoA ligases, thus suggesting a role in the formation of the taxol lateral chain. Functional analysis confirmed that the TB768 gene encodes an acyl‐CoA ligase that localizes to the cytoplasm and is able to convert β‐phenylalanine, as well as coumaric acid, into their respective derivative CoA esters. β‐phenylalanyl‐CoA is attached to baccatin III in one of the last steps of the taxol biosynthetic pathway. The identification of this gene will contribute to the establishment of sustainable taxol production systems through metabolic engineering or synthetic biology approaches.     

Tissue‐specific profiling of the Arabidopsis thaliana auxin metabolome

The plant hormone auxin is believed to influence almost every aspect of plant growth and development. Auxin transport, biosynthesis and degradation combine to form gradients of the hormone that influence a range of key developmental and environmental response processes. There is abundant genetic evidence for the existence of multiple pathways for auxin biosynthesis and degradation. The complexity of these pathways makes it difficult to obtain a clear picture of the relative importance of specific metabolic pathways during development. We have developed a sensitive mass spectrometry‐based method to simultaneously profile the majority of known auxin precursors and conjugates/catabolites in small amounts of Arabidopsis tissue. The method includes a new derivatization technique for quantification of the most labile of the auxin precursors. We validated the method by profiling the auxin metabolome in root and shoot tissues from various Arabidopsis thaliana ecotypes and auxin over‐producing mutant lines. Substantial differences were shown in metabolite patterns between the lines and tissues. We also found differences of several orders of magnitude in the abundance of auxin metabolites, potentially indicating the relative importance of these compounds in the maintenance of auxin levels and activity. The method that we have established will enable researchers to obtain a better understanding of the dynamics of auxin metabolism and activity during plant growth and development.