The Metabolic Response of Arabidopsis Roots to Oxidative Stress is Distinct from that of Heterotrophic Cells in Culture and Highlights a Complex Relationship between the Levels of Transcripts, Metabolites, and Flux

Metabolic adjustments are a significant, but poorly understood, part of the response of plants to oxidative stress. In a previous study （Baxter et al., 2007）, the metabolic response of Arabidopsis cells in culture to induction of oxidative stress by menadione was characterized. An emergency survival strategy was uncovered in which anabolic primary metabolism was largely down-regulated in favour of catabolic and antioxidant metabolism. The response in whole plant tissues may be different and we have therefore investigated the response of Arabidopsis roots to menadione treatment, analyzing the transcriptome, metabolome and key metabolic fluxes with focus on primary as well as secondary metabolism. Using a redox-sensitive GFP, it was also shown that menadione causes redox perturbation, not just in the mitochondrion, but also in the cytosol and plastids of roots. In the first 30 min of treatment, the response was similar to the cell culture： there was a decrease in metabolites of the TCA cycle and amino acid biosynthesis and the transcriptomic response was dominated by up-regulation of DNA regulatory proteins. After 2 and 6 h of treatment, the response of the roots was different to the cell culture. Metabolite levels did not remain depressed, but instead recovered and, in the case of pyruvate, some amino acids and aliphatic glucosinolates showed a steady increase above control levels. However, no major changes in fluxes of central carbon metabolism were observed and metabolic transcripts changed largely independently of the corresponding metabolites. Together, the results suggest that root tissues can recover metabolic activity after oxidative inhibition and highlight potentially important roles for glycolysis and the oxidative pentose phosphate pathway.

Combined Transcriptomics and Metabolomics of Arabidopsis thaliana Seedlings Exposed to Exogenous GABA Suggest Its Role in Plants Is Predominantly Metabolic

Dear Editor, y-Aminobutyric acid （GABA） is a non-protein amino acid （AA） metabolized via the GABA shunt; a three-enzyme pathway that includes glutamate decarboxylase （GAD）, GABA transaminases （GABA-T）, and succinic semialdehyde dehydrogenase （SSADH） （Bown and Shelp, 1997; Bouch~ and Fromm, 2004）. The majority of work on GABA, to date, has focused on its role as an inhibitory neurotransmitter in mammals. In plants, however, due to its intermediate posi- tion between carbon （C） and nitrogen （N）, the metabolism of GABA has been suggested as a modulator of C-N balance （Fair et al., 2008）. In addition, the numerous observations of a rapid accumulation of GABA in response to （a）biotic stresses has prompted different hypotheses to be postu- lated on its role including a regulator of cytosolic pH and a herbivore deterrent （Bouche and Fromm, 2004; Roberts, 2007）. Additionally, a signaling role has been hypothesized in plants, though evidence remains elusive and the debate on the dual function of GABA as a signaling molecule and as a metabolite in plants remains ongoing. In the present work, we report changes in the metabolite and transcript profiles in Arabidopsis seedlings exposed to exogenous GABA. Liquid-grown seedlings were cultured under C and N limitation to reveal a possible role of GABA as either C or N substrate and to ascertain its influence on transcriptional programs.

Metabolic and Phenotypic Responses of Greenhouse-Grown Maize Hybrids to Experimentally Controlled Drought Stress

Adaptation to abiotic stresses like drought is an important acquirement of agriculturally relevant crops like maize. Development of enhanced drought tolerance in crops grown in climatic zones where drought is a very dominant stress factor therefore plays an essential role in plant breeding. Previous studies demonstrated that corn yield potential and enhanced stress tolerance are associated traits. In this study, we analyzed six different maize hybrids for their ability to deal with drought stress in a greenhouse experiment. We were able to combine data from morphophysiological parameters measured under well-watered conditions and under water restriction with metabolic data from different organs. These different organs possessed distinct metabolite compositions, with the leaf blade displaying the most considerable metabolome changes following water deficiency. Whilst we could show a general increase in metabolite levels under drought stress, including changes in amino acids, sugars, sugar alcohols, and intermediates of the TCA cycle, these changes were not differential between maize hybrids that had previously been designated based on field trial data as either drought-tolerant or susceptible. The fact that data described here resulted from a greenhouse experiment with rather different growth conditions compared to natural ones in the field may explain why tolerance groups could not be confirmed in this study. We were, however, able to highlight several metabolites that displayed conserved responses to drought as well as metabolites whose levels correlated well with certain physiological traits.

Induction of the AOX1D Isoform of Alternative Oxidase in A. thaliana T-DNA Insertion Lines Lacking Isoform AOX1A Is Insufficient to Optimize Photosynthesis when Treated with Antimycin A

Plant respiration is characterized by two pathways for electron transfer to O2, namely the cytochrome pathway （CP） that is linked to ATP production, and the alternative pathway （AP）, where electrons from ubiquinol are directly transferred to O2 via an alternative oxidase （AOX） without concomitant ATP production. This latter pathway is well suited to dispose of excess electrons in the light, leading to optimized photosynthetic performance. We have characterized T- DNA-insertion mutant lines of Arabidopsis thaliana that do not express the major isoform, AOXIA. In standard growth conditions, these plants did not show any phenotype, but restriction of electron flow through CP by antimycin A, which induces AOXIA expression in the wild-type, led to an increased expression of AOXID in leaves of the aoxla-knockout mutant. Despite the increased presence of the AOX1D isoform in the mutant, antimycin A caused inhibition of photosyn- thesis, increased ROS, and ultimately resulted in amplified membrane leakage and necrosis when compared to the wild- type, which was only marginally affected by the inhibitor. It thus appears that AOX1 D was unable to fully compensate for the loss of AOXIA when electron flow via the CP is restricted. A combination of inhibition studies, coupled to metabolite profiling and targeted expression analysis of the P-protein of glycine decarboxylase complex （GDC）, suggests that the aoxla mutants attempt to increase their capacity for photorespiration. However, given their deficiency, it is intriguing that increase in expression neither of AOX1D nor of GDC could fully compensate for the lack of AOXIA to optimize pho- tosynthesis when treated with antimycin A. We suggest that the aoxla mutants can further be used to substantiate the current models concerning the influence of mitochondrial redox on photosynthetic performance and gene expression.

Catabolism of Branched Chain Amino Acids Supports Respiration but Not Volatile Synthesis in Tomato Fruits

The branched-chain amino acid transaminases （BCATs） have a crucial role in metabolism of the branched-chain amino acids leucine, isoleucine, and valine. These enzymes catalyze the last step of synthesis and the initial step of degradation of these amino acids. Although the biosynthetic pathways of branched chain amino acids in plants have been extensively investigated and a number of genes have been characterized, their catabolism in plants is not yet completely understood. We previously characterized the branched chain amino acid transaminase gene family in tomato, revealing both the subcellular localization and kinetic properties of the enzymes encoded by six genes. Here, we examined possible functions of the enzymes during fruit development. We further characterized transgenic plants differing in the expression of branched chain amino acid transaminases I and 3, evaluating the rates of respiration in fruits deficient in BCAT1 and the levels of volatiles in lines overexpressing either BCAT1 or BCAT3. We quantitatively tested, via precursor and isotope feeding experiments, the importance of the branched chain amino acids and their corresponding keto acids in the formation of fruit volatiles. Our results not only demonstrate for the first time the importance of branched chain amino acids in fruit respiration, but also reveal that keto acids, rather than amino acids, are the likely precursors for the branched chain flavor volatiles.

Mapping theArabidopsis Metabolic Landscape by Untargeted Metabolomics at Different Environmental Conditions

Metabolic genome-wide association studies （mGWAS）, whereupon metabolite levels are regarded as traits, can help unravel the genetic basis of metabolic networks. A total of 309Arabidopsis accessions were grown under two independent environmental conditions （control and stress） and subjected to untargeted LC-MS- based metabolomic profiling; levels of the obtained hydrophilic metabolites were used in GWAS. Our two- condition-based GWAS for more than 3000 semi-polar metabolites resulted in the detection of 123 highly resolved metabolite quantitative trait loci （p ≤ 1.0E-08）, 24.39% of which were environment-specific. Interestingly, differently from natural variation in Arabidopsis primary metabolites, which tends to be controlled by a large number of small-effect loci, we found several major large-effect loci alongside a vast number of small-effect loci controlling variation of secondary metabolites. The two-condition-based GWAS was fol- lowed by integration with network-derived metabolite-transcript correlations using a time-course stress experiment. Through this integrative approach, we selected 70 key candidate associations between struc- tural genes and metabolites, and experimentally validated eight novel associations, two of them showing differential genetic regulation in the two environments studied. We demonstrate the power of combining large-scale untargeted metabolomics-based GWAS with time-course-derived networks both performed under different ablotic environments for identifying metabollte-gene associations, providing novel global insights into the metabolic landscape of Arabidopsis.

Quantitative Trait Loci Analysis Identifies a Prominent Gene Involved in the Production of Fatty Acid-Derived Flavor Volatiles in Tomato

To gain insight into the genetic regulation of lipid metabolism in tomato, we conducted metabolic trait loci （mQTL） analysis following the lipidomic profiling of fruit pericarp and leaf tissue of the Solanum pennellii introgression lines （IL）. To enhance mapping resolution for selected fruit-specific mQTL, we profiled the lipids in a subset of independently derived S. pennellii backcross inbred lines, as well as in a nearsogenic sub-iL population. We identified a putative lecithin：cholesterol acyltransferase that controls the levels of several lipids, and two members of the class III lipase family, LIP1 and LIP2, that were associated with decreased levels of diacylglycerols （DAGs） and triacylglycerols （TAGs）. Lipases of this class cleave fatty acids from the glycerol backbone of acylglycerols. The released fatty acids serve as precursors of flavor volatiles. We show that LIP1 expression correlates with fatty acid-derived volatile levels. We further confirm the function of LIP1 in TAG and DAG breakdown and volatile synthesis using transgenic plants. Taken together, our study extensively characterized the genetic architecture of Upophilic compounds in tomato and demonstrated at molecular level that release of free fatty acids from the glycerol backbone can have a major impact on downstream volatile synthesis.

Complex Assembly and Metabolic Profiling of Arabidopsis thaliana PlantsOverexpressing Vitamin B6 Biosynthesis Proteins

In plants, vitamin B6 biosynthesis requires the activity of PDX1 and PDX2 proteins. Arabidopsis thaliana encodes for three PDX1 proteins, named PDXI.1, 1.2, and 1.3, but only one PDX2. Here, we show in planta complex assembly of PDX proteins, based on split-YFP and FPLC assays, and can demonstrate their presence in higher complexes of around 750 kDa. Metabolic profiling of plants ectopically expressing the different PDX proteins indicates a negative influence of PDX1.2 on vitamin Be biosynthesis and a correlation between aberrant vitamin B6 content, PDX1 gene expression, and light sensitivity specifically for PDX1.3. These findings provide first insights into in planta vitamin B6 synthase complex assembly and new information on how the different PDX proteins affect plant metabolism.

Analysis of Short-Term Metabolic Alterations in Arabidopsis Following Changes in the Prevailing Environmental Conditions

Although a considerable increase in our knowledge concerning the importance of metabolic adjustments to unfavorable growth conditions has been recently provided, relatively little is known about the adjustments which occur in response to fluctuation in environmental factors. Evaluating the metabolic adjustments occurring under changing environmental conditions thus offers a good opportunity to increase our current understanding of the crosstalk between the major pathways which are affected by such conditions. To this end, plants growing under normal conditions were transferred to different light and temperature conditions which were anticipated to affect （amongst other processes） the rates of photosynthesis and photorespiration and characterized at the physiological, molecular, and metabolic levels following this transition. Our results revealed similar behavior in response to both treatments and imply a tight connec- tivity of photorespiration with the major pathways of plant metabolism. They further highlight that the majority of the regulation of these pathways is not mediated at the level of transcription but that leaf metabolism is rather pre-poised to adapt to changes in these input parameters.

Mild Reductions in Mitochondrial NAD- Dependent Isocitrate Dehydrogenase Activity Result in Altered Nitrate Assimilation and Piqmentation But Do Not Impact Growth

Transgenic tomato （Solanum lycopersicum） plants were generated expressing a fragment of the mitochon- drial NAD-dependent isocitrate dehydrogenase gene （SIIDH1） in the antisense orientation. The transgenic plants displayed a mild reduction in the activity of the target enzyme in the leaves but essentially no visible alteration in growth from the wild-type. Fruit size and yield were, however, reduced. These plants were characterized by relatively few changes in pho- tosynthetic parameters, but they displayed a minor decrease in maximum photosynthetic efficiency （Fv/Fm）. Furthermore, a clear reduction in flux through the tricarboxylic acid （TCA） cycle was observed in the transformants. Additionally, bio- chemical analyses revealed that the transgenic lines exhibited considerably altered metabolism, being characterized by slight decreases in the levels of amino acids, intermediates of the TCA cycle, photosynthetic pigments, starch, and NAD（P）H levels, but increased levels of nitrate and protein. Results from these studies show that even small changes in mitochon- drial NAD-dependent isocitrate dehydrogenase activity lead to noticeable alterations in nitrate assimilation and suggest the presence of different strategies by which metabolism is reprogrammed to compensate for this deficiency.

Is There a Metabolic Requirement for Photorespiratory Enzyme Activities in Heterotrophic Tissues？

Photorespiration is an essential metabolic process in leaves that facilitates recovery of carbon lost by the oxygenase reaction of Rubisco and avoids the accumulation of the toxic product, 2-phosphoglycolate （2PG） of this reaction （Bauwe et al., 2012）. However, there is also evidence to suggest that photorespiration has a more complex role during normal growth than the mere detoxification of 2PG and the recovery of 3-phosphoglycerate （3PGA） （Bauwe et al., 2012）.

At Long Last： Evidence for Pexophagy in Plants

Peroxisomes are highly dynamic single-membrane-bound eukaryotic organelles displaying great variability in enzymatic content （Platta and Erdmann, 2007）. Plant peroxisomes function in a plethora of crucial development and stress ameliorating processes. Among the functions of plant peroxisomes are 13-oxidation of fatty acids, phytohormone production, participation in photorespiration, the glyoxyalte cycle, detoxification processes, and signal molecule generation （Hu et al., 2012）.

Analysis of the Interface between Primary and Secondary Metabolism in Catharanthus roseus Cell Cultures Using ^13C-Stable Isotope Feeding and Coupled Mass Spectrometry

Dear Editor, The tropical plant Madagascar periwinkle Catharanthus roseus （L.） G.Don is a rich source of plant-derived medicinal ter-penoid indole alkaloids （TIAs）, including the anti-hypertensive ajmalicine, the sedative compound serpentine, and the anti-cancer drugs vinblastine and vincristine. However, the latter two compounds are produced in C. roseus plants only in very low amounts. Elicitors such as hormones （e.g. jasmonates or salicylic acid） activate plant natural defense responses, includ-ing increased secondary metabolite production （EI-Sayed and Verpoorte, 2007; Lackman et al.. 2011）.

Photorespiration Is Crucial for Dynamic Response of Photosynthetic Metabolism and Stomatal Movement to Altered C02 Availability

The photorespiratory pathway or photorespiration is an essential process in oxygenic photosynthetic or- ganisms, which can reduce the efficiency of photosynthetic carbon assimilation and is hence frequently considered as a wasteful process. By comparing the response of the wild-type plants and mutants impaired in photorespiration to a shift in ambient C02 concentrations, we demonstrate that photorespiration also plays a beneficial role during short-term acclimation to reduced C02 availability. The wild-type plants re- sponded with few differentially expressed genes, mostly involved in drought stress, which is likely a conse- quence of enhanced opening of stomata and concomitant water loss upon a shift toward low C02. In contrast, mutants with impaired activity of photorespiratory enzymes were highly stressed and not able to adjust stomatal conductance to reduced external C02 availability. The transcriptional response of mutant plants was congruent, indicating a general reprogramming to deal with the consequences of reduced C02 availability, signaled by enhanced oxygenation of ribulose-l,5-bisphosphate and amplified by the artificially impaired photorespiratory metabolism. Central in this reprogramming was the pro- nounced reallocation of resources from growth processes to stress responses. Taken together, our results indicate that unrestricted photorespiratory metabolism is a prerequisite for rapid physiological acclimation to a reduction in C02 availability.

Involvement of the Hexose Transporter Gene LeHT1 and of Sugars in Resistance of Tomato to Tomato Yellow Leaf Curl Virus

Dear Editor, Tomato yellow leaf curl virus （TYLCV） is a whitefly-trans- mitted geminivirus infecting tomato crops （Czosnek, 2007）. TYLCV-resistant （R） and susceptible （S） lines with the same genetic background have been bred using Solanum habro- chaites as the resistance source. The gene（s） conferring resist- ance are unknown. Previously, we demonstrated that the hexose transporter gene LeHT1 is up-regulated upon infec- tion in R plants and its silencing in R plants （RH） leads to the collapse of resistance （Eybishtz et al., 2010）. To uncover the role of LeHT1 in resistance, we （1） analyzed the transcriptome reprogramming in leaves of S, R, and RH plants using a home- designed microarray, before and 7 d after TYLCV inoculation （0, 7 dpi）, and （2） measured the concentration of sugars and their derivatives in S, R, and RH leaves at 1 and 7 dpi because LeHT1 is transporting both glucose and fructose （McCurdy et al., 2010）.

Establishment of a Photoautotrophic Cell Suspension Culture of Arabidopsis thaliana for Photosynthetic, Metabolic, and Signaling Studies

Dear Editor, Plant cell suspension cultures have been used as model systems to circumvent the problems associated with the analyses of a multi-factorial plant that is composed of multiple tissue and cell types exposed to diverse signals. A number of plant suspension cultures have proven to be valuable to study various topics including defense response, secondary metabolite formation, ion transport, gene regulation, and signal duction （Roitsch and Sinha, 2002 and references therein）. However, most cultures reported to date, including the cultures from model species such as Arabidopsis （Christie and Jenkins,