Non-aqueous fractionation revealed changing subcellular metabolite distribution during apple fruit development

In developing apple fruit,metabolic compartmentation is poorly understood due to the lack of experimental data.Distinguishing subcellular compartments in fruit using non-aqueous fractionation has been technically difficult due to the excess amount of sugars present in the different subcellular compartments limiting the resolution of the technique.The work described in this study represents the first attempt to apply non-aqueous fractionation to developing apple fruit,covering the major events occurring during fruit development(cell division,cell expansion,and maturation).Here we describe the non-aqueous fractionation method to study the subcellular compartmentation of metabolites during apple fruit development considering three main cellular compartments(cytosol,plastids,and vacuole).Evidence is presented that most of the sugars and organic acids were predominantly located in the vacuole,whereas some of the amino acids were distributed between the cytosol and the vacuole.The results showed a shift in the plastid marker from the lightest fractions in the early growth stage to the dense fractions in the later fruit growth stages.This implies that the accumulation of starch content with progressing fruit development substantially influenced the distribution of plastidial fragments within the non-aqueous density gradient applied.Results from this study provide substantial baseline information on assessing the subcellular compartmentation of metabolites in apple fruit in general and during fruit growth in particular.

An Oryza-specific hydroxycinnamoyl tyramine gene cluster contributes to enhanced disease resistance

Genomic clustering of non-homologous genes for the biosynthesis of plant defensive compounds is an emerging theme, but insights into their formation and physiological function remain limited. Here we report the identification of a newly discovered hydroxycinnamoyl tyramine(HT) gene cluster in rice.This cluster contains a pyridoxamine 50-phosphate oxidase(Os PDX3) producing the cofactor pyridoxal50-phosphate(PLP), a PLP-dependent tyrosine decarboxylase(Os Ty DC1), and two duplicated hydroxycinnamoyl transferases(Os THT1 and Os THT2). These members were combined to represent an enzymological innovation gene cluster. Natural variation analysis showed that the abundance of the toxic tyramine intermediate of the gene cluster among different rice accessions is mainly determined by the coordinated transcription of Os Ty DC1 and Os THT1. Further pathogen incubation assays demonstrated that the end products of the HT gene cluster displayed enhanced resistance to the bacterial pathogen Xanthomonas oryzae pv. Oryzae(Xoo) and fungal pathogen Magnaporthe oryzae(M. oryzae), and the enhanced resistance is associated with the boost of phytoalexins and the activation of defense response. The unique presence of the HT gene cluster in Oryza AA genome, together with the enrichment of transposon elements within this gene cluster region, provides an evolutionary background to accelerate cluster member combinations. Our study not only discovered a gene cluster involved in the phenylpropanoid metabolism but also addressed the key aspects of gene cluster formation. In addition, our results provide a new metabolic pool for plant defense against pathogens.

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.

The polyketide synthase OsPKS2 is essential for pollen exine and Ubisch body patterning in rice

Lipid and phenolic metabolism are important for pollen exine formation. In Arabidopsis, polyketide synthases （PKSs） are essential for both sporopollenin biosynthesis and exine formation. Here, we characterized the role of a polyketide synthase （OsPKS2） in male reproduction of rice （Oryza sativa）. Recombinant OsPKS2 catalyzed the condensation of fatty acyl-CoA with malonyl- CoA to generate triketide and tetraketide α-pyrones, the main components of pollen exine. Indeed, the ospks2 mutant had defective exine patterning and was male sterile. However, the mutant showed no significant reduction in sporopollenin accumulation. Compared with the WT （wild type）, ospks2 displayed unconfined and amorphous tectum and nexine layers in the exine, and less organized Ubisch bodies. Like the pksb/lap5 mutant of the Arabidopsis ortholog, ospks2 showed broad alterations in the profiles of anther-related phenolic compounds. However, unlike pksb/laps, in which most detected phenolics were substantially decreased, ospks2 accumu- lated higher levels of phenolics. Based on these results and our observation that OsPKS2 is unable to fully restore the exine defects in the pksb/laps, we propose that PKS proteins have functionally diversified during evolution. Collectively, our results suggest that PKSs represent a conserved and diversified biochemical pathway for anther and pollen development in higher plants.

Exploiting Natural Variation in Tomato to Define Pathway Structure and Metabolic Regulation of Fruit Polyphenolics in the Lycopersicum Complex

While the structures of plant primary metabolic pathways are generally well defined and highly conserved across species,those defining specialized metabolism are less well characterized and more highly variable across species.In this study,we investigated polyphenolic metabolism in the lycopersicum complex by characterizing the underlying biosynthetic and decorative reactions that constitute the metabolic network of polyphenols across eight different species of tomato.For this purpose,GC-MS-and LC-MS-based metabolomics of different tissues of Solatium lycopersicum and wild tomato species were carried out,in concert with the evaluation of cross-hybridized microarray data for MapMan-based transcriptomic analysis,and publicly available RNA-sequencing data for annotation of biosynthetic genes.The combined data were used to compile species-specific metabolic networks of polyphenolic metabolism,allowing the establishment of an entire pan-species biosynthetic framework as well as annotation of the functions of decoration enzymes involved in the formation of metabolic diversity of the flavonoid pathway.The combined results are discussed in the context of the current understanding of tomato flavonol biosynthesis as well as a global view of metabolic shifts during fruit ripening.Our results provide an example as to how large-scale biology approaches can be used for the definition and refinement of large specialized metabolism pathways.

Metabolomics-assisted refinement of the pathways of steroidal glycoalkaloid biosynthesis in the tomato clade

Steroidal glycoalkaloids（SGAs） are nitrogen-containing secondary metabolites of the Solanum species,which are known to have large chemical and bioactive diversity in nature.While recent effort and development on LC/MS techniques for SGA profiling have elucidated the main pathways of SGA metabolism in tomato,the problem of peak annotation still remains due to the vast diversity of chemical structure and similar on overlapping of chemical formula.Here we provide a case study of peak classification and annotation approach by integration of species and tissue specificities of SGA accumulation for provision of comprehensive pathways of SGA biosynthesis.In order to elucidate natural diversity of SGA biosynthesis,a total of 169 putative SGAs found in eight tomato accessions（Soianum lycopersicum, S.pimpinellifolium, S.cheesmaniae, S.chmielewskii, S.neorickii,S.peruvianum,S.habrochaites,S.pennellii） and four tissue types were used for correlation analysis.The results obtained in this study contribute annotation and classification of SGAs as well as detecting putative novel biosynthetic branch points.As such this represents a novel strategy for peak annotation for plant secondary metabolites.

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.

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.

Analysis of metabolic alterations in Arabidopsis following changes in the carbon dioxide and oxygen partial pressures

As sessile organisms,plants are subject to a multitude of environmental variations including several which directly affect their interaction with the atmosphere.Given the indiscriminant nature of Rubisco,the relative rates of photosynthesis and photorespiration are known to be responsive to changes in gas composition.However,comprehensive profiling methods have not yet been applied in order to characterize the wider consequences of these changes on primary metabolism in general.Moreover,although transcriptional profiling has revealed that a subset of photorespiratory enzymes are co-expressed,whether transcriptional responses play a role in short-term responses to atmospheric compositional changes remains unknown.To address these questions,plants Arabidopsis thaliana（Arabidopsis） ecotype Columbia（Col-O） grown under normal air conditions were transferred to different CO_2 and O_2 concentrations and characterized at the physiological,molecular,and metabolic levels following this transition.The results reveal alterations in the components,which are directly involved in,or supporting,photorespiration,including transcripts and metabolite levels.The results further highlight that the majority of the regulation of these pathways is not mediated at the level of transcription and that the photorespiratory pathway is essential also in conditions in which flux through the pathway is minimized,yet suggest that flux through this pathway is not mediated at the level of transcription.

From Fruit Omics to Fruiting Omics: Systematic Studies of Tomato Fruiting by Metabolic Networks

Understanding metabolic shifts and the regulatory network during fleshy fruiting in fruit crops is highly valuable for the design of crop improvement targeting better plant growth,fruit yield,stress tolerance,fruit shelf-life,nutritional quality,and health benefits.The"fruit omics"approach,which is an integrative omics analysis focusing on fruit development and ripening,has been highly developed for tomato species after its genome was sequenced.With the current advantage of next-generation sequencing for transcriptomics and the significant development of mass spectrometry-based metabolomics,tomato fruit omics studies have provided many key insights on metabolic shifts and networks during fruit ripening with fruit organ specificities.These studies have focused on primary metabolites as well as"specialized(secondary)metabolites,"which are phytochemicals with bioactivities that can confer health benefits and act as stress protectants,because of their usability in crop improvement.

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.