Medicago sativa and Medicago truncatula Show Contrasting Root Metabolic Responses to Drought.

Drought is an environmental stressor that affects crop yield worldwide. Understanding plant physiological responses to stress conditions is needed to secure food in future climate conditions. In this study, we applied a combination of plant physiology and metabolomic techniques to understand plant responses to progressive water deficit focusing on the root system. We chose two legume plants with contrasting tolerance to drought, the widely cultivated alfalfa Medicago sativa (Ms) and the model legume Medicago truncatula (Mt) for comparative analysis. Ms taproot (tapR) and Mt fibrous root (fibR) biomass increased during drought, while a progressive decline in water content was observed in both species. Metabolomic analysis allowed the identification of key metabolites in the different tissues tested. Under drought, carbohydrates, abscisic acid, and proline predominantly accumulated in leaves and tapRs, whereas flavonoids increased in fibRs in both species. Raffinose-family related metabolites accumulated during drought. Along with an accumulation of root sucrose in plants subjected to drought, both species showed a decrease in sucrose synthase (SUS) activity related to a reduction in the transcript level of SUS1, the main SUS gene. This study highlights the relevance of root carbon metabolism during drought conditions and provides evidence on the specific accumulation of metabolites throughout the root system.

Comprehensive Metabolite Profiling in Genetic Resources of Garlic (Allium sativum L.) Collected from Different Geographical Regions.

Garlic (Allium sativum) is the second most important Allium crop that has been used as a vegetable and condiment from ancient times due to its characteristic flavor and taste. Although garlic is a sterile plant that reproduces vegetatively through cloves, garlic shows high biodiversity, as well as phenotypic plasticity and environmental adaptation capacity. To determine the possible mechanism underlying this phenomenon and to provide new genetic materials for the development of a novel garlic cultivar with useful agronomic traits, the metabolic profiles in the leaf tissue of 30 garlic accessions collected from different geographical regions, with a special focus on the Asian region, were investigated using LC/MS. In addition, the total saponin and fructan contents in the roots and cloves of the investigated garlic accessions were also evaluated. Total saponin and fructan contents did not separate the garlic accessions based on their geographical origin, implying that saponin and fructan contents were clone-specific and agroclimatic changes have affected the quantitative and qualitative levels of saponins in garlic over a long history of cultivation. Principal component analysis (PCA) and dendrogram clustering of the LC/MS-based metabolite profiling showed two major clusters. Specifically, many Japanese and Central Asia accessions were grouped in cluster I and showed high accumulations of flavonol glucosides, alliin, and methiin. On the other hand, garlic accessions grouped in cluster II exhibited a high accumulation of anthocyanin glucosides and amino acids. Although most of the accessions were not separated based on country of origin, the Central Asia accessions were clustered in one group, implying that these accessions exhibited distinct metabolic profiles. The present study provides useful information that can be used for germplasm selection and the development of new garlic varieties with beneficial biotic and abiotic stress-adaptive traits.

Development of a Pteris vittata L. compound database by widely targeted metabolomics profiling.

The objective of this work was the development of a detailed, extensive and reliable database of the metabolomes of P. vittata. Using an ultra-performance liquid chromatography-triple quadrupole tandem mass spectrometry system (UPLC-QqQ-MS/MS) and based on the knowledge of retention time and mass spectral characteristics of an in-house collection of authentic standards, we screened for the presence of a large collection of natural compounds. The database represents 359 authenticated metabolites, comprising 220 primary and 139 secondary metabolites (70 flavonoids, 16 phenylpropanoic acid derivatives, five coumarins, two stilbenoids, 14 benzoic acids, nine phenols, 20 alkaloids and three terpenoids). Comparison of the accumulation of these compounds in two tissues showed that the aerial parts were enriched in flavonols, whereas the subterranean parts were enriched in anthocyanins. The comprehensive database developed here will be beneficial in improving the understanding of the chemical basis of plant therapeutic profile using multivariate analysis, with a particular example of antioxidant activity.

OryzaGenome2.1: Database of Diverse Genotypes in Wild Oryza Species.

BACKGROUND: OryzaGenome ( http://viewer.shigen.info/oryzagenome21detail/index.xhtml ), a feature within Oryzabase ( https://shigen.nig.ac.jp/rice/oryzabase/ ), is a genomic database for wild Oryza species that provides comparative and evolutionary genomics approaches for the rice research community. RESULTS: Here we release OryzaGenome2.1, the first major update of OryzaGenome. The main feature in this version is the inclusion of newly sequenced genotypes and their meta-information, giving a total of 217 accessions of 19 wild Oryza species (O. rufipogon, O. barthii, O. longistaminata, O. meridionalis, O. glumaepatula, O. punctata, O. minuta, O. officinalis, O. rhizomatis, O. eichingeri, O. latifolia, O. alta, O. grandiglumis, O. australiensis, O. brachyantha, O. granulata, O. meyeriana, O. ridleyi, and O. longiglumis). These 19 wild species belong to 9 genome types (AA, BB, CC, BBCC, CCDD, EE, FF, GG, and HHJJ), representing wide genomic diversity in the genus. Using the genotype information, we analyzed the genome diversity of Oryza species. Other features of OryzaGenome facilitate the use of information on single nucleotide polymorphisms (SNPs) between O. sativa and its wild progenitor O. rufipogon in rice research, including breeding as well as basic science. For example, we provide Variant Call Format (VCF) files for genome-wide SNPs of 33 O. rufipogon accessions against the O. sativa reference genome, IRGSP1.0. In addition, we provide a new SNP Effect Table function, allowing users to identify SNPs or small insertion/deletion polymorphisms in the 33 O. rufipogon accessions and to search for the effect of these polymorphisms on protein function if they reside in the coding region (e.g., are missense or nonsense mutations). Furthermore, the SNP Viewer for 446 O. rufipogon accessions was updated by implementing new tracks for possible selective sweep regions and highly mutated regions that were potentially exposed to selective pressures during the process of domestication. CONCLUSION: OryzaGenome2.1 focuses on comparative genomic analysis of diverse wild Oryza accessions collected around the world and on the development of resources to speed up the identification of critical trait-related genes, especially from O. rufipogon. It aims to promote the use of genotype information from wild accessions in rice breeding and potential future crop improvements. Diverse genotypes will be a key resource for evolutionary studies in Oryza, including polyploid biology.

Analysis of carbon flow at the metabolite level reveals that starch synthesis from hexose is a limiting factor in a high-yielding rice cultivar.

Understanding the limiting factors of grain filling is essential for the further improvement of grain yields in rice (Oryza sativa). The relatively slow grain growth of the high-yielding cultivar 'Momiroman' is not improved by increasing carbon supply, and hence low sink activity (i.e. the metabolic activity of assimilate consumption/storage in sink organs) may be a limiting factor for grain filling. However, there is no metabolic evidence to corroborate this hypothesis, partly because there is no consensus on how to define and quantify sink activity. In this study, we investigated the carbon flow at a metabolite level from photosynthesis in leaves to starch synthesis in grains of three high-yielding cultivars using the stable isotope 13C. We found that a large amount of newly fixed carbon assimilates in Momiroman was stored as hexose instead of being converted to starch. In addition, the activity of ADP-glucose pyrophosphorylase and the expression of AGPS2b, which encodes a subunit of the ADP-glucose pyrophosphorylase enzyme, were both lower in Momiroman than in the other two cultivars in grains in superior positions on panicle branches. Hence, slower starch synthesis from hexose, which is partly explained by the low expression level of AGPS2b, may be the primary metabolic reason for the lower sink activity observed in Momiroman.

Abscisic acid-mediated induction of FLAVIN-CONTAINING MONOOXYGENASE 2 leads to reduced accumulation of methylthioalkyl glucosinolates in Arabidopsis thaliana.

Side-chain modification contributes to the structural diversity of aliphatic glucosinolates (GSLs), a class of sulfur-containing secondary metabolites found in Brassicales. The first step in side-chain modification of aliphatic GSLs is the S-oxygenation of the methylthioalkyl (MT) moiety to the methylsulfinylalkyl (MS) moiety. This reaction is catalyzed by flavin-containing monooxygenase (FMOGS-OX), which is encoded by seven genes in Arabidopsis thaliana. Therefore, the regulation of FMOGS-OX gene expression is key to controlling side-chain structural diversity. In this study, we demonstrated that the expression of FMOGS-OX2 and FMOGS-OX4 was induced by glucose treatment, independent of MYB28/29 and MYC2/3/4, the transcription factors that positively regulate aliphatic GSL biosynthesis. Glucose treatment of the abscisic acid (ABA)-related mutants indicated that glucose-triggered upregulation of FMOGS-OX2 and FMOGS-OX4 was partially regulated by ABA through the key negative regulators ABI1 and ABI2, and the positive regulator SnRK2, but not via the transcription factor ABI5. In wild-type plants, glucose treatment drastically reduced the accumulation of 4-methylthiobutyl (4MT) GSL, whereas a decrease in 4MT GSL was not observed in the fmogs-ox2, abi1-1, abi2-1, aba2-1, or aba3-1 mutants. This result indicated that the decreased accumulation of 4MT GSL by glucose treatment was attributed to upregulation of FMOGS-OX2 via the ABA signaling pathway.

Syringic Acid Alleviates Cesium-Induced Growth Defect in Arabidopsis.

Syringic acid, a phenolic compound, serves a variety of beneficial functions in cells. Syringic acid increases in plants in response to cesium, and exogenous application of syringic acid resulted in a significant attenuation of cesium-induced growth defects in Arabidopsis. In addition, cesium or syringic acid application to plants also resulted in increased lignin deposition in interfascicular fibers. To better understand the role of lignin and syringic acid in attenuating cesium-induced growth defects, two mutants for Arabidopsis REDUCED EPIDERMAL FLUORESCENE 4 (REF4) and fourteen laccase mutants, some of which have lower levels of lignin, were evaluated for their response to cesium. These mutants responded differently to cesium stress, compared to control plants, and the application of syringic acid alleviated cesium-induced growth defects in the laccase mutants but not in the ref4 mutants. These findings imply that lignin plays a role in cesium signaling but the attenuation of cesium stress defects by syringic acid is mediated by regulatory components of lignin biosynthesis and not lignin biosynthesis itself. In contrast, syringic acid did not alleviate any low potassium-induced growth defects. Collectively, our findings provide the first established link between lignin and cesium stress via syringic acid in plants.

Metabolome-Based Discrimination Analysis of Shallot Landraces and Bulb Onion Cultivars Associated with Differences in the Amino Acid and Flavonoid Profiles.

Shallot landraces and varieties are considered an important genetic resource for Allium breeding due to their high contents of several functional metabolites. Aiming to provide new genetic materials for the development of a novel bulb onion cultivar derived from intraspecific hybrids with useful agronomic traits from shallots, the metabolic profiles in the bulbs of 8 Indonesian shallot landraces and 7 short-day and 3 long-day bulb onion cultivars were established using LC-Q-TOF-MS/MS. Principal component analysis, partial least squares discriminant analysis, and dendrogram clustering analysis showed two major groups; group I contained all shallot landraces and group II contained all bulb onion cultivars, indicating that shallots exhibited a distinct metabolic profile in comparison with bulb onions. Variable importance in the projection and Spearman's rank correlation indicated that free and conjugated amino acids, flavonoids (especially metabolites having flavonol aglycone), and anthocyanins, as well as organic acids, were among the top metabolite variables that were highly associated with shallot landraces. The absolute quantification of 21 amino acids using conventional HPLC analysis showed high contents in shallots rather than in bulb onions. The present study indicated that shallots reprogrammed their metabolism toward a high accumulation of amino acids and flavonoids as an adaptive mechanism in extremely hot tropical environments.

Identification of a Unique Type of Isoflavone O-Methyltransferase, GmIOMT1, Based on Multi-Omics Analysis of Soybean under Biotic Stress.

Isoflavonoids are commonly found in leguminous plants. Glycitein is one of the isoflavones produced by soybean. The genes encoding the enzymes in the isoflavone biosynthetic pathway have mostly been identified and characterized. However, the gene(s) for isoflavone O-methyltransferase (IOMT), which catalyzes the last step of glycitein biosynthesis, has not yet been identified. In this study, we conducted multi-omics analyses of fungal-inoculated soybean and indicated that glycitein biosynthesis was induced in response to biotic stress. Moreover, we identified a unique type of IOMT, which participates in glycitein biosynthesis. Soybean seedlings were inoculated with Aspergillus oryzae or Rhizopus oligosporus and sampled daily for 8 d. Multi-omics analyses were conducted using liquid chromatography-tandem mass spectrometry and RNA sequencing. Metabolome analysis revealed that glycitein derivatives increased following fungal inoculation. Transcriptome co-expression analysis identified two candidate IOMTs that were co-expressed with the gene encoding flavonoid 6-hydroxylase (F6H), the key enzyme in glycitein biosynthesis. The enzymatic assay of the two IOMTs using respective recombinant proteins showed that one IOMT, named as GmIOMT1, produced glycitein. Unlike other IOMTs, GmIOMT1 belongs to the cation-dependent OMT family and exhibited the highest activity with Zn2+ among cations tested. Moreover, we demonstrated that GmIOMT1 overexpression increased the levels of glycitein derivatives in soybean hairy roots when F6H was co-expressed. These results strongly suggest that GmIOMT1 participates in inducing glycitein biosynthesis in response to biotic stress.

Morphological characterization of domatium development in Callicarpa saccata.

BACKGROUND AND AIMS: Domatia are plant structures within which organisms reside. Callicarpa saccata (Lamiaceae) is the sole myrmecophyte, or 'ant plant', that develops foliar (leaf-borne) myrmeco-domatia in this genus. In this work we examined domatium development in C. saccata to understand the developmental processes behind pouch-like domatia. METHODS: Scanning electron microscopy, sectioning and microcomputed tomography were carried out to compare the leaves of C. saccata with those of the closely related but domatia-less myrmecophyte Callicarpa subaequalis, both under cultivation without ants. KEY RESULTS: Callicarpa saccata domatia are formed as a result of excess cell proliferation at the blade/petiole junctions of leaf primordia. Blade/petiole junctions are important meristematic sites in simple leaf organogenesis. We also found that the mesophyll tissue of domatia does not clearly differentiate into palisade and spongy layers. CONCLUSIONS: Rather than curling of the leaf margins, a perturbation of the normal functioning of the blade/petiole junction results in the formation of domatium tissue. Excess cell proliferation warps the shape of the blade and disturbs the development of the proximal-distal axis. This process leads to the generation of distinct structures that facilitate interaction between C. saccata and ants.

Regulation of Sugar and Storage Oil Metabolism by Phytochrome during De-etiolation.

Exposure of dark-grown (etiolated) seedlings to light induces the heterotrophic-to-photoautotrophic transition (de-etiolation) processes, including the formation of photosynthetic machinery in the chloroplast and cotyledon expansion. Phytochrome is a red (R)/far-red (FR) light photoreceptor that is involved in the various aspects of de-etiolation. However, how phytochrome regulates metabolic dynamics in response to light stimulus has remained largely unknown. In this study, to elucidate the involvement of phytochrome in the metabolic response during de-etiolation, we performed widely targeted metabolomics in Arabidopsis (Arabidopsis thaliana) wild-type and phytochrome A and B double mutant seedlings de-etiolated under R or FR light. The results revealed that phytochrome had strong impacts on the primary and secondary metabolism during the first 24 h of de-etiolation. Among those metabolites, sugar levels decreased during de-etiolation in a phytochrome-dependent manner. At the same time, phytochrome upregulated processes requiring sugars. Triacylglycerols are stored in the oil bodies as a source of sugars in Arabidopsis seedlings. Sugars are provided from triacylglycerols through fatty acid ß-oxidation and the glyoxylate cycle in glyoxysomes. We examined if and how phytochrome regulates sugar production from oil bodies. Irradiation of the etiolated seedlings with R and FR light dramatically accelerated oil body mobilization in a phytochrome-dependent manner. Glyoxylate cycle-deficient mutants not only failed to mobilize oil bodies but also failed to develop thylakoid membranes and expand cotyledon cells upon exposure to light. Hence, phytochrome plays a key role in the regulation of metabolism during de-etiolation.

Inter-Laboratory Comparison of Metabolite Measurements for Metabolomics Data Integration.

BACKGROUND: One of the current problems in the field of metabolomics is the difficulty in integrating data collected using different equipment at different facilities, because many metabolomic methods have been developed independently and are unique to each laboratory. METHODS: In this study, we examined whether different analytical methods among 12 different laboratories provided comparable relative quantification data for certain metabolites. Identical samples extracted from two cell lines (HT-29 and AsPc-1) were distributed to each facility, and hydrophilic and hydrophobic metabolite analyses were performed using the daily routine protocols of each laboratory. RESULTS: The results indicate that there was no difference in the relative quantitative data (HT-29/AsPc-1) for about half of the measured metabolites among the laboratories and assay methods. Data review also revealed that errors in relative quantification were derived from issues such as erroneous peak identification, insufficient peak separation, a difference in detection sensitivity, derivatization reactions, and extraction solvent interference. CONCLUSION: The results indicated that relative quantification data obtained at different facilities and at different times would be integrated and compared by using a reference materials shared for data normalization.

Metabolome-based discrimination of chrysanthemum cultivars for the efficient generation of flower color variations in mutation breeding.

INTRODUCTION: The color variations of ornamental flowers are often generated by ion-beam and gamma irradiation mutagenesis. However, mutation rates differ significantly even among cultivars of the same species, resulting in high cost and intensive labor for flower color breeding. OBJECTIVES: We aimed to establish a metabolome-based strategy to identify biomarkers and select promising parental lines with high mutation rates using Chrysanthemum as the case study. METHODS: The mutation rates associated with flower color were measured in 10 chrysanthemum cultivars with pink, yellow, or white flowers after soft X-ray irradiation at the floret-formation stage. The metabolic profiles of the petals of these cultivars were clarified by widely targeted metabolomics and targeted carotenoid analysis using liquid chromatography-tandem quadrupole mass spectrometry. Metabolome and carotenoid data were subjected to an un-supervised principal component analysis (PCA) and a supervised logistic regression with least absolute shrinkage and selection operator (LASSO). RESULTS: The PCA of the metabolic profile data separated chrysanthemum cultivars according to flower color rather than mutation rates. By contrast, logistic regression with LASSO generated a discrimination model to separate cultivars into two groups with high or low mutation rates, and selected 11 metabolites associated with mutation rates that can be biomarkers candidates for selecting parental lines for mutagenesis. CONCLUSION: This metabolome-based strategy to identify metabolite markers for mutation rates associated with flower color might be applied to other ornamental flowers to accelerate mutation breeding for generating new cultivars with a wider range of flower colors.

Differential regulation of host plant adaptive genes in Pieris butterflies exposed to a range of glucosinolate profiles in their host plants.

Specialist herbivores have often evolved highly sophisticated mechanisms to counteract defenses mediated by major plant secondary-metabolites. Plant species of the herbivore host range often display high chemical diversity and it is not well understood how specialist herbivores respond to this chemical diversity. Pieris larvae overcome toxic products from glucosinolate hydrolysis, the major chemical defense of their Brassicaceae hosts, by expressing nitrile-specifier proteins (NSP) in their gut. Furthermore, Pieris butterflies possess so-called major allergen (MA) proteins, which are multi-domain variants of a single domain major allergen (SDMA) protein expressed in the guts of Lepidopteran larvae. Here we show that Pieris larvae fine-tune NSP and MA gene expression depending on the glucosinolate profiles of their Brassicaceae hosts. Although the role of MA is not yet fully understood, the expression levels of NSP and MA in larvae that fed on plants whose glucosinolate composition varied was dramatically changed, whereas levels of SDMA expression remained unchanged. In addition, we found a similar regulation pattern among these genes in larvae feeding on Arabidopsis mutants with different glucosinolate profiles. Our results demonstrate that Pieris larvae appear to use different host plant adaptive genes to overcome a wide range of glucosinolate profiles in their host plants.

Interspecific Differences in the Larval Performance of Pieris Butterflies (Lepidoptera: Pieridae) Are Associated with Differences in the Glucosinolate Profiles of Host Plants.

The tremendous diversity of plants and herbivores has arisen from a coevolutionary relationship characterized by plant defense and herbivore counter adaptation. Pierid butterfly species feed on Brassicales plants that produce glucosinolates as a chemical deterrent against herbivory. In turn, the larvae of pierids have nitrile specifier proteins (NSPs) that are expressed in their gut and disarm glucosinolates. Pierid butterflies are known to have diversified in response to glucosinolate diversification in Brassicales. Therefore, each pierid species is expected to have a spectrum of host plants characterized by specific glucosinolate profiles. In this study, we tested whether the larval performance of different Pieris species, a genus in Pieridae (Lepidoptera: Pieridae), was associated with plant defense traits of putative host plants. We conducted feeding assays using larvae of three Pieris species and 10 species of the Brassicaceae family possessing different leaf physical traits and glucosinolate profile measurements. The larvae of Pieris rapae responded differently in the feeding assays compared with the other two Pieris species. This difference was associated with differences in glucosinolate profiles but not with variations in physical traits of the host plants. This result suggests that individual Pieris species are adapted to a subset of glucosinolate profiles within the Brassicaceae. Our results support the idea that the host ranges of Pieris species depend on larval responses to glucosinolate diversification in the host species, supporting the hypothesis of coevolution between butterflies and host plants mediated by the chemical arms race.

Genetic Variation for Seed Metabolite Levels in Brachypodium distachyon.

Metabolite composition and concentrations in seed grains are important traits of cereals. To identify the variation in the seed metabolotypes of a model grass, namely Brachypodium distachyon, we applied a widely targeted metabolome analysis to forty inbred lines of B. distachyon and examined the accumulation patterns of 183 compounds in the seeds. By comparing the metabolotypes with the population structure of these lines, we found signature metabolites that represent different accumulation patterns for each of the three B. distachyon subpopulations. Moreover, we found that thirty-seven metabolites exhibited significant differences in their accumulation between the lines Bd21 and Bd3-1. Using a recombinant inbred line (RIL) population from a cross between Bd3-1 and Bd21, we identified the quantitative trait loci (QTLs) linked with this variation in the accumulation of thirteen metabolites. Our metabolite QTL analysis illustrated that different genetic factors may presumably regulate the accumulation of 4-pyridoxate and pyridoxamine in vitamin B6 metabolism. Moreover, we found two QTLs on chromosomes 1 and 4 that affect the accumulation of an anthocyanin, chrysanthemin. These QTLs genetically interacted to regulate the accumulation of this compound. This study demonstrates the potential for metabolite QTL mapping in B. distachyon and provides new insights into the genetic dissection of metabolomic traits in temperate grasses.

Effects of Thiosulfate as a Sulfur Source on Plant Growth, Metabolites Accumulation and Gene Expression in Arabidopsis and Rice.

Plants are considered to absorb sulfur from their roots in the form of sulfate. In bacteria like Escherichia coli, thiosulfate is a preferred sulfur source. It is converted into cysteine (Cys). This transformation consumes less NADPH and ATP than sulfate assimilation into Cys. In Saccharomyces cerevisiae, thiosulfate promoted growth more than sulfate. In the present study, the availability of thiosulfate, the metabolite transformations and gene expressions it induces were investigated in Arabidopsis and rice as model dicots and monocots, respectively. In Arabidopsis, the thiosulfate-amended plants had lower biomass than those receiving sulfate when sulfur concentrations in the hydroponic medium were above 300 µM. In contrast, rice biomass was similar for plants raised on thiosulfate and sulfate at 300 µM sulfur. Therefore, both plants can use thiosulfate but it is a better sulfur source for rice. In both plants, thiosulfate levels significantly increased in roots following thiosulfate application, indicating that the plants absorbed thiosulfate into their root cells. Thiosulfate is metabolized in plants by a different pathway from that used for sulfate metabolism. Thiosulfate increases plant sulfide and cysteine persulfide levels which means that plants are in a more reduced state with thiosulfate than with sulfate. The microarray analysis of Arabidopsis roots revealed that 13 genes encoding Cys-rich proteins were upregulated more with thiosulfate than with sulfate. These results together with those of the widely targeted metabolomics analysis were used to proposes a thiosulfate assimilation pathway in plants.

Widely targeted metabolome and transcriptome landscapes of Allium fistulosum-A. cepa chromosome addition lines revealed a flavonoid hot spot on chromosome 5A.

Here, we report a comprehensive analysis of the widely targeted metabolome and transcriptome profiles of Allium fistulosum L. (FF) with the single extra chromosome of shallot [A. cepa L. Aggregatum group (AA)] to clarify the novel gene functions in flavonoid biosynthesis. An exhaustive metabolome analysis was performed using the selected reaction monitoring mode of liquid chromatography-tandem quadrupole mass spectrometry, revealing a specific accumulation of quercetin, anthocyanin and flavone glucosides in AA and FF5A. The addition of chromosome 5A from the shallot to A. fistulosum induced flavonoid accumulation in the recipient species, which was associated with the upregulation of several genes including the dihydroflavonol 4-reductase, chalcone synthase, flavanone 3-hydroxylase, UDP-glucose flavonoid-3-O-glucosyltransferase, anthocyanin 5-aromatic acyltransferase-like, pleiotropic drug resistance-like ATP binding cassette transporter, and MYB14 transcriptional factor. Additionally, an open access Allium Transcript Database (Allium TDB, http://alliumtdb.kazusa.or.jp ) was generated by using RNA-Seq data from different genetic stocks including the A. fistulosum-A. cepa monosomic addition lines. The functional genomic approach presented here provides an innovative means of targeting the gene responsible for flavonoid biosynthesis in A. cepa. The understanding of flavonoid compounds and biosynthesis-related genes would facilitate the development of noble Allium varieties with unique chemical constituents and, subsequently, improved plant stress tolerance and human health benefits.

Functional screening of salt tolerance genes from a halophyte Sporobolus virginicus and transcriptomic and metabolomic analysis of salt tolerant plants expressing glycine-rich RNA-binding protein.

Sporobolus virginicus is a halophytic C4 grass found worldwide, from tropical to warm temperate regions. One Japanese genotype showed a salinity tolerance up to 1.5 M NaCl, a three-fold higher concentration than the salinity of sea water. To identify the key genes involved in the regulation of salt tolerance in S. virginicus, we produced 3500 independent transgenic Arabidopsis lines expressing random cDNA from S. virginicus and screened 10 lines which showed enhanced salt tolerance compared with the wild type in a medium containing 150 mM NaCl. Among the selected lines, two contained cDNA coding glycine-rich RNA-binding proteins (SvGRP1 and SvGRP2). This is the first reports on the function of GRPs from halophytes in salt tolerance though reports have shown GRPs are involved in diverse biological and biochemical processes including salt tolerance in Arabidopsis and some other glycophytes. Transcriptomic analysis and GO enrichment analysis of SvGRP1-expressing Arabidopsis under salt stress revealed upregulation of polyol and downregulation of glucosinolate and indole acetic acid biosynthesis/metabolic pathways. Metabolomic analysis of the SvGRP1-transformant suggested that the increase in 3-aminoppropanoic acid, citramalic acid, and isocitric acid content was associated with enhanced salt tolerance. These findings could provide novel insight into the roles of GRPs in plant salt tolerance.

Arabidopsis molybdenum cofactor sulfurase ABA3 contributes to anthocyanin accumulation and oxidative stress tolerance in ABA-dependent and independent ways.

Arabidopsis ABA3 is an enzyme involved in the synthesis of the sulfurated form of the molybdenum (Mo) cofactor (MoCo), which is required for the enzymatic activity of so-called Mo enzymes such as aldehyde oxidase (AO) and xanthine dehydrogenase (XDH). It has been reported that AO and XDH are essential for the biosynthesis of the bioactive compounds, ABA and allantoin, respectively. However, aba3 mutants often exhibit pleiotropic phenotypes that are not explained by defects in ABA and/or allantoin biosynthesis, leading us to hypothesize that ABA3 regulates additional metabolic pathways. To reveal the currently unidentified functions of ABA3 we compared transcriptome and metabolome of the Arabidopsis aba3 mutant with those of wild type and a typical ABA-deficient mutant aba2. We found that endogenous levels of anthocyanins, members of the flavonoid group, were significantly lower in the aba3 mutant than in the wild type or the aba2 mutant under oxidative stress. In contrast, mutants defective in the AO and XDH holoenzymes accumulated significantly higher levels of anthocyanins when compared with aba3 mutant under the same conditions. Our findings shed light on a key role of ABA3 in the ABA- and allantoin-independent accumulation of anthocyanins during stress responses.