Dynamic relationships among pathways producing hydrocarbons and fatty acids of maize silk cuticular waxes.

The hydrophobic cuticle is the first line of defense between aerial portions of plants and the external environment. On maize (Zea mays L.) silks, the cuticular cutin matrix is infused with cuticular waxes, consisting of a homologous series of very long-chain fatty acids (VLCFAs), aldehydes, and hydrocarbons. Together with VLC fatty-acyl-CoAs (VLCFA-CoAs), these metabolites serve as precursors, intermediates, and end-products of the cuticular wax biosynthetic pathway. To deconvolute the potentially confounding impacts of the change in silk microenvironment and silk development on this pathway, we profiled cuticular waxes on the silks of the inbreds B73 and Mo17, and their reciprocal hybrids. Multivariate interrogation of these metabolite abundance data demonstrates that VLCFA-CoAs and total free VLCFAs are positively correlated with the cuticular wax metabolome, and this metabolome is primarily affected by changes in the silk microenvironment and plant genotype. Moreover, the genotype effect on the pathway explains the increased accumulation of cuticular hydrocarbons with a concomitant reduction in cuticular VLCFA accumulation on B73 silks, suggesting that the conversion of VLCFA-CoAs to hydrocarbons is more effective in B73 than Mo17. Statistical modeling of the ratios between cuticular hydrocarbons and cuticular VLCFAs reveals a significant role of precursor chain length in determining this ratio. This study establishes the complexity of the product-precursor relationships within the silk cuticular wax-producing network by dissecting both the impact of genotype and the allocation of VLCFA-CoA precursors to different biological processes and demonstrates that longer chain VLCFA-CoAs are preferentially utilized for hydrocarbon biosynthesis.

Defective cytokinin signaling reprograms lipid and flavonoid gene-to-metabolite networks to mitigate high salinity in Arabidopsis.

Cytokinin (CK) in plants regulates both developmental processes and adaptation to environmental stresses. Arabidopsis histidine phosphotransfer ahp2,3,5 and type-B Arabidopsis response regulator arr1,10,12 triple mutants are almost completely defective in CK signaling, and the ahp2,3,5 mutant was reported to be salt tolerant. Here, we demonstrate that the arr1,10,12 mutant is also more tolerant to salt stress than wild-type (WT) plants. A comprehensive metabolite profiling coupled with transcriptome analysis of the ahp2,3,5 and arr1,10,12 mutants was conducted to elucidate the salt tolerance mechanisms mediated by CK signaling. Numerous primary (e.g., sugars, amino acids, and lipids) and secondary (e.g., flavonoids and sterols) metabolites accumulated in these mutants under nonsaline and saline conditions, suggesting that both prestress and poststress accumulations of stress-related metabolites contribute to improved salt tolerance in CK-signaling mutants. Specifically, the levels of sugars (e.g., trehalose and galactinol), amino acids (e.g., branched-chain amino acids and γ-aminobutyric acid), anthocyanins, sterols, and unsaturated triacylglycerols were higher in the mutant plants than in WT plants. Notably, the reprograming of flavonoid and lipid pools was highly coordinated and concomitant with the changes in transcriptional levels, indicating that these metabolic pathways are transcriptionally regulated by CK signaling. The discovery of the regulatory role of CK signaling on membrane lipid reprogramming provides a greater understanding of CK-mediated salt tolerance in plants. This knowledge will contribute to the development of salt-tolerant crops with the ability to withstand salinity as a key driver to ensure global food security in the era of climate crisis.

Excretion of triacylglycerol as a matrix lipid facilitating apoplastic accumulation of a lipophilic metabolite shikonin.

Plants produce a large variety of lipophilic metabolites, many of which are secreted by cells and accumulated in apoplasts. These compounds often play a role to protect plants from environmental stresses. However, little is known about how these lipophilic compounds are secreted into apoplastic spaces. In this study, we used shikonin-producing cultured cells of Lithospermum erythrorhizon as an experimental model system to analyze the secretion of lipophilic metabolites, taking advantage of its high production rate and the clear inducibility in culture. Shikonin derivatives are lipophilic red naphthoquinone compounds that accumulate exclusively in apoplastic spaces of these cells and also in the root epidermis of intact plants. Microscopic analysis showed that shikonin is accumulated in the form of numerous particles on the cell wall. Lipidomic analysis showed that L. erythrorhizon cultured cells secrete an appreciable portion of triacylglycerol (24-38% of total triacylglycerol), composed predominantly of saturated fatty acids. Moreover, in vitro reconstitution assay showed that triacylglycerol encapsulates shikonin derivatives with phospholipids to form lipid droplet-like structures. These findings suggest a novel role for triacylglycerol as a matrix lipid, a molecular component involved in the secretion of specialized lipophilic metabolites.

Dual-Localized Enzymatic Components Constitute the Fatty Acid Synthase Systems in Mitochondria and Plastids.

Plant fatty acid biosynthesis occurs in both plastids and mitochondria. Here, we report the identification and characterization of Arabidopsis (Arabidopsis thaliana) genes encoding three enzymes shared between the mitochondria- and plastid-localized type II fatty acid synthase systems (mtFAS and ptFAS, respectively). Two of these enzymes, ß-ketoacyl-acyl carrier protein (ACP) reductase and enoyl-ACP reductase, catalyze two of the reactions that constitute the core four-reaction cycle of the FAS system, which iteratively elongates the acyl chain by two carbon atoms per cycle. The third enzyme, malonyl-coenzyme A:ACP transacylase, catalyzes the reaction that loads the mtFAS system with substrate by malonylating the phosphopantetheinyl cofactor of ACP. GFP fusion experiments revealed that the these enzymes localize to both chloroplasts and mitochondria. This localization was validated by characterization of mutant alleles, which were rescued by transgenes expressing enzyme variants that were retargeted only to plastids or only to mitochondria. The singular retargeting of these proteins to plastids rescued the embryo lethality associated with disruption of the essential ptFAS system, but these rescued plants displayed phenotypes typical of the lack of mtFAS function, including reduced lipoylation of the H subunit of the glycine decarboxylase complex, hyperaccumulation of glycine, and reduced growth. However, these latter traits were reversible in an elevated-CO2 atmosphere, which suppresses mtFAS-associated photorespiration-dependent chemotypes. Sharing enzymatic components between mtFAS and ptFAS systems constrains the evolution of these nonredundant fatty acid biosynthetic machineries.

Maize Glossy2 and Glossy2-like Genes Have Overlapping and Distinct Functions in Cuticular Lipid Deposition.

Plant epidermal cells express unique molecular machinery that juxtapose the assembly of intracellular lipid components and the unique extracellular cuticular lipids that are unidirectionally secreted to plant surfaces. In maize (Zea mays), mutations at the glossy2 (gl2) locus affect the deposition of extracellular cuticular lipids. Sequence-based genome scanning identified a new Gl2 homolog in the maize genome, namely Gl2-like Both the Gl2-like and Gl2 genes are members of the BAHD superfamily of acyltransferases, with close sequence similarity to the Arabidopsis (Arabidopsis thaliana) CER2 gene. Transgenic experiments demonstrated that Gl2-like and Gl2 functionally complement the Arabidopsis cer2 mutation, with differential influences on the cuticular lipids and the lipidome of the plant, particularly affecting the longer alkyl chain acyl lipids, especially at the 32-carbon chain length. Site-directed mutagenesis of the putative BAHD catalytic HXXXDX-motif indicated that Gl2-like requires this catalytic capability to fully complement the cer2 function, but Gl2 can accomplish complementation without the need for this catalytic motif. These findings demonstrate that Gl2 and Gl2-like overlap in their cuticular lipid function, but have evolutionarily diverged to acquire nonoverlapping functions.

Differential expression of SlKLUH controlling fruit and seed weight is associated with changes in lipid metabolism and photosynthesis-related genes.

The sizes of plant organs such as fruit and seed are crucial yield components. Tomato KLUH underlies the locus fw3.2, an important regulator of fruit and seed weight. However, the mechanism by which the expression levels of KLUH affect organ size is poorly understood. We found that higher expression of SlKLUH increased cell proliferation in the pericarp within 5 d post-anthesis in tomato near-isogenic lines. Differential gene expression analyses showed that lower expression of SlKLUH was associated with increased expression of genes involved in lipid metabolism. Lipidomic analysis revealed that repression of SlKLUH mainly increased the contents of certain non-phosphorus glycerolipids and phospholipids and decreased the contents of four unknown lipids. Co-expression network analyses revealed that lipid metabolism was possibly associated with but not directly controlled by SlKLUH, and that this gene instead controls photosynthesis-related processes. In addition, many transcription factors putatively involved in the KLUH pathway were identified. Collectively, we show that SlKLUH regulates fruit and seed weight which is associated with altered lipid metabolism. The results expand our understanding of fruit and seed weight regulation and offer a valuable resource for functional studies of candidate genes putatively involved in regulation of organ size in tomato and other crops.

HEAT INDUCIBLE LIPASE1 Remodels Chloroplastic Monogalactosyldiacylglycerol by Liberating α-Linolenic Acid in Arabidopsis Leaves under Heat Stress.

Under heat stress, polyunsaturated acyl groups, such as α-linolenate (18:3) and hexadecatrienoate (16:3), are removed from chloroplastic glycerolipids in various plant species. Here, we showed that a lipase designated HEAT INDUCIBLE LIPASE1 (HIL1) induces the catabolism of monogalactosyldiacylglycerol (MGDG) under heat stress in Arabidopsis thaliana leaves. Using thermotolerance tests, a T-DNA insertion mutant with disrupted HIL1 was shown to have a heat stress-sensitive phenotype. Lipidomic analysis indicated that the decrease of 34:6-MGDG under heat stress was partially impaired in the hil1 mutant. Concomitantly, the heat-induced increment of 54:9-triacylglycerol in the hil1 mutant was 18% lower than that in the wild-type plants. Recombinant HIL1 protein digested MGDG to produce 18:3-free fatty acid (18:3-FFA), but not 18:0- and 16:0-FFAs. A transient assay using fluorescent fusion proteins confirmed chloroplastic localization of HIL1. Transcriptome coexpression network analysis using public databases demonstrated that the HIL1 homolog expression levels in various terrestrial plants are tightly associated with chloroplastic heat stress responses. Thus, HIL1 encodes a chloroplastic MGDG lipase that releases 18:3-FFA in the first committed step of 34:6 (18:3/16:3)-containing galactolipid turnover, suggesting that HIL1 has an important role in the lipid remodeling process induced by heat stress in plants.

Discovery and Characterization of the 3-Hydroxyacyl-ACP Dehydratase Component of the Plant Mitochondrial Fatty Acid Synthase System.

We report the characterization of the Arabidopsis (Arabidopsis thaliana) 3-hydroxyacyl-acyl carrier protein dehydratase (mtHD) component of the mitochondrial fatty acid synthase (mtFAS) system, encoded by AT5G60335. The mitochondrial localization and catalytic capability of mtHD were demonstrated with a green fluorescent protein transgenesis experiment and by in vivo complementation and in vitro enzymatic assays. RNA interference (RNAi) knockdown lines with reduced mtHD expression exhibit traits typically associated with mtFAS mutants, namely a miniaturized morphological appearance, reduced lipoylation of lipoylated proteins, and altered metabolomes consistent with the reduced catalytic activity of lipoylated enzymes. These alterations are reversed when mthd-rnai mutant plants are grown in a 1% CO2 atmosphere, indicating the link between mtFAS and photorespiratory deficiency due to the reduced lipoylation of glycine decarboxylase. In vivo biochemical feeding experiments illustrate that sucrose and glycolate are the metabolic modulators that mediate the alterations in morphology and lipid accumulation. In addition, both mthd-rnai and mtkas mutants exhibit reduced accumulation of 3-hydroxytetradecanoic acid (i.e. a hallmark of lipid A-like molecules) and abnormal chloroplastic starch granules; these changes are not reversible by the 1% CO2 atmosphere, demonstrating two novel mtFAS functions that are independent of photorespiration. Finally, RNA sequencing analysis revealed that mthd-rnai and mtkas mutants are nearly equivalent to each other in altering the transcriptome, and these analyses further identified genes whose expression is affected by a functional mtFAS system but independent of photorespiratory deficiency. These data demonstrate the nonredundant nature of the mtFAS system, which contributes unique lipid components needed to support plant cell structure and metabolism.

Ancient rice cultivar extensively replaces phospholipids with non-phosphorus glycolipid under phosphorus deficiency.

Recycling of phosphorus (P) from P-containing metabolites is an adaptive strategy of plants to overcome soil P deficiency. This study was aimed at demonstrating differences in lipid remodelling between low-P-tolerant and -sensitive rice cultivars using lipidome profiling. The rice cultivars Akamai (low-P-tolerant) and Koshihikari (low-P-sensitive) were grown in a culture solution with [2 mg l-1 (+P)] or without (-P) phosphate for 21 and 28 days after transplantation. Upper and lower leaves were collected. Lipids were extracted from the leaves and their composition was analysed by liquid chromatography/mass spectrometry (LC-MS). Phospholipids, namely phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG) and phosphatidylinositol (PI), lysophosphatidylcholine (lysoPC), diacylglycerol (DAG), triacylglycerol (TAG) and glycolipids, namely sulfoquinovosyl diacylglycerol (SQDG), digalactosyldiacylglycerol (DGDG), monogalactosyldiacylglycerol (MGDG) and 1,2-diacyl-3-O-alpha-glucuronosyl glycerol (GlcADG), were detected. GlcADG level was higher in both cultivars grown in -P than in +P and the increase was larger in Akamai than in Koshihikari. DGDG, MGDG and SQDG levels were higher in Akamai grown in -P than in +P and the increase was larger in the upper leaves than in the lower leaves. PC, PE, PG and PI levels were lower in both cultivars grown in -P than in +P and the decrease was larger in the lower leaves than in the upper leaves and in Akamai than in Koshihikari. Akamai catabolised more phospholipids in older leaves and synthesised glycolipids in younger leaves. These results suggested that extensive phospholipid replacement with non-phosphorus glycolipids is a mechanism underlying low-P-tolerance in rice cultivars.

Deficiency of Starch Synthase IIIa and IVb Alters Starch Granule Morphology from Polyhedral to Spherical in Rice Endosperm.

Starch granule morphology differs markedly among plant species. However, the mechanisms controlling starch granule morphology have not been elucidated. Rice (Oryza sativa) endosperm produces characteristic compound-type granules containing dozens of polyhedral starch granules within an amyloplast. Some other cereal species produce simple-type granules, in which only one starch granule is present per amyloplast. A double mutant rice deficient in the starch synthase (SS) genes SSIIIa and SSIVb (ss3a ss4b) produced spherical starch granules, whereas the parental single mutants produced polyhedral starch granules similar to the wild type. The ss3a ss4b amyloplasts contained compound-type starch granules during early developmental stages, and spherical granules were separated from each other during subsequent amyloplast development and seed dehydration. Analysis of glucan chain length distribution identified overlapping roles for SSIIIa and SSIVb in amylopectin chain synthesis, with a degree of polymerization of 42 or greater. Confocal fluorescence microscopy and immunoelectron microscopy of wild-type developing rice seeds revealed that the majority of SSIVb was localized between starch granules. Therefore, we propose that SSIIIa and SSIVb have crucial roles in determining starch granule morphology and in maintaining the amyloplast envelope structure. We present a model of spherical starch granule production.

Isotopic Combinatomer Analysis Provides in Vivo Evidence of the Direct Epimerization of Monoglucosyl Diacylglycerol in Cyanobacteria.

Galactolipids constitute the majority of photosynthetic membranes called thylakoid membranes in cyanobacteria and chloroplasts of land plants and algae. The galactolipids, although identical in headgroup structure, are synthesized by significantly different pathways in cyanobacteria and chloroplasts. In the cyanobacterial pathway, monoglucosyl diacylglycerol (GlcDG) is synthesized first and then converted to monogalactosyl diacylglycerol (MGDG). On the basis of circumstantial evidence, the mechanism of conversion was thought to be epimerization at C-4, but no direct evidence has yet been provided, because there is no in vitro enzymatic system of the putative membrane-bound reaction. Labeling studies with 14C and 13C suggested that the labels in the headgroup and the acyl groups were kept at a reasonably constant ratio before and after the conversion. We then provide in vivo evidence of the direct epimerization based on detailed isotopomer analysis of the conversion, named "combinatomer analysis". The different types of molecules formed by the combination of labeled or unlabeled parts (sn-1 acyl, sn-2 acyl, glycerol, and hexose) are called here "combinatomers". Combinatomer analysis of the experiments with pulse labeling with 13C and chase in Anabaena sp. PCC 7118 indicated that the composition of combinatomers in the precursor GlcDG was kept unchanged in the product MGDG. Production of combinatomers resulting from exchange of hexose was minimal. This provides solid evidence of the epimerization of the glucose moiety of GlcDG, as well as the direct desaturation of acyl groups at the sn-1 position.

Metabolome-genome-wide association study dissects genetic architecture for generating natural variation in rice secondary metabolism.

Plants produce structurally diverse secondary (specialized) metabolites to increase their fitness for survival under adverse environments. Several bioactive compounds for new drugs have been identified through screening of plant extracts. In this study, genome-wide association studies (GWAS) were conducted to investigate the genetic architecture behind the natural variation of rice secondary metabolites. GWAS using the metabolome data of 175 rice accessions successfully identified 323 associations among 143 single nucleotide polymorphisms (SNPs) and 89 metabolites. The data analysis highlighted that levels of many metabolites are tightly associated with a small number of strong quantitative trait loci (QTLs). The tight association may be a mechanism generating strains with distinct metabolic composition through the crossing of two different strains. The results indicate that one plant species produces more diverse phytochemicals than previously expected, and plants still contain many useful compounds for human applications.

Conferring high-temperature tolerance to nontransgenic tomato scions using graft transmission of RNA silencing of the fatty acid desaturase gene.

We investigated graft transmission of high-temperature tolerance in tomato scions to nontransgenic scions from transgenic rootstocks, where the fatty acid desaturase gene (LeFAD7) was RNA-silenced. Tomato was transformed with a plasmid carrying an inverted repeat of LeFAD7 by Agrobacterium. Several transgenic lines showed the lower amounts of LeFAD7 RNA and unsaturated fatty acids, while nontransgenic control did not, and siRNA was detected in the transgenic lines, but not in control. These lines grew under conditions of high temperature, while nontransgenic control did not. Further, the nontransgenic plants were grafted onto the silenced transgenic plants. The scions showed less of the target gene RNA, and siRNA was detected. Under high-temperature conditions, these grafted plants grew, while control grafted plants did not. Thus, it was shown that high-temperature tolerance was conferred in the nontransgenic scions after grafting onto the silenced rootstocks.

Roles of lipids as signaling molecules and mitigators during stress response in plants.

Lipids are the major constituents of biological membranes that can sense extracellular conditions. Lipid-mediated signaling occurs in response to various environmental stresses, such as temperature change, salinity, drought and pathogen attack. Lysophospholipid, fatty acid, phosphatidic acid, diacylglycerol, inositol phosphate, oxylipins, sphingolipid, and N-acylethanolamine have all been proposed to function as signaling lipids. Studies on these stress-inducible lipid species have demonstrated that each lipid class has specific biological relevance, biosynthetic mechanisms and signaling cascades, which activate defense reactions at the transcriptional level. In addition to their roles in signaling, lipids also function as stress mitigators to reduce the intensity of stressors. To mitigate particular stresses, enhanced syntheses of unique lipids that accumulate in trace quantities under normal growth conditions are often observed under stressed conditions. The accumulation of oligogalactolipids and glucuronosyldiacylglycerol has recently been found to mitigate freezing and nutrition-depletion stresses, respectively, during lipid remodeling. In addition, wax, cutin and suberin, which are not constituents of the lipid bilayer, but are components derived from lipids, contribute to the reduction of drought stress and tissue injury. These features indicate that lipid-mediated defenses against environmental stress contributes to plant survival.

Leaf oil body functions as a subcellular factory for the production of a phytoalexin in Arabidopsis.

Oil bodies are intracellular structures present in the seed and leaf cells of many land plants. Seed oil bodies are known to function as storage compartments for lipids. However, the physiological function of leaf oil bodies is unknown. Here, we show that leaf oil bodies function as subcellular factories for the production of a stable phytoalexin in response to fungal infection and senescence. Proteomic analysis of oil bodies prepared from Arabidopsis (Arabidopsis thaliana) leaves identified caleosin (CLO3) and α-dioxygenase (α-DOX1). Both CLO3 and α-DOX1 were localized on the surface of oil bodies. Infection with the pathogenic fungus Colletotrichum higginsianum promoted the formation of CLO3- and α-DOX1-positive oil bodies in perilesional areas surrounding the site of infection. α-DOX1 catalyzes the reaction from α-linolenic acid (a major fatty acid component of oil bodies) to an unstable compound, 2-hydroperoxy-octadecatrienoic acid (2-HPOT). Intriguingly, a combination of α-DOX1 and CLO3 produced a stable compound, 2-hydroxy-octadecatrienoic acid (2-HOT), from α-linolenic acid. This suggests that the colocalization of α-DOX1 and CLO3 on oil bodies might prevent the degradation of unstable 2-HPOT by efficiently converting 2-HPOT into the stable compound 2-HOT. We found that 2-HOT had antifungal activity against members of the genus Colletotrichum and that infection with C. higginsianum induced 2-HOT production. These results defined 2-HOT as an Arabidopsis phytoalexin. This study provides, to our knowledge, the first evidence that leaf oil bodies produce a phytoalexin under a pathological condition, which suggests a new mechanism of plant defense.

Induced accumulation of glucuronosyldiacylglycerol in tomato and soybean under phosphorus deprivation.

Glucuronosyldiacylglycerol (GlcADG) is a plant glycolipid that accumulates in Arabidopsis and rice in response to phosphorus (P) starvation. It has been suggested that GlcADG functions to mitigate the stress induced by P depletion. Biosynthesis of GlcADG requires sulfolipid (SQDG) synthase, which is coded for in plant genomes. This indicates the possibility that GlcADG may be a general constituent of membrane lipids in plants. In this study, we investigated the SQDG synthases found in the genomes of higher plants, ferns, mosses, algae and cyanobacteria. In addition, we analyzed GlcADG accumulation, and the expression of SQDG synthase homologs in tomato and soybean plants grown under P-limited conditions. LC-MS analysis of lipids from these plants confirmed that GlcADG accumulated during P deprivation, as previously observed in Arabidopsis and rice. We also observed upregulation of SQDG synthase transcripts in these plants during P deprivation. These data suggest that GlcADG is present not only in model plants, but also in various other plant species, and that this lipid molecule performs an important physiological function as a mitigator of P-deprivation stress in plants.

LipidBlast templates as flexible tools for creating new in-silico tandem mass spectral libraries.

Tandem mass spectral libraries (MS/MS) are usually built by acquiring experimentally measured mass spectra from chemical reference compounds. We here show the versatility of in-silico or computer generated tandem mass spectra that are directly obtained from compound structures. We use the freely available LipidBlast development software to generate 15 000 MS/MS spectra of the glucuronosyldiacylglycerol (GlcADG) lipid class, recently discovered for the first time in plants. The generation of such an in-silico MS/MS library for positive and negative ionization mode took 5 h development time, including the validation of the obtained mass spectra. Such libraries allow for high-throughput annotations of previously unknown glycolipids. The publicly available LipidBlast templates are universally applicable for the development of MS/MS libraries for novel lipid classes.

The phosphorylated pathway of serine biosynthesis affects sperm, embryo, and sporophyte development, and metabolism in Marchantia polymorpha.

Serine metabolism is involved in various biological processes. Here we investigate primary functions of the phosphorylated pathway of serine biosynthesis in a non-vascular plant Marchantia polymorpha by analyzing knockout mutants of MpPGDH encoding 3-phosphoglycerate dehydrogenase in this pathway. Growth phenotypes indicate that serine from the phosphorylated pathway in the dark is crucial for thallus growth. Sperm development requires serine from the phosphorylated pathway, while egg formation does not. Functional MpPGDH in the maternal genome is necessary for embryo and sporophyte development. Under high CO2 where the glycolate pathway of serine biosynthesis is inhibited, suppressed thallus growth of the mutants is not fully recovered by exogenously-supplemented serine, suggesting the importance of serine homeostasis involving the phosphorylated and glycolate pathways. Metabolomic phenotypes indicate that the phosphorylated pathway mainly influences the tricarboxylic acid cycle, the amino acid and nucleotide metabolism, and lipid metabolism. These results indicate the importance of the phosphorylated pathway of serine biosynthesis in the dark, in the development of sperm, embryo, and sporophyte, and metabolism in M. polymorpha.

Dissection of genotype-phenotype associations in rice grains using metabolome quantitative trait loci analysis.

A comprehensive and large-scale metabolome quantitative trait loci (mQTL) analysis was performed to investigate the genetic backgrounds associated with metabolic phenotypes in rice grains. The metabolome dataset consisted of 759 metabolite signals obtained from the grains of 85 lines of rice (Oryza sativa, Sasanishiki × Habataki back-crossed inbred lines). Metabolome analysis was performed using four mass spectrometry pipelines to enhance detection of different classes of metabolites. This mQTL analysis of a wide range of metabolites highlighted an uneven distribution of 802 mQTLs on the rice genome, as well as different modes of metabolic trait (m-trait) control among various types of metabolites. The levels of most metabolites within rice grains were highly sensitive to environmental factors, but only weakly associated with mQTLs. Coordinated control was observed for several groups of metabolites, such as amino acids linked to the mQTL hotspot on chromosome 3. For flavonoids, m-trait variation among the experimental lines was tightly governed by genetic factors that alter the glycosylation of flavones. Many loci affecting levels of metabolites were detected by QTL analysis, and plausible gene candidates were evaluated by in silico analysis. Several mQTLs profoundly influenced metabolite levels, providing insight into the control of rice metabolism. The genomic region and genes potentially responsible for the biosynthesis of apigenin-6,8-di-C-α-l-arabinoside are presented as an example of a critical mQTL identified by the analysis.

Studies on vacuolar membrane microdomains isolated from Arabidopsis suspension-cultured cells: local distribution of vacuolar membrane proteins.

The local distribution of both the vacuolar-type proton ATPase (V-ATPase) and the vacuolar-type proton pyrophosphatase (V-PPase), the main vacuolar proton pumps, was investigated in intact vacuoles isolated from Arabidopsis suspension-cultured cells. Fluorescent immunostaining showed that V-PPase was distributed evenly on the vacuolar membrane (VM), but V-ATPase localized to specific regions of the VM. We hypothesize that there may be membrane microdomains on the VM. To confirm this hypothesis, we prepared detergent-resistant membranes (DRMs) from the VM in accordance with well established conventional methods. Analyses of fatty acid composition suggested that DRMs had more saturated fatty acids compared with the whole VM in phosphatidylcholine and phosphatidylethanolamine. In the proteomic analyses of both DRMs and detergent-soluble mebranes (DSMs), we confirmed the different local distributions of V-ATPase and V-PPase. The observations of DRMs with an electron microscope supported the existence of different areas on the VM. Moreover, it was observed using total internal reflection fluorescent microscopy (TIRFM) that proton pumps were frequently immobilized at specific sites on the VM. In the proteomic analyses, we also found that many other vacuolar membrane proteins are distributed differently in DRMs and DSMs. Based on the results of this study, we discuss the possibility that VM microdomains might contribute to vacuolar dynamics.