Yozo Okazaki

A Chloroplastic UDP-Glucose Pyrophosphorylase from Arabidopsis Is the Committed Enzyme for the First Step of Sulfolipid Biosynthesis

Plants synthesize a sulfur-containing lipid, sulfoquinovosyldiacylglycerol, which is one of three nonphosphorus glycerolipids that provide the bulk of the structural lipids in photosynthetic membranes. Here, the identification of a novel gene, UDP-glucose pyrophosphorylase3 (UGP3), required for sulfolipid biosynthesis is described. Transcriptome coexpression analysis demonstrated highly correlated expression of UGP3 with known genes for sulfolipid biosynthesis in Arabidopsis thaliana. Liquid chromatography–mass spectrometry analysis of leaf lipids in two Arabidopsis ugp3 mutants revealed that no sulfolipid was accumulated in these mutants, indicating the participation of UGP3 in sulfolipid biosynthesis. From the deduced amino acid sequence, UGP3 was presumed to be a UDP-glucose pyrophosphorylase (UGPase) involved in the generation of UDP-glucose, serving as the precursor of the polar head of sulfolipid. Recombinant UGP3 was able to catalyze the formation of UDP-glucose from glucose-1-phosphate and UTP. A transient assay using fluorescence fusion proteins and UGPase activity in isolated chloroplasts indicated chloroplastic localization of UGP3. The transcription level of UGP3 was increased by phosphate starvation. A comparative genomics study on UGP3 homologs across different plant species suggested the structural and functional conservation of the proteins and, thus, a committing role for UGP3 in sulfolipid synthesis.

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.

Recent advances of metabolomics in plant biotechnology

Biotechnology, including genetic modification, is a very important approach to regulate the production of particular metabolites in plants to improve their adaptation to environmental stress, to improve food quality, and to increase crop yield. Unfortunately, these approaches do not necessarily lead to the expected results due to the highly complex mechanisms underlying metabolic regulation in plants. In this context, metabolomics plays a key role in plant molecular biotechnology, where plant cells are modified by the expression of engineered genes, because we can obtain information on the metabolic status of cells via a snapshot of their metabolome. Although metabolome analysis could be used to evaluate the effect of foreign genes and understand the metabolic state of cells, there is no single analytical method for metabolomics because of the wide range of chemicals synthesized in plants. Here, we describe the basic analytical advancements in plant metabolomics and bioinformatics and the application of metabolomics to the biological study of plants.

A new class of plant lipid is essential for protection against phosphorus depletion

Phosphorus supply is a major factor responsible for reduced crop yields. As a result, plants utilize various adaptive mechanisms against phosphorus depletion, including lipid remodelling. Here we report the involvement of a novel plant lipid, glucuronosyldiacylglycerol, against phosphorus depletion. Lipidomic analysis of Arabidopsis plants cultured in phosphorus-depleted conditions revealed inducible accumulation of glucuronosyldiacylglycerol. Investigation using a series of sulfolipid sulfoquinovosyldiacylglycerol synthesis-deficient mutants of Arabidopsis determined that the biosynthesis of glucuronosyldiacylglycerol shares the pathway of sulfoquinovosyldiacylglycerol synthesis in chloroplasts. Under phosphorus-depleted conditions, the Arabidopsis sqd2 mutant, which does not accumulate either sulfoquinovosyldiacylglycerol or glucuronosyldiacylglycerol, was the most severely damaged of three sulfoquinovosyldiacylglycerol-deficient mutants. As glucuronosyldiacylglycerol is still present in the other two mutants, this result indicates that glucuronosyldiacylglycerol has a role in the protection of plants against phosphorus limitation stress. Glucuronosyldiacylglycerol was also found in rice, and its concentration increased significantly following phosphorus limitation, suggesting a shared physiological significance of this novel lipid against phosphorus depletion in plants.

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.

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.

OsATG7 is required for autophagy-dependent lipid metabolism in rice postmeiotic anther development

In flowering plants, the tapetum, the innermost layer of the anther, provides both nutrient and lipid components to developing microspores, pollen grains, and the pollen coat. Though the programmed cell death of the tapetum is one of the most critical and sensitive steps for fertility and is affected by various environmental stresses, its regulatory mechanisms remain mostly unknown. Here we show that autophagy is required for the metabolic regulation and nutrient supply in anthers and that autophagic degradation within tapetum cells is essential for postmeiotic anther development in rice. Autophagosome-like structures and several vacuole-enclosed lipid bodies were observed in postmeiotic tapetum cells specifically at the uninucleate stage during pollen development, which were completely abolished in a retrotransposon-insertional OsATG7 (autophagy-related 7)-knockout mutant defective in autophagy, suggesting that autophagy is induced in tapetum cells. Surprisingly, the mutant showed complete sporophytic male sterility, failed to accumulate lipidic and starch components in pollen grains at the flowering stage, showed reduced pollen germination activity, and had limited anther dehiscence. Lipidomic analyses suggested impairment of editing of phosphatidylcholines and lipid desaturation in the mutant during pollen maturation. These results indicate a critical involvement of autophagy in a reproductive developmental process of rice, and shed light on the novel autophagy-mediated regulation of lipid metabolism in eukaryotic cells.

Leaf Oil Body Functions as a Subcellular Factory for the Production of a Phytoalexin in Arabidopsis

Leaf oil bodies are the source of a novel phytoalexin produced in response to fungal infection and senescence. 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.

Toward better annotation in plant metabolomics: isolation and structure elucidation of 36 specialized metabolites from Oryza sativa (rice) by using MS/MS and NMR analyses

Metabolomics plays an important role in phytochemical genomics and crop breeding; however, metabolite annotation is a significant bottleneck in metabolomic studies. In particular, in liquid chromatography–mass spectrometry (MS)-based metabolomics, which has become a routine technology for the profiling of plant-specialized metabolites, a substantial number of metabolites detected as MS peaks are still not assigned properly to a single metabolite. Oryza sativa (rice) is one of the most important staple crops in the world. In the present study, we isolated and elucidated the structures of specialized metabolites from rice by using MS/MS and NMR. Thirty-six compounds, including five new flavonoids and eight rare flavonolignan isomers, were isolated from the rice leaves. The MS/MS spectral data of the isolated compounds, with a detailed interpretation of MS fragmentation data, will facilitate metabolite annotation of the related phytochemicals by enriching the public mass spectral data depositories, including the plant-specific MS/MS-based database, ReSpect.

Landscape of the lipidome and transcriptome under heat stress in Arabidopsis thaliana

Environmental stress causes membrane damage in plants. Lipid studies are required to understand the adaptation of plants to climate change. Here, LC-MS-based lipidomic and microarray transcriptome analyses were carried out to elucidate the effect of short-term heat stress on the Arabidopsis thaliana leaf membrane. Vegetative plants were subjected to high temperatures for one day, and then grown under normal conditions. Sixty-six detected glycerolipid species were classified according to patterns of compositional change by Spearman’s correlation coefficient. Triacylglycerols, 36:4- and 36:5-monogalactosyldiacylglycerol, 34:2- and 36:2-digalactosyldiacylglycerol, 34:1-, 36:1- and 36:6-phosphatidylcholine, and 34:1-phosphatidylethanolamine increased by the stress and immediately decreased during recovery. The relative amount of one triacylglycerol species (54:9) containing α-linolenic acid (18:3) increased under heat stress. These results suggest that heat stress in Arabidopsis leaves induces an increase in triacylglycerol levels, which functions as an intermediate of lipid turnover, and results in a decrease in membrane polyunsaturated fatty acids. Microarray data revealed candidate genes responsible for the observed metabolic changes.