Wenyun Shen

Arabidopsis AtGPAT1, a Member of the Membrane-Bound Glycerol-3-Phosphate Acyltransferase Gene Family, Is Essential for Tapetum Differentiation and Male Fertility

Membrane-bound glycerol-3-phosphate acyltransferase (GPAT; EC 2.3.1.15) mediates the initial step of glycerolipid biosynthesis in the extraplastidic compartments of plant cells. Here, we report the molecular characterization of a novel GPAT gene family from Arabidopsis, designated AtGPAT. The corresponding polypeptides possess transmembrane domains and GPAT activity when expressed heterologously in a yeast lipid mutant. The functional significance of one isoform, AtGPAT1, is the focus of the present study. Disruption of the AtGPAT1 gene causes a massive pollen development arrest, and subsequent introduction of the gene into the mutant plant rescues the phenotype, illustrating a pivotal role for AtGPAT1 in pollen development. Microscopic examinations revealed that the gene lesion results in a perturbed degeneration of the tapetum, which is associated with altered endoplasmic reticulum profiles and reduced secretion. In addition to the sporophytic effect, AtGPAT1 also exerts a gametophytic effect on pollen performance, as the competitive ability of a pollen grain to pollinate is dependent on the presence of an AtGPAT1 gene. Deficiency in AtGPAT1 correlates with several fatty acid composition changes in flower tissues and seeds. Unexpectedly, however, a loss of AtGPAT1 causes no significant change in seed oil content.

Metabolic Interactions between the Lands Cycle and the Kennedy Pathway of Glycerolipid Synthesis in Arabidopsis Developing Seeds

The Kennedy pathway and the Lands cycle are two principal metabolic modules of glycerolipid metabolism. This work examines the crosstalk of these two pathways and shows that loss of Lands cycle activity leads to an enhanced de novo phosphatidylcholine PC synthesis through the Kennedy pathway and PC turnover in Arabidopsis developing seeds. It has been widely accepted that the primary function of the Lands cycle is to provide a route for acyl remodeling to modify fatty acid (FA) composition of phospholipids derived from the Kennedy pathway. Lysophosphatidylcholine acyltransferase (LPCAT) is an evolutionarily conserved key enzyme in the Lands cycle. In this study, we provide direct evidence that the Arabidopsis thaliana LPCATs, LPCAT1 and LPCAT2, participate in the Lands cycle in developing seeds. In spite of a substantially reduced initial rate of nascent FA incorporation into phosphatidylcholine (PC), the PC level in the double mutant lpcat1 lpcat2-2 remained unchanged. LPCAT deficiency triggered a compensatory response of de novo PC synthesis and a concomitant acceleration of PC turnover that were attributable at least in part to PC deacylation. Acyl-CoA profile analysis revealed complicated metabolic alterations rather than merely reduced acyl group shuffling from PC in the mutant. Shifts in FA stereo-specific distribution in triacylglycerol of the mutant seed suggested a preferential retention of saturated acyl chains at the stereospecific numbering (sn)-1 position from PC and likely a channeling of lysophosphatidic acid, derived from PC, into the Kennedy pathway. Our study thus illustrates an intricate relationship between the Lands cycle and the Kennedy pathway.

Analysis of a Blumeria graminis-Secreted Lipase Reveals the Importance of Host Epicuticular Wax Components for Fungal Adhesion and Development

The biotrophic powdery mildew fungus Blumeria graminis releases extracellular materials to the surface of fungal infection structures that facilitate anchoring them to hydrophobic plant surfaces prior to infection; however, the chemistry of fungal adhesives and the mechanism of adhesion remain largely unclear. Expressed sequence tag analysis led to identification of a secreted lipase, Lip1, from B. graminis. Expression of LIP1 is dramatically upregulated during the early stages of fungal development. Lip1, secreted to the surface of fungal cell walls, possesses lipolytic activity against a broad range of glycerides and releases alkanes and primary fatty alcohols from the epicuticular wax of wheat leaves. Of the epicuticular wax components released by Lip1 activity, long-chain alkanes are the most efficient cues for triggering appressorium formation. Pretreatment of wheat leaves with Lip1, thereby removing leaf surface wax, severely compromises components of fungal pathogenicity, including conidial adhesion, appressorium formation, and secondary hypha growth. Our data suggest that Lip1 activity releases cues from the host surface to promote pathogen development and infection.

Understanding the Biochemical Basis of Temperature-Induced Lipid Pathway Adjustments in Plants

Analysis of three plant species with distinct patterns of lipid profiles addresses how glycerolipid pathways in the ER and chloroplast are coordinated under temperature stress at the metabolite and transcript levels. Glycerolipid biosynthesis in plants proceeds through two major pathways compartmentalized in the chloroplast and the endoplasmic reticulum (ER). The involvement of glycerolipid pathway interactions in modulating membrane desaturation under temperature stress has been suggested but not fully explored. We profiled glycerolipid changes as well as transcript dynamics under suboptimal temperature conditions in three plant species that are distinctively different in the mode of lipid pathway interactions. In Arabidopsis thaliana, a 16:3 plant, the chloroplast pathway is upregulated in response to low temperature, whereas high temperature promotes the eukaryotic pathway. Operating under a similar mechanistic framework, Atriplex lentiformis at high temperature drastically increases the contribution of the eukaryotic pathway and correspondingly suppresses the prokaryotic pathway, resulting in the switch of lipid profile from 16:3 to 18:3. In wheat (Triticum aestivum), an 18:3 plant, low temperature also influences the channeling of glycerolipids from the ER to chloroplast. Evidence of differential trafficking of diacylglycerol moieties from the ER to chloroplast was uncovered in three plant species as another layer of metabolic adaptation under temperature stress. We propose a model that highlights the predominance and prevalence of lipid pathway interactions in temperature-induced lipid compositional changes.

Metabolic and transcriptional responses of glycerolipid pathways to a perturbation of glycerol 3-phosphate metabolism in Arabidopsis.

Glycerolipid synthesis in plants involves two major metabolic pathways compartmentalized in the chloroplasts and cytosol, respectively. Although these two parallel pathways are regulated with considerable flexibility, the factors mediating this process remain unclear. To investigate the influence of glycerol 3-phosphate (Gly-3-P) on the interactions of the glycerolipid pathways, we generated transgenic Arabidopsis lines with a feedback-resistant Gly-3-P dehydrogenase gene (gpsAFR) from Escherichia coli. gpsAFR was detected in the cytosol, but augmented Gly-3-P levels were observed in the cytosol as well as in chloroplasts. Glycerolipid composition and fatty acid positional distribution analyses revealed an altered fatty acid flux that affected not only the molar ratios of glycerolipid species but also their fatty acid composition. To decipher this complex pathway, a transgenic line was subjected to lipidomic analysis and a global gene-expression survey. The results revealed that changes in Gly-3-P metabolism caused altered expression of a broad array of genes. When viewed from the perspective of glycerolipid metabolism, coherent networks emerged, revealing that many enzymatic components of the glycerolipid pathways operate in a modular manner under the influence of Gly-3-P. Transcript levels of the enzymes involved in the prokaryotic pathway were mostly induced, whereas genes of the eukaryotic pathway enzymes were largely suppressed. Hence, the gene-expression changes were consistent with the detected biochemical phenotype. Our results suggest that Gly-3-P modulates the balance of the two glycerolipid pathways in Arabidopsis by influencing both metabolic flux and gene transcription.

Deciphering the roles of Arabidopsis LPCAT and PAH in phosphatidylcholine homeostasis and pathway coordination for chloroplast lipid synthesis

Phosphatidylcholine (PC) is a key intermediate in the metabolic network of glycerolipid biosynthesis. Lysophosphatidylcholine acyltransferase (LPCAT) and phosphatidic acid phosphatase (PAH) are two key enzymes of PC homeostasis. We report that LPCAT activity is markedly induced in the Arabidopsis pah mutant. The quadruple pah lpcat mutant, with dual defects in PAH and LPCAT, had a level of lysophosphatidylcholine (LPC) that was much higher than that in the lpcat mutants and a PC content that was higher than that in the pah mutant. Comparative molecular profile analysis of monogalactosyldiacylglycerol and digalactosyldiacylglycerol revealed that both the pah and pah lpcat mutants had increased proportions of 34:6 from the prokaryotic pathway despite differing levels of LPCAT activity. We show that a decreased representation of the C16:0 C18:2 diacylglycerol moiety in PC was a shared feature of pah and pah lpcat, and that this change in PC metabolic profile correlated with the increased prokaryotic contribution to chloroplast lipid synthesis. We detected increased PC deacylation in the pah lpcat mutant that was attributable at least in part to the induced phospholipases. Increased LPC generation was also evident in the pah mutant, but the phospholipases were not induced, raising the possibility that PC deacylation is mediated by the reverse reaction of LPCAT. We discuss possible roles of LPCAT and PAH in PC turnover that impacts lipid pathway coordination for chloroplast lipid synthesis.

Adjustments of lipid pathways in plant adaptation to temperature stress

Modulation of membrane lipid composition under varying environmental conditions is an important part of plant stress adaptation. Most notably, proportional changes of lipid composition in response to temperature changes are a major cellular response to requirements of membrane fluidity adjustment. In higher plants, synthesis of glycerolipids is accomplished by 2 major pathways, the prokaryotic and eukaryotic pathway, located in the chloroplast and the endoplasmic reticulum (ER), respectively. Recently, we systematically investigated the re-adjustments of glycerolipid pathways under temperature stress at the metabolite and transcript levels using 3 plant species with distinct lipid profiles. The relative contributions of 2 pathways and lipid channeling from the ER and chloroplast were both observed in plants under temperature stress. Potential factors controlling the lipid flux were identified through transcriptome analysis.

Effects of eIFiso4G1 mutation on seed oil biosynthesis

Fatty acid biosynthesis is a primary metabolic pathway that occurs in plastids, whereas the formation of glycerolipid molecules for the majority of cellular membrane systems and the deposition of storage lipid in seeds takes place in the cytosolic compartment. In this report, we present a study of an Arabidopsis mutant, ar21, with a novel seed fatty acid phenotype showing higher contents of eicosanoic acid (20:1) and oleic acid (18:1) and a reduced level of α-linolenic acid (18:3). A combination of map-based cloning and whole-genome sequencing identified the genetic basis underlying the fatty acid phenotype as a lesion in the plant-specific eukaryotic translation initiation factor eIFiso4G1. Transcriptome analysis on developing seeds revealed a reduced level of plastid-encoded genes. Specifically, decreases in both transcript and protein levels of an enzyme involved in fatty acid biosynthesis, the β-subunit of the plastidic heteromeric acetyl-CoA carboxylase (htACCase) encoded by accD, were evident in the mutant. Biochemical assays showed that the developing seeds of the mutant possessed a decreased htACCase activity in the plastid but an elevated activity of homomeric acetyl-CoA carboxylase (hmACCase). These results suggested that the increased 20:1 was attributable at least in part to the enhanced cytosolic hmACCase activity. We also detected a significant repression of FATTY ACID DESATURASE 3 (FAD3) during seed development, which correlated with a decreased 18:3 level in seed oil. Together, our study on a mutant of eIFiso4G1 uncovered multifaceted interactions between the cytosolic and plastidic compartments in seed lipid biosynthesis that impact major seed oil traits.

CIPK9 is involved in seed oil regulation in Brassica napus L. and Arabidopsis thaliana (L.) Heynh.

BackgroundAccumulation of storage compounds during seed development plays an important role in the life cycle of oilseed plants; these compounds provide carbon and energy resources to support the establishment of seedlings.ResultsIn this study, we show that BnCIPK9 has a broad expression pattern in Brassica napus L. tissues and that wounding stress strongly induces its expression. The overexpression of BnCIPK9 during seed development reduced oil synthesis in transgenic B. napus compared to that observed in wild-type (WT) plants. Functional analysis revealed that seed oil content (OC) of complementation lines was similar to that of WT plants, whereas OC in Arabidopsis thaliana (L.) Heynh. Atcipk9 knockout mutants (cipk9) was higher than that of WT plants. Seedling of cipk9 mutants failed to establish roots on a sugar-free medium, but root establishment could be rescued by supplementation of sucrose or glucose. The phenotype of complementation transgenic lines was similar to that of WT plants when grown on sugar-free medium. Mutants, cipk9, cbl2, and cbl3 presented similar phenotypes, suggesting that CIPK9, CBL2, and CBL3 might work together and play similar roles in root establishment under sugar-free condition.ConclusionThis study showed that BnCIPK9 and AtCIPK9 encode a protein kinase that is involved in sugar-related response and plays important roles in the regulation of energy reserves. Our results suggest that AtCIPK9 negatively regulates lipid accumulation and has a significant effect on early seedling establishment in A. thaliana. The functional characterization of CIPK9 provides insights into the regulation of OC, and might be used for improving OC in B. napus. We believe that our study makes a significant contribution to the literature because it provides information on how CIPKs coordinate stress regulation and energy signaling.

The D-lactate dehydrogenase MoDLD1-mediated cellular metabolism is essential for growth and infection-related development in Magnaporthe oryzae

Rice blast disease caused by Magnaporthe oryzae is initiated by the attachment of conidia to plant surfaces. Germ tubes emerging from conidia develop melanized appressoria to physically penetrate the host surface. Previous studies revealed that appressorium development requires the breakdown of storage lipids and glycogen that occur in peroxisomes and the cytosol respectively, culminating in production of pyruvate. However, the downstream product(s) entering the mitochondria for further oxidation is unclear. In this study, we aimed to investigate the molecular basis underlying the metabolic flux towards the mitochondria associated with the infectious-related development in M. oryzae. We showed that D-lactate is a key intermediate metabolite of the mobilization of lipids and glycogen, and its oxidative conversion to pyruvate is catalysed by a mitochondrial D-lactate dehydrogenase MoDLD1. Deletion of MoDLD1 caused defects in conidiogenesis and appressorium formation, and subsequently the loss of fungal pathogenicity. Further analyses demonstrated that MoDLD1 activity is involved in the maintenance of redox homeostasis during conidial germination. Thus, MoDLD1 is a critical modulator that channels metabolite flow to the mitochondrion coupling cellular redox state, and contributes to development and virulence of M. oryzae.