A Pair of Arabidopsis Diacylglycerol Kinases Essential for Gametogenesis and Endoplasmic Reticulum Phospholipid Metabolism in Leaves and Flowers.

Phosphatidic acid (PA) is a key phospholipid in glycerolipid metabolism and signaling. Diacylglycerol kinase (DGK) produces PA by phosphorylating diacylglycerol, a crucial step in PA metabolism. Although DGK activity is known to be involved in plant development and stress response, how specific DGK isoforms function in development and phospholipid metabolism remains elusive. Here, we showed that Arabidopsis (Arabidopsis thaliana) DGK2 and DGK4 are crucial for gametogenesis and biosynthesis of phosphatidylglycerol and phosphatidylinositol in the endoplasmic reticulum (ER). With comprehensive transcriptomic data of seven DGKs and genetic crossing, we found that dgk2-1/- dgk4-1/- plants were gametophyte lethal, although parental single homozygous plants were viable. The dgk2-1/+ dgk4-1/+ double heterozygote showed defective pollen tube growth and seed development because of nonviable mutant gametes. DGK2 and DGK4 were localized to the ER and were involved in PA production for pollen tube growth. Transgenic knockdown lines of DGK2 and DGK4 confirmed the gametophyte defect and also revealed defective leaf and root growth. Glycerolipid analysis in the knockdown lines showed that phosphatidylglycerol and phosphatidylinositol metabolism was affected differently in floral buds and leaves. These results suggest that DGK2 and DGK4 are essential during gametogenesis and are required for ER-localized phospholipid metabolism in vegetative and reproductive growth.

Membrane lipid polyunsaturation mediated by FATTY ACID DESATURASE 2 (FAD2) is involved in endoplasmic reticulum stress tolerance in Arabidopsis thaliana.

Unsaturation of membrane glycerolipid classes at their hydrophobic fatty acid tails critically affects the physical nature of the lipid molecule. In Arabidopsis thaliana, 7 fatty acid desaturases (FADs) differently desaturate each glycerolipid class in plastids and the endoplasmic reticulum (ER). Here, we showed that polyunsaturation of ER glycerolipids is required for the ER stress response. Through systematic screening of FAD mutants, we found that a mutant of FAD2 resulted in a hypersensitive response to tunicamycin, a chemical inducer of ER stress. FAD2 converts oleic acid to linoleic acid of the fatty acyl groups of ER-synthesized phospholipids. Our functional in vivo reporter assay revealed the ER localization and distinct tissue-specific expression patterns of FAD2. Moreover, glycerolipid profiling of both mutants and overexpressors of FAD2 under tunicamycin-induced ER stress conditions, along with phenotypic screening of the mutants of the FAD family, suggested that the ratio of monounsaturated fatty acids to polyunsaturated fatty acids, particularly 18:1 to 18:2 species, may be an important factor in allowing the ER membrane to cope with ER stress. Therefore, our results suggest that membrane lipid polyunsaturation mediated by FAD2 is involved in ER stress tolerance in Arabidopsis.

LYSOPHOSPHATIDIC ACID ACYLTRANSFERASES 4 and 5 are involved in glycerolipid metabolism and nitrogen starvation response in Arabidopsis.

Nitrogen (N) deficiency triggers an accumulation of a storage lipid triacylglycerol (TAG) in seed plants and algae. Whereas the metabolic pathway and regulatory mechanism to synthesize TAG from diacylglycerol are well known, enzymes involved in the supply of diacylglycerol remain elusive under N starvation. Lysophosphatidic acid acyltransferase (LPAT) catalyzes an important step of the de novo phospholipid biosynthesis pathway and thus has a strong flux control in the biosynthesis of phospholipids and TAG. Five LPAT isoforms are known in Arabidopsis; however, the functions of LPAT4 and LPAT5 remain elusive. Here, we show that LPAT4 and LPAT5 are functional endoplasmic-reticulum-localized LPATs. Seedlings of the double knockout mutant lpat4-1 lpat5-1 showed reduced content of phospholipids and TAG under normal growth condition. Under N starvation, lpat4-1 lpat5-1 seedlings showed severer growth defect than the wild-type in shoot. The phenotype was similar to dgat1-4, which affects a major TAG biosynthesis pathway and showed similarly reduced TAG content as the lpat4-1 lpat5-1. We suggest that LPAT4 and LPAT5 may redundantly function in endoplasmic-reticulum-localized de novo glycerolipid biosynthesis for phospholipids and TAG, which is important for the N starvation response in Arabidopsis.

Membrane glycerolipid equilibrium under endoplasmic reticulum stress in Arabidopsis thaliana.

Endoplasmic reticulum (ER) is an indispensable organelle for secretory protein synthesis as well as metabolism of phospholipids and their derivatives in eukaryotic cells. Various external and internal factors may cause an accumulation of aberrant proteins in the ER, which causes ER stress and activates cellular ER stress responses to cope with the stress. In animal research, molecular mechanisms for protein quality control upon ER stress are well documented; however, how cells maintain lipid homeostasis under ER stress is an emerging issue. The ratio of phosphatidylcholine (PC) to phosphatidylethanolamine (PE), two major phospholipid classes, is important under ER stress in animal cells. However, in seed plants, no study has reported on the changes in membrane lipid content under ER stress, although a number of physiologically important environmental stresses, such as heat and salinity, induce ER stress. Here, we investigated membrane glycerolipid metabolism under ER stress in Arabidopsis. ER stress transcriptionally affected PC and PE biosynthesis pathways differentially, with no significant changes in membrane glycerolipid content. Our results suggest that higher plants maintain membrane lipid equilibrium during active transcription of phospholipid biosynthetic genes under ER stress.

Enhanced root growth in phosphate-starved Arabidopsis by stimulating de novo phospholipid biosynthesis through the overexpression of LYSOPHOSPHATIDIC ACID ACYLTRANSFERASE 2 (LPAT2).

Upon phosphate starvation, plants retard shoot growth but promote root development presumably to enhance phosphate assimilation from the ground. Membrane lipid remodelling is a metabolic adaptation that replaces membrane phospholipids by non-phosphorous galactolipids, thereby allowing plants to obtain scarce phosphate yet maintain the membrane structure. However, stoichiometry of this phospholipid-to-galactolipid conversion may not account for the massive demand of membrane lipids that enables active growth of roots under phosphate starvation, thereby suggesting the involvement of de novo phospholipid biosynthesis, which is not represented in the current model. We overexpressed an endoplasmic reticulum-localized lysophosphatidic acid acyltransferase, LPAT2, a key enzyme that catalyses the last step of de novo phospholipid biosynthesis. Two independent LPAT2 overexpression lines showed no visible phenotype under normal conditions but showed increased root length under phosphate starvation, with no effect on phosphate starvation response including marker gene expression, root hair development and anthocyanin accumulation. Accompanying membrane glycerolipid profiling of LPAT2-overexpressing plants revealed an increased content of major phospholipid classes and distinct responses to phosphate starvation between shoot and root. The findings propose a revised model of membrane lipid remodelling, in which de novo phospholipid biosynthesis mediated by LPAT2 contributes significantly to root development under phosphate starvation.

Porcine respiratory and reproductive syndrome virus variants, Vietnam and China, 2007.

We characterized isolates from porcine respiratory and reproductive syndrome virus epidemics in Vietnam and China in 2007. These isolates showed approximately 99% identity at the genomic level. Genetic analysis indicated that they share a discontinuous deletion of 30 aa in nonstructural protein 2, which indicates that identical variants emerged in Vietnam and China.

Molecular characterization of serotype A foot-and-mouth disease viruses circulating in Vietnam in 2009.

Foot-and-mouth disease (FMD) is a major cause of endemic outbreaks in Vietnam in recent years. In this work, six serotype A foot-and-mouth disease viruses (FMDV), collected from endemic outbreaks during January and February of 2009 in four different provinces in Vietnam, were genetically characterized for their complete genome sequences. Genetic analysis based on the complete viral genome sequence indicated that they were closely related to each other and shared 99.0-99.8% amino acid (aa) identity. Genetic and deduced aa analysis of the capsid coding gene VP1 showed that the six Vietnamese strains were all classified into the genotype IX from a total of 10 major genotypes worldwide, sharing 98.1-100% aa identity each other. They were most closely related to the type A strains recently isolated in Laos (A/LAO/36/2003, A/LAO/1/2006, A/LAO/6/2006, A/LAO/7/2006, and A/LAO/8/2006), Thailand (A/TAI/2/1997 and A/TAI/118/1987), and Malaysia (A/MAY/2/2002), sharing 88.3-95.5% nucleotide (nt) identities. In contrast, Vietnamese type A strains showed low nt identities with the two old type A FMDVs, isolated in 1960 in Thailand (a15thailand iso43) and in 1975 in the Philippines (aphilippines iso50), ranging from 77.3 to 80.9% nt identity. A multiple alignment based on the deduced amino acid sequences of the capsid VP1 coding gene of type A FMDV revealed three amino acid substitutions between Vietnamese strains and the strains of other Southeast Asian countries (Laos, Thailand, Malaysia, and the Philippines). Alanine was replaced by valine at residue 24, asparagine by arginine at residue 85, and serine by threonine at residue 196. Furthermore, type A FMDV strains recently isolated in Vietnam, Laos, Thailand, and Malaysia all have one amino acid deletion at residue 140 of the capsid VP1 protein compared with the two old type A FMDV strains from Thailand and the Philippines as well as most other type A representatives worldwide. This article is the first to report on the comprehensive genetic characterization of type A FMDV circulating in Vietnam.

Heterogeneity and genetic variations of serotypes O and Asia 1 foot-and-mouth disease viruses isolated in Vietnam.

Six field foot-and-mouth disease viruses (FMDVs), including four serotype O and two serotype Asia 1 strains, were collected from endemic outbreaks in 2005, 2006, and 2007 from four different provinces in Vietnam. The viruses were isolated and genetically characterized for their complete genomic sequences. The genetic analysis based on the complete genomic coding sequences revealed that the four serotype O FMDVs were related to each other, sharing 95.2% nucleotide (nt) identity and 97.5-97.6% amino acid (aa) identity. Genetic analysis and a phylogenetic tree, based on the VP1 gene of FMDV, showed that the four present Vietnamese serotype O strains have a high level of identity with other serotype O representatives of the Mya-98 lineage of the Southeast Asian (SEA) topotype. The four viruses were all clustered into the Mya-98 lineage of the SEA topotype, sharing 92.3-95.6% nt and 93.4-96.7% aa identity. This finding of the Mya-98 lineage was different from previous reports that the Vietnamese serotype O strains belonged to the Cam-94 lineage of the SEA topotype and two other topotypes, Middle East-South Asia (ME-SA) and Cathay. For the two serotype Asia 1 FMDVs, the genetic analysis based on the complete genomic coding sequences as well as on the VP1 gene revealed that they belonged to two genogroups, IV and V. Of note, the As1/VN/QT03/2007 strain of genogroup V, isolated in 2007, was very closely related to the pandemic Asia 1 strain which caused FMD outbreaks in China (Asia1/WHN/CHA/06, FJ906802) and Mongolia (Asia1/MOG/05, EF614458) in 2005, sharing 99.0-99.3% nt and 99.5-100% aa identity. In contrast, the second strain As1/VN/LC04/2005 of genogroup IV, isolated in 2005, was closely related to all referenced Vietnamese serotype Asia 1 strains found in the GenBank databases, sharing 86.4-100% nt and 90.9-100% aa identity with each. This study is the first description of the full-length genomic sequence of Vietnamese FMDV serotypes O and Asia 1 and may provide the evidence of the concurrent circulation of different serotypes and subtypes of FMDV in recent years in Vietnam.