GDSL Lipases Research Articles

BrUFO positively regulates the infection of Chinese cabbage by Plasmodiophora brassicae.

Chinese cabbage is one of the most important vegetable crops in China. However, the clubroot disease caused by the infection of Plasmodiophora brassicae (P. brassicae) has seriously affected the yield and quality of Chinese cabbage. In our previous study, BrUFO gene was found to be significantly up-regulated in diseased roots of Chinese cabbage after inoculation with P. brassicae. UFO (UNUSUAL FLORAL ORGANS) have the properties of substrate recognition during ubiquitin-mediated proteolysis. A variety of plant can activate immunity response through the ubiquitination pathway. Therefore, it is very important to study the function of UFO in response to P. brassicae. In this study, The expression pattern of BrUFO Gene was measured by qRT-PCR and In situ Hybridization (ISH). The expression location of BrUFO in cells was determined by subcellular localization. The function of BrUFO was verified by Virus-induced Gene Silencing (VIGS). proteins interacting with BrUFO protein were screened by yeast two-hybrid. Quantitative real-time polymerase chain reactions (qRT-PCR) and in situ hybridization analysis showed that expression of BrUFO gene in the resistant plants was lower than that in susceptible plants. Subcellular localization analysis showed that BrUFO gene was expressed in the nucleus. Virus-induced gene silencing (VIGS) analysis showed that silencing of BrUFO gene reduced the incidence of clubroot disease. Six proteins interacting with BrUFO protein were screened by Y2H assay. Two of them (Bra038955, a B-cell receptor-associated 31-like protein and Bra021273, a GDSL-motif esterase/acyltransferase/lipase Enzyme) were confirmed to strongly interact with BrUFO protein. BrUFO gene should be a key gene of chinese cabbage against the infection of P. brassicae. BrUFO gene silencing improves the resistance of plants to clubroot disease. BrUFO protein may interact with CUS2 to induce ubiquitination in PRR-mediated PTI reaction through GDSL lipases, so as to achieve the effect of Chinese cabbage against the infection of P. brassicae.

Key Amino Acids for Transferase Activity of GDSL Lipases

The Gly-Asp-Ser-Leu (GDSL) motif of esterase/lipase family proteins (GELPs) generally exhibit esterase activity, whereas transferase activity is markedly preferred in several GELPs, including the Tanacetum cinerariifolium GDSL lipase TciGLIP, which is responsible for the biosynthesis of the natural insecticide, pyrethrin I. This transferase activity is due to the substrate affinity regulated by the protein structure and these features are expected to be conserved in transferase activity-exhibiting GELPs (tr-GELPs). In this study, we identified two amino acid residues, [N/R]208 and D484, in GELP sequence alignments as candidate key residues for the transferase activity of tr-GELPs by two-entropy analysis. Molecular phylogenetic analysis demonstrated that each tr-GELP is located in the clusters for non-tr-GELPs, and most GELPs conserve at least one of the two residues. These results suggest that the two conserved residues are required for the acquisition of transferase activity in the GELP family. Furthermore, substrate docking analyses using ColabFold-generated structure models of both natives and each of the two amino acids-mutated TciGLIPs also revealed numerous docking models for the proper access of substrates to the active site, indicating crucial roles of these residues of TciGLIP in its transferase activity. This is the first report on essential residues in tr-GELPs for the transferase activity.

Expression Pattern and Functional Analyses of Arabidopsis Guard Cell-Enriched GDSL Lipases.

There are more than 100 GDSL lipases in Arabidopsis, but only a few members have been functionally investigated. Moreover, no reports have ever given a comprehensive analysis of GDSLs in stomatal biology. Here, we systematically investigated the expression patterns of 19 putative Guard-cell-enriched GDSL Lipases (GGLs) at various developmental stages and in response to hormone and abiotic stress treatments. Gene expression analyses showed that these GGLs had diverse expression patterns. Fifteen GGLs were highly expressed in guard cells, with seven preferentially in guard cells. Most GGLs were localized in endoplasmic reticulum, and some were also localized in lipid droplets and nucleus. Some closely homologous GGLs exhibited similar expression patterns at various tissues and in response to hormone and abiotic stresses, or similar subcellular localization, suggesting the correlation of expression pattern and biological function, and the functional redundancy of GGLs in plant development and environmental adaptations. Further phenotypic identification of ggl mutants revealed that GGL7, GGL14, GGL22, and GGL26 played unique and redundant roles in stomatal dynamics, stomatal density and morphology, and plant water relation. The present study provides unique resources for functional insights into these GGLs to control stomatal dynamics and development, plant growth, and adaptation to the environment.

GDSL-domain proteins have key roles in suberin polymerization and degradation.

Plant roots acquire nutrients and water, while managing interactions with the soil microbiota. Their endodermis provides an extracellular diffusion barrier via a network of lignified cell walls, called Casparian strips, supported by subsequent formation of suberin lamellae. Whereas lignification is thought to be irreversible, suberin lamellae display plasticity, which is crucial for root adaptative responses in the plant. Despite suberin being a major plant polymer, fundamental aspects of its biosynthesis and turnover have remained obscure. Plants shape their root system via lateral root formation, an auxin-induced process requiring local breaking and re-sealing of endodermal lignin and suberin barriers. Here, we show that differentiated endodermal cells have a specific, auxin-mediated transcriptional response, dominated by cell wall remodelling genes. We identified two sets of auxin-regulated GDSL-lipases. One is required for suberin synthesis, while the other can drive suberin degradation. These enzymes constitute novel core players of suberisation, driving root suberin plasticity.

Functional Omics Identifies Serine Hydrolases That Mobilize Storage Lipids during Rice Seed Germination.

Elucidating proteolipidome dynamics is crucial for understanding the roles of these molecules in plant physiology and disease. Sequence-based functional annotation of the protein is inadequate, since protein activities depend on posttranslational modification. In this study, we applied a gel-free activity-based protein profiling approach to unravel the active lipases, including other Serine hydrolases (SHs), expressed during seed germination in rice (Oryza sativa). We successfully mapped the active sites of 43 active SHs encompassing lipases/esterases, GDSL lipases, proteases, Ser carboxypeptidases, ABHD protein, pectin acetylesterase, and other SHs. The mRNA expression levels of those genes encoding the identified SHs were monitored using microarray analysis. The lipidome analysis revealed distinct patterns of molecular species distribution in individual lipid classes and displayed the metabolic connections between lipid mobilization and rice seedling growth. Changes in the mobilization of storage lipids and their molecular species remodeling were correlated with the expression of the identified lipases and their lipase activity in a time-dependent manner. The physiological significance of the identified SHs was explored during biotic stress with Fusarium verticillioides infection. The fungal infection significantly reduced lipase activity and lipid mobilization, thus impairing the rice seedling. Collectively, our data demonstrate application of the functional proteome strategy along with the shotgun lipidome approach for the identification of active SHs, and thus for deciphering the role of lipid homeostasis during rice seed germination.

Elevating seed oil content in a polyploid crop by induced mutations in SEED FATTY ACID REDUCER genes.

SummaryPlant‐based oils are valuable agricultural products, and seed oil content (SOC) is the major yield component in oil crops. Increasing SOC has been successfully targeted through the selection and genetic modification of oil biosynthesis. The SOC in rapeseed declined during the seed maturation and eventually caused the final accumulated seed oil quantity. However, genes involved in oil degradation during seed maturity are not deeply studied so far. We performed a candidate gene association study using a worldwide collection of rapeseed germplasm. We identified SEED FATTY ACID REDUCER (SFAR) genes, which had a significant effect on SOC and fatty acid (FA) composition. SFAR genes belong to the GDSL lipases, and GDSL lipases have a broad range of functions in plants. After quantification of gene expression using RNA‐seq and quantitative PCR, we used targeted (CRISPR‐Cas mediated) and random (chemical) mutagenesis to modify turnover rates of seed oil in winter rapeseed. For the first time, we demonstrate significant increase of SOC in a crop after knocking out members of the BnSFAR4 and BnSFAR5 gene families without pleiotropic effects on seed germination, vigour and oil mobilization. Our results offer new perspectives for improving oil yield by targeted mutagenesis.

Advances in plant GDSL lipases: from sequences to functional mechanisms

GDSL lipases form multigene families in Arabidopsis, rice and some other sequenced plant species. Unlike classical GxSxG motif-containing lipases, this new sub-family of lipolytic enzymes possesses a GDSL sequence motif (GDSxxDxG), and members are active in the hydrolysis and synthesis of a variety of lipids. Research on plant GDSL lipases started later than studies of microbial and animal GDSL lipases; therefore, our knowledge regarding plant GDSL lipases, from sequences to functional mechanisms, is limited. Recently, some GDSL lipase genes have been cloned and identified in different plant species. In this paper, we present a comprehensive review of the advances in research on plant GDSL lipases, which include their structures, distributions, biochemical activities, expression patterns and biological functions. Plant GDSL lipases have very flexible enzyme active sites, which can lead to extensive substrate binding and result in multifunctional properties. They play important roles in many physiological and biochemical processes, such as plant growth and development, organ morphogenesis, adversity stress and lipid metabolism. In addition, their potential applications in agriculture and industry are discussed.

Rv0518, a nutritive stress inducible GDSL lipase of Mycobacterium tuberculosis, enhanced intracellular survival of bacteria by cell wall modulation

Rv0518, a hypothetical protein of Mycobacterium tuberculosis, was designated as possible exported protein. In-silico analysis suggested that the protein belonged to the family of GDSL lipases. In this study, rv0518 gene was cloned and expressed in E. coli followed by purification and characterization. It possessed lipolytic activity, preferably hydrolyzed pNP-decanoate at pH 9.0 and 40 °C. The enzyme was stable till 50 °C and wider pH range (5.0–11.0). The predicted active site residues, Ser-46, Asp-205, His-208, Gly-87, Asn-120 were confirmed by site directed mutagenesis. rv0518 gene expression in M. tuberculosis H37Ra was up-regulated under nutrient starvation and the protein was detected in membrane fraction. The expression of rv0518 in M. smegmatis altered the colony morphology/growth kinetics, provided resistance to SDS, lysozyme, anti-TB drugs and enhanced in vitro survival of M. smegmatis under nutritive stress. The total lipid content and trehalose dimycolate was elevated in M. smegmatis expressing rv0518 gene. The presence of Rv0518 enhanced infection ability and intracellular survival capability of M. smegmatis. Hence, Rv0518 is a cell wall associated GDSL lipase might helped bacteria to utilize glycerol/lipids for its growth as well as provided resistance to various intracellular stresses by cell wall modulation resulting in its enhanced intracellular survival.

GDSL lipases modulate immunity through lipid homeostasis in rice.

Lipids and lipid metabolites play important roles in plant-microbe interactions. Despite the extensive studies of lipases in lipid homeostasis and seed oil biosynthesis, the involvement of lipases in plant immunity remains largely unknown. In particular, GDSL esterases/lipases, characterized by the conserved GDSL motif, are a subfamily of lipolytic enzymes with broad substrate specificity. Here, we functionally identified two GDSL lipases, OsGLIP1 and OsGLIP2, in rice immune responses. Expression of OsGLIP1 and OsGLIP2 was suppressed by pathogen infection and salicylic acid (SA) treatment. OsGLIP1 was mainly expressed in leaf and leaf sheath, while OsGLIP2 showed high expression in elongating internodes. Biochemical assay demonstrated that OsGLIP1 and OsGLIP2 are functional lipases that could hydrolyze lipid substrates. Simultaneous down-regulation of OsGLIP1 and OsGLIP2 increased plant resistance to both bacterial and fungal pathogens, whereas disease resistance in OsGLIP1 and OsGLIP2 overexpression plants was significantly compromised, suggesting that both genes act as negative regulators of disease resistance. OsGLIP1 and OsGLIP2 proteins mainly localize to lipid droplets and the endoplasmic reticulum (ER) membrane. The proper cellular localization of OsGLIP proteins is indispensable for their functions in immunity. Comprehensive lipid profiling analysis indicated that the alteration of OsGLIP gene expression was associated with substantial changes of the levels of lipid species including monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG). We show that MGDG and DGDG feeding could attenuate disease resistance. Taken together, our study indicates that OsGLIP1 and OsGLIP2 negatively regulate rice defense by modulating lipid metabolism, thus providing new insights into the function of lipids in plant immunity.

Functional Characterization of a Novel Marine Microbial GDSL Lipase and Its Utilization in the Resolution of (±)-1-Phenylethanol.

A novel GDSL lipase (MT6) was cloned from the genome of Marinactinospora thermotolerans SCSIO 00652 identified from the South China Sea. MT6 showed its maximum identity of 59% with a putative lipase from Nocardiopsis dassonville. MT6 was heterologously expressed in E. coli BL21(DE3) and further functionally characterized. MT6 could efficiently resolve racemic 1-phenylethanol and generate (R)-1-phenylethanol with high enantiomeric excess (99%) and conversion rate (54%) through transesterification reactions after process optimization. Our report was the first one report about the utilization of one GDSL lipase in the preparation of chiral chemicals by transesterification reactions, and the optical selectivity of MT6 was interestingly opposite to those of other common lipases. GDSL lipases represented by MT6 possess great potential for the generation of valuable chiral chemicals in industry.

The extracellular lipase EXL4 is required for efficient hydration of Arabidopsis pollen

Pollination in species with dry stigmas begins with the hydration of desiccated pollen grains on the stigma, a highly regulated process involving the proteins and lipids of the pollen coat and stigma cuticle. Self-incompatible species of the Brassicaceae block pollen hydration, and while the early signaling steps of the self-incompatibility response are well studied, the precise mechanisms controlling pollen hydration are poorly understood. Both lipids and proteins are important for hydration; loss of pollen coat lipids and proteins results in defective or delayed hydration on the stigma surface. Here, we examine the role of the pollen coat protein extracellular lipase 4 (EXL4), in the initial steps of pollination, namely hydration on the stigma. We identify a mutant allele, exl4-1, that shows a reduced rate of pollen hydration. exl4-1 pollen is normal with respect to pollen morphology and the downstream steps in pollination, including pollen tube germination, growth, and fertilization of ovules. However, owing to the delay in hydration, exl4-1 pollen is at a disadvantage when competed with wild-type pollen. EXL4 also functions in combination with GRP17 to promote the initiation of hydration. EXL4 is similar to GDSL lipases, and we show that it functions in hydrolyzing ester bonds. We report a previously unknown function for EXL4, an abundant pollen coat protein, in promoting pollen hydration on the stigma. Our results indicate that changes in lipid composition at the pollen-stigma interface, possibly mediated by EXLs, are required for efficient pollination in species with dry stigmas.

The Salmonella SPI-2 effector SseJ exhibits eukaryotic activator-dependent phospholipase A and glycerophospholipid : cholesterol acyltransferase activity

Intracellular replication of Salmonella enterica serovar Typhimurium within membrane-bound compartments, called Salmonella-containing vacuoles, depends on the activities of several effector proteins translocated by the Salmonella pathogenicity island 2 (SPI-2)-encoded type III secretion system. The SPI-2 effector protein SseJ shows similarity at the amino acid level to several GDSL lipases with glycerophospholipid : cholesterol acyltransferase (GCAT) activity. In this study, we show that catalytic serine-dependent phospholipase A (PLA) and GCAT activity of recombinant SseJ is potentiated by factor(s) present in HeLa cells, RAW macrophages and Saccharomyces cerevisiae. SseJ activity was enhanced with increasing amounts of, or preincubation with, eukaryotic cell extracts. Analysis of the activating factor(s) shows that it is soluble and heat- and protease-sensitive. We conclude that PLA and GCAT activities of SseJ are potentiated by proteinaceous eukaryotic factor(s).

Sequence Analysis of GDSL Lipase Gene Family in Arabidopsis thaliana

To analyze sequence characters of GDSL lipase gene family in Arabidopsis thaliana, 108 members of GDSL lipases were analyzed using data mining. The gene structures display remarkable diversity, consisting of zero to 13 introns. And the genes are asymmetrically distributed in chromosome 1-5, some of which are arranged in tandem. Phylogenetically, they were classified into three groups. Lipase-GDSL domain (PF00478) is housed at or close to N-terminus, or in the middle of amino acid sequences, additionally in which other domains and replicates were also found. Most GDSL lipases contain a signal peptide for conducting the secretary pathway. They are predicted to be extracellularly secreted, or target to mitochondria, chloroplast or any other parts of the cells. Functionally, these lipases are potentially involved in multiple physiological roles including seed germination, flowering and defense reactions. This study will help further understand the sequences and functions of Arabidopsis GDSL lipases.

Role of a GDSL lipase‐like protein as sinapine esterase in Brassicaceae

The seeds of most members of the Brassicaceae accumulate high amounts of sinapine (sinapoylcholine) that is rapidly hydrolyzed during early stages of seed germination. One of three isoforms of sinapine esterase activity (BnSCE3) has been isolated from Brassica napus seedlings and subjected to trypsin digestion and spectrometric sequencing. The peptide sequences were used to isolate BnSCE3 cDNA, which was shown to contain an open reading frame of 1170 bp encoding a protein of 389 amino acids, including a leader peptide of 25 amino acids. Sequence comparison identified the protein as the recently cloned BnLIP2, i.e. a GDSL lipase-like protein, which displays high sequence identity to a large number of corresponding plant proteins, including four related Arabidopsis lipases. The enzymes belong to the SGNH protein family, which use a catalytic triad of Ser-Asp-His, with serine as the nucleophile of the GDSL motif. The corresponding B. napus and Arabidopsis genes were heterologously expressed in Nicotiana benthamiana leaves and proved to confer sinapine esterase activity. In addition to sinapine esterase activity, the native B. napus protein (BnSCE3/BnLIP2) showed broad substrate specificity towards various other choline esters, including phosphatidylcholine. This exceptionally broad substrate specificity, which is common to a large number of other GDSL lipases in plants, hampers their functional analysis. However, the data presented here indicate a role for the GDSL lipase-like BnSCE3/BnLIP2 as a sinapine esterase in members of the Brassicaceae, catalyzing hydrolysis of sinapine during seed germination, leading, via 1-O-sinapoyl-beta-glucose, to sinapoyl-l-malate in the seedlings.

Lipolytic Enzymes inMyxococcus xanthus

The genome of Myxococcus xanthus encodes lipolytic enzymes in three different families: patatin lipases, alpha/beta hydrolases, and GDSL lipases. One member of each family was characterized. The protein encoded by MXAN_3852 contains motifs characteristic of patatins. MXAN_5522 encodes a protein with the G-X-S-X-G motif characteristic of the lipase subfamily of alpha/beta hydrolases. MXAN_4569 encodes a member of the GDSL family of lipolytic enzymes. Strains with deletions of MXAN_5522 and MXAN_4569 undergo faster development and earlier myxospore formation than the wild-type strain. The MXAN_5522 mutation results in spore yields substantially higher than those seen for wild-type cells. Gene expression analysis using translational lacZ fusions indicates that while all three genes are expressed during development, only MXAN_5522 and MXAN_4569 are expressed during vegetative growth. The proteins encoded by these genes were overexpressed using a T7 RNA polymerase transcription (pET102/D-TOPO) system in Escherichia coli BL21 Star (DE3) cells. The substrate specificities of the purified enzymes were investigated using p-nitrophenyl esters with chain lengths from C(2) to C(16). These enzymes preferentially hydrolyzed esters of short-chain fatty acids, yielding the highest activity with p-nitrophenyl acetate.

Isolation and Expression Analysis of a GDSL-like Lipase Gene from Brassica napus L.

As lipolytic enzymes, GDSL lipases play an important role in plant growth and development. In order to identify their functions and roles, the full-length cDNA of a GDSL lipase gene, designated BnLIP2, was isolated from Brassica napus L. BnLIP2 was 1,300 bp long, with 1,122 bp open reading frame (ORF) encoding 373 amino acid residues. Sequence analysis indicated that BnLIP2 belonged to GDSL family. Southern blot analysis indicated that BnLIP2 belonged to a small gene family in rapeseed genome. RT-PCR analysis revealed that BnLIP2 was a tissue-specific expressing gene during reproductive growth and strongly expressed during seed germination. BnLIP2 expression could not be detected until three days after germination, and it subsequently became stronger. The transcript of this gene was deficient in root of seedlings growing at different stages. When juvenile seedlings were treated by methyl jasmonate (MeJ), salicylic acid (SA) and naphthalene acetic acid (NAA), BnLIP2 expression could not be induced in root. Our study implicates that BnLIP2 probably plays an important role in rapeseed germination, morphogenesis, flowering, but independent of root growth and development.

AraPerox. A database of putative Arabidopsis proteins from plant peroxisomes.

To identify unknown proteins from plant peroxisomes, the Arabidopsis genome was screened for proteins with putative major or minor peroxisome targeting signals type 1 or 2 (PTS1 or PTS2), as defined previously (Reumann S [2004] Plant Physiol 135: 783-800). About 220 and 60 proteins were identified that carry a putative PTS1 or PTS2, respectively. To further support postulated targeting to peroxisomes, several prediction programs were applied and the putative targeting domains analyzed for properties conserved in peroxisomal proteins and for PTS conservation in homologous plant expressed sequence tags. The majority of proteins with a major PTS and medium to high overall probability of peroxisomal targeting represent novel nonhypothetical proteins and include several enzymes involved in beta-oxidation of unsaturated fatty acids and branched amino acids, and 2-hydroxy acid oxidases with a predicted function in fatty acid alpha-oxidation, as well as NADP-dependent dehydrogenases and reductases. In addition, large protein families with many putative peroxisomal isoforms were recognized, including acyl-activating enzymes, GDSL lipases, and small thioesterases. Several proteins are homologous to prokaryotic enzymes of a novel aerobic hybrid degradation pathway for aromatic compounds and proposed to be involved in peroxisomal biosynthesis of plant hormones like jasmonic acid, auxin, and salicylic acid. Putative regulatory proteins of plant peroxisomes include protein kinases, small heat shock proteins, and proteases. The information on subcellular targeting prediction, homology, and in silico expression analysis for these Arabidopsis proteins has been compiled in the public database AraPerox to accelerate discovery and experimental investigation of novel metabolic and regulatory pathways of plant peroxisomes.

The pore size of the autotransporter domain is critical for the active translocation of the passenger domain

The autotransporter mode of surface presentation in Gram-negative bacteria requires a hypothetical C-terminal β-barrel which makes up an aqueous channel in the outer membrane. PalA is a Pseudomonas sp. autotransporter lipolytic protein. PalA is a 66 kDa protein that is composed of two parts, the N-terminal region (Ala 1–Ala 296) similar to the GDSL lipases and the C-terminal region (Leu 320–Phe 612) to the autotransporter. In this report, we provide biochemical and structural evidence demonstrating that the pore size of the β-barrel conduit is important in delivering the N-terminal domain to the cell surface. Among all the autotransporter domains two strictly conserved residues (Pro 478 and Gly 576 in PalA) are converted to other various residues using site-directed mutagenesis. This investigation was made into the different pore-size mutants, affecting the folding of N-terminal domain. Wild β-domain contains a cavity of ∼2 nm diameter that is optimal for the active conformation of the N-terminal domains. However, deviation from the proper size of the pore, whether it is larger or smaller, is not suitable for the proper folding of the N-terminal catalytic domain.