Bioinformatics and Expression Profiling of the DHHC-CRD S-Acyltransferases Reveal Their Roles in Growth and Stress Response in Woodland Strawberry ().

Protein S-acyl transferases (PATs) are a family of enzymes that catalyze protein S-acylation, a post-translational lipid modification involved in protein membrane targeting, trafficking, stability, and protein-protein interaction. S-acylation plays important roles in plant growth, development, and stress responses. Here, we report the genome-wide analysis of the family genes in the woodland strawberry (), a model plant for studying the economically important Rosaceae family.

A Global Identification of Protein Disulfide Isomerases from 'duli' Pear () and Their Expression Profiles under Salt Stress.

Protein disulfide isomerases (PDIs) and PDI-like proteins catalyze the oxidation and reduction in protein disulfide bonds, inhibit aggregation of misfolded proteins, and participate in isomerization and abiotic stress responses. The wild type 'duli' pear () is an important rootstock commonly used for commercial pear tree grafting in northern China. In this study, we identified 24 genes, named , from the genome of 'duli' pear.

Transcriptome analysis reveals key genes involved in the resistance to Cryphonectria parasitica during early disease development in Chinese chestnut.

Background: Chestnut blight, one of the most serious branch diseases in Castanea caused by Cryphonectria parasitica, which has ravaged across American chestnut and most of European chestnut since the early twentieth century. Interestingly, the Chinese chestnut is strongly resistant to chestnut blight, shedding light on restoring the ecological status of Castanea plants severely affected by chestnut blight. To better explore the early defense of Chinese chestnut elicited in response to C.

Genome-Wide Analysis of Genes Involved in the GA Signal Transduction Pathway in '' Pear ( Bunge).

Gibberellic acid (GA) is an important phytohormone that regulates every aspect of plant growth and development. While elements involved in GA signaling have been identified and, hence, their functions have been well studied in model plants, such as Arabidopsis and rice, very little is known in pear. We, therefore, analyzed the genes related to GA signaling from the recently sequenced genome of the wildtype '' pear ( Bunge), a widely used rootstock for grafting in pear cultivation in China due to its vigorous growth and resistance to abiotic and biotic stress.

Genome-Wide Identification of Genes Encoding for Rho-Related Proteins in '' Pear ( Bunge) and Their Expression Analysis in Response to Abiotic Stress.

Twelve Rho-related proteins (ROPs), namely PbROPs, were identified from the genome of the recently sequenced '' pear ( Bunge), a wild-type pear variety routinely used for rootstocks in grafting in China. The length and molecular weight of these proteins are between 175 and 215 amino acids and 19.46 and 23.

FvWRKY48 binds to the pectate lyase FvPLA promoter to control fruit softening in Fragaria vesca.

The regulatory mechanisms that link WRKY gene expression to fruit ripening are largely unknown. Using transgenic approaches, we showed that a WRKY gene from wild strawberry (Fragaria vesca), FvWRKY48, may be involved in fruit softening and ripening. We showed that FvWRKY48 is localized to the nucleus and that degradation of the pectin cell wall polymer homogalacturonan, which is present in the middle lamella and tricellular junction zones of the fruit, was greater in FvWRKY48-OE (overexpressing) fruits than in empty vector (EV)-transformed fruits and less substantial in FvWRKY48-RNAi (RNA interference) fruits.

Knockout of the S-acyltransferase Gene, , Confers the Dwarf Yellowing Phenotype in First Generation Pear by ABA Accumulation.

The development of dwarf fruit trees with smaller and compact characteristics leads to significantly increased fruit production, which is a major objective of pear () breeding. We identified the S-acylation activity of , an S-acyltransferase gene related to plant development, using a yeast () complementation assay, and also could rescue the growth defect of the mutant . We further studied the function of by designing three guide RNAs for to use in the CRISPR/Cas9 system.

Investigations into a putative role for the novel BRASSIKIN pseudokinases in compatible pollen-stigma interactions in Arabidopsis thaliana.

Background: In the Brassicaceae, the early stages of compatible pollen-stigma interactions are tightly controlled with early checkpoints regulating pollen adhesion, hydration and germination, and pollen tube entry into the stigmatic surface. However, the early signalling events in the stigma which trigger these compatible interactions remain unknown.

Results: A set of stigma-expressed pseudokinase genes, termed BRASSIKINs (BKNs), were identified and found to be present in only core Brassicaceae genomes.

Both male and female gametogenesis require a fully functional protein S-acyl transferase 21 in Arabidopsis thaliana.

S-Acylation is a reversible post-translational lipid modification in which a long chain fatty acid covalently attaches to specific cysteine(s) of proteins via a thioester bond. It enhances the hydrophobicity of proteins, contributes to their membrane association and plays roles in protein trafficking, stability and signalling. A family of Protein S-Acyl Transferases (PATs) is responsible for this reaction.

Protein S-acyl transferase 15 is involved in seed triacylglycerol catabolism during early seedling growth in Arabidopsis.

Seeds of Arabidopsis contain ~40% oil, which is primarily in the form of triacylglycerol and it is converted to sugar to support post-germination growth. We identified an Arabidopsis T-DNA knockout mutant that is sugar-dependent during early seedling establishment and determined that the β-oxidation process involved in catabolising the free fatty acids released from the seed triacylglycerol is impaired. The mutant was confirmed to be transcriptional null for Protein Acyl Transferase 15, AtPAT15 (At5g04270), one of the 24 protein acyl transferases in Arabidopsis.

Production of eicosapentaenoic acid (EPA, 20:5n-3) in transgenic peanut (Arachis hypogaea L.) through the alternative Δ8-desaturase pathway.

An important alternative source of fish oil is its production by plants through metabolic engineering. To produce eicosapentaenoic acid (EPA, 20:5n-3) in peanut through the alternative Δ8-pathway, a plant expression vector containing five heterologous genes driven by the constitutive 35S promoter respectively, namely, ∆9-elongase (Isochrysis galbana), ∆8-desaturase (Euglena gracilis), ∆5-desaturase (Mortierella alpina), ∆15-desaturase (Arabidopsis thaliana) and ∆17-desaturase (Phytophthora infestans) were transferred into peanut through Agrobacterium-mediated transformation method. The gas chromatography results indicated that the average content of EPA in the leaves of the transgenic lines was 0.

Progress toward Understanding Protein S-acylation: Prospective in Plants.

S-acylation, also known as S-palmitoylation or palmitoylation, is a reversible post-translational lipid modification in which long chain fatty acid, usually the 16-carbon palmitate, covalently attaches to a cysteine residue(s) throughout the protein via a thioester bond. It is involved in an array of important biological processes during growth and development, reproduction and stress responses in plant. S-acylation is a ubiquitous mechanism in eukaryotes catalyzed by a family of enzymes called Protein S-Acyl Transferases (PATs).

PCP-B class pollen coat proteins are key regulators of the hydration checkpoint in Arabidopsis thaliana pollen-stigma interactions.

The establishment of pollen-pistil compatibility is strictly regulated by factors derived from both male and female reproductive structures. Highly diverse small cysteine-rich proteins (CRPs) have been found to play multiple roles in plant reproduction, including the earliest stages of the pollen-stigma interaction. Secreted CRPs found in the pollen coat of members of the Brassicaceae, the pollen coat proteins (PCPs), are emerging as important signalling molecules that regulate the pollen-stigma interaction.

Elevation of the Yields of Very Long Chain Polyunsaturated Fatty Acids via Minimal Codon Optimization of Two Key Biosynthetic Enzymes.

Eicosapentaenoic acid (EPA, 20:5Δ5,8,11,14,17) and Docosahexaenoic acid (DHA, 22:6Δ4,7,10,13,16,19) are nutritionally beneficial to human health. Transgenic production of EPA and DHA in oilseed crops by transferring genes originating from lower eukaryotes, such as microalgae and fungi, has been attempted in recent years. However, the low yield of EPA and DHA produced in these transgenic crops is a major hurdle for the commercialization of these transgenics.

Protein S-Acyltransferase 14: A Specific Role for Palmitoylation in Leaf Senescence in Arabidopsis.

The Asp-His-His-Cys-Cys-rich domain-containing Protein S-Acyl Transferases (PATs) are multipass transmembrane proteins that catalyze S-acylation (commonly known as S-palmitoylation), the reversible posttranslational lipid modification of proteins. Palmitoylation enhances the hydrophobicity of proteins, contributes to their membrane association, and plays roles in protein trafficking and signaling. In Arabidopsis (Arabidopsis thaliana), there are at least 24 PATs; previous studies on two PATs established important roles in growth, development, and stress responses.

Accumulation of eicosapolyenoic acids enhances sensitivity to abscisic acid and mitigates the effects of drought in transgenic Arabidopsis thaliana.

IgASE1, a C₁₈ Δ(9)-specific polyunsaturated fatty acid elongase from the marine microalga Isochrysis galbana, is able to convert linoleic acid and α-linolenic acid to eicosadienoic acid and eicosatrienoic acid in Arabidopsis. Eicosadienoic acid and eicosatrienoic acid are precursors of arachidonic acid, eicosapentaenoic acid, and docosahexaenoic acid, which are synthesized via the Δ(8) desaturation biosynthetic pathways. This study shows that the IgASE1-expressing transgenic Arabidopsis exhibited altered morphology (decreased leaf area and biomass) and enhanced drought resistance compared to wild-type plants.

Putative DHHC-cysteine-rich domain S-acyltransferase in plants.

Protein S-acyltransferases (PATs) containing Asp-His-His-Cys within a Cys-rich domain (DHHC-CRD) are polytopic transmembrane proteins that are found in eukaryotic cells and mediate the S-acylation of target proteins. S-acylation is an important secondary and reversible modification that regulates the membrane association, trafficking and function of target proteins. However, little is known about the characteristics of PATs in plants.

Creation and validation of a widely applicable multiple gene transfer vector system for stable transformation in plant.

Multiple gene transfer (MGT) technology has become a powerful tool for basic and applied plant biology research in recent years. Despite some notable successes in obtaining plant lines harbouring multiple transgenes, these methods are still generally unwieldy and costly. We report here a straightforward and cost effective strategy, utilizing commonly available restriction enzymes for the transfer of multiple genes into plants, hence greatly widening the accessibility of MGT.

A Golgi and tonoplast localized S-acyl transferase is involved in cell expansion, cell division, vascular patterning and fertility in Arabidopsis.

S-acylation of eukaryotic proteins is the reversible attachment of palmitic or stearic acid to cysteine residues, catalysed by protein S-acyl transferases that share an Asp-His-His-Cys (DHHC) motif. Previous evidence suggests that in Arabidopsis S-acylation is involved in the control of cell size, polarity and the growth of pollen tubes and root hairs. Using a combination of yeast genetics, biochemistry, cell biology and loss of function genetics the roles of a member of the protein S-acyl transferase PAT family, AtPAT10 (At3g51390), have been explored.

Characterization of three novel desaturases involved in the delta-6 desaturation pathways for polyunsaturated fatty acid biosynthesis from Phytophthora infestans.

Phytophthora infestans is the causative agent of potato blight that resulted in the great famine in Ireland in the nineteenth century. This microbe can release large amounts of the C20 very long-chain polyunsaturated fatty acids arachidonic acid (ARA; 20:4Δ(5, 8, 11, 14)) and eicosapentaenoic acid (EPA; 20:5Δ(5, 8, 11, 14, 17)) upon invasion that is known to elicit a hypersensitive response to their host plant. In order to identify enzymes responsible for the biosynthesis of these fatty acids, we blasted the recently fully sequenced P.

The alternative pathway C20 Delta8-desaturase from the non-photosynthetic organism Acanthamoeba castellanii is an atypical cytochrome b5-fusion desaturase.

A cDNA encoding a C20 Delta8-desaturase was isolated from the free-living soil amoeba, Acanthamoeba castellanii and functionally characterised by heterologous expression. The open reading frame of the A. castellanii C20 Delta8-desaturase showed similarity to other microsomal front-end desaturases, but the N-terminal domain contained a variant form of the conserved heme-binding motif in which H-P-G-G is replaced by H-P-A-G.

Progress toward the production of long-chain polyunsaturated fatty acids in transgenic plants.

Long-chain PUFA such as eicosapentaenoic and docosahexaenoic acids are prevalent in fish oils, and these compounds have been demonstrated to play important roles in human health and nutrition. In particular, these n-3/omega-3 long-chain PUFA provide protection from cardiovascular disease and a collection of symptoms (termed metabolic syndrome) associated with progression toward type 2 diabetes and obesity. Within Western populations, a large increase in the occurrence of these conditions represents a major public health concern.

Expression of the Isochrysis C18-delta9 polyunsaturated fatty acid specific elongase component alters Arabidopsis glycerolipid profiles.

A cDNA isolated from the prymnesiophyte micro-alga Isochrysis galbana, designated IgASE1, encodes a fatty acid elongating component that is specific for linoleic acid (C18:2n-6) and alpha-linolenic acid (C18:3n-3). Constitutive expression of IgASE1 in Arabidopsis resulted in the accumulation of eicosadienoic acid (EDA; C20:2n-6) and eicosatrienoic acid (ETrA; C20:3n-3) in all tissues examined, with no visible effects on plant morphology. Positional analysis of the various lipid classes indicated that these novel fatty acids were largely excluded from the sn-2 position of chloroplast galactolipids and seed triacylglycerol, whereas they were enriched in the same position in phosphatidylcholine.

Production of very long chain polyunsaturated omega-3 and omega-6 fatty acids in plants.

We report the production of two very long chain polyunsaturated fatty acids, arachidonic acid (AA) and eicosapentaenoic acid (EPA), in substantial quantities in a higher plant. This was achieved using genes encoding enzymes participating in the omega3/6 Delta8 -desaturation biosynthetic pathways for the formation of C20 polyunsaturated fatty acids. Arabidopsis thaliana was transformed sequentially with genes encoding a Delta9 -specific elongating activity from Isochrysis galbana, a Delta8 -desaturase from Euglena gracilis and a Delta5 -desaturase from Mortierella alpina.