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Vegetable oils are indispensable in human and animal diets and have been widely used for the production of detergents, lubricants, cosmetics, and biofuels. The seeds of an allotetraploid Perilla frutescens contain approximately 35 to 40% oils with high levels of polyunsaturated fatty acids (PUFAs). WRINKELD1 (WRI1) encoding an AP2/ERF-type transcription factor is known to upregulate the expression of genes involved in glycolysis and fatty acid biosynthesis and TAG assembly. In this study, two WRI1 isoforms, PfWRI1A, and PfWRI1B were isolated from Perilla and predominantly expressed in developing Perilla seeds. The fluorescent signals from PfWRI1A:eYFP and PfWRI1B:eYFP driven by the CaMV 35S promoter were detected in the nucleus of the Nicotiana benthamiana leaf epidermis. Ectopic expression of each of PfWRI1A and PfWRI1B increased the levels of TAG by approximately 2.9- and 2.7-fold in N. benthamiana leaves and particularly, the enhanced levels (mol%) of C18:2, and C18:3 in the TAGs were prominent with the concomitant reduction in the amounts of saturated fatty acids. The expression levels of NbPl-PKβ1, NbKAS1, and NbFATA, which were known to be target genes of WRI1, significantly increased in tobacco leaves overexpressing PfWRI1A or PfWRI1B. Therefore, newly characterized PfWRI1A and PfWRI1B can be potentially useful for the enhanced accumulation of storage oils with increased PUFAs in oilseed crops.

Plants are sessile organisms that have developed hydrophobic cuticles that cover their aerial epidermal cells to protect them from terrestrial stresses. The cuticle layer is mainly composed of cutin, a polyester of hydroxy and epoxy fatty acids, and cuticular wax, a mixture of very-long-chain fatty acids (>20 carbon atoms) and their derivatives, aldehydes, alkanes, ketones, alcohols, and wax esters. During the last 30 years, forward and reverse genetic, transcriptomic, and biochemical approaches have enabled the identification of key enzymes, transporters, and regulators involved in the biosynthesis of cutin and cuticular waxes. In particular, cuticular wax biosynthesis is significantly influenced in an organ-specific manner or by environmental conditions, and is controlled using a variety of regulators. Recent studies on the regulatory mechanisms underlying cuticular wax biosynthesis have enabled us to understand how plants finely control carbon metabolic pathways to balance between optimal growth and development and defense against abiotic and biotic stresses. In this review, we summarize the regulatory mechanisms underlying cuticular wax biosynthesis at the transcriptional, post-transcriptional, post-translational, and epigenetic levels.

The cuticular wax layer on leaf surfaces limits non-stomatal water loss to the atmosphere and protects against pathogen invasion. Although many genes associated with wax biosynthesis and wax transport in plants have been identified, their regulatory mechanisms remain largely unknown. Here, we show that the MYB transcription factor OsMYB60 positively regulates cuticular wax biosynthesis and this helps rice (Oryza sativa) plants tolerate drought stress. Compared with the wild type (japonica cultivar 'Dongjin'), osmyb60 null mutants (osmyb60-1 and osmyb60-2) exhibited increased drought sensitivity, with more chlorophyll leaching and higher rates of water loss. Quantitative reverse-transcription PCR showed that the loss of function of OsMYB60 led to downregulation of wax biosynthesis genes, leading to reduced amounts of total wax components on leaf surfaces under normal conditions. Yeast one-hybrid, luciferase transient transcriptional activity, and chromatin immunoprecipitation assays revealed that OsMYB60 directly binds to the promoter of OsCER1 (a key gene involved in very-long-chain alkane biosynthesis) and upregulates its expression. Taken together, these results demonstrate that OsMYB60 enhances rice resilience to drought stress by promoting cuticular wax biosynthesis on leaf surfaces.

Very long-chain fatty acids (VLCFAs) are essential precursors of membrane lipids, such as phospholipids and sphingolipids, cuticular waxes, suberins, and Brassica seed oils. The first step of VLCFA synthesis is mediated by 3-ketoacyl-CoA synthase (KCS), which catalyzes the condensation of a C2 unit from malonyl-CoA to acyl-CoA. In the present study, we investigated the role of KCS4 in pollen tube and root growth. KCS4 was predominantly expressed in shoot and root apical meristems, leaf veins, mature and germinated pollen grains, and developing embryos. The fluorescent signals of KCS4 fused with enhanced yellow fluorescent protein (KCS4:eYFP) were detected in the endoplasmic reticulum of tobacco epidermis. KCS4 disruption inhibited pollen tube elongation and root growth, whereas KCS4 promoter-driven KCS4 expression rescued the growth-retarded phenotype to wild type (WT) in kcs4 complementation lines. Root growth assay of WT and kcs4 lines treated with metazachlor and mefluidide, which are specific KCS inhibitors, and fatty acid analysis of their roots and seeds revealed that KCS4 is involved in the elongation of longer than C24 VLCFAs, which are essential for root and pollen tube growth.

Malonyl-acyl carrier protein (ACP) is a key building block for the synthesis of fatty acids, which are important components of cell membranes, storage oils and lipid-signaling molecules. Malonyl CoA-ACP malonyltransferase (MCAMT) catalyzes the production of malonyl-ACP and CoA from malonyl-CoA and ACP. Here, we report that MCAMT plays a critical role in cell division and has the potential to increase the storage oil content in Arabidopsis. The quantitative real-time PCR and MCAMT promoter:GUS analyses showed that MCAMT is predominantly expressed in shoot and root apical meristems, leaf hydathodes and developing embryos. The fluorescent signals of MCAMT:eYFP were observed in both chloroplasts and mitochondria of tobacco leaf protoplasts. In particular, the N-terminal region (amino acid residues 1-30) of MCAMT was required for mitochondrial targeting. The Arabidopsis mcamt-1 and -2 mutants exhibited an embryo-lethal phenotype because of the arrest of embryo development at the globular stage. The transgenic Arabidopsis expressing antisense MCAMT RNA showed growth retardation caused by the defects in cell division. The overexpression of MCAMT driven by the promoter of the senescence-associated 1 (SEN1) gene, which is predominantly expressed in developing seeds, increased the seed yield and storage oil content of Arabidopsis. Taken together, the plastidial and mitochondrial MCAMT is essential for Arabidopsis cell division and is a novel genetic resource useful for enhancing storage oil content in oilseed crops.

Linking flowering to ambient temperature In the small mustard plant Arabidopsis , the florigen FLOWERING LOCUS T (FT) mobilizes to initiate flowering at the shoot apical meristem. Susila et al . now show that FT, which is produced in leaf cells, can be held in reserve if ambient temperatures are not favorable (see the Perspective by Jaillais and Parcy). At low temperatures, FT binds a membrane phosopholipid and is thus restricted in mobility. At higher temperatures, such binding is less favored, and FT is released to mobilize into the shoot apical meristem to drive flowering. Thus, temperature-sensitive lipid binding helps the plant time flowering with favorable ambient temperatures. —PJH

Drought is a critical environmental stress that limits growth and development of plants and reduces crop productivity. The aerial part of land plants is covered with cuticular waxes to minimize water loss. To understand the regulatory mechanisms underlying cuticular wax biosynthesis in Arabidopsis under drought stress conditions, we characterized the role of an AP2/DREB type transcription factor, RAP2.4. RAP2.4 expression was detected in one-week-old seedlings and rosette leaves, stems, stem epidermis, cauline leaves, buds, flowers, and siliques of 6-week-old Arabidopsis. The levels of RAP2.4 transcripts increased with treatments of abscisic acid (ABA), mannitol, NaCl, and drought stress. Under drought, total wax loads decreased by approximately 11% and 10%, and in particular, the levels of alkanes, which are a major wax component, decreased by approximately 11% and 12% in rap2.4-1 and rap2.4-2 leaves, respectively, compared with wild type (WT) leaves. Moreover, the transcript levels of cuticular wax biosynthetic genes, KCS2 and CER1, decreased by approximately 15-23% and 32-40% in rap2.4-1 and rap2.4-2 leaves, respectively, relative to WT 4 h after drought treatment, but increased by 2- to 12-fold and 3- to 70-fold, respectively, in three independent RAP2.4 OX leaves relative to WT. Epicuticular wax crystals were observed on the leaves of RAP2.4 OX plants, but not on the leaves of WT. Total wax loads increased by 1.5- to 3.3-fold in leaves of RAP2.4 OX plants relative to WT. Cuticular transpiration and chlorophyll leaching occurred slowly in the leaves of RAP2.4 OX plants relative to WT. Transcriptional activation assay in tobacco protoplasts showed that RAP2.4 activates the expression of KCS2 and CER1 through the involvement of the consensus CCGAC or GCC motifs present in the KCS2 and CER1 promoter regions. Overall, our results revealed that RAP2.4 is a transcription factor that activates cuticular wax biosynthesis in Arabidopsis leaves under drought stress conditions.

Germination requires sufficient water absorption by seeds, but excessive water in the soil inhibits plant growth. We therefore hypothesized that tolerance mechanisms exist that help young seedlings survive and develop in waterlogged conditions. Many ATP-BINDING CASSETTE TRANSPORTER subfamily G (ABCG) proteins protect terrestrial plants from harsh environmental conditions. To establish whether any of these proteins facilitate plant development under waterlogged conditions, we observed the early seedling growth of many ABCG transporter mutants under waterlogged conditions. abcg5 seedlings exhibited severe developmental problems under waterlogged conditions: the shoot apical meristem was small, and the seedling failed to develop true leaves. The seedlings had a high water content and reduced buoyancy on water, suggesting that they were unable to retain air spaces on and inside the plant. Supporting this possibility, abcg5 cotyledons had increased cuticle permeability, reduced cuticular wax contents, and a much less dense cuticle layer than the wild-type. These results indicate that proper development of plants under waterlogged conditions requires the dense cuticle layer formed by ABCG5 activity.

The hydrophobic biopolymer suberin, which is deposited in the root endodermis and seed coats, functions as an extracellular barrier against uncontrolled water, gas, and ion loss. Suberin monomers synthesized in the endoplasmic reticulum (ER) are exported through the plasma membrane to the apoplast. However, limited information is available about the molecular mechanisms underlying suberin monomer export and assembly. In this study, we investigated the in planta role of LTPG15 encoding a glycosylphosphatidylinositol (GPI)-anchored lipid transfer protein. LTPG15 was predominantly expressed in the root endodermis and seed coat. Fluorescent signals from LTPG15:eYFP were detected in the plasma membrane in tobacco epidermis. Disruption of LTPG15 caused a significant decrease in the levels of fatty acids (C20-C24), primary alcohols (C20 and C22), ω-hydroxy fatty acids (C22 and C24), and α,ω-alkanediols (C20 and C22), but an increase in the amounts of primary alcohols and hydroxy fatty acids with C16 and C18 in seed coats. The mutant phenotype was restored to that of the wild type (WT) by the expression of LTPG15 driven by its own promoter. Seed coats of ltpg15 had an increase in permeability to tetrazolium salts compared with WT seed coats. ltpg15 seeds were more sensitive than WT seeds to inhibition of germination and seedling establishment by salt and osmotic stress treatments. Taken together, our results indicate that LTPG15 is involved in suberin monomer export in seed coats, and this highlights the role of Type G non-specific lipid transfer proteins (LTPGs) in very-long-chain fatty acids and their derivatives' export for suberin polyester formation.

The LATERAL ORGAN BOUNDARIES (LOB) DOMAIN/ASYMMETRIC LEAVES2-LIKE (LBD/ASL) gene family members play key roles in diverse aspects of plant development. Previous studies have shown that LBD16, 18, 29 and 33 are critical for integrating the plant hormone auxin to control lateral root development in Arabidopsis thaliana. In the present study, we show that LBD14 is expressed exclusively in the root where it promotes lateral root (LR) emergence. Repression of LBD14 expression by ABA correlates with the inhibitory effects of ABA on LR emergence. Transient gene expression assays with Arabidopsis protoplasts demonstrated that LBD14 is a nuclear-localized transcriptional activator. The knock-down of LBD14 expression by RNA interference (RNAi) resulted in reduced LR formation by delaying both LR primordium development and LR emergence, whereas overexpression of LBD14 in Arabidopsis enhances LR formation. We show that ABA (but not other plant hormones such as auxin, brassinosteroids and cytokinin) specifically down-regulated β-glucuronidase (GUS) expression under the control of the LBD14 promoter in transgenic Arabidopsis during LR development from initiation to emergence and endogenous LBD14 transcript levels in the root. Moreover, RNAi of LBD14 enhanced the LR suppression in response to ABA, whereas LBD14 overexpression did not alter the ABA-mediated suppression of LR formation. Taken together, these results suggest that LBD14 promoting LR formation is one of the critical factors regulated by ABA to inhibit LR growth, contributing to the regulation of the Arabidopsis root system architecture in response to ABA.

An increased permeability of the cuticle is closely associated with downregulation of genes involved in cuticular lipid synthesis in hypoxia-stressed Arabidopsis and may allow plants to cope with oxygen deficiency. The hydrophobic cuticle layer consisting of cutin polyester and cuticular wax is the first barrier to protect the aerial parts of land plants from environmental stresses. In the present study, we investigated the role of cuticle membrane in Arabidopsis responses to oxygen deficiency. TEM analysis showed that the epidermal cells of hypoxia-treated Arabidopsis stems and leaves possessed a thinner electron-translucent cuticle proper and a more electron-dense cuticular layer. A reduction in epicuticular wax crystal deposition was observed in SEM images of hypoxia-treated Arabidopsis stem compared with normoxic control. Cuticular transpiration was more rapid in hypoxia-stressed leaves than in normoxic control. Total wax and cutin loads decreased by approximately 6-12 and 12-22%, respectively, and the levels of C29 alkanes, secondary alcohols, and ketones, C16:0 ω-hydroxy fatty acids, and C18:2 dicarboxylic acids were also prominently reduced in hypoxia-stressed Arabidopsis leaves and/or stems relative to normoxic control. Genome-wide transcriptome and quantitative RT-PCR analyses revealed that the expression of several genes involved in the biosynthesis and transport of cuticular waxes and cutin monomers were downregulated more than fourfold, but no significant alterations were detected in the transcript levels of fatty acid biosynthetic genes, BCCP2, PDH-E1α, and ENR1 in hypoxia-treated Arabidopsis stems and leaves compared with normoxic control. Taken together, an increased permeability of the cuticle is closely associated with downregulation of genes involved in cuticular lipid synthesis in hypoxia-stressed Arabidopsis. The present study elucidates one of the cuticle-related adaptive responses that may allow plants to cope with low oxygen levels.

Drought is a critical environmental stress that limits growth and development of plants and reduces crop productivity. The aerial part of land plants is covered with cuticular waxes to minimize water loss. To understand the regulatory mechanisms underlying cuticular wax biosynthesis in Arabidopsis under drought stress conditions, we characterized the role of an AP2/DREB type transcription factor, RAP2.4. RAP2.4 expression was detected in one-week-old seedlings and rosette leaves, stems, stem epidermis, cauline leaves, buds, flowers, and siliques of 6-week-old Arabidopsis. The levels of RAP2.4 transcripts increased with treatments of abscisic acid (ABA), mannitol, NaCl, and drought stress. Under drought, total wax loads decreased by approximately 11% and 10%, and in particular, the levels of alkanes, which are a major wax component, decreased by approximately 11% and 12% in rap2.4-1 and rap2.4-2 leaves, respectively, compared with wild type (WT) leaves. Moreover, the transcript levels of cuticular wax biosynthetic genes, KCS2 and CER1, decreased by approximately 15–23% and 32–40% in rap2.4-1 and rap2.4-2 leaves, respectively, relative to WT 4 h after drought treatment, but increased by 2- to 12-fold and 3- to 70-fold, respectively, in three independent RAP2.4 OX leaves relative to WT. Epicuticular wax crystals were observed on the leaves of RAP2.4 OX plants, but not on the leaves of WT. Total wax loads increased by 1.5- to 3.3-fold in leaves of RAP2.4 OX plants relative to WT. Cuticular transpiration and chlorophyll leaching occurred slowly in the leaves of RAP2.4 OX plants relative to WT. Transcriptional activation assay in tobacco protoplasts showed that RAP2.4 activates the expression of KCS2 and CER1 through the involvement of the consensus CCGAC or GCC motifs present in the KCS2 and CER1 promoter regions. Overall, our results revealed that RAP2.4 is a transcription factor that activates cuticular wax biosynthesis in Arabidopsis leaves under drought stress conditions.

During the evolution of land plants from aquatic to terrestrial environments, their aerial surfaces were surrounded by cuticle composed of cutin and cuticular waxes to protect them from environmental stresses. Glycerol-3-phosphate acyltransferase (GPAT) harboring bifunctional sn-2 acyltransferase/phosphatase activity produces 2-monoacylglycerol, a precursor for cutin synthesis. Here, we report that bifunctional sn-2 GPATs play roles in cuticle biosynthesis and gametophore development of Physcomitrella patens. Land plant-type cuticle was observed in gametophores but not in protonema. The expression of endoplasmic reticulum-localized PpGPATs was significantly upregulated in gametophores compared with protonema. Floral organ fusion and permeable cuticle phenotypes of Arabidopsis gpat6-2 petals were rescued to the wild type (WT) by the expression of PpGPAT2 or PpGPAT4. Disruption of PpGPAT2 and PpGPAT4 caused a significant reduction of total cutin loads, and a prominent decrease in the levels of palmitic and 10,16-dihydroxydecanoic acids, which are major cutin monomers in gametophores. Δppgpat2 mutants displayed growth retardation, delayed gametophore development, increased cuticular permeability, and reduced tolerance to drought, osmotic and salt stresses compared to the WT. Genome-wide analysis of genes encoding acyltransferase or phosphatase domains suggested that the occurrence of sn-2 GPATs with both domains may be a key event in cuticle biogenesis of land plants.

Strigolactone (SL) is a recently discovered class of phytohormone that inhibits shoot branching. The molecular mechanism underlying SL biosynthesis, perception, and signal transduction is vital to the plant branching phenotype. Some aspects of their biosynthesis, perception, and signaling include the role of four MORE AXILLARY GROWTH genes, MAX3, MAX4, MAX1, and MAX2. It is important to identify downstream genes that are involved in SL signaling. To achieve this, we studied the genomic aspects of the strigolactone biosynthesis pathway using microarray analysis of four max mutants. We identified SL signaling candidate genes that showed differential expression patterns in max mutants. More specifically, 1-AMINOCYCLOPROPANE-1-CARBOXYLATE SYNTHASE 4 (ACC4) and PROTEIN KINASE 3 (PKS3) displayed contrasting expression patterns, indicating a regulatory mechanism in SL signaling pathway to control different phenotypes apart from branching phenotype.

Since vegetable oils (usually triacylglycerol [TAG]) are extensively used as food and raw materials, an increase in storage oil content and production of valuable polyunsaturated fatty acids (PUFAs) in transgenic plants is desirable. In this study, a gene encoding glycerol-3-phosphate acyltransferase 9 (GPAT9), which catalyzes the synthesis of lysophosphatidic acid (LPA) from a glycerol-3-phosphate and acyl-CoA, was isolated from Physcomitrella patens, which produces high levels of very-long-chain PUFAs in protonema and gametophores. P. patens GPAT9 shares approximately 50%, 60%, and 70% amino acid similarity with GPAT9 from Chlamydomonas reinhardtii, Klebsormidium nitens, and Arabidopsis thaliana, respectively. PpGPAT9 transcripts were detected in both the protonema and gametophores. Fluorescent signals from the eYFP:PpGPAT9 construct were observed in the ER of Nicotiana benthamiana leaf epidermal cells. Ectopic expression of PpGPAT9 increased the seed oil content by approximately 10% in Arabidopsis. The levels of PUFAs (18:2, 18:3, and 20:2) and saturated FAs (16:0, 18:0, and 20:0) increased by 60% and 43%, respectively, in the storage oil of the transgenic seeds when compared with the wild type. The transgenic embryos with increased oil content contained larger embryonic cells than the wild type. Thus, PpGPAT9 may be a novel genetic resource to enhance storage oil yields from oilseed crops.

Cuticular waxes, which cover the aboveground parts of land plants, are essential for plant survival in terrestrial environments. However, little is known about the regulatory mechanisms underlying cuticular wax biosynthesis in response to changes in ambient humidity. Here, we report that the Arabidopsis (Arabidopsis thaliana) Kelch repeat F-box protein SMALL AND GLOSSY LEAVES1 (SAGL1) mediates proteasome-dependent degradation of ECERIFERUM3 (CER3), a biosynthetic enzyme involved in the production of very long chain alkanes (the major components of wax), thereby negatively regulating cuticular wax biosynthesis. Disruption of SAGL1 led to severe growth retardation, enhanced drought tolerance, and increased wax accumulation in stems, leaves, and roots. Cytoplasmic SAGL1 physically interacts with CER3 and targets it for degradation. β‑glucuronidase (GUS) expression was observed in the roots of pSAGL1:GUS plants but was barely detected in aerial organs. High humidity-induced GUS activity and SAGL1 transcript levels were reduced in response to abscisic acid treatment and water deficit. SAGL1 levels increase under high humidity, and the stability of this protein is regulated by the 26S proteasome. These findings indicate that the SAGL1-CER3 module negatively regulates cuticular wax biosynthesis in Arabidopsis in response to changes to humidity, and they highlight the importance of permeable cuticle formation in terrestrial plants under high humidity conditions.

Monoacylglycerol lipase (MAGL) catalyzes the hydrolysis of monoacylglycerols (MAG) to free fatty acids and glycerol, which is the last step of triacylglycerol breakdown. Among sixteen members, Arabidopsis thaliana MAGL6 (AtMAGL6) and AtMAGL8 showed strong lipase activities, but several AtMAGLs including AtMAGL16 displayed very weak activities (Kim et al. in Plant. J 85:758–771, 2016). To understand the internal factors that influence Arabidopsis MAGL activities, this study investigated the significance of ‘GxSxS motif,’ which is conserved in MAGLs. First, we observed that the presence of a serine protease inhibitor, phenylmethylsulfonyl fluoride, decreased the enzyme activity of AtMAGL6 and AtMAGL8 by IC50 values of 2.30 and 2.35, respectively. Computational modeling showed that amino acid changes of the GxSxG motif in AtMAGL6 and AtMAGL8 altered the nucleophilic elbow structure, which is the active site of MAGLs. Mutating the GxSxG motif in the recombinant maltose binding protein (MBP):AtMAGL6 and MBP:AtMAGL8 proteins to SxSxG, GxAxG, and GxSxS motifs completely demolished the activities of the mutant MAGLs. In contrast, no significant differences were observed between the activities of AtMAGL16 wild type form harboring the SxSxG motif, and mutant AtMAGL16 containing the GxSxG motif. These results revealed that the glycine and serine residues of the GxSxG motif are essential for AtMAGL6 and AtMAGL8 enzyme activities, and that AtMAGL16 may not be involved in the hydrolysis of lipid substrates.

Vegetable oil is not only important for its edibility but for industrial purposes. High-oleic vegetable oil is especially useful for making biodiesel because it is highly stable against oxidation. Transgenic oilseed crops with modified fatty acid compositions have been developed with several biotechnological gene-silencing methods. The seed oils with the most successfully altered fatty acid compositions are produced from high-oleic oilseed crops in which FAD2 gene expression is suppressed. Vegetable oil from oilseed rape (Brassica napus) is one of the most commonly used vegetable oils throughout the world and its oleic acid content is moderately high (about 65 %). Therefore, oilseed rape can be modified to produce high-oleic oilseed crops. Oilseed rape has four FAD2 genes, originating from B. rapa and B. oleracea, with nucleotide identities of 88–97 %. To produce transgenic high-oleic oilseed rape, plant transformation vectors were constructed using antisense RNA and RNA interference (RNAi) to modify the BrFAD2-1 gene, and canola-type cultivar Youngsan was transformed with Agrobacterium carrying the vectors. The transgenic lines generated, AS9A, HP15, and HPAS29, showed high-oleic phenotypes, which were stably inherited. Their oleic acid contents increased from 67 (Youngsan) to 78, 85, and 86 %, respectively, and their polyunsaturated fatty acid (PUFA) contents decreased from 24 (Youngsan) to 13, 8, and 6 %, respectively. HPAS29, developed with a combined antisense RNA–RNAi method, produced seed oil with the highest oleic acid and lowest PUFA contents. These transgenic high-oleic oilseed rapes could be useful in the manufacture of high-temperature frying oils and high-quality biodiesel fuel.

Camelina (Camelina sativa), which belongs to the Brassicaeae family, is an emerging oilseed crop with the potential to expand biodiesel production to arid land. During storage oil synthesis, diacylglycerol acyltrasferase1 (DGAT1) catalyzes the conversion of diacylglycerol (DAG) and free fatty acids to triacylglycerol (TAG). In this study, three DGAT1 genes (CsDGAT1A, CsDGAT1B, and CsDGAT1C) were isolated from developing C. sativa seeds. The deduced amino acid sequences of the three CsDGAT1 genes shared more than 84 % identity with those of DGAT1 genes from Arabidopsis thaliana and Brassica napus. CsDGAT1A, B, and C transcripts were detected in various C. sativa organs, including developing seeds. Fluorescent protein-fused CsDGAT1A, B, and C were localized in the endoplasmic reticulum (ER) of tobacco epidermal cells. When the CsDGAT1A, B, and C genes under the control of the BnNapin promoter were expressed in an Arabidopsis AS11 mutant, which is defective in DGAT1, the amounts and composition of total fatty acids in dry seeds were restored to those of the wild type, indicating the three CsDGAT1 genes to be functionally active. In transgenic C. sativa plants overexpressing CsDGAT1B, the levels of total seed oils were increased by ~24 % compared with non-transgenic lines. Transgenic C. sativa embryos with enhanced seed oil contents harbored larger embryonic cells and a greater number of cells compared with the wild type. Transgenic Camelina plants with increased oil contents can be used as renewable resources for the production of biodiesel and non-petroleum-based biomaterials.

Determining the role of castor lysophosphatidic acid acyltransferases (RcLPATs) provides information that aid in understanding the biosynthesis mechanism of castor oil (triacylglycerols, TAG), which contains 90 % ricinoleic acid (18:1OH), a hydroxy fatty acid (HFA) with numerous industrial applications. The entire family of seven RcLPATs was shown to encode functional enzymes using in vitro assays. Gene expression analysis suggested that RcLPATs play roles in various vegetative and reproductive organs by associating with membrane lipid and TAG biosynthesis. To identify isoforms of RcLPATs capable of acylating 18:1OH, individual RcLPATs were expressed in CL37, an Arabidopsis line containing approximately 17 % HFA in seed TAG. Transgenics expressing RcLPAT2, RcLPAT3B, or RcLPATB increased total HFA to 18.2 %–20.3 %. Furthermore, different accumulation levels of 18:1OH and densipolic acid (18:2OH) were detected among these three transgenic backgrounds. The mechanisms of substrate selectivity among RcLPAT2, RcLPAT3B, and RcLPATB are discussed.

A Kelch repeat F-box containing protein, SMALL AND GLOSSY LEAVES1 (SAGL1) regulates phenylpropanoid biosynthesis as a post-translational regulator for PAL1 (phenylalanine ammonia-lyase) and an indirect transcriptional regulator for ANTHOCYANIDIN SYNTHASE. Phenylpropanoid biosynthesis in plants produces diverse aromatic metabolites with important biological functions. Phenylalanine ammonia-lyase (PAL) catalyzes the first step in phenylpropanoid biosynthesis by converting l-phenylalanine to trans-cinnamic acid. Here, we report that SMALL AND GLOSSY LEAVES1 (SAGL1), a Kelch repeat F-box protein, interacts with PAL1 protein for proteasome-mediated degradation to regulate phenylpropanoid biosynthesis in Arabidopsis. Mutations in SAGL1 caused high accumulation of anthocyanins and lignin derived from the phenylpropanoid biosynthesis pathway. We found that PAL enzyme activity increased in SAGL1-defective mutants, sagl1, but decreased in SAGL1-overexpressing Arabidopsis (SAGL1OE) without changes in the transcript levels of PAL genes, suggesting protein-level regulation by SAGL1. Indeed, the levels of PAL1-GFP fusion protein were reduced when both SAGL1 and PAL1-GFP were transiently co-expressed in leaves of Nicotiana benthamiana. In addition, bimolecular fluorescence complementation analysis suggested an interaction between SAGL1 and PAL1. We also found that the transcript levels of ANTHOCYANIDIN SYNTHASE (ANS) increased in the sagl1 mutants but decreased in SAGL1OE. Our results suggest that SAGL1 regulates phenylpropanoid biosynthesis post-translationally at PAL1 and transcriptionally at ANS.

The importance of the cuticular layer in regulating a plant’s water status and providing protection from environmental challenges has been recognized for a long time. The cuticular layer in plants restricts non-stomatal water loss and protects plants against damage from pathogen infection and UV radiation. Much genetic and biochemical research has been done about cutin and wax transportation in Arabidopsis thaliana, but little is known about it in rice. Here, we report that a rice ATP-binding cassette (ABC) transporter, OsABCG9, is essential for normal development during vegetative growth and could play a critical role in the transportation of epicuticular wax in rice. Rice phenotypes with mutated OsABCG9 exhibited growth retardation and sensitivity to low humidity. The total amount of cuticular wax on the leaves of the osabcg9-1 mutant diminished by 53% compared with the wild type, and wax crystals disappeared completely in osabcg9-2 mutant leaves. However, OsABCG9 does not seem to be involved in cutin transportation, even though its ortholog in Arabidopsis, AtABCG11, transports both wax and cutin. Furthermore, the osabcg9-1 mutant had increased leaf chlorophyll leaching and more severe drought susceptibility. This study provides new insights about differences between rice and A. thaliana in wax and cutin transportation associated with the ABCG family during evolution.

Advances in molecular biology have improved crops through transferring genes from one organism to new hosts, and these efforts have raised concerns about potential unexpected outcomes. Here, we provide evidence that a gene with a specific function in one organism can yield completely different effects in a new host. CaRZFP1 is a C3HC4-type RING zinc finger protein gene previously isolated from a cDNA library for heat-stressed hot pepper. In our previous work investigating in vivo CaRZFP1 function, we transferred CaRZFP1 into tobacco; transgenic tobacco exhibited enhanced growth and tolerance to abiotic stresses. As further analysis of CaRZFP1 ectopic expression in a heterologous host plant, here we mobilized and constitutively overexpressed CaRZFP1 in lettuce. In contrast to tobacco, transgenic lettuce exhibited poorer growth and delayed flowering compared with vector-only controls. To identify genes that might be involved in this phenotypic effect, transcriptome analyses on transgenic plants of both species were performed, uncovering dozens of genes that reflect the different outcomes between tobacco and lettuce. These included protein kinase, transcriptional factor, transporter protein, hormone and metabolism-related genes, and some unannotated genes. The opposite effects of CaRZFP1 ectopic expression in lettuce and tobacco address concerns of unexpectedly different outcomes in different host species. Electronic supplementary material The online version of this article (10.1007/s11032-018-0812-3) contains supplementary material, which is available to authorized users.

Maturing seeds stimulate fatty acid (FA) biosynthesis and triacylglycerol (TAG) accumulation to ensure carbon and energy reserves. Transcriptional reprogramming is a key regulatory scheme in seed oil accumulation. In particular, TAG assembly is mainly controlled by the transcriptional regulation of two key enzymes, acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) and phospholipid:diacylglycerol acyltransferase 1 (PDAT1), in Arabidopsis seeds. However, the transcriptional regulators of these enzymes are as yet unknown. Here, we report that the R2R3-type MYB96 transcription factor regulates seed oil accumulation by activating the genes encoding DGAT1 and PDAT1, the rate-limiting enzymes of the last step of TAG assembly. Total FA levels are significantly elevated in MYB96-overexpressing transgenic seeds, but reduced in MYB96-deficient mutant seeds. Notably, MYB96 regulation of TAG accumulation is independent of WRINKLED 1 (WRI1)-mediated FA biosynthesis. Taken together, our findings indicate that FA biosynthesis and TAG accumulation are under independent transcriptional control, and MYB96 is mainly responsible for TAG assembly in seeds.

Under heavy-metal stress, CsHMA3 overexpressing transgenic Camelina plants displayed not only a better quality, but also a higher quantity of unsaturated fatty acids in their seeds compared with wild type. Camelina sativa L. belongs to the Brassicaceae family and is frequently used as a natural vegetable oil source, as its seeds contain a high content of fatty acids. In this study, we observed that, when subjected to heavy metals (Cd, Co, Zn and Pb), the seeds of CsHMA3 (Heavy-Metal P1B-ATPase 3) transgenic lines retained their original golden yellow color and smooth outline, unlike wild-type seeds. Furthermore, we investigated the fatty acids content and composition of wild type and CsHMA3 transgenic lines after heavy metal treatments compared to the control. The results showed higher total fatty acid amounts in seeds of CsHMA3 transgenic lines compared with those in wild-type seeds under heavy-metal stresses. In addition, the compositions of unsaturated fatty acids—especially 18:1 (oleic acid), 18:2 (linoleic acid; only in case of Co treatment), 18:3 (linolenic acid) and 20:1 (eicosenoic acid)—in CsHMA3 overexpressing transgenic lines treated with heavy metals were higher than those of wild-type seeds under the same conditions. Furthermore, reactive oxygen species (ROS) contents in wild-type leaves and roots when treated with heavy metal were higher than in CsHMA3 overexpressing transgenic lines. These results indicate that overexpression of CsHMA3 affects fatty acid composition and content—factors that are responsible for the fuel properties of biodiesel—and can alleviate ROS accumulation caused by heavy-metal stresses in Camelina. Due to these factors, we propose that CsHMA3 transgenic Camelina can be used for phytoremediation of metal-contaminated soil as well as for oil production.

The synthesis of fatty acids and glycerolipids in wild-type Arabidopsis leaves does not typically lead to strong triacylglycerol (TAG) accumulation. LEAFY COTYLEDON2 (LEC2) is a master regulator of seed maturation and oil accumulation in seeds. Constitutive ectopic LEC2 expression causes somatic embryogenesis and defects in seedling growth. Here, we report that senescence-inducible LEC2 expression caused a threefold increase in TAG levels in transgenic leaves compared with that in the leaves of wild-type plants. Plant growth was not severely affected by the accumulation the TAG in response to LEC2 expression. The levels of plastid-synthesized lipids, mono- and di-galactosyldiacylglycerol and phosphatidylglycerol were reduced more in senescence-induced LEC2 than in endoplasmic reticulum-synthesized lipids, including phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol. Senescence-induced LEC2 up-regulated the expression of many genes involved in fatty acid and TAG biosynthesis at precise times in senescent leaves, including WRINKLED1 (WRI1), which encodes a fatty acid transcription factor. The expressions of glycerol-3-phosphate dehydrogenase 1 and phospholipid:diacylglycerol 2 were increased in the transgenic leaves. Five seed-type oleosin-encoding genes, expressed during oil-body formation, and the seed-specific FAE1 gene, which encodes the enzyme responsible for the synthesis of C20:1 and C22:1 fatty acids, were also expressed at higher levels in senescing transgenic leaves than in wild-type leaves. Senescence-inducible LEC2 triggers the key metabolic steps that increase TAG accumulation in vegetative tissues.

The aerial parts of terrestrial plants are covered with hydrophobic wax layers, which represent the primary barrier between plant cells and the environment and act to protect plants from abiotic and biotic stresses. Although total wax loads are precisely regulated in an environmental- or organ-specific manner, regulatory mechanisms underlying cuticular wax biosynthesis remain largely unknown. In this study, we characterized DEWAX2 (DECREASE WAX BIOSYNTHESIS2) which encodes an APETALA 2 (AP2)/ethylene response element-binding factor (ERF)-type transcription factor and is predominantly expressed in young seedlings, and rosette and cauline leaves. Total wax loads increased by approximately 12% and 16% in rosette and cauline leaves of dewax2, respectively, but were not significantly altered in the stems of dewax2 relative to the wild type (WT). The excess wax phenotype of dewax2 leaves was rescued upon expression of DEWAX2 driven by its own promoter. Overexpression of DEWAX2 decreased total wax loads by approximately 15% and 26% in the stems and rosette leaves compared with those of the WT, respectively. DEWAX2:eYFP (enhanced yellow fluorescent protein) was localized to the nucleus in Arabidopsis roots and hypocotyls. DEWAX2 possessed transcriptional repression activity in tobacco protoplasts. Transcriptome and quantitative real-time PCR analyses showed that the transcript levels of CER1, ACLA2, LACS1, LACS2 and KCS12 were down-regulated in DEWAX2 overexpression lines compared with the WT. Transient transcriptional assays showed that DEWAX2 represses the expression of its putative target genes. Quantitative chromatin immunoprecipitation-PCR revealed that DEWAX2 binds directly to the GCC motifs of the LACS1, LACS2, KCS12 and CER1 promoters. These results suggest that DEWAX2-mediated transcriptional repression may contribute to the total wax load in Arabidopsis leaves.

The cuticle of land plants is the first physical barrier to protect their aerial parts from biotic and abiotic stresses. DEWAX, an AP2/ERF-type transcription factor, negatively regulates cuticular wax biosynthesis. In this study, we investigated the resistance to Botrytis cinerea in Arabidopsis thaliana and Camelina sativa overexpressing DEWAX and in Arabidopsis dewax mutant. Compared to wild type (WT) leaves, Arabidopsis DEWAX OX and dewax leaves were more and less permeable to toluidine blue dye, respectively. The ROS levels increased in DEWAX OX leaves, but decreased in dewax relative to WT leaves. Compared to WT, DEWAX OX was more resistant, while dewax was more sensitive to B. cinerea; however, defense responses to Pseudomonas syringae pv. tomato DC3000:GFP were inversely modulated. Microarray and RT-PCR analyses indicated that the expression of defense-related genes was upregulated in DEWAX OX, but downregulated in dewax relative to WT. Transactivation assay showed that DEWAX upregulated the expression of PDF1.2a, IGMT1, and PRX37. Chromatin immunoprecipitation assay revealed that DEWAX directly interacts with the GCC-box motifs of PDF1.2a promoter. In addition, ectopic expression of DEWAX increased the tolerance to B. cinerea in C. sativa. Taken together, we suggest that increased ROS accumulation and DEWAX-mediated upregulation of defense-related genes are closely associated with enhanced resistance to B. cinerea in Arabidopsis and C. sativa.

The cuticle of land plants is the first physical barrier to protect their aerial parts from biotic and abiotic stresses. DEWAX, an AP2/ERF-type transcription factor, negatively regulates cuticular wax biosynthesis. In this study, we investigated the resistance to Botrytis cinerea in Arabidopsis thaliana and Camelina sativa overexpressing DEWAX and in Arabidopsis dewax mutant. Compared to wild type (WT) leaves, Arabidopsis DEWAX OX and dewax leaves were more and less permeable to toluidine blue dye, respectively. The ROS levels increased in DEWAX OX leaves, but decreased in dewax relative to WT leaves. Compared to WT, DEWAX OX was more resistant, while dewax was more sensitive to B. cinerea; however, defense responses to Pseudomonas syringae pv. tomato DC3000:GFP were inversely modulated. Microarray and RT-PCR analyses indicated that the expression of defense-related genes was upregulated in DEWAX OX, but downregulated in dewax relative to WT. Transactivation assay showed that DEWAX upregulated the expression of PDF1.2a, IGMT1, and PRX37. Chromatin immunoprecipitation assay revealed that DEWAX directly interacts with the GCC-box motifs of PDF1.2a promoter. In addition, ectopic expression of DEWAX increased the tolerance to B. cinerea in C. sativa. Taken together, we suggest that increased ROS accumulation and DEWAX-mediated upregulation of defense-related genes are closely associated with enhanced resistance to B. cinerea in Arabidopsis and C. sativa.

Cuticular wax is an important hydrophobic layer that covers the plant aerial surface. Cuticular wax biosynthesis is shaped by multiple layers of regulation. In particular, a pair of R2R3-type MYB transcription factors, MYB96 and MYB30, are known to be the main participants in cuticular wax accumulation. Here, we report that the MYB30-INTERACTING E3 LIGASE 1 (MIEL1) E3 ubiquitin ligase controls the protein stability of the two MYB transcription factors and thereby wax biosynthesis in Arabidopsis. MIEL1-deficient miel1 mutants exhibit increased wax accumulation in stems, with up-regulation of wax biosynthetic genes targeted by MYB96 and MYB30. Genetic analysis reveals that wax accumulation of the miel1 mutant is compromised by myb96 or myb30 mutation, but MYB96 is mainly epistatic to MIEL1, playing a predominant role in cuticular wax deposition. These observations indicate that the MIEL1-MYB96 module is important for balanced cuticular wax biosynthesis in developing inflorescence stems.

The aerial parts of all land plants are covered with hydrophobic cuticular wax layers that act as the first barrier against the environment. The MYB94 transcription factor gene is expressed in abundance in aerial organs and shows a higher expression in the stem epidermis than within the stem. When seedlings were subjected to various treatments, the expression of the MYB94 transcription factor gene was observed to increase approximately 9-fold under drought, 8-fold for ABA treatment and 4-fold for separate NaCl and mannitol treatments. MYB94 harbors the transcriptional activation domain at its C-terminus, and fluorescent signals from MYB94:enhanced yellow fluorescent protein (eYFP) were observed in the nucleus of tobacco epidermis and in transgenic Arabidopsis roots. The total wax loads increased by approximately 2-fold in the leaves of the MYB94-overexpressing (MYB94 OX) lines, as compared with those of the wild type (WT). MYB94 activates the expression of WSD1, KCS2/DAISY, CER2, FAR3 and ECR genes by binding directly to their gene promoters. An increase in the accumulation of cuticular wax was observed to reduce the rate of cuticular transpiration in the leaves of MYB94 OX lines, under drought stress conditions. Taken together, a R2R3-type MYB94 transcription factor activates Arabidopsis cuticular wax biosynthesis and might be important in plant response to environmental stress, including drought.

카멜리나(Camelina sativa)는 십자화과(Brassicaceae)에 속하는 유지작물이다. 카멜리나 종자에는 건물 중의 약 40%에 해당하는 저장 오일을 가지고 있고, 이러한 저장오일은 식품뿐만 아니라 산업재료로 이용이 가능하다. Microsomal delta-12 fatty acid desaturase2 (FAD2) 효소는 oleic acid를 linoleic acid로 전환시키는데, 종자 내 oleic acid의 함량 차이를 보이는 품종들에서 FAD2 유전자의 polymorphism이 보고되었다. 본 연구에서는 카멜리나(Camelina sativa L. 품종 CAME)에 존재하는 3개의 FAD2 유전자를 발달하는 종자로부터 분리하였다. 3개의 카멜리나 FAD2 유전자의 염기서열 및 아미노산 서열은 카멜리나 품종 Sunesone과 SRS933으로부터 확인된 FAD2 유전자들과 여러 단자엽 및 쌍자엽 식물의 FAD2 유전자들의 염기서열 및 아미노산 서열과 상동성을 비교하였다. FAD2 효소의 활성을 결정짓는다고 알려진 histidine motif (HECGHH, HRRHH 그리고 HVAHH)와 효소 활성에 영향을 주는 SNP (single nucleotide polymorphism) 마커라고 알려진 소수성 아미노산 계열인 valine 혹은 isoleucine이 3 개의 카멜리나 FAD2에서도 잘 보존되어 있음을 확인하였다. 세개의 카멜리나 FAD2 유전자들 중 CsFAD2-1의 경우 카멜리나의 발달하는 조직에서 전반적으로 높은 발현 양상을 보이는 반면 CsFAD2-2와 CsFAD2-3.1은 꽃과 발달하는 종자에서 특이적인 발현을 보였다. 애기장대 fad2-2 돌연변이체에 3개의 카멜리나 FAD2를 각각 도입한 형질전환 식물체의 종자에는 애기장대 fad2-2 돌연변이체 종자대비 oleic acid의 함량이 감소하고, linoleic acid 함량은 증가하는 표현형이 관찰되었다. 이러한 결과는 카멜리나로부터 분리된 3개의 FAD2가 효소로서 활성을 가지고 있다는 것을 의미한다. 더불어 분리된 카멜리나의 FAD2 유전자는 종자 오일 성분이 변화된 유지작물을 개발하는데 응용될 수 있을 것이다.

Camelina has been highlighted as an emerging oilseed crop. Transgenic Camelina plants overexpressing Arabidopsis MYB96 exhibited drought resistance by activating expression of Camelina wax biosynthetic genes and accumulating wax load. Camelina (Camelina sativa L.) is an oilseed crop in the Brassicaeae family with potential to expand biofuel production to marginal land. The aerial portion of all land plants is covered with cuticular wax to protect them from desiccation. In this study, the Arabidopsis MYB96 gene was overexpressed in Camelina under the control of the CaMV35S promoter. Transgenic Camelina plants overexpressing Arabidopsis MYB96 exhibited normal growth and development and enhanced tolerance to drought. Deposition of epicuticular wax crystals and total wax loads increased significantly on the surfaces of transgenic leaves compared with that of non-transgenic plants. The levels of alkanes and primary alcohols prominently increased in transgenic Camelina plants relative to non-transgenic plants. Cuticular transpiration occurred more slowly in transgenic leaves than that in non-transgenic plants. Genome-wide identification of Camelina wax biosynthetic genes enabled us to determine that the expression levels of CsKCS2, CsKCS6, CsKCR1-1, CsKCR1-2, CsECR, and CsMAH1 were approximately two to sevenfold higher in transgenic Camelina leaves than those in non-transgenic leaves. These results indicate that MYB96-mediated transcriptional regulation of wax biosynthetic genes is an approach applicable to generating drought-resistant transgenic crops. Transgenic Camelina plants with enhanced drought tolerance could be cultivated on marginal land to produce renewable biofuels and biomaterials.

The aerial parts of plants are protected from desiccation and other stress by surface cuticular waxes. The total cuticular wax loads and the expression of wax biosynthetic genes are significantly downregulated in Arabidopsis thaliana under dark conditions. We isolated Decrease Wax Biosynthesis (DEWAX), which encodes an AP2/ERF-type transcription factor that is preferentially expressed in the epidermis and induced by darkness. Disruption of DEWAX leads to an increase in total leaf and stem wax loads, and the excess wax phenotype of dewax was restored to wild type levels in complementation lines. Moreover, overexpression of DEWAX resulted in a reduction in total wax loads in leaves and stems compared with the wild type and altered the ultrastructure of cuticular layers. DEWAX negatively regulates the expression of alkane-forming enzyme, long-chain acyl-CoA synthetase, ATP citrate lyase A subunit, enoyl-CoA reductase, and fatty acyl-CoA reductase, and chromatin immunoprecipitation analysis suggested that DEWAX directly interacts with the promoters of wax biosynthesis genes. Cuticular wax biosynthesis is negatively regulated twice a day by the expression of DEWAX, throughout the night and at stomata closing. Significantly higher levels (10- to 100-fold) of DEWAX transcripts were found in leaves than in stems, suggesting that DEWAX-mediated transcriptional repression may be an additional mechanism contributing to the different total wax loads in leaves and stems.

Camelina (Camelina sativa, Cs) is an emerging crop for the production of biodiesel and biofeedstock. This study aims to develop transgenic plants that have the advantage of two traits: improved drought resistance and oil content. Three genes, CsMYB96A, CsMYB96B, and CsMYB96C, were isolated from camelina stem. The deduced amino acid sequence of the three CsMYB96s showed at least 93% identity with Arabidopsis MYB96. CsMYB96A, B, and C transcripts were detected in various camelina tissues. Fluorescence signal from the fusion of CsMYB96A: enhanced yellow fluorescent protein was confined to the nucleus of tobacco epidermal cells. Transactivation analysis of tobacco protoplasts revealed that CsMYB96A was a transcription activator. Wax biosynthesis genes such as camelina β-ketoacyl-CoA synthase 2, β-ketoacyl-CoA synthase 6, β-ketoacyl-CoA reductase 1-1, β-ketoacyl-CoA reductase 1-2, enoyl-CoA reductase, ECERIFERUM 1 and ECERIFERUM 3 were upregulated approximately 2 to 120 times by CsMYB96A, indicating that CsMYB96A was involved in the activating of cuticular wax synthesis on plant epidemics. Camelina diacylglycerol acyltransferase 1C (CsDGAT1C) has been shown to increased oil synthesis genes in Arabidopsis. When CsDGAT1C and CsMYB96A were co-overexpressed in camelina, total fatty acid levels in transgenic seeds increased by approximately 21%. In addition, the transgenic camelina plants showed improved resistance to drought stress. This result suggests that the transgenic camelina can be grown to produce biodiesel and biofeedstock in arid or semi-arid lands.

Strigolactone (SL) is a recently discovered class of phytohormone that inhibits shoot branching. The molecular mechanism underlying SL biosynthesis, perception, and signal transduction is vital to the plant branching phenotype. Some aspects of their biosynthesis, perception, and signaling include the role of four MORE AXILLARY GROWTH genes, MAX3, MAX4, MAX1, and MAX2. It is important to identify downstream genes that are involved in SL signaling. To achieve this, we studied the genomic aspects of the strigolactone biosynthesis pathway using microarray analysis of four max mutants. We identified SL signaling candidate genes that showed differential expression patterns in max mutants. More specifically, 1-AMINOCYCLOPROPANE-1-CARBOXYLATE SYNTHASE 4 (ACC4) and PROTEIN KINASE 3 (PKS3) displayed contrasting expression patterns, indicating a regulatory mechanism in SL signaling pathway to control different phenotypes apart from branching phenotype.

Since vegetable oils (usually triacylglycerol [TAG]) are extensively used as food and raw materials, an increase in storage oil content and production of valuable polyunsaturated fatty acids (PUFAs) in transgenic plants is desirable. In this study, a gene encoding glycerol-3-phosphate acyltransferase 9 (GPAT9), which catalyzes the synthesis of lysophosphatidic acid (LPA) from a glycerol-3-phosphate and acyl-CoA, was isolated from Physcomitrella patens, which produces high levels of very-long-chain PUFAs in protonema and gametophores. P. patens GPAT9 shares approximately 50%, 60%, and 70% amino acid similarity with GPAT9 from Chlamydomonas reinhardtii, Klebsormidium nitens, and Arabidopsis thaliana, respectively. PpGPAT9 transcripts were detected in both the protonema and gametophores. Fluorescent signals from the eYFP:PpGPAT9 construct were observed in the ER of Nicotiana benthamiana leaf epidermal cells. Ectopic expression of PpGPAT9 increased the seed oil content by approximately 10% in Arabidopsis. The levels of PUFAs (18:2, 18:3, and 20:2) and saturated FAs (16:0, 18:0, and 20:0) increased by 60% and 43%, respectively, in the storage oil of the transgenic seeds when compared with the wild type. The transgenic embryos with increased oil content contained larger embryonic cells than the wild type. Thus, PpGPAT9 may be a novel genetic resource to enhance storage oil yields from oilseed crops.

Fatty acid desaturase 2 (FAD2), which resides in the endoplasmic reticulum (ER), plays a crucial role in producing linoleic acid (18:2) through catalyzing the desaturation of oleic acid (18:1) by double bond formation at the delta 12 position. FAD2 catalyzes the first step needed for the production of polyunsaturated fatty acids found in the glycerolipids of cell membranes and the triacylglycerols in seeds. In this study, four FAD2 genes from amphidiploid Brassica napus genome were isolated by PCR amplification, with their enzymatic functions predicted by sequence analysis of the cDNAs. Fatty acid analysis of budding yeast transformed with each of the FAD2 genes showed that whereas BnFAD2-1, BnFAD2-2, and BnFAD2-4 are functional enzymes, and BnFAD2-3 is nonfunctional. The four FAD2 genes of B. napus originated from synthetic hybridization of its diploid progenitors Brassica rapa and Brassica oleracea, each of which has two FAD2 genes identical to those of B. napus. The BnFAD2-3 gene of B. napus, a nonfunctional pseudogene mutated by multiple nucleotide deletions and insertions, was inherited from B. rapa. All BnFAD2 isozymes except BnFAD2-3 localized to the ER. Nonfunctional BnFAD2-3 localized to the nucleus and chloroplasts. Four BnFAD2 genes can be classified on the basis of their expression patterns.

Very-long-chain fatty acids (VLCFAs) with chain lengths from 20 to 34 carbons are involved in diverse biological functions such as membrane constituents, a surface barrier, and seed storage compounds. The first step in VLCFA biosynthesis is the condensation of two carbons to an acyl-coenzyme A, which is catalyzed by 3-ketoacyl-coenzyme A synthase (KCS). In this study, amino acid sequence homology and the messenger RNA expression patterns of 21 Arabidopsis (Arabidopsis thaliana) KCSs were compared. The in planta role of the KCS9 gene, showing higher expression in stem epidermal peels than in stems, was further investigated. The KCS9 gene was ubiquitously expressed in various organs and tissues, including roots, leaves, and stems, including epidermis, silique walls, sepals, the upper portion of the styles, and seed coats, but not in developing embryos. The fluorescent signals of the KCS9::enhanced yellow fluorescent protein construct were merged with those of BrFAD2::monomeric red fluorescent protein, which is an endoplasmic reticulum marker in tobacco (Nicotiana benthamiana) epidermal cells. The kcs9 knockout mutants exhibited a significant reduction in C24 VLCFAs but an accumulation of C20 and C22 VLCFAs in the analysis of membrane and surface lipids. The mutant phenotypes were rescued by the expression of KCS9 under the control of the cauliflower mosaic virus 35S promoter. Taken together, these data demonstrate that KCS9 is involved in the elongation of C22 to C24 fatty acids, which are essential precursors for the biosynthesis of cuticular waxes, aliphatic suberins, and membrane lipids, including sphingolipids and phospholipids. Finally, possible roles of unidentified KCSs are discussed by combining genetic study results and gene expression data from multiple Arabidopsis KCSs.

The aerial parts of land plants are covered with cuticular waxes that limit non-stomatal water loss and gaseous exchanges, and protect plants from ultraviolet radiation and pathogen attacks. They are composed of very-long-chain fatty acids (VLCFAs; C20 to C34) in addition to their derivatives, aldehydes, alkanes, primary and secondary alcohols, and wax esters. Due to their physical properties, such as solidity at room temperature and a translucency ranging from transparent to opaque, plant waxes have been used as raw materials in the production of cosmetics, detergents, plastics, soaps, paints, drugs, lubricants, and high-value renewable fuels.

Triacylglycerol (TAG) is an energy-rich reserve in plant seeds that is composed of glycerol esters with three fatty acids. Since TAG can be used as a feedstock for the production of biofuels and bio-chemicals, producing TAGs in vegetative tissue is an alternative way of meeting the increasing demand for its usage. The WRINKLED1 (WRI1) gene is a well-established key transcriptional regulator involved in the upregulation of fatty acid biosynthesis in developing seeds. WRI1s from Arabidopsis and several other crops have been previously employed for increasing TAGs in seed and vegetative tissues. In the present study, we first identified three functional CsWRI1 genes (CsWRI1A. B, and C) from the Camelina oil crop and tested their ability to induce TAG synthesis in leaves. The amino acid sequences of CsWRI1s exhibited more than 90% identity with those of Arabidopsis WRI1. The transcript levels of the three CsWRI1 genes showed higher expression levels in developing seeds than in vegetative and floral tissues. When the CsWRI1A. B, or C was introduced into Arabidopsis wri1-3 loss-of-function mutant, the fatty acid content was restored to near wild-type levels and percentages of the wrinkled seeds were remarkably reduced in the transgenic lines relative to wri1-3 mutant line. In addition, the fluorescent signals of the enhanced yellow fluorescent protein (eYFP) fused to the CsWRI1 genes were observed in the nuclei of Nicotiana benthamiana leaf epidermal cells. Nile red staining indicated that the transient expression of CsWRI1A. B, or C caused an enhanced accumulation of oil bodies in N. benthamiana leaves. The levels of TAGs was higher by approximately 2.5- to 4.0-fold in N. benthamiana fresh leaves expressing CsWRI1 genes than in the control leaves. These results suggest that the three Camelina WRI1s can be used as key transcriptional regulators to increase fatty acids in biomass.

Cuticular waxes act as a protective barrier against environmental stresses. In the present study, we investigated developmental and genotypic variation in wax formation of cabbage lines, with a view to understand the related morphology, genetics and biochemistry. Our studies revealed that the relative expression levels of wax biosynthetic genes in the first-formed leaf of the highest-wax line remained constantly higher but were decreased in other genotypes with leaf aging. Similarly, the expression of most of the tested genes exhibited decrease from the inner leaves to the outer leaves of 5-month-old cabbage heads in the low-wax lines in contrast to the highest-wax line. In 10-week-old plants, expression of wax biosynthetic genes followed a quadratic function and was generally increased in the early developing leaves but substantially decreased at the older leaves. The waxy compounds in all cabbage lines were predominately C29-alkane, -secondary alcohol, and -ketone. Its deposition was increased with leaf age in 5-month-old plants. The high-wax lines had dense, prominent and larger crystals on the leaf surface compared to low-wax lines under scanning electron microscopy. Principal component analysis revealed that the higher expression of LTP2 genes in the lowest-wax line and the higher expression of CER3 gene in the highest-wax line were probably associated with the comparatively lower and higher wax content in those two lines, respectively. This study furthers our understanding of the relationships between the expression of wax biosynthetic genes and the wax deposition in cabbage lines. Highlight: In cabbage, expression of wax-biosynthetic genes was generally decreased in older and senescing leaves, while wax deposition was increased with leaf aging, and C29-hydrocarbon was predominant in the wax crystals.

Triacylglycerol (TAG) is an energy-rich reserve in plant seeds that is composed of glycerol esters with three fatty acids. Since TAG can be used as a feedstock for the production of biofuels and bio-chemicals, producing TAGs in vegetative tissue is an alternative way of meeting the increasing demand for its usage. The WRINKLED1 (WRI1) gene is a well-established key transcriptional regulator involved in the upregulation of fatty acid biosynthesis in developing seeds. WRI1s from Arabidopsis and several other crops have been previously employed for increasing TAGs in seed and vegetative tissues. In the present study, we first identified three functional CsWRI1 genes (CsWRI1A. B, and C) from the Camelina oil crop and tested their ability to induce TAG synthesis in leaves. The amino acid sequences of CsWRI1s exhibited more than 90% identity with those of Arabidopsis WRI1. The transcript levels of the three CsWRI1 genes showed higher expression levels in developing seeds than in vegetative and floral tissues. When the CsWRI1A. B, or C was introduced into Arabidopsis wri1-3 loss-of-function mutant, the fatty acid content was restored to near wild-type levels and percentages of the wrinkled seeds were remarkably reduced in the transgenic lines relative to wri1-3 mutant line. In addition, the fluorescent signals of the enhanced yellow fluorescent protein (eYFP) fused to the CsWRI1 genes were observed in the nuclei of Nicotiana benthamiana leaf epidermal cells. Nile red staining indicated that the transient expression of CsWRI1A. B, or C caused an enhanced accumulation of oil bodies in N. benthamiana leaves. The levels of TAGs was higher by approximately 2.5- to 4.0-fold in N. benthamiana fresh leaves expressing CsWRI1 genes than in the control leaves. These results suggest that the three Camelina WRI1s can be used as key transcriptional regulators to increase fatty acids in biomass.

Cuticular waxes act as a protective barrier against environmental stresses. In the present study, we investigated developmental and genotypic variation in wax formation of cabbage lines, with a view to understand the related morphology, genetics and biochemistry. Our studies revealed that the relative expression levels of wax biosynthetic genes in the first-formed leaf of the highest-wax line remained constantly higher but were decreased in other genotypes with leaf aging. Similarly, the expression of most of the tested genes exhibited decrease from the inner leaves to the outer leaves of 5-month-old cabbage heads in the low-wax lines in contrast to the highest-wax line. In 10-week-old plants, expression of wax biosynthetic genes followed a quadratic function and was generally increased in the early developing leaves but substantially decreased at the older leaves. The waxy compounds in all cabbage lines were predominately C29-alkane, -secondary alcohol, and -ketone. Its deposition was increased with leaf age in 5-month-old plants. The high-wax lines had dense, prominent and larger crystals on the leaf surface compared to low-wax lines under scanning electron microscopy. Principal component analysis revealed that the higher expression of LTP2 genes in the lowest-wax line and the higher expression of CER3 gene in the highest-wax line were probably associated with the comparatively lower and higher wax content in those two lines, respectively. This study furthers our understanding of the relationships between the expression of wax biosynthetic genes and the wax deposition in cabbage lines. Highlight: In cabbage, expression of wax-biosynthetic genes was generally decreased in older and senescing leaves, while wax deposition was increased with leaf aging, and C29-hydrocarbon was predominant in the wax crystals.

Aerial plant surfaces are coated by a cuticular wax layer to protect against environmental stresses, such as desiccation. In this study, we investigated the functional relationship between MYB94 and MYB96 transcription factors involved in cuticular wax biosynthesis. Both MYB94 and MYB96 transcripts were abundantly expressed in the aerial organs of Arabidopsis, and significantly induced at the same or similar time points under conditions of drought. MYB94 complemented the wax-deficient phenotype of the myb96 loss-of-function mutant under both well-watered and drought stress conditions. The magnitude of decrease in total wax load in the myb94 myb96 double mutant was almost equal to the sum of the reduced wax loads in the individual myb94 and myb96 mutants under both conditions. Leaves of the myb94 myb96 mutant lost water through the cuticle faster than those of myb94 or myb96 plants. Transcript levels of wax biosynthetic genes were decreased in the single mutants, and further reduced in the double mutant, relative to the wild type, under drought and ABA treatment conditions. MYB94 and MYB96 interact with the same regions containing MYB consensus motifs in the promoter regions of wax biosynthetic genes. The data collectively indicate that MYB94 and MYB96 exert an additive effect on cuticular wax biosynthesis, which may represent an efficient adaptive mechanism of response to drought in plants.

Summary The aerial surfaces of terrestrial plants are covered by a cuticular wax layer, which protects the plants from environmental stresses such as desiccation, high irradiance, and UV radiation. Cuticular wax deposition is regulated in an organ-specific manner; Arabidopsis stems have more than 10-fold higher wax loads than leaves. In this study, we found that WRINKLED4 (WRI4), encoding an AP2/ERF transcription factor (TF), is predominantly expressed in stem epidermis, is upregulated by salt stress, and is involved in activating cuticular wax biosynthesis in Arabidopsis stems. WRI4 harbors a transcriptional activation domain at its N-terminus, and fluorescent signals from a WRI4:eYFP construct were localized to the nuclei of tobacco leaf protoplasts. Deposition of epicuticular wax crystals on stems was reduced in wri4-1 and wri4-3 knockout mutants. Total wax loads were reduced by ~28% in wri4 stems but were not altered in wri4 siliques or leaves compared to the wild type. The levels of 29-carbon long alkanes, ketones, and secondary alcohols, which are the most abundant components of stem waxes, were significantly reduced in wri4 stems relative to the wild type. A transactivation assay in tobacco protoplasts and a chromatin immunoprecipitation (ChIP) assay showed that the expression of long-chain acyl-CoA synthetase1 (LACS1), β–ketoacyl-CoA reductase1 (KCR1), PASTICCINO2 (PAS2), trans-2,3-enoyl-CoA reductase (ECR), and bifunctional wax synthase/acyl-CoA: diacylglycerol acyltransferase (WSD1) is positively regulated by direct binding of WRI4 to their promoters. Taken together, these results suggest that WRI4 is a transcriptional activator that specifically controls cuticular wax biosynthesis in Arabidopsis stems. This article is protected by copyright. All rights reserved.