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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.

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.

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.

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.

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.

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.

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.

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 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.

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.

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.

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.

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.

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.

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.

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.

Seed storage oils are essential resources for not only human and animal diets but also industrial applications. The primary goal of this study was to increase seed oil content through comparative analysis of two seed-specific promoters, AtFAE1 from Arabidopsis Fatty Acid Elongase 1 gene and BnNapin from Brassica napus seed storage protein gene. AtWRI1 and AtDGAT1 genes encoding an AP2-type transcription factor and a Diacylglycerol Acyltransferase 1 enzyme, respectively, were expressed under the control of AtFAE1 and BnNapin promoters in Arabidopsis. The total seed oil content in all transgenic plants was increased by 8-11% compared with wild-type seeds. The increased level of oil content in AtWRI1 and AtDGAT1 transgenic lines under the control of both promoters was similar, although the activity of the BnNapin promoter is much stronger than that of AtFAE1 promoter in the mature stage of developing seeds where storage oil biosynthesis occurs at a maximum rate. This result demonstrates that the AtFAE1 promoter as well as the BnNapin promoter can be used to increase the seed oil content in transgenic plants.

The synthesis of polyunsaturated fatty acids (PUFAs), the most abundant fatty acids in plants, begins with a reaction catalyzed by fatty acid desaturase 2 (FAD2; EC 1.3.1.35), also called microsomal oleate Δ12-desaturase. Since the FAD2 gene was first identified in Arabidopsis thaliana, FAD2 research has gained wide interest as the essential enzyme for synthesizing PUFA. Grapes are one of the most frequently cultivated fruits in the world, with most commercial growers cultivating Vitis vinifera and V. labrusca. Grapeseed oil contains a high proportion, 60-70% of linoleic acid (18:2). We cloned two putative FAD2 genes from V. labrusca cv. Campbell Early based on V. vinifera genome sequences. Deduced amino acid sequences of two putative genes showed that VlFAD2s show high similarity to Arabidopsis FAD2 and commonly contain six transmembrane domain, three histidine boxes and endoplasmic reticulum (ER) retrieval motif representing the characteristics of fatty acid desaturase. Phylogenetic analyses of various plant FAD2s showed that VlFAD2-1 and VlFAD2-2 are separately grouped with constitutive and seed-type FAD2s, respectively. Southern blot showed that one or two bands are found in each lane. Because Campbell Early is a hybrid cultivar, FAD2-1 and FAD2-2 genes may exist as one copy in V. labrusca. Expression analysis in different tissues indicated that VlFAD2-1 is a constitutive gene but VlFAD2-2 is a seed-type gene. Complementation experiments of fad2-1 mutant Arabidopsis with VlFAD2-1 or VlFAD2-2 demonstrated that VlFAD2-1 and VlFAD2-2 can restore low PUFA proportion of fad2 to normal PUFA proportion.

Plants produce structurally diverse triterpenoids, which are important for their life and survival. Most triterpenoids and sterols share a common biosynthetic intermediate, 2,3-oxidosqualene (OS), which is cyclized by 2,3-oxidosqualene cyclase (OSC). To investigate the role of an OSC, marneral synthase 1 (MRN1), in planta, we characterized a Arabidopsis mrn1 knock-out mutant displaying round-shaped leaves, late flowering, and delayed embryogenesis. Reduced growth of mrn1 was caused by inhibition of cell expansion and elongation. Marnerol, a reduced form of marneral, was detected in Arabidopsis overexpressing MRN1, but not in the wild type or mrn1. Alterations in the levels of sterols and triterpenols and defects in membrane integrity and permeability were observed in the mrn1. In addition, GUS expression, under the control of the MRN1 gene promoter, was specifically detected in shoot and root apical meristems, which are responsible for primary growth, and the mRNA expression of Arabidopsis clade II OSCs was preferentially observed in roots and siliques containing developing seeds. The eGFP:MRN1 was localized to the endoplasmic reticulum in tobacco protoplasts. Taken together, this report provides evidence that the unusual triterpenoid pathway via marneral synthase is important for the growth and development of Arabidopsis.

Ricinoleic acid (12-hydroxy-octadeca-9-enoic acid) is a major unusual fatty acid in castor oil. This hydroxy fatty acid is useful in industrial materials. This unusual fatty acid accumulates in triacylglycerol (TAG) in the seeds of the castor bean (Ricinus communis L.), even though it is synthesized in phospholipids, which indicates that the castor plant has an editing enzyme, which functions as a phospholipid:diacylglycerol acyltransferase (PDAT) that is specific to ricinoleic acid. Transgenic plants containing fatty acid Δ12-hydroxylase encoded by the castor bean FAH12 gene produce a limited amount of hydroxy fatty acid, a maximum of around 17% of TAGs present in Arabidopsis seeds, and this unusual fatty acid remains in phospholipids of cell membranes in seeds. Identification of ricinoleate-specific PDAT from castor bean and manipulation of the phospholipid editing system in transgenic plants will enhance accumulation of the hydroxy fatty acid in transgenic seeds. The castor plant has three PDAT genes; PDAT1-1 and PDAT2 are homologs of PDAT, which are commonly found in plants; however, PDAT1-2 is newly grouped as a castor bean-specific gene. PDAT1-2 is expressed in developing seeds and localized in the endoplasmic reticulum, similar to FAH12, indicating its involvement in conversion of ricinoleic acid into TAG. PDAT1-2 significantly enhances accumulation of total hydroxy fatty acid up to 25%, with a significant increase in castor-like oil, 2-OH TAG, in seeds of transgenic Arabidopsis, which is an identification of the key gene for oilseed engineering in production of unusual fatty acids.

The exine of the pollen wall shows an intricate pattern, primarily comprising sporopollenin, a polymer of fatty acids and phenolic compounds. A series of enzymes synthesize sporopollenin precursors in tapetal cells, and the precursors are transported from the tapetum to the pollen surface. However, the mechanisms underlying the transport of sporopollenin precursors remain elusive. Here, we provide evidence that strongly suggests that the Arabidopsis ABC transporter ABCG26/WBC27 is involved in the transport of sporopollenin precursors. Two independent mutations at ABCG26 coding region caused drastic decrease in seed production. This defect was complemented by expression of ABCG26 driven by its native promoter. The severely reduced fertility of the abcg26 mutants was caused by a failure to produce mature pollen, observed initially as a defect in pollen-wall development. The reticulate pattern of the exine of wild-type microspores was absent in abcg26 microspores at the vacuolate stage, and the vast majority of the mutant pollen degenerated thereafter. ABCG26 was expressed specifically in tapetal cells at the early vacuolate stage of pollen development. It showed high co-expression with genes encoding enzymes required for sporopollenin precursor synthesis, i.e. CYP704B1, ACOS5, MS2 and CYP703A2. Similar to two other mutants with defects in pollen-wall deposition, abcg26 tapetal cells accumulated numerous vesicles and granules. Taken together, these results suggest that ABCG26 plays a crucial role in the transfer of sporopollenin lipid precursors from tapetal cells to anther locules, facilitating exine formation on the pollen surface.

An interest in the production of seed-oil based fuel and raw materials, which comes from renewable plant sources, has been intrigued by the phenomenon of global warming and shortage of fossil fuels. Rapeseed (Brassica napus) is the most important oilseed crop, which produces seeds with 40% oil. It is desirable to develop genetically modified rapeseed producing oils, which can be easily converted to biodiesel. As an initial step for development of genetically modified rapeseed for the production of biofuels or bio-based materials, Korean rapeseed cultivars, Naehan, Youngsan, Tammi and Halla, were analyzed. Four Korean rapeseed cultivars produce 32 to 40% oil of seed dry weight, which is rich in oleic acid (more than 60 mole%). The cotyledonary petioles of rapeseed cultivar, Halla, were transformed using Agro- bacterium tumefaciens strain GV3101, carrying the uidA gene encoding β-glucuronidase (GUS) as a reporter gene and the phosphinothricin acetyltransferase (PAT) gene as a selectable marker. The stable integration of PAT gene in the genome of transgenic rapeseeds was confirmed by PCR analysis. Expression of uidA gene in various rapeseed organs was determined by fluorometric assay and histo- chemical staining. Transformation efficiency of a Korean rapeseed Halla cultivar was 10.4%. Genetic inheritance of transgenes was confirmed in T2 generation.

The FCA gene, which is a component of the autonomous pathway that regulates flowering time, is an important example of how alternative processing can control plant development. We have previously characterized the FCA homolog, OsFCA, from a japonica-type rice cultivar and demonstrated that the polyadenylation site within intron 3, which can generate non-functional FCA-β, was conserved in rice. In this study, we detected five alternatively processed variants of OsFCA pre-mRNA, four of which were equivalents of FCA-α, -β, -γ, and -δ, in japonica-type Korean rice cultivars. The fifth transcript, referred to as OsFCA-ɛ, was similar to OsFCA-γ, except a part of the OsFCA intron 16 was retained. Unlike the FCA-γ protein, the OsFCA-γ protein contains a glycine-rich region at its N-terminus. We detected the OsFCA transcripts missing the region encoding the glycine-rich domain in the indica-type rice, but not in the japonica-type rice. We also found that the OsFCA-δ and OsFCA-ɛ transcripts were expressed in almost all of the different tissue types examined. Taken together, these results indicate that the alternative processing of the OsFCA transcript is more complex than its Arabidopsis counterpart.

Functional characterization of the fatty acid desaturase genes and seed-specific promoters is prerequisite for altering the unsaturated fatty acid content of oilseeds by genetic manipulation. The ω-6 fatty acid desaturase (FAD2) and ω-3 fatty acid desaturase (FAD3) catalyze extra-plastidial desaturation of oleic acid to linoleic acid and linoleic acid to linolenic acid, respectively. These are major constituents in seed storage oils. Here, we report the complementation of a perilla linoleic acid desaturase (PrFAD3) cDNA under the seed-specific sesame FAD2 (SeFAD2) promoter in the Arabidopsis fad3 mutant. PrFAD3 is functionally active and the SeFAD2 promoter is applicable for modifying fatty acid composition in developing seeds. Transient expression of the GUS gene under that promoter in the developing seeds and leaves of sesame, soybean, and corn via microprojectile bombardment indicated that the SeFAD2 promoter likely will be useful for altering the seed phenotypes of dicot and monocot crops.

Sesame ( Sesamum indicum ) is an important oilseed crop; approximately 50% of the seed dry weight is storage oil. In a previous report, developing sesame seed expressed sequence tags (ESTs) revealed that ESTs encoding lipid transfer protein (LTPs) were one of the most abundant groups of sesame ESTs. LTP functions in the transfer of wax or cutin monomers and in the defense response against pathogen attack. To study the biological role of the abundant LTP isoforms in developing seeds, 122 ESTs out of 3328 sesame ESTs were analyzed against Arabidopsis and rice proteome databases. LTP fraction, which was partially purified from developing sesame seeds, actively transferred fluorescent phospholipids and bound to fatty acids. Full-length cDNAs of five out of 21 LTP isoforms were isolated and named SiLTP1 – SiLTP5 . The predicted amino acid sequences of the five SiLTPs harbor typical characteristics of LTPs, including conserved arrangement of cysteine residues. Northern blot analysis revealed that the five SiLTP isoforms were most abundantly expressed in developing seeds, but were also detected in flower tissues. Also, SiLTP3 and SiLTP4 transcripts were expressed in leaves and seed-pot walls, respectively. In addition, SiLTP2 and SiLTP4 transcripts were significantly induced in 6-day-old sesame seedlings by application of NaCl, mannitol, and abscisic acid (ABA). Transient expression of green fluorescent protein (GFP)-fusion constructs in Arabidopsis protoplasts revealed that SiLTP1 and SiLTP2 were secreted by different pathways. Taken together, the abundant LTPs in developing sesame seeds are involved in lipid transfer into the extracellular matrix. Possible biological roles of SiLTPs related to organ-specific expression and abiotic stresses are discussed.

The aerial parts of plants are covered with a cuticle, a hydrophobic layer consisting of cutin polyester and cuticular waxes that protects them from various environmental stresses. Cuticular waxes mainly comprise very long chain fatty acids and their derivatives such as aldehydes, alkanes, secondary alcohols, ketones, primary alcohols, and wax esters that are also important raw materials for the production of lubricants, adhesives, cosmetics, and biofuels. The major function of cuticular waxes is to control non-stomatal water loss and gas exchange. In recent years, the in planta roles of many genes involved in cuticular wax biosynthesis have been characterized not only from model organisms like Arabidopsis thaliana and saltwater cress (Eutrema salsugineum), but also crop plants including maize, rice, wheat, tomato, petunia, Medicago sativa, Medicago truncatula, rapeseed, and Camelina sativa through genetic, biochemical, molecular, genomic, and cell biological approaches. In this review, we discuss recent advances in the understanding of the biological functions of genes involved in cuticular wax biosynthesis, transport, and regulation of wax deposition from Arabidopsis and crop species, provide information on cuticular wax amounts and composition in various organs of nine representative plant species, and suggest the important issues that need to be investigated in this field of study.

Rice lipid transfer protein(LTP) genes belong to a complex multigene family and are differentially regulated. The organ specificity of expression for the different classes of riceLTP gene Subfamily I was investigated here.LTPC1 (Class I) was highly expressed in flowers and stems, whileLTPC2 (Class II) showed higher expression in leaves and stems.LTPC3 (Class III) showed higher expression in stems, whereas corresponding mRNA was barely detected in leaves. Treatment of 3-wk-old plants with ABA induced the expression ofLTPC1 andLTPC3 after 1 d. Expression of all threeLTP isoforms was induced in response to salicylic acid. Because some of these genes are reportedly induced by pathogens, we also investigated whether they might be responsive to infection byMagnaporthe grisea. Transcripts ofLTPC1 started to accumulate in the leaves at 8 h after incompatible inoculation, reaching a maximum level at 48 h. In contrast, the response was much slower and weaker after compatible inoculation.

The seed-specific or seed-predominant promoters of acyl carrier protein (Cs-ACP1) and Δ4-palmitoyl-acyl carrier protein desaturase (Cs-4PAD) genes, which are involved in the biosynthesis of petroselinic acid, were isolated from coriander (Coriandrum sativum) and analyzed in coriander endosperms and transgenic Arabidopsis. The expression of Cs-ACP1 and Cs-4PAD genes was coordinately regulated during seed development.

Flower development is a complex process mediated by a cascade of transcriptional regulation and signal transduction. Differentially expressed genes in two different developmental stages of carnation flowers were isolated. For this purpose, the suppression subtractive hybridization (SSH) technique followed by the differential hybridization screening was employed to identify rarely transcribed flower maturation-inducible genes. Using the reverse Northern blot analysis we screened 85 positive clones from a total 274 clones obtained by SSH. Among these 85 cDNAs, 60 genes showed obvious distinction of signal intensity. Thirty-five out of 60 clones were analyzed by Northern blot, and 60% (21 of 35) of the clones revealed the true positives of flower maturation-related genes, but others were not certain. Thirteen clones (CFMI-3, 5, 6, 8, 9, 10, 11, 16, 38, 61, 230, 237, 263) were only expressed in mature flowers. None of 13 clones exhibited any visible expression in the driver populations, but were distinctly induced during flower maturation (tester populations). However, the other eight clones (CFMI-7, 14, 49, 66, 203, 205, 243, 388) showed a basal level of expression in the flower bud and increased expression in the mature flower.

Small heat shock proteins (sHSPs) function as ATP-independent molecular chaperones, and although the production and function of sHSPs have often been described under heat stress, the expression and function of sHSPs in fundamental developmental processes, such as pollen and seed development, have also been confirmed. Seed germination involves the breaking of dormancy and the resumption of embryo growth that accompany global changes in transcription, translation, and metabolism. In many plants, germination is triggered simply by imbibition of water; however, different seeds require different conditions in addition to water. For small-seeded plants, like Arabidopsis (Arabidopsis thaliana), lettuce (Lactuca sativa), tomato (Solanum lycopersicum), and tobacco (Nicotiana tabacum), light is an important regulator of seed germination. The facts that sHSPs accumulate during seed development, sHSPs interact with various client proteins, and seed germination accompanies synthesis and/or activation of diverse proteins led us to investigate the role of sHSPs in seed germination, especially in the context of light dependence. In this study, we have built transgenic tobacco plants that ectopically express sHSP, and the effect was germination of the seeds in the dark. Administering heat shock to the seeds also resulted in the alleviation of light dependence during seed germination. Subcellular localization of ectopically expressed sHSP was mainly observed in the cytoplasm, whereas heat shock-induced sHSPs were transported to the nucleus. We hypothesize that ectopically expressed sHSPs in the cytoplasm led the status of cytoplasmic proteins involved in seed germination to function during germination without additional stimulus and that heat shock can be another signal that induces seed germination.

To investigate the signal transduction pathway of cytokinin, we incubated seven-day-old maize leaves in distilled water with or without 0.1 mM benzylaminopurine (BAP). Several gene fragments were either induced or repressed by this exogenous treatment, and were then isolated by differential display-polymerase chain reaction (DD-PCR). One fragment (IBC4) had significant sequence homology with the N-terminal portion of the chloroplast ATP synthase IV subunit gene (atpl) of higher plants. After the stimulated expression of IBC4 by BAP was confirmed using orthern blot hybridization, we isolated the full-length gene, including 1BC4, from the chloroplast genome. We verified it as anatpl gene, using BLAST search analysis. Thisatpl gene, designated asZm- atpl (Zea mays- atpl), is 744 nucleotides long and encodes 247 amino adds. Its deduced amino acid sequence does not vary among maize cultivars, and it shares >90% identity with theatpl genes fromOryza sativa, Triticum aestivum, Nkotiana tabacum, Spinacia oleracea, andPisum sativum. We also confirmed, by northern blot analysis, that expression ofZm- atpl transcripts was significantly stimulated by the exogenous application of BAP.

Summary Previously we generated transgenic tobacco plants overexpressing sweet potato ADP-glucose pyrophosphorylase (AGPase) small subunit cDNA under control of CaMV 35S promoter. In order to understand the role of small subunit AGPase in pollen development, these transgenic tobacco plants were studied. Nine out of ten transgenic tobacco plants produced more than 50 percnt; aborted pollen grains. The two transgenic lines T4 and T27 produced more than 75 percnt; aborted pollen grains. Analysis of inheritance of the transgene inserts in T4 and T27 revealed that transgene co-segregated with the pollen abortion phenotype. Microscopic examination of aborted pollen grains showed that their cytoplasm did not contain starch granules, nuclei or any other organelles. Transcript levels of the transgene were very high in stamen tissues of transgenic plants. Transcript levels were correlated with the severity of the pollen abortion phenotype in transgenic plants, indicating that the pollen abortion phenotype probably resulted from overproduction of sweet potato small subunit AGPase mRNAs. Western blot analysis revealed that AGPase small subunit polypeptide levels were significantly reduced in stamen tissues of T4 and T27 compared with control plant. These results suggest that AGPase small subunit gene may play an important role in pollen development.

Lipids function as the structural components of cell membranes, which serve as permeable barriers to the external environment of cells. In plants, lipids play especially important roles as signaling and energy storage compounds. Plant lipids include triacylglycerols, phospholipids, galactolipids, and sphingolipids. Extracellular lipids are especially important in plants and are comprised of cutin, suberin, and waxes. Jasmonic acid is a particularly important signaling compound in plants (Li-Beisson et al. 2013). In particular, storage oils that accumulate in seeds or fruits as triacylglycerol have been widely used for industrial applications including in paints, soaps, cosmetics, detergents, and pharmaceuticals (Dyer et al. 2008). In the bioeconomy, plant storage oils are used as renewables for the production of biodiesel and non-petroleum-based biomaterials (Durrett et al. 2008). Beyond bioenergy and industrial bioproducts, plant lipids are altered to improve the quantity and quality of oils for food and feed. Thus, our understanding about plant lipid chemistry and biosynthesis is necessary for downstream manipulation of lipids via biotechnology. This special issue focuses on current topics that are currently important for basic and applied plant lipid research. While plant lipid research has been active for centuries (Ohlrogge and Browse 1995), our understanding about plant lipid biosynthesis and metabolism has more recently been advanced by the availability of the Arabidopsis thaliana genome sequence, followed by genomics of crop plants. Genome-scale research has greatly facilitated the identification of genes that underlie acyl lipid biosynthesis and metabolism (Li-Beisson et al. 2013). Even more recently, technological developments in lipidomics have allowed the analysis of the complete lipid profile within cells, tissues, organs, and organisms (Horn and Chapman 2012). In this special issue, papers sum up our current knowledge about plant lipids and present modern tools that will be of broad interest to plant researchers. McGlew et al. (2014) describe the integration of Arabidopsis mutant information within the ARALIP plant acyl lipid metabolism website (http://aralip.plantbiology.msu.edu). This tool allows facile and rapid access to information about each mutant. In addition, there is a compendium of literature sources that describe the basis for lipid composition of relevant mutants. Both Parveez et al. (2014) and Goold et al. (2014) review cover storage and neutral lipids in oil palm and microalgae, respectively. Parveez et al. describe advances in biotechnology of oil palm that includes biochemical studies, gene and promoter isolation, vector construction for transformation, and genetic transformation technology. Goold et al. report the biogenesis, structure, and function of lipid droplets in the green microalga Chlamydomonas reinhardtii and model microalgae, such as species in Haematococcus and Nannochloropsis. The three review articles written by Lee and Suh (2015), Vishwanath et al. (2014), and Molina and Kosma (2014) are related with the extracellular lipids, cuticular waxes, cutin, and suberins. Lee and Suh specifically describe recent advances & Mi Chung Suh mcsuh@chonnam.ac.kr

Five cDNA clones encoding the catalytic subunits of Ser/Thr protein phosphatases (PP) of Nicotiana tabacum were described. Among them, three clones (NPP1, NPP2, and NPP3) encoded type 1 PP (PP1), whereas the rest of the clones (NPP4 and NPP5) encoded type 2A PP (PP2A). These cDNA clones exhibited high sequence identity in the PP core region to the corresponding genes from animals and plants. NPP1 mRNA was predominantly expressed in flowers, whereas NPP5 mRNA was mainly detected in leaves and flowers. In contrast, the transcripts of NPP2, NPP3, and NPP4 genes were present in all tissues examined. Throughout flower development, NPP1, NPP2, NPP5 mRNAs were expressed without any significant variation at the steady-state level. Genomic Southern blot showed that tobacco genome contained multiple genes and/or pseudogenes for both type 1 and type 2A phosphatases.

Although the functional roles of zinc finger-containing glycine-rich RNA-binding proteins (RZs) have been characterized in several plant species, including Arabidopsis thaliana and rice (Oryza sativa), the physiological functions of RZs in wheat (Triticum aestivum) remain largely unknown. Here, the functional roles of the three wheat RZ family members, named TaRZ1, TaRZ2, and TaRZ3, were investigated using transgenic Arabidopsis plants under various abiotic stress conditions. Expression of TaRZs was markedly regulated by salt, dehydration, or cold stress. The TaRZ1 and TaRZ3 proteins were localized to the nucleus, whereas the TaRZ2 protein was localized to the nucleus, endoplasmic reticulum, and cytoplasm. Germination of all three TaRZ-expressing transgenic Arabidopsis seeds was retarded compared with that of wild-type seeds under salt stress conditions, whereas germination of TaRZ2- or TaRZ3-expressing transgenic Arabidopsis seeds was retarded under dehydration stress conditions. Seedling growth of TaRZ1-expressing transgenic plants was severely inhibited under cold or salt stress conditions, and seedling growth of TaRZ2-expressing plants was inhibited under salt stress conditions. By contrast, expression of TaRZ3 did not affect seedling growth of transgenic plants under any of the stress conditions. In addition, expression of TaRZ2 conferred freeze tolerance in Arabidopsis. Taken together, these results suggest that different TaRZ family members play various roles in seed germination, seedling growth, and freeze tolerance in plants under abiotic stress.

We report a novel approach for enhanced accumulation of fatty acids and triacylglycerols for utilization as biodiesel in transgenic tobacco stems through xylem-specific expression of Arabidopsis DGAT1 and LEC2 genes. The use of plant biomass for production of bioethanol and biodiesel has an enormous potential to revolutionize the global bioenergy outlook. Several studies have recently been initiated to genetically engineer oil production in seeds of crop plants to improve biodiesel production. However, the “food versus fuel” issues have also sparked some studies for enhanced accumulation of oils in vegetative tissues like leaves. But in the case of bioenergy crops, use of woody stems is more practical than leaves. Here, we report the enhanced accumulation of fatty acids (FAs) and triacylglycerols (TAGs) in stems of transgenic tobacco plants expressing Arabidopsis diacylglycerol acyltransferase 1 (DGAT1) and LEAFY COTYLEDON2 (LEC2) genes under a developing xylem-specific cellulose synthase promoter from aspen trees. The transgenic tobacco plants accumulated significantly higher amounts of FAs in their stems. On an average, DGAT1 and LEC2 overexpression showed a 63 and 80 % increase in total FA production in mature stems of transgenic plants over that of controls, respectively. In addition, selected DGAT1 and LEC2 overexpression lines showed enhanced levels of TAGs in stems with higher accumulation of 16:0, 18:2 and 18:3 TAGs. In LEC2 lines, the relative mRNA levels of the downstream genes encoding plastidic proteins involved in FA synthesis and accumulation were also elevated. Thus, here, we provide a proof of concept for our approach of enhancing total energy yield per plant through accumulation of higher levels of FAs in transgenic stems for biodiesel production.

Hot pepper (Capsicum annuum), one of the oldest domesticated crops in the Americas, is the most widely grown spice crop in the world. We report whole-genome sequencing and assembly of the hot pepper (Mexican landrace of Capsicum annuum cv. CM334) at 186.6× coverage. We also report resequencing of two cultivated peppers and de novo sequencing of the wild species Capsicum chinense. The genome size of the hot pepper was approximately fourfold larger than that of its close relative tomato, and the genome showed an accumulation of Gypsy and Caulimoviridae family elements. Integrative genomic and transcriptomic analyses suggested that change in gene expression and neofunctionalization of capsaicin synthase have shaped capsaicinoid biosynthesis. We found differential molecular patterns of ripening regulators and ethylene synthesis in hot pepper and tomato. The reference genome will serve as a platform for improving the nutritional and medicinal values of Capsicum species.

Microsomal oleic acid desaturase (FAD2) catalyzes the first extra-plastidial desaturation in plants, converting oleic acid to linoleic acid, which is a major constituent in all cellular membranes as well as in seed storage oils. Seed-specific FAD2 (SeFAD2) produced 40% of linoleic acids in the total fatty acids of sesame (Sesamum indicum) seeds. The expression of SeFAD2 transcripts was spatially and temporally controlled during seed development. To investigate the regulatory mechanism controlling seed-specific SeFAD2 expression, we isolated a well-matched sequence homologous to the basic region/helix-loop-helix proteins by yeast one-hybrid screening and named it SebHLH. SebHLH transcripts were expressed in developing seeds and roots of sesame. SebHLH:GFP fusion protein localized in the nucleus. Recombinant SebHLH protein bound E-box (CANNTG) and G-box (CACGTG) elements in the region from −179 to −53 of the SeFAD2 gene promoter, and the external C and G nucleotides in the E- and G-box motifs were essential for SebHLH protein binding. The SebHLH gene, under the CaMV35S promoter, and the GUS reporter gene driven by E- and G-box motifs were co-expressed in developing sesame seeds and Arabidopsis transgenic leaves. This co-expression demonstrated that SebHLH protein mediates transactivation of the SeFAD2 gene promoter through binding to E- and G-box elements. E- or G-box elements frequently occur in the 5′-flanking region of genes that are involved in triacylglycerol biosynthesis and that exhibit seed-specific expression in Arabidopsis and other plants, suggesting that bHLH transcription factors play a key role in the transcriptional regulation of genes related to storage lipid biosynthesis and accumulation during seed development.