Young Sam Go

The MYB96 Transcription Factor Regulates Cuticular Wax Biosynthesis under Drought Conditions in Arabidopsis

This work provides evidence that deposition of cuticular waxes is intimately associated with plant responses to drought. The Arabidopsis MYB96 transcription factor functions as a regulator of ABA-mediated cuticular wax biosynthesis under drought conditions by binding directly to the promoters of genes encoding very-long-chain fatty acid–condensing enzymes involved in cuticular wax biosynthesis. Drought stress activates several defense responses in plants, such as stomatal closure, maintenance of root water uptake, and synthesis of osmoprotectants. Accumulating evidence suggests that deposition of cuticular waxes is also associated with plant responses to cellular dehydration. Yet, how cuticular wax biosynthesis is regulated in response to drought is unknown. We have recently reported that an Arabidopsis thaliana abscisic acid (ABA)–responsive R2R3-type MYB transcription factor, MYB96, promotes drought resistance. Here, we show that transcriptional activation of cuticular wax biosynthesis by MYB96 contributes to drought resistance. Microarray assays showed that a group of wax biosynthetic genes is upregulated in the activation-tagged myb96-1D mutant but downregulated in the MYB96-deficient myb96-1 mutant. Cuticular wax accumulation was altered accordingly in the mutants. In addition, activation of cuticular wax biosynthesis by drought and ABA requires MYB96. By contrast, biosynthesis of cutin monomers was only marginally affected in the mutants. Notably, the MYB96 protein acts as a transcriptional activator of genes encoding very-long-chain fatty acid–condensing enzymes involved in cuticular wax biosynthesis by directly binding to conserved sequence motifs present in the gene promoters. These results demonstrate that ABA-mediated MYB96 activation of cuticular wax biosynthesis serves as a drought resistance mechanism.

Disruption of Glycosylphosphatidylinositol-Anchored Lipid Transfer Protein Gene Altered Cuticular Lipid Composition, Increased Plastoglobules, and Enhanced Susceptibility to Infection by the Fungal Pathogen Alternaria brassicicola

All aerial parts of vascular plants are covered with cuticular waxes, which are synthesized by extensive export of intracellular lipids from epidermal cells to the surface. Although it has been suggested that plant lipid transfer proteins (LTPs) are involved in cuticular lipid transport, the in planta evidence is still not clear. In this study, a glycosylphosphatidylinositol-anchored LTP (LTPG1) showing higher expression in epidermal peels of stems than in stems was identified from an Arabidopsis (Arabidopsis thaliana) genome-wide microarray analysis. The expression of LTPG1 was observed in various tissues, including the epidermis, stem cortex, vascular bundles, mesophyll cells, root tips, pollen, and early-developing seeds. LTPG1 was found to be localized in the plasma membrane. Disruption of the LTPG1 gene caused alterations of cuticular lipid composition, but no significant changes on total wax and cutin monomer loads were seen. The largest reduction (10 mass %) in the ltpg1 mutant was observed in the C29 alkane, which is the major component of cuticular waxes in the stems and siliques. The reduced content was overcome by increases of the C29 secondary alcohols and C29 ketone wax loads. The ultrastructure analysis of ltpg1 showed a more diffuse cuticular layer structure, protrusions of the cytoplasm into the vacuole in the epidermis, and an increase of plastoglobules in the stem cortex and leaf mesophyll cells. Furthermore, the ltpg1 mutant was more susceptible to infection by the fungus Alternaria brassicicola than the wild type. Taken together, these results indicated that LTPG1 contributed either directly or indirectly to cuticular lipid accumulation.

Two Arabidopsis 3-ketoacyl CoA synthase genes, KCS20 and KCS2/DAISY, are functionally redundant in cuticular wax and root suberin biosynthesis, but differentially controlled by osmotic stress

Very-long-chain fatty acids (VLCFAs) are essential precursors of cuticular waxes and aliphatic suberins in roots. The first committed step in VLCFA biosynthesis is condensation of C(2) units to an acyl CoA by 3-ketoacyl CoA synthase (KCS). In this study, two KCS genes, KCS20 and KCS2/DAISY, that showed higher expression in stem epidermal peels than in stems were isolated. The relative expression of KCS20 and KCS2/DAISY transcripts was compared among various Arabidopsis organs or tissues and under various stress conditions, including osmotic stress. Although the cuticular waxes were not significantly altered in the kcs20 and kcs2/daisy-1 single mutants, the kcs20 kcs2/daisy-1 double mutant had a glossy green appearance due to a significant reduction of the amount of epicuticular wax crystals on the stems and siliques. Complete loss of KCS20 and KCS2/DAISY decreased the total wax content in stems and leaves by 20% and 15%, respectively, and an increase of 10-34% was observed in transgenic leaves that over-expressed KCS20 or KCS2/DAISY. The stem wax phenotype of the double mutant was rescued by expression of KSC20. In addition, the kcs20 kcs2/daisy-1 roots exhibited growth retardation and abnormal lamellation of the suberin layer in the endodermis. When compared with the single mutants, the roots of kcs20 kcs2/daisy-1 double mutantss exhibited significant reduction of C(22) and C(24) VLCFA derivatives but accumulation of C(20) VLCFA derivatives in aliphatic suberin. Taken together, these findings indicate that KCS20 and KCS2/DAISY are functionally redundant in the two-carbon elongation to C(22) VLCFA that is required for cuticular wax and root suberin biosynthesis. However, their expression is differentially controlled under osmotic stress conditions.

Endoplasmic Reticulum-Located PDAT1-2 from Castor Bean Enhances Hydroxy Fatty Acid Accumulation in Transgenic Plants

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.

Arabidopsis Cuticular Wax Biosynthesis Is Negatively Regulated by the DEWAX Gene Encoding an AP2/ERF-Type Transcription Factor

This work identifies a negative transcriptional regulator, DEWAX, that represses the expression of genes involved in Arabidopsis cuticular wax biosynthesis. The results suggest that DEWAX-mediated negative regulation of the wax biosynthetic genes might be involved in determining the total wax loads produced in Arabidopsis during daily dark and light cycles. 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.

Arabidopsis 3-Ketoacyl-Coenzyme A Synthase9 Is Involved in the Synthesis of Tetracosanoic Acids as Precursors of Cuticular Waxes, Suberins, Sphingolipids, and Phospholipids

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. 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.

Senescence‐inducible LEC2 enhances triacylglycerol accumulation in leaves without negatively affecting plant growth

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.

Identification of marneral synthase, which is critical for growth and development in Arabidopsis

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.

Expression of Camelina WRINKLED1 Isoforms Rescue the Seed Phenotype of the Arabidopsis wri1 Mutant and Increase the Triacylglycerol Content in Tobacco Leaves

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.

Seed-expressed casein kinase I acts as a positive regulator of the SeFAD2 promoter via phosphorylation of the SebHLH transcription factor

Microsomal oleic acid desaturase (FAD2) catalyzes the first committed step of the biosynthesis of polyunsaturated fatty acids via extra-plastidial desaturation of oleic acid to linoleic acid. In the regulatory mechanism controlling seed-specific SeFAD2 expression, trans-activation of the seed-specific SeFAD2 promoter is mediated by the SebHLH transcription factor (Kim et al. in Plant Mol Biol 64:453–466, 2007). In this study, a protein interacting with SebHLH was isolated from yeast two-hybrid analysis. The protein shares approximately 80% sequence identity with other putative casein kinases and was named SeCKI (Sesame Casein Kinase I). SeCKI transcripts were predominantly expressed in developing sesame seeds and were induced approximately threefold by exogenous application of ABA. eGFP:SeCKI fusion protein was localized to the nucleus. The SeCKI protein specifically bound to SebHLH. The SeCKI protein was autophosphorylated in a calcium-independent manner and transphosphorylated the SebHLH protein. Both the SebHLH and the SeCKI genes or both the SebHLH and mutated SemCKI (K182G) genes, under the control of CaMV 35S promoter, and the GUS reporter gene driven by SeFAD2 promoter containing E- and G-Box motifs were co-expressed in developing sesame seeds. This co-expression revealed that SeCKI enhanced the SebHLH-mediated transactivation of the SeFAD2 gene promoter via phosphorylation of the SebHLH transcription factor.