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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.

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.

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.

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.

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.

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.

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.

Cuticular waxes are synthesized by the extensive export of intracellular lipids from epidermal cells. However, it is still not known how hydrophobic cuticular lipids are exported to the plant surface through the hydrophilic cell wall. The LTPG2 gene was isolated based on Arabidopsis microarray analysis; this gene is predominantly expressed in stem epidermal peels as compared with in stems. The expression of LTPG2 transcripts was observed in various organs, including stem epidermis and silique walls. The composition of the cuticular wax was significantly altered in the stems and siliques of the ltpg2 mutant and ltpg1 ltpg2 double mutant. In particular, the reduced level of the C29 alkane, which is the major component of cuticular waxes in ltpg1 ltpg2 stems and siliques, was similar to the sum of reduced values of either parent. The total cuticular wax load was reduced by approximately 13% and 20% in both ltpg2 and ltpg1 ltpg2 siliques, respectively, and by approximately 14% in ltpg1 ltpg2 stems when compared with the wild-type. Similarly, severe alterations in the cuticular layer structure of epidermal cells of ltpg2 and ltpg1 ltpg2 stems and silique walls were observed. In tobacco epidermal cells, intracellular trafficking of the fluorescent LTPG/LTPG1 and LTPG2 to the plasma membrane was prevented by a dominant-negative mutant form of ADP-ribosylation factor 1, ARF1(T31N). Taken together, these results indicate that LTPG2 is functionally overlapped with LTPG/LTPG1 during cuticular wax export or accumulation and LTPG/LTPG1 and LTPG2 are targeted to the plasma membrane via the vesicular trafficking system.

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 FCA protein is involved in controlling fl owering time and plays more general roles in RNA-mediated chromatin silencing in Arabidopsis. It contains two RNA-binding domains and a WW domain. The FCA protein interacts with FY, a polyadenylation factor, via its WW domain. We previously characterized a rice gene, OsFCA , which was homologous to FCA . Here, we found that the OsFCA protein could interact through its WW domain with the following proteins: OsFY, a protein containing a CID domain present in RNA-processing factors such as Pcf11 and Nrd1; a protein similar to splicing factor SF1; a protein similar to FUSE splicing factor; and OsMADS8. The FY protein is associated with the 3 ′ end processing machinery in Arabidopsis. Thus, we examined interactions between OsFY and the rice homologs (OsCstF-50, -64 and -77) of the AtCstF-50, -64 and -77 proteins. We found that OsFY could bind OsCstF50, whereas the OsCstF77 protein could bridge the interaction between OsCstF50 and OsCstF64. Taken together, our data suggest that OsFCA could interact with several proteins other than OsFY through its WW domain and may play several roles in rice.

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.

All vascular plants are protected from the environment by a cuticle, a lipophilic layer synthesized by epidermal cells and composed of a cutin polymer matrix and waxes. The mechanism by which epidermal cells accumulate and assemble cuticle components in rapidly expanding organs is largely unknown. We have begun to address this question by analyzing the lipid compositional variance, the surface micromorphology, and the transcriptome of epidermal cells in elongating Arabidopsis (Arabidopsis thaliana) stems. The rate of cell elongation is maximal near the apical meristem and decreases steeply toward the middle of the stem, where it is 10 times slower. During and after this elongation, the cuticular wax load and composition remain remarkably constant (32 μg/cm2), indicating that the biosynthetic flux into waxes is closely matched to surface area expansion. By contrast, the load of polyester monomers per unit surface area decreases more than 2-fold from the upper (8 μg/cm2) to the lower (3 μg/cm2) portion of the stem, although the compositional variance is minor. To aid identification of proteins involved in the biosynthesis of waxes and cutin, we have isolated epidermal peels from Arabidopsis stems and determined transcript profiles in both rapidly expanding and nonexpanding cells. This transcriptome analysis was validated by the correct classification of known epidermis-specific genes. The 15% transcripts preferentially expressed in the epidermis were enriched in genes encoding proteins predicted to be membrane associated and involved in lipid metabolism. An analysis of the lipid-related subset is presented.

A cDNA encoding a chitinase-related receptor-like kinase, designated CHRK1, was isolated from tobacco (Nicotiana tabacum). The C-terminal kinase domain (KD) of CHRK1 contained all of the conserved amino acids of serine/threonine protein kinases. The putative extracellular domain was closely related to the class V chitinase of tobacco and to microbial chitinases.CHRK1 mRNA accumulation was strongly stimulated by infection with fungal pathogen and tobacco mosaic virus. Amino acid-sequence analysis revealed that the chitinase-like domain of CHRK1 lacked the essential glutamic acid residue required for chitinase activity. The recombinant chitinase-like domain did not show any catalytic activity for either oligomeric or polymeric chitin substrates. The recombinant KD of CHRK1 exhibited autophosphorylation, but the mutant KD with a mutation in the essential ATP-binding site did not, suggesting that CHRK1 encoded a functional kinase. CHRK1 was detected in membrane fractions of tobacco BY2 cells. Furthermore, CHRK1-GFP fusion protein was localized in plasma membranes when it was expressed in animal cells. This is the first report of a new type of receptor-like kinase containing a chitinase-like sequence in the putative extracellular domain.