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Abstract Seed triacylglycerol (TAG), a major component of vegetable oil, consists of a glycerol esterified with three fatty acids. Vegetable oil has industrial applications and is widely used as edible oil. The increasing demand for plant oils, owing to population growth, it is crucial to enhance the oil content in seeds. We found castor WRINKLED1A (RcWRI1A) and R2R3-type MYB domain protein 306 (RcMYB306) which have homology with Arabidopsis WRI1 (AtWRI1) and AtMYB96 which regulate genes involved in fatty acid and TAG synthesis, respectively. These castor genes were separately and jointly overexpressed using seed-specific promoters in an oil crop, camelina (Camelina sativa). Overexpression of RcWRI1A, RcMYB306, or RcWRI1A + RcMYB306 increased the total seed oil content in camelina. However, this increase was not significantly different from that observed during the overexpression of RcWRI1A or/and RcMYB306. RcWRI1A overexpression increased the fatty acid content, including 16:0, 18:2, 18:3. Contrastingly, RcMYB306 overexpression increased the 18:1, 18:2, 18:3, 20:0 and 20:1 fatty acid. In the RcWRI1A + RcMYB306 lines, changes in fatty acid composition demonstrated the combined effects of these transcription factors. These results suggest that RcWRI1A and RcMYB306 can be used to improve the productivity of oil crops.

Suberin, a complex polyester deposited in the seed coat outer integument, acts as a hydrophobic barrier to control the movement of water, ions, and gas. However, relatively little is known about the signal transduction involved in suberin layer formation during seed coat development. In this study, the effect of the plant hormone abscisic acid (ABA) on suberin layer formation in seed coats was investigated by characterizing mutations in Arabidopsis related to ABA biosynthesis and signaling. Seed coat permeability to tetrazolium salt was noticeably elevated in aba1-1 and abi1-1 mutants, but not significantly altered in snrk2.2/3/6, abi3-8, abi5-7, and pyr1pyl1pyl2pyl4 quadruple mutants compared with that in the wild-type (WT). ABA1 encodes a zeaxanthin epoxidase that functions in the first step of ABA biosynthesis. aba1-1 and aba1-8 mutant seed coats showed reduced autofluorescence under UV light and increased tetrazolium salt permeability relative to WT levels. ABA1 disruption resulted in decreased total seed coat polyester levels by approximately 3%, with a remarkable reduction in levels of C24:0 ω-hydroxy fatty acids and C24:0 dicarboxylic acids, which are the most abundant aliphatic compounds in seed coat suberin. Consistent with suberin polyester chemical analysis, RT-qPCR analysis showed a significant reduction in transcript levels of KCS17, FAR1, FAR4, FAR5, CYP86A1, CYP86B1, ASFT, GPAT5, LTPG1, LTPG15, ABCG2, ABCG6, ABCG20, ABCG23, MYB9, and MYB107, which are involved in suberin accumulation and regulation in developing aba1-1 and aba1-8 siliques, as compared with WT levels. Together, seed coat suberization is mediated by ABA and partially processed through canonical ABA signaling.

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

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.

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.

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.

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

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

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.