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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.

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 downregulated 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 ABAmediated 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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-offunction 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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, ). 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. [ABSTRACT FROM AUTHOR]

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 C2 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 C22 and C24 VLCFA derivatives but accumulation of C20 VLCFA derivatives in aliphatic suberin. Taken together, these findings indicate that KCS20 and KCS2/DAISY are functionally redundant in the two-carbon elongation to C22 VLCFA that is required for cuticular wax and root suberin biosynthesis. However, their expression is differentially controlled under osmotic stress conditions. [ABSTRACT FROM AUTHOR]

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. hi this study, a glycosylphosphatidylinositol-anchored LTP (LTPG1) showing higher expression in epidermal peels of stems than in stems was identified from an Arabidopsis (Arabidopsis thuliana) 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 LTPGT1 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. [ABSTRACT FROM AUTHOR]

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 lipid-related subset is presented. [ABSTRACT FROM AUTHOR]

Sesame (Sesamum indicum) is an important oilseed crop which produces seeds with 50% oil that have a distinct flavor and contains antioxidant lignans. Because sesame lignans are known to have antioxidant and health-protecting properties, metabolic pathways for lignans have been of interest in developing sesame seeds. As an initial approach to identify genes involved in accumulation of storage products and in the biosynthesis of antioxidant lignans, 3328 expressed sequence tags (ESTs) were obtained from a cDNA library of immature seeds 5–25 days old. ESTs were clustered and analyzed by the BLASTX or FASTAX program against the GenBank NR and Arabidopsis proteome databases. To compare gene expression profiles during development of green and non-green seeds, a comparative analysis was carried out between developing sesame and Arabidopsis seed ESTs. Analyses of these two seed EST sets have helped to identify similar and different gene expression profiles during seed development, and to identify a large number of sesame seed-specific genes. In particular, we have identified EST candidates for genes possibly involved in biosynthesis of sesame lignans, sesamin and sesamolin, and also suggest a possible metabolic pathway for the generation of cofactors required for synthesis of storage lipid in non-green oilseeds. Seed-specific expression of several candidate genes has been confirmed by northern blot analysis. [ABSTRACT FROM AUTHOR]

To understand the function of metallothioneins (MTs) in plants, we introduced the Nicotiana gilltinosa MT gene into tobacco (N. tabacum) plants via an Agrobacterium mediated transformation. Full-length MT cDNA was fused between the cauliflower mosaic virus 35S (CaMV 35S) promoter and the nopaline synthase (nos) terminator of the pMBP1 binary vector in sense orientation. Tobacco leaf discs which were cocultivated with Agrobacterium carrying the chimeric MT gene, formed kanamycin-resistant shoots on medium containing kanamycin. The kanamycin-resistant shoots were subsequently rooted on medium containing 200 μM CdSO4• Approximately 30% of individual transgenic plants developed normally. Nontransgenic plants promptly underwent leaf chlorosis, and their growth and development were inhibited on MS medium containing 50 μM CdSO4• Genomic Southern blot analysis showed that the MT gene was stably integrated into the nuclear genome of transgenic tobacco plants. The expression level of MT transcripts was analyzed by RNA gel blot analysis. Selfpollinated seeds obtained from transgenic tobacco plants showing cadmium tolerance were germinated on a medium containing 100 μM CdSO4• PCR analysis from sensitive and stably resistant T2 seedlings for cadmium sulfate confirmed a high correlation between the phenotypic expression of the MT gene and the transgenic genotype, indicating that the MT gene is inherited in the next generation. [ABSTRACT FROM AUTHOR]