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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.

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.

Cuticular waxes act as a protective barrier against environmental stresses. In the present study, we investigated developmental and genotypic variation in wax formation of cabbage lines, with a view to understand the related morphology, genetics and biochemistry. Our studies revealed that the relative expression levels of wax biosynthetic genes in the first-formed leaf of the highest-wax line remained constantly higher but were decreased in other genotypes with leaf aging. Similarly, the expression of most of the tested genes exhibited decrease from the inner leaves to the outer leaves of 5-month-old cabbage heads in the low-wax lines in contrast to the highest-wax line. In 10-week-old plants, expression of wax biosynthetic genes followed a quadratic function and was generally increased in the early developing leaves but substantially decreased at the older leaves. The waxy compounds in all cabbage lines were predominately C29-alkane, -secondary alcohol, and -ketone. Its deposition was increased with leaf age in 5-month-old plants. The high-wax lines had dense, prominent and larger crystals on the leaf surface compared to low-wax lines under scanning electron microscopy. Principal component analysis revealed that the higher expression of LTP2 genes in the lowest-wax line and the higher expression of CER3 gene in the highest-wax line were probably associated with the comparatively lower and higher wax content in those two lines, respectively. This study furthers our understanding of the relationships between the expression of wax biosynthetic genes and the wax deposition in cabbage lines. Highlight: In cabbage, expression of wax-biosynthetic genes was generally decreased in older and senescing leaves, while wax deposition was increased with leaf aging, and C29-hydrocarbon was predominant in the wax crystals.

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.

Cuticular waxes act as a protective barrier against environmental stresses. In the present study, we investigated developmental and genotypic variation in wax formation of cabbage lines, with a view to understand the related morphology, genetics and biochemistry. Our studies revealed that the relative expression levels of wax biosynthetic genes in the first-formed leaf of the highest-wax line remained constantly higher but were decreased in other genotypes with leaf aging. Similarly, the expression of most of the tested genes exhibited decrease from the inner leaves to the outer leaves of 5-month-old cabbage heads in the low-wax lines in contrast to the highest-wax line. In 10-week-old plants, expression of wax biosynthetic genes followed a quadratic function and was generally increased in the early developing leaves but substantially decreased at the older leaves. The waxy compounds in all cabbage lines were predominately C29-alkane, -secondary alcohol, and -ketone. Its deposition was increased with leaf age in 5-month-old plants. The high-wax lines had dense, prominent and larger crystals on the leaf surface compared to low-wax lines under scanning electron microscopy. Principal component analysis revealed that the higher expression of LTP2 genes in the lowest-wax line and the higher expression of CER3 gene in the highest-wax line were probably associated with the comparatively lower and higher wax content in those two lines, respectively. This study furthers our understanding of the relationships between the expression of wax biosynthetic genes and the wax deposition in cabbage lines. Highlight: In cabbage, expression of wax-biosynthetic genes was generally decreased in older and senescing leaves, while wax deposition was increased with leaf aging, and C29-hydrocarbon was predominant in the wax crystals.

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.

Summary The aerial surfaces of terrestrial plants are covered by a cuticular wax layer, which protects the plants from environmental stresses such as desiccation, high irradiance, and UV radiation. Cuticular wax deposition is regulated in an organ-specific manner; Arabidopsis stems have more than 10-fold higher wax loads than leaves. In this study, we found that WRINKLED4 (WRI4), encoding an AP2/ERF transcription factor (TF), is predominantly expressed in stem epidermis, is upregulated by salt stress, and is involved in activating cuticular wax biosynthesis in Arabidopsis stems. WRI4 harbors a transcriptional activation domain at its N-terminus, and fluorescent signals from a WRI4:eYFP construct were localized to the nuclei of tobacco leaf protoplasts. Deposition of epicuticular wax crystals on stems was reduced in wri4-1 and wri4-3 knockout mutants. Total wax loads were reduced by ~28% in wri4 stems but were not altered in wri4 siliques or leaves compared to the wild type. The levels of 29-carbon long alkanes, ketones, and secondary alcohols, which are the most abundant components of stem waxes, were significantly reduced in wri4 stems relative to the wild type. A transactivation assay in tobacco protoplasts and a chromatin immunoprecipitation (ChIP) assay showed that the expression of long-chain acyl-CoA synthetase1 (LACS1), β–ketoacyl-CoA reductase1 (KCR1), PASTICCINO2 (PAS2), trans-2,3-enoyl-CoA reductase (ECR), and bifunctional wax synthase/acyl-CoA: diacylglycerol acyltransferase (WSD1) is positively regulated by direct binding of WRI4 to their promoters. Taken together, these results suggest that WRI4 is a transcriptional activator that specifically controls cuticular wax biosynthesis in Arabidopsis stems. This article is protected by copyright. All rights reserved.

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.

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.

Microsomal delta-12 fatty acid desaturase (FAD2) functions in the first committed step of the biosynthesis of polyunsaturated fatty acids via the desaturation of oleic acid to linoleic acid. In this study, two FAD2 genes were identified through genome-wide analysis of Brassica rapa. One BrFAD2-1 gene harbors functional sequence information, but another BrFAD2-2 gene has mutations that generated a premature stop codon, rendering it nonfunctional. From a database of 120,000 B. rapa expressed sequence tags, we determined that all sequences coding for FAD2 corresponded to the BrFAD2-1 gene. The BrFAD2-1 protein was shown to share high sequence homology (71-99%) with FAD2 proteins from other plant species. An intron in the 5'-untranslated region and three histidine boxes in the protein, which are characteristic of plant FAD2 genes, have been well-conserved. BrFAD2-1 transcripts were detected in various organs of B. rapa. When a pBrFAD2-1:mRFP construct was introduced into tobacco epidermal cells, the fluorescent signal was noted in the endoplasmic reticulum. Ectopic expression of BrFAD2-1:mRFP complemented the Arabidopsis fad2-2 mutant. Finally, transgenic Korean rapeseed Tammi containing high oleic acid contents (78 mol%) was developed via the expression of the BrFAD2-1 gene in an antisense orientation. The data demonstrate that B. rapa harbors only one functional FAD2 that can be utilized for the development of the high-oleic acid Korean rapeseed cultivar Tammi, which might be useful for both human consumption and industrial applications.

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.

국내 육성 추파 품종인 ``영산`` 유채의 자엽 부착 잎자루 부위를 이용하여 재분화와 형질전환의 효율 증진에 영향을 주는 요인을 살펴보았다. 줄기 형성율은 NAA 0.5 mg/L와 Kinetin 2~4mg/L가 혼용 첨가된 배지에서 61~71%로 가장 양호하였다. 또한 질산은(Silver nitrate) 5~9 mg/L 첨가는 재분화에 필수적이었고, GA3 0.01 mg/L 첨가는 재분화에 긍정적인 영향을 주는 것으로 관찰되었다. 아그로박테리움의 Strain 종류, 공동배양시간, 그리고 형질전환체 초기 선발시 항생제 농도 조절(저→고농도)에 따른 ``영산`` 유채의 형질전환 효율을 살펴본 결과, 아그로박테리움의 Strain 선정이 가장 중요한 요인으로 조사되었다. 특히, EHA105 succinamopine strain을 사용하였을 때 형질전환 효율이 26.8%로 가장 효과적이었다. Bar 유전자와 GUS유전자가 내재된 형질전환체는 제초제 ``바스타`` 살포시 생존하고, X-Gluc으로 염색되었다. 그리고 Southern 분석을 통해 후대로 유전자가 안정적으로 유전됨을 확인하였다.

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 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.

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.

Transcription factors (TFs) are the key regulators of gene expression and play crucial roles during plant growth and development. A novel TF family, containing one or two BSD domains in a variety of organisms ranging from prokaryotes to human, was newly identified by computational analysis. In this study, one Arabidopsis gene encoding the BSD domain, designated as AtBSD1, was characterized. The AtBSD1 transcript was expressed in all Arabidopsis tissues tested. The expression level of the AtBSD1 transcripts was not controlled by exogenous application of abiotic stresses and plant hormones. When the AtBSD1::GFP construct under the control of the CaMV35S promoter was introduced into onion epidermal cells, a GFP signal was detected in the nucleus. A transcriptional activity assay of the AtBSD1 protein in yeast revealed that the AtBSD1 protein functions as a transcriptional activator, and the N-terminal region (1–204 amino acids) of the AtBSD1 protein contains a transcriptional activation domain. These results suggest that Arabidopsis genes encoding the BSD1 domain might be classified as a novel transcription factor family.

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 cuticular wax layer, which is the first barrier between a plant and its environment. Although cuticular wax deposition increases more in the light than in the dark, little is known about the molecular mechanisms underlying the regulation of cuticular wax biosynthesis. Recently DEWAX (Decrease Wax Biosynthesis) encoding an AP2/ERF transcription factor was found to be preferentially expressed in the epidermis and induced by darkness. Wax analysis of the dewax knockout mutant, wild type, and DEWAX overexpression lines (OX) indicates that DEWAX is a negative regulator of cuticular wax biosynthesis. DEWAX represses the expression of wax biosynthetic genes CER1, LACS2, ACLA2, and ECR via direct interaction with their promoters. Cuticular wax biosynthesis is negatively regulated twice a day by the expression of DEWAX; throughout the night and another for stomata closing. Taken together, it is evident that DEWAX-mediated negative regulation of the wax biosynthetic genes plays role in determining the total wax loads produced in Arabidopsis during daily dark and light cycles. In addition, significantly higher levels of DEWAX transcripts in leaves than stems suggest that DEWAX-mediated transcriptional repression might be involved in the organ-specific regulation of total wax amounts on plant surfaces.

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.

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.

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.

The nucleotide sequence for NgCDM1 was deposited in GenBank database under the accession number AF208022. Incompatible plant-pathogen interactions result in rapid cell death known as the hypersensitive response (HR) and activation of host defense-related genes. To understand cellular mechanisms controlling HR against pathogen attack, we previously isolated a pool of tobacco genes that are induced or repressed by infection of TMV. In this study, expression of a TMV-induced gene, NgCDM1, which has low sequence homology to known genes was investigated. NgCDM1 mRNA accumulated not only during HR by inoculation of the avirulent pathogen Pseudomonas syringae pv. syringae 61, but also in necrotic cell death caused by infiltration of the virulent pathogen P. s. pv. tabaci. However, induction of the NgCDM1 transcripts was not triggered by cell death caused by senescence or freezing stress. Expression of NgCDM1 mRNA was independent of the plant defense signaling pathways induced by salicylic acid, methyl jasmonate, or H2O2 and also not associated with systemic acquired resistance. Immunoblot analysis revealed that NgCDM1 protein coincidently accumulated with induction of NgCDM1 transcript and was localized primarily in the vicinity of HR-lesion. These results indicate that expression of NgCDM1 gene is preferentially associated with pathogen-induced cell death.

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.

Key message We have constructed and annotated a web-based database of over 280 Arabidopsis genes that have characterized mutants associated with Arabidopsis acyl lipid metabolism. Abstract Mutants have played a fundamental role in gene discovery and in understanding the function of genes involved in plant acyl lipid metabolism. The first mutant in Arabidopsis lipid metabolism (fad4) was described in 1985. Since that time, characterization of mutants in more than 280 genes associated with acyl lipid metabolism has been reported. This review provides a brief background and history on identification of mutants in acyl lipid metabolism, an analysis of the distribution of mutants in different areas of acyl lipid metabolism and presents an annotated database (ARALIPmutantDB) of these mutants. The database provides information on the phenotypes of mutants, pathways and enzymes/proteins associated with the mutants, and allows rapid access via hyperlinks to summaries of information about each mutant and to literature that provides information on the lipid composition of the mutants. In addition, the database of mutants is integrated within the ARALIP plant acyl lipid metabolism website (http://aralip.plantbiology.msu.edu) so that information on mutants is displayed on and can be accessed from metabolic pathway maps. Mutants for at least 30 % of the genes in the database have multiple names, which have been compiled here to reduce ambiguities in searches for information. The database should also provide a tool for exploring the relationships between mutants in acyl lipid-related genes and their lipid phenotypes and point to opportunities for further research. Electronic supplementary material The online version of this article (doi:10.1007/s00299-014-1710-8) contains supplementary material, which is available to authorized users.

The membrane-anchored Arabidopsis thaliana ACYL-COA-BINDING PROTEIN1 (AtACBP1) plays important roles in embryogenesis and abiotic stress responses, and interacts with long-chain (LC) acyl-CoA esters. Here, AtACBP1 function in stem cuticle formation was investigated. Transgenic Arabidopsis transformed with an AtACBP1pro::GUS construct revealed β-glucuronidase (GUS) expression on the stem (but not leaf) surface, suggesting a specific role in stem cuticle formation. Isothermal titration calorimetry results revealed that (His)6-tagged recombinant AtACBP1 interacts with LC acyl-CoA esters (18:1-, 18:2-, and 18:3-CoAs) and very-long-chain (VLC) acyl-CoA esters (24:0-, 25:0-, and 26:0-CoAs). VLC fatty acids have been previously demonstrated to act as precursors in wax biosynthesis. Gas chromatography (GC)–flame ionization detector (FID) and GC–mass spectrometry (MS) analyses revealed that an acbp1 mutant showed a reduction in stem and leaf cuticular wax and stem cutin monomer composition in comparison with the wild type (Col-0). Consequently, the acbp1 mutant showed fewer wax crystals on the stem surface in scanning electron microscopy and an irregular stem cuticle layer in transmission electron microscopy in comparison with the wild type. Also, the mutant stems consistently showed a decline in expression of cuticular wax and cutin biosynthetic genes in comparison with the wild type, and the mutant leaves were more susceptible to infection by the necrotrophic pathogen Botrytis cinerea. Taken together, these findings suggest that AtACBP1 participates in Arabidopsis stem cuticle formation by trafficking VLC acyl-CoAs., link_to_OA_fulltext

By means of differential display, a pool of salicylic acid (SA)-induced mRNAs were identified and subsequently their cDNAs were isolated from a cDNA library prepared from SA-induced leaf tissues of hot pepper. One of these cDNA clones, designated CaSIG4, was 1900 bp and contained an open reading frame encoding 523 amino acids with a calculated molecular mass of 56.3 kDa. The predicted amino acid sequence of CaSIG4 showed high sequence similarity to the AMP-binding protein family of both prokaryotic and eukaryotic acyl-CoA synthetases. CaSIG4 transcripts accumulated rapidly after SA treatment and in response to both incompatible and compatible interactions with Xanthomonas campestris pv. vesicatoria race 1. To investigate the cis-acting elements mediating CaSIG4 expression, the CaSIG4 5′-flanking region was isolated by inverse PCR. Database searches indicated that a potential cis-regulatory element is almost identical to the consensus core sequences ACC(A/T)ACC(A/C) which are conserved among promoters of other phenylpropanoid biosynthetic genes. The subcellular localization of the CaSIG4 protein was studied by using a soluble modified GFP gene fusion delivered into epidermal cells of onion by biolistic bombardment. The CaSIG4-smGFP fusion protein was localized to the plasma membrane. Taken together, CaSIG4 encoding a putative acyl-CoA synthetase could function as a plasma membrane-bound protein with a role in signaling in plant defense.

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.

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.

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.

Summary During initial spliceosome assembly, SF1 binds to intron branch points and interacts with U2 snRNP auxiliary factor 65 (U2AF65). Here, we present evidence indicating that AtSF1, the Arabidopsis SF1 homolog, interacts with AtU2AF65a and AtU2AF65b, the Arabidopsis U2AF65 homologs. A mutant allele of AtSF1 (At5g51300) that contains a T–DNA insertion conferred pleiotropic developmental defects, including early flowering and abnormal sensitivity to abscisic acid. An AtSF1 promoter-driven GUS reporter assay showed that AtSF1 promoter activity was temporally and spatially altered, and that full AtSF1 promoter activity required a significant proportion of the coding region. DNA chip analyses showed that only a small proportion of the transcriptome was altered by more than twofold in either direction in the AtSF1 mutant. Expression of the mRNAs of many heat shock proteins was more than fourfold higher in the mutant strain; these mRNAs were among those whose expression was increased most in the mutant strain. An RT–PCR assay revealed an altered alternative splicing pattern for heat shock transcription factor HsfA2 (At2g26150) in the mutant; this altered splicing is probably responsible for the increased expression of the target genes induced by HsfA2. Altered alternative splicing patterns were also detected for the transcripts of other genes in the mutant strain. These results suggest that AtSF1 has functional similarities to its yeast and metazoan counterparts.

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.

Glycine-rich proteins (GRPs) belong to a large family of heterogenous proteins that are enriched in glycine residues. The expression of two GRP genes of Arabidopsis thaliana, AtGRP5 and AtGRP23, was induced by 16-hydroxypalmitic acid (HPA), a major component of cutin. The expression of AtGRP3, which encodes a GRP protein that is structurally different from AtGRP5 and AtGRP23, was not responsive to HPA application. Treatment with HPA also induced expression of the pathogen-related PR-1 and PR-4 genes. Abscisic acid and salicylic acid treatments enhanced the transcript levels of AtGRP5 and AtGRP23 as well as those of AtGRP3. It was also demonstrated that HPA effectively elicited the accumulation of H2O2 in rosette leaves of Arabidopsis. Results suggest the possible role of some species of GRPs, such as AtGRP5 and AtGRP23, in response to the pathogenic invasion mediated by cutin monomers in plants.

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.

The regulation of genes involved in primary lipid metabolism in plants is much less well understood than that in many other pathways in plant biology. In the investigation reported here, we have characterized transcriptional regulatory mechanisms controlling seed-specific FAD2 expression in sesame (Sesamum indicum). FAD2 codes for extra-plastidial FAD2 desaturase, which catalyzes the conversion of oleic acid to linoleic acid. Promoter analysis of the sesame FAD2 gene (SeFAD2) using the beta-glucuronidase (GUS) reporter system demonstrated that the - 660 to - 180 promoter region functions as a negative cis-element in the seed-specific expression of the SeFAD2 gene. Sesame and Arabidopsis FAD2 genes harbor one large intron within their 5'-untranslated region. These introns conferred up to 100-fold enhancement of GUS expression in transgenic Arabidopsis tissues as compared with intron-less controls. Prerequisite cis-elements for the SeFAD2 intron-mediated enhancement of gene expression and the promoter-like activity of SeFAD2 intron were identified. SeFAD2 transcripts were induced by abscisic acid (ABA) in developing sesame seeds, and the - 660 to - 548 and - 179 to - 53 regions in the SeFAD2 promoter were implicated in ABA-responsive signaling. Theses observations indicate that an intron-mediated regulatory mechanism is involved in controlling not only the seed-specific expression of the SeFAD2 gene but also the expression of plant FAD2 genes, which are essential for the synthesis of polyunsaturated fatty acids.