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The cuticular wax layer on leaf surfaces limits non-stomatal water loss to the atmosphere and protects against pathogen invasion. Although many genes associated with wax biosynthesis and wax transport in plants have been identified, their regulatory mechanisms remain largely unknown. Here, we show that the MYB transcription factor OsMYB60 positively regulates cuticular wax biosynthesis and this helps rice (Oryza sativa) plants tolerate drought stress. Compared with the wild type (japonica cultivar 'Dongjin'), osmyb60 null mutants (osmyb60-1 and osmyb60-2) exhibited increased drought sensitivity, with more chlorophyll leaching and higher rates of water loss. Quantitative reverse-transcription PCR showed that the loss of function of OsMYB60 led to downregulation of wax biosynthesis genes, leading to reduced amounts of total wax components on leaf surfaces under normal conditions. Yeast one-hybrid, luciferase transient transcriptional activity, and chromatin immunoprecipitation assays revealed that OsMYB60 directly binds to the promoter of OsCER1 (a key gene involved in very-long-chain alkane biosynthesis) and upregulates its expression. Taken together, these results demonstrate that OsMYB60 enhances rice resilience to drought stress by promoting cuticular wax biosynthesis on leaf surfaces.

The hydrophobic biopolymer suberin, which is deposited in the root endodermis and seed coats, functions as an extracellular barrier against uncontrolled water, gas, and ion loss. Suberin monomers synthesized in the endoplasmic reticulum (ER) are exported through the plasma membrane to the apoplast. However, limited information is available about the molecular mechanisms underlying suberin monomer export and assembly. In this study, we investigated the in planta role of LTPG15 encoding a glycosylphosphatidylinositol (GPI)-anchored lipid transfer protein. LTPG15 was predominantly expressed in the root endodermis and seed coat. Fluorescent signals from LTPG15:eYFP were detected in the plasma membrane in tobacco epidermis. Disruption of LTPG15 caused a significant decrease in the levels of fatty acids (C20-C24), primary alcohols (C20 and C22), ω-hydroxy fatty acids (C22 and C24), and α,ω-alkanediols (C20 and C22), but an increase in the amounts of primary alcohols and hydroxy fatty acids with C16 and C18 in seed coats. The mutant phenotype was restored to that of the wild type (WT) by the expression of LTPG15 driven by its own promoter. Seed coats of ltpg15 had an increase in permeability to tetrazolium salts compared with WT seed coats. ltpg15 seeds were more sensitive than WT seeds to inhibition of germination and seedling establishment by salt and osmotic stress treatments. Taken together, our results indicate that LTPG15 is involved in suberin monomer export in seed coats, and this highlights the role of Type G non-specific lipid transfer proteins (LTPGs) in very-long-chain fatty acids and their derivatives' export for suberin polyester formation.

A Kelch repeat F-box containing protein, SMALL AND GLOSSY LEAVES1 (SAGL1) regulates phenylpropanoid biosynthesis as a post-translational regulator for PAL1 (phenylalanine ammonia-lyase) and an indirect transcriptional regulator for ANTHOCYANIDIN SYNTHASE. Phenylpropanoid biosynthesis in plants produces diverse aromatic metabolites with important biological functions. Phenylalanine ammonia-lyase (PAL) catalyzes the first step in phenylpropanoid biosynthesis by converting l-phenylalanine to trans-cinnamic acid. Here, we report that SMALL AND GLOSSY LEAVES1 (SAGL1), a Kelch repeat F-box protein, interacts with PAL1 protein for proteasome-mediated degradation to regulate phenylpropanoid biosynthesis in Arabidopsis. Mutations in SAGL1 caused high accumulation of anthocyanins and lignin derived from the phenylpropanoid biosynthesis pathway. We found that PAL enzyme activity increased in SAGL1-defective mutants, sagl1, but decreased in SAGL1-overexpressing Arabidopsis (SAGL1OE) without changes in the transcript levels of PAL genes, suggesting protein-level regulation by SAGL1. Indeed, the levels of PAL1-GFP fusion protein were reduced when both SAGL1 and PAL1-GFP were transiently co-expressed in leaves of Nicotiana benthamiana. In addition, bimolecular fluorescence complementation analysis suggested an interaction between SAGL1 and PAL1. We also found that the transcript levels of ANTHOCYANIDIN SYNTHASE (ANS) increased in the sagl1 mutants but decreased in SAGL1OE. Our results suggest that SAGL1 regulates phenylpropanoid biosynthesis post-translationally at PAL1 and transcriptionally at ANS.

Advances in molecular biology have improved crops through transferring genes from one organism to new hosts, and these efforts have raised concerns about potential unexpected outcomes. Here, we provide evidence that a gene with a specific function in one organism can yield completely different effects in a new host. CaRZFP1 is a C3HC4-type RING zinc finger protein gene previously isolated from a cDNA library for heat-stressed hot pepper. In our previous work investigating in vivo CaRZFP1 function, we transferred CaRZFP1 into tobacco; transgenic tobacco exhibited enhanced growth and tolerance to abiotic stresses. As further analysis of CaRZFP1 ectopic expression in a heterologous host plant, here we mobilized and constitutively overexpressed CaRZFP1 in lettuce. In contrast to tobacco, transgenic lettuce exhibited poorer growth and delayed flowering compared with vector-only controls. To identify genes that might be involved in this phenotypic effect, transcriptome analyses on transgenic plants of both species were performed, uncovering dozens of genes that reflect the different outcomes between tobacco and lettuce. These included protein kinase, transcriptional factor, transporter protein, hormone and metabolism-related genes, and some unannotated genes. The opposite effects of CaRZFP1 ectopic expression in lettuce and tobacco address concerns of unexpectedly different outcomes in different host species. Electronic supplementary material The online version of this article (10.1007/s11032-018-0812-3) contains supplementary material, which is available to authorized users.

Fatty acid desaturase 2 (FAD2), which resides in the endoplasmic reticulum (ER), plays a crucial role in producing linoleic acid (18:2) through catalyzing the desaturation of oleic acid (18:1) by double bond formation at the delta 12 position. FAD2 catalyzes the first step needed for the production of polyunsaturated fatty acids found in the glycerolipids of cell membranes and the triacylglycerols in seeds. In this study, four FAD2 genes from amphidiploid Brassica napus genome were isolated by PCR amplification, with their enzymatic functions predicted by sequence analysis of the cDNAs. Fatty acid analysis of budding yeast transformed with each of the FAD2 genes showed that whereas BnFAD2-1, BnFAD2-2, and BnFAD2-4 are functional enzymes, and BnFAD2-3 is nonfunctional. The four FAD2 genes of B. napus originated from synthetic hybridization of its diploid progenitors Brassica rapa and Brassica oleracea, each of which has two FAD2 genes identical to those of B. napus. The BnFAD2-3 gene of B. napus, a nonfunctional pseudogene mutated by multiple nucleotide deletions and insertions, was inherited from B. rapa. All BnFAD2 isozymes except BnFAD2-3 localized to the ER. Nonfunctional BnFAD2-3 localized to the nucleus and chloroplasts. Four BnFAD2 genes can be classified on the basis of their expression patterns.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Although several genes have been identified in rice which are functionally equivalent to the flowering time genes in Arabidopsis, primarily genes involved in the photoperiod pathway, little data is available regarding the genes that function in the autonomous pathway in rice. In order to acquire further insight into the control of heading dates in rice, we isolated and conducted an expression analysis on OsFCA, which exhibited 38% sequence homology with Arabidopsis FCA. The N-terminal region of the OsFCA protein appears to be unusually rich in glycine-residues, unlike the N-terminal region found in FCA. However, the genetic structure of OsFCA is, in general, similar to that of FCA. RT-PCR and in silico analyses also showed that alternative splicing and polyadenylation at intron3 were conserved in the genetic expression of OsFCA. We were able to detect alpha, beta, and gamma transcripts, but not the delta transcript, of the OsFCA gene. The beta and gamma transcripts of the OsFCA gene were detected via Northern analysis in the leaves, roots, and flowers of the plant. Flowers in younger stages exhibited higher transcript levels. These data suggest that intron3 may constitute a primary control point in the OsFCA pre-mRNA processing of rice. The overexpression of OsFCA cDNA, driven by the 35S promoter, was shown to partially rescue the late flowering phenotype of the fca mutant, suggesting that the functions of the OsFCA and the FCA are partially overlapped, despite the lack of an apparent FLC homologue in the rice genome. The constitutive expression of OsFCA resulted in no downregulation of FLC, but did result in the weak upregulation of SOC1 in the transgenic Arabidopsis. OsFCA overexpression did not result in a reduction of the gamma transcript levels of FCA in the transgenic Arabidopsis either, thereby suggesting that OsFCA had no effects on the autoregulation of Arabidopsis FCA. All of these results imply conservation and divergence in the functions of FCA between rice and Arabidopsis.

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.

Unusual monounsaturated fatty acids are major constituents (greater than 80%) in seeds of Coriandrum sativum L. (coriander) and Thunbergia alata Bojer, as well as in glandular trichomes (greater than 80% derived products) of Pelargonium x hortorum (geranium). These diverged fatty acid structures are produced via distinct plastidial acyl-acyl carrier protein (ACP) desaturases. When expressed in Arabidopsis thaliana (L.) Heynh. under strong seed-specific promoters the unusual acyl-ACP desaturases resulted in accumulation of unusual monoene fatty acids at 1-15% of seed fatty acid mass. In this study, we have examined several factors that potentially limit higher production of unusual monoenes in transgenic oilseeds. (i) Immunoblots indicated that the introduced desaturases were expressed at levels equivalent to or higher than the endogenous delta9 18:0-ACP desaturase. However, the level of unusual fatty acid produced in transgenic plants was not correlated with the level of desaturase expression. (ii) The unusual desaturases were expressed in several backgrounds, including antisense 18:0-ACP desaturase plants, in fab1 mutants, and co-expressed with specialized ACP or ferredoxin isoforms. None of these experiments led to high production of expected products. (iii) No evidence was found for degradation of the unusual fatty acids during seed development. (iv) Petroselinic acid added to developing seeds was incorporated into triacylglycerol as readily as oleic acid, suggesting no major barriers to its metabolism by enzymes of glycerolipid assembly. (v) In vitro and in situ assay of acyl-ACP desaturases revealed a large discrepancy of activity when comparing unusual acyl-ACP desaturases with the endogenous delta9 18:0-ACP desaturase. The combined results, coupled with the sensitivity of acyl-ACP desaturase activity to centrifugation and low salt or detergent suggests low production of unusual monoenes in transgenic plants may be due to the lack of, or incorrect assemble of, a necessary multi-component enzyme association.

Acyl-acyl carrier protein (ACP) desaturases function to position a single double bond into an acyl-ACP substrate and are best represented by the ubiquitous Δ9 18:0-ACP desaturase. Several variant acyl-ACP desaturases have also been identified from species that produce unusual monoenoic fatty acids. All known acyl-ACP desaturase enzymes use ferredoxin as the electron-donating cofactor, and in almost all previous studies the photosynthetic form of ferredoxin rather than the non-photosynthetic form has been used to assess activity. We have examined the influence of different forms of ferredoxin on acyl-ACP desaturases. Using combinations of in vitro acyl-ACP desaturase assays and [14C]malonyl-coenzyme A labeling studies, we have determined that heterotrophic ferredoxin isoforms support up to 20-fold higher unusual acyl-ACP desaturase activity in coriander (Coriandrum sativum), Thunbergia alata, and garden geranium (Pelargonium ×hortorum) when compared with photosynthetic ferredoxin isoforms. Heterotrophic ferredoxin also increases activity of the ubiquitous Δ9 18:0-ACP desaturase 1.5- to 3.0-fold in both seed and leaf extracts. These results suggest that ferredoxin isoforms may specifically interact with acyl-ACP desaturases to achieve optimal enzyme activity and that heterotrophic isoforms of ferredoxin may be the in vivo electron donor for this reaction.

A gene (NgPI) encoding a novel proteinase inhibitor (PI) has been isolated from tobacco leaves. Protein encoded by the gene consists of 241 amino acid residues having a predicted molecular mass of 26.7 kDa and a calculated pI of 8.7. A predicted N-terminal signal sequence followed by a vacuolar targeting signal and a peptide conserved in the Kunitz type PIs were identified. The deduced NgPI protein has sequence homology with aspartic and cysteine protease inhibitors. The gene is present as double copies in the Nicotiana glutinosa genome. Expression of the NgPI gene is rapidly and transiently induced by tobacco mosaic virus infection at a time earlier than apparent lesions of hypersensitive responses appear on the leaves.

Seeds of coriandrum sativum (coriander) and Thunbergia alata (black-eyed Susan vine) produce unusual monoenoic fatty acids which constitute over 80% of the total fatty acids of the seed oil. The initial step in the formation of these fatty acids is the desaturation of palmitoyl-ACP (acyl carrier protein) at the delta(4) or delta(6) positions to produce delta(4)-hexadecenoic acid (16:1(delta(4)) or delta(6)-hexadecenoic acid (16:1(delta(6)), respectively. The involvement of specific forms of ACP in the production of these novel monoenoic fatty acids was studied. ACPs were partially purified from endosperm of coriander and T. alata and used to generate 3H- and 14C-labelled palmitoyl-ACP substrates. In competition assays with labelled palmitoyl-ACP prepared from spinach (Spinacia oleracea), delta(4)-acyl-ACP desaturase activity was two- to threefold higher with coriander ACP than with spinach ACP. Similarly, the T. alata delta(6) desaturase favoured T. alata ACP over spinach ACP. A cDNA clone, Cs-ACP-1, encoding ACP was isolated from a coriander endosperm cDNA library. Cs-ACP-1 mRNA was predominantly expressed in endosperm rather than leaves. The Cs-ACP-1 mature protein was expressed in E. coli and comigrated on SDS-PAGE with the most abundant ACP expressed in endosperm tissues. In in vitro delta(4)-palmitoyl-ACP desaturase assays, the Cs-ACP-1 expressed from E. coli was four- and 10-fold more active than spinach ACP or E. coli ACP, respectively, in the synthesis of delta(4)-hexadecenoic acid from palmitoyl-ACP. In contrast, delta(9)-stearoyl-ACP desaturase activity from coriander endosperm did not discriminate strongly between different ACP species. These results indicate that individual ACP isoforms are specifically involved in the biosynthesis of unusual seed fatty acids and further suggest that expression of multiple ACP isoforms may participate in determining the products of fatty acid biosynthesis.

Hypersensitive response (HR) in plants is generally characterized by a rapid and localized cell death, cell wall strengthening and synthesis of phytoalexins and PR proteins [1]. In order to understand the molecular and cellular defense mechanisms better during HR that results from the interaction between plant pathogen and its non-host, we inoculated hot pepper (Capsicum annuum cv. Pukang) leaves with soybean pustule pathogen, Xanthomonas campestris pv. glycines8ra. A pool of genes induced or repressed by infection of the pathogen has been isolated by using a diierential displaypolymerase chain reaction (DD-PCR) technique (Yi et al., unpublished data). Approximately one half of the isolated genes had no sequence similarity in existing databases and the major portion of identi¢ed DNA fragments consisted of enzymes involved in primary or secondary metabolic pathways. One of the fragments was found to have signi¢cant sequence homology with ATP:citrate lyase (ACL) known from diverse organisms including alga, fungi, and mammals. ACL is known to catalyze the following reaction: citrate+ CoA+ATPCoxaloacetate+ADP+Pi+acetyl-CoA. In the cytosol of animals, oleaginous yeast, and fungi, acetyl-CoA produced by the action of ACL is the major precursor for fatty acid and sterol biosynthesis [2]. However, in plants, the biosynthetic origin of cytosolic acetyl-CoA that is needed for biosynthesis of phytochemicals is not established. In sweet potato, ACL activity has been observed to increase 10-fold in root tissues response to infection by the fungal pathogen, Ceratocystis ¢mbriata, and to correspond to the accumulation of phytoalexin ipomeamarone [3], but the molecular characterization of a plant ACL has not yet been reported. In order to isolate a full-length cDNA clone encoding ACL, a cDNA library was constructed from hot pepper leaves inoculated with X. campestris pv. glycines8ra. After screening of the cDNA library by using a cDNA fragment of ACL produced by DD-PCR as a probe, a cDNA clone with an open reading frame containing 608 amino acids was ¢nally isolated and designated as Ca-ACL1 (GenBank accession number: AF290958). The deduced amino acid of Ca-ACL1 gene has signi¢cant sequence homology with the C-terminal part of known animal ACLs. Calculated molecular mass of hot pepper ACL polypeptide was 66 kDa, which is only half of the animal ACLs. The ACL catalytic center, GHAGA, and the histidine residue that is autophosphorylated by ATP during catalysis are highly conserved in Ca-ACL1, but none of the three additional phosphorylation sites (Tyr446, Ser450, Ser454) that are involved in the regulation of rat ACL activity exists [2]. To investigate the expression kinetics of the Ca-ACL1 transcripts during inoculation with X. campestris pv. glycines8ra, total RNA was isolated from hot pepper leaves 0, 4, 8, and 12 h after inoculation, and Northern blot analysis was performed by using 32P-labeled Ca-ACL1 as a probe. HR cell death on pepper leaves appeared at approximately 15 h post-in¢ltration, whereas no visible symptom was detected from the leaves in¢ltrated with buier (0.9% NaCl). Pathogenesis-related protein 4 (PR4) probe was included as a positive marker of pathogen inoculation. Approximately 2 kb mRNA corresponding for Ca-ACL1 was signi¢cantly induced 4^8 h after inoculation of X. campestris pv. glycines8ra, but not in buierin¢ltrated tissues. In the duplicated blots, PR4 transcript was only expressed in leaf tissues inoculated with the pathogen (Fig. 1A). To analyze whether induction of Ca-ACL1 mRNA level is speci¢c to incompatible plant^microbe interaction, we inoculated two nearly isogenic pepper lines, C. annuum cv. ECW2DR (BS2) and C. annuum cv. ECW (bs2) with a natural pepper pathogen X. campestris pv. vesicatoria race3 (avrBS2). In Fig. 1B, signi¢cant accumulation of the Ca-ACL1 transcript was observed in the resistant (C. annuum cv. ECW2DR) but not in the susceptible (C. annuum cv. ECW) strain. In the identical blot, a higher level of PR4 mRNA was only detected in the resistant strain. In both cases, induced levels of Ca

To identify genes related to initiation of storage root development in sweetpotato, a cDNA library was constructed with early stage storage roots (0.3–1 cm in diameter). Single-pass sequences of the 5′ ends of 2859 sweetpotato cDNA clones were assembled into 483 clusters and 442 singletons. Comparison of sweetpotato expressed sequence tags (ESTs) to nodulation/tumorigenesis-related sequence databases (nodule-, tumor-, potato tuber- and development-related sequences) revealed that homologs of 39 sweetpotato EST sequences potentially involved in gene regulation, signal transduction and development were present in at least one of the nodulation/tumorigenesis-related sequence databases. Northern blot analyses of these 39 sequences identified 22 differentially expressed genes in early stage storage root and fibrous root. These differentially expressed genes will be potential candidates for research to elucidate the molecular processes related to sweetpotato storage root induction.