Arabidopsis Small Rubber Particle Protein Homolog SRPs Play Dual Roles as Positive Factors for Tissue Growth and Development and in Drought Stress Responses.

Lipid droplets (LDs) act as repositories for fatty acids and sterols, which are used for various cellular processes such as energy production and membrane and hormone synthesis. LD-associated proteins play important roles in seed development and germination, but their functions in postgermination growth are not well understood. Arabidopsis (Arabidopsis thaliana) contains three SRP homologs (SRP1, SRP2, and SRP3) that share sequence identities with small rubber particle proteins of the rubber tree (Hevea brasiliensis). In this report, the possible cellular roles of SRPs in postgermination growth and the drought tolerance response were investigated. Arabidopsis SRPs appeared to be LD-associated proteins and displayed polymerization properties in vivo and in vitro. SRP-overexpressing transgenic Arabidopsis plants (35S:SRP1, 35S:SRP2, and 35S:SRP3) exhibited higher vegetative and reproductive growth and markedly better tolerance to drought stress than wild-type Arabidopsis. In addition, constitutive over-expression of SRPs resulted in increased numbers of large LDs in postgermination seedlings. In contrast, single (srp1, 35S:SRP2-RNAi, and srp3) and triple (35S:SRP2-RNAi/srp1srp3) loss-of-function mutant lines exhibited the opposite phenotypes. Our results suggest that Arabidopsis SRPs play dual roles as positive factors in postgermination growth and the drought stress tolerance response. The possible relationships between LD-associated proteins and the drought stress response are discussed.

Constitutive expression of CaPLA1 conferred enhanced growth and grain yield in transgenic rice plants.

Phospholipids are not only important components of cell membranes, but participate in diverse processes in higher plants. In this study, we generated Capsicum annuum phospholipiase A1 (CaPLA1) overexpressing transgenic rice (Oryza sativa L.) plants under the control of the maize ubiquitin promoter. The T4 CaPLA1-overexpressing rice plants (Ubi:CaPLA1) had a higher root:shoot mass ratio than the wild-type plants in the vegetative stage. Leaf epidermal cells from transgenic plants had more cells than wild-type plants. Genes that code for cyclin and lipid metabolic enzymes were up-regulated in the transgenic lines. When grown under typical paddy field conditions, the transgenic plants produced more tillers, longer panicles and more branches per panicle than the wild-type plants, all of which resulted in greater grain yield. Microarray analysis suggests that gene expressions that are related with cell proliferation, lipid metabolism, and redox state were widely altered in CaPLA1-overexpressing transgenic rice plants. Ubi:CaPLA1 plants had a reduced membrane peroxidation state, as determined by malondialdehyde and conjugated diene levels and higher peroxidase activity than wild-type rice plants. Furthermore, three isoprenoid synthetic genes encoding terpenoid synthase, hydroxysteroid dehydrogenase and 3-hydroxy-3-methyl-glutaryl-CoA reductase were up-regulated in CaPLA1-overexpressing plants. We suggest that constitutive expression of CaPLA1 conferred increased grain yield with enhanced growth in transgenic rice plants by alteration of gene activities related with cell proliferation, lipid metabolism, membrane peroxidation state and isoprenoid biosynthesis.

Cooperation and functional diversification of two closely related galactolipase genes for jasmonate biosynthesis.

Jasmonic acid (JA) plays pivotal roles in diverse plant biological processes, including wound response. Chloroplast lipid hydrolysis is a critical step for JA biosynthesis, but the mechanism of this process remains elusive. We report here that DONGLE (DGL), a homolog of DEFECTIVE IN ANTHER DEHISCENCE1 (DAD1), encodes a chloroplast-targeted lipase with strong galactolipase and weak phospholipase A(1) activity. DGL is expressed in the leaves and has a specific role in maintaining basal JA content under normal conditions, and this expression regulates vegetative growth and is required for a rapid JA burst after wounding. During wounding, DGL and DAD1 have partially redundant functions for JA production, but they show different induction kinetics, indicating temporally separated roles: DGL plays a role in the early phase of JA production, and DAD1 plays a role in the late phase of JA production. Whereas DGL and DAD1 are necessary and sufficient for JA production, phospholipase D appears to modulate wound response by stimulating DGL and DAD1 expression.

Constitutive expression of CaRma1H1, a hot pepper ER-localized RING E3 ubiquitin ligase, increases tolerance to drought and salt stresses in transgenic tomato plants.

UNLABELLED: CaRma1H1, an endoplasmic reticulum (ER)-localized hot pepper really interesting new genes (RING) E3 Ub ligase, was previously reported to be a positive regulator of drought stress responses. To address the possibility that CaRma1H1 can be used to improve tolerance to abiotic stress in crop plants, CaRma1H1 was constitutively expressed in transgenic tomato (Solanum lycopersicum) plants. CaRma1H1-overexpressing tomato plants (35S:CaRma1H1) exhibited greatly enhanced tolerance to high-salinity treatments compared with wild-type plants. Leaf chlorophyll and proline contents in CaRma1H1 overexpressors were 4.3- to 8.5-fold and 1.2- to 1.5-fold higher, respectively, than in wild-type plants after 300 mM NaCl treatment. Transgenic cotyledons developed and their roots elongated in the presence of NaCl up to 200 mM. In addition, 35S:CaRma1H1 lines were markedly more tolerant to severe drought stress than were wild-type plants. Detached leaves of CaRma1H1 overexpressors preserved water more efficiently than did wild-type leaves during a rapid dehydration process. The ER chaperone genes LePDIL1, LeBIP1, and LeCNX1 were markedly up-regulated in 35S:CaRma1H1 tomatoes compared with wild-type plants. Therefore, overexpression of CaRma1H1 may enhance tomato plant ER responses to drought stress by effectively removing nonfunctional ubiquitinated proteins. Collectively, constitutive expression of CaRma1H1 in tomatoes conferrred strongly enhanced tolerance to salt- and water-stress. This raises the possibility that CaRma1H1 may be useful for developing abiotic stress-tolerant tomato plants. KEY MESSAGE: CaRma1H1 increases drought tolerance in transgenic tomato plants.

The Arabidopsis sn-1-specific mitochondrial acylhydrolase AtDLAH is positively correlated with seed viability.

Lipid-derived molecules produced by acylhydrolases play important roles in the regulation of diverse cellular functions in plants. In Arabidopsis, the DAD1-like phospholipase A1 family consists of 12 members, all of which possess a lipase 3 domain. In this study, the biochemical and cellular functions of AtDLAH, an Arabidopsis thaliana DAD1-like acylhydrolase, were examined. Bacterially expressed AtDLAH contained phospholipase A1 activity for catalysing the hydrolysis of phospholipids at the sn-1 position. However, AtDLAH displayed an even stronger preference for 1-lysophosphatidylcholine, 1-monodiacylglycerol, and phosphatidic acid, suggesting that AtDLAH is a sn-1-specific acylhydrolase. The AtDLAH gene was highly expressed in young seedlings, and its encoded protein was exclusively localized to the mitochondria. AtDLAH-overexpressing transgenic seeds (35S:AtDLAH) were markedly tolerant to accelerated-ageing treatment and thus had higher germination percentages than wild-type seeds. In contrast, the atdlah loss-of-function knockout mutant seeds were hypersusceptible to accelerated-ageing conditions. The 35S:AtDLAH seeds, as opposed to the atdlah seeds, exhibited a dark red staining pattern following tetrazolium treatment under both normal and accelerated-ageing conditions, suggesting that AtDLAH expression is positively correlated with seed viability. The enhanced viability of 35S:AtDLAH seeds was accompanied by more densely populated epidermal cells, lower levels of accumulated lipid hydroperoxides, and higher levels of polar lipids as compared with wild-type and atdlah mutant seeds. These results suggest that AtDLAH, a mitochondrial-localized sn-1-specific acylhydrolase, plays an important role in Arabidopsis seed viability.

Constitutive expression of CaXTH3, a hot pepper xyloglucan endotransglucosylase/hydrolase, enhanced tolerance to salt and drought stresses without phenotypic defects in tomato plants (Solanum lycopersicum cv. Dotaerang).

The hot pepper xyloglucan endo-trans-gluco-sylase/hydrolase (CaXTH3) gene that was inducible by a broad spectrum of abiotic stresses in hot pepper has been reported to enhance tolerance to drought and high salinity in transgenic Arabidopsis. To assess whether CaXTH3 is a practically useful target gene for improving the stress tolerance of crop plants, we ectopically over-expressed the full-length CaXTH3 cDNA in tomato (Solanum lycopersicum cv. Dotaerang) and found that the 35S:CaXTH3 transgenic tomato plants exhibited a markedly increased tolerance to salt and drought stresses. Transgenic tomato plants exposed to a salt stress of 100 mM NaCl retained the chlorophyll in their leaves and showed normal root elongation. They also remained green and unwithered following exposure to 2 weeks of dehydration. A high proportion of stomatal closures in 35S:CaXTH3 was likely to be conferred by increased cell-wall remodeling activity of CaXTH3 in guard cell, which may reduce transpirational water loss in response to dehydration stress. Despite this increased stress tolerance, the transgenic tomato plants showed no detectable phenotype defects, such as abnormal morphology and growth retardation, under normal growth conditions. These results raise the possibility that CaXTH3 gene is appropriate for application in genetic engineering strategies aimed at improving abiotic stress tolerance in agriculturally and economically valuable crop plants.

Constitutive expression of CaSRP1, a hot pepper small rubber particle protein homolog, resulted in fast growth and improved drought tolerance in transgenic Arabidopsis plants.

Transient and long-term shortages of fresh water are major adverse environmental factors that cause dramatic reductions in crop production and distribution globally. In this study, we isolated a full-length CaSRP1 (Capsicum annuum stress-related protein 1) cDNA, which was rapidly induced by dehydration in hot pepper plants. The predicted CaSRP1 protein sequence exhibited significant amino acid identity to putative stress-related proteins and the small rubber particle protein (SRPP) found in rubber trees (Hevea brasiliensis). To study the cellular functions of CaSRP1, transgenic Arabidopsis plants (35S:CaSRP1) that constitutively expressed the CaSRP1 gene were constructed. Overexpression of CaSRP1 resulted in enhanced root and shoot growth and earlier bolting in the transgenic plants relative to wild-type plants. In addition, 35S:CaSRP1 overexpressors exhibited enhanced tolerance to drought stress as compared to the control plants. These results suggest that CaSRP1 plays dual functions as a positive factor for tissue growth and development and for drought-defensive responses. A possible cellular function of SRPP homologs in non-rubber-producing plants in relation to drought stress tolerance is discussed.

Heterologous expression, and biochemical and cellular characterization of CaPLA1 encoding a hot pepper phospholipase A1 homolog.

Phospholipid signaling has been recently implicated in diverse cellular processes in higher plants. We identified a cDNA encoding the phospholipase A1 homolog (CaPLA1) from 5-day-old early roots of hot pepper. The deduced amino acid sequence showed that the lipase-specific catalytic triad is well conserved in CaPLA1. In vitro lipase assays and site-directed mutagenesis revealed that CaPLA1 possesses PLA1 activity, which catalyzes the hydrolysis of phospholipids at the sn-1 position. CaPLA1 was selectively expressed in young roots, at days 4-5 after germination, and rapidly declined thereafter, suggesting that the expression of CaPLA1 is subject to control by a development-specific mechanism in roots. Because transgenic work was extremely difficult in hot peppers, in this study we overexpressed CaPLA1 in Arabidopsis so as to provide cellular information on the function of this gene. CaPLA1 overexpressors had significantly longer roots, leaves and petioles, and grew more rapidly than the wild-type plants, leading to an early bolting phenotype with prolonged inflorescence. Microscopic analysis showed that the vegetative tissues of 35S:CaPLA1 plants contained an increased number of small-sized cells, which resulted in highly populated cell layers. In addition, mRNAs for cell cycle-controlled proteins and fatty acid catabolizing enzymes were coordinately upregulated in CaPLA1-overexpressing plants. These results suggest that CaPLA1 is functionally relevant in heterologous Arabidopsis cells, and hence might participate in a subset of positive control mechanisms of cell and tissue growth in transgenic lines. We discuss possible biochemical and cellular functions of CaPLA1 in relation to the phospholipid signaling pathway in hot pepper and transgenic Arabidopsis plants.

Overexpression of a Brassica rapa NGATHA gene in Arabidopsis thaliana negatively affects cell proliferation during lateral organ and root growth.

In an effort to elucidate biological functions of transcription factors of Brassica rapa L. (ssp. pekinensis), an NGATHA homolog, BrNGA1, that belongs to the B3-type transcription factor superfamily was identified and expressed in Arabidopsis thaliana under the control of the cauliflower mosaic virus (CaMV) 35S promoter. Arabidopsis plants overexpressing BrNGA1, named BrNGA1ox, displayed markedly reduced organ growth compared with the wild type: lateral organs, such as leaves, flowers and cotyledons, were small and distinctively narrow, and their root growth was also severely retarded. Reduced sizes of BrNGA1ox organs were mainly due to reduction in cell numbers. Kinematic analysis of leaf growth revealed that both the rate and duration of cell proliferation declined during organogenesis, which was consistent with the reduced expression of cyclin genes. Reduction in organ growth was strongly correlated with the small size of meristematic cell pools in the shoot and root meristems. Taken together, these data indicate that BrNGA1 acts as a negative regulator of cell proliferation and may do so, in part, by regulating the size of the meristematic cell pool.

The active site and substrate-binding mode of 1-aminocyclopropane-1-carboxylate oxidase determined by site-directed mutagenesis and comparative modelling studies.

The active site and substrate-binding mode of MD-ACO1 (Malus domestica Borkh. 1-aminocyclopropane-1-carboxylate oxidase) have been determined using site-directed mutagenesis and comparative modelling methods. The MD-ACO1 protein folds into a compact jelly-roll motif comprised of eight a-helices, 12 b-strands and several long loops. The active site is well defined as a wide cleft near the C-terminus. The co-substrate ascorbate is located in cofactor Fe2+-binding pocket, the so-called '2-His-1-carboxylate facial triad'. In addition, our results reveal that Arg244 and Ser246 are involved in generating the reaction product during enzyme catalysis. The structure agrees well with the biochemical and site-directed mutagenesis results. The three-dimensional structure together with the steady-state kinetics of both the wild-type and mutant MD-ACO1 proteins reveal how the substrate specificity of MD-ACO1 is involved in the catalytic mechanism, providing insights into understanding the fruit ripening process at atomic resolution.

Overexpression of CaDSR6 increases tolerance to drought and salt stresses in transgenic Arabidopsis plants.

The partial CaDSR6 (Capsicum annuum Drought Stress Responsive 6) cDNA was previously identified as a drought-induced gene in hot pepper root tissues. However, the cellular role of CaDSR6 with regard to drought stress tolerance was unknown. In this report, full-length CaDSR6 cDNA was isolated. The deduced CaDSR6 protein was composed of 234 amino acids and contained an approximately 30 amino acid-long Asp-rich domain in its central region. This Asp-rich domain was highly conserved in all plant DSR6 homologs identified and shared a sequence identity with the N-terminal regions of yeast p23(fyp) and human hTCTP, which contain Rab protein binding sites. Transgenic Arabidopsis plants overexpressing CaDSR6 (35S:CaDSR6-sGFP) were tolerant to high salinity, as identified by more vigorous root growth and higher levels of total chlorophyll than wild type plants. CaDSR6-overexpressors were also more tolerant to drought stress compared to wild type plants. The 35S:CaDSR6-sGFP leaves retained their water content and chlorophyll more efficiently than wild type leaves in response to dehydration stress. The expression of drought-induced marker genes, such as RD20, RD22, RD26, RD29A, RD29B, RAB18, KIN2, ABF3, and ABI5, was markedly increased in CaDSR6-overexpressing plants relative to wild type plants under both normal and drought conditions. These results suggest that overexpression of CaDSR6 is associated with increased levels of stress-induced genes, which, in turn, conferred a drought tolerant phenotype in transgenic Arabidopsis plants. Overall, our data suggest that CaDSR6 plays a positive role in the response to drought and salt stresses.

Genome sequence of the hot pepper provides insights into the evolution of pungency in Capsicum species.

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.

AtDSEL, an Arabidopsis cytosolic DAD1-like acylhydrolase, is involved in negative regulation of storage oil mobilization during seedling establishment.

Mobilization of seed storage reserves is essential for seed germination and seedling establishment. Here, we report that AtDSEL, an Arabidopsis thalianaDAD1-like Seedling Establishment-related Lipase, is involved in the mobilization of storage oils for early seedling establishment. AtDSEL is a cytosolic member of the DAD1-like acylhydrolase family encoded by At4g18550. Bacterially expressed AtDSEL preferentially hydrolyzed 1,3-diacylglycerol and 1-monoacylglycerol, suggesting that AtDSEL is an sn-1-specific lipase. AtDSEL-overexpressing transgenic Arabidopsis plants (35S:AtDSEL) were defective in post-germinative seedling growth in medium without an exogenous carbon source. This phenotype was rescued by the addition of sucrose to the growth medium. In contrast, loss-of-function mutant plants (atdsel-1 and atdsel-2) had a mildly fast-growing phenotype regardless of the presence of an exogenous carbon source. Electron microscopy revealed that 5-day-old 35S:AtDSEL cotyledons retained numerous peroxisomes and oil bodies, which were exhausted in wild-type and mutant cotyledons. The impaired seedling establishment of 35S:AtDSEL was not rescued by the addition of an exogenous fatty acid source, and 35S:AtDSEL seedling growth was insensitive to 2,4-dichlorophenoxybutyric acid, indicating that ß-oxidation was blocked in AtDSEL-overexpressers. These results suggest that AtDSEL is involved in the negative regulation of seedling establishment by inhibiting the breakdown of storage oils.

Ectopic expression of apple fruit homogentisate phytyltransferase gene (MdHPT1) increases tocopherol in transgenic tomato (Solanum lycopersicum cv. Micro-Tom) leaves and fruits.

Homogentisate phytyltransferase (HPT) is an important enzyme in the biosynthesis of tocopherols (vitamin E). Herein, an HPT homolog (MdHPT1) was isolated from apple (Malus domestica Borkh. cv. Fuji) fruits, whose gene expression level gradually decreased during fruit ripening, reaching a background level in ripened apple fruits. The amounts of α- and γ-tocopherols, two major tocopherols in plant organs, were 5- to 14-fold lower in the fruits than in the leaves and flowers of apple plants. Transgenic tomato plants (Solanum lycopersicum cv. Micro-Tom) overexpressing MdHPT1 were next constructed. Transgenic independent T(1) leaves contained ∼1.8- to 3.6-fold and ∼1.6- to 2.9-fold higher levels of α-tocopherol and γ-tocopherol, respectively, than those in control plants. In addition, the levels of α-tocopherol and γ-tocopherol in 35S:MdHPT1 T(1) fruits increased up to 1.7-fold and 3.1-fold, respectively, as compared to the control fruits, indicating that an increase in α-tocopherol in fruits (maximal 1.7-fold) was less evident than that in leaves (maximal 3.6-fold). This finding suggests that the apple MdHPT1 plays a role in tocopherol production in transgenic tomatoes.

Enzymatic characterization of class I DAD1-like acylhydrolase members targeted to chloroplast in Arabidopsis.

In Arabidopsis, there are at least seven class I acylhydrolase members, which have a putative N-terminal chloroplast-targeting signal. Here, we show that all seven class I proteins are localized to the chloroplasts and hydrolyze phosphatidylcholine at the sn-1 position. However, based on their activities toward various lipids, Arabidopsis class I enzymes could be further divided into three sub-groups by substrate specificity, one with phospholipase-specific activity, another with phospholipase and galactolipase activities, and the other with broad lipolytic activity toward phosphatidylcholine, galactolipids, and triacylglycerol. These results suggest that the three sub-groups of class I acylhydrolases have specific roles in chloroplasts.

Structure and expression of MdFBCP1, encoding an F-box-containing protein 1, during Fuji apple (Malus domestica Borkh.) fruit ripening.

From database comparisons of 1,117 expressed sequence tags (ESTs) generated from ripened Fuji apple fruits, we identified ten ubiquitin (Ub)-related genes. RNA gel-blot analysis suggests that these Ub-related genes are induced by at least four distinct signaling pathways in fruits. In this study, we analyzed structure and expression of MdFBCP1, encoding an F-box-containing protein 1, in Fuji apples. MdFBCP1 transcript was predominantly expressed in the fully ripened climacteric fruits, in which serge of ethylene production occurred. The MdFBCP1 gene was also activated effectively in response to exogenous ethylene treatment, with the induction pattern being comparable to those of ACC oxidase and beta-cyanoalanine synthase. Thus, it seems likely that the expression of MdFBCP1 is closely associated with a climacteric ethylene production and ACC oxidase activity and, hence, MdFBCP1 may play a role in the ripening process of Fuji apple fruits. Yeast two hybrid and in vitro pull-down assays revealed that MdFBCP1 physically interacted with MdSkp1 and N-terminal F-box motif was essential for this interaction. These results suggest that MdFBCP1 indeed functions as an F-box-containing protein and participates in the formation of SCF complex, which acts as E3 Ub ligase. Genomic Southern blot analysis showed that MdFBCP1 exhibited different pattern of restriction enzyme digestion in three cultivars (Tsugaru, Golden Delicious and Fuji) that produce different amount of ethylene, suggesting that the MdFBCP1 gene is organized in a cultivar specific manner. Collectively, our data suggest that Ub degradation pathway may play an important role in the ripening of Fuji apple fruits.

Expression of MdCAS1 and MdCAS2, encoding apple beta-cyanoalanine synthase homologs, is concomitantly induced during ripening and implicates MdCASs in the possible role of the cyanide detoxification in Fuji apple (Malus domestica Borkh.) fruits.

Fruit ripening involves complex biochemical and physiological changes. Ethylene is an essential hormone for the ripening of climacteric fruits. In the process of ethylene biosynthesis, cyanide (HCN), an extremely toxic compound, is produced as a co-product. Thus, most cyanide produced during fruit ripening should be detoxified rapidly by fruit cells. In higher plants, the key enzyme involved in the detoxification of HCN is beta-cyanoalanine synthase (beta-CAS). As little is known about the molecular function of beta-CAS genes in climacteric fruits, we identified two homologous genes, MdCAS1 and MdCAS2, encoding Fuji apple beta-CAS homologs. The structural features of the predicted polypeptides as well as an in vitro enzyme activity assay with bacterially expressed recombinant proteins indicated that MdCAS1 and MdCAS2 may indeed function as beta-CAS isozymes in apple fruits. RNA gel-blot studies revealed that both MdCAS1 and MdCAS2 mRNAs were coordinately induced during the ripening process of apple fruits in an expression pattern comparable with that of ACC oxidase and ethylene production. The MdCAS genes were also activated effectively by exogenous ethylene treatment and mechanical wounding. Thus, it seems like that, in ripening apple fruits, expression of MdCAS1 and MdCAS2 genes is intimately correlated with a climacteric ethylene production and ACC oxidase activity. In addition, beta-CAS enzyme activity was also enhanced as the fruit ripened, although this increase was not as dramatic as the mRNA induction pattern. Overall, these results suggest that MdCAS may play a role in cyanide detoxification in ripening apple fruits.

Microarray analysis of apple gene expression engaged in early fruit development.

To evaluate gene expressions mostly engaged in early development of apple fruit, we performed the identification of transcripts differentially expressed in young fruit by using microarrays spotted with 6,253 cDNAs collected from young and mature apple fruits of the cultivar Fuji (Malus domestica Borkh. cv. Fuji). A total of 3,484 cDNAs out of 6,253 were selected after quality control of microarray spots and analyzed for differential gene expression patterns between young fruit and other tissues (mature fruit, leaf and flower). Among them, 192 cDNAs displayed a signal value higher than twofold in young fruit compared with other tissues. Blast analysis of the 192 cDNA clones identified 88 non-redundant groups encoding proteins with known function and 50 non-redundant groups with unknown function. The putative protein products were classified into the following categories: photosynthesis (16.7%), protein synthesis (12.3%), cell proliferation and differentiation (10.9%), cell enlargement (5.8%), metabolism (8.0%), stress response (7.2%), others (2.9%), and unknown functions (32.2%). Furthermore, confirming the microarray data by reverse transcription-polymerase chain reaction revealed that the wide range of transcripts differentially expressed in young fruit was expressed in other organs but not in the mature fruit. The data presented suggested that apple fruit development depends on the tight regulation of the expression of a number of genes, which are also expressed in other organs.

Lys296 and Arg299 residues in the C-terminus of MD-ACO1 are essential for a 1-aminocyclopropane-1-carboxylate oxidase enzyme activity.

The 1-aminocyclopropane-1-carboxylate (ACC) oxidase catalyzes the last step in the biosynthesis of ethylene from ACC in higher plants. The complex structure of ACC oxidase/Fe(2+)/H(2)O derived from Petunia hybrida has recently been established by X-ray crystallography and it provides a vast structural information for ACC oxidase. Our mutagenesis study shows that both Lys296 and Arg299 residues in the C-terminal helix play important roles in enzyme activity. Both K296R and R299K mutant proteins retain only 30-15% of their enzyme activities with respect to that of the wild-type, implying that the positive charges of C-terminal residues are involved in enzymatic reaction. Furthermore, the sequence alignment of ACC oxidases from 24 different species indicates an existence of the exclusively conserved motif (Lys296-Glu301) especially in the C-terminus. The structure model based on our findings suggests that the positive-charged surface in the C-terminal helix of the ACC oxidase could be a major stabilizer in the spatial arrangement of reactants and that the positive-charge network between the active site and C-terminus is critical for ACC oxidase activity.

OsEIN2 is a positive component in ethylene signaling in rice.

EIN2 is a central signal transducer in the ethylene-signaling pathway, and a unique membrane-anchored protein. By screening a cDNA library, we have isolated a cDNA clone (OsEIN2) that encodes the rice EIN2 homolog. The full-length ORF clone was obtained by reverse transcriptase-polymerase chain reaction. OsEIN2 shares significant amino acid sequence similarity with Arabidopsis EIN2 (57% similarity and 42% identity). Both the numbers and positions of introns and exons in the OsEIN2 and AtEIN2 coding regions are also conserved. To address whether this structural similarity is indicative of functional conservation of the corresponding proteins, we also generated transgenic lines expressing the antisense construct of OsEIN2. Those plants were stunted and shoot elongation was severely inhibited. Their phenotypes were similar to that found with wild-type rice seedlings that were treated with AgNO3, an ethylene signal inhibitor. In the OsEIN2 antisense plants, the expression levels of two ethylene-responsive genes, SC129 and SC255, were decreased compared with the wild types. These results suggest that OsEIN2 is a positive component of the ethylene-signaling pathway in rice, just as AtEIN2 is in Arabidopsis: Our antisense transgenic plants produced approximately 3.5 times more ethylene than the wild-type plants. Expression analysis of rice ACS and ACO genes showed that the transcript levels of OsACS1 and OsACO1 were elevated in the transgenic plants.