Young Sam Seo

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.

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.

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.

Two polyphenol oxidases are differentially expressed during vegetative and reproductive development and in response to wounding in the Fuji apple

Abstract Polyphenol oxidase (PPO), a copper-containing metalloprotein, catalyzes the oxidation of phenolics to quinones which make brown pigments in wounded tissues. Because the phenomena decrease fruit quality, PPO has been regarded to be a critical enzyme in food technology. In the course of expressed sequence tags (ESTs) analysis of the Fuji apple ( Malus domesticus Borkh.), we identified two partial PPO cDNA clones; F114 corresponded to the previously isolated Granny Smith apple pAPO5 (Plant Mol. Biol. 27 (1995) 429), while F226 was a new clone. Using F226 as a probe, we isolated a full length PPO clone, pMD-PPO2, from a cDNA library prepared from young fruits of the Fuji apple. The deduced amino acid sequences of pMD-PPO2 and pAPO5 share 55% identity, and display a high degree of sequence identity (43–58%) with previously identified PPO from various species. RNA gel blot analysis using gene-specific probes showed that two apple PPO genes display unique patterns of expression in a tissue- and developmental-specific manner. In the process of flower development, the APO5 transcript was detectable only at the post-anthesis stage. In contrast, MD-PPO2 was expressed in all stages of flower development, with the abundance of mRNA being the highest at the pre-anthesis stage and then receding as the flower developed. Both genes are expressed in the early stages of fruit development. The expression was dramatically reduced as the fruit ripened. In leaf tissue, the APO5 gene was highly expressed in young and immature leaves, while MD-PPO2 was transcriptionally more active in both immature and mature leaves. Upon wounding, APO5 was significantly induced in leaves and fruits, whereas the level of MD-PPO2 mRNA was not affected by mechanical damage. Thus, it appears that two Fuji PPO genes are differentially expressed during vegetative and reproductive development and in response to wounding in the Fuji apple plants. The possible molecular mechanism of differential regulation of PPO gene expression in apple plant and its physiological significance are discussed.

Structure and stress-related expression of two cDNAs encoding proteinase inhibitor II of Nicotiana glutinosa L.

Two cDNAs, pNGPI-1 and pNGPI-2, encoding Nicotiana glutinosa proteinase inhibitor II (PI-II) have been cloned, sequenced and identified. The deduced amino acid sequences are 54-82% identical to those of other plant PI-II. The NGPI-1 protein is composed of eight repeated domains, while NGPI-2 contains six repeated regions, each with a putative reactive site. The expression of NGPI-1 is highly regulated in a developmental- and tissue-specific manner, with the transcript being detected in young leaves and floral organs of N. glutinosa plants. In mature leaves, the NGPI-1 gene is rapidly activated by distinct temporal induction patterns in response to pathogen-related (biotic) and wound-related (abiotic) stresses.

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.

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.

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

A small rubber particle protein homolog plays dual roles as positive factors in post-germination growth and the drought stress tolerance response 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.

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.

Expression of MdCAS1 and MdCAS2, encoding apple β-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 β-cyanoalanine synthase (β-CAS). As little is known about the molecular function of β-CAS genes in climacteric fruits, we identified two homologous genes, MdCAS1 and MdCAS2, encoding Fuji apple β-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 β-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, β-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.