Cheol Seong Jang

A gene family encoding RING finger proteins in rice: their expansion, expression diversity, and co-expressed genes

The proteins harboring RING finger motif(s) have been shown to mediate protein–protein interactions that are relevant to a variety of cellular processes. In an effort to elucidate the evolutionary dynamics of the rice RING finger protein family, we have attempted to determine their genomic locations, expression diversity, and co-expressed genes via in silico analysis and semi-quantitative RT–PCR. A total of 425 retrieved genes appear to be distributed over all 12 of the chromosomes of rice with different distributions, and are reflective of the evolutionary dynamics of the rice genome. A genome-wide dataset harboring 155 gene expression omnibus sample plates evidenced some degree of differential evolutionary fates between members of RING-H2 and RING-HC types. Additionally, responses to abiotic stresses, such as salinity and drought, demonstrated that some degree of expression diversity existed between members of the RING finger protein genes. Interestingly, we determined that one RING-H2 finger protein gene (Os04g51400) manifested striking differences in expression patterns in response to abiotic stresses between leaf and culm-node tissues, further revealing responses highly similar to the majority of randomly selected co-expressed genes. The gene network of genes co-expressed with Os04g51400 may suggest some role in the salt response of the gene. These findings may shed further light on the evolutionary dynamics and molecular functional diversity of these proteins in complex cellular regulations.

The rice RING finger E3 ligase, OsHCI1, drives nuclear export of multiple substrate proteins and its heterogeneous overexpression enhances acquired thermotolerance

Thermotolerance is very important for plant survival when plants are subjected to lethally high temperature. However, thus far little is known about the functions of RING E3 ligase in response to heat shock in plants. This study found that one rice gene encoding the RING finger protein was specifically induced by heat and cold stress treatments but not by salinity or dehydration and named it OsHCI1 (Oryza sativa heat and cold induced 1). Subcellular localization results showed that OsHCI1 was mainly associated with the Golgi apparatus and moved rapidly and extensively along the cytoskeleton. In contrast, OsHCI1 may have accumulated in the nucleus under high temperatures. OsHCI1 physically interacted with nuclear substrate proteins including a basic helix-loop-helix transcription factor. Transient co-overexpression of OsHCI1 and each of three nuclear proteins showed that their fluorescent signals moved into the cytoplasm as punctuate formations. Heterogeneous overexpression of OsHCI1 in Arabidopsis highly increased survival rate through acquired thermotolerance. It is proposed that OsHCI1 mediates nuclear–cytoplasmic trafficking of nuclear substrate proteins via monoubiquitination and drives an inactivation device for the nuclear proteins under heat shock.

Molecular dissection of the response of a rice leucine-rich repeat receptor-like kinase (LRR-RLK) gene to abiotic stresses

Leucine-rich repeat (LRR) receptor-like kinase (RLK) proteins play key roles in a variety of biological pathways. In a previous study, we analyzed the members of the rice LRR-RLK gene family using in silico analysis. A total of 23 LRR-RLK genes were selected based on the expression patterns of a genome-wide dataset of microarrays. The Oryza sativa gamma-ray induced LRR-RLK1 (OsGIRL1) gene was highly induced by gamma irradiation. Therefore, we studied its expression pattern in response to various different abiotic and phytohormone treatments. OsGIRL1 was induced on exposure to abiotic stresses such as salt, osmotic, and heat, salicylic acid (SA), and abscisic acid (ABA), but exhibited downregulation in response to jasmonic acid (JA) treatment. The OsGIRL1 protein was clearly localized at the plasma membrane. The truncated proteins harboring juxtamembrane and kinase domains (or only harboring a kinase domain) exhibited strong autophosphorylation. The biological function of OsGIRL1 was investigated via heterologous overexpression of this gene in Arabidopsis plants subjected to gamma-ray irradiation, salt stress, osmotic stress, and heat stress. A hypersensitive response was observed in response to salt stress and heat stress, whereas a hyposensitive response was observed in response to gamma-ray treatment and osmotic stress. These results provide critical insights into the molecular functions of the rice LRR-RLK genes as receptors of external signals.

Expression diversity and evolutionary dynamics of rice duplicate genes

Duplicate genes are believed to be a major source of new gene functions over evolutionary time. In order to evaluate the evolutionary dynamics of rice duplicate genes, formed principally by paleoployploidization prior to the speciation of the Poaceae family, we have employed a public microarray dataset including 155 gene expression omnibus sample plates and bioinformatics tools. At least 57.4% of old ~70 million years ago (MYA) duplicate gene pairs exhibit divergences in expression over the given experimental set, whereas at least 50.9% of young ~7.7-MYA duplicate gene pairs were shown to be divergent. When grouping the rice duplicate genes according to functional categories, we noted a striking and significant enrichment of divergent duplicate metabolism-associated genes, as compared to that observed in non-divergent duplicate genes. While both non-synonymous substitution (Ka) and synonymous substitution (Ks) values between non- and divergent duplicate gene pairs evidenced significant differences, the Ka/Ks values between them exhibited no significant differences. Interestingly, the average numbers of conserved motifs of the duplicate gene pairs revealed a pattern of decline along with an increase in expression diversity, partially supporting the subfunctionalization model with degenerative complementation in regulatory motifs. Duplicate gene pairs with high local similarity (HLS) segments, which might be formed via conversion between rice paleologs, evidenced higher expression correlations than were observed in the gene pairs without the HLS segments; this probably resulted in an increased likelihood of gene conversion in promoters of the gene pairs harboring HLS segments. More than 60% of the rice gene families exhibited similar high expression diversity between members as compared to that of randomly selected gene pairs. These findings are likely reflective of the evolutionary dynamics of rice duplicate genes for gene retention.

Comparative analysis of evolutionary dynamics of genes encoding leucine-rich repeat receptor-like kinase between rice and Arabidopsis

The leucine-rich repeat (LRR) receptor kinase (RLK) proteins constitute a large superfamily in the plant genome, and carry out key functions in a variety of biological pathways. In an effort to determine the evolutionary fate of members of a large gene family such as plant LRR RLK proteins we conducted in silico analysis using complete genome sequencing datasets, genome-wide transcriptome databases, and bioinformatics tools. A total of 292 and 165 LRR RLK genes were retrieved from the rice and Arabidopsis genomes, respectively, formed by diverse duplication events for gene expansion. The phylogenic analyses of the LRR RLK genes suggested combinations of LRR domains and RLK domains in the ancient plant genome prior to the divergence of rice and Arabidopsis, followed by massive independent expansions during speciation. The somewhat high frequencies (50–73%) of expressional divergence of members of duplicate gene pairs formed by whole/segmental genome duplication (W/SGD) and tandem duplication (TD) events of Arabidopsis and TD events of rice support the idea of their functional diversity for gene retention. By contrast, a relatively low degree (at least 20%) of members of rice LRR RLK gene pairs formed by W/SGD appear to be divergent in expression following the duplication event. At least 7 pairs of co-expressed gene clusters, including each of the tentative orthologous LRR RLK genes between rice and Arabidopsis, were enriched to an orthologous set between members of each of the pairs as compared to those of the random pairs, suggesting some degree of functional conservation of individual genes. These results may shed some light on the crucial functions of the plant LRR RLK genes with regard to a variety of biological processes.

The rice RING E3 ligase, OsCTR1, inhibits trafficking to the chloroplasts of OsCP12 and OsRP1, and its overexpression confers drought tolerance in Arabidopsis.

Plant growth under low water availability adversely affects many key processes with morphological, physiological, biochemical and molecular consequences. Here, we found that a rice gene, OsCTR1, encoding the RING Ub E3 ligase plays an important role in drought tolerance. OsCTR1 was highly expressed in response to dehydration treatment and defense-related phytohormones, and its encoded protein was localized in both the chloroplasts and the cytosol. Intriguingly, the OsCTR1 protein was found predominantly targeted to the cytosol when rice protoplasts transfected with OsCTR1 were treated with abscisic acid (ABA). Several interacting partners were identified, which were mainly targeted to the chloroplasts, and interactions with OsCTR1 were confirmed by using biomolecular fluorescence complementation (BiFC). Interestingly, two chloroplast-localized proteins (OsCP12 and OsRP1) interacted with OsCTR1 in the cytosol, and ubiquitination by OsCTR1 led to protein degradation via the Ub 26S proteasome. Heterogeneous overexpression of OsCTR1 in Arabidopsis exhibited hypersensitive phenotypes with respect to ABA-responsive seed germination, seedling growth and stomatal closure. The ABA-sensitive transgenic plants also showed improvement in their tolerance against severe water deficits. Taken together, our findings lend support to the hypothesis that the molecular functions of OsCTR1 are related to tolerance to water-deficit stress via ABA-dependent regulation and related systems.

Insight into the Molecular Evolution of Non-Specific Lipid Transfer Proteins via Comparative Analysis Between Rice and Sorghum

Phylogenetic analysis was conducted on 9 kDa non-specific lipid transfer protein (nsLTP) genes from nine plant species. Each of the five classified types in angiosperms exhibited eight conserved cysteine patterns. The most abundant nsLTP genes fell into the type I category, which was particularly enriched in a grass-specific lineage of clade I.1. Six pairs of tandem copies of nsLTP genes on the distal region of rice chromosomes 11 and 12 were well-preserved under concerted evolution, which was not observed in sorghum. The transgenic promoter–reporter assay revealed that both rice and sorghum nsLTP genes of type I displayed a relatively conserved expression feature in the epidermis of growing tissue, supporting its functional roles in cutin synthesis or defence against phytopathogens. For type I, the frequent expression in the stigma and seed are indicative of functional involvement in pistil–pollen interactions and seed development. By way of contrast, several type V genes were observed, mainly in the vascular bundle of the rosette as well as the young shoots, which might be related with vascular tissue differentiation or defence signalling. Compared with sorghum, the highly redundant tissue-specific expression pattern among members of rice nsLTP genes in clade I.1 suggests that concerted evolution via gene conversion favours the preservation of crucial expression motifs via the homogenization of proximal promoter sequences under high selection constraints. However, extensive regulatory subfunctionalization might also have occurred under relative low selection constraints, resulting in functional divergence at the expression level.

PLANEX: the plant co-expression database

BackgroundThe PLAnt co-EXpression database (PLANEX) is a new internet-based database for plant gene analysis. PLANEX (http://planex.plantbioinformatics.org) contains publicly available GeneChip data obtained from the Gene Expression Omnibus (GEO) of the National Center for Biotechnology Information (NCBI). PLANEX is a genome-wide co-expression database, which allows for the functional identification of genes from a wide variety of experimental designs. It can be used for the characterization of genes for functional identification and analysis of a gene’s dependency among other genes. Gene co-expression databases have been developed for other species, but gene co-expression information for plants is currently limited.DescriptionWe constructed PLANEX as a list of co-expressed genes and functional annotations for Arabidopsis thaliana, Glycine max, Hordeum vulgare, Oryza sativa, Solanum lycopersicum, Triticum aestivum, Vitis vinifera and Zea mays. PLANEX reports Pearson’s correlation coefficients (PCCs; r-values) that distribute from a gene of interest for a given microarray platform set corresponding to a particular organism. To support PCCs, PLANEX performs an enrichment test of Gene Ontology terms and Cohen’s Kappa value to compare functional similarity for all genes in the co-expression database. PLANEX draws a cluster network with co-expressed genes, which is estimated using the k-mean method. To construct PLANEX, a variety of datasets were interpreted by the IBM supercomputer Advanced Interactive eXecutive (AIX) in a supercomputing center.ConclusionPLANEX provides a correlation database, a cluster network and an interpretation of enrichment test results for eight plant species. A typical co-expressed gene generates lists of co-expression data that contain hundreds of genes of interest for enrichment analysis. Also, co-expressed genes can be identified and cataloged in terms of comparative genomics by using the ‘Co-expression gene compare’ feature. This type of analysis will help interpret experimental data and determine whether there is a common term among genes of interest.

Selection of 5-methyltryptophan resistant rice mutants from irradiated calli derived from embryos

For increasing the contents of specificfree amino acids in rice (Oryzasativa L.) cultivar, Donganbyeo, mutantcell lines resistant to growth inhibitionby 5-methyltryptophan (5MT) were selectedfrom embryo cultured callus irradiated with50 Gy of gamma rays. Two lines, M2-1and M2-2, were obtained from theregenerated plants by selfing. Thesegregation ratios of resistance andsensitivity in these lines fitted 9:7 and3:1 ratios, respectively. Considering theagronomic traits, M2-1 was about thesame as the original variety in culm lengthand a little longer or higher in paniclelength and number of tillers. However, thefertility of M2-1 significantlyincreased compared to the originalvariety. M2-2 had an extremely highculm length and tillering capacity. Four5MT-resistant homozygous M3lines (MRclasses), M3-1-40 and M3-1-116from M2-1 (MR I),and M3-2-8 and M3-2-12from M2-2 (MR II), were obtained inthe same manner as with the M2generation. Protein contents of brown ricewere increased about 19% and 32% and the total contents of 9 free essentialamino acids were 71% and 34% greater thanthe original variety in the MR I andMR II groups, respectively. However, forthe free essential amino acids of polishedrice, there was no difference from theoriginal variety.

Development of AFLP and STS markers for identifying wheat-rye translocations possessing 2RL

Wheat-rye translocation line (2BS/2RL) has been developed for resistance to biotype L of Hessian fly and agronomically useful traits. AFLP analysis using 64 primer pairs was conducted in order to identify 2RL-specific polymorphisms between “Coker 797” (non-2RL), near-isogenic line (NIL) carrying 2RL, and “Hamlet”. Nine primer combinations identified twelve reproducible polymorphic fragments in the NIL carrying 2RL. These twelve fragments were cloned and sequenced with an aim towards converting AFLP markers into sequence tagged sites (STS). A comparison of the 12 sequences with non-redundant accessions in the NCBI database using the BLAST search option indicated that one fragment of approximately 200 bp in length (amplified using primer combination E+AAC / M+CTA) was highly homologous with the rye-specific repetitive sequence R173-1 and Wis-2-lA, a retrotransposon-like element in wheat. Two STS primers (SJ07 and SJ09) out of twelve STS primer sets enabled the detection of polymorphisms between Coker 797 and NIL carrying 2RL. In order to verify whether the polymorphism detected by primers SJ07 and SJ09 was in fact the result of the presence of 2RL, additional plant material was examined. Amplified products of about 260 bp fragment with the SJ07 primer set were generated in rye cvs.“Chilbohomil” and “Jochunhomil”, triticale experimental line Suwon 15, and wheat experimental line K-14 (1AL/1RS & 2BS/2RL), as well as NIL carrying 2RL and Hamlet, but not in Coker 797 (non-2RL), “Keumgangmil” (non-translocation wheat), KS92WGRC17 (PI592729 /;/ 6BS/6BL-6RL), KS92WGRC19 (P1592731 /;/ 4BS/4BL-6RL), “TAM200” (1AL/1RS), and “Siouxland” (1BL/1RS). Our data suggest that primer set SJ07 amplifies a “2RL-specific” fragment of diagnostic value.