Functional analyses of the novel salt-inducible genes from Korean halophytes

Selvam Ayarpadikannan,Eunsook Chung,Hyun-A So,Kyoung-Mee Kim,Kenneth Ryan Schraufnagle,Kim Hyo Young,Jae-Sung Kwak,Hai Yang Yu,Soon-Ok Kim,Joo-Min Jeon,Myoung-HaeKwak,Jai-Heon Lee 한국육종학회 2012 한국육종학회 심포지엄 Vol.2012 No.07

Salinity stress severely affects plant growth and development causing crop loss worldwide. Suaeda asparagoides is a salt-marsh euhalophyte widely distributed in southwestern foreshore of Korea. To isolate salt tolerance genes from S. asparagoides, we constructed a cDNA library from leaf tissues of S. asparagoides that was treated with 200 mM NaCl. A total of 1,056 clones were randomly selected for EST sequencing, and 932 of them produced readable sequence. By sequence analysis, we identified 538 unigenes and registered each in National Center for Biotechnology Information. The 80 salt stress related genes were selected to study their differential expression. Reverse Transcriptase-PCR and Northern blot analysis revealed that 23 genes were differentially expressed under the high salinity stress conditions in S. asparagoides. They are functionally diverse including transport, signal transduction, transcription factor, metabolism and stress associated protein, and unknown function. Among them dehydrin (SaDhn) and RNA binding protein (SaRBP1) were examined for their abiotic stress tolerance in yeast (Saccharomyces cerevisiae). Yeast overexpressing SaDhn and SaRBP1 showed enhanced tolerance to osmotic, freezing and heat shock stresses. This study provides the evidence that SaRBP1 and SaDhn from S.asparagoides exert abiotic stress tolerance in yeast. Information of salt stress related genes from S. asparagoides will contribute for the accumulating genetic resources to improve osmotic tolerance in plants.

Predicting family-wide consensus sequence of pre-mRNA splicing signals in legumes and their potential utility as phylogenetic markers

최홍규,이채영,Goon-Bo Kim,남영우,Kenneth R. Schraufnagle,정영수,이재헌,김도훈,정호원 한국식물생명공학회 2013 Plant biotechnology reports Vol.7 No.2

Recent studies have shown that an intron is notmerely ‘‘junk’’, but something that plays important roles inmany biological processes such as gene expression regulationand alternative splicing. For purposes of studyingintron structures and predicting consensus splice motifs, atotal of 102 legume species were used to isolate intronsacross the family. Of 196 gene-targeted PCR primer pairs,we successfully amplified 118 intron-containing genes(60.2 %) and obtained a total of 1,870 introns with anaverage size of 143 nucleotides, ranging from 61 to 1,036. Species-based compilation of 50- and 30-splicing motifsshowed, to some extent, lineage-specific conservation ineach splicing motif. Compilation of the entire intron setpermitted prediction of the consensus sequences of splicingsignal motifs in legumes, AYGWGTABABGH and TVNC/TAGGHTV for the 50SS and 30SS, respectively. Interestingly,these consensus motifs are very similar to the correspondinggenome-wide splicing signals of two modelsystems, Arabidopsis and rice. This result suggests conservationof pre-mRNA splicing mechanism occurring inhigher plants. Multiple alignments of CALTL intronsdemonstrated that the BP-30SS region was relatively moreconserved than the 50SS-BP region. We speculate thatlength of the BP-30SS region needs to be retained for theinteraction with U2AF protein. Phylogenetic analysisdemonstrates that each of three splicing motifs is not onlyphylogenetically informative, but also relevant to evolutionarydivergence of species. This result suggests that thesplice signal sequences would be a useful tool for themolecular phylogenetic analysis. We also anticipate thatgene-targeted amplification in multiple genomes, describedin this study, would facilitate studies on intron-locatedfunctional elements involved in gene expressionregulations.

Overexpression of SaRBP1 enhances tolerance of Arabidopsis to salt stress

Ayarpadikannan, Selvam,Chung, Eun Sook,So, Hyun Ah,Kim, Kyoung Mi,Schraufnagle, Kenneth Ryan,Lee, Jai Heon Springer-Verlag 2014 Plant cell, tissue and organ culture Vol.118 No.2

RsERF1 derived from wild radish (Raphanus sativus) confers salt stress tolerance in Arabidopsis

Ayarpadikannan, S.,Chung, E.,Kim, K.,So, H. A.,Schraufnagle, K. R.,Lee, J. H. POLISH ACADEMY SCIENCES WARSAW 2014 ACTA PHYSIOLOGIAE PLANTARUM Vol.36 No.4

The change in environmental parameters affects normal growth of plants, eventually reduces agricultural production. Ethylene plays vital roles in plant stress responses, germination, fruit ripening, organ abscission, pathogen response, and senescence. Expression of an ethylene-responsive transcription factor (ERF) was induced in Korean halophyte, Raphanus sativus var. hortensis f. raphanistroides (wild radish) by 200-mM sodium chloride (NaCl). Raphanus sativus ethylene-responsive transcription factor 1 (RsERF1) is also localized to nucleus, similar to other transcription factors. In yeast, RsERF1 showed transcriptional activation property, by expressing the reporter gene. Being a TF, RsERF1 specifically bound to the cis-acting elements, GCC box and DRE/CRT in vitro, to initiate transcription. Homozygous T3 transgenic Arabidopsis, overexpressing RsERF1, showed significant tolerance against salt stress in soil-grown conditions. The tolerance was also marked by an increased germination rate of RsERF1 transgenics in salt-containing media. In RsERF1 overexpression lines, abiotic stress-related genes such as ABF3, ABF4, ADH, Rab18, and SUS1 were upregulated by 200-mM NaCl. ERFs have been studied and proven for their tolerance potential against various abiotic stresses, but RsERF1 belongs to an ERF subgroup called ethylene-responsive transcription factor related to AP2 (ERF-RAP2). Thus, this is a first report for ERF-RAP2 from Korean halophyte cDNA library. We believe that extensive posttranslational modification studies will reveal the role and location of RsERF1 in stress tolerance pathway.

Ectopic expression of mungbean ubiquitin conjugating enzyme E2 enhances resistance to osmotic stress in Arabidopsis

Eunsook Chung,Hyun-A So,Kyoung-Mee Kim,Selvam Ayarpadikannan,Kenneth Ryan Schraufnagle,Kim Hyo Young,Jae-Sung Kwak,Hai Yang Yu,Jai-Heon Lee 한국육종학회 2012 한국육종학회 심포지엄 Vol.2012 No.07

A low temperature-inducible cDNA designated as VrUBC1 from mungbean (Vigna radiata) was isolated by subtractive hybridization method. By rapid amplification of cDNA end technique, the full-length cDNA of VrUBC1 was obtained. The full-length cDNA of VrUBC1 contains an open reading frame of 444 nucleotides in length and capable of specifying a 16.5-kDa protein of 148 amino acids (aa) with an isoelectric point of 7.72. VrUBC1 mRNA was induced by NaCl and ABA, but not by wounding and low temperature stress. It was shown that VrUBC1-GFP was localized to the cytoplasm in tobacco cell. To examine the function of VrUBC1, VrUBC1 was expressed in Escherichia coli as His-fusion protein. Purified VrUBC1-His recombinant protein was shown to have ubiquitination activity in vitro. For the in vivo functional analysis of VrUBC1, VrUBC1 was expressed in yeast ubc4/5 double mutant. Stress tolerance was tested in the VrUBC1 overexpressing Arabidopsis transgenic plants. We propose that VrUBC1 play an important role in protein degradation processes during abiotic stress in plants.

Functional analysis of a novel gene encoding DnaJ-like protein from soybean

Hyun-A So,Eunsook Chung,Kyoung-Mee Kim,Selvam Ayarpadikannan,Kenneth Ryan Schraufnagle,Kim Hyo Young,Jae-Sung Kwak,Hai Yang Yu,Jai-Heon Lee 한국육종학회 2012 한국육종학회 심포지엄 Vol.2012 No.07

We have isolated wound-inducible genes from soybean using suppression subtractive hybridization (SSH) method and were able to obtain the full-length clone of GmDjp1 gene encoding DnaJ-like protein. The full-length cDNA of GmDjp1 is 689 bp with an open reading frame (ORF) consisting of 163 amino acid (aa). Genomic southern blot confirmed that soybean genome has two copies of GmDjp1 gene. Northern blot analysis showed that the RNA expression of GmDjp1 gene is specifically induced by heat, NaCl, wounding and drought stresses. It was demonstrated that GmDjp1-GFP was targeted to the nucleus in tobacco cell. GmDjp1 overexpression plants showed more susceptible to salt and heat stress compared to WT. RNA expression level of Hsp18.2 and Hsp25.3-P was lower than that of WT during recovery after heat hock in plants. This indicates that GmDjp1 may play a negative regulator to stress responses in plants.

Predicting family-wide consensus sequence of pre-mRNA splicing signals in legumes and their potential utility as phylogenetic markers

Choi, Hong-Kyu,Lee, Chae-Young,Kim, Goon-Bo,Nam, Young-Woo,Schraufnagle, Kenneth R.,Chung, Young-Soo,Lee, Jai-Heon,Kim, Doh-Hoon,Jung, Ho-Won 한국식물생명공학회 2013 Plant biotechnology reports Vol.7 No.2

Recent studies have shown that an intron is not merely "junk", but something that plays important roles in many biological processes such as gene expression regulation and alternative splicing. For purposes of studying intron structures and predicting consensus splice motifs, a total of 102 legume species were used to isolate introns across the family. Of 196 gene-targeted PCR primer pairs, we successfully amplified 118 intron-containing genes (60.2 %) and obtained a total of 1,870 introns with an average size of 143 nucleotides, ranging from 61 to 1,036. Species-based compilation of 5'- and 3'-splicing motifs showed, to some extent, lineage-specific conservation in each splicing motif. Compilation of the entire intron set permitted prediction of the consensus sequences of splicing signal motifs in legumes, $A^YG^W\underline{GT}A^BA^BG^H$ and $T^VNC/T\underline{AG}G^HT^V$ for the 50SS and 30SS, respectively. Interestingly, these consensus motifs are very similar to the corresponding genome-wide splicing signals of two model systems, Arabidopsis and rice. This result suggests conservation of pre-mRNA splicing mechanism occurring in higher plants. Multiple alignments of CALTL introns demonstrated that the BP-3'SS region was relatively more conserved than the 5'SS-BP region. We speculate that length of the BP-3'SS region needs to be retained for the interaction with U2AF protein. Phylogenetic analysis demonstrates that each of three splicing motifs is not only phylogenetically informative, but also relevant to evolutionary divergence of species. This result suggests that the splice signal sequences would be a useful tool for the molecular phylogenetic analysis. We also anticipate that gene-targeted amplification in multiple genomes, described in this study, would facilitate studies on intron-located functional elements involved in gene expression regulations.

Molecular characterization of type III DnaJ-like proteins from Arabidopsis

Hyun-A So,Eunsook Chung,Kyoung-Mee Kim,Selvam Ayarpadikannan,Kenneth Ryan Schraufnagle,Kim Hyo Young,Jae-Sung Kwak,Hai Yang Yu,Jai-Heon Lee 한국육종학회 2012 한국육종학회 심포지엄 Vol.2012 No.07

Arabidopsis atDjC53 and atDjC32 gene DnaJ-like protein homologous to DnaJ-like protein was characterized for the functional analysis of DnaJ-like protein. It was shown that atDjC53 and atDjC32 RNA expression is induced by heat shock stress and atDjC53- and atDjC32-GFP was targeted to the nucleus of protoplasts. The atDjC53 and atDjC32 promoter (1 kb) was isolated and fused to the GUS reporter gene to investigate gene regulation of atDjC53 and atDjC32 specific to heat shock stress or to developmental organ in the transgenic lines. RNAi and overexpression construct was employed to generate atDjC53 and atDjC32 knock-out plants for the study of their function. Molecular function of atDjC53 and atDjC32 is discussed in relation to heat shock and also developmental stages in Arabidopsis.

Genome wide survey and molecular characterization of heat shock transcription factor gene family in Glycine max

Kyoung-Mee Kim,Eunsook Chung,Selvam Ayarpadikannan,Hyun-A So,Kenneth Ryan Schraufnagle,Ji Hae Park,Se Hyun Park,Jai-Heon Lee 한국육종학회 2013 한국육종학회 심포지엄 Vol.2013 No.07

Heat shock transcription factors (HSFs) are the major heat shock factors regulating the heat stress response. They participate in regulating the expression of heat shock proteins (HSPs), which are critical in the protection against stress damage and many other important biological processes. In this study, a genome-wide analysis was carried out to identify all HSFs soybean genes. Twenty six nonredundant HSF genes (GmHsf) were identified in the latest soybean genome sequence. Chromosomal location, protein domain and motif organization of GmHsfs were analyzed in soybean genome. The phylogenetic relationships, gene duplications and expression profiles of GmHsf genes were also presented in this study. According to their structural features, the predicted members were divided into the previously defined classes A–C, as described in Arabidopsis. Using RT-PCR, the expression patterns of 26 GmHsf genes were investigated under heat stress. The data revealed that these genes presented different expression levels in response to heat stress conditions. Real-time (q)RT-PCR was performed to investigate transcript levels of five GmHsfs in response to multiple abiotic stresses. Differential expression of five GmHsfs implies their role during abiotic stresses. Subcellular localization using GFP-fusion protein demonstrated that GmHsf12 and GmHsf34 were restricted to the nucleus and GmHsf28 was localized in the nucleus and cytoplasm in plant. The results provide a fundamental clue for understanding of the complexity of the soybean HSF gene family and cloning specific function genes in further studies and applications.

Functional analyses of the novel salt-inducible genes from Korean halophytes

Selvam Ayarpadikannan,Eunsook Chung,Hyun-A So,Kyoung-Mee Kim,Kenneth Ryan Schraufnagle,Ji Hae Park,Se Hyun Park,Jai-Heon Lee 한국육종학회 2013 한국육종학회 심포지엄 Vol.2013 No.07

Salinity stress severely affects plant growth and development causing crop loss worldwide. Suaeda asparagoides is a salt-marsh euhalophyte widely distributed in southwestern foreshore of Korea. To isolate salt tolerance genes from S. asparagoides, we constructed a cDNA library from leaf tissues of S. asparagoides that was treated with 200 mM NaCl. A total of 1,056 clones were randomly selected for EST sequencing, and 932 of them produced readable sequence. By sequence analysis, we identified 538 unigenes and registered each in National Center for Biotechnology Information. The 80 salt stress related genes were selected to study their differential expression. Reverse Transcriptase-PCR and Northern blot analysis revealed that 23 genes were differentially expressed under the high salinity stress conditions in S. asparagoides. They are functionally diverse including transport, signal transduction, transcription factor, metabolism and stress associated protein, and unknown function. Among them dehydrin (SaDhn) and RNA binding protein (SaRBP1) were examined for their abiotic stress tolerance in yeast (Saccharomyces cerevisiae). Yeast overexpressing SaDhn and SaRBP1 showed enhanced tolerance to osmotic, freezing and heat shock stresses. This study provides the evidence that SaRBP1 and SaDhn from S.asparagoides exert abiotic stress tolerance in yeast. Information of salt stress related genes from S. asparagoides will contribute for the accumulating genetic resources to improve osmotic tolerance in plants.