6(4) 2013 issue
July 2013 issue
Southern Cross Publishing Group©2013

Plant Omics Journal | July 2013 issue
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Molecular cloning, characterization and expression of the caffeic acid O-methyltransferase (COMT) ortholog from kenaf (Hibiscus cannabinus)

Jonggeun Kim1, Bosung Choi1, Byoung-Kwan Cho2, Hyoun-Sub Lim3, Joon Bum Kim4, Savithiry Natarajan5, Eunjung Kwak6*, Hanhong Bae1*

1School of Biotechnology, Yeungnam University, Gyeongsan 712-749, Republic of Korea
2Department of Biosystems and Machinery Engineering, Chungnam National University, Daejeon 305-764, Republic of Korea
3Department of Applied Biology, Chungnam National University, Daejeon 305-764, Republic of Korea
4Warm-Temperate Forest Research Center, Korea Forest Research Institute, Seogwipo 697-050, Korea
5US Department of Agriculture, Agricultural Research Service, Soybean Genomics and Improvement Laboratory, 10300, Baltimore Avenue, Beltsville, MD 20705, USA
6Department of Food Science and Technology, Yeungnam University, Gyeongsan 712-749, Republic of Korea

We cloned the full-length of the gene putatively encoding caffeic acid O-methyltransferase (COMT) from kenaf (Hibiscus cannabinus L.) using degenerate primers and the RACE (rapid amplification of cDNA ends) method. COMT is an important methylating enzyme in the phenylpropanoid pathway that belongs to the SAM (S-adenosyl L-methionine)-dependent methyltransferases family. We investigated the expression pattern of kenaf COMT during developmental stages in different tissues and organs as well as in response to diverse abiotic stresses [wounding, salicylic acid (SA), NaCl, cold, H2O2 and methyl jasmonate (MeJA)]. The full-length COMT ortholog is composed of a 1,098-bp open reading frame (ORF) encoding 365 amino acids. The deduced amino acid sequence indicated that kenaf COMT had the highest similarity (95%) with that of Gossypium hirsutum. Three-week-old stem tissues were used to analyze COMT ortholog expression upon abiotic stresses. The highest level of COMT transcript (32%, relative to ACTIN) was detected at an early stage (4-week old) during stem development. The transcript levels of COMT ortholog were almost constant during the developmental stages of leaves and flowers, while early induction of the COMT ortholog was detected following wounding, SA, cold and H2O2 treatments, and MeJA led late induction and NaCl led to intermediate induction of the COMT ortholog.

Pages 246-253 | Full Text PDF
Regulation of 4CL, encoding 4-coumarate: coenzyme A ligase, expression in kenaf under diverse stress conditions

Md. Emran Khan Chowdhury1, Bosung Choi1, Byoung-Kwan Cho2, Joon Bum Kim3, Sang Un Park4, Savithiry Natarajan5, Hyoun-Sub Lim6*, Hanhong Bae1*

1School of Biotechnology, Yeungnam University, Gyeongsan712-749, Republic of Korea
2Department of Biosystems and Machinery Engineering, Chungnam National University, Daejeon 305-764, Republic of Korea
3Warm-Temperate Forest Research Center, Korea Forest Research Institute, Seogwipo 697-050, Korea
4Department of Crop Science, Chungnam National University, Daejeon 305-754, Korea
5US Department of Agriculture, Agricultural Research Service, Soybean Genomics and Improvement Laboratory, 10300, Baltimore Avenue, Beltsville, MD 20705, USA
6Department of Applied Biology, Chungnam National University, Daejeon 305-764, Republic of Korea

We cloned the full length 4CL ortholog encoding 4-coumarate:coenzymeA ligase from kenaf  (Hibiscus cannabiuns, GenBank Accession No. JX548316) using degenerate primers and RACE (rapid amplification of cDNA ends) method. The 4CL is a key regulatory enzyme of the phenylpropanoid pathway that regulates the activation of cinnamic acid, leading to the synthesis of flavonoids and lignin. The 1,704-bp full length of 4CL ortholog had a 1,623-bp open reading frame (ORF) encoding a predicted protein of 540 amino acids. The predicted molecular weight and isoelectric point (pI) of the deduced protein was 59.56 kDa and 6.58, respectively. The sequence of the deduced amino acid shared 57-79% identities with other 4CL sequences. 4CL ortholog had two conserved putative AMP (adenosine monophosphate)-binding motifs, the SSGTTGLPKGV and GEICIRG domains. A BlastP analysis showed that kenaf 4CL ortholog showed 79% identity with ri4CL2 of Rubus idaeus (AAF91309), which is a class I 4CL involved in lignin synthesis. 4CL ortholog showed differential expression in all tissues during the developmental stages and was highly expressed in stem and root tissues. However, the lowest expression of 4CL ortholog was observed in leaf and mature flower tissues. 4CL ortholog was responsive to various stress conditions in the stem tissues of 3-week-old kenaf plants. Wounding caused biphasic expression at 6 h and 24 h after treatment. Taken together, the results of this study contribute to the knowledge of the presence of 4CL ortholog and its possible role in lignin biosynthesis, as well as its differential expression during developmental stages.

Pages 254-262 | Full Text PDF
Accumulation of g-aminobutyric acid and transcription of glutamate decarboxylase in Brassica juncea (L.) Czern.

Yeon Bok Kim1, Min-Ki Lee2, Sun-Ju Kim2, Haeng Hoon Kim3
, Eunsook Chung4, Jai-Heon Lee4*, Sang Un Park1*

1Department of Crop Science, Chungnam National University, Daejeon 305-764, Korea
2Department of Bio Environmental Chemistry, Chungnam National University, Deajeon 305-764, Korea
3Department of Well-being Resources, Sunchon National University, 413 Jungangno, Suncheon, Jeollanam-do, 540-742, Korea
Department of Genetic Engineering, Dong-A University, Busan 604-714, Korea

Mustard leaf (Brassica juncea (L.) Czern.) using as material of a traditional fermented vegetable food (Kimchi) in Korea, is one of the important vegetables. Two cultivars of mustard (red and green) were grown in the experimental farm of Chungnam National University and allowed them to grow until flowering stage. In this study, the variability of
g-aminobutyric acid (GABA) content and mRNA transcript levels of genes involved in GABA biosynthesis were investigated in 2 cultivars (green and red mustard) of Brassica juncea. We observed that the transcript levels of glutamate decarboxylase (GAD) 1, 2, and 4a in roots were the highest, whereas those in seeds were the lowest compared with other organs harvested at flowering stage in green mustard. The transcript levels in green and red mustards displayed similar transcription patterns among a variety of organs, except for those of BjGAD2 and 4a in roots. In red mustard, the GABA content in flower buds was the highest (179.8 mg 100 g-1 FW), whereas roots contained the lowest amount (1.77 mg 100 g-1 FW). In green mustard, the GABA content in young leaves was the highest (97.76 mg 100 g-1 FW), whereas seeds had the lowest (1.23 mg 100 g-1 FW). Green and red mustard contained varying amounts of GABA in different organs. Specially, because green and red mustard leaf has high GABA content compared with other vegetables, it can be used as a good vegetable for healthy. 

Pages 263-267 | Full Text PDF
Comparative proteomic analysis of tomato (Solanum lycopersicum) leaves under salinity stress

Arafet Manaa1*, Hajer Mimouni1, Salma Wasti1, Emna Gharbi1,2, Samira Aschi-Smiti1, Mireille Faurobert2, and Hela Ben Ahmed1

1Unité d’Ecophysiologie et Nutrition des Plantes, Département de Biologie, Faculté des Sciences de Tunis. Université Tunis El Manar. 2092. Tunisia
2Groupe de Recherche en Physiologie Végétale (GRPV), Earth and Life Institute-Agronomy (ELI-A), Université catholique de Louvain, 5 (Bte L 7.07.13) Place Croix du Sud, 1348 Louvain-la-Neuve, Belgium

Salinity is a major constraint to crop productivity. The mechanisms responsible for the effects of salt stress on tomato plants were examined by means of proteomic analysis. Two contrasting tomato genotypes (cv. Roma and cv. SuperMarmande) seedlings were cultivated using a hydroponic system in the controlled environment growth chamber. The salt stress (NaCl) was applied (0, 100 and 200 mM) and maintained for 14 days. Leaf osmotic potential significantly decreased with NaCl treatment in genotype SuperMarmande. However, genotype Roma maintained its leaf osmotic adjustment under salt treatment. Lipid peroxidation (estimated by MDA content) significantly increased under salt in both cultivars, but the rate of increment was higher in genotype SuperMarmande. Leaves of control and salt-stressed plants were also sampled for phenol protein extraction. Proteins were separated by two-dimensional gel electrophoresis (2-DE). 26 protein spots exhibited significant abundance variations between samples. Our results indicated that some proteins exhibited variation strictly related to salt stress whatever the genotype, while some other proteins also showed variation which could be related to the degrees of genotype tolerance. The up-regulation of Rubisco activases and RuBisCO large subunit under salt treatment, was correlated with an increase in abundance level of proteins that are involved in energy metabolism (pyruvate dehydrogenase, Glucose-6-phosphate dehydrogenase, Malate dehydrogenase), especially in salt-tolerant genotype (Roma). Accumulation of antioxidants enzymes (ascorbate peroxidase, glutathione peroxidase, peroxidase and mitochondrial peroxiredoxin) in the leaves of salt tolerant genotype (Roma) was well correlated with the level of lipid peroxidation of membranes. Up-regulation of Heat Shock Proteins and maintenance of water status as reflected by leaf osmotic potential are considered to be strongly correlated with to the degrees of tomato genotype tolerance in this study.

Pages 268-277 | Full Text PDF | Supplementary data PDF
In silico analysis of putative transcription factor binding sites in differentially expressed genes: Study of the turnover of TFBSs under salt stress responsiveness in solanaceae family

Sanchita*, Blessy M. Baby, Ashok Sharma

Biotechnology Division, CSIR-Central Institute of Medicinal and Aromatic Plants, Post Office CIMAP, Lucknow- 226015, India

The binding of transcription factors in transcription factor binding sites (TFBSs) play a key role in the regulation of different biological processes showing change in the expression of genes in presence of adverse conditions. In this study, an in silico analysis of the data of differentially expressed genes of seven plants of family solanaceae under different time periods of salt stress was performed to find out the TFBSs. The data used for the study was retrieved from the public domain. Our analysis revealed up and down expression of genes that might result due to binding of transcription factors in promoter region. The promoter regions of differentially expressed genes were utilized for the prediction of TFBSs. The prediction was done using position weight matrices (PWMs) constructed by taking the data of experimentally validated transcription factors and their respective binding sites. The PWMs were scanned over promoter sequences present in the upstream region of differentially expressed genes. The TFBSs with a threshold of similarity score = 2.97 were selected to get highly up and down expressed genes. These predicted TFBSs would be of help to understand the role of turnover of TFBSs responsible for the change in expression of genes under salt stress.

Pages 278-285 | Full Text PDF | Supplementary data PDF
Proteomic analysis of leaves of different wheat genotypes subjected to PEG 6000 stress and rewatering

Jingxiu Ye1*, Shuping Wang2, Fengjun Zhang1, Deqing Xie1, Youhua Yao1

1State Key Laboratory Breeding Base for innovation and utilization of plateau crop germplasm, Qinghai Academy of Agriculture and Forestry Science, Xining, Qinghai, China
2Northwest A&F University, Yangling, Shaanxi, China

Drought is an abiotic stress that strongly influences plant growth, development and productivity. To understand the drought tolerance mechanism at the protein level in wheat, a differential proteomics study was carried out on young spring wheat leaves of different genotypes in PEG-stressed and rewatered, using two-dimensional polyacrylamide gel electrophoresis (2-DE). A 2-DE pattern with high resolution and good reproducibility was obtained after staining with Coomassie brilliant blue G-250. Using PDQuest software, 600 protein spots were clearly identified from the treatment and control groups with isoelectric points ranging from 4.0 to 7.0. Thirty-eight differentially expressed protein spots were MALDI-TOF/TOF-MS fingerprinted using 2-DE gel and 35 spots were identified by search through the NCBInr database using Mascot software. Of 35 proteins, twenty-one proteins changed in abundance after PEG stress, with 15 proteins up-regulated, whereas 6 proteins down-regulated. Twenty four hour after rewatering, there were 5 proteins up-regulated and 9 proteins down-regulated compared to the well-watered control. Twenty-two differentially expressed proteins were detected in Qingchun 38 and 13 proteins in Abbondanza. They were involved in photosynthesis, protein biosynthesis, energy pathway, carbon metabolism, cell defense, oxidation reduction, transportation and signal transduction. Our proteomics results suggested that drought stress significantly affects wheat photosynthesis.

Pages 286-294 | Full Text PDF | Supplementary data Excel | Supplementary data PDF
Development of EST-SSR markers to study genetic diversity in hyacinth bean (Lablab purpureus L.)

Guwen Zhang1+, Shengchun Xu1+, Weihua Mao2, Yaming Gong1*, Qizan Hu1

1Institute of Vegetables, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, People’s Republic of China
2Center of Analysis and Measurement, Zhejiang University, Hangzhou 310023, People’s Republic of China

The development of expressed sequence tags (ESTs) has provided a useful source for the mining of simple sequence repeat (SSR) markers. The hyacinth bean (Lablab purpureus L.) is one of the most ancient and important legume crops in the world, and is widely distributed in tropical and sub-tropical regions, but currently only limited molecular markers are available. The aim of the present study is to develop EST-SSRs for hyacinth beans to investigate the genetic structure and diversity of different populations originating from China and Africa. A total of 459 hyacinth bean ESTs from the National Center for Biotechnology Information (NCBI) database were downloaded and analyzed to search for SSRs with a minimum of 12 repeating nucleotides. Finally, 22 microsatellites were identified in 420 unigenes, indicating that merely 4.79% of the sequences contained SSRs. EST-SSR loci were subsequently screened on 24 hyacinth bean accessions collected from both China and Africa. Among 22 EST-SSRs, 11 loci showed polymorphism and revealed two to four alleles per locus. Tri-nucleotide motifs were the most common type of repeats, accounting for 54.55% of the total, followed by di-nucleotide repeats. The polymorphic information content (PIC) values ranged from 0.0767 to 0.4864, with a mean of 0.286. Furthermore, both principal coordinate analysis (PCA) and phylogenetic tree analysis <javascript:showjdsw('showjd_0','j_0')> indicated that all accessions were clustered into two main groups, and that all 19 Chinese accessions were clustered into the single group. These results suggest that there is a narrow genetic basis for Chinese hyacinth bean accessions. In summary, the results of this study suggest that the data mining of EST databases is a feasible method for generating EST-SSR markers for hyacinth bean, and these newly developed EST-SSR markers will be useful for further taxonomy, molecular breeding and comparative mapping studies.

Pages 295-301 | Full Text PDF
Cloning and expression profiling of polycomb gene, DNA Polymerase Alpha (POLa) from tomato Solanum lycopersicum L.

Zainab M. Almutairi1*, Monther T. Sadder2,3

Biology Department, College of Science and Humanity Studies, P. O. Box 83, Salman Bin Abdulaziz University, Alkharj 11942, Saudi Arabia
2Center of Excellence in Biotechnology Research, King Saud University, PO Box 2455, Riyadh 11451, Saudi Arabia
3Plant Biotechnology Lab, Faculty of Agriculture, The University of Jordan, Amman, 11942, Jordan

The decision to replicate its DNA is of crucial importance for every cell and, in many organisms, is decisive for the progression through the entire cell cycle. DNA polymerases are required to maintain the integrity of genome during DNA replication.
DNA Polymerase Alpha (POLa) encodes the putative catalytic subunit of the DNA polymerase a which plays an essential role in the initiation of DNA replication. Here we present the cloning, characterization and expression of a putative SlPOLa gene in tomato by isolating cDNA clones corresponding to SlPOLa gene from tomato using primers designed from available ESTs based on conserved sequences between PcG genes in Arabidopsis thaliana and tomato. The SlPOLa cDNAs were cloned into pBS plasmid and sequenced. Both 5' and 3' RACE were generated and sequenced. The flcDNA for SlPOLa gene length was composed of 4,944 bp, containing 103 bp in the 5'UTR; 4,683 bp in the ORF; and 158 bp in 3'UTR. The translated ORF encodes a polypeptide of 1,561 amino acids. Alignment of deduced amino acids indicated that there are highly conserved regions between tomato SlPOLa predicted protein and hypothetical plant POLa gene family members. Both unrooted phylogenetic trees, constructed using maximum parsimony and maximum likelihood methods indicated that there is a close relationship between SlPOLa predicted protein and POLa protein of Mimulus guttatus. SlPOLa was strongly expressed in proliferating tissues (closed floral buds) and moderately expressed in flower tissues, whereas, it was weakly expressed in non-proliferating tissues (unripe fruit).

Pages 302-310 | Full Text PDF | Supplementary data PDF
Analysis of expressed sequence tags and alternative splicing genes in sacred lotus (Nelumbo nucifera Gaertn.)

Robert VanBuren1, Braden Walters2, Ray Ming1, Xiang Jia Min2*

1Department of Plant Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA
2Center for Applied Chemical Biology, Department of Biological Sciences, Youngstown State University, Youngstown, OH 44555, USA

Sacred lotus, Nelumbo nucifera (Gaertn.), a basal eudicot of agricultural, medicinal, cultural, and religious importance, is an under-studied species at the molecular level.  Expressed sequence tags (ESTs) are particularly useful for gene identification, genome annotation and alternative splicing (AS) study.  We generated 626,137 reads by 454 pyrosequencing of a cDNA library, which was constructed from mRNA collected from 7 different tissues of sacred lotus.  The reads were assembled into 16,349 non-redundant EST contigs with an average length of 715 bp.  Aligning ESTs to sacred lotus genome revealed the internal exons and introns had an average size of 140 and 2,005 bp, respectively. The average intron length in the sacred lotus is much larger than most sequenced plant genomes. We also identified 174 AS events involving 161 genes.  Classification of the AS events showed that 62.6% of the AS events were retained introns, 8.0% were alternative acceptor sites, 7.5% were alternative donor sites, 4.0% were skipped exons, and 17.8% had “complex events” which had more than one basic event.  Comparison of AS genes in sacred lotus, rice and Arabidopsis identified 7 commonly conserved genes between lotus and rice and 9 between sacred lotus and Arabidopsis. A gene encoding a 40S ribosomal protein was commonly conserved in all three species.  These AS events and annotated ESTs are available through the Plant Alternative Splicing Database website (   

Pages 311-317 | Full Text PDF | Supplementary data Excel | Supplementary data PDF