8(6) 2015 issue
November 2015 issue
Southern Cross Publishing Group©2015
Molecular characterization of stress-inducible PLATZ gene from soybean (Glycine max L.)
Hyun-A So, Su Jin Choi, Eunsook Chung, Jai-Heon Lee*
Department of Applied Bioscience, College of Natural Resources and Life Science, Dong-A University, Busan 604-714, Republic of Korea
PLATZ (plant AT-rich sequence and zinc-binding protein) is a novel class of DNA-binding proteins; however, the function of the PLATZ gene has not yet been identified in plants. This study aims to isolate, sequence, and analyse the PLATZ gene responsive to abiotic stress in soybean. We isolated a stress-inducible gene encoding the PLATZ from soybean (Glycine max L.). This gene, designated as GmPLATZ1, was specifically induced by drought, high salinity, or abscisic acid (ABA) in soybean. GmPLATZ1 cDNA is composed of 711 bp nucleotide sequences encoding a PLATZ protein (236 amino acids) with 9.41 pI and 26.75 kDa. Multiple sequence alignment analysis showed that the N-terminal region of GmPLATZ1 shares the highly conserved zinc-finger motifs with other PLATZ proteins. The subcellular localization of the GmPLATZ1 protein was analysed via the green fluorescent protein (GFP)-GmPLATZ1 fusion protein in tobacco plant cell. The GFP-GmPLATZ1 protein was shown to be targeted to the nucleus. The function of the GmPLATZ1 gene was further investigated using the overexpression transgenic Arabidopsis. The germination rate in transgenic plants overexpressing GmPLATZ1 was significantly delayed in media supplemented with mannitol compared with that of wild-type (WT) plants. Moreover, cotyledon development in the GmPLATZ1-overexpression transgenic plants was remarkably retarded in the presence of ABA compared to WT. This shows that GmPLATZ1 is implicated in developmental processes such as germination under osmotic stress conditions in plants.
Pages 479-484 | Full Text PDF | Supplementary Data PDF
Stress-induced expression in Arabidopsis with a Dehydrin LEA protein from Cleistogenes songorica, a xerophytic desert grass
Jiyu Zhang, Lingfang Kong, Zhipeng Liu, Zulfi Jahufer, Zhen Duan, Yaxin Huo, Hongyan Di, Yanrong Wang*
State Key Laboratory of Grassland Agro-ecosystems, College of Pastoral Agriculture Science and Technology, Lanzhou University, Lanzhou, 730020, China
Grasslands Research Centre, AgResearch Ltd., Palmerston North 4442, New Zealand
Late embryogenesis abundant (LEA) proteins were confirmed to be involved in the acquisition of tolerance to drought, cold and high salinity in many different organisms. In this paper, we report on the isolation and characterization of the cDNA clone for a LEA protein (CsLEA) and its putative promoter sequence from Cleistogenes songorica, a xerophytic desert grass. CsLEA was predicted as a hydrophilic LEA protein. In addition, semi-quantitative RT-PCR and relative quantitative RT-PCR assays showed that CsLEA was only expressed in dehydrated roots samples, while absolute quantitative RT-PCR showed that accumulation of CsLEA transcripts was constitutively increased both in leaves and roots during the time-course of drought stress. Transgenic plants of Arabidopsis with stress inducible expression of rd29A::CsLEA were generated and used to study the role of this LEA protein in stress tolerance. Transgenic plants exhibited higher viability than that of WT plants on MS media supplemented with 300mM sorbitol or 50mM NaCl. Significant differences for CO2 assimilation rate (P<0.05) and proline concentration (P<0.01) were shown between rd29A::CsLEA transgenics and WT during dehydration and rehydration of the plants. Data presented here suggest that CsLEA expression is modulated by drought stress and therefore affects osmotic regulation during water deficit.
Pages 485-492 | Full Text PDF
The first insight into transcriptome profile of herbaceous plant Nervilia fordii based on RNA-seq
Qionglin HUANG, Lingling LIANG, Rui HE*, Xinye MA, Ruoting ZHAN, Weiwen CHEN*
Research Center of Chinese Medicinal Resource Science and Engineering, Guangzhou University of Chinese Medicine; Key Laboratory of Chinese Medicinal Resource from Lingnan, Ministry of Education, Guangzhou 510006, P.R. China
Guangdong Medical University, Zhanjiang 524023, P.R. China
Nervilia fordii is a famous and valuable herbaceous plant that has been used to cure pulmonary and respiratory diseases for hundred years. However, the wild resource of N. fordii has been virtually exhausted, and the available approaches such as artificial cultivation and tissue proliferation cannot produce enough plants to satisfy the clinic and market. Utilization of biological technique to regulate some physiological processes of the species is a considerable method to solve the scarcity of N. fordii. To date, little is known about functional genes from N. fordii, especially the genes involved in biosynthesis pathways of effective components. Herein, a transcriptomic study was performed using leaf and corm of N. fordii as material. A total of 102,258,558 high-quality reads were produced by Illumina RNA-seq platform and finally 142,220 unigenes were assembled with an average length of 518 bp. The distinct genes were searched against an Nr database and 37.9% (53,970) of them had at least one hit. We also found 38,640, 110,029, and 28,970 of the unigenes assigned in COG, GO and KEGG databases, respectively. We discovered almost every important gene participated in flavonoids and terpenoids biosynthesis pathways and illustrated their expression difference between leaf and corm of N. fordii. We presented the first transcriptome insight for N. fordii and provide a firm foundation for genetic manipulation of the endangered plant.
Pages 493-499 | Full Text PDF | Supplementary Data PDF
An efficient protein extraction method for proteomic analysis of black pepper (Piper nigrum L.) and generation of protein map using nano LC-LTQ Orbitrap mass spectrometry
Palaniyandi Umadevi*, Muthuswamy Anandaraj
Division of Crop Improvement and Biotechnology, ICAR- Indian Institute of Spices Research, Kozhikode, Kerala, India
ICAR-Indian Institute of Spices Research, Kozhikode, Kerala, India
Information on black pepper proteins can accelerate the basic understanding of crop response to various biotic, abiotic stresses as well as the crop improvement programmes. Here, we present the protocols for total leaf protein extraction from this non model, recalcitrant plant species P. nigrum after evaluating 5 different standard methods. The efficiency and suitability of the methods were evaluated using important parameters such as protein quantity and quality, solubility in IEF compatible buffer and streak free results, number of spots in 2DE gel. Modified lysis buffer and phenol method were found to be applicable methods for protein extraction from black pepper. Among two applicable methods, the phenol protocol is time-consuming, demanding the toxic phenol. Hence, we propose the modified lysis buffer method as superior, rapid method with no polyphenol contamination for total leaf protein extraction. This is a very first report which evaluated different methods and optimized the standards for the analysis of black pepper leaf proteome. Successful identification of randomly selected spots by nano LC-LTQ Orbitrap proves its compatibility to high throughput MS analysis which leads to the generation of leaf protein map, the first of its kind.
Pages 500-507 | Full Text PDF
Co-suppression of NbClpC1 and NbClpC2 in Nicotiana benthamiana lowers photosynthetic capacity via altered leaf structure
Md. Sarafat Ali, Youngjae Yu, Wook Oh, Ji Yoon Cho, Jinhee Choi, Radhika Dhakal, Youn-Il Park, Kwang-Hyun Baek*
School of Biotechnology, Yeungnam University, Gyeongsan 712-749, Republic of Korea
Department of Biological Sciences, Chungnam National University, Daejeon 305-764, Republic of Korea
Department of Horticulture & Life Science, Yeungnam University, Gyeongsan 712-749, Republic of Korea
The Clp protease composed of more than 15 proteins, is the central protein degradation machinery in the plastids of a plant cell which plays a similar role as proteasomes in cytoplasm and nucleus. ClpC1 and ClpC2 are two chaperonic proteins for the Clp protease and share more than 90% similarities in the nucleotide and amino acid sequences. In this study, we investigated the functions of ClpC1 and ClpC2 in photosynthesis by co-suppression of both genes (NbClpC1 and NbClpC2, NbClpC1/C2) in Nicotiana benthamiana using virus-induced gene silencing (VIGS) technique. Co-suppression of NbClpC1/C2 in N. benthamiana resulted in aberrant structure with severe chlorotic leaves, stunted growth and reduced numbers of stomata and in lowered photosynthetic capacity. Leaf CO2 assimilation rate, the maximum quantum yield of photosystem II (Fv/Fm), chlorophyll and carotenoid contents were also significantly lower in the leaves of NbClpC1/C2 co-suppressed N. benthamiana than those in the control. Microscopic analysis revealed that NbClpC1/C2 co-suppressed leaves had coarsely packed mesophyll cells. Our findings strongly suggest that ClpC1 and ClpC2 play a pivotal role in photosynthetic competence by affecting the proper structure of leaves and numbers of stomata.
Pages 508-516 | Full Text PDF | Supplementary Data PDF
Yeast antagonist and microwave treatment control blue mold rots of harvested jujube fruits
Chongzhi Xu, Shan Gao*, Cuiyun Wu, Hui Jing, Hongbo Shao*
College of Plant Science, Tarim University, Alar843300, Xinjiang, China
Key Laboratory of Production and Utilization of Biological Resources in Tarim Basin, Alar 843300, Xinjiang, China
College of Life Science, Tarim University, Alar843300, Xinjiang, China
Institute of Agro-biotechnology, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China
In this study, the functions of antagonistic yeast alone or in combination with microwave treatment in controlling blue mold rot of jujube fruit were investigated. Meanwhile, their effects on the quality of the harvested fruits were also detected. Therefore, in vitro and vivo experiments were both adopted for the study. For in vitro test, the growth of Penicillium citrinum was almost completely inhibited (98%) by 2 and 3 min 2450 MHz microwave treatments, respectively. In addition, the population density of the pathogenic bacteria in the surface wounds of fruit was also significantly lower than that of the control in vivo test. The results showed that the microwave treatment reduced disease incidence and lesion diameter of infected wounds from 100% to 36.0% and 1.92 cm to 1.38 cm, respectively. Similarly, the Metschnikowia pulcherrima were capable of reducing incidence from 100% to 45.0% and lesion diameter from 1.92 cm to 1.50 cm, respectively. The disease incidence and lesion diameter were only 21.0% and 1.00 cm, respectively, when treated with the combination of microwave and antagonist yeast. Meanwhile, the incidence of the natural decay on treated fruits was similar to that of the inoculated fruits. Therefore, the results also demonstrated that all the treatments did not influence the quality parameters of Jujube fruit. In short, our research suggested that yeast antagonist in combination with microwave treatment could be an alternative to synthetic fungicides for controlling the postharvest blue mold rot of jujube fruits.
Pages 517-522 | Full Text PDF
Metabolite profiling of four different tissue locations in grape leaf of brown spot disease caused by Pseudocercospora vitis
Su-Jin Kim, Se-Hyun Jeong, Youn-Young Hur, Sung-Min Jung*
Fruit Research Division National Institute of Horticultural & Herbal Science, RDA, Suwon 440-310, Korea
Infected Campbell Early grape leaf tissues were collected according to four different tissue locations of brown spot disease: center of the lesion (A), lesion border (C) the uninfected leaf tissue (D) and tissue from A and C as well as the lesion halo (B). A total of 118 metabolites were identified using polar phase GC-MS analysis with sample derivatization and annotated four different sampling tissues. Metabolites were clustered into three groups according to tissues (tissue A, tissue B, and tissues C and D) in the PCA score plot. Tissues C and D were separated in the PCA plot; therefore, they had similar metabolite contents variations. 20 of the metabolites were significantly related to tissues A and C in the S-plot of OPLS-DA. Most of the metabolites, including caffeic acid, succinic acid, and citric acid, were increased in related contents in uninfected tissues (C and D) compared with lesions (A and B). In contrast, D-mannitol, xylitol, and raffinose were increased in the lesion tissues (A and B). Neohesperidin, lactulose, and dehydroascorbic acid were found in the tissue B. As a result, those three metabolites can be expected to relate to the defense mechanism of grapes against brown leaf spot disease.
Pages 523-528 | Full Text PDF
Optimization of Agrobacterium-mediated transformation conditions for tomato (Solanum lycopersicum L.)
Jie Ma, Tingting Liu, Dongliang Qiu*
College of Horticulture, Fujian Agriculture and Forestry University, Fuzhou, 350002, China
An orthogonal experimental design L16 (45) was used to optimize Agrobacterium-mediated transformation of cotyledon and stem explants of Solanum lycopersicum L. cv Zheza No.905. Five factors influencing transformation efficiency including stage of explants, preculture, Agrobacterium density, infection time and co-cultivation duration were evaluated using the carotenoid biosynthetic gene CsZCD (Crocus zeaxanthin 7,8-cleavage dioxygenase), with an aim to establish a high-throughput transformation protocol for tomato. The result showed that the optimal transformation protocol for cotyledon explants consisted of 8-9 day age of seedling, 2 days of preculture, an Agrobacterium suspension of OD600 = 0.6, 3 days of con-cultivation and an infection time of 20 min. For stem explants, the protocol included 4-5 day age of seedling, 3 days of preculture, an Agrobacterium suspension of OD600 = 0.6, 4-day con-cultivation and an infection time of 15 min. Under these conditions, the transformation efficiency of the cotyledon and stem reached 26.33% and 28.00%, respectively. This protocol is also suitable to another local cultivar Shengyawith transformation efficiency of 19.33% and 23.33%, respectively.
Pages 529-536 | Full Text PDF
GC-MS based metabolomics and multivariate statistical analysis of Wedelia trilobata extracts for the identification of potential phytochemical properties
Kamalrul Azlan Azizan*, Nurul Haizun Abdul Ghani, Mohammad Firdaus Nawawi
Metabolomics Research Laboratory, Institute of Systems Biology (INBIOSIS), Universiti Kebangsaan Malaysia, 43600, Bangi Selangor, Malaysia
National Science Centre (PSN), Persiaran Bukit Kiara, 50662 Bukit Kiara, Kuala Lumpur, Malaysia
Wedelia trilobata is known as a noxious weed with potential pharmaceutical properties that can be used to treat inflammation and bacterial infections. Despite its impacts and potentials, the characterization of W. trilobatas metabolite profiles using metabolomics approach has never been described. In this study, we used a non-targeted gas chromatography mass spectrometer (GC-MS) and multivariate statistical analysis (MVA) to determine the metabolic content of W. trilobata. Metabolite extraction was carried out using solvents of methanol/water, methanol/chloroform/water, ethanol and water. Unsupervised principle component analysis (PCA) and partial least square discriminant analysis (PLSDA) were applied to evaluate grouping trends between the different solvents extracts. Upon evaluation of four different extraction solvents systems, ethanol was found to have good extraction efficiency based on metabolites contribution and separation trend observed in PCA and PLSDA. Variable importance in projection (VIP) scores revealed that separation between solvents extract were largely contributed by monosaccharides and diterpenes of resin acids of 13-cis-retinoid acid and isopimaric acid. High abundance of resin acids in W. trilobata suggested potential allelopathy properties that can have beneficial herbicides. This study presents a simple non-targeted metabolomics approach to determine the metabolite differences in W. trilobata. The findings can be used to further optimise metabolite extraction from W. trilobata.
Pages 537-543 | Full Text PDF | Supplementary Data PDF
Genome-wide in silico screening of genetic variability of microRNA genes in seven plant species
Fekonja Simon, Zorc Minja, Kunej Tanja*
Department of Animal Science, Biotechnical Faculty, University of Ljubljana, Groblje 3, 1230 Domzale, Slovenia
MicroRNAs (miRNAs) are non-coding RNAs, about 21 nucleotides in length, with a significant role in post-transcriptional regulation of gene expression. MicroRNA genes play an important role in regulatory functions, affect disease susceptibility and are associated with almost all biological and metabolic processes. It has been shown that miRNA polymorphisms are linked with important plant traits for agricultural production. Genetic variability of plant miRNA genes has been systematically studied only in rice. In this study we used the upgraded online tool miRNA SNiPer version 5.0 (http://integromics-time.com/miRNA-SNiPer/) which now enables analysis in 22 genomes, including seven plant species. Genome-wide in silico screening of the seven plant genomes revealed that miRNA genes are most polymorphic in tomato (100%; 70/70) and mouse-ear cress, followed by sorghum, maize, rice, grape and false purple brome (2.6%; 8/310). In average 13.8% of miRNA genes have variations within seed region in seven plant species, the highest number in mouse-ear cress, having 43.4% (141/325). Several miRNA genes had long regions of consecutive single nucleotide polymorphisms (SNPs), the longest being ath-MIR5998a with 11 consecutive SNPs. Mouse-ear cress miRNA genes also contained a large number of insertions and deletions (indels), multiple nucleotide polymorphisms (MNPs) and substitutions. Our study presents a baseline for further functional research and associational studies to explore the importance of genetic variability of microRNA genes and lead to a new miRNA-based biotechnology, for improving plant yield, quality and tolerance to environmental biotic and abiotic stresses.
Pages 544-548 | Full Text PDF | Supplementary Data PDF
QTL analysis of rice yield components at two different environmental circumstances
Ya-chun Yang, Peng-cheng Wei, Hao Li, Juan Li, Rui-ying Qin, Da-hu Ni, Feng-shun Song, Li Li*, Jian-bo Yang*
Rice Research Institute, Anhui Academy of Agricultural Sciences, Hefei 230031, PR China
College of Agriculture, Anhui Agricultural University, Hefei 230031, PR China
The 190 recombinant inbred lines were created by the single-seed descent method using 93-11 and Nippinbare rice varieties as parents. A genetic linkage map was made employing 202 SSR, CAPs, and STS markers. Applying composite interval mapping, we carried out quantitative trait loci (QTL) analysis at two different ecological sites (Sanya and Hefei) on 6 rice yield traits. The traits included panicle number per seed (PNP), total grains per plant (TGP), filled grain per plant (FGP), blighted grains per plant (BGP), seed rate per plant (SRP), as well as 1000-grains weigh per plant (TGW). A total of 33 QTLs were identified. The single trait QTLs ranged from 2 to 9, indicating the yield-related traits are controlled by multiple genes. 4 QTLs were detected in both sites, in which two of them (qBGP-3 and qTGP-10a) detected twice with the contribution rate of 35.85% and 27.33%, respectively. 11 QTLs were pleiotropic, controlling up to 4 yield traits. As example, RM5493-RM458 on chromosome 8 control BGP, TGP, SRP and TGW.
Pages 549-555 | Full Text PDF
Metabolic engineering of SK2-type of dehydrin1 (DHN1) gene isolated from Sorghum bicolor enhances tolerance to water-deficit and NaCl stresses in transgenic tobacco
Tekula Vijaya Lakshmi, Yellisetty Varalaxmi, Sushil Kumar Yadav, Manchikatla Venkat Rajam, Mandapaka Maheswari*
Division of Crop Sciences, ICAR-Central Research Institute for Dryland Agriculture, Hyderabad - 500 059, India
Department of Genetics, University of Delhi South Campus, Benito Juarez Road, Dhaula Kuan, New Delhi - 110 021, India
A stress-inducible dehydrin (DHN1) gene was isolated from S. bicolor and its role in water-deficit and NaCl stresses was studied in transgenic tobacco. The cDNA sequence of SbDHN1 gene was 638bp in length and the ORF encoded a predicted polypeptide of 153 amino acid residues. Multiple alignment of amino acid sequence of SbDHN1 with DHN from other plant species revealed the presence of two K-segments and an S-segment, revealing it to be an SK2 type of dehydrin. Induction of SbDHN1 under water-deficit, salinity, high and low temperature stresses indicated its role in various abiotic stresses. Assays carried out using transformed E.coli cells expressing SbDHN1 showed enhanced tolerance to multiple abiotic stresses. Transgenic tobacco plants expressing SbDHN1 gene were generated. GUS histochemical assay and PCR analysis indicated the transgenic nature of the putative T0 transgenics. Molecular characterization of the T1 and T2 transgenics using PCR, RT-PCR and Southern analysis confirmed the stable integration, expression and carrying forward of the transgene. When subjected to water-deficit (-1.0 MPa) and salt (300 mM NaCl) stresses, the transgenics exhibited higher germination rate as well as shoot and root lengths. Also the leaf discs of the transgenics retained more leaf water content (10.12%) and chlorophyll content (22.5%) in comparison to their untransformed controls. Water-deficit stress tolerance of the T2 transgenics was revealed by maintenance of higher relative water content, membrane stability and superoxide dismutase activity. Thus, these results indicate the role of SbDHN1 gene in imparting tolerance to water-deficit and NaCl stresses.
Pages 556-564 | Full Text PDF
Recombination analysis and in silico structural characterization of ίC1 protein gene from Okra leaf curl betasatellite
Sayed Sartaj Sohrab, Muhammad Yasir, Sherif Ali El-Kafrawy, Zeenat Mirza, Hassan S.M. Al-Zahrani, Parthasarthi Bhattacharya, Debashis Rana
King Fahd Medical Research Center, King Abdulaziz University, Post Box No 80216, Jeddah, 21589, Saudi Arabia
Biology Department, Faculty of Sciences, King Abdulaziz University, Post Box No 800054, Jeddah, 21589, Saudi Arabia
Division of Biotechnology, JK Agri-Genetics Ltd., Hyderabad, A.P. India
Okra leaf curl and yellow vein mosaic disease caused by begomovirus and satellite DNA complexes is a major constraint of okra (Abelmoschus esculentus L.) production in India. We performed recombination analysis, in silico structural characterization of ίC1 protein gene from okra leaf curl betasatellites isolated from Hyderabad and discussed the recombination events, hot spots and break points. The results of recombination and in silico structural characterization indicates that the virus associated with Okra leaf curl disease in Southern India could be a variant of Bhendi yellow vein mosaic virus associated betasatellite. Six major and minor inter-specific recombination events, hot spots and breakpoints were identified in Okra leaf curl betasatellites, Hyderabad isolate. The closest major and minor recombination was observed with Bhendi yellow vein betasatellite-Muthupatti and Malvastrum yellow vein betasatellite-China. Recombination was also observed with Cotton leaf curl betasatellite-Rajasthan and Cotton leaf curl Gezira betasatellite-Sudan. The predicted secondary structure indicates a mixed class with helices (29.7%), extended sheets (33.9%) and loops (36.4%).The predicted tertiary model of Okra leaf curl betasatellite has three a helix: one short helix, H1 (37-41) and two long helices, H2 (62-74) & H3 (84-97). This is the first structural characterization for the ίC1 protein gene associated with Okra leaf curl disease.
Pages 565-571 | Full Text PDF
The host pathogen interactions observed between Fusarium sp. F2 and rice
Kalaivani Nadarajah*, Hamdia Z. Ali, Nurulhidayah Mat Muni
School of Environmental and Natural Resources Sciences, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43000 UKM Bangi, Selangor, Malaysia
School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43000 UKM Bangi, Selangor, Malaysia
An ITS verification was conducted on a fungal isolate (Fusarium sp F2) obtained from diseased culms of MR219. The ITS sequence analysis determined the isolate be a member of the Fusarium incarnatum-equisetti complex (FIEC). This isolate was then used to study cellular interactions in three rice varieties (Pongsu Seribu, Maswangi and MR219) where the microscopic observation of DAB stained infected tissues displayed inter and intracellular proliferation in all varieties with low to high levels of ROIs detected in and around infected tissues. Further to the cellular observation, we studied the variation in expression levels of a defense related gene, PR-1b where the level of ROIs expressed within the infected tissue corresponds directly to the levels of gene expressed. Pongsu Seribu was the most resistant rice variety to Fusarium sp F2 infections based on disease severity, ROI levels, gene expression profiles and fold increase.
Pages 572- 580 | Full Text PDF
HCRA: a hybrid colour-space read-additive method for de novo transcriptomic assembly integrating Illumina and SOLiD datasets
Adriano Viegas, Artur Silva, Sylvain Darnet*
Federal University of Para, Institute of Biological Sciences, Laboratory of Plant Biotechnology, Belem, Para, Brazil
Federal University of Para, Institute of Biological Sciences, Laboratory of DNA Polymorphism, Belem, Para, Brazil
Hybrid approaches have been developed to mix large datasets from different next-generation sequencing (NGS) platforms, such as Illumina and SOLiD, for optimizing de novo transcriptomic assembly. The classical approach (CA) is to form supercontigs from contigs obtained by the de novo assembly of each dataset. We have developed a new hybrid colour-space read-additive (HCRA) approach to assemble both datasets encoded in colour-space reads, using a multiple k-mer Velvet/Oases method. All reads are combined in colour-space and subjected together to the assembler. To evaluate both CA and HCRA methods, we used assembly statistics such as total base pairs, N50, reads mapped back to transcripts, and percentage of unique transcripts partially and completely identified. These approaches were tested using SOLiD and Illumina simulated runs, generated from 41392 sequences of Arabidopsis cDNA, totalling 64.8 Mb and with an N50 of 1913 bp. The CA and HCRA methods generated contig datasets with 225811 and 172835 transcripts with N50s of 931 and 1617 bp, respectively. Compared with the initial Arabidopsis dataset, 35960 contigs were reconstructed with CA, totalling 35.4 Mb, and 35240 contigs were reconstructed with HCRA, totalling 52.3 Mb. The HCRA method generated approximately 2-fold longer contigs than CA, and 40% more transcripts were completely identified. The proposed pipeline was applied to a real dataset of the Piper nigrum transcriptome, generating 60645 unigenes with an N50 of 1653 bp and representing about 71 Mb of its transcriptome. This method improves the integration of SOLiD datasets with those from other NGS platforms and should open new perspectives to add colour-space datasets to Illumina runs to improve de novo transcriptomic assembly in non-model organisms.
Pages 581-586 | Full Text PDF
Growth stage-based metabolite profiling of drought-tolerant transgenic rice under well-watered and deficit conditions
Kyong-Hee Nam, Hee Jae Shin, In-Soon Pack, Jung-Ho Park, Ho Bang Kim, Chang-Gi Kim*
Bio-Evaluation Center, KRIBB, Cheongju 363-883, Republic of Korea
Life Sciences Research Institute, Biomedic Co., Ltd., Bucheon 420-020, Republic of Korea
Metabolite profiling of transgenic crops is useful when evaluating the intended and unintended effects of genetic modifications. Our study objective was to investigate variations in metabolites from drought-tolerant transgenic rice (Oryza sativa L.) that over-expresses AtCYP78A7, a gene encoding cytochrome P450 protein. Two transgenic Lines, 10B-5 and 18A-4, plus the wild-type Hwayoung, were cultivated under either well-watered or water-deficit conditions in a rainout shelter. Their shoots were collected at the tillering, heading, and ripening stages and polar extracts were subsequently analyzed by 1H-NMR and GC-MS. Principal component analysis revealed that the metabolite profiles could be clearly distinguished during those stages. Soil-water conditions also contributed to the variation in profiles. However, a marked discrimination of metabolites between transgenic and non-transgenic rice was apparent only under water-deficit conditions at the heading stage. This was mainly a result of differences in the sugar-related NMR profiles among genotypes. Our data suggested that the genetic contribution to metabolite profiles is constrained by growth stage and water status. In addition, sugar content is of great importance when separating metabolite profiles in shoots from rice plants that over-express AtCYP78A7.
Pages 587-594 | Full Text PDF | Supplementary Data PDF
Phosphoproteomic profiling of microsomal fractions in leaves of Cogon grass
Ting-Ying Wu, Ping Kao, Chia-Lun Chang, Pang-Hung Hsu, Chang-Hung Chou, Ing-Feng Chang*
Department of Life Science, National Taiwan University, Taipei, Taiwan
Institute of Plant Biology, National Taiwan University, Taipei, Taiwan
Institute of Bioscience and Biotechnology, National Taiwan Ocean University, Keelung, Taiwan
Graduate Institute of Ecology and Evolutionary Biology, China Medical University, Taichung, Taiwan
Genome and Systems Biology Degree Program, National Taiwan University and Academia Sinica, Taipei Taiwan
Cogon grass [Imperata cylindrica (L.) Beauv. var. major] is one of the top-ten weeds. It is a widespread distributed C4 plant. In particular, one ecotype (Chuwei) is salt and flooding tolerant. Imperata leaves harvested from Chuwei and Sarlun control site were subjected to microsomal fractionation followed by membrane shaving using trypsin. The resulting peptides were further enriched by columns and mass spectrometry analyses for phosphopeptide identification. The phosphoproteomic analysis identified 20 phosphorylation sites from 16 unique phosphorylated peptides in the microsomal fractions isolated from leaves of Imperata, of which 5 are specific to Chuwei ecotype. Out of these 5 phosphopeptides, 2 belong to sugar transporters, including a sucrose transporter SUT1 which appears to be a novel and C4-specific phosphorylation site. Whereas the other 3 belong to photosynthesis proteins of which 2 are C4 enzymes. Interestingly, two independent phosphopeptides of tonoplast monosaccharide transporter 2 (TMT2) were identified in Chuwei ecotype and the other salt-intolerant Sarlun population individually. Our phosphoproteomic study identified differentially phosphorylated peptides of membrane proteins which may play an important role in the salt stress response of Imperata.
Pages 595-603 | Full Text PDF