May 2013 issue | Plant Omics Journal

Involvement of nitric oxide signal in Alternaria alternata toxin induced defense response in Rauvolfia serpentina Benth. ex Kurz calli

Namrata Shanu Gupta¹, Maitreyi Banerjee², Saikat Kumar Basu³ and Krishnendu Acharya¹*
¹Molecular and Applied Mycology and Plant Pathology Laboratory, Department of Botany, University of Calcutta, Kolkata, WB, 700 019, India
²West Bengal State Council of Science and Technology, Bikash Bhavan, Salt Lake, Kolkata, WB, India 700 091
³Department of Biological Sciences, University of Lethbridge, Lethbridge, AB, T1K 3M4; Canada

Abstract
This study elucidates Alternaria alternata toxin induced defense enzyme activity and phenolics accumulation and its correlation with nitric oxide (NO) generation. Role of NO in plant defense signaling to biotic stress has been strongly established by earlier investigators. Here, in vitro treatment of Rauvolfia serpentina Benth. ex Kurz callus with A. alternata toxin induced a rapid production of NO at 50 mg L^-1 toxin concentration. This increase in toxin induced NO production was further confirmed by real time visualization of NO burst using a fluorescent probe, 4, 5-diaminofluorescein diacetate (DAF-2DA) in cells of R. serpentina. Parallely, the same concentration of toxin also induced the production of peroxidase (PO), polyphenol oxidase (PPO), phenylalanine ammonia-lyase (PAL), ß-1, 3 glucanase and phenolics accumulation in the callus tissue of R. serpentina. Further, inhibition of NO production by co-treatment with NO synthase inhibitor, N^G-nitro-L-arginine-methyl ester (L-NAME) at 10 µM and NO scavenger, 2-(4- carboxyphenyl)- 4,4,5,5- tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) at 100 µM reduced the accumulation of all the defense enzymes and total phenols in the callus signifying the regulatory role of NO in the induction of defense enzyme and phenol accumulation. The effect of NO in host defense response was crosschecked further by sole application of NO donor sodium nitroprusside (SNP). Treatment of R. serpentina callus with 100 µM SNP showed the similar pattern of defense responses. So, the overall results demonstrated that increased production of NO in R. serpentina callus treated with fungal toxin, might act as an essential signaling molecule for triggering the activation of PO, PPO, PAL, ß-1, 3 glucanase and phenolics accumulation.

Pages 157-164 | Full Text PDF

Establishment of molecular markers for germplasm management in a worldwide provenance Ribes spp. collection

Luisa Palmieri*, Maria Stella Grando, Maddalena Sordo, Marcella Grisenti, Stefan Martens, Lara Giongo

Fondazione Edmund Mach, Research and Innovation Centre, 38010 San Michele all’Adige (TN), Italy

Abstract
The variability of a Ribes spp. germplasm collection was characterized using molecular markers as a complementary strategy to traditional phenotyping approach to redefine the plant genetic resources within the Fondazione Edmund Mach (FEM) Ribes collection. With 91 accessions, the FEM Ribes spp. collection contains a major part of the European and some extra-European germplasm. Molecular markers were used for its characterization and to study possible genetic relationships between species and cultivars. A total of 138 blackcurrant (Ribes nigrum L.), six jostaberry (R. nigrum L. × nidigrolaria Bauer and Ribes nidigrolaria Bauer × nidigrolaria Bauer) and 147 red, white and pink currant (Ribes rubrum L., R. petraeum Wulf., R. longeracemosum Franch., R. multiflorum Kit.) clones belonging to the 91 accessions, were genotyped using ten simple sequence repeat (SSR) markers. Seventy-five of these accessions were evaluated using 17 phenological descriptors. The SSR characterization identified 87 unique multilocus genotypes (MLGs), allowing 96% of samples analyzed to be uniquely discriminated. Phenotypic and genetic diversity were related to the taxonomy and geographical origin of provenience. We demonstrated that a limited set of markers has proved highly informative for studying the diversity and relationships within our collection and to identify synonyms, homonyms and mislabeling. The knowledge derived from genotyping associated with phenological and agronomic traits will be the background for the development of marker-assisted breeding strategies for Ribes, to improve fruit quality and to expedite the process through which new cultivars are developed.

Pages 165-174 | Full Text PDF

Genetic divergence of Malaysian upland rices revealed by microsatellite markers

Mehdi Sohrabi¹, Mohd Y. Rafii^1,2*, Mohamed M. Hanafi¹ and Md. Abdul Latif^{2, 3}
¹Institute of Tropical Agriculture, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia
²Department of Crop Science, Faculty of Agriculture, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia
³Bangladesh Rice Research Institute (BRRI), Gazipur-1701, Bangladesh

Abstract
Molecular markers are useful tools for evaluating genetic diversity and determining cultivar identity. A total of 50 upland rice accessions, 24 from Peninsular Malaysia and 26 from West Malaysia (Sabah state) were investigated using 10 microsatellite (SSR) markers distributed across the rice genome to study the genetic diversity. A total of 49 alleles were detected across the 50 accessions. The number of alleles per locus ranged from 2 to 11 with an average of 4.9. The average Polymorphism Information Content (PIC) value was 0.710. A dendrogram was constructed using Jaccard’s similarity coefficient, and accessions were clustered into 7 groups. The most of the accessions were clustered according to their geographical origin. Shannon’s information index ranged from 0.5269 to 2.0050. Nei's gene diversity (h) ranged from 0.3432 to 0.8273. Overall gene flow was 0.0011. In order to develop suitable upland rice varieties, accessions 03838, 03835, 07537, 07538, 03826, 07574, 07588, 07585, 07540, 07575, 07541, 07543, 07544, 07576, 07571, 07539, 03825, 03830 could be used as parents for future breeding program in Malaysian environment.

Pages 175-182 | Full Text PDF | Supplementary data PDF

Nitric oxide (NO) regulates the expression of single-domain cystatins in Glycine max (soybean)

Marshall Keyster², Ruqaiyah Adams¹, Ashwil Klein¹, Ndiko Ludidi¹*

¹Plant Biotechnology Research Group, Department of Biotechnology, University of the Western Cape, Private Bag X17, BELLVILLE, 7535, South Africa
²Environmental Biotechnology Laboratory, Department of Biotechnology, University of the Western Cape, Private Bag X17, BELLVILLE, 7535, South Africa

Abstract
Plant cystatins inhibit cysteine proteases and are important in regulating plant development and plant responses to biotic and abiotic stress. Furthermore, nitric oxide plays a signaling role in regulating plant responses to developmental processes, biotic and abiotic stress. With the aim of determining if nitric oxide is involved in the regulation of the expression of single-domain cystatins, we have identified single-domain cystatin genes in soybean (Glycine max cv. PAN626) on the basis of sequence homology to a nitric oxide-inducible cystatin (AtCYS1, At5g12140) from Arabidopsis thaliana. Analysis of the expression of the four cystatin genes revealed that transcript levels of these cystatins are altered by exogenously applied nitric oxide and a nitric oxide synthase inhibitor. Altered expression of these cystatins by nitric oxide and the nitric oxide synthase inhibitor implies that changes in cellular nitric oxide content, which have previously been shown to occur during development and/or biotic and abiotic stress, influence soybean physiological processes that are regulated by cysteine proteases. Recombinant protein expression of one of the cystatins (as a glutathione-S-transferase fusion protein) showed that it has inhibitory activity against the model cysteine protease papain but not the model serine protease trypsin and that it inhibits caspase-like activity in soybean nodule extracts. This serves as evidence that these four plant cystatins are functional cysteine protease inhibitors because of their high degree of primary sequence identity. It also indicates that the single-domain cystatins regulate caspase-like activity, which is known to participate in plant responses to biotic and abiotic stress. We thus conclude that nitric oxide and nitric oxide synthase-like activity regulate the expression of these cystatins, thus influencing soybean caspase-like activity. We also propose a role for this nitric oxide-mediated regulation of cystatin gene expression in the mediation of developmental processes and responses to abiotic stress in soybean.

Pages 183-192 | Full Text PDF | Supplementary data PDF

Development of microsatellite markers in Cocos nucifera and their application in evaluating the level of genetic diversity of Cocos nucifera

Yong Xiao^1#,Yi Luo^1#, Yaodong Yang^1*, Haikuo Fan¹³,Wei Xia¹, Annaliese S. Mason⁵, Songlin Zhao¹, Ross Sager⁴, Fei Qiao²
¹Hainan Key Laboratory of Tropical Oil Crops Biology/Coconuts Research Institute, Chinese Academy of Tropical Agricultural Sciences, Wenchang, Hainan 571339, P.R. China
²Key Laboratory of Crop Gene Resources and Germplasm Enhancement in Southern China, Ministry of Agriculture/Tropical Crops Genetic Resources Institute, Chinese Academy of Tropical Agricultural Sciences, Danzhou, Hainan, 571101, China
³College of Landscape Architecture, Sichuan Agriculture University, Yaan, Sichuan, 625000, P.R.China
⁴Delaware Biotechnology Institute, University of Delaware, USA, Newark, Delaware, 19711, USA
⁵School of Agriculture and Food Sciences and Centre for Integrative Legume Research, the University of Queensland, Brisbane, Australia

^#The authors have equal contribution to the manuscript

Abstract
Cocos nucifera (coconut) is an economically important tropical crop, but opportunities for molecular breeding are limited by lack of DNA sequence information for this species. Previous assessments of coconut germplasm have been conducted based solely on phenotypic data for agronomic and quality traits, due to lack of available molecular markers. In this study, we developed 30 novel microsatellite markers from Illumina transcriptome sequence data, and used these markers to evaluate coconut genetic diversity in 30 individuals representing accessions from China (12 samples) and Southeast Asia (18 samples). The microsatellite markers displayed low to high genetic polymorphism across the population: observed heterozygosity varied from 0.06 to 0.79, with an average of 0.39 ± 0.15. Our results indicated that the Southeast Asian population had a significantly higher number of alleles (p = 0.02), but not significantly different (p<0.05) observed heterozygosity (p = 0.08) or Shannon’s diversity index score (p = 0.12) compared to the population from China. Population structure analysis showed that Chinese coconuts were genetically similar to a subset of coconut germplasm from Southeast Asia, suggesting that Chinese coconuts did not evolve independently of the Southeast Asian populations. Combining population structural analyses and historic information, we have devised a possible explanation for coconut dispersal patterns from Southeast Asia to China: sea currents could have carried coconuts into the Hainan province, while human dispersal from Southeast Asia may have brought coconuts to the Yunnan province.

Pages 193-200 | Full Text PDF

Genome-wide bioinformatics analysis of DELLA-family proteins from plants

Jinhui Chen^1,#, Tielong Cheng^2,#, Pengkai Wang¹, Lin Tian², Guangping Wang¹, Yuming Luo^3,*, Junjie Wang³, Liming Yang³ and Jisen Shi^1*
¹Key Laboratory of Forest Genetics and Biotechnology, Ministry of Education, Nanjing Forestry University, Nanjing 210037, China
²Division of Research Management, Chinese Academy of Forestry, Beijing 100091, China
³School of Life Sciences, Huaiyin Normal University, Huaian 223300, Jiangsu, China

^#These authors contributed equally to this work.

Abstract
DELLA-family proteins have been implicated as negative modulators of the gibberellin signaling pathway, which regulates many aspects of plant growth and development. Despite the importance of DELLA proteins, a genome-wide overview of the DELLA gene family is not yet available. Here, based on conserved domain searching, we identified 60 different DELLA-encoding genes from 29 plant genomes, including 49 complete length sequences. Phylogenetic analysis indicated that these DELLAs can be grouped into four different subfamilies, including Algae, Bryophyte, monocots and dicots. Analysis of domains and motifs in the DELLA gene family showed the following domains including DELLA, TVHYNP, VHIID, RKVATYFGEALARR, AVNSVFELH, RVER, and SAW are strictly conserved in the DELLAs. Gene duplication events were the main reason for expansion of the DELLA family; selective pressure operated on the DELLAs after gene duplication, resulting in the formation of distinct DELLA groups. Our results provide new insights into the evolutionary relationships of DELLA proteins.

Pages 201-207 | Full Text PDF | Supplementary data xls

Synergistic effect of C2H2 type Zinc-finger protein with LEA promoter to enhance abiotic stress tolerance in Brassica juncea

Shiv S. Verma^1*, Viswanathan Chinnusamy², Muhammad Asif^1,3, K. C. Bansal^2,4

¹Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta, T6G 2P5, Canada
²Divisions of Plant Physiology, Indian Agricultural Research Institute, New Delhi, 110012, India
³Ex-Research Scientist, Pakistan Agricultural Research Council, Islamabad, Pakistan
⁴National Bureau of Plant Genetic Resources, Indian Agricultural Research Institute Campus, New Delhi, 110012, India

Abstract
The Cys2/His2 (C2H2) type zinc finger (ZF), and Late-embryogenesis abundant (LEA) proteins are associated with various cellular processes that play an important role in plant development and abiotic stress tolerance. The study was designed to evaluate the role of P_LEA1:BcZF1 to enhance abiotic stress tolerance in Brassica juncea. The Group 4 LEA, LEA4-1, and ZF proteins isolated from B. napus and B. carinata respectively; were expressed in B. juncea cv. varuna. Expression of ZF protein in B. juncea under the control of LEA promoter showed increased tolerance against multiple abiotic stresses: salt, oxidative and drought. The increased level in the stability of total cellular membrane was observed in the transgenic lines (ZL1, ZL2 and ZL4) of B. juncea. The phenotypic analysis of transgenic lines also showed increased level of root and shoot length as compared to wild type (WT) plants under abiotic stresses. Our study suggest that cDNA encoding BcZF1 and the promoter LEA1 function as regulatory molecules involved in stabilizing and modulating the optimal plant growth under various abiotic stresses.

Pages 208-214 | Full Text PDF

Comprehensive and evolutionary analysis of protein tyrosine phosphatases (PTP) in the green plants

Lujun Yu^#, Dongru Feng^#, Wenyan Li, Hongbin Wang, Jinfa Wang and Bing Liu*

Guangdong Key Laboratory of Plant Resources and State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, 510275, Guangzhou, P.R. China

^#These authors contributed equally to this work.

Abstract
Protein tyrosine phosphatases (PTP) exhibit specific functions in regulation of organism growth and development, cooperating with protein tyrosine kinase (PTK) to balance tyrosine phosphorylation. In the current study, we investigated a comprehensive analysis of PTP complements (PTPome) in five representative major plant lineages, including green algae, bryophytes, pteridophytes, monocots and eudicots, and classified them into ten subfamilies according to the catalysis manner, sequence phylogenetic and domain organization analysis. Furthermore, we proposed a PTP subfamily stepwise shift model, which was consistent with plant species complexity. An original PTP kit containing seven subfamilies was detected in Chlamydomomas reinhardtii, with one acquisition and one reduction step followed by species radiation. Lipid phosphatases were only detected in land plants, but not aquatic plants. Additionally, comparisons of green plant PTPome with human PTPome revealed significant differences in gene number, subfamilies and domain combinations. Comprehensive analysis provides insight into PTP evolution in plants and the basis for functional genomics.

Pages 215-223 | Full Text PDF | Supplementary data PDF | xls

Metabolite profiling approach for assessing the effects of colored light-emitting diode lighting on the adventitious roots of ginseng (Panax ginseng C. A. Mayer)

Soo-Yun Park¹, Jae Geun Lee², Hyun Suk Cho¹, Eun Soo Seong², Hee Young Kim², Chang Yeon Yu^2,* and Jae Kwang Kim^1,*
¹National Academy of Agricultural Science, Rural Development Administration, Suwon 441-707, Republic of Korea
²Bioherb Research Institute, Kangwon National University, Chunchon 200-701, Republic of Korea

Abstract
Metabolome analysis was carried out to evaluate the effects of light-emitting diode (LED) spectra on the metabolic processes of ginseng (Panax ginseng C. A. Mayer) adventitious roots. In total, 35 hydrophilic and 11 lipophilic metabolites were identified in ginseng roots irradiated with red (630 nm), blue (465 nm) LED light or fluorescent lamp (FL) light, by gas chromatography-time-of-flight mass spectrometry. Principal components analysis results using the 46 metabolites showed differentiation between the metabolomes of ginseng roots irradiated with red LED, blue LED, and FL light, indicating that metabolic changes were occurred in the ginseng roots by light spectral quality. The corresponding loading indicated that LED light-irradiated ginseng roots had higher sucrose and lower amino acids compared to FL-irradiated ginseng roots. The quantitative results revealed that ginseng roots irradiated with blue LED light had higher concentrations of a-tocopherol and ß-amyrin, as well as phenolic acids compared with FL-irradiated ginseng roots. This is the first study to determine the comprehensive metabolic changes in response to LED light in ginseng adventitious roots and to demonstrate the utility of the metabolite profiling approach employed in this study for detecting environmental effects on the plant metabolome.

Pages 224-230 | Full Text PDF

Optimization of Agrobacterium-mediated genetic transformation in gherkin (Cucumis anguria L.)

Muthu Thiruvengadam¹, Jerome Jeyakumar², Malaiyandi Kamaraj², Ill-Min Chung¹, Jong-Jin Kim³*

¹Department of Applied Bioscience, College of Life and Environmental Sciences, Konkuk University, Seoul - 143 701, South Korea
²Post Graduate Department of Botany, Jamal Mohamed College (Autonomous), Tiruchirappalli - 620 020, Tamil Nadu, India
³Department of Environmental Sciences, Konkuk University, Seoul, 143-701, South Korea.

Abstract
The present work was aimed to study various factors influencing Agrobacterium tumefaciens mediated genetic transformation of gherkin (Cucumis anguria L). Agrobacterium strain LBA4404 harboring binary vector pBAL2 carrying the reporter gene ß-glucuronidase intron (gus) and the marker gene neomycin phosphotransferase (nptII) was used for transformation. Factors affecting transformation efficiency, such as Agrobacterium concentration, effect of acetosyringone, pre-cultivation, infection and co-cultivation time of Agrobacterium were studied. After co-cultivation, explants were transferred into MS medium plus B5 vitamins (MSB₅) containing 1.5 µMbenzylaminopurine (BAP) with 0.5 µM naphthalene acetic acid (NAA), 100 mg L^-1 kanamycin and 300 mg L^-1carbenicillin for callus induction. Regeneration of adventitious shoots from callus was achieved on MSB₅ medium containing 3.0 µM BAP, 100 mg L^-1 kanamycin and 300 mg L^-1 carbenicillin. Transgenic shoots were elongated in MSB₅ medium fortified with 2.0 µM gibberellic acid (GA₃), 100 mg L^-1 kanamycin and 300 mg L^-1 carbenicillin. The transgenic elongated shoots were rooted in MSB₅ medium supplemented with 3.0 µM indole 3-butyric acid (IBA) and 100 mg L^-1 kanamycin. The putative transgenic plants were acclimatized in the greenhouse. A strong ß-glucuronidase activity was detected in the transformed plants by histochemical assay. Integration of T-DNA into the nuclear genome of transgenic plants was confirmed by polymerase chain reaction and southern hybridization. The nptII gene expression in transgenic plants was confirmed by RT-PCR. A transformation efficiency of 15% was obtained. This protocol allows effective transformation and direct regeneration of C. anguria.

Pages 231-239 | Full Text PDF

Comparative proteomics analysis revealed increased expression of photosynthetic proteins in transgenic tobacco by overexpression of AtCBF1 gene

Han Cheng, Haibin Cai, Zewei An, Huasun Huang*

Rubber Research Institute, Chinese Academy of Tropical Agricultural Science, Danzhou City, Hainan, 571737, China

Abstract
CRT/DRE Binding Factor 1 (CBF1) overexpression enhanced freezing tolerance in Nicotiana tabacum. To unravel the proteomics changes caused by the ectopic overexpression of AtCBF1 genes, we utilized 2-DE along with MALDI-TOF-MS and LC ESI-MS/MS to study the proteome profiles. We also tried to identify the proteins that significantly change in abundance in the AtCBF1 transgenic tobacco compared to wild type plant. Totally, 68 proteins were up-regulated in AtCBF1 transgenic tobacco, while 150 were down-regulated when the plants were cultured at room temperature. Sixteen up-regulated proteins were successfully identified and functionally annotated. These proteins mainly fell into 4 categories, including photosynthetic proteins, metabolic proteins, stress related proteins and chaperons. In all identified proteins, the photosynthetic proteins were the major category, suggesting AtCBF1 overexpression has substantial positive effects on photosynthesis system. The chlorophll a and b content were substantially increased by 20% and 40% in transgenic tobacco than in wild type plant, respectively. These data suggested that AtCBF1 ectopic overexpression protected tobacco photosynthetic system under cold stress, which contributes the cold tolerance of this non-cold acclimation plant.

Pages 240-245 | Full Text PDF | Supplementary data