Generation and Analysis of Expressed Sequence Tags from Chimonanthus praecox (Wintersweet) Flowers for Discovering Stress-Responsive and Floral Development-Related Genes
A complementary DNA library was constructed from the flowers of Chimonanthus praecox, an ornamental perennial shrub blossoming in winter in China. Eight hundred sixty-seven high-quality expressed sequence tag sequences with an average read length of 673.8？bp were acquired. A nonredundant set of 479 unigenes, including 94 contigs and 385 singletons, was identified after the expressed sequence tags were clustered and assembled. BLAST analysis against the nonredundant protein database and nonredundant nucleotide database revealed that 405 unigenes shared significant homology with known genes. The homologous unigenes were categorized according to Gene Ontology hierarchies (biological, cellular, and molecular). By BLAST analysis and Gene Ontology annotation, 95 unigenes involved in stress and defense and 19 unigenes related to floral development were identified based on existing knowledge. Twelve genes, of which 9 were annotated as “cold response,” were examined by real-time RT-PCR to understand the changes in expression patterns under cold stress and to validate the findings. Fourteen genes, including 11 genes related to floral development, were also detected by real-time RT-PCR to validate the expression patterns in the blooming process and in different tissues. This study provides a useful basis for the genomic analysis of C. praecox. 1. Introduction Chimonanthus praecox (L.) Link, wintersweet, belongs to the Calycanthaceae family. It is a perennial deciduous shrub and blossoms in winter, from late November to March. Its unique flowering time and long blooming period make it one of most popular ornamental plants in China [1]. C. praecox is mainly a garden plant that also provides cut flowers. The flower is strongly fragrant and may be used as a source of essential oil, which has received much attention in New Zealand [2]. C. praecox thrives in cold environments and blooms in low-temperature seasons with little rainfall. The plant is assumed to be rich in genes related to floral development and adversities, especially those responding to environmental stress factors. However, the molecular mechanism that regulates floral development and copes with stresses in C. praecox flowers remains unclear. Expressed sequence tags (ESTs) have been proven to be an efficient and rapid means to identify novel genes (and proteins) induced by environmental changes or stresses [3–7]. Genes related to flower form, longevity, and scent from roses, Phalaenopsis equestris, and Pandanus fascicularis were identified by ESTs [8–10]. The present study used transcriptomic analysis of
References
[1]
K. G. Zhao, M. Q. Zhou, L. Q. Chen, D. Zhang, and G. W. Robert, “Genetic diversity and discrimination of Chimonanthus praecox (L.) link germplasm using ISSR and RAPD markers,” HortScience, vol. 42, no. 5, pp. 1144–1148, 2007.
[2]
J. Q. Feng, “New Zealand flower industry—with special reference to wintersweet introduction and commercialization,” Journal of Beijing Forestry University, vol. 29, supplement 1, pp. 4–8, 2007.
[3]
H. Wei, A. L. Dhanaraj, L. J. Rowland, Y. Fu, S. L. Krebs, and R. Arora, “Comparative analysis of expressed sequence tags from cold-acclimated and non-acclimated leaves of Rhododendron catawbiense Michx,” Planta, vol. 221, no. 3, pp. 406–416, 2005.
[4]
R. Mahalingam, A. Gomez-Buitrago, N. Eckardt et al., “Characterizing the stress/defense transcriptome of Arabidopsis,” Genome Biology, vol. 4, no. 3, article R20, 2003.
[5]
G. Iturriaga, M. A. F. Cushman, and J. C. Cushman, “An EST catalogue from the resurrection plant Selaginella lepidophylla reveals abiotic stress-adaptive genes,” Plant Science, vol. 170, no. 6, pp. 1173–1184, 2006.
[6]
J. Zhang, U. P. John, Y. Wang et al., “Targeted mining of drought stress-responsive genes from EST resources in Cleistogenes songorica,” Journal of Plant Physiology, vol. 168, no. 15, pp. 1844–1851, 2011.
[7]
N. O. Ozgenturk, F. Oru, U. Sezerman et al., “Generation and analysis of expressed sequence tags from Olea europaea L,” Comparative and Functional Genomics, vol. 2010, Article ID 757512, 9 pages, 2010.
[8]
S. Channelière, S. Rivière, G. Scalliet et al., “Analysis of gene expression in rose petals using expressed sequence tags,” FEBS Letters, vol. 515, no. 1–3, pp. 35–38, 2002.
[9]
M. S. Vinod, H. M. Sankararamasubramanian, R. Priyanka, G. Ganesan, and A. Parida, “Gene expression analysis of volatile-rich male flowers of dioecious Pandanus fascicularis using expressed sequence tags,” Journal of Plant Physiology, vol. 167, no. 11, pp. 914–919, 2010.
[10]
W. C. Tsai, Y. Y. Hsiao, S. H. Lee et al., “Expression analysis of the ESTs derived from the flower buds of Phalaenopsis equestris,” Plant Science, vol. 170, no. 3, pp. 426–432, 2006.
[11]
C. L. Wu and N. Z. Hu, “Studies on the flower form and blooming characteristics of the wintersweet,” Acta Horticulturae Sinica, vol. 22, no. 3, pp. 277–282, 1995.
[12]
Q. K. Gao and C. Hu, “Construction of a cDNA library of host recognition kairomone for telenomus theophilae,” Insect Science, vol. 9, no. 1, pp. 35–39, 2002.
[13]
Y. Ogihara, K. Mochida, Y. Nemoto et al., “Correlated clustering and virtual display of gene expression patterns in the wheat life cycle by large-scale statistical analyses of expressed sequence tags,” Plant Journal, vol. 33, no. 6, pp. 1001–1011, 2003.
[14]
F. Sterky, S. Regan, J. Karlsson et al., “Gene discovery in the wood-forming tissues of poplar: analysis of 5,692 expressed sequence tags,” Proceedings of the National Academy of Sciences of the United States of America, vol. 95, no. 22, pp. 13330–13335, 1998.
[15]
S. Carbon, A. Ireland, C. J. Mungall et al., “AmiGO: online access to ontology and annotation data,” Bioinformatics, vol. 25, no. 2, pp. 288–289, 2009.
[16]
P. Fernández, N. Paniego, S. Lew, H. E. Hopp, and R. A. Heinz, “Differential representation of sunflower ESTs in enriched organ-specific cDNA libraries in a small scale sequencing project,” BMC Genomics, vol. 4, article no. 40, 2003.
[17]
T. Lotan, N. Ori, and R. Fluhr, “Pathogenesis-related proteins are developmentally regulated in tobacco flowers,” The Plant cell, vol. 1, no. 9, pp. 881–887, 1989.
[18]
A. D. Neale, J. A. Wahleithner, M. Lund et al., “Chitinase, β-1,3-glucanase, osmotin, and extensin are expressed in tobacco explants during flower formation,” Plant Cell, vol. 2, no. 7, pp. 673–684, 1990.
[19]
Q. Gu, E. E. Kawata, M. J. Morse, H. M. Wu, and A. Y. Cheung, “A flower-specific cDNA encoding a novel thionin in tobacco,” Molecular and General Genetics, vol. 234, no. 1, pp. 89–96, 1992.
[20]
A. H. Atkinson, R. L. Heath, R. J. Simpson, A. E. Clarke, and M. A. Anderson, “Proteinase inhibitors in Nicotiana alata stigmas are derived from a precursor protein which is processed into five homologous inhibitors,” Plant Cell, vol. 5, no. 2, pp. 203–213, 1993.
[21]
U. Wagner, R. Edwards, D. P. Dixon, and F. Mauch, “Probing the diversity of the Arabidopsis glutathione S-transferase gene family,” Plant Molecular Biology, vol. 49, no. 5, pp. 515–532, 2002.
[22]
A. Moons, “Osgstu3 and osgtu4, encoding tau class glutathione S-transferases, are heavy metal- and hypoxic stress-induced and differentially salt stress-responsive in rice roots,” FEBS Letters, vol. 553, no. 3, pp. 427–432, 2003.
[23]
A. D. O. Carvalho and V. M. Gomes, “Role of plant lipid transfer proteins in plant cell physiology—a concise review,” Peptides, vol. 28, no. 5, pp. 1144–1153, 2007.
[24]
C. Chen, G. Chen, X. Hao, et al., “CaMF2, an anther-specific lipid transfer protein (LTP) gene, affects pollen development in Capsicum annuum L.,” Plant Science, vol. 181, no. 4, pp. 439–448, 2011.
[25]
A. Kie？bowicz-Matuk, P. Rey, and T. Rorat, “The organ-dependent abundance of a Solanum lipid transfer protein is up-regulated upon osmotic constraints and associated with cold acclimation ability,” Journal of Experimental Botany, vol. 59, no. 8, pp. 2191–2203, 2008.
[26]
C. Ndong, J. Danyluk, K. E. Wilson, T. Pocock, N. P. A. Huner, and F. Sarhan, “Cold-regulated cereal chloroplast late embryogenesis abundant-like proteins. Molecular characterization and functional analyses,” Plant Physiology, vol. 129, no. 3, pp. 1368–1381, 2002.
[27]
K. N. Dramé, D. Clavel, A. Repellin, C. Passaquet, and Y. Zuily-Fodil, “Water deficit induces variation in expression of stress-responsive genes in two peanut (Arachis hypogaea L.) cultivars with different tolerance to drought,” Plant Physiology and Biochemistry, vol. 45, no. 3-4, pp. 236–243, 2007.
[28]
S. C. Park, Y. H. Kim, J. C. Jeong et al., “Sweetpotato late embryogenesis abundant 14 (IbLEA14) gene influences lignification and increases osmotic- and salt stress-tolerance of transgenic calli,” Planta, vol. 233, no. 3, pp. 621–634, 2011.
[29]
J. Grelet, A. Benamar, E. Teyssier, M. H. Avelange-Macherel, D. Grunwald, and D. Macherel, “Identification in pea seed mitochondria of a late-embryogenesis abundant protein able to protect enzymes from drying,” Plant Physiology, vol. 137, no. 1, pp. 157–167, 2005.
[30]
L. Gramzow and G. Theissen, “A hitchhiker's guide to the MADS world of plants,” Genome Biology, vol. 11, no. 6, article no. 214, 2010.
[31]
A. Becker and G. Thei？en, “The major clades of MADS-box genes and their role in the development and evolution of flowering plants,” Molecular Phylogenetics and Evolution, vol. 29, no. 3, pp. 464–489, 2003.
[32]
W. S. Chao, M. E. Foley, and D. P. Horvath et al., “Signals regulating dormancy in vegetative buds,” International Journal of Plant Developmental Biology, vol. 1, no. 1, pp. 49–56, 2007.
[33]
E. S. Dennis and W. J. Peacock, “Epigenetic regulation of flowering,” Current Opinion in Plant Biology, vol. 10, no. 5, pp. 520–527, 2007.
[34]
P. Chouard, “Vernalization and its relations to dormancy,” Annual Review of Plant Physiology, vol. 11, pp. 191–238, 1960.
[35]
A. Rohde and R. P. Bhalerao, “Plant dormancy in the perennial context,” Trends in Plant Science, vol. 12, no. 5, pp. 217–223, 2007.
[36]
D. Callard, M. Axelos, and L. Mazzolini, “Novel molecular markers for late phases of the growth cycle of arabidopsis thaliana cell-suspension cultures are expressed during organ senescence,” Plant Physiology, vol. 112, no. 2, pp. 705–715, 1996.
[37]
P. C. Bailey, G. W. Lycett, and J. A. Roberts, “A molecular study of dormancy breaking and germination in seeds of Trollius ledebouri,” Plant Molecular Biology, vol. 32, no. 3, pp. 559–564, 1996.
