Isolation and expression analysis of low temperature-induced genes in white poplar (Populus alba)
Introduction
Temperature is one of the abiotic factors that limit plant growth. In plants, exposure to low temperatures induces reduction of membrane fluidity and affects enzyme kinetic parameters and protein folding. As a consequence, developmental, morphological, physiological, and biochemical processes are altered, affecting productivity, quality, and survival (Alberdi and Corcuera, 1991; Vergnolle et al., 2005). In plants living in temperate climates, low non-freezing temperature triggers physiological changes that increase the tolerance of plants to cold stress, a process known as cold acclimation (Alberdi and Corcuera, 1991; Fowler and Thomashow, 2002; Welling, 2003).
Forest crops are especially susceptible to cold stress after dormancy release; the newly flushed shoots are vulnerable, and their death causes serious loss in biomass production (Sauter et al., 1999). During last decades, an increased incidence of warm episodes has determined early onset of spring phenophases. For example, it has been calculated that an early onset equating to 6 d has occurred from the sixties to the present in western Canada (Beaubien and Freeland, 2000). The predicted global warming should further induce earlier dormancy release, and consequently, forest crops will be more exposed to frost injury in the spring.
Breeding of forest crops for cold tolerance is usually based on the development of late flushing genotypes (Tsarouhas et al., 2003), for which the possibility to encounter spring frost is reduced. Knowledge of the molecular bases of low temperature tolerance in forest trees can be useful to improve the selection process.
A number of different molecules are involved in cold tolerance in plants. For example, the accumulation of cryoprotectants such as proline, sugars, and polyamines (Strand et al., 1997), and changes in membrane lipid composition (Miquel et al., 1993), ion channel activities (Cao et al., 2005) have been observed. These metabolic processes are due to distinct changes in gene expression and in enzyme activities (Thomashow et al., 2001; Renaut et al., 2004, Renaut et al., 2005). Similar changes in plant metabolism and gene expression, and modifications of the signaling pathways, often occur in different plant species and under different stress conditions, such as dehydration, salt and water stress (Seki et al., 2001; Rabbani et al., 2003; Chinnusamy et al., 2004).
Recent full-genome transcript profiling studies in Arabidopsis have led to the identification of a core set of cold-regulated plant genes, the so-called CBF-DREB1 regulon (C-repeat binding factors, also known as dehydration-responsive-element-binding proteins, Nakashima and Yamaguchi-Shinozaki, 2006). Defense proteins such as cold-regulated proteins (COR), cold-induced (KIN), responsive to dehydration (RD), low-temperature induced (LTI) are the final products of this gene pathway. CBF-DREBs are upstream transcription factors that bind to the promoter CCGAC cis-element and activate the expression of cold-responsive genes (Thomashow, 1999). Cold induction of CBF genes is controlled by Inducer of CBF Expression1 (ICE1) and other related transcription factors (Chinnusamy et al., 2003).
With respect to functional genomics analyses of stress responses, similarities between Arabidopsis and forest trees have been ascertained. EST libraries generated from poplar leaves exposed to different abiotic stresses were enriched for genes that were shown to be stress inducible in Arabidopsis (Nanjo et al., 2004). Microarray transcriptome analysis of the poplar interspecific hybrid Populus tremula×Populus alba overexpressing Arabidopsis CBF1 gene revealed similarity between poplar and Arabidopsis CBF regulons; some cold-regulated genes were orthologs to Arabidopsis CBF3 regulon genes (Benedict et al., 2006). Benedict et al. (2006) also reported a list of genes up- and down-regulated after 7 d cold treatment in leaves and stem of the untransformed poplar hybrid, indicating different gene regulation in annual and perennial tissues.
The aim of this study was to identify up-regulated genes in white poplar (P. alba) plants subjected to low non-freezing temperature. The genus Populus is an important crop and a model system to understand molecular processes of growth, development, and responses to environmental stimuli in trees. While in Northern America and Northern Europe the most cultivated poplar species are Populus deltoides, Popolus nigra, Populus trichocarpa, as well as different interspecific hybrids, in Mediterranean regions, P. alba is often preferred because it is a fast growing species and has the capacity to adapt to stress conditions (Kuzminsky et al., 1999; Sixto et al., 2006).
Suppression subtractive hybridization (SSH) libraries were performed to isolate differentially expressed sequences from white poplar plants exposed to 6 or 48 h of low non-freezing temperature. As noted by Cao et al. (2004), such an approach is complementary to microarray analysis in identifying new and low abundant genes involved in biological processes.
Section snippets
Plant materials
Rooted cuttings of Populus alba L., clone Villafranca were cultivated in 20×20 cm2 pots in the open. In the autumn of their second growing season, plants were cut at 50 cm in height. In the subsequent spring, plants were transferred for two weeks in a growth chamber, at 20 °C, under a light intensity of 160 μmol m−2 s−1 (Sylvania Grolux Fluorescent lamps) and a 16/8 h light–dark photoperiod to induce dormancy release and leaf emergence. Then, some healthy, well-hydrated plants were transferred to a
Isolation of cold-induced genes and gene ontology
Using suppression subtractive hybridization, we constructed two cDNA libraries from P. alba plants exposed to low non-freezing temperature, constituted by genes activated by cold treatment. Six- and 48-h cold treatments were chosen to examine, in depth, the stages at which gene expression patterns definitively change to respond to cold. At these two stages, regulatory genes and defense protein coding genes, respectively, are expected to be induced. We isolated 225 differential clones from the
Conclusions
Subtracted suppressive hybridization is a powerful tool in functional genomics because the generated cDNA subtractive library represents not only a high abundance of transcripts, but also a low abundance of mRNAs differentially expressed between two samples. Consequently, SSH can be used as an alternative and complementary transcript profiling tool with GeneChip microarrays, especially in identifying novel genes and transcripts of low abundance. One hundred sixty-two different cDNA fragments
Acknowledgements
This research was supported by PRIN-MIUR, Italy, Projects “Strategie per l’adattamento ai cambiamenti ambientali: identificazione di geni coinvolti nella risposta a stress abiotici in pioppo” and “Genomica funzionale in genotipi di pioppo bianco sottoposti a stress abiotici”.
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