Heterology Expression of the ArabidopsisC-Repeat/Dehydration Response Element Binding Factor 1 Gene Confers Elevated Tolerance to Chilling and Oxidative Stresses in Transgenic Tomato

Abstract

In an attempt to improve stress tolerance of tomato (Lycopersicon esculentum) plants, an expression vector containing an Arabidopsis C-repeat/dehydration responsive element binding factor 1 (CBF1) cDNA driven by a cauliflower mosaic virus 35S promoter was transferred into tomato plants. Transgenic expression of CBF1 was proved by northern-and western-blot analyses. The degree of chilling tolerance of transgenic T 1 and T 2 plants was found to be significantly greater than that of wild-type tomato plants as measured by survival rate, chlorophyll fluorescence value, and radical elongation. The transgenic tomato plants exhibited patterns of growth retardation; however, they resumed normal growth after GA 3 (gibberellic acid) treatment. More importantly, GA 3 -treated transgenic plants still exhibited a greater degree of chilling tolerance compared with wild-type plants. Subtractive hybridization was performed to isolate the responsive genes of heterologous Arabidopsis CBF1 in transgenic tomato plants. CATALASE1 (CAT1) was obtained and showed activation in transgenic tomato plants. The CAT1 gene and catalase activity were also highly induced in the transgenic tomato plants. The level of H 2 O 2 in the transgenic plants was lower than that in the wild-type plants under either normal or cold conditions. The transgenic plants also exhibited considerable tolerance against oxidative damage induced by methyl viologen. Results from the current study suggest that heterologous CBF1 expression in transgenic tomato plants may induce several oxidative-stress responsive genes to protect from chilling stress.Many tropical and subtropical crops, e.g. tomato (Lycopersicon esculentum), bell pepper (Capsicum annuum), and avocado (Persea americana), are sensitive to cold (Saltveit and Morris, 1990). Because of the susceptibility to chilling, the growing season of the crops is limited, and the quality of produce is affected. On the other hand, plants originating in the temperate zone are generally more tolerant to cold and have protective processes, such as cold acclimation (Thomashow, 1999). It has been demonstrated that the ability to cold acclimate is related to specific signal transduction pathways resulting in the activation of many cold-regulated (COR) genes (Thomashow, 1998).The COR genes, including RD29A (COR78), COR15a, KIN1, and COR6.6 of Arabidopsis, are inducible in response to cold treatment, ABA, and water-deficit stress (Thomashow, 1998). The C-repeat (CRT) and dehydration responsive element (DRE)-related motifs have been reported in the promoter sequences of these genes (Horvath et al., 1993;Nordin et al., 1993; Baker et al., 1994;Wang et al., 1995). The CRT/DRE binding factor 1 (CBF1) has been isolated using a yeast (Saccharomyces cerevisiae) onehybrid system (Stockinger et al., 1997). Overexpression of CBF1 can induce COR gene expression and result in increased tolerance to freezing temperature treatment without cold-acclimation (Jaglo-Ottosen et al., 1998). Arabidopsis CBF1 is also heterologously effective in canola ...

Tomato Plants Ectopically Expressing Arabidopsis CBF1 Show Enhanced Resistance to Water Deficit Stress

“…Recent findings indicate that there is cross talk between two stress-signaling pathways in Arabidopsis (Shinozaki and Yamaguchi-Shinozaki, 2000). Recently, we also observed that transgenic CBF1 tomato plants have enhanced chilling tolerance as compared with wild-type plants (Hsieh et al, 2002). These results in combination with the results in this study confirm that the enhancement of stress tolerance phenomenon in transgenic tomato plants might be conferred by multiple defense systems.…”

“…Previously, internode length has been reported to have a positive correlation with GA content (Ross et al, 1989). Recently, we found that the internode length of transgenic plants could be recovered by applying GA 3 exogenously (Hsieh et al, 2002). It was of interest to know whether the resistance to water deficit stress of transgenic tomato plants would be affected after GA 3 treatment.…”

“…The GUS DNA, excised from the BamHI-SacI restriction fragment of plasmid pBI221 (CLONTECH Laboratories, Palo Alto, CA), and the CBF1 gene, isolated from pT7Blue-CBF1 (Hsieh et al, 2002), were used as probes. Tomato ␤-TUBULIN cDNA fragment was isolated by reverse transcriptase-PCR from 3-month-old tomato plant leaves.…”

“…All fragments were labeled with [␣-32 P]dCTP using the random primer method (Feinberg and Vogelstein, 1983). Tomato seeds produced from transgenic tomato plants were collected and selection procedures were performed as described previously (Hsieh et al, 2002).…”

Cloning and characterization of cold, salt and drought inducible C-repeat binding factor gene from a highly cold adapted ecotype of Lepidium latifolium L.

“…Adaptation processes include an array of biochemical and physiological alterations such as changes in lipid composition from saturated to unsaturated form, increased sugar content and other osmoltytes, as well as protein contents and enzyme activities (Guy 1990;Thomashow 1999). Specific proteins are induced in compliance to low temperature that help cells to cope up with chilling and freezing stress (Thomashow 1999;Knight et al 1999;Tahtiharju and Palva 2001;Gong et al 2002;Hsieh et al 2002). The functions of cold stressinduced proteins include metabolism of carbohydrates, lipids, and phenylpropanoids, as antioxidants, molecular chaperones, mRNA binding proteins, proteinase inhibitors and antifreeze proteins, and also tolerance to cellular dehydration caused by extra-cellular freezing (Mohapatra et al 1989;Guy 1990;Thomashow 1999).…”