Heterology expression of the Arabidopsis C-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.

Figure 1

RNA and protein analyses of the transgenic plants overexpressing CBF1. A, Total RNA (10 μg) was extracted from wild type (WT; lane 1) and three lines of transgenic tomatoes overexpressing CBF1 (C5, C15, and C21). cDNA probes used were ³²P-labeled Arabidopsis CBF1, GUS, tomato CATALASE 1 (CAT1), and β-TUBULIN. B, Detection of the CBF1 protein by western-blot analysis in leaf protein extracts (20 μg per lane) of WT and the transgenic tomato plants (C5, C15, and C21).

Figure 2

Overexpression of CBF1 enhances tomato chilling tolerance. Tomato T₁ plants were exposed to various temperatures (24°C, 6°C, 4°C, 2°C, and 0°C) for 7 d and a photoperiod of 16 h. F_v/F_m values (A) and percent leakage of ions (B) were measured. Results were obtained from an average of five measurements with less than 5% sd.

Figure 3

Transgenic tomato plants overexpressing CBF1 display high level of tolerance to chilling stresses. A wild-type (WT) plant and three transgenic plants (C5, C15, and C21) were incubated at 0°C for 2 d. The photograph shows whole plants. Leaves of C5, C15, and C21 did not significantly curl and wilt, a sign of tolerance shown in the photograph. For survival rate test, WT, C5, C15, and C21 were incubated at 0°C for 7 d and returned to 24°C for 5 d. Numbers of surviving plants per total number of tested plants are indicated at the top of the image.

Figure 4

Growth retardation of CBF1 transgenic plants can be recovered by treatment with exogenous GA₃, without affecting chilling tolerance. The average height (A) of each line grown for 60 d was measured. Three-week-old wild-type and transgenic T₁ tomato plants were treated with GA₃ or buffer (control) three times within 1 week, and the height was measured after treatment for 39 d. After GA₃ treatment, whole plants were incubated at 0°C for 1, 3, 5, and 7 d, and then the F_v/F_m values (B) and percent leakage of ions (C) were measured. After incubation at 0°C for various time, the wild-type and transgenic tomato plants were transferred to room temperature for another 5 d. The results are an average of seven measurements with less than 5% sd.

Figure 5

Overexpression of CBF1 conferred chilling tolerance in the transgenic tomato T₂ seedlings. Chilling tolerance was expressed by radical elongation assay as described in “Materials and Methods.” The results are an average of 10 to 15 measurements with less than 5% sd.

Figure 6

Constitutive expression of CBF1 increases CAT1 and GST-like transcripts and CAT activity and reduces the H₂O₂ concentration in the transgenic plants. Total RNA (10 μg) was extracted from leaves of wild-type and transgenic tomato plant C15 line grown at 24°C (N) and was exposed to chilling for 3 d (C), respectively. The northern blots were hybridized with the probes for CAT1 and β-TUBULIN (A). The activities of CAT (B) and H₂O₂ concentration (C) in the transgenic tomato plants are shown. Tomato plants were grown at 24°C growth condition (control) and exposed to 0°C for 7 d (chilling), respectively. The hydrogen peroxide concentration was also measured in the same condition.

Figure 7

Improved tolerance of CBF1 transgenic tomato plants under methyl viologen-induced oxidative stress condition. Five leaf discs were incubated in water or 10 μm methyl viologen for 2 d. Light-activated fluorescence (A) and loss of chlorophyll (B) in transgenic and wild-type tomato plants were measured after treatment. The results are an average of five measurements with less than 5% sd.