Tomato plants ectopically expressing Arabidopsis CBF1 show enhanced resistance to water deficit stress

Abstract

A DNA cassette containing an Arabidopsis C repeat/dehydration-responsive element binding factor 1 (CBF1) cDNA and a nos terminator, driven by a cauliflower mosaic virus 35S promoter, was transformed into the tomato (Lycopersicon esculentum) genome. These transgenic tomato plants were more resistant to water deficit stress than the wild-type plants. The transgenic plants exhibited growth retardation by showing dwarf phenotype, and the fruit and seed numbers and fresh weight of the transgenic tomato plants were apparently less than those of the wild-type plants. Exogenous gibberellic acid treatment reversed the growth retardation and enhanced growth of transgenic tomato plants, but did not affect the level of water deficit resistance. The stomata of the transgenic CBF1 tomato plants closed more rapidly than the wild type after water deficit treatment with or without gibberellic acid pretreatment. The transgenic tomato plants contained higher levels of Pro than those of the wild-type plants under normal or water deficit conditions. Subtractive hybridization was used to isolate the responsive genes to heterologous CBF1 in transgenic tomato plants and the CAT1 (CATALASE1) was characterized. Catalase activity increased, and hydrogen peroxide concentration decreased in transgenic tomato plants compared with the wild-type plants with or without water deficit stress. These results indicated that the heterologous Arabidopsis CBF1 can confer water deficit resistance in transgenic tomato plants.

Figure 1

Northern-blot analysis of transgenic tomato plants. Total RNA (10 μg) was extracted from the wild-type (WT; lane 1) and transgenic T₁ plants overexpressing CBF1 (lane 2–9). Probes used were ³²P-labeled Arabidopsis CBF1 cDNA, the GUS reporter gene from pCAMBIA2301, tomato CAT1, and β-TUBULIN.

Figure 2

Transgenic tomato plants exhibited more resistance to water deficit stress than wild-type plants. Wild-type and three transgenic T₁ plants (WT, C5, C15, and C21) were grown at 24°C without watering for 21 d. Leaves of the wild-type plant significantly curled and wilted. For the survival rate test, wild-type (WT) and three T₁ transgenic plants (C5, C15, and C21) were grown at 24°C without watering for 28 d. Numbers of plants alive per total number of tested plants are indicated in the middle of the photograph.

Figure 3

Improved resistance of CBF1 transgenic tomato T₁ plants to water deficit stress not affected by gibberellic acid (GA₃) treatment. GA₃-treated and non-treated tomato plants were deprived of water for various times. F_v/F_m values (A) and water content of leaves (B) were measured on d 0, 7, 21, and 28. The water content of the roots (C) of water deficit-stressed transgenic tomato and wild-type plants was measured on d 0 and 28. Each value is the mean ± sd (n = 5 individual plants).

Figure 4

Transgenic tomato T₂ plants rapidly close stomata compared with wild-type plants under water deficit condition. Transgenic tomato and wild-type plants were grown at 24°C with regular watering (A) or no watering for 7 d (B). Horizontal bars in the axis of abscissas represent the dark period. The 2-h dark period extended from 10 to 12 h. Each value is the mean ± sd (n = 15 individual plants).

Figure 5

Transgenic tomato plants contain more Pro than wild-type plants. Wild-type and transgenic T₁ plants with or without GA₃ treatment were grown at 24°C with daily watering (control) or without watering for 28 d (water deficit). Pro content was measured. Each value is the mean ± sd (n = 5 individual plants).

Figure 6

Transgenic tomatoes exhibit increased catalase activity but a reduction in H₂O₂ concentrations under normal and water deficit conditions. Ten micrograms of RNA was extracted from wild-type (WT) and three transgenic plants (C5, C15, and C21) grown under control conditions (N) or without watering for 12 d (D), were used to for northern-blot analysis. Probes used were the ³²P-labeled tomato CAT1 gene and β-TUBULIN (A). Plants were grown at 24°C with regular watering (control) or without watering for 28 d (water deficit), catalase activity (B), and H₂O₂ concentration (C) were measured.