Description

       Drought, high salinity, and low temperatures are adverse environmental conditions that affect plant growth and markedly decrease crop productivity. To increase crop yields under stressed conditions, it is important to improve the stress tolerance of crop plants. Plants naturally respond and adapt to these stresses in order to survive. These stresses induce various biochemical and physiological changes, including growth inhibition, to achieve stress tolerance. A number of genes have been described that respond at the transcriptional level to stress. Although various genes are induced by these stresses, many stress-downregulated genes are also reported. Analysis of stressdownregulated as well as stress-upregulated genes is important to gain an understanding of molecular responses to abiotic stresses.
　　Some members of Cys2/His2-type zinc-finger transcription factors have been reported to be upregulated by abiotic stress in Arabidopsis . To characterize the role of these types of proteins, we analyzed the function of Arabidopsis genes encoding four different Cys2/His2-type zinc-finger proteins (AZF1, AZF2, AZF3, and STZ). Using gel-shift analysis, we found that all these proteins recognize A(G/C)T repeats within an EP2 sequence, known as a target sequence for some petunia Cys2/His2-type zinc-finger proteins. Using transient analysis, we showed that the EP2 sequence is a negative cis-element, and these four zinc-finger proteins act as transcriptional repressors mediated by binding to this negative cis-element in Arabidopsis protoplasts.
　　Using RNA gel-blot analysis, we showed that among the four genes, AZF2 and STZ were clearly in Arabidopsis plants induced by abiotic stresses such as drought, cold, and high salinity. Our results show that these proteins function under abiotic stress conditions. To gain further understanding of the function of these proteins, we generated transgenic Arabidopsis plants overexpressing STZ under the control of the constitutive CaMV 35S promoter. We analyzed two independent lines of STZ overexpressors. The growth of the STZ overexpressors on GM agar plates or in soil was compared with that of the wild-type plants. Both transgenic lines showed growth retardation (Figs. 1A and B), and the level of the growth retardation was correlated with that of STZ expression in the transgenic plants (Fig. 1C).
　　To examine whether overexpression of STZ affects tolerance to drought stress, the wild-type and transgenic plants grown in pots were not watered for two weeks. Almost all of the wild-type plants died within this two-week period, whereas nearly all the transgenic plants of both STZ lines survived this level of drought stress and continued to grow when rewatered (Fig. 1D). We also tried to explore the differences in recovery after desiccation using plants grown on agar plates. Wild-type and transgenic plants were removed from the agarplates and kept on plastic plates for four hours, followed by rehydration overnight. Only 5.5% of the wild-type plants survived, whereas 88.9% and 63.9% of the transgenic plants survived (Fig. 1E). Leakage of electrolytes is a sensitive measure of loss of membrane integrity and it is commonly used to assay osmotic pressure-related injury. When plants were dehydrated for four hours, the ion leakage of the wild-type plants was 88.1%, whereas for the transgenic plants the ion leakage was 48.0% and 63.4%, (Fig. 1F). These results indicate that both STZ transgenic lines clearly showed higher tolerance to drought stress than control plants.
　　Since both independent lines of the STZ overexpressors showed growth retardation and tolerance to drought stress, the target downregulated genes might both promote plant tolerance and inhibit plant growth. Because the expression of AZF2 was also induced by drought stress, AZF2 might regulate similar target genes to those of STZ. By using microarray analysis, we have shown that many photosynthesis-related genes and genes for carbohydrate metabolism are downregulated under drought, high salinity, and cold stress conditions. Under stressed conditions, the energy balance changes and photosynthesis in plants is reduced. The expression of certain photosynthesis-related genes and genes for carbohydrate metabolism becomes unnecessary and is reduced at the transcription level under stressed conditions. Reduction of these proteins may lead to a better energy balance for plants under stressed conditions. Therefore, plant growth is inhibited and stress tolerance is increased. These photosynthesis-related genes and genes for carbohydrate metabolism may be the target genes of STZ and AZF2, and reduction of these proteins may increase stress tolerance in the STZ overexpressors. Further elucidation of the roles of these genes in relation to plant stress adaptation will show us a new way to improve plant tolerance to environmental stress conditions.

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   Fig. 1. Phenotypes and drought-stress tolerance of the 35S::STZ and wild-type plants
   (A) Transgenic and wild-type plants grown on agar plates for 21 days. We used plants transformed with vector pBI121 as controls.
   (B) Plants grown in soil pots for 36 days. (C) Expression of the STZ gene in the transgenic and wild-type plants. (D) Droughtstress tolerance of the transgenic and control plants. Both 35S::STZa and 35S::STZb plants were highly tolerant to drought stress (P < 0.001; χ 2 test). Number codes = number of surviving plants out of total number. (E) Difference in recovery after rehydration among the wildtype, 35S::STZa and 35S::STZb plants. Photographs show the plants dehydrated on dry plastic plates in air for 4 hours and then rehydrated overnight. (F) Electrolyte leakage was evaluated after dehydration treatment. A 17-day-old plant was used in each experiment. Plants were removed from the agar plates and dehydrated on dry plastic plates for 4 hours. The values are means of 15 independent samples. Statistical significance compared with the value of the control plants was determined by Welch’s test (p < 0.005).