Thursday, 24 November 2016

Dehydration responsive element binding protein (DREB)1-type transcription factors in transgenic rice improve tolerance to drought, salt, and freezing

  Drought, salt and freezing are environmental conditions that dramatically affect the growth of plants and crop yields. Genetic engineering has high potential for improving the stress tolerance of crops using gene transfer technology. A cis-acting promoter element, DRE/C-repeat (CRT), plays an important role in regulating gene expression in response to these environmental stresses. The Arabidopsis transcription factors DREB1s/C-repeat binding factor (CBF)s bind to DRE and control the expression of many stress-responsive genes. In rice, we have isolated cDNAs for DREB1 homologs and named them OsDREB1s. We have reported previously that overexpression of the DREB1 or OsDREB1 genes induces strong expression of the many stress-responsive genes in transgenic Arabidopsis plants, which in turn boost stress tolerance to high salt and freezing. In this study, we analyzed the functions of the OsDREB1A and OsDREB1B genes in stress tolerance using transgenic rice and compared it with that of DREB1A. We observed improved tolerance to drought, high-salt and cold stresses in the transgenic rice plants.

  To analyze the function of rice OsDREB1A and OsDREB1B proteins, we generated transgenic rice (Oryza sativa cv. Kita-ake) overexpressing OsDREB1A or OsDREB1B under the control of the constitutive maize ubiquitine promoter. To compare the function of the rice OsDREB1 family with that of Arabidopsis DREB1/CBF family in rice, we also generated transgenic rice overexpressing Arabidopsis DREB1A, DREB1B or DREB1C. These transgenic rice plants showed growth retardation under normal growth conditions in the same way as did the transgenic Arabidopsisplants overexpressing OsDREB1 or DREB1. Both OsDREB1 and DREB1 showed a similar effect of growth retardation under normal growth conditions. The tolerance of the transgenic rice overexpressing OsDREB1 or DREB1 to drought, high-salt and cold stresses was compared with that of the control plants (Fig. 1). Although 0%, 5% and 5% of the wild-type plants survived under drought, high-salinity and cold conditions, respectively, 17 to 80%, 13 to 83% and 25 to 60% of the plants overexpressing OsDREB1 or DREB1 respectively survived under the same conditions.

  Many plants accumulate a number of compatible osmolytes such as proline and various sugars under drought, high-salt and cold stress conditions, and these osmolytes function as osmoprotectants that contribute to tolerance to stress. Transgenic Arabidopsis plants overexpressing DREB1A have been reported to accumulate proline and various sugars even under unstressed control conditions. We examined whether transgenic rice overexpressing OsDREB1A or DREB1Aalso accumulated proline and various sugars under control conditions. The free proline levels in the wild-type rice under high-salt and cold stress conditions were approximately 5- and 7-fold higher than those under the control conditions, respectively. Even under control conditions, the transgenic plants overexpressing OsDREB1A and DREB1A accumulated 4- to 5-fold and 6- to 15-fold higher levels of proline, respectively, as compared with the wild-type rice. Similarly, wild-type rice plants accumulated high levels of sugars such as raffinose, sucrose, glucose and fructose in response to high salt or cold stress. Even under unstressed conditions, the transgenic rice plants overexpressing DREB1A accumulated high levels of these sugars.

  The whole genome sequence of rice (cv. Nipponbare) has recently been determined. In addition, approximately 22,000 sequences of independent full-length cDNAs of rice (cv. Nipponbare) were registered in a public database, and an oligoarray containing approximately 21,500 rice probes was produced based on these sequences. To analyze the upregulated genes in the transgenic rice overexpressing OsDREB1A or DREB1A, we generated transgenic rice plants overexpressing OsDREB1A or DREB1A using cv. Nipponbare. We identified 12 target genes of OsDREB1A in the transgenic rice using microarray and RNA gel blot analyses (Fig. 2). These genes encode proteins that are thought to function in stress tolerance in plants and most of these genes were induced by at least one of drought, high-salt and cold stresses; many promoters of the upregulated genes contain DRE sequences. These results indicate that the DREB1/CBF cold-responsive pathway is conserved in rice and the DREB1-type genes are quite useful for improvement of tolerance to environmental stresses in various kinds of transgenic plants, including rice.
(Y. Ito, K. Maruyama and K. Yamaguchi-Shinozaki)

Fig. 1. Drought, high-salt and cold stress tolerance of the transgenic rice plants (cv. Kita-ake) overexpressing OsDREB1 or DREB1 genes, and wild-type plants. The stress treatments were conducted as described below. Drought = 17-day-old plants with water withheld for 9 days and then supplied with water again for 13 days. High-salt = 17-day-old plants soaked in 250 mM NaCl solution for 3 days and transferred to pots under normal growth conditions for 19 days. Cold = 17-day-old plants were exposed to a temperature of 2 ?C for 93 h and then to 28 ?C for 18 days. ‘Wild’, ‘Os1A’, ‘Os1B’, ‘At1A’, ‘At1B’ and ‘At1C’ mean wild-type plants and transgenic rice plants overexpressing OsDREB1A, OsDREB1B, DREB1A, DREB1B and DREB1C, respectively.
Fig. 1. Drought, high-salt and cold stress tolerance of the transgenic rice plants (cv. Kita-ake) overexpressing OsDREB1 or DREB1 genes, and wild-type plants. The stress treatments were conducted as described below. Drought = 17-day-old plants with water withheld for 9 days and then supplied with water again for 13 days. High-salt = 17-day-old plants soaked in 250 mM NaCl solution for 3 days and transferred to pots under normal growth conditions for 19 days. Cold = 17-day-old plants were exposed to a temperature of 2 ˚C for 93 h and then to 28 ˚C for 18 days. ‘Wild’, ‘Os1A’, ‘Os1B’, ‘At1A’, ‘At1B’ and ‘At1C’ mean wild-type plants and transgenic rice plants overexpressing OsDREB1A, OsDREB1B, DREB1A, DREB1B and DREB1C, respectively.
Fig. 2. Expression of the OsDREB1Aupregulated genes in the transgenic rice plants (cv. Nipponbare) overexpressing the OsDREB1A or DREB1A genes and in the wild-type plants. Sixteen-day-old transgenic rice (cv. Nipponbare) seedlings grown hydroponically were sampled under unstressed conditions (control) to prepare total RNAs. The wild-type plants were kept in 250 mM NaCl for 5 h (NaCl), water withheld for 5 h (dry) or kept at 4 ?C for 24 h (cold). Each lane was loaded with 10 mg of total RNA.
Fig. 2. Expression of the OsDREB1Aupregulated genes in the transgenic rice plants (cv. Nipponbare) overexpressing the OsDREB1A or DREB1Agenes and in the wild-type plants. Sixteen-day-old transgenic rice (cv. Nipponbare) seedlings grown hydroponically were sampled under unstressed conditions (control) to prepare total RNAs. The wild-type plants were kept in 250 mM NaCl for 5 h (NaCl), water withheld for 5 h (dry) or kept at 4 ˚C for 24 h (cold). Each lane was loaded with 10 mg of total RNA.

For further details log on website :
https://www.jircas.affrc.go.jp/english/publication/highlights/2005/2005_04.html

No comments:

Post a Comment