- Plant growth and crop productivity are largely affected by environmental stresses such as drought, salinity and low temperature. To date, many stress-related genes have been isolated and characterized from various plants. These genes encode products either directly protecting plant cells from abotic stresses or regulating expression of other genes to enhance plant tolerance to the stresses. Under the stress conditions, the C-repeat binding factor/dehydration-responsive element binding factor (CBF/DREB) transcription factors induce the expression of downstream genes containing C-repeat/dehydration response elements (CRT/DRE) in their promoters to improve plant tolerance. Many other transcription factors such as NAC, MYB, bZIP and zinc finger proteins have been well characterized with their roles in the regulation of stress-re-sponses.
- The SCOF-1, a TFIIIA-type zinc finger protein from soybean, functions as a positive regulator of cold-regulated (COR) gene expression mediated by abscisic acid responsive element (ABRE) via protein–protein interaction, which in turn enhances cold tolerance of transgenic plants. A petunia zinc finger protein gene, ZPT2-3, was induced by cold and drought stresses, and its overexpression in transgenic petunia increased plant tolerance to drought stress. The Arabidopsis STZ/ZAT10 functions as a transcription repressor under abiotic stresses and its gain and loss-of-function mutants both improved plant tolerance to abiotic stresses.
- Recently, we isolated three TFIIIA-type zinc finger protein genes ZFP245, ZFP182 and ZFP252 (renamed from RZF71) from rice and found that these genes were induced by various abiotic stresses. As the expression of ZFP182 in transgenic tobacco or overexpression in rice plants increased their tolerance to salt stress, ZFP182 might play a curial role in plant tolerance to salt. Here we report the functional analysis of ZFP252 using gain- and loss-of-function strategies. By stress assays, we found that overexpression of ZFP252 in rice increased tolerance to salt and drought stresses. The contents of free proline and soluble sugars in sense-ZFP252 transgenic rice plants were higher than those in the WT (wild-type) and antisense-ZFP252 transgenic rice plants under salt and drought stress. Our results suggest that ZFP252 might play a key role in stress-responsive signal transduction pathway, and be useful in engineering crop plants with enhanced tolerance to salinity and drought stresses.
- Agarwal et al. identified a total of 189 C2H2-type zinc finger proteins in the indica rice genome and found 26 genes that were upregulated by cold, drought or salt stress . By gene expression analysis, Xu et al. identified several stress-related C2H2-type zinc finger proteins, such as ZFP182, ZFP252 and ZFP245, in japonica rice.
- Using gain- and loss-of-function strategies, Xu et al. found that overexpression of ZFP252 in rice increased tolerance to salt and drought stresses. The contents of free proline and soluble sugars in sense-ZFP252 transgenic rice plants were higher than those in the WT (wild-type) and antisense-ZFP252 transgenic rice plants under salt and drought stress. ZFP252 might play a key role in stress-responsive signal transduction pathway, and be useful in engineering crop plants with enhanced tolerance to salinity and drought stresses.
- ZFP252 and Transgenic rice plants:
- After salt treatment with 100 mM NaCl for 10 d when the top leaves of WT plants began to slightly roll, ZFP252-ox transgenic lines S3, S6, S7 and S10 accumulated more free proline and soluble sugars than WT and ZFP252-kd transgenic lines A14 and A17.After water withholding for 10 d when the top leaves of WT plants started to slightly roll, it was observed that the contents of free proline and soluble sugars in ZFP252-ox transgenic lines were also significantly higher than those in WT and ZFP252-kd lines.There was no significant difference in the contents of free proline and soluble sugars between WT, ZFP252-ox and ZFP252-kd transgenic lines before the drought stress.
- Expression of ZFP252 in transgenic rice plants: Both semi-quantitative RTPCR and quantitative real-time PCR showed that the expression level of ZFP252 in the four ZFP252-ox lines(S3, S6, S7 and S10) was significantly higher than that in WT plants. The expression of ZFP252 in two ZFP252-kd lines (A14 and A17) was hardly detected.
- In mZFPs, the expression level of ZFP252 was enhanced under salt treatment but not under cold treatment (Figure 1). In mZFP+, the expression level of ZFP252 under normal conditions was equal to that of mZFP- as the insertion of mPing did not change the position of the TATA box or the Y Patch (Figure 1). In mZFP+, the expression level of ZFP252 was also increased only under salt stress, and its expression level was higher than that of mZFP-. The database search indicated that the REG of ZFP252 contained the auxinand salicylic acid-responsive cis-element ASF1MOTIFCAMV.
- Overexpression of ZFP252 increases rice tolerance to drought stresses: Almost all leaves of the WT and transgenic lines A14 and A17 rolled after 14 d of un-watered, while only a few of leaves of transgenic lines S3, S6, S7 and S10 rolled (Fig. 2A).The survival rates of lines S3, S6, S7 and S10 were 85.32%–90.32%, and significantly higher than those of WT plants(11.23%) and ZFP252-kd lines A14 (17.62%), A17 (15.32%)(Fig. 2B). It was found that the third leaves from top of WT and ZFP252-kd transgenic plants severely rolled after 10 d of water withholding and their relative electrolyte leakages were higher than those from ZFP252-ox (Fig. 2C). These results suggested that damage degree to the cell membrane of WT and ZFP252-kd transgenic plants was higher than that to the ZFP252-ox transgenic under drought stress.
- Expression of stress-related genes in ZFP252 transgenic rice plants: Xu et al. analyzed the expression of several known stress-related genes in ZFP252 transgenic lines and WT, including OsDREB1A, Oslea3, OsP5CS and OsProT. There was no significant difference in the expression levels of Oslea3, OsP5CS and OsProT between ZFP252 transgenic lines and WT plants under non-treated conditions (Fig. 3B–D). However the OsDREB1A mRNA was much more accumulated in ZFP252-ox transgenic lines S3, S6, S7 and S10 as compared with that in ZFP252-kd lines A14, A17 or WT plants under normal conditions (Fig. 3A). Under salt or drought treatments the expression levels of all four stress-related genes in ZFP252-ox transgenic lines was increased more than that in ZFP252-kd lines and WT plants(Fig. 3A–D). It suggested that ZFP252 might be one upstream regulator of these genes mediating expression of some stress-related genes upon rice treated with salt or drought stresses. It acted as a master switch in stress tolerance, and was involved in the complicated network controlling stress responsive genes.
- OsDREB1A encoding a DREB protein in rice was responsive to overexpression of ZFP252 in ZFP252-ox plants, suggesting ZFP252 might be an upstream regulator of OsDREB1A.
- PCR was performed with ZFP252-specific primers of 5'-GGTGGAGGCGGTTCTTGAGG-3' and 5'-CGTCGTAGTGGCATCGCTTGT-3'.
- To investigate the evolutionary relationship among plant C2H2-type zinc finger proteins involved in stress responses, a phylogenetic tree was constructed using Neighbor–Joining method with the full-length amino acid sequences (Figure 4). The result revealed that ZFP179 was clustered with ZFP182, ZFP150 and ZAT12, whereas other stress responsive zinc finger proteins were categorized into another big branch.
- Schematic Presentation of the Signaling Relationships among the Genes Described in this Review
Yang et al. focuses mainly on the recent studies of genes that are involved in molecular or biochemical processes affecting drought tolerance and that have been used successfully in the genetic engineering of staple crop species such as rice, maize, wheat (Triticum aestivum), soybean, and canolafor improvement of drought tolerance(Figure 5).
- The movement of guard cells might be controlled through the actions of activation/inactivation by a specific pair of kinase–phosphatase.
The identification of commercial grade transgenes that enhance crop performance under both drought and optimal conditions is a lengthy, tedious, and expensive process. Nevertheless, the successful genetic engineering of canola and maize for improved drought tolerance as reviewed herein confirms that the approach is feasible(Figure 5).
- The ZFP252 is a TFIIIA-type zinc finger protein that was structurally similar with STZ/ZAT10, an extensively studied zinc finger protein in Arabidopsis. Overexpression of STZ/ZAT10 improved plant tolerance to salt and dehydration stresses in transgenic Arabidopsis plants and resulted in the growth retardation. In this study, we generated transgenic plants overexpressing ZFP252 (ZFP252-ox) or knocking-down ZFP252 (ZFP252-kd) and found that ZFP252-ox plants were more tolerant to salt and drought stress as compared with WT and ZFP252-kd plants but not to cold (data not shown). There were no significant changes in morphological or agronomic traits among ZFP252-ox plants and ZFP252-kd and WT plants (Supplementary Fig.S1, Tables S1 and S2). It indicated that ZFP252 gene might be more effective in engineering crops with enhanced stress tolerance. The STZ/ZAT10 has been suggested as one of the downstream regulator of DRE-B1A, a CBF/DREB transcription factor in Arabidopsis. As there were two CRT/DRE elements within ZFP252 promoter region (data not shown), ZFP252 might likely be a downstream target of rice CBF/DREB proteins.
- When suffered with abiotic stresses, many plants can accumulate more compatible osmolytes, such as free proline and soluble sugars. These osmolytes function as osmoprotectants in the stress tolerance of the plants. Our results showed that the salt and drought stress-induced increases of the contents of free proline and soluble sugars in the ZFP252-ox transgenic plants were higher than those in WT and ZFP252-kd transgenic plants. Further, the expressions of OsP5CS encoding proline synthetase and OsProT encoding proline transporter in ZFP252-ox plants were higher than that in WT and ZFP252-kd transgenic plants under salt and drought stress conditions by real-time qPCR analysis. These findings suggest that enhanced stress tolerance of ZFP252-ox plants might partially be through activating proline synthesis and transport pathways by ZFP252 in rice under salt and drought stresses.
Labs working on this gene
- State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing 210095, PR China
- Graduate School of Agriculture, Kyoto University, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan
- Genebank, National Institute of Agrobiological Sciences, Kannondai 2-1-2, Tsukuba, Ibaraki 305-8602, Japan
- Xu D Q, Huang J, Guo S Q, et al. Overexpression of a TFIIIA-type zinc finger protein gene ZFP252 enhances drought and salt tolerance in rice ( Oryza sativa L.)[J]. FEBS letters, 2008, 582(7): 1037-1043.
- Yasuda K, Ito M, Sugita T, et al. Utilization of transposable element mPing as a novel genetic tool for modification of the stress response in rice[J]. Molecular Breeding, 2013, 32(3): 505-516.
- Huang J, Sun S J, Xu D Q, et al. Increased tolerance of rice to cold, drought and oxidative stresses mediated by the overexpression of a gene that encodes the zinc finger protein ZFP245[J]. Biochemical and biophysical research communications, 2009, 389(3): 556-561.
- Agarwal P, Arora R, Ray S, et al. Genome-wide identification of C2H2 zinc-finger gene family in rice and their phylogeny and expression analysis[J]. Plant molecular biology, 2007, 65(4): 467-485.
- Sun S J, Guo S Q, Yang X, et al. Functional analysis of a novel Cys2/His2-type zinc finger protein involved in salt tolerance in rice[J]. Journal of experimental botany, 2010: erq120.
- Yang S, Vanderbeld B, Wan J, et al. Narrowing down the targets: towards successful genetic engineering of drought-tolerant crops[J]. Molecular Plant, 2010, 3(3): 469-490.