Function of Gene OsASIE1 in Response to Abiotic Stress in Rice

ACTA AGRONOMICA SINICA Volume 37, Issue 10, October 2011 Online English edition of the Chinese language journal Cite this article as: Acta Agron Sin, 2011, 37(10): 1771–1778.

RESEARCH PAPER

Function of Gene OsASIE1 in Response to Abiotic Stress in Rice WU Hui-Min, HUANG Li-Yu, PAN Ya-Jiao, JIN Peng, and FU Bin-Ying* Institute of Crop Sciences/ National Key Facility for Crop Gene Resources and Genetic Improvement, Chinese Academy of Agricultural Sciences, Beijing 100081, China

Abstract: AP2/EREBP transcription factors play an important role in plant development, hormone response, and responses to biotic and abiotic stress. OsASIE1, a member of the EREBP subfamily of AP2/EREBP transcription factors in rice (Oryza sativa L.), is determined to be involved in abiotic stress response. OsASIE1 expression was induced by drought and salt stresses. In addition, an electrophoresis mobility shift assay (EMSA) revealed that the AP2 domain of the OsASIE1 protein can bind to both DRE (dehydration-responsive element) and GCC box (ethylene response element, ERE) in vitro. All these results indicate that OsASIE1 may participate in abiotic stress response by regulating the expression of downstream genes with DRE and GCC box binding. Overexpression of OsASIE1 in transgenic rice plants might be used to improve tolerance to salt stress. Keywords: rice; AP2/EREBP transcription factor; salt tolerance; overexpression

Various biotic and abiotic stresses have a major influence on the growth and yield of rice (Oryza sativa L.). An exploration of genes conferring resistance to abiotic stress may provide gene resources for breeding experiments and lead to improvements in rice stress tolerance. Over time, plants have evolved a variety of response mechanisms to stress at the molecular, cellular, physiological, and biochemical levels. As upstream gene regulators, transcription factors play an important role in plant stress response pathways [1]. The primary genes participating in plant stress responses include members of the AP2/EREBP, bZIP, NAC, MYB, and WRKY transcription factor families [2]. AP2/EREBP transcription factor genes are widely involved in plant growth, hormone signal transduction, pathogen responses, and responses to stresses such as drought, salt, and low temperature. This family features an AP2 domain consisting of about 60 amino acids, which has an important role in DNA binding. Based on characteristics of the AP2 domain, the AP2/EREBP transcription factor family is divided into 4 subcategories: AP2, EREBP, RAV, and others. AP2 transcription factors contain 2 repetitive AP2 domains, while RAV subfamily members have one AP2 domain and one B3-like domain.

EREBP transcription factors, with a single AP2 domain, are further subdivided into ERF and DREB/CBF branches [3, 4]. Studies have shown that EREBP transcription factors are extensively involved in responses to biotic and abiotic stresses. ERF transcription factors are mainly associated with plant biotic stress responses, including pathogenic reaction, injury reaction, and ethylene signaling pathways [1, 5]. The first ERF transcription factors to be discovered were the 4 induced pathogenesis-related (PR) proteins EREBP1–4 from tobacco (Nicotiana tabacum L.); they regulate gene expression by binding with the ethylene-response element, or GCC box, in the promoter region of downstream genes. The GCC box, with core sequence AGCCGCC, is commonly found in the 5-UTR region of PR proteins in tobacco and other plants [6]. Three ERF proteins (Pti-4, Pti-5, and Pti-6) have been isolated from tomato (Lycopersicon esculentum Mill.) and may interact with the Pto disease resistance protein [7, 8]. ERF transcription factors have been further confirmed to play an important role in plant responses to pathogens. Overexpression of ERF genes, including ERF1, Pti4, and ATERF1, may increase plant resistance [5]. Recent studies have demonstrated that ERF transcription factors play positive regulatory roles in many

Received: 14 March 2011; Accepted: 25 June 2011. * Corresponding author. E-mail: [email protected] Copyright © 2011, Crop Science Society of China and Institute of Crop Sciences, Chinese Academy of Agricultural Sciences. Published by Elsevier BV. All rights reserved. Chinese edition available online at http://www.chinacrops.org/zwxb/ DOI: 10.1016/S1875-2780(11)60048-5

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biological pathways in rice, including responses to ethylene [9], flooding stress [10, 11], and drought stress [12]. DREB transcription factors participate in absicisic acid (ABA)-independent stress responses, including responses to stress induced by cold, drought, salt, and peroxides. DREBs can regulate expressions of downstream genes by binding to cis-acting DRE/CRT elements located in the promoter region of downstream drought-responsive genes. A DRE element, whose core sequence is A/GCCGAC, has been found in the promoter region of the drought-responsive rd29A gene [13], while a CRT element with core sequence TGGCCGA has been discovered in the promoter region of the cold-inducible cor15a gene [14, 15]. DRE/CRT elements generally exist in promoter regions of drought- and low-temperature-inducible genes [16–18]. An increasing number of DREB genes have been cloned from various plants [19]. DREB genes cloned from rice include OsDREB1 to OsDREB1G, OsDREB2A, and OsDREB2B, although only OsDREB1A, OsDREB1E, OsDREB1G, OsDREB2A, and OsDREB2B can specifically bind to DRE elements [17, 18]. Overexpressions of DREB genes in Arabidopsis and rice result in improved resistance to abiotic stresses such as low temperature, salt, and drought [13, 17, 18]. ERF and DREB transcription factors mainly differ from their cis-acting elements combining and regulating downstream genes. Some EREBP transcription factors, such as TINY [20], TINY2 [21], BnDREB III [22], AtERF1, AtERF4, AtEBP, and CBF1 [23], can bind to both DRE and GCC box elements, but the majority, including CBF2/DREB1C and CBF3/DREB1A, can only bind to DRE. In general, ERF transcription factors tend to bind to the GCC box, whereas DREB transcription factors usually bind to DRE. Using alignment analysis in Arabidopsis, Sakuma et al. [3] found 2 conserved amino acids in the AP2 domains of DERF and ERF transcription factors, i.e., the 14th valine (V14) and the 19th glutamic acid (E19) in DREB proteins, and the 14th alanine (A14) and the 19th aspartic acid (D19) in ERF proteins. V14 and E19, particularly V14, are important for the DNA-binding specificity of DRE, but have no effect on DNA-binding of the GCC box, resulting in the different functions of ERF and DREB transcription factors [3]. To determine the amino acids crucial to binding on the GCC box, Sun et al. [20] analyzed DREB AP2/ERF domain sequences. The most noticeable difference detected among these sequences was the identity of the 15th amino acid, which was Ser in TINY but Cys in the other types. It is thus reasonable to deduce that Ser-15 is crucial for specific binding to the ERE element. TINY might play a role in the cross-talk between gene expressions in response to abiotic and biotic stress by connecting DRE- and ERE-mediated signaling pathways. Some AP2/EREBP transcription factor genes, mainly DREB and ERF, were obviously up-regulated under abiotic stress in rice [24]. Although abiotic stress response mechanisms involving EREBPs in higher plants have been relatively well

studied, the functions of many EREBP genes in rice are unknown. To explore EREBP genes responsive to abiotic stress in rice, the OsASIE1 (Os08g0408500) gene, which was induced by abiotic stress during preliminary experiments in our laboratory, was selected for further study. The relationship between OsASIE1 and response to abiotic stress was analyzed by examining differential expression patterns in various rice varieties under drought, salt, and low temperature stresses, binding to cis-acting elements (DRE and GCC box), and phenotypic variations in OsASIE1-overexpressing plants.

1 1.1

Materials and methods Plant materials

Four japonica rice varieties were used in this experiment, including Nipponbare and 3 varieties with tolerance to drought (IRAT109), salt (FL478), and cold (LTH). IRAT109 is a typical upland variety and LTH is a landrace from Yunnan Province, China. 1.2 Low temperature, salt, and drought stress treatments 1.2.1 Treatments of Nipponbare Disinfected Nipponbare seeds were subjected to accelerated germination at 37 qC for 3 d. The uniformly germinated seeds were sown, 2 seeds per hole, into foam supports in a plastic box. The resulting seedlings were incubated with water until the 2-leaf stage, and thereafter with Yoshida nutrient solution. Seedlings at the 5-leaf stage were exposed to low temperature (4 °C), salt (150 mmol L1 NaCl), or drought (20% PEG-6000, W/V). Leaves were sampled from plants 0, 0.5, 3, 6, 12, and 24 h after stress treatments, immediately frozen in liquid nitrogen, and stored at 70 qC. 1.2.2 Treatments of stress-resistant varieties Seeds were disinfected and subjected to accelerated germination. Germinated seeds to be subsequently exposed to drought were sown into soil-filled plastic pots; those undergoing future cold and salt stress treatments were sown into foam supports in a plastic box. Five-leaf seedlings were exposed to drought (withholding water until all leaves curled), salt (150 mmol L1 NaCl, 48 h), or low temperature (4 °C, 48 h). Leaves were sampled both before and after stress treatments, immediately frozen in liquid nitrogen, and stored at 70 qC. 1.3

Real-time quantitative RT-PCR

Total RNA was isolated from leaf samples using TRIzol reagent (Invitrogen). Using the isolated RNA, cDNA was synthesized as described in Jin et al. [24]. The OsASIE1 region was amplified from cDNA using gene-specific primers (F: 5-TGGTCTGATTTGGTAGCC-3, R: 5-TCCAAGAACTGG CAGACGA-3). The Actin1 gene (F: 5-GACTCTGGTGAT GGTGTCAGC-3, R: 5-GGCTGGAAGAGGACCTCAGG-3)

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was used as an internal reference. 1.4

Electrophoretic mobility shift assay

PCR was performed to amplify the OsASIE1 AP2 domain (67–193 aa) coding region. The resulting fragment was cloned into a pET-32a vector (Novagen, USA, 69015-3), and the constructs were transformed into Escherichia coli strain BL21 (DE3) to produce protein. This protein was separated using a His-trap column, concentrated by Millipore filtration, and desalted. The concentration of purified protein was measured using the Bradford method [25]. Cis-acting elements, i.e., the GCC box (CATAAGAGC CGCCACT) and its mutant (CATAAGATCCTCCACT), and DRE (ATACTACCGACATGAG) and its mutant (ATACTA CTGATATGAG), were chemically synthesized by 3 tandem repeats, respectively. The double-stranded synthesized DNAs were then combined in equal proportions (final concentration of 100 nmol L1 single-stranded DNA). The mixture was incubated for 10 min at 95 qC, and the double-stranded DNA was then allowed to renature as the solution slowly cooled. An electrophoretic mobility shift assay (EMSA) was carried out using an EMSA kit (Invitrogen) following the manufacturer’s protocol. 1.5

Alignment analysis and clustering

Alignment analysis between OsASIE1 and selected EREBPs was performed using BioEdit 7.0 (http://www.mbio.ncsu.edu/ bioedit/bioedit.html). A phylogenetic tree was constructed according to the neighbor-joining method using MEGA4 software with the complete deletion option [26]. Support for individual nodes in the tree was assessed by bootstrap analysis. 1.6 Identification of transgenic plants and phenotypic evaluation 1.6.1 Vector construction and plant transformation The full-length cDNA of OsASIE1 was amplified from IRAT109 using gene-specific primers (F: 5-GCACTGCAGATGGCA GCTGCTATAGAAGG-3, R: 5-GCACTGCAGATGGCAGC

Fig. 1

TGCTATAGAAGG-3) that incorporated Pst I and BamH I restriction sites at the 5 and 3 ends. After enzyme digestion, the resulting product was cloned into a pCUbi1390 vector driven by a ubiquitin 1 promoter. The vector was introduced into Agrobacterium tumefaciens strain EHA105, and then transferred into Nipponbare plants via Agrobacterium-mediated transformation following the protocol of Chen et al. [27]. 1.6.2 Phenotypic identification of transgenic plants Seeds of T1 plants were germinated in half-strength MS medium supplemented with hygromycin (100 mg L1) for removal of non-transgenic seeds. The germinated seeds were sown into the wells of a 96-well PCR plate and cultured using Yoshida solution for 4 weeks. The seedlings were then subjected to salt stress (150 mmol L1 NaCl). Nipponbare wild type seedlings were used as controls. Each treatment had 3 replicates.

2

Results

2.1 OsASIE1 expression patterns in rice under various stresses 2.1.1 OsASIE1 expression patterns in Nipponbare under various stresses To uncover stress-induced OsASIE1 expression patterns, expression level changes were analyzed under low temperature, salt, and drought conditions using RT-PCR. Although OsASIE1 was up-regulated during all stress conditions, expression patterns varied greatly (Fig. 1). When subjected to low temperature and drought stresses, OsASIE1 expression gradually increased, reaching the highest levels 3 h after treatments. Compared with levels seen during low temperature stress, however, the increase in OsASIE1 expression under PEG stress was higher and remained high until the end of the treatment. Under salt stress, OsASIE1 expression also gradually increased; the extent of the increase was much higher, more than 20 times that of the other treatments. These results provide strong evidence that OsASIE1 is an important gene in response to cold, salt, and drought stresses; it serves as an early regulator in plant abiotic stress defense systems and may have different regulatory mechanisms under different stresses.

Relative expressions of OsASIE1 under cold (4 qC), NaCl (150 mmol L1), and osmotic (20% PEG) stresses

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Transcription factors play important roles in abiotic stress response to abiotic stresses, and their concentrations generally increase rapidly in early stages of stress to regulate expression of related downstream genes. In rice seedlings, OsASIE1 expression levels markedly increased following PEG and NaCl treatments, but only increased slightly upon exposure to low temperature. 2.1.2 OsASIE1 differential expression before and after stress treatments in typical stress-resistant varieties To further clarify the relationship between OsASIE1 and stress resistance in rice, expression levels in Nipponbare and typical highly resistant varieties were analyzed under different abiotic stresses. Although OsASIE1 expression levels in the resistant varieties prior to stress treatments were slightly different than those in Nipponbare, expression patterns were similar in all 4 resistant varieties under stress conditions (Fig. 2). OsASIE1 expression was clearly different, however, in

leaves and roots before and after stress. Under low temperature and drought conditions, expression levels were higher in roots than in leaves; the converse was true during salt stress. 2.2 Analysis of DNA-binding affinity of OsASIE1 AP2 domain with GCC box and DRE elements By binding to cis-acting elements (DRE or GCC box) in downstream gene promoters, EREBP transcription factors can regulate gene expression in response to biotic and abiotic stress [6, 13]. To detect the target sequences of the OsASIE1 protein AP2 domain, an EMSA was performed. The AP2 of the OsASIE1 protein bound effectively to the GCC box and DRE, but did not bind to the mutant sequence (Fig. 3). We therefore conclude that OsASIE1 regulates gene expression in response to biotic and abiotic stress by specifically binding to GCC box and DRE elements.

Fig. 2 Relative expressions of OsASIE1 in typical rice varieties before and after cold (4 qC), NaCl (150 mmol L1), and osmotic (20% PEG) stresses N: Nipponbare; L: LTH; F: FL478; I: IRAT109.

Fig. 3 EMSA analysis of bindings between the AP2 domain of OsASIE1 and GCC box or DRE element A: EMSA of the AP2 domain of OsASIE1 binding on GCC box. Lane 1: Free DNA; Lanes 2–5: 80 ng GCC box incubated with 0.25, 0.5, 1.0, 1.5 μg target protein, respectively; Lane 6: 80 ng mGCC box incubated with 1.5 μg target protein. B: EMSA of the AP2 domain of OsASIE1 binding on DRE. Lane 1: Free DNA; Lanes 2–4: 80 ng GCC box incubated with 0.5, 1.0, and 1.5 μg target proteins, respectively; Lane 5: 80 ng mDRE incubated with 1.5 μg target protein. C: GCC box and DRE sequence information in wild and mutant types.

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2.3 Comparison between OsASIE1 and EREBPs in rice and Arabidopsis To explain the mechanisms underlying binding of OsASIE1 to the GCC box and DRE, OsASIE1 and previously-published EREBP transcription factor sequences of rice and Arabidopsis were aligned and used to construct a polygenetic tree (Fig. 4). Based on alignment and phylogenetic analyses, OsASIE1 is closely related to DREB transcription factors. In both OsASIE1 and in DREB transcription factors, the 14th amino acid is valine (Val). The 19th amino acid, which is typically glutamic acid (Glu) in other transcription factors, has been replaced by leucine (Leu) in OsASIE1 and by Val in OsDREB1A and OsDREB1B. Similarly, the 16th amino acid, which is involved in binding to GCC box and DRE, has undergone substitution from serine (Ser) in OsASIE1 as well as in AtERF1, AtERF4, and AtEBP. These results indicate that the amino acids in the AP2/EREBP DNA-binding domain that are crucial for binding sequence recognition have not

been conserved in OsASIE1. 2.4 Improvement of salt-tolerance in plants overexpressing OsASIE1 To further analyze the function of OsASIE1 in response to stress, an overexpression vector was constructed and transferred into Nipponbare plants. Compared with wild-type plants, plants of the 14 resulting transgenic lines were shorter (Fig. 5-A). According to RT-PCR, OsASIE1 expression levels in the

Fig. 4

transgenic lines were significantly higher than in the wild type (Fig. 5-B). After 2 d of salt stress treatment, the leaves of wild-type plants wilted; in contrast, transgenic plants exhibited healthy growth (Fig. 5-C). Three days after termination of salt treatment, most transgenic plants, but only a few of the wild-type ones, sprouted new leaves (Fig. 5-D and 5-E).

3

Discussion

Mechanisms of abiotic stress response are very complicated and involve many genes. In recent years, many stress-inducible genes have been detected using various techniques. Some genes responsive to drought and cold are induced by ABA while others are not, indicating the presence of 2 stress response signal pathways—one ABA-dependent and one ABA-independent—in higher plants. Transcription factors involved in stress response have accordingly been divided into 2 categories: ABA-dependent, such as ABRE, and ABA-independent, for example, DREB [1]. OsASIE1 is induced by low temperature, salt, and drought stress, but not by ABA. This suggests that OsASIE1, like OsDREBs, may be associated with an ABA-independent pathway. In addition, the identical expression patterns observed for OsASIE1 in both Nipponbare and typical highly-resistant varieties indicate that this gene plays a basic regulatory role in stress response and is not the major source of high stress resistance in IRAT109, FL478, and LTH.

Multiple alignment and phylogenic analysis of AP2 domains of OsASIE1 and EREBP transcription factors Arrows indicate the 15th, 16th, and 20th amino acids of the AP2 domain.

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Fig. 5 Overexpression of OsASIE1 enhanced salt tolerance of rice A: Phenotypes of transgenic lines and wild type (WT); B: Expression identification of OsASIE1 in transgenic plants; C: Phenotypes of transgenic lines and wild types treated by 150 mmol L1 NaCl for 2 d; D and E: Phenotypes of transgenic lines and wild types in 3 d recovery after treatment.

EREB transcription factors, all of which are involved in plant abiotic stress response, can be divided into two classes, i.e., EREB1 and DREB2. Their regulatory mechanisms are different [28–31], however. Divergent expression patterns have also been observed under different abiotic stresses; for example, the DREB1 gene is induced by low temperature, while the DREB2 gene is induced by drought and salt [13]. In this study, OsASIE1 expression levels increased only slightly under low temperature conditions, but increased significantly and remained elevated in response to salt and drought stress. Phylogenetic analysis revealed that OsASIE1 is closely related to DREB2 transcription factors. OsASIE1 may thus belong to the DREB2 transcription factor subfamily. In rice and Arabidopsis, DREB transcription factors can all bind to the DRE element, but the binding mechanisms are different in the 2 species. For example, in one study, DREB1A in Arabidopsis was found to effectively bind to the DRE core sequences GCCGAC and ACCGAC, while OsDREB1A in rice could only bind preferentially to GCCGAC. As a consequence, OsDREB1A was unable to regulate expression of genes containing the sequence ACCGAC [17]. Alignment analysis of EREBP transcription factors indicated that the 14th and 19th amino acids, which are involved in binding to the GCC box and DRE, are not completely conserved. This demonstrates that the crucial amino acids for binding

sequence recognition are different in the DNA-binding domains of various DREBPs, resulting in transcription factors with different functions. This may be an adaptability mechanism that has arisen over the course of evolution in plants. In this study, OsASIE1 was confirmed to be involved in response to high salt stress. OsASIE1 expression levels rapidly increased early in the stress period to regulate expression of downstream stress response genes. Transgenic plants overexpressing OsASIE1 were shorter than the wild type, similar to phenotypes observed for OsDREB-overexpressing transgenic plants [17, 18]. In Arabidopsis, overexpression of ERF1 and OsERF1 has been found to up-regulate ethyleneresponsive genes and produce similar phenotypes, such as short, developmentally stunted plants [9]. Further study is needed, however, to confirm whether the altered phenotypes observed in the transgenic lines can be attributed to OsASIE1 overexpression. Like other EREBP transcription factors, OsASIE1 can bind to DRE and the GCC box, indicating overlapping functions for ERF and DREB. Of course, additional research is required to compare functional differences between these two types of transcription factors and further elucidate stress response mechanisms in plants. As previously mentioned, EREBP transcription factors play an important role in biotic and abiotic stress response, and

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EREBP gene overexpression might thus be used to enhance plant resistance. On the other hand, EREBP overexpression can also restrain plant growth and development, such that EREBPs cannot be directly used for genetic engineering breeding. Some method for eliminating the restraining effect of EREBP overexpression is therefore required. The stress-inducible promoter would eliminate this adverse effect, but its regulating ability, response rate, and expression level could not satisfy the plant’s stress response needs. New, more efficient stress-inducible promoters therefore need to be developed to satisfy the needs of plant breeders.

[8]

4

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Conclusions

OsASIE1 is a member of the EREB2 subfamily of AP2/EREBP transcription factors in rice. OsASIE1 expression levels rapidly increased under high salt and drought stress, but increased only slightly under low temperature conditions. All results indicate that OsASIE1 can regulate expression of downstream stress-responsive genes by binding to GCC box and DRE elements in the promoter. Overexpression of OsASIE1 might be used to improve the salt tolerance of transgenic rice plants.

[9]

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[14]

Acknowledgment This study was supported by the National Transgenic Plant Program of China (2008ZX001-003 and 2009ZX08009-007B).

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