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1 A Novel MYBS3-dependent Pathway Confers Cold Tolerance in Rice Chin-Fen Su1, Yi-Chieh Wang2a, Tsai-Hung Hsieh3a, Chung-An Lu4, Tung-Hai Tseng5, and Su-May Yu2* 1 Institute of Biotechnology, National Cheng Kung University, University Road, Tainan 701, Taiwan, ROC 2 Institute of Molecular Biology, Academia Sinica, Nangang, Taipei 115, Taiwan, ROC 3 Institute of Plant and Microbial Biology, Acadmia Sinica, Nankang, Taipei 115, Taiwan, ROC 4 Department of Life Science, National Central University, Zhongli, Taoyuan County 320, Taiwan, ROC 5 Division of Biotechnology, Taiwan Agricultural Research Institute, Wu-Fong, Taichung County 413, Taiwan, ROC a Equal contributors Running title: Rice cold tolerant gene Key words: rice, cold tolerance, MYBS3, DREB1, microarray *Corresponding author: Su-May Yu Phone: 886-2-2788-2695 FAX: 886-2-2788-2695 or 886-2-2782-6085 e-mail: sumay@imb.sinica.edu.tw Plant Physiology Preview. Published on February 3, 2010, as DOI:10.1104/pp.110.153015 Copyright 2010 by the American Society of Plant Biologists 2 ABSTRACT Rice seedlings are particularly sensitive to chilling in early spring in temperate and subtropical zones and in high elevation areas. Improvement of chilling tolerance in rice may significantly increase rice production. MYBS3 is a single DNA-binding repeat (1R) MYB transcription factor previously shown to mediate sugar signaling in rice. In the present study, we observed that MYBS3 also plays a critical role in cold adaptation in rice. Gain- and loss-of-function analyses indicated that MYBS3 was sufficient and necessary for enhancing cold tolerance in rice. Transgenic rice constitutively over-expressing MYBS3 tolerated 4°C for at least 1 week, and exhibited no yield penalty in normal field conditions. Transcription profiling of transgenic rice over- or under-expressing MYBS3 led to identification of many genes in the MYBS3-mediated cold signaling pathway. Several genes activated by MYBS3 as well as inducible by cold have previously been implicated in various abiotic stress response and/or tolerance in rice and other plant species. Surprisingly, MYBS3 repressed the well-known DREB1/CBF-dependent cold signaling pathway in rice, and the repression appears to act at the transcriptional level. DREB1 responded quickly and transiently while MYBS3 responded slowly to cold stress, which suggests distinct pathways act sequentially and complementarily for adapting short- and long-term cold stress in rice. Our studies thus reveal a hitherto undiscovered novel pathway which controls cold adaptation in rice. 3 INTRODUCTION Rice is one of the most important food crops in the world, and increases in rice yield could significantly ease the pressure on world food production. Rice is also a powerful model for functional genomics study for dissecting genetic networks of stress responses in cereal crops. Low temperatures are one of the major environmental stresses that adversely affect rice productivity in temperate and subtropical zones and in high elevation areas. Rice seedlings are particularly sensitive to chilling in early spring in these areas, leading to slow seedling development, yellowing, withering, reduced tillering and stunted growth (Andaya and Mackill, 2003). Rice can not be grown in approximately 7,000,000 hectares of land in south and south-east Asia due to cold stress (Sthapit and Witcombe, 1998); in temperate regions such as California (USA), cold is an important stress that results in delayed heading and yield reduction due to spikelet sterility (Peterson et al., 1974). Thus, improvement of chilling tolerance may significantly increase rice production. Plants respond and adapt to cold stress at the molecular and cellular levels as well as induce an array of biochemical and physiological alterations that enable them to survive (Bohnert et al., 1995; Browse and Xin, 2001). Under cold stress, the expression of many genes is induced in various plant species (Hughes and Dunn, 1996; Thomashow, 1999), and the products of these genes function not only in adaptations promoting stress tolerance, e.g., biosynthesis of osmotica (Chen and Murata, 2002; Taji et al., 2002), generation of antioxidants (Prasad et al., 1994), and increased membrane fluidity (Murata and Los, 1997; Orvar et al., 2000), but also in the regulation of gene expression and signaling transduction in stress responses, e.g., transcription factors and proteins involved in RNA processing and nuclear export (Yamaguchi-Shinozaki and Shinozaki, 2006; Chinnusamy et al., 2007). Deciphering 4 the mechanisms by which plants perceive and transmit cold signals to cellular machinery to activate adaptive responses is of critical importance for developing breeding strategies to enhance cold stress tolerance in crops. In Arabidopsis and rice, the CBF/DREB1-dependent cold response pathway has been shown to play a predominant role in freezing-tolerance through the process of cold acclimation (Thomashow, 1999; Yamaguchi-Shinozaki and Shinozaki, 2006; Chinnusamy et al., 2007). The DREB1/CBF family, including DREB1A/CBF3, DREB1B/CBF1, and DREB1C/CBF2, are able to bind to and activate the cis-acting elements DRE (dehydration-responsive element) (Yamaguchi-Shinozaki and Shinozaki, 1994; Stockinger et al., 1997) or CRT (C-repeat) (Baker et al., 1994) on promoters of several cold-responsive genes (CORs) (Gilmour et al., 1998; Jaglo-Ottosen et al., 1998; Liu et al., 1998; Medina et al., 1999). Rice DREB1A and DREB1B are induced by cold stress, and constitutive over-expression of these genes leads to induction of stress-responsive genes, increased tolerance to high-salt and cold, and growth retardation under normal conditions in transgenic Arabidopsis and rice (Dubouzet et al., 2003; Ito et al., 2006), indicating the evolutionary conservation of the DREB1/CBF cold-responsive pathway in monocots and dicots. However, in comparison to Arabidopsis and other cereals like wheat and barley that cold acclimate (Wen et al., 2002), rice does not undergo acclimation process and is more sensitive to low temperature exposures. Microarray analysis demonstrated the existence of 22 cold-regulated genes in rice, which have not been reported in Arabidopsis (Rabbani et al., 2003). These studies also indicate that plant species vary in their abilities to adapt to cold stress. 5 Other rice proteins have also been shown to be involved in cold tolerance. For example, a zinc-finger protein iSAP1 confers cold, dehydration, and salt tolerance in transgenic tobacco (Mukhopadhyay et al., 2004); the rice MYB4 transcription factor confers chilling and freezing tolerances by enhancing the COR gene expression and proline accumulation in Arabidopsis (Vannini et al., 2004), and improves cold and drought tolerances by accumulating osmolyte in transgenic apples (Pasquali et al., 2008). Overexpression of the rice cold-, drought, and salt-inducible MYB3R-2 (an R1R2R3 MYB) gene enhances cold, drought, and salt tolerance by regulating some stress-responsive genes involved in the CBF-dependent or CBF-independent pathways in Arabidopsis (Dai et al., 2007; Ma et al., 2009). The expression of DREB1 is subjected to regulation by several factors. For example, it is affected by members in the same DREB1 family. The Arabidopsis cbf2 mutant, in which CBF2/DREB1C is disrupted, shows higher freezing, dehydration and salt tolerance than the wild-type plant, indicating that DREB1C/CBF2 acts as a repressor of CBF1/DREB1B and CBF3/DREB1A expression (Novillo et al., 2004). The expression of DREB1/CBF is activated by Inducer of CBF Expression 1, ICE1 (a MYC-like basic helix-loop-helix-type transcription factor) (Chinnusamy et al., 2003), CAX1 (a Ca2+/H+ transporter) (Catala et al., 2003), CBL1 (a Ca2+ sensor) (Albrecht et al., 2003), and LOS4 (a DEAD-box RNA helicase) (Gong et al., 2002), and repressed by FRY2 (a transcription factor) (Xiong et al., 2002), HOS1 (a putative RING finger E3 ligase) (Lee et al., 2001), and ZAT12 (a C2H2 zinc finger transcription factor) (Vogel et al., 2005), during cold acclimation in Arabidopsis. The mechanism by which these factors affect the expression of CBF /DREB1 is not clear. 6 Previously, three MYB transcription factors, MYBS1, MYBS2 and MYBS3 each with a single DNA binding domain (1R MYB), were identified in rice and shown to bind specifically to the TA box (TATCCA) in the sugar response complex (SRC) of α-amylase gene (αAmy3) promoter (Lu et al., 2002). MYBS1 and MYBS2 transactivate, while MYBS3 represses, the sugar starvation-inducible αAmy3 SRC activity in rice (Lu et al., 2002). The rice MYBS3 homologue in Arabidopsis (AGI code: At5g47390) is activated by ABA, CdCl2 and NaCl (Yanhui et al., 2006). Recently, we found that the expression of MYBS3 was induced by cold, which prompted us to study its functions in rice in more detail. In the present report, by both gain- and loss-of-function analysis, we show that MYBS3 is essential for cold stress tolerance in rice. Transcription profiling of transgenic rice over- or under-expressing MYBS3 led to identification of genes that are activated or repressed by MYBS3 and play diverse functions. The DREB1-dependent cold response signaling pathway is among those repressed by MYBS3 in rice. Our studies suggest that the DREB1- and MYBS3-dependent pathways may complement each other and act sequentially to adapt to immediate and persistent cold stress in rice. 7 RESULTS Expression of MYBS3 Is Ubiquitous and Activated by Cold Stress Expression of MYBS3 was found to be ubiquitous in all tissues in seedlings and mature plants and in cultured suspension cells of rice (Fig. 1A). The regulation of MYBS3 expression by various stresses was investigated by subjecting rice seedlings to ABA (20 μM), drought (air dry), cold (4°C), salt (200 mM NaCl), and heat (45°C) treatments. The accumulation of MYBS3 mRNA was induced by cold in roots and by cold and salt in shoots (Fig. 1B), but reduced by ABA in shoots (Fig. 1C). The expression pattern of MYBS3 and DREB1A under cold stress was further compared. The amount of MYBS3 mRNA was detectable at 28°C, and increased 5-fold at 4°C after 72 h; in contrast, the accumulation of DREB1A mRNA was barely detectable at 28°C, increased drastically after shifting to 4°C and peaked at 6 h, but declined to one fifth after 72 h (Fig. 1D). To determine whether MYBS3 is regulated by cold at the transcriptional level, the 2.5-kb MYBS3 promoter was fused to the reporter gene GFP encoding a green fluorescence protein and introduced into the rice genome. Ubi promoter fused to GFP was used as a control. Transgenic rice seedlings were grown at 4°C. Under the control of MYBS3 promoter, the accumulation of GFP mRNA was 2.5 times higher at 12 h and stayed high up to 24 h (Fig. 2A, upper panel). In contrast, under the control of Ubi promoter, the accumulation of GFP mRNA decreased by nearly 50% at 6 h, and then stayed at similar levels up to 24 h (Fig. 2A, lower panel). This result indicates that the MYBS3 promoter is activated by cold. 8 Previous study has shown that MYBS3 is a transcriptional repressor of αAmy3 SRC in rice suspension cells (Lu et al., 2002). To determine whether MYBS3 is localized in nucleus, the Ubi promoter was fused to the MYBS3-GFP fusion DNA. The Ubi::MYBS3-GFP and Ubi::GFP constructs were introduced into the rice genome. Protoplasts were isolated from transformed calli, incubated at 4°C or 28°C, and examined. Accumulation of MYBS3-GFP was detected mainly in the nucleus, whereas GFP alone was distributed throughout the cell except the vacuole, at both 4°C and 28°C (Fig. 2B), suggesting that MYBS3 is constitutively localized in the nucleus. MYBS3 Is Sufficient and Necessary for Cold Tolerance in Rice Since MYBS3 was induced by cold, its role in cold tolerance in rice was explored by gain- and loss-of-function approaches. Constructs Ubi::MYBS3 and Ubi::MYBS3(RNAi) (RNA interference) (Fig. S1) were introduced into the rice genome, and several transgenic lines were obtained. Compared to the untransformed wild type (WT) rice, the accumulation of MYBS3 mRNA was higher in MYBS3-overexpression [MYBS3(Ox)] lines S3(Ox)-110-1 and S3(Ox)-112-7, and lower in MYBS3-underexpression [MYBS3(Ri)] lines S3(Ri)-42-10 and S3(Ri)-52-7 (Fig. 3A). Each of these lines contained only one copy of inserted DNA. To test the cold tolerance of transgenic rice, seedlings were shifted from 28°C to 4°C. MYBS3(Ox) lines and WT remained normal while MYBS3(Ri) lines started to show leaf rolling at 4°C after 8 h (Fig. 3B), and both WT and MYBS3(Ri) lines showed leaf rolling and wilting at 4°C after 24 h in hydroponic culture (Fig. 3C and Fig. S2) or 1 week in soil (Fig. 4). Seedlings seemed to be more cold sensitive in hydroponic culture, probably due to weaker growth in hydroponic culture than in soil. Line S3(Ox)-110-1, which accumulated three 9 times more MYBS3 mRNA than S3(Ox)-112-7 (Fig. 3A), conferred higher cold tolerance than line S3(Ox)-112-7 (Fig. 4C). Quantitative analysis also indicated that MYBS3(Ox) lines were more cold tolerant than WT and MYBS3(Ri) lines, and WTs were more cold tolerant than MYBS3(Ri) lines (Table 1). These observations suggest that MYBS3 is sufficient and necessary for cold tolerance in rice, and the degree of cold tolerance correlates with the MYBS3 expression level. The morphology of transgenic rice was similar to the WT, except under greenhouse growth conditions, where plants of the MYBS3(Ox) lines were 20% shorter, had 30% lower tiller numbers, and headed 1 week later than the WT and MYBS3(Ri) lines (Fig. S3). However, in field conditions, most agronomic traits and yield of MYBS3(Ox) lines were similar to those of the WT (Table 2). MYBS3 Regulates the Expression of Genes with Diverse Functions To identify downstream genes regulated by MYBS3 under cold stress, seedlings of S3(Ox)-110-1, S3(Ri)-52-7 and WT were grown at 4 and 28 °C for 24 h. Total RNAs were isolated for microarray analysis using the Affymetrix rice gene chip array containing 55,515 probe sets. Relative change was calculated by comparing the data for MYBS3(Ox) line or MYBS3(Ri) line against those for WT grown at 4°C and 28°C, generating six comparisons. Only relative changes of 3-fold or more were taken to be significantly different. Based on a Venn diagram analysis, 89 genes were up-regulated in the MYBS3(Ox) line (compared with WT) at either 4 or 28 °C, and 1466 genes were up-regulated in WT at 4°C (compared with 28 °C) (Fig. S4A, left panel). Among these genes, 17 genes were up-regulated by over-expression of MYBS3 as well as up-regulated by cold in WT (Table S1). On the other 10 hand, 291 genes were down-regulated in the MYBS3(Ox) line (compared with WT) at either 4 or 28 °C, and 871 genes were down-regulated in WT at 4°C (compared with 28 °C) (Fig. S4A, right panel). Among these genes, 53 genes were down-regulated by over-expression of MYBS3 as well as down-regulated by cold in WT (Table S1). Another analysis revealed that 389 genes were up-regulated in the MYBS3(Ri) line (compared with WT) at either 4 or 28°C (Fig. S4B, left panel). Among these genes, 17 genes were up-regulated by under-expression of MYBS3 as well as up-regulated by cold in WT (Table S2). On the other hand, 124 genes were down-regulated in the MYBS3(Ri) line (compared with WT) at either 4 or 28°C (Fig. S4B, right panel). Among these genes, 37 genes were down-regulated by over-expression of MYBS3 as well as by cold in WT (Table S2). The cold- and MYBS3-regulated genes seem to be involved in diverse functions, and many of them have also been shown to be regulated by drought and salt stresses (Tables S1 and S2). Among the 17 genes up-regulated by over-expression of MYBS3 as well as up-regulated by cold in WT, five genes that have also been shown to be up-regulated by drought (Table S1) and cold (Jain et al., 2007), such as genes encoding glutamate decarboxylase, WRKY77, multidrug resistance protein 4, and trehalose-6-phosphate phosphatase (TPP1 and TPP2), were selected for further quantitative real-time RT-PCR analysis. The accumulation of mRNA of all five genes was significantly increased in WT and further increased in the MYBS3(Ox) line but reduced in the MYBS3(Ri) line at 4°C (Fig. 5 and Table S3), indicating that these genes are downstream of the MYBS3-mediated cold signaling pathway. 11 MYBS3 Suppresses the DREB1-dependent pathway under Prolonged Cold Stress We noticed that in the microarray analysis, the DREB1 family, including DREB1A, DREB1B and DREB1C, and another two DREB1-like genes (ERF#025 and ERF#104) were up-regulated in WT at 4°C, but the induction was surprisingly reduced or abolished in the MYBS3(Ox) line at 4°C (Fig. S5). To investigate how MYBS3 regulates DREB1 gene expression, the accumulation of mRNAs of three DREB1 genes was further analyzed with the quantitative real-time RT-PCR analysis. As shown in Fig. 6, compared with WT, accumulation of MYBS3 mRNA increased significantly at 28°C and was further induced 2-fold at 4°C in the MYBS3(Ox) line. The accumulation of MYBS3 mRNA was reduced in the MYBS3(Ri) line at both 4 and 28°C. In contrast, the cold-induced DREB1A, DREB1B and DREB1C expression was significantly suppressed in the MYBS3(Ox) line at 4°C. Furthermore, the cold inducibility of aAmy3/RAmy3D and a cytochrome P450 gene, both members of the cold-inducible DREB1A regulon (Ito et al., 2006), were also significantly reduced in the MYBS3(Ox) line at 4°C. The accumulation of DREB1, αAmy3/RAmy3D and cytochrome P450 mRNAs were significantly higher in the MYBS3(Ri) line than in the MYBS3(Ox) line at 4°C, although levels did not reach to that in WT at 4°C. Our previous study has shown that MYBS3 represses αAmy3 SRC through the TA box (Lu et al., 2002). Examination of promoter regions within 1 kb upstream of the translation start codon (ATG) revealed the presence of TA box and/or its variants in DREB1 genes (Fig. 7). To determine whether MYBS3 represses DREB1 promoters, a rice embryo transient expression assay was performed. Rice embryos were cotransfected with the effector construct containing Ubi promoter fused to MYBS3 cDNA and the reporter construct containing DREB1A (1054 bp), DREB1B (747 bp) or αAmy3 SRC (105 bp) promoter 12 sequence fused to luciferase cDNA (Luc). Both DREB1 promoters were significantly induced at 4°C, but only the DREB1B promoter was repressed by over-expression of MYBS3 at 4°C (Fig. 8). The αAmy3 SRC was repressed by over-expression of MYBS3 at both 4 and 28°C, consistent with the role of MYBS3 as a repressor of αAmy3 SRC (Lu et al., 2002). These results indicate that MYBS3 could repress DREB1B promoter and αAmy3 SRC at 4°C. 13 DISCUSSION A Novel MYBS3-mediated Cold Signaling Pathway In the present study, both gain- and loss-of-function analyses demonstrated that the MYBS3-mediated pathway is essential for cold stress tolerance in rice. We showed that DREB1A responds early and transiently, which is consistent with previous reports in Arabidopsis and rice (Liu et al., 1998; Shinwari et al., 1998; Dubouzet et al., 2003; Vogel et al., 2005), whereas MYBS3 responds relatively slowly, to cold stress in rice (Fig. 9). The DREB1-mediated process is most likely crucial in respon