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Brassinosteroid Signaling and Application in Rice
Available online at www.sciencedirect.com
Journal of Genetics and Genomics 39 (2012) 3e9
JGG REVIEW
Brassinosteroid Signaling and Application in Rice Hongning Tong, Chengcai Chu* State Key Laboratory of Plant Genomics and Center for Plant Gene Research (Beijing), Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China Received 28 October 2011; revised 14 December 2011; accepted 14 December 2011 Available online 20 December 2011
ABSTRACT Combined approaches with genetics, biochemistry, and proteomics studies have greatly advanced our understanding of brassinosteroid (BR) signaling in Arabidopsis. However, in rice, a model plant of monocot and as well an important crop plant, BR signaling is not as well characterized as in Arabidopsis. Recent studies by forward and reverse genetics have identified a number of either conserved or specific components of rice BR signaling pathway, bringing new ideas into BR signaling regulation mechanisms. Genetic manipulation of BR level or BR sensitivity to improve rice yield has established the great significance of BR research achievements. KEYWORDS: Brassinosteroid; BR signaling; Rice; Yield
1. INTRODUCTION Brassinosteroids (BRs) are a class of steroid hormones involved in diverse biological processes. In recent years, rapid progress has been made in BR signaling pathway in Arabidopsis (Fig. 1) (Clouse, 2002; Li and Jin, 2007; Kim and Wang, 2010; Clouse, 2011; Yang et al., 2011). In the absence of BR, BKI1 binds membrane-located BR receptor BRI1 and inhibits its function (Wang and Chory, 2006). In the presence of BR, BR binding leads to BRI1 dissociation with BKI1, BRI1 autophosphorylation and association/transphosphorylation with BAK1 kinase (Li et al., 2002; Nam and Li, 2002; Kinoshita et al., 2005; Wang et al., 2005a, 2005b). The hormone signal is transduced through CDG1 and BSK1 kinases and then BSU1 phosphatase to repress the negative regulator BIN2 kinase, as well somehow activate PP2A phosphatase (Li et al., 2001; Choe et al., 2002; Li and Nam, 2002; Mora-Garcia et al., 2004; Tang et al., 2008, 2011; Kim et al., 2009). Inhibition of BIN2 and activation of PP2A will promote the transformation of phosphorylated BZR1 and BES1 to dephosphorylated forms, which subsequently transfer * Corresponding author. Tel/fax: þ86 10 6487 7570. E-mail address: [email protected] (C. Chu).
to nucleus and form diverse transcriptional complexes to regulate a large number of BR-responsive genes (He et al., 2002; Wang et al., 2002; Yin et al., 2002, 2005; Vert and Chory, 2006; Ryu et al., 2007; Peng et al., 2010; Sun et al., 2010; Tang et al., 2011; Yu et al., 2011). This primary signaling pathway tends to be regulated by complicated mechanisms. For example, at transcriptional level, BRI1 is negatively feedback regulated by BZR1 and BES1 (Sun et al., 2010; Yu et al., 2011), whereas at protein level, BRI1 is also negatively feedback regulated by PP2A (Di Rubbo et al., 2011; Wu et al., 2011). In addition, BIN2 and BZR1 were suggested to be regulated by proteasome-mediated degradation processes (He et al., 2002; Peng et al., 2008). Early in 1980’s, physiological experiment has proved the active role of BRs in promoting rice leaf blade bending (Wada et al., 1981). The tests using either dark-grown or light-grown lamina inclination responsive to BRs have been developed (Takeno and Pharis, 1982; Wada et al., 1984) and were widely used to characterize the sensitivity of rice mutants or varieties to BRs. However, the in vivo roles of BRs in rice growth and development begin to be unveiled until the identification of the BR-deficient mutants, d61, which carry mutations in a gene orthologous to BR receptor gene BRI1 (Yamamuro et al., 2000). Recently, a number of either conserved or specific
1673-8527/$ - see front matter Copyright Ó 2012, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Limited and Science Press. All rights reserved. doi:10.1016/j.jgg.2011.12.001
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Fig. 1. Integrated diagrams of BR signaling pathway in Arabidopsis (A) and rice (B). For BR signaling in Arabidopsis, only the primary components mentioned in this article are involved. Arrows show promotion actions and bar ends show inhibitory actions. Solid lines show direct regulation, dotted lines indicate hypothetical regulation, and curve lines indicate feedback regulations. Roles of OsGSK1 need to be further testified.
components of rice BR signaling pathway have been identified by forward and reverse genetics, bringing new ideas into BR signaling regulation mechanisms. 2. BR SIGNALING COMPONENTS IDENTIFIED BY FORWARD GENETICS 2.1. OsBRI1 Phenotypic analysis of d61 suggests the function of endogenous BRs in promoting internode elongation, lamina inclination, and skotomorphogenesis (Yamamuro et al., 2000). It is interesting to note that the second internode of rice appears to be more sensitive to BR-deficiency as the mutant has extremely decreased percentage of the second internode. This phenomenon was also observed in BR-biosynthetic mutants (Hong et al., 2003; Tanabe et al., 2005), and it could be caused by the relatively lower expression levels of BR sensing components, such as OsBRI1 and DLT, in the second internode (Yamamuro et al., 2000; Tong et al., 2009). Observation of a severe d61 allele implied rice root is likely to have an alternative BR sensing system. In d61-4, the most severe allele, the shoot is seriously affected while the root has little phenotype (Nakamura et al., 2006). Expression analyses suggest two homologous OsBRI1-like genes might play, at least, the redundant roles in rice root to sense BRs (Nakamura et al., 2006). Therefore, it seems that the different tissues or organs could have varied BR sensitivities or signaling pathways. 2.2. OsGSK1 In a screening of abiotic stress-related mutants, a T-DNA tagged knock-out mutant of OsGSK1 was identified (Koh
et al., 2007). OsGSK1 encodes a GSK3-like kinase, which is an ortholog of BIN2, the key negative regulator of BR signaling (Li et al., 2001; Choe et al., 2002; Li and Nam, 2002). Thus, it seems that the higher resistance to abiotic stress of the mutant should be conferred by the increased BR sensitivity. Indeed, coleoptiles of the mutant were found to be more sensitive to BR and overexpression of OsGSK1 in Arabidopsis led to plant dwarfism (Koh et al., 2007). However, considering the critical role of BIN2 in BR signaling, further study is essential to determine whether OsGSK1 is the real counterpart of BIN2. 2.3. DLT dlt appears to be a typical BR-deficient mutant and was found insensitive to BR by lamina inclination tests (Tong et al., 2009). However, detailed analyses discovered that dlt has normal skotomorphogenesis as well as normal root response to BR treatment. In addition, dlt has obviously decreased tiller numbers, which has not been quite emphasized in other studies of BR-deficient mutants. Only one report described a severe BR-biosynthetic mutant, brd1, normally develops fewer tillers than wild-type (Mori et al., 2002). Actually, in our observation, both d2 and d11, two BRbiosynthetic mutants (Hong et al., 2003; Tanabe et al., 2005), have decreased tiller numbers, while m107, a D11activation mutant (Wan et al., 2009), develops increased tiller numbers. Although the extent of the changes in these mutants are not comparable to that in dlt, BR should play a role in tiller happening. DLT encodes a GRAS-family protein, famous members of which, including SLR1, MOC1 and SCR, play crucial roles in gibberellin (GA) signaling, tiller happening, and root development, respectively (Di Laurenzio et al., 1996; Ikeda et al., 2001; Li et al., 2003). While DLT has already
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been indicated to function in GA signaling by an independent characterization of an allele mutant d62 (Li et al., 2010), it remains uncertain whether it has a functional connection to MOC1 and SCR1. DLT is localized predominantly in nucleus (unpublished data), which is consistent with the putative function of GRASfamily proteins as transcriptional factor (Bolle, 2004). Three BR-responsive elements (BRREs) were discovered in the promoter region of DLT, and OsBZR1 can bind to at least one of the BRREs in vitro, putting DLT under the control of OsBZR1 (He et al., 2005; Tong et al., 2009). While DLT is repressed by BRs, the inhibition of DLT by OsBZR1 does not match the positive roles of both of them. Thus, it has been presumed that DLT should be regulated by BR through a hypothetic GSK3-kinase at the protein level (Tong and Chu, 2009). 2.4. D1/RGA1 d1 was initially identified as a GA signaling mutant (Ueguchi-Tanaka et al., 2000). However, the specifically shortened second internode, the obviously decreased seed size and the defective skotomorphogenesis strongly suggest that it should also be a BR-related mutant (Wang et al., 2006; Oki et al., 2009). D1 encodes an alpha-subunit of rice heterotrimeric G protein, RGA1. Plant G protein alpha-subunit has been suggested to play important roles in many signaling transduction processes (Suharsono et al., 2002; Joo et al., 2005; Huang et al., 2006; Pandey et al., 2006; Warpeha et al., 2007). Thus, the role of D1/RGA1 in BR signaling pathway might not be specific and D1/RGA1 could be the factor acting at a very upstream position of many different signaling pathways. Double mutant analysis between d1 and d61-7 also suggests that D1/RGA1 should be involved in an alternative BR signaling pathway independent of OsBRI1 (Oki et al., 2009). 2.5. ILI1 and IBH1 ili1-d has a striking phenotype with greatly enlarged leaf angles and is hypersensitive to brassinolide (BL), the most active BR (Zhang et al., 2009). The mutant carries a T-DNA insertion which has activated a flanking HLH protein (bHLH protein lacking basic region) encoding gene, leading to the constitutive BR response phenotype. ILI1 can interact with another bHLH protein, IBH1. However, IBH1 was found to play a negative role in BR signaling, and its expression is inhibited by BRs through OsBZR1, which is just contrasted to ILI1. Thus, it was presumed that ILI1 and IBH1 act antagonistically downstream of OsBZR1 to mediate BR signaling (Zhang et al., 2009). 2.6. RAVL1 RAVL1 encodes a B3-domain protein, and ravl1 mutants show several typical BR-related defects and are insensitive to BRs (Je et al., 2010). Expression analysis discovered that the
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transcript levels of OsBRI1 and several BR-biosynthetic genes are quite well correlated with that of RAVL1. Further promoter binding experiments and genetic analysis strongly suggest a novel mechanism that RAVL1 functions in BR response through directly activating the expression of OsBRI1 and BR biosynthetic genes (Je et al., 2010). Unlike OsBZR1, RAVL1 is not involved in feedback suppression of these genes and it appears to play an important role in ensuring the basal activity of endogenous BRs (Je et al., 2010). 3. BR SIGNALING COMPONENTS IDENTIFIED BY REVERSE GENETICS Genetic screening is not very favorable in rice compared to Arabidopsis, as rice has a larger body, longer growth period and requires appropriate field growth condition. Taking reverse genetics approach, certain interested genes can be studied and evaluated whether they are relative to BR signaling. However, it is a little difficult to connect those BR signaling components identified by reverse genetics to the primary BR signaling pathway. 3.1. OsBZR1 BZR1 and BES1/BZR2, two highly similar transcriptional factors, play critical roles in regulating the expression of downstream BR response genes in Arabidopsis (Wang et al., 2002; Yin et al., 2002; He et al., 2005; Li and Deng, 2005). OsBZR1 was identified by homology comparison, and suppression of OsBZR1 leads to erect rice leaves, BRinsensitive and defective feedback regulation on BRbiosynthetic genes by exogenous BR application (Bai et al., 2007). In addition, similar to that in Arabidopsis, nucleocytoplasmic shuttling of OsBZR1 with the help of 14-3-3 proteins is also essential for BR response (Bai et al., 2007). Thus, although functional mechanisms of OsBZR1 (such as the protein phosphorylation) need to be further testified, OsBZR1 should play a conserved role as BES1/BZR1 in Arabidopsis. 3.2. OsMDP1/OsMADS47, OsMADS22 and OsMADS55 Transgenic analysis of three SVP-group MADS-box genes in rice proved that all of them have negative roles in BR responses. Overexpression or suppression of OsMDP1/ OsMADS47 leads to relevant change of leaf angle and BR sensitivity (Duan et al., 2006; Lee et al., 2008). It should be noted that OsMDP1 does not seem to play a role in root response to BR, although it is indeed involved in root development (Duan et al., 2006). Similar results have been suggested in OsBRI1 and DLT studies (Nakamura et al., 2006; Tong et al., 2009). The three MADS-box genes function redundantly each other, but it appears that OsMADS22 has only a minor supportive function in lamina inclination response to BRs (Lee et al., 2008). As the two studies have got inconsistent results about the BR effects on OsMDP1/OsMADS47
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expression (Duan et al., 2006; Lee et al., 2008), it remains unknown how and where they function in BR signaling pathway. 3.3. OsGSR1 OsGSR1 was firstly identified as a GA induced gene (Wang et al., 2009b). Further analysis revealed that its expression is repressed by BR treatment. Knock-down of OsGSR1 led to the plants having decreased GA sensitivity but increased BR sensitivity, suggesting OsGSR1 might be involved in GA and BR crosstalk (Wang et al., 2009b). Interestingly, screening for OsGSR1 interacted protein by yeast two-hybrid has identified DIM/DWF1, a BR-biosynthetic enzyme (Hong et al., 2005), and the interaction was further proved by both in vitro and in vivo experiments. Thus, OsGSR1 might be involved in BR signaling through regulating DIM1/DWF1 activity (Wang et al., 2009b). 3.4. OsBU1 Many bHLH proteins have been found involved in BR response, including ILI1 and IBH1 in rice (Friedrichsen et al., 2002; Yin et al., 2005; Chandler et al., 2009; Wang et al., 2009a; Zhang et al., 2009). OsBU1, encoding an HLH protein homologous to ILI1, was isolated as a BR-upregulated gene in a microarray analysis (Tanaka et al., 2009). Overexpression of OsBU1 leads to greatly enlarged leaf angle and increased seed size. Genetic analysis suggests OsBU1 acts downstream of OsBRI1 (Tanaka et al., 2009). Interestingly, in d1 mutant, OsBU1 is obviously less induced by BR, indicating OsBU1 is downstream of D1/RGA1 in BR response (Tanaka et al., 2009). As D1 and OsBRI1 have been implicated function independent of the upstream BR signaling pathways (Oki et al., 2009), OsBU1 could be regarded as a much downstream integrator of BR responses. 3.5. OsBAK1 OsBAK1 is the closest relative of AtBAK1 and thus was characterized using biochemistry, genetic and transgenic approaches (Li et al., 2009). OsBAK1 can directly interact with OsBRI1, and overexpression of OsBAK1 can suppress both Arabidopsis bri1-5 and rice d61-1 mutants (Li et al., 2009). Also, OsBAK1-overexpressors are hypersensitive to BRs, while suppression of OsBAK1 leads to mild BRinsensitive phenotype (Li et al., 2009). These results strongly suggest OsBAK1 has a conserved role in rice as in Arabidopsis. 3.6. Other BR response genes High throughput analyses have identified a number of BRresponsive genes including OsBU1, OsGSR1, OsBLE1, OsBLE2, and OsCKI1 etc. (Liu et al., 2003; Yang et al., 2003; Yang and Komatsu, 2004; Tanaka et al., 2009; Wang et al., 2009b). Except that OsBU1 has been found specifically
induced by BL (Tanaka et al., 2009), others are generally regulated by more than one hormone. Although most of these genes are taken for granted to be downstream targets of transcriptional factors, they could also probably function in BR signaling pathway like OsBU1. Detailed studies of these genes will greatly improve our knowledge of the BR functional mechanisms. 4. APPLICATION OF BR GENES IN RICE BREEDING BR was known to control many agronomic traits, such as seed germination, flowering time, stress tolerance, seed size, leaf erectness and plant height. Early in 1980’s, BR was already widely used as a plant growth modulator in agriculture (Ikekawa and Zhao, 1991; Khripach et al., 2000). After identifying BR-biosynthetic and signaling genes, a number of studies have verified the huge potential of modulating expression of BR genes in plant breeding (Divi and Krishna, 2009). In rice, these applications have mainly taken the advantage of the compact structure of weak BR-deficient mutants. osdwarf4-1 has a very weak phenotype because OsDWARF4 functions redundantly to its homolog D11 (Sakamoto et al., 2006). Rice leaf angle is very sensitive to BR level, so osdwarf4-1 has only obviously erected leaves but no other alterations. At high plant density, erect leaf can effectively avoid leaf shading, enhance light capture efficiency, and finally increase grain yield (Sakamoto et al., 2006). A similar strategy was used on BR receptor gene OsBRI1. At high plant density, d61-7, the weakest allele of d61 mutant, has its biomass increased about 35% compared to wild-type, but without increased grain yield due to small grains of the mutant (Morinaka et al., 2006). Further, much weaker BRdeficient transgenic rice was obtained by partial suppression of OsBRI1 expression, which has only erect leaves but without changes in grain size, finally making about 30% higher grain yield than wild-type at high plant density (Morinaka et al., 2006). OsBAK1 was considered as a good target used to engineer for high yield as it acts as a co-receptor of OsBRI1 and should not be crucial for BR signaling (Li et al., 2009). Similarly, by antisense suppression of OsBAK1, desired weak BR-deficient transgenic plants with erect leaves and normal reproduction were obtained, and were thought to have potential in increasing yield at high plant density (Li et al., 2009). An opposite way has been taken to enhance rice yield by improvement of BR level through overexpressing a BRbiosynthetic gene encoding sterol C-22 hydroxylases (Wu et al., 2008). The transgenic plants grow much better than wild-type, with higher stature, thick culm, longer leaf, enlarged leaf angles and more tillers, which is consistent with the higher photosynthetic efficiency and increased sugar content in leaves. The plants also bear obviously large seeds, leading to a higher yield. Because a vegetative tissue specific promoter was used, the increase of seed size should be resulted by the acceleration of grain filling that BR regulates (Wu et al., 2008).
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Function of BR in stress tolerance has been suggested by the better performance of plant under stress with BR treatment and BR regulated expression of stress-related genes (Dhaubhadel et al., 1999; Krishna, 2003; Kagale et al., 2007). Consistently, knock-out mutant of OsGSK1, a rice ortholog of BR negative regulator BIN2, has obviously better performance under different stress conditions (Koh et al., 2007). In addition, a subset of stress responsive genes has increased expression in the mutant, suggesting OsGSK1 is involved in anti-stress process (Koh et al., 2007). However, present data show weak evidence for the role of OsGSK1 in rice BR signaling. Thus, further stress treatment studies using either BRbiosynthetic mutants or BR signaling mutants are essential to make certain of the role of BR in stress tolerance. 5. PERSPECTIVE The identification of OsBRI1, OsBAK1, OsGSK1, and OsBZR1 suggest that rice should share a conserved primary pathway with Arabidopsis (Yamamuro et al., 2000; Bai et al., 2007; Li et al., 2009). However, other components, such as the presumed cytoplasmic OsBSK1 and OsBSU1 (Kim et al., 2009; Kim and Wang, 2010), remain to be identified in rice. In addition, a number of questions remain to be addressed concerning BR signaling pathway. Is OsGSK1 the real counterpart of BIN2? How is the BR response released? What are the proteins responsible for the degradation of BIN2, BZR1, and BES1 (Peng et al., 2008; Kim et al., 2009)? Are there other BR signaling pathways and how do they work? Does BIN2 have other substrates except BZR1, BES1 and ARF2 (Zhao et al., 2002; Vert et al., 2008)? How are the phosphorylation specificities determined? Although it is yet far from drawing a complete rice BR signaling transduction pathway, the tough problem in rice BR signaling research is not only to identify more components, but also how these components function and integrate, and how they are regulated at different levels, especially at the protein level, which is not well studied in the function characterization of most rice genes. To achieve this goal, biochemistry studies of those known components or screening for those unidentified proteins by approaches such as yeast two-hybrid and proteomics will be helpful. Considering rice as an important crop plant as well as the model plant for monocot and BR as an important plant hormone with great potential in biotechnology, elucidation of BR signaling and BR response mechanisms is particularly significant in rice. With more attentions been paid on rice BR studies recently, we will expect a rapid progress on understanding rice BR signaling pathway and further application of the BR knowledge in rice breeding. ACKNOWLEDGEMENTS This work was supported by grants from the National Natural Science Foundation of China (Nos. 31170715, 30825029, and 30621001) and the Ministry of Agriculture of China (No. 2011ZX08009-003).
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Warpeha, K.M., Upadhyay, S., Yeh, J., Adamiak, J., Hawkins, S.I., Lapik, Y.R., Anderson, M.B., Kaufman, L.S., 2007. The GCR1, GPA1, PRN1, NF-Y signal chain mediates both blue light and abscisic acid responses in Arabidopsis. Plant Physiol. 143, 1590e1600. Wu, C.Y., Trieu, A., Radhakrishnan, P., Kwok, S.F., Harris, S., Zhang, K., Wang, J., Wan, J., Zhai, H., Takatsuto, S., Matsumoto, S., Fujioka, S., Feldmann, K.A., Pennell, R.I., 2008. Brassinosteroids regulate grain filling in rice. Plant Cell 20, 2130e2145. Wu, G., Wang, X.L., Li, X.B., Kamiya, Y.J., Otegui, M.S., Chory, J., 2011. Methylation of a phosphatase specifies dephosphorylation and degradation of activated brassinosteroid receptors. Sci. Signal. 4, ra29. Yamamuro, C., Ihara, Y., Wu, X., Noguchi, T., Fujioka, S., Takatsuto, S., Ashikari, M., Kitano, H., Matsuoka, M., 2000. Loss of function of a rice brassinosteroid insensitive1 homolog prevents internode elongation and bending of the lamina joint. Plant Cell 12, 1591e1606. Yang, C.J., Zhang, C., Lu, Y.N., Jin, J.Q., Wang, X.L., 2011. The mechanisms of brassinosteroids’ action: from signal transduction to plant development. Mol. Plant 4, 588e600. Yang, G., Matsuoka, M., Iwasaki, Y., Komatsu, S., 2003. A novel brassinolideenhanced gene identified by cDNA microarray is involved in the growth of rice. Plant Mol. Biol. 52, 843e854. Yang, G.X., Komatsu, S., 2004. Molecular cloning and characterization of a novel brassinolide enhanced gene OsBLE1 in Oryza sativa seedlings. Plant Physiol. Biochem. 42, 1e6. Yin, Y., Vafeados, D., Tao, Y., Yoshida, S., Asami, T., Chory, J., 2005. A new class of transcription factors mediates brassinosteroid-regulated gene expression in Arabidopsis. Cell 120, 249e259. Yin, Y., Wang, Z.Y., Mora-Garcia, S., Li, J., Yoshida, S., Asami, T., Chory, J., 2002. BES1 accumulates in the nucleus in response to brassinosteroids to regulate gene expression and promote stem elongation. Cell 109, 181e191. Yu, X.F., Li, L., Zola, J., Aluru, M., Ye, H.X., Foudree, A., Guo, H.Q., Anderson, S., Aluru, S., Liu, P., Rodermel, S., Yin, Y.H., 2011. A brassinosteroid transcriptional network revealed by genome-wide identification of BESI target genes in Arabidopsis thaliana. Plant J. 65, 634e646. Zhang, L.Y., Bai, M.Y., Wu, J., Zhu, J.Y., Wang, H., Zhang, Z., Wang, W., Sun, Y., Zhao, J., Sun, X., Yang, H., Xu, Y., Kim, S.H., Fujioka, S., Lin, W.H., Chong, K., Lu, T., Wang, Z.Y., 2009. Antagonistic HLH/bHLH transcription factors mediate brassinosteroid regulation of cell elongation and plant development in rice and Arabidopsis. Plant Cell 21, 3767e3780. Zhao, J., Peng, P., Schmitz, R.J., Decker, A.D., Tax, F.E., Li, J., 2002. Two putative BIN2 substrates are nuclear components of brassinosteroid signaling. Plant Physiol. 130, 1221e1229.
Journal of Genetics and Genomics 39 (2012) 3e9
JGG REVIEW
Brassinosteroid Signaling and Application in Rice Hongning Tong, Chengcai Chu* State Key Laboratory of Plant Genomics and Center for Plant Gene Research (Beijing), Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China Received 28 October 2011; revised 14 December 2011; accepted 14 December 2011 Available online 20 December 2011
ABSTRACT Combined approaches with genetics, biochemistry, and proteomics studies have greatly advanced our understanding of brassinosteroid (BR) signaling in Arabidopsis. However, in rice, a model plant of monocot and as well an important crop plant, BR signaling is not as well characterized as in Arabidopsis. Recent studies by forward and reverse genetics have identified a number of either conserved or specific components of rice BR signaling pathway, bringing new ideas into BR signaling regulation mechanisms. Genetic manipulation of BR level or BR sensitivity to improve rice yield has established the great significance of BR research achievements. KEYWORDS: Brassinosteroid; BR signaling; Rice; Yield
1. INTRODUCTION Brassinosteroids (BRs) are a class of steroid hormones involved in diverse biological processes. In recent years, rapid progress has been made in BR signaling pathway in Arabidopsis (Fig. 1) (Clouse, 2002; Li and Jin, 2007; Kim and Wang, 2010; Clouse, 2011; Yang et al., 2011). In the absence of BR, BKI1 binds membrane-located BR receptor BRI1 and inhibits its function (Wang and Chory, 2006). In the presence of BR, BR binding leads to BRI1 dissociation with BKI1, BRI1 autophosphorylation and association/transphosphorylation with BAK1 kinase (Li et al., 2002; Nam and Li, 2002; Kinoshita et al., 2005; Wang et al., 2005a, 2005b). The hormone signal is transduced through CDG1 and BSK1 kinases and then BSU1 phosphatase to repress the negative regulator BIN2 kinase, as well somehow activate PP2A phosphatase (Li et al., 2001; Choe et al., 2002; Li and Nam, 2002; Mora-Garcia et al., 2004; Tang et al., 2008, 2011; Kim et al., 2009). Inhibition of BIN2 and activation of PP2A will promote the transformation of phosphorylated BZR1 and BES1 to dephosphorylated forms, which subsequently transfer * Corresponding author. Tel/fax: þ86 10 6487 7570. E-mail address: [email protected] (C. Chu).
to nucleus and form diverse transcriptional complexes to regulate a large number of BR-responsive genes (He et al., 2002; Wang et al., 2002; Yin et al., 2002, 2005; Vert and Chory, 2006; Ryu et al., 2007; Peng et al., 2010; Sun et al., 2010; Tang et al., 2011; Yu et al., 2011). This primary signaling pathway tends to be regulated by complicated mechanisms. For example, at transcriptional level, BRI1 is negatively feedback regulated by BZR1 and BES1 (Sun et al., 2010; Yu et al., 2011), whereas at protein level, BRI1 is also negatively feedback regulated by PP2A (Di Rubbo et al., 2011; Wu et al., 2011). In addition, BIN2 and BZR1 were suggested to be regulated by proteasome-mediated degradation processes (He et al., 2002; Peng et al., 2008). Early in 1980’s, physiological experiment has proved the active role of BRs in promoting rice leaf blade bending (Wada et al., 1981). The tests using either dark-grown or light-grown lamina inclination responsive to BRs have been developed (Takeno and Pharis, 1982; Wada et al., 1984) and were widely used to characterize the sensitivity of rice mutants or varieties to BRs. However, the in vivo roles of BRs in rice growth and development begin to be unveiled until the identification of the BR-deficient mutants, d61, which carry mutations in a gene orthologous to BR receptor gene BRI1 (Yamamuro et al., 2000). Recently, a number of either conserved or specific
1673-8527/$ - see front matter Copyright Ó 2012, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Limited and Science Press. All rights reserved. doi:10.1016/j.jgg.2011.12.001
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H. Tong, C. Chu / Journal of Genetics and Genomics 39 (2012) 3e9
Fig. 1. Integrated diagrams of BR signaling pathway in Arabidopsis (A) and rice (B). For BR signaling in Arabidopsis, only the primary components mentioned in this article are involved. Arrows show promotion actions and bar ends show inhibitory actions. Solid lines show direct regulation, dotted lines indicate hypothetical regulation, and curve lines indicate feedback regulations. Roles of OsGSK1 need to be further testified.
components of rice BR signaling pathway have been identified by forward and reverse genetics, bringing new ideas into BR signaling regulation mechanisms. 2. BR SIGNALING COMPONENTS IDENTIFIED BY FORWARD GENETICS 2.1. OsBRI1 Phenotypic analysis of d61 suggests the function of endogenous BRs in promoting internode elongation, lamina inclination, and skotomorphogenesis (Yamamuro et al., 2000). It is interesting to note that the second internode of rice appears to be more sensitive to BR-deficiency as the mutant has extremely decreased percentage of the second internode. This phenomenon was also observed in BR-biosynthetic mutants (Hong et al., 2003; Tanabe et al., 2005), and it could be caused by the relatively lower expression levels of BR sensing components, such as OsBRI1 and DLT, in the second internode (Yamamuro et al., 2000; Tong et al., 2009). Observation of a severe d61 allele implied rice root is likely to have an alternative BR sensing system. In d61-4, the most severe allele, the shoot is seriously affected while the root has little phenotype (Nakamura et al., 2006). Expression analyses suggest two homologous OsBRI1-like genes might play, at least, the redundant roles in rice root to sense BRs (Nakamura et al., 2006). Therefore, it seems that the different tissues or organs could have varied BR sensitivities or signaling pathways. 2.2. OsGSK1 In a screening of abiotic stress-related mutants, a T-DNA tagged knock-out mutant of OsGSK1 was identified (Koh
et al., 2007). OsGSK1 encodes a GSK3-like kinase, which is an ortholog of BIN2, the key negative regulator of BR signaling (Li et al., 2001; Choe et al., 2002; Li and Nam, 2002). Thus, it seems that the higher resistance to abiotic stress of the mutant should be conferred by the increased BR sensitivity. Indeed, coleoptiles of the mutant were found to be more sensitive to BR and overexpression of OsGSK1 in Arabidopsis led to plant dwarfism (Koh et al., 2007). However, considering the critical role of BIN2 in BR signaling, further study is essential to determine whether OsGSK1 is the real counterpart of BIN2. 2.3. DLT dlt appears to be a typical BR-deficient mutant and was found insensitive to BR by lamina inclination tests (Tong et al., 2009). However, detailed analyses discovered that dlt has normal skotomorphogenesis as well as normal root response to BR treatment. In addition, dlt has obviously decreased tiller numbers, which has not been quite emphasized in other studies of BR-deficient mutants. Only one report described a severe BR-biosynthetic mutant, brd1, normally develops fewer tillers than wild-type (Mori et al., 2002). Actually, in our observation, both d2 and d11, two BRbiosynthetic mutants (Hong et al., 2003; Tanabe et al., 2005), have decreased tiller numbers, while m107, a D11activation mutant (Wan et al., 2009), develops increased tiller numbers. Although the extent of the changes in these mutants are not comparable to that in dlt, BR should play a role in tiller happening. DLT encodes a GRAS-family protein, famous members of which, including SLR1, MOC1 and SCR, play crucial roles in gibberellin (GA) signaling, tiller happening, and root development, respectively (Di Laurenzio et al., 1996; Ikeda et al., 2001; Li et al., 2003). While DLT has already
H. Tong, C. Chu / Journal of Genetics and Genomics 39 (2012) 3e9
been indicated to function in GA signaling by an independent characterization of an allele mutant d62 (Li et al., 2010), it remains uncertain whether it has a functional connection to MOC1 and SCR1. DLT is localized predominantly in nucleus (unpublished data), which is consistent with the putative function of GRASfamily proteins as transcriptional factor (Bolle, 2004). Three BR-responsive elements (BRREs) were discovered in the promoter region of DLT, and OsBZR1 can bind to at least one of the BRREs in vitro, putting DLT under the control of OsBZR1 (He et al., 2005; Tong et al., 2009). While DLT is repressed by BRs, the inhibition of DLT by OsBZR1 does not match the positive roles of both of them. Thus, it has been presumed that DLT should be regulated by BR through a hypothetic GSK3-kinase at the protein level (Tong and Chu, 2009). 2.4. D1/RGA1 d1 was initially identified as a GA signaling mutant (Ueguchi-Tanaka et al., 2000). However, the specifically shortened second internode, the obviously decreased seed size and the defective skotomorphogenesis strongly suggest that it should also be a BR-related mutant (Wang et al., 2006; Oki et al., 2009). D1 encodes an alpha-subunit of rice heterotrimeric G protein, RGA1. Plant G protein alpha-subunit has been suggested to play important roles in many signaling transduction processes (Suharsono et al., 2002; Joo et al., 2005; Huang et al., 2006; Pandey et al., 2006; Warpeha et al., 2007). Thus, the role of D1/RGA1 in BR signaling pathway might not be specific and D1/RGA1 could be the factor acting at a very upstream position of many different signaling pathways. Double mutant analysis between d1 and d61-7 also suggests that D1/RGA1 should be involved in an alternative BR signaling pathway independent of OsBRI1 (Oki et al., 2009). 2.5. ILI1 and IBH1 ili1-d has a striking phenotype with greatly enlarged leaf angles and is hypersensitive to brassinolide (BL), the most active BR (Zhang et al., 2009). The mutant carries a T-DNA insertion which has activated a flanking HLH protein (bHLH protein lacking basic region) encoding gene, leading to the constitutive BR response phenotype. ILI1 can interact with another bHLH protein, IBH1. However, IBH1 was found to play a negative role in BR signaling, and its expression is inhibited by BRs through OsBZR1, which is just contrasted to ILI1. Thus, it was presumed that ILI1 and IBH1 act antagonistically downstream of OsBZR1 to mediate BR signaling (Zhang et al., 2009). 2.6. RAVL1 RAVL1 encodes a B3-domain protein, and ravl1 mutants show several typical BR-related defects and are insensitive to BRs (Je et al., 2010). Expression analysis discovered that the
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transcript levels of OsBRI1 and several BR-biosynthetic genes are quite well correlated with that of RAVL1. Further promoter binding experiments and genetic analysis strongly suggest a novel mechanism that RAVL1 functions in BR response through directly activating the expression of OsBRI1 and BR biosynthetic genes (Je et al., 2010). Unlike OsBZR1, RAVL1 is not involved in feedback suppression of these genes and it appears to play an important role in ensuring the basal activity of endogenous BRs (Je et al., 2010). 3. BR SIGNALING COMPONENTS IDENTIFIED BY REVERSE GENETICS Genetic screening is not very favorable in rice compared to Arabidopsis, as rice has a larger body, longer growth period and requires appropriate field growth condition. Taking reverse genetics approach, certain interested genes can be studied and evaluated whether they are relative to BR signaling. However, it is a little difficult to connect those BR signaling components identified by reverse genetics to the primary BR signaling pathway. 3.1. OsBZR1 BZR1 and BES1/BZR2, two highly similar transcriptional factors, play critical roles in regulating the expression of downstream BR response genes in Arabidopsis (Wang et al., 2002; Yin et al., 2002; He et al., 2005; Li and Deng, 2005). OsBZR1 was identified by homology comparison, and suppression of OsBZR1 leads to erect rice leaves, BRinsensitive and defective feedback regulation on BRbiosynthetic genes by exogenous BR application (Bai et al., 2007). In addition, similar to that in Arabidopsis, nucleocytoplasmic shuttling of OsBZR1 with the help of 14-3-3 proteins is also essential for BR response (Bai et al., 2007). Thus, although functional mechanisms of OsBZR1 (such as the protein phosphorylation) need to be further testified, OsBZR1 should play a conserved role as BES1/BZR1 in Arabidopsis. 3.2. OsMDP1/OsMADS47, OsMADS22 and OsMADS55 Transgenic analysis of three SVP-group MADS-box genes in rice proved that all of them have negative roles in BR responses. Overexpression or suppression of OsMDP1/ OsMADS47 leads to relevant change of leaf angle and BR sensitivity (Duan et al., 2006; Lee et al., 2008). It should be noted that OsMDP1 does not seem to play a role in root response to BR, although it is indeed involved in root development (Duan et al., 2006). Similar results have been suggested in OsBRI1 and DLT studies (Nakamura et al., 2006; Tong et al., 2009). The three MADS-box genes function redundantly each other, but it appears that OsMADS22 has only a minor supportive function in lamina inclination response to BRs (Lee et al., 2008). As the two studies have got inconsistent results about the BR effects on OsMDP1/OsMADS47
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expression (Duan et al., 2006; Lee et al., 2008), it remains unknown how and where they function in BR signaling pathway. 3.3. OsGSR1 OsGSR1 was firstly identified as a GA induced gene (Wang et al., 2009b). Further analysis revealed that its expression is repressed by BR treatment. Knock-down of OsGSR1 led to the plants having decreased GA sensitivity but increased BR sensitivity, suggesting OsGSR1 might be involved in GA and BR crosstalk (Wang et al., 2009b). Interestingly, screening for OsGSR1 interacted protein by yeast two-hybrid has identified DIM/DWF1, a BR-biosynthetic enzyme (Hong et al., 2005), and the interaction was further proved by both in vitro and in vivo experiments. Thus, OsGSR1 might be involved in BR signaling through regulating DIM1/DWF1 activity (Wang et al., 2009b). 3.4. OsBU1 Many bHLH proteins have been found involved in BR response, including ILI1 and IBH1 in rice (Friedrichsen et al., 2002; Yin et al., 2005; Chandler et al., 2009; Wang et al., 2009a; Zhang et al., 2009). OsBU1, encoding an HLH protein homologous to ILI1, was isolated as a BR-upregulated gene in a microarray analysis (Tanaka et al., 2009). Overexpression of OsBU1 leads to greatly enlarged leaf angle and increased seed size. Genetic analysis suggests OsBU1 acts downstream of OsBRI1 (Tanaka et al., 2009). Interestingly, in d1 mutant, OsBU1 is obviously less induced by BR, indicating OsBU1 is downstream of D1/RGA1 in BR response (Tanaka et al., 2009). As D1 and OsBRI1 have been implicated function independent of the upstream BR signaling pathways (Oki et al., 2009), OsBU1 could be regarded as a much downstream integrator of BR responses. 3.5. OsBAK1 OsBAK1 is the closest relative of AtBAK1 and thus was characterized using biochemistry, genetic and transgenic approaches (Li et al., 2009). OsBAK1 can directly interact with OsBRI1, and overexpression of OsBAK1 can suppress both Arabidopsis bri1-5 and rice d61-1 mutants (Li et al., 2009). Also, OsBAK1-overexpressors are hypersensitive to BRs, while suppression of OsBAK1 leads to mild BRinsensitive phenotype (Li et al., 2009). These results strongly suggest OsBAK1 has a conserved role in rice as in Arabidopsis. 3.6. Other BR response genes High throughput analyses have identified a number of BRresponsive genes including OsBU1, OsGSR1, OsBLE1, OsBLE2, and OsCKI1 etc. (Liu et al., 2003; Yang et al., 2003; Yang and Komatsu, 2004; Tanaka et al., 2009; Wang et al., 2009b). Except that OsBU1 has been found specifically
induced by BL (Tanaka et al., 2009), others are generally regulated by more than one hormone. Although most of these genes are taken for granted to be downstream targets of transcriptional factors, they could also probably function in BR signaling pathway like OsBU1. Detailed studies of these genes will greatly improve our knowledge of the BR functional mechanisms. 4. APPLICATION OF BR GENES IN RICE BREEDING BR was known to control many agronomic traits, such as seed germination, flowering time, stress tolerance, seed size, leaf erectness and plant height. Early in 1980’s, BR was already widely used as a plant growth modulator in agriculture (Ikekawa and Zhao, 1991; Khripach et al., 2000). After identifying BR-biosynthetic and signaling genes, a number of studies have verified the huge potential of modulating expression of BR genes in plant breeding (Divi and Krishna, 2009). In rice, these applications have mainly taken the advantage of the compact structure of weak BR-deficient mutants. osdwarf4-1 has a very weak phenotype because OsDWARF4 functions redundantly to its homolog D11 (Sakamoto et al., 2006). Rice leaf angle is very sensitive to BR level, so osdwarf4-1 has only obviously erected leaves but no other alterations. At high plant density, erect leaf can effectively avoid leaf shading, enhance light capture efficiency, and finally increase grain yield (Sakamoto et al., 2006). A similar strategy was used on BR receptor gene OsBRI1. At high plant density, d61-7, the weakest allele of d61 mutant, has its biomass increased about 35% compared to wild-type, but without increased grain yield due to small grains of the mutant (Morinaka et al., 2006). Further, much weaker BRdeficient transgenic rice was obtained by partial suppression of OsBRI1 expression, which has only erect leaves but without changes in grain size, finally making about 30% higher grain yield than wild-type at high plant density (Morinaka et al., 2006). OsBAK1 was considered as a good target used to engineer for high yield as it acts as a co-receptor of OsBRI1 and should not be crucial for BR signaling (Li et al., 2009). Similarly, by antisense suppression of OsBAK1, desired weak BR-deficient transgenic plants with erect leaves and normal reproduction were obtained, and were thought to have potential in increasing yield at high plant density (Li et al., 2009). An opposite way has been taken to enhance rice yield by improvement of BR level through overexpressing a BRbiosynthetic gene encoding sterol C-22 hydroxylases (Wu et al., 2008). The transgenic plants grow much better than wild-type, with higher stature, thick culm, longer leaf, enlarged leaf angles and more tillers, which is consistent with the higher photosynthetic efficiency and increased sugar content in leaves. The plants also bear obviously large seeds, leading to a higher yield. Because a vegetative tissue specific promoter was used, the increase of seed size should be resulted by the acceleration of grain filling that BR regulates (Wu et al., 2008).
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Function of BR in stress tolerance has been suggested by the better performance of plant under stress with BR treatment and BR regulated expression of stress-related genes (Dhaubhadel et al., 1999; Krishna, 2003; Kagale et al., 2007). Consistently, knock-out mutant of OsGSK1, a rice ortholog of BR negative regulator BIN2, has obviously better performance under different stress conditions (Koh et al., 2007). In addition, a subset of stress responsive genes has increased expression in the mutant, suggesting OsGSK1 is involved in anti-stress process (Koh et al., 2007). However, present data show weak evidence for the role of OsGSK1 in rice BR signaling. Thus, further stress treatment studies using either BRbiosynthetic mutants or BR signaling mutants are essential to make certain of the role of BR in stress tolerance. 5. PERSPECTIVE The identification of OsBRI1, OsBAK1, OsGSK1, and OsBZR1 suggest that rice should share a conserved primary pathway with Arabidopsis (Yamamuro et al., 2000; Bai et al., 2007; Li et al., 2009). However, other components, such as the presumed cytoplasmic OsBSK1 and OsBSU1 (Kim et al., 2009; Kim and Wang, 2010), remain to be identified in rice. In addition, a number of questions remain to be addressed concerning BR signaling pathway. Is OsGSK1 the real counterpart of BIN2? How is the BR response released? What are the proteins responsible for the degradation of BIN2, BZR1, and BES1 (Peng et al., 2008; Kim et al., 2009)? Are there other BR signaling pathways and how do they work? Does BIN2 have other substrates except BZR1, BES1 and ARF2 (Zhao et al., 2002; Vert et al., 2008)? How are the phosphorylation specificities determined? Although it is yet far from drawing a complete rice BR signaling transduction pathway, the tough problem in rice BR signaling research is not only to identify more components, but also how these components function and integrate, and how they are regulated at different levels, especially at the protein level, which is not well studied in the function characterization of most rice genes. To achieve this goal, biochemistry studies of those known components or screening for those unidentified proteins by approaches such as yeast two-hybrid and proteomics will be helpful. Considering rice as an important crop plant as well as the model plant for monocot and BR as an important plant hormone with great potential in biotechnology, elucidation of BR signaling and BR response mechanisms is particularly significant in rice. With more attentions been paid on rice BR studies recently, we will expect a rapid progress on understanding rice BR signaling pathway and further application of the BR knowledge in rice breeding. ACKNOWLEDGEMENTS This work was supported by grants from the National Natural Science Foundation of China (Nos. 31170715, 30825029, and 30621001) and the Ministry of Agriculture of China (No. 2011ZX08009-003).
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