J Plant Res (2015) 128:995–1005 DOI 10.1007/s10265-015-0755-2
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Identification and characterization of TIFY family genes in Brachypodium distachyon Lihua Zhang1 · Jun You1 · Zhulong Chan1
Received: 8 June 2015 / Accepted: 30 July 2015 / Published online: 30 September 2015 © The Botanical Society of Japan and Springer Japan 2015
Abstract The TIFY family is a plant-specific gene family encoding proteins characterized by a conserved TIFY domain. This family encodes four subfamilies of proteins, including ZIM-like (ZML), TIFY, PPD and JASMONATE ZIM-Domain (JAZ) proteins. TIFY proteins play important roles in plant development and stress responses. In this study, 21 BdTIFYs were identified in Brachypodium distachyon through genome-wide analysis, including 15 JAZ and 6 ZML genes. Analysis of the distribution of conserved domains showed that there are three additional domains (CCT domain, GATA domain and Jas domain) in the BdTIFY proteins besides the TIFY domain. Phylogenetic analysis indicated that these 21 proteins were classified into two major groups. Expression profile of BdTIFY genes in response to abiotic stresses and phytohormones was analyzed using quantitative real-time RT-PCR. Among 21 BdTIFY genes, 12 of them were induced by JA treatment, and 4 of them were induced by ABA treatment. Most of BdTIFY genes were responsive to one or more abiotic stresses including drought, salinity, low temperature and heat. Especially, BdTIFY5, 9a, 9b, 10c and 11a were significantly up-regulated by multiple abiotic stresses. These
L. Zhang and J. You have contributed equally to this work. Electronic supplementary material The online version of this article (doi:10.1007/s10265-015-0755-2) contains supplementary material, which is available to authorized users. * Zhulong Chan
[email protected] 1
Key Laboratory of Plant Germplasm Enhancement and Specialty Agriculture, Wuhan Botanical Garden, Chinese Academy of Sciences, Wuhan 430074, China
results provided important clues for functional analysis of TIFY family genes in B. distachyon. Keywords Brachypodium distachyon · TIFY · JAZ · ZIM · Abiotic stress · Expression profile
Introduction TIFY is a plant-specific gene family that was firstly characterized in Arabidopsis (Vanholme et al. 2007). This family owes its name to a conserved motif TIF[F/Y]XG located in TIFY domain which can be divided into four groups based on structural and phylogenetic analyses. The TIFY domain approximately contains 36 amino acids, but exhibits variant forms including TII[F/Y]XG, TIS[F/Y]XG, TLF[F/Y]XG, TLL[F/Y]XG, TLS[F/Y]XG, TLV[F/Y]XG, TMF[F/Y]XG and VIF[F/Y]XG (Bai et al. 2011; Vanholme et al. 2007). The TIFY family is comprised of four major groups, including the Zinc-finger expressed in Inflorescence Meristem (ZIM)/ZIM-like (ZML), TIFY, PEAPOD (PPD) and JAZ subfamilies (Staswick 2008). ZIM/ZML proteins contain a C2C2-GATA zinc-finger DNA-binding domain and a CCT domain (CONSTANS, CO-like, TOC1), while both PPD and JAZ protein subfamilies are short of GATA and CCT domains (Chung et al. 2009). The JAZ subfamily contains a conserved Jas domain besides the TIFY domain, which is a sequence of approximately 27 amino acids near the C-terminus (Chung et al. 2009; Staswick 2008). The PPD protein has a particular N-terminal PPD domain which lacks the conserved PY in its C-terminus when compared with Jas motif (Chung et al. 2009). Finally, proteins from TIFY subfamily contain nothing but the TIFY domain (Staswick 2008).
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In Arabidopsis and rice, 18 and 20 TIFY proteins were identified, respectively (Vanholme et al. 2007; Ye et al. 2009). To date, there is a paucity of functional analyses of TIFY proteins in Arabidopsis and rice. The expression of AtTIFY1/AtZIM was detected in all tissues (Nishii et al. 2000) and over-expression of AtTIFY1/AtZIM resulted in petiole and hypocotyl elongation because of increased cell elongation, which was independent of gibberellin and brassinosteroids (Shikata et al. 2004). Loss-of-function mutations of AtTIFY4a/AtPPD1 and its closest homolog AtTIFY4b/AtPPD2 caused multiple phenotypic changes including leaf shape, silique length, and stomata number (White 2006). Recently, JAZ subfamily proteins have been investigated as key regulators of jasmonate (JA) response in Arabidopsis. In the absence of JA, JAZ proteins repress the activity of MYC transcription factors through interaction with MYCs (e.g.MYC2 and MYC3), which regulate the expression of early response genes (Bai et al. 2011; Melotto et al. 2008; Thines et al. 2007; Yan et al. 2007). In response to developmental or environmental cues that activate JA synthesis, high levels of JA promote SCFCOI1-mediated ubiquitination and activate subsequent degradation of JAZs via the 26S proteasomes. Following its release from JAZ-mediated repression, MYC2 positively regulates the expression of primary JA-responsive genes, including JAZ genes, which contain a G-box in the promoter region (Chung et al. 2009). Interestingly, both developmental and environmental cues induce the expression of JAZ genes, indicating that JA-induced JAZ expression may establish a negative feedback loop by binding MYC2 and attenuating expression of JA response genes (Chini et al. 2007; Katsir et al. 2008; Thines et al. 2007). Over-expression of truncated JAZ proteins, including AtTIFY6b/JAZ3 and AtTIFY9/ JAZ10, resulted in a jasmonate-insensitive phenotype (Chini et al. 2007; Chung and Howe 2009; Thines et al. 2007; Yan et al. 2007). In rice, Cai et al. (2014) reported that OsTIFY3/OsJAZ1 has an important role in spikelet development through interaction with OsCOI1b and OsMYC2 at the reproductive stage. Another rice JAZ gene, OsTIFY11b/OsJAZ10 was identified as a growth-promoting gene that increased grain-size through enhanced accumulation of carbohydrates in the stem (Hakata et al. 2012). In addition to the roles in plant development and phytohormone responses, many TIFY proteins also function in environmental stresses, such as drought, low temperature and salinity (Bai et al. 2011; Chung et al. 2008; Ye et al. 2009). AtTIFY10a, 10b and their homolog in wild soybean, GsTIFY10a, were positive regulators of plant alkaline stress (Zhu et al. 2014). Over-expression of OsTIFY11a/OsJAZ9 showed a highly increased tolerance to salt and dehydration stresses in rice (Ye et al. 2009). OsTIFY11a/OsJAZ9 and OsTIFY11c/OsJAZ11 are involved in
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J Plant Res (2015) 128:995–1005
salt tolerance through interaction with RSS3 and regulating the expression of JA-induced genes (Toda et al. 2013). Recently, OsTIFY11a/OsJAZ9 was characterized as a transcriptional regulator by forming a transcriptional regulation complex with OsNINJA and OsbHLH062 to fine tune the expression of JA-responsive genes involved in salt stress tolerance in rice (Wu et al. 2015). In other studies, Seo et al. (2011) reported that OsJAZ1 [corresponding to OsTIFY11d/OsJAZ12 reported by Ye et al. (2009)] acted as a transcriptional regulator of the OsbHLH148-mediated JA signaling pathway leading to drought tolerance in rice. OsTIFY10c/OsJAZ8 was reported as a repressor of JA signaling and plays an important role in resistance to bacterial blight in rice (Yamada et al. 2012). Moreover, JA-induced volatile compounds, which are involved in plant defense systems, are regulated by OsTIFY10c/OsJAZ8 (Taniguchi et al. 2014a, b). All these results indicated that TIFY proteins played important roles in plant stress responses. B. distachyon has become a model system for functional genomics studies in temperate cereals (such as wheat and barley), forage and turf grasses due to its small genome size and plant size, short life cycle, efficient cultivation and transformation systems (Opanowicz et al. 2008; The International Brachypodium Initiative 2010). TIFY proteins play important roles in plant growth and stress responses. However, limited information on the TIFY gene family is available in B. distachyon. In this study, we identified 21 genes encoding TIFY proteins in B. distachyon, including 15 JAZ and 6 ZML genes. Duplication analysis showed that both segmental and tandem duplication events have contributed to the evolution of the B. distachyon TIFY gene family. In order to analyze the possible functions of the TIFY proteins in abiotic stress responses of B. distachyon, we further analyzed the expression profiles of the BdTIFYs under various abiotic stress conditions, as well as in response to different phytohormone treatments. These results provided a foundation for further evolutionary and functional characterization of TIFY genes in B. distachyon and temperate cereals.
Materials and methods Identification of B. distachyon TIFY genes In order to identify all members of the TIFY gene family in B. distachyon, Arabidopsis TIFY gene sequences were first submitted to the Pfam database (http://pfam.sanger.ac.uk) (Finn et al. 2010) to acquire the conserved domain architecture of this family. The Pfam accession number of the TIFY domain, Jas and CCT motif were PF06200, PF09425 and PF06203, respectively. HMMER (Eddy 1998) was further used for domain search with an E-value
