Analysis of rice Snf2 family proteins and their potential roles in epigenetic regulation

Plant Physiology and Biochemistry 70 (2013) 33e42

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Research article

Analysis of rice Snf2 family proteins and their potential roles in epigenetic regulation Yongfeng Hu a, *, Ning Zhu b, Xuemin Wang a, Qingping Yi a, Deyan Zhu a, Yan Lai a, Yu Zhao b, ** a b

Jingchu University of Technology, 448000 Jingmen, China National Key Laboratory for Crop Genetic Improvement, Huazhong Agricultural University, 430070 Wuhan, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 1 April 2013 Accepted 2 May 2013 Available online 17 May 2013

Snf2 family proteins are ATP-dependent chromatin remodeling factors that control many aspects of DNA events such as transcription, replication, homologous recombination and DNA repair. In animals several members in this family have been revealed to control gene expression in concert with other epigenetic mechanisms including histone modification, histone variants and DNA methylation. Their function in regulating genome expression in plant has hardly been disclosed before except in Arabidopsis. Here we identified 40 members of this family in the rice (Oryza Sativa) genome and constructed a phylogenetic tree together with Arabidopsis 41 Snf2 proteins. Sequence alignment of the Snf2 helicase regions revealed conserved motifs and blocks in most proteins. Expression profile analysis indicates that many rice Snf2 family genes show a tissue-specific expression pattern and some of them respond to abiotic stresses including drought, salt and cold. The results provide a basis for further analysis of their roles in epigenetic regulation to control rice development. Ó 2013 Elsevier Masson SAS. All rights reserved.

Keywords: Abitotic stress Epigenetic regulation Expression profile Rice development Snf2 family

1. Introduction Eukaryotic DNA is associated with core histones and packed into higher order fibers called chromatin. Recent analysis reveals that the chromatin structure may function as an important platform to regulate gene expression. The mechanism of this epigenetic regulation involves histone modification, DNA methylation, histone variants and chromatin remodeling. Many proteins have been identified to mediate these processes, among which Snf2 family proteins are responsible for chromatin remodeling by consuming energy provided by ATP. The Snf2 family proteins are defined by several unique features including a number of conserved motifs and blocks within the helicase-like region [1]. Domain analysis

Abbreviations: AtBRM, Arabidopsis thaliana BRAHMA; BPTF, bromodomain PHD finger transcription factor; CHD3, chromodomain helicase DNA binding protein3; DDM1, decrease in DNA methylation 1; DRD1, defective in RNA-directed DNA methylation 1; H3K27me3, trimethylated histone H3 lysine 27; H3K4me3, trimethylated histone H3 lysine 4; H3K79, histone H3 lysine 79; H3K9, histone H3 lysine 9; HP1, heterochromatin protein 1; ING2, inhibitor of growth 2; MOM1, Morpheus molecule 1; PC, polycomb; PIE1, photoperiod-independent early flowering 1; Snf2, sucrose nonfermentable; SYD, SPLAYED. * Corresponding author. Tel.: þ86 13677246318. ** Corresponding author. Tel.: þ86 27 87281800. E-mail address: [email protected] (Y. Hu). 0981-9428/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.plaphy.2013.05.001

indicates that there are two conserved domains, SNF2_N and Helicase_C, in the helicase-like region. The proteins in this family are classified into six groups based on the helicase-like region [1]. Each group could be subdivided into several subfamilies in which the proteins show specific properties such as distinct extra insertions between conserved blocks in the helicase-like region and additional domains outside or inside the helicase-like region [1]. Some of these subfamilies are unique to specific organisms and the others are ubiquitous [1]. Forty one Snf2 family proteins have been identified in Arabidopsis and they fall into 18 subfamilies [2]. Functional analysis indicates that many of these proteins play important roles in plant development and stress response [2]. Moreover, some of them are involved in epigenetic regulation such as DRD1 and DDM1 required for DNA methylation [3,4], PIE1 responsible for H2A.Z deposition [5,6] and PICKLE involved in the regulation of H3K27me3-enriched genes [7e9]. In rice, few Snf2 family proteins have been studied hitherto except DDM1 and CHR729 and their roles in epigenetic regulation are still obscure [10,11]. Here we characterized the rice Snf2 family proteins and analyzed expression profiles of these genes in different tissues and in responding to salt, drought and cold stress. The results provide a wealth of information for further exploring the developmental and regulatory function of these family proteins.

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2. Results and discussion 2.1. Identification of Snf2 protein in rice Forty two proteins belonging to the Snf2 family in rice are listed in chromDB database. CHR723 and CHR744, homologs of Arabidopsis MOM1 (CHR15), which contain a chromodomain and a PHD domain are evolved from CHD3 chromatin remodelers (Mi-2 subfamily in this paper) and were considered to be categorized into this group (subfamily in this paper) [12]. However, domain architecture analysis indicates that these proteins do not have the conserved Helicase_C domain which is an important part of the Snf2 proteins. Here these proteins are not considered to be Snf2 family members, thus in rice there are totally 40 Snf2 family proteins which are listed in Table 1. Phylogenetic analysis indicates that these proteins belong to 18 subfamilies as in Arabidopsis (Fig. 1). Sequence alignment of the Snf2 helicase-like regions of all proteins in rice and Arabidopsis revealed that most of the Snf2

Table 1 Snf2 family genes in Arabidopsis and rice. Group

Subfamily

Arabidopsis thaliana

Oryza sativa

Snf2-like

Snf2

CHR2 (AtBRM, AT2G46020) CHR3 (SYD, AT2G28290) CHR12 (AT3G06010) CHR23 (AT5G19310) CHR11 (AT3G06400) CHR17 (AT5G18620) CHR1 (DDM1, AT5G66750)

CHR707 (Os02g02290) CHR720 (Os06g14406) CHR719 (Os05g05230)

ISWI Lsh1 ALC1 Chd1 Mi-2

Swr1like

Rad54like

Rad5/16like

CHD7 Swr1 EP400 Ino80 Etl1 Rad54 ATRX Arip4 DRD1

JBP2 Rad5/16

Ris1

Lodestar SHPRH SSO1653like

Mot1 ERCC6

Distant

SSO1653 SMARCAL1

CHR10 (AT2G44980) CHR5 (AT2G13370) CHR6 (PICKLE, AT2G25170) CHR4 (AT5G44800) CHR7 (AT4G31900) e CHR13 (PIE1, AT3G12810) e CHR21 (INO80, AT3G57300) CHR19 (AT2G02090) CHR25 (RAD54, AT3G19210) CHR20 (AT1G08600) e CHR35 (DRD1, AT2G16390) CHR34 (AT2G21450) CHR38 (AT3G42670) CHR42 (AT5G20420) CHR31 (AT1G05490) CHR40 (AT3G24340) e CHR22 (AT5G05130) CHR29 (AT5G22750) CHR32 (AT5G43530) CHR37 (AT1G05120) CHR41 (AT1G02670) CHR26 (AT3G16600) CHR27 (AT3G20010) CHR28 (AT1G50410) CHR30 (AT1G11100) CHR33 (AT1G61140) e CHR36 (AT2G40770) CHR39 (AT3G54460) CHR16 (AT3G54280) CHR8 (AT2G18760) CHR9 (AT1G03750) CHR24 (AT5G63950) e CHR14 (AT5G07810) CHR18 (AT1G48310)

CHR727 CHR728 CHR741 CHR746 CHR711 CHR705 CHR702 CHR729 CHR703 e CHR709 e CHR732 CHR714 CHR733 CHR717 e CHR722 CHR730 CHR736 CHR737 CHR740 CHR742 CHR743 e CHR724 CHR710 CHR735 CHR731

(Os05g05780) (Os01g27040) (Os03g51230) (Os09g27060) (Os03g01200) (Os07g46590) (Os06g08480) (Os07g31450) (Os01g65850) (Os02g46450) (Os03g22900) (Os04g47830) (Os02g52510) (Os10g31970) (Os07g49210) (Os03g06920) (Os07g25390) (Os06g14440) (Os02g43460) (Os05g32610) (Os08g14610) (Os07g44800) (Os02g32570) (Os04g09800) (Os07g32730)

CHR706 (Os01g57110) CHR715 (Os04g53720) CHR725 (Os08g08220)

e CHR739 CHR708 CHR701 CHR704 CHR713 CHR712 CHR745 e CHR726 CHR721

(Os07g48270) (Os01g72310) (Os02g06592) (Os01g01312) (Os05g15890) (Os04g59620) (Os01g44990) (Os07g40730) (Os07g44210)

family proteins in these two species have 8 highly conserved motif (Q, I, Ia, II, III, IV, V and VI) and 13 less conserved blocks (A to N without I) which show characteristic patterns of individual subfamilies as in other organisms (Fig. S1) [1]. “Minor insertion site” located between motifs I and Ia and/or “major insertion site” between blocks C and K are also found respectively, where additional sequences are inserted in a few subfamilies such as Ris1, Rad5/16, SHPRH, Swr1 and Ino80 (Fig. S1). Domain analysis of these proteins and the distribution of rice Snf2 family genes in the genome are shown in Figs. 2 and 3 respectively. Most of the proteins have additional conserved domains which are specific to their subfamily such as PHD domain, chromodomain, bromodomain, SANT domain and RING domain. Eight subfamiles of them are described in detail below as these subfamily members have been reported to be involved in epigenetic regulation. 2.1.1. Snf2 subfamily The SWI/SNF ATP-dependent chromatin remodeling complex translocates DNA within the nucleosome by interaction with transcription factors and nucleosomal substrates to facilitate transcription [13]. Acetylation of histone H3 and transcription factors have combined effect on the recruitment of the complex [13]. Human Snf2 subfamily protein BRG1 has the HSA domain and the bromodomain in addition to the SNF2_N and Helicase_C domains [14,15]. However, in rice all three Snf2 subfamily proteins (CHR707, CHR720 and CHR719) lack the conserved HSA domain and only one protein (CHR707) has the bromodomain (Fig. 2). The HSA domain was reported to mediate interaction with the other protein such as BAF250 which was required for SWI/SNF2-dependent transcriptional activation [14,16]. Absence of this domain in rice homologs indicates that a novel mechanism may be adopted by these proteins to regulate gene expression. The bromodomain which recognizes acetylated lysines on the N-terminal tails of histones is a conserved domain present in many transcription activators such as histone acetyltransferases and BET family proteins [17]. Arabidopsis AtBRM (CHR2), a CHR707 homolog, and the other Snf2 subfamily member SYD (CHR3) which is homologous to CHR720 and lacks the bromodomain are redundantly required to overcome polycomb repression and activate target gene expression [18]. It is not known whether the bromodomain of AtBRM is involved in the redundant function. 2.1.2. ISWI subfamily ISWI is involved in many events such as transcription regulation, chromosome organization and DNA replication [19]. Chromatin remodeling activity of this protein could be stimulated by incorporation of histone variant H2A.Z [20]. In rice there are two members in this subfamily: CHR727 and CHR728. Both proteins have conserved domains: HAND, SANT and SLIDE which are located in the C-terminal half of protein (Fig. 2). These domains are important for them to recognize substrates: DNA and histone [19]. Sequence analysis indicates that the two proteins show high homology with each other (86% identities), which implies that they may have redundant function. 2.1.3. Lsh subfamily In Arabidopsis the sole member of this subfamily is DDM1 (CHR1) which is required for DNA methylation as its homolog in mammalian [4,21]. In rice two members OsDDM1a (CHR746) and OsDDM1b (CHR741) have been identified recently [10]. The sequences of the genes show 81% similarity to each other even including intron. Down-regulation of both genes by expressing antisense OsDDM1a cDNA in rice results in DNA hypomethylation mostly in repeated DNA region as in Arabidopsis ddm1 plants and

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Fig. 1. Phylogenetic relationship of Snf2 family proteins from Oryza sativa (CHR7**) and Arabidopsis thaliana (CHR*). The division into subfamilies (names are indicated) is marked by wedge backgrounds with different colors representing different groups of subfamilies. Yellow: Snf2-like group; red: Swr1-like group; blue: Rad54-like group; green: Rad5/16-like group; purple: SSO1653-like group; gray: Distant group. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

many transposons are activated in the transgenic plants which might be due to reduced DNA methylation at these loci [10]. However, it is still not known whether this effect is mediated by decreased expression of OsDDM1a or OsDDM1b or both. Further analysis needs to be conducted in the mutants of these two genes respectively to answer this question. 2.1.4. Chd1 subfamily Chd1 subfamily proteins have additional double chromodomains upstream to the helicase-like region. In rice CHR705 was identified to be this subfamily member. The chromodomains from both HP1 and Pc act as methylated histone binding modules and recognize methylated-lysines by an aromatic cage formed by three residues [22e24]. The tandem chromodomains of CHD1 in human but not in budding yeast has also been reported to recognize methylated H3K4 by two aromatic residues Trp 64 and Trp 67 [25]. However, lack of Trp 67 in the CHR705 chromodomain as in Chd1 of budding yeast suggests that rice CHR705 might not interact with methylated H3 tail either (Fig. 4A). Recent paper indicates that Chd1 is required for nucleosome reassembly and the maintenance of high levels of H2B monoubiquitination which is essential for H3K4 and H3K79 trimethylation [26]. But loss of Chd1 has little

effect on the genome-wide H3K4 and H3K79 trimethylation suggesting a complicated role of this protein in the regulation of histone modification to control gene transcription [26]. It would be intriguing to discover whether rice CHR705 regulates gene expression by a similar mechanism as in budding yeast. 2.1.5. Mi-2 subfamily The proteins of Mi-2 subfamily, Chd1 subfamily and CHD7 subfamily are designated CHD (chromodomain helicase DNAbinding) proteins [27]. All of these proteins contain two double chromodomains while some of them have other different domains [27]. The additional PHD domain in Mi-2 subfamily proteins is located at N-terminal part of the proteins [27]. Different from CHD1 the chromodomains of dMi-2 display DNA but not histone binding activity [28]. However, the chromodomains of a rice Mi-2 homolog, CHR729, have been analyzed to bind to methylated H3K4 suggesting that Mi-2 proteins may function differently in animals and plants [11]. PHD domain shows trimethylated-H3K4 binding activity in many proteins such as ING2, YNG1 and BPTF [29e31]. The recognition of methyl group by PHD domains also involves two or four aromatic residues as chromodomains. In other proteins such as SMCX, an H3K4me3 demethylase, its PHD domain is also found to

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CHR727 CHR728 CHR741 Lsh1 CHR746 CHR707 CHR719 Snf2 CHR720 CHR705 Chd1 CHR702 Mi2 CHR703 CHR729 CHR711 ALC1 CHR732 Ino80 CHR709 Swr1 Etl1 CHR714 CHR717 Rad54 ATRX CHR733 CHR722 CHR730 CHR736 DRD1 CHR737 CHR740 CHR742 CHR743 CHR710 CHR724 Rad5/16 CHR731 CHR735 CHR706 CHR715 Ris1 CHR725 CHR708 SHPRH CHR739 CHR701 Mot1 CHR704 CHR712 ERCC6 CHR713 CHR745 CHR726 SMARCAL1 CHR721

1158 1107

ISWI

775 773 2197 1128 4221 1731 1361 1150 2259 898 1483 1984 800 1476 955 1366 935 876 946 1406 1425 766 1028 821 862 1132 1213 1051 1030 1298

SNF2-N PHD QLQ HAND

HELICc HIRAN HNHc SLIDE

RING HSA SANT

Chromodomain Bromodomain RRM

1668 2034 1187 v

v

987 856 875 1166

700

Fig. 2. Domain architectures of rice Snf2 family proteins. Different domains are showed by rectangle with different colors. The number at the end represents the length of the protein and all proteins are displayed in proportion.

bind to H3K9me3 but the molecular mechanism of recognition is not clear [32]. Human Mi-2 homolog CHD4 has two PHD domains (PHD1 and PHD2) and both domains exhibit binding activities to Nterminus of H3 including unmodified H3K4 and methylated H3K9 [33]. In contrast, plant Mi-2 homologs have only one PHD domain. The PHD domain of rice CHR729 could bind to trimethylated H3K27 indicating the multiple binding activities of this domain and diverse functions of Mi-2 homologs in animal and plant [11]. In rice there are three Mi-2 homologs: CHR729, CHR702 and CHR703. All three proteins possess the PHD domain and two tandem chromodomains (Fig. 2). Sequence comparison indicates that some residues in CHD1 chromodomains involved in histone binding are also found in these proteins demonstrating a potential histone binding activity of the chromodomains in rice Mi-2 homologs (Fig. 4A). Lack of conserved aromatic residues in the PHD domain of these proteins including CHR729, which make cation-p contacts with the methylated lysine, suggests a novel mechanism might be used to recognize methylated histones (Fig. 4B). The regulatory function of Mi-2 proteins is still obscure. Drosophila Mi-2 was initially found to repress Hox gene expression but later results showed that this protein also bound to actively transcribed genes suggesting both repressive and active function of this protein [34,35]. The same scenario has been found in

Arabidopsis that PICKLE may affect transcription both negatively and positively to control plant development [7e9]. In contrast to Arabidopsis, mutation of rice PICKLE homolog CHR702 could not affect rice development but CHR729 T-DNA insertion mutants displayed severe phenotypes including dwarf, later flowering, less tiller and narrow leaf [11]. This demonstrates that Mi-2 homologs in dicot and monocots play different roles in plant development. 2.1.6. Swr1 subfamily Swr1 protein is responsible for deposition of H2A.Z which is a histone variant involved in transcription regulation. In Arabidopsis Swr1 homolog PIE1 has been identified to be required for the deposition of H2A.Z at multiple loci to regulate genes expression [5]. Blast result showed that rice homolog CHR709 was highly homologous to Arabidopsis PIE1 at N terminus containing the HSA domain and the helicase region which are important for the function of the proteins [36]. This implies that CHR709 might be involved in the incorporation of H2A.Z in rice. 2.1.7. Ino80 subfamily INO80 complex has been reported to contribute to various events including transcription, replication and repair partly by controlling genome-wide distribution of the histone variant H2A.Z

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Fig. 3. Chromosome distribution of rice Snf2 family genes.

[37]. Arabidopsis INO80 controls homologous recombination to play a dual role in transcription and DNA repair [38]. The protein sequence of rice homolog CHR732 displayed 58% identities to Arabidopsis INO80. 2.1.8. DRD1 subfamily DRD1 is the first SNF2-like protein implicated in an RNA-guided, epigenetic modification of the genome [3]. Mutation of DRD1 in Arabidopsis leads to elimination of RNA-induced non-CpG methylation at a target promoter but DDM1-mediated cytosine methylation of repetitive sequences was not affected in drd1 plants [3]. However, DRD1 is also required for the complete demethylation on the target sites when the RNA trigger is withdrawn indicating the complicated roles of this protein in regulating DNA methylation [39]. There are seven members of this subfamily in rice compared to six members in Arabidopsis. But rice homolog of DRD1 could not

be identified according to the sequence alignment and phylogenetic analysis. It seems that DRD1 subfamily members could be subdivided into two subgroups based on the sequence homology. Four rice genes (CHR730, CHR736, CHR737 and CHR743) are in the first subgroup which contains DRD1 and the others (CHR722, CHR740 and CHR742) belong to the second subgroup. Functional distinction between two subgroups needs to be further studied. It would be interesting to disclose which one functions as Arabidopsis DRD1 to control RNA-directed DNA methylation in rice. 2.2. Expression profile of rice Snf2 family genes To study expression profiles of the rice Snf2 family genes, we analyzed the Affimetrix microarray data from the Collection of Rice Expression Profiles (http://crep.ncpgr.cn) where transcripts from various tissues at different developmental stage are detected. Many

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Fig. 4. Sequence comparison of chromodomains and PHD domains. A. Sequence alignment of chromdomains from various proteins. Chromodomain sequences of all CHD proteins in rice (CHR7**) and Arabidopsis(CHR*), human CHD1 (hCHD1),budding yeast Chd1(ScCHD1), Drosophila Mi-2 (dMi-2), Drosophila HP1 (dHP1) and Drosophila Pc (dPc) were aligned by ClustalX program. B. Sequence alignment of PHD domains from various proteins. These proteins includes Mi-2 homologs in rice and Arabidopsis except CHR7, two PHD domains of human CHD4 and SMCX (PHD1 represents the first one and PHD2 represents the second), ING2, YNG1 and BPTF. Aromatic residues reported to be responsible for the recognition of methylated lysine within chromodomain and PHD domain are marked by red rectangle. The conserved residues involved in the interaction with histone in human CHD1 chromodomain are marked by green rectangle. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

genes displayed specific expression patterns. For example, most Snf2 family genes were expressed relatively higher in panicles and some of them also in callus (Fig. 5A). The transcripts of CHR740 and CHR703 were largely accumulated in endosperm (Fig. 5A). The data also showed that the transcripts of some genes such as CHR702, CHR713, CHR722, CHR729, CHR730, CHR739 and CHR743 were accumulated in developing endosperm and the expression of other genes such as CHR703, CHR715, CHR720, CHR733 and CHR746 was decreased, suggesting a possible involvement of these genes in the development of endosperm (Fig. 5A). Interestingly, the expression of all three Mi-2 subfamily members was regulated during this developmental process: two of them were up-regulated and the other one was down-regulated. Arabidopsis PICKLE has been reported to repress embryogenesis genes directly and rice CHR729 mutants also showed developmental abnormality of seed (data not shown) [40], which implicates that Mi-2 homologs in plant might have a conserved function to regulate seed development genes. We also tested expression pattern of several genes, homologs of which have been characterized in Arabidopsis, by RT-PCR. Eleven tissues were selected and expression levels of eight genes were tested by Real-time PCR and then were compared with Actin expression level. We found that most of these genes were expressed in a high level comparable to Actin except CHR714 and CHR746 which are DDM1 homologs (Fig. 6). CHR702, CHR705, CHR707, CHR709 and CHR720 showed similar expression patterns which were expressed higher in leaf and flag leaf whereas CHR741 and CHR746 transcripts were relatively abundant in panicles and CHR728 transcripts were accumulated in both of these tissues, suggesting different Snf2 family proteins have specific function in different tissues (Fig. 6).

In order to know whether Snf2 family genes possibly regulate abiotic stress responses, we analyzed the expression of these genes responding to different stresses including drought, salt and cold by collecting microarray data from GEO database (http://www.ncbi. nlm.nih.gov/geo/). We found that only a small number of the genes were affected by stress treatment. For example, CHR720 was clearly induced by all three treatments. CHR712, CHR735 and CHR742 were repressed by drought and salt stress and CHR728 was repressed only by salt stress (Fig. 5B). To confirm the microarray data we treated two-week-old rice seedling in the different stress conditions and harvested the samples at different time points. RTPCR was performed using gene specific primers corresponding to these five genes. The results are not completely consistent with the microarray data. CHR720 and CHR728 expression was increased whereas CHR735 expression was decreased by drought and salt treatment but not cold treatment (Fig. 7). Surprisingly, in contrast to microarray data CHR712 and CHR742 were not repressed but induced by drought and salt stress (Fig. 7). The results indicate that these genes may play different roles in salt and drought stress response. 2.3. Conclusion Snf2 family ATP-dependent chromatin remodeling factors affect gene transcription either by translocating nucleosomes or by exchanging histone variants. Some of them could recognize and bind to specific modified histones by conserved domains demonstrating the important roles played by these proteins in the epigenetic regulation to control development. Our results indicate

Fig. 5. Expression analysis of the rice Snf2 family genes. (A) A hierarchical cluster display of relative expression levels of 38 rice Snf2 family genes in 33 samples representing different organs or tissues at different developmental stages of the Minghui 63 cultivar. The Affymetrix probe set IDs which have perfect probe matches to the other two genes (CHR736 and CHR741) could not be found out.1e5: Calli at different induction stages from cultured embryos. 6: Plumule at 48 h after emergence in the dark. 7: Plumule at 48 h after emergence in the light. 8: Radical at 48 h after emergence in the dark. 9: Radical at 48 h after emergence in the light. 10: 72 h imbibed seed. 11: Embryo and radicle after germination. 12: Seedling leaves and roots at three-leaf stage. 13: Shoots of seedlings with two tillers. 14: Roots of seedlings with two tillers. 15: Stem at day 5 before heading. 16: Stems at heading stage. 17: Leaves from plants at young panicle stage 3. 18: Leaves at young panicle stage 7. 19: Flag leaves at day 5 before heading. 20: Flag leaf at day 14 after heading. 21: Sheaths at young panicle stage 3. 22: Sheaths at young panicle stage 7. 23 : Hulls one day before flowering. 24: Stamens one day before flowering. 25: Young panicle at stage 3. 26: Young panicles at stage 4. 27: Young panicles at stage 5. 28: Panicles at stage 7. 29: Panicles at heading stage. 30: Spikelets, 3 days after pollination. 31: Endosperm, 7 days after pollination. 32: Endosperm, 14 days after pollination. 33: Endosperm, 21 days after pollination. B. Expression changes of Snf2 genes responding to abiotic stresses including drought, salt and cold in seven-day-old light-grown seedlings. In addition to CHR736 and CHR741, the expression values of the other six genes (CHR703, CHR726, CHR730, CHR733, CHR740 and CHR743) in all tested samples are extremely low and not displayed in the figure. Color bar at the base represents log2 expression values: Green, representing low expression; black, medium expression; red, high expression. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Fig. 6. Expression pattern of eight Snf2 family genes tested by RT-PCR.

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Fig. 7. Expression profile of five Snf2 family genes responding to drought, salt and cold stress tested by RT-PCR.

that many rice Snf2 family members have conserved domains as in animal counterparts implying that they may function similarly to control gene expression. However, sequence alignment showed that some key residues involved in histone binding were absent in rice homologs indicating that distinct mechanisms may be adopted by rice proteins. In addition, we found that some of Snf2 family genes showed tissue-specific expression patterns and some respond to abiotic stresses which demonstrate that they are probably involved in rice development and response to environmental changes. 3. Materials and methods 3.1. Bioinformatic analysis Rice Snf2 family protein sequences were collected from ChromDB database (http://www.chromdb.org/) and confirmed in the MSU Rice Genome Annotation Project Database (http://rice. plantbiology.msu.edu/) with their locus numbers. Conserved domains of these proteins were analyzed in Pfam database (http:// pfam.sanger.ac.uk/) and confirmed in SMART database (http:// smart.embl-heidelberg.de/). Sequence alignment of Snf2 helicase region was performed using Muscle program and phylogenetic tree was constructed by MEGA 3.1 using neighborhood-joining method and a bootstrap test of 1000 replications. 3.2. Expression profile analysis of rice Snf2 family genes Expression data of Snf2 family genes were downloaded from two databases (CREP for expression profile analysis and GEO for analysis of gene expression responding to abiotic stress). Expression values

were obtained by searching the data using Affymetrix probe set ID of each gene for gene cluster analysis by the Cluster 3.0 program and the results were visualized by the Treeview program. 3.3. Plant materials, growth and stress treatment Zhonghua11 rice variety was used in this experiment for various stress treatment. Rice seeds were germinated on agar plates and grown for two weeks under 14 h of light, 28  C/10 h of dark, 24  C. The seedlings were transferred to the 300 mM NaCl solution for salt treatment, to 4  C plant incubator for cold treatment and to the filter papers for drought treatment using water treatment under room temperature as a control. Samples were harvested at different time points and stored in 80  C for further analysis. 3.4. RNA isolation, reverse transcription and real-time PCR Total RNA was extracted using TRIzol reagents (Invitrogen). For RT-PCR analysis, 4 mg total RNA was treated first with 2 units DNase I (Invitrogen) and then reverse transcribed in a total volume of 20 mL with 0.5 mg oligo(dT)15, 0.75 mM dNTPs, 10 mM dithiothreitol, and 200 units SuperScripTM III RNase He reverse transcriptase (Invitrogen). The resulting products were tested by Real-Time PCR with gene specific primers (Table S1). Real-time PCR was performed in a total volume of 25 mL with 1.0 mL of the RT, 0.25 mM primers, and 12.5 mL SYBR Green Master mix (TAKARA) on a 7500 real-time PCR machine (Applied Biosystems) according to the manufacturer’s instructions. The rice actin gene was used as the internal control. All primers were annealed at 60  C and run 42 cycles. The expression level of target genes was also normalized with that of actin: 2(Ct of actinCt of target).

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