Gene 549 (2014) 266–274
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The rice WUSCHEL-related homeobox genes are involved in reproductive organ development, hormone signaling and abiotic stress response Saifeng Cheng, Yulan Huang, Ning Zhu, Yu Zhao ⁎ National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan 430070, China
a r t i c l e
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Article history: Received 2 May 2014 Received in revised form 21 July 2014 Accepted 1 August 2014 Available online 6 August 2014 Keywords: Abiotic stress Development Hormone response Rice WOX genes
a b s t r a c t The WUSCHEL-related homeobox (WOX) genes are important transcription regulators participated in plant development processes. Rice (Oryza sativa L.) genome encodes at least 13 WOX members. In this study, a systematic microarray-based gene expression profiling of eleven WOX genes was performed for the whole life cycle of rice at 16 different tissues/organs of MH63 (rice indica cultivar), which included eight reproductive organs and eight vegetative tissues. The results demonstrated that four genes (OsWUS, OsNS1/OsNS2, OsWOX3 and OsWOX9A) were specifically expressed in panicle and endosperm development, and six genes (OsWOX5, OsWOX9B, OsWOX9D, OsWOX11, OsWOX12A and OsWOX12B) were preferentially expressed in seeds (72 h after imbibitions) during root emergence or growth. In situ hybridization analysis revealed differential transcript levels of OsWOX4, OsWOX5, OsWOX9A and OsWOX12B during panicle development and embryogenesis. Results of qRT-PCR showed that expression of four rice WOX genes (OsWOX5, OsWOX11, OsWOX12B and OsWOX12A) was up- or downregulated by plant hormones (auxin, cytokinin and gibberellin). More interestingly, most WOX genes were responsive to abiotic stress stimuli of drought, salt and cold. The molecular studies presented here will further provide insight in understanding the functions of rice WOX gene family in rice development, hormone signaling, and abiotic stress response. © 2014 Elsevier B.V. All rights reserved.
1. Introduction The WOX (WUSCHEL related homeobox) gene family is one of plant homeobox (HB) transcription factor families, which is characterized by the presence of a short stretch of amino acid residues with the helix– loop–helix–turn–helix structure. It is distinguished by the phylogenetic relatedness of its homeodomains from other HB transcription factor (Gehring et al., 1990). Compared with the animal HOX homeodomain, homology modeling of the plant WOX homeodomain reveals two extended loops between helices 1 and 2 within a generally highly conserved structure (Haecker et al., 2004). In addition to homeodomain, some WOX proteins contain the distinct WUS-box motif that locates carboxy-terminal to the homeodomain (van der Graaff et al., 2009). In Arabidopsis, the WUS-box motif is shown to be essential for WUS function in the shoot stem-cell population maintenance or differentiation, lateral organ formation, floral patterning, and embryogenesis (Haecker et al., 2004; Kamiya et al., 2003; Skylar et al., 2010).
Abbreviations: WOX gene, WUSCHEL homeobox gene; SAM/RAM, shoot/root apical meristem; PAT, polar auxin transport; DAP, day after pollination. ⁎ Corresponding author. E-mail addresses: [email protected] (S. Cheng), [email protected] (Y. Huang), [email protected] (N. Zhu), [email protected] (Y. Zhao).
http://dx.doi.org/10.1016/j.gene.2014.08.003 0378-1119/© 2014 Elsevier B.V. All rights reserved.
Analysis of WOX gene expression, function and evolution in a variety of plant species indicates that they play important roles in regulating key development of plant tissues and organs by determining cell fate, such as shoot/root apical meristem (SAM/RAM) cell maintenance or differentiation, lateral organ formation, and embryogenesis (Chandler et al., 2008; Hedman et al., 2013; Nardmann and Werr, 2006; Ten Hove and Heidstra, 2008; Zhang et al., 2010). For instance, Arabidopsis WUSCHEL (WUS) is the founding member of this family, which is found to be specifically required in maintaining central meristem identity of shoot and floral meristem structural and functional integrity (Haecker et al., 2004; Laux et al., 1996; Mayer et al., 1998). AtWOX5 performs a similar function in the root apical meristem and is specifically expressed in the cells of quiescent center (QC) of root (Gonzali et al., 2005; Sarkar et al., 2007). PRESSED FLOWER1 (PRS1/AtWOX3) is involved in the development of lateral and marginal regions of leaves and leaf orthologs of the flower by recruiting founder cells from all meristem (Matsumoto and Okada, 2001; Shimizu et al., 2009). While another member of Arabidopsis WOX genes, PRETTY FEW SEEDS2/WOX6 affects either ovules patterning by regulating cell proliferation of the maternal integuments or differentiation of megaspore mother cell (MMC) (Park et al., 2005). STIMPY1/WOX9 is required for maintaining cell division and preventing premature differentiation in vegetative shoot apical meristem (SAM) and early embryogenesis in Arabidopsis (Skylar et al., 2010; Wu et al., 2007, 2005). Arabidopsis WOX2 and STIMPY-LIKE1/ WOX8 (STPL1) are important cell fate regulators of early pre-embryo
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and cotyledon boundary formation (Breuninger et al., 2008; Haecker et al., 2004; Lie et al., 2012; Wu et al., 2007). In Arabidopsis and tomato, accumulation of WOX4 mRNA is found in the developing vascular bundle of root and shoot lateral organs, which promotes differentiation and/ or maintains the vascular procambium (Ji et al., 2010). WUS homolog ZmWUS1 is expressed in a few cells underlying the emerging coleoptile and in the seedling SAM after germination, while ZmWUS2 is activated in the P1 leaf primordium in Maize (Nardmann and Werr, 2006). PaWOX2 is found to be regulated by auxin polar transport (PAT), and PaWOX8 and PaWOX9 are involved in zygote and embryo development in conifers (Palovaara and Hakman, 2009; Palovaara et al., 2010). Recent results also show that WOX genes have redundant functions in plant development. For example, WOX4 acts redundantly with WOX14 in regulating vascular cell division (Etchells et al., 2013). WOX11 acts redundantly with its homolog WOX12 to function in the first-step cell fate transition during de novo root organogenesis (Liu et al., 2014). In green algae, bryophytes, lycophytes, fern and gymnosperm, many WOX complete genome sequences provide an unprecedented opportunity to explore the diversity among this class of protein in various organisms and to study their roles in regulating critical developmental processes at the whole genome level. Based on the above-mentioned observations that WOX genes play important roles in regulating plant development in a variety of species, presumably rice WOX gene function would be also important for programming rice development and growth regulation. By far at least 13 WOX genes are found in rice (Oryza sativa L.) genome (Zhang et al., 2010). Among them, WOX5, also named QHB/OsWOX9, which is a homolog of AtWOX5, is involved in specification and maintenance of QC cell in root apical meristem (Cho et al., 2013; Kamiya et al., 2003). WOX11 is shown to be induced by exogenous auxin or cytokinin and involved in activation of crown root growth and development (Zhao et al., 2009). OsWUS is expressed at the abaxial face of the auxiliary bud, not expressed in the organizing center (OC) of the vegetative SAM (Nardmann and Werr, 2006). OsWOX3 (or OsNS2), which is homolog of AtWOX3 (or PRS), is expressed in the leaf and floral organ primordia and its expression is shown to be required for leaf development (Cho et al., 2013; Dai et al., 2007; Ishiwata et al., 2013). Recently, WUSCHELrelated homeobox4 (WOX4), which is associated with cytokinin action, is involved in the maintenance of vegetative and reproductive meristem in rice (Ohmori et al., 2013). DAWAR TILLER, a WUSCHEL-related homeobox transcription factor is required for rice tiller growth (Wang et al., 2014). However, the functions of the other rice WOX genes are not clear. Because plant growth and development are also controlled by external conditions such as abiotic stress and hormones that work through impacting the expression of development-related genes, it would be interesting to know whether WOX gene expression is regulated by abiotic stress and plant hormones. In the present study, an attempt was made to gain insight into the expression pattern of rice WOX genes during seed germination, panicle development, and embryogenesis, responsiveness to plant hormones and abiotic stresses. Utilizing a microarray-based gene expression data, expression profiles of eleven WOX genes were compiled from tissues of the seed, vegetative organs, panicle and endosperm, together with seedlings subjected to plant hormones (GA, NAA and 6-BA) and abiotic stresses including cold, NaCl and drought. These results will be greatly useful for further exploring the developmental and regulatory function aimed at understanding the roles of rice WOX genes. 2. Results 2.1. Expression profiles of rice WOX genes during the entire life cycle To gain insight into the developmental windows during which rice WOX genes are expressed, spatial and temporal expression profiles of these genes were analyzed in different tissues/organs. For this purpose,
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affymetrix microarray data were collected from Rice Expression Profiles database (http://crep.ncpgr.cn), in which the expression profiles of whole rice genome were available for various tissues/organs collected during the entire life cycle of rice. In this study, a total of 16 representative tissues/organs, including eight from vegetative tissues and eight from reproductive organs, were selected for expression level analysis of WOX genes. Eleven WOX genes had corresponding probe sets on the Affymetrix Genechips. Based on the normalized hybridization signals in 16 tissues/organs including the seed (72 h after imbibitions), radicle (48 h after emergence), root, leaf, flag leaf, panicle and endosperm, almost all of the studied WOX genes showed tissue-/organ-specific expression patterns (Fig. 1). According to the analysis, expression patterns of the eleven genes could be classified into two types: reproductive organpreferential and vegetative organ-preferential. OsWUS, OsNS1/OsNS2, OsWOX3, OsWOX4 and OsWOX9A predominantly expressed in reproductive tissues such as young panicles at stages 3–5 and heading stage, spikelet at 3 days after pollination, and endosperm (7, 14 and 21 days after pollination, DAP) (Fig. 1). In reproductive tissues/organs, WOX genes had different expression levels. For example, OsWOX3 was mainly expressed in the spikelet (3 DAP) and endosperm (7 and 14 DAP). Expression pattern of OsWUS was similar to that of OsWOX4, they had higher transcription level in panicle at stages 4 and 5, and heading time. OsNS1/OsNS2 and OsWOX9A were expressed during reproductive organ formation processes except 21-day endosperm after pollination. Additionally, OsWOX4 accumulated more mRNA in flag leaf and OsWOX9A was highly expressed in the seed (72 h after imbibitions) (Fig. 1). Six genes (OsWOX9D, OsWOX12B, OsWOX12A, OsWOX5, OsWOX11 and OsWOX9B) seemed to be preferentially expressed in the seed at 72 h after imbibitions, and radicle (48 h after emergence) and root (seedling with two tillers) in comparison with panicle and endosperm (Fig. 1). The results indicated that expression of WOX genes in rice were specific to tissues/organs. 2.2. Rice WOX genes involved in floral organ development To further validate the microarray data, panicle at different developmental stages were selected and detail tissue expression levels of ten genes were tested by qRT-PCR. The results showed that OsWUS, OsNS1/ OsNS2, OsWOX4, OsWOX5 and OsWOX9B were found to be expressed highly in young panicle at early stage (3–5 and 4–5 cm), while transcripts for OsWOX3, OsWOX9A and OsWOX11 showed maximum accumulation in spikelet (3 DAP), stamen at one day before flowering (Fig. 2). OsWOX4, OsWOX5, OsWOX9A, and OsWOX12B were further confirmed by in situ hybridization. The transcript accumulation patterns for all four genes observed were similar to those observed from microarray analysis (Figs. 1 and 3). OsWOX4, OsWOX9A and OsWOX12B mRNA had more accumulation in floral primordium (Fig. 3A, G, J) and in gynoecium primordium (gp) and developing stamen (st) (Fig. 3B, H and K). OsWOX5 transcripts were only observed in floral primordium (fp) (Fig. 3D). In mature flower, only OsWOX9A had weaker mRNA signal in anther (Fig. 3I). These results suggested that WOX family proteins played different roles in the panicle formation process and might have redundancy functions. 2.3. Expression pattern of rice WOX genes in embryogenesis In order to know the functions of WOX family genes in rice embryogenesis, four WOX gene (OsWOX4, OsWOX5, OsWOX9A and OsWOX12B) transcripts during embryogenesis were detected by in situ hybridization. OsWOX4 and OsWOX12B mRNA had similar distribution, and were transcribed specifically in leaf primordium (lp) and margin of root primordium (rp) in mature embryo (7-DAP) (Fig. 4C, L). Weaker expressions of OsWOX4 and OsWOX12B mRNA were detected in 1-DAP and 3-DAP embryos (Fig. 4A, B, J and K). OsWOX5 was expressed in early embryo development, such as in embryo proper of 1-DAP embryo (Fig. 4D) and in
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Fig. 1. Hierarchical analysis of expression profiles of eleven rice WOX genes in 16 different organs/tissues and developmental stages. The signal value is log10-transformed and subjected to complete linkage hierarchical clustering with Treeview program. The color bar at the bottom represents the z-score values transformed from log2-based expression values with green color representing low level, black indicating medium level, and red signifying high level. The 16 organs/tissues are as follows: S1, seed at 72 h after imbibition; S2, radicle at 48 h after emergence, dark; S3, radicle at 48 h after emergence, light; S4, root (seedling with 2 tillers); S5, leaf from plants with young panicle at stage 3; S6, leaf from young panicle 4–5 cm in length; S7, flag leaf at 5 days before heading; S8, flag leaf at 5 days after heading; S9, young panicle at stage 4; S10, young panicle at stage 5; S11, panicle at heading stage; S12, panicle 4–5 cm in length; S13, spikelet at 3 days after pollination; S14, endosperm at 7 days after pollination; S15, endosperm at 14 days after pollination; S16, endosperm at 21 days after pollination.
Fig. 2. WOX gene expression profiles in floral developmental stages by qRT-PCR. T1, young panicle at stage 3; T2, young panicle at stage 4; T3 young panicle at stage 5; T4, 4–5 cm young panicle; T5, panicle at heading stage; T6, stamen, one day before flowering; T7, spikelet, 3 days after pollination.
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Fig. 3. In situ hybridization detection of OsWOX4, OsWOX5, OsWOX9A, and OsWOX12B gene transcripts in a floral organ primordium and mature flower. OsWOX4 (A–C), OsWOX5 (D–F), OsWOX9A (G–I) and OsWOX12B (J–L) expression was investigated. fp, flower primordium; st, stamen; gy, gynoecium; bar = 50 μm.
coleoptile of 3-DAP embryo (Fig. 4E). In mature embryo (7-DAP), a small amount of OsWOX5 mRNA was detectable in root primordium, shoot apical meristem and leaf primordium (Fig. 4F). OsWOX9A mRNA was distributed evenly in 1-DAP and 3-DAP embryos (Fig. 4G, H). However in 7-DAP embryo, the mRNA was mainly restricted in shoot primordium (sp), leaf primordium (lp), hypocotyl (hy) and root primordium (rp) (Fig. 4I). These results suggested that OsWOX4 and OsWOX12B might have a function in shoot, root and leaf primordium development during embryogenesis. OsWOX5 might play roles in embryo formation and polar construction. OsWOX9A was involved in organ development during embryogenesis. The results indicated that some rice WOX genes might play key roles during embryogenesis including embryo pattern formation and differentiation. 2.4. Response of WOX genes to hormone treatments in seedling stages Plant hormones such as auxin, gibberellin (GA), cytokinin, abscisic acid (ABA) and ethylene have been shown to be involved in the regulation of plant development. Analysis of the promoter regions of the WOX genes by PLACE program or by TOUCAN software showed that all WOX genes included cytokinin response elements (NAGATT) and auxin
Fig. 4. In situ hybridization detection of WOX gene transcripts during embryogenesis. OsWOX4 (A–C), OsWOX5 (D–F), OsWOX9A (G–I), and OsWOX12B (J–L) mRNA were detected in 1-DAP, 3-DAP, and 7-DAP embryos. Antisense line (M–O) was used as control. em, embryo; en, endosperm; lp, leaf primordia; sp, shoot primordium; rp, root primordium; s, suspensor; sc, scutellum; st, stigma; c, coleoptilar. Bar = 50 μm.
response element (TGTATC or GAGACA) in their promoters (Fig. 5, Table 1). ABA response element (MACGYGB) existed in ten WOX gene (OsWOX3, OsNS1/OsNS2, OsWOX4, OsWOX5, OsWOX9A, OsWOX9B, OsWOX9D, OsWOX11, OsWOX12A and OsWOX12B) promoter regions. Ethylene response element existed in six WOX genes (OsWOX3, OsNS1/OsNS2, OsWOX4, OsWOX9A, OsWOX9B and OsWOX12A), while GA response element existed in eight WOX genes (OsWUS, OsWOX2, OsWOX3, OsWOX4, OsWOX5, OsWOX9A, OsWOX11 and OsWOX12A). This suggested that WOX genes in rice might be involved in distinct and overlapping roles in organ development mediated by different plant hormones.
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family genes possibly responded to abiotic stress. Two week-old rice seedlings in the different stress conditions were treated and harvested as samples at different time points. The expression of these genes responding to different stresses such as drought, salt and cold was analyzed by qRT-PCR. We found that the expression of most WOX genes was induced by stress treatment (Fig. 7). For example, eight WOX genes (OsWUS, OsWOX3, OsWOX4, OsWOX5, OsWOX9B, OsWOX11, OsWOX12A, and OsWOX12B) were up-regulated to 4-fold by drought after 3 h induction (Fig. 7). The expression of OsWOX3 and OsWOX5 was quickly increased to 3–8 folds after 1 h treated with 150 mM NaCl, while other WOX genes had no response to NaCl stress (Fig. 7). OsWOX5, OsWOX9B, OsWOX12A and OsWOX12B had a higher transcription level after 6 h cold treatment (Fig. 7). The results indicate that WOX genes might involve in abiotic stresses that are dependent on ABA signal pathway. 3. Discussion
Fig. 5. Analysis of hormone response elements in the promoter of WOX genes. WOX genes possessed a number of cis-elements in their promoter regions. Computational analyses of promoter sequences were performed using analysis tools from the PLACE website (http:// www.dna.affrc.go.jp/htdocs/PLACE/) and manual searches for cis-regulatory elements are performed by TOUCAN software. Elements of TGTATC, ACGTSSC, AGATT, AWTTCAAA, CTCTGG, GAGACA, GCC box, and MACGYGB are shown.
Furthermore, we examined the expression levels of six WOX genes (OsWOX5, OsWOX4, OsWOX9B, OsWOX11, OsWOX12A, OsWOX12B) in 7-day-old seedling treated with cytokinin, auxin and GA by qRT-PCR. The results showed that expression of OsWOX12A and OsWOX12B could be greatly increased by GA after 3 h treatment, consisting with their highest expression level in the seed at 72 h after imbibitions (Figs. 1 and 6). All of these root-specific expressed WOX genes, such as OsWOX5, OsWOX11, OsWOX12A and OsWOX12B could respond rapidly and be induced 2 to 4 times at 1 h treatment by NAA and 6-BA, respectively (Fig. 6). However, OsWOX4 and OsWOX9B did not respond to all of the three hormones (Fig. 6). Collectively, these results indicated that some WOX genes were possibly involved in different hormone-mediated organ development. 2.5. Functional analysis of WOX genes in abiotic stresses Given that ten WOX genes contain ABA response element in their promoter regions (Fig. 5, Table 1), we wanted to know whether WOX
Table 1 Auxin-, ABA-, cytokinin-, and ethylene-regulated cis-elements in promoters of WOX genes. Gene
MACGYGB
NAGATT
GCCGCC
TGTCTC
GAGAC
TAACAAR
WUS OsWOX2 OsWOX3 OsNS1/OsNS2 OsWOX4 OsWOX5 OsWOX9A OsWOX9B OsWOX11 OsWOX12A OsWOX12B
0 0 3 2 2 2 3 3 2 3 1
28 11 23 26 32 25 34 34 23 17 26
0 0 2 3 1 0 6 6 0 2 0
1 1 9 1 0 2 2 2 2 1 0
2 1 0 6 4 7 4 4 5 2 2
4 1 2 0 1 2 0 0 1 1 0
Numbers of cis-elements are listed. ABA-response element: MACGYGB. Cytokininresponse element: NAGATT. Ethylene-response element: GCCGCC. Auxin-response element: TGTATC and GAGAC. GA-response element: TAACAAR.
The expression pattern of a gene is often indicative of its functional relevance. Genome-wide profiling of the WOX family was performed using a whole genome rice microarray for 16 different tissues/organs. Our results showed that three genes (OsWOX4, OsWOX9B and OsWOX9D) were widely expressed at different tissues/organs, suggesting that they might have pleiotropic functions at multiple developmental stages in rice. Ohmori et al. found that WUSCHEL-related homeobox4 (WOX4) promoted the undifferentiated state of the vegetative shoot apex and reproductive meristem in rice and was associated with cytokinin action (Ohmori et al., 2013). Our results also showed that OsWOX4 could be induced by 6-BA after 3 h treatment (Fig. 6), and its RNAi transgenic plants displayed many phenotypes, such as dwarf, narrow and short leaf, abnormal flower branches (Fig. 8), which further confirmed that OsWOX4 was involved in cytokinin-regulated rice development. In contrast to OsWOX4 broad expression, OsWOX12A (also named WOX11 in Zhao et al.), OsWOX5 (also named QHB in Kamyia et al.), OsWOX11 and OsWOX12B were similar and predominantly confined to radicle and root, indicating that they might be implicated in the development of radicle and root regulated by auxin and cytokinin (Figs. 1, 5, 6; Table 1). In the previous reports, OsWOX5 (QHB/OsWOX9) and OsWOX12A (WOX11) were found to be involved in stem cell maintenance in root apical meristem and cell proliferation during crown root development, respectively (Kamiya et al., 2003; Zhao et al., 2009). Zhang et al. also showed that in rice OsWOX5, OsWOX11, OsWOX12A and OsWOX12B were in the same clade (Zhang et al., 2010), further suggesting that they had similar function in rice. However, OsWOX11 and OsWOX12B RNAi transgenic plants did not exhibit any obvious phenotypes especially during root development in the current study (data not shown), indicating that they may have redundant functions in rice root development. In Arabidopsis, the functions of many WOX genes studied so far can be related to either promotion of cell division and/or prevention of premature flower organ development (Ikeda et al., 2009; Lenhard et al., 2001; Lohmann et al., 2001). In this study, microarray data and qRTPCR results revealed that almost all WOX genes were involved in different processes of flower development (Figs. 1, 2 and 3). In situ hybridization results showed that in rice OsWOX4, OsWOX5, OsWOX9A and OsWOX12B were expressed in flower primordia (Fig. 3A, D, G, and J), suggesting that they may stimulate cell division and differentiation in floral organ apical meristem in rice. Accordingly, abnormal panicle development of OsWOX3 over-expression transgenic plants with most florets degenerated, no recognizable anther structure and the absence of ovules were reported (Dai et al., 2007). Similarly, down-regulation of OsWOX4 in rice displayed abnormal spikelets (no second branched panicles formed) (Fig. 8C, Ohmori et al., 2013). These results suggested that WOX genes played key roles in the rice floral organ formation and development. Embryogenesis is the first developmental phase during which morphogenetic events occur to establish fundamental body plan and shoot
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Fig. 6. Kinetics of induction of WOX genes in response to plant hormones NAA, 6-BA and GA3. The transcription levels of OsWOX3, OsWOX4, OsWOX11, OsWOX9B, OsWOX12B and OsWOX12A under treatments of exogenous NAA, 6-BA, and GA3 were examined by qRT-PCR analysis. Ten-day-old seedlings were treated and harvested for analysis. The relative transcription levels were shown by setting the expression of corresponding WOX genes without treatment as 1.0. The experiments were repeated three times with biological replicates, and the data were presented as means ± SD.
and root apical meristem in plants. The complicated regulatory processes are operating during embryogenesis (Dolfini et al., 2007; Ito et al., 2002; Sato et al., 1996). WOX genes play very important roles during embryo formation in Arabidopsis and Confiers (Berleth and Jurgens, 1993; Haecker et al., 2004; Mayer et al., 1993; Palovaara and Hakman, 2009; Palovaara et al., 2010), but the regulatory mechanisms of WOX genes in rice embryogenesis are almost unknown. Our data suggested that OsWOX5 may function in early stage and OsWOX4 and OsWOX12B in mature stage of embryo development, while OsWOX9A may regulate the whole process of embryogenesis. Possibly, rice WOX gene expressions in the embryogenesis might be related to the complexity of cell division or organ formation in this process. So far, there were no studies about WOX gene expression under abiotic stress conditions. Our data showed that expression of WOX family members were responsive to hormones and abiotic stresses (Figs. 6, 7), suggesting that WOX regulated rice developmental processes might be related to plant hormones and stress. It has been shown that WOX gene can be regulated by growth hormones such as auxin, cytokinin and gibberellin (Liu et al., 2014; Ohmori et al., 2013; Wang et al., 2014; Zhao et al., 2009). In addition to the similar observation of WOX gene expression in response to hormone exposure, the present work further noticed that cis-elements of stress hormone abscisic acid (ABA) extensively existed in the promoter region of WOX genes (Table 1; Fig. 5), and ten WOX genes responded to abiotic stress such as drought (Fig. 7). We identified WOX family members by using forward-genetics approaches, but some insertion lines or RNAi transgenic plants of WOX genes did not display obvious phenotypes, possibly because of functional redundancy existed among rice WOX genes. It is also likely that many phenotypes will be apparent only under specific environmental conditions such as biotic or abiotic stress. The expression data we reported here will help define conditions in which WOX functional defects may be expected. Ultimately, these results will provide a foundation for indepth investigation of WOX gene functions. Further investigation of the roles of these proteins in rice development response to hormones/ abiotic stress and molecular mechanisms underlying these activities could extend our knowledge and understanding of the roles of rice WOX genes.
germinated at 28 °C (in light) and 24 °C (in dark) with a 14-h-light/ 10-h-dark cycle and collected panicle, embryo at different developmental stages. 4.2. DNA chip data resources for expression profiling analysis The expression profile data of rice WOX gene family were extracted from the Collection of Rice Expression Profiles (CREP) database established in our laboratory (Wang et al., 2010). 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. 4.3. In situ hybridization analysis The hybridization and immunological detection were performed as described by Dai et al. (2007). Probes for OsWOX4, OsWOX5, OsWOX9A, and OsWOX12B were amplified using the gene-specific primers listed in Supplemental Table 1. Images were photographed using a LEICA MZFL111 microscope with a color CCD camera. 4.4. Cis-element analysis of promoter regions of rice WOX genes Promoter regions of rice WOX genes were analyzed for known consensus cis-regulatory elements using Signal Scan Search (http://www. dna.affrc.go.jp/htdocs/PLACE/). The 3-kb upstream sequences of ATG were extracted and analyzed. In addition, manual searches were performed for the auxin response element (TGTCTC, GAGACA, CTCTGT), ABA response element (MACGYGB), cytokinin response element (AGATT), and ethylene response element (GCC box) by TOUCAN software (http://homes.esat.kuleuven.be/~saerts/software/toucan. php). 4.5. Plant hormone and abiotic treatment
4. Materials and methods
Seeds of MH63 were sowed and germinated on agar medium. After 10 days, the seedlings were transferred to media with or without 10−6 M NAA, 10−5 M 6-BA, or 10−4 M GA3. Total RNA was extracted after 0, 1 and 3 h of treatment and analyzed by qRT-PCR.
4.1. Plant materials and growth conditions
4.6. RNA isolation, reverse transcription and qRT-PCR
The indica rice cultivar MH 63 (O. sativa L. spp. indica) was used to study the expression of WOX genes at different developmental stages by rice Affymetrix microarray data analysis. To further identify tissuespecifically expressed genes, seeds of MH63 were soaked in water and
Total RNA was extracted using TRIzol reagents (Invitrogen, Carlsbad, CA, USA). For Reverse transcription, 4 μg total RNA was treated first with 2 units DNase I (Invitrogen) and then reverse transcribed in a total volume of 20 μL with 0.5 μg oligo (dT)15, 0.75 mM dNTPs, 10 mM
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dithiothreitol, and 200 units SuperScripTMIIIRNase H–reverse transcriptase (Invitrogen). The resulting products were tested by RealTime PCR with gene specific primers (Supplemental Table 1).
qRT-PCR was performed previously by Huang et al. (2007). The rice actin gene was used as the internal control. All primers (Supplemental Table) were annealed at 60 °C and run 42 cycles. The
Fig. 7. Expression of WOX genes under NaCl, cold and drought stresses. The transcription levels of WOX genes under treatments of NaCl, cold and drought stress were examined by qRT-PCR analysis. Ten-day-old seedlings were treated and harvested for analysis. The relative transcription levels were shown by setting the expression of corresponding WOX without treatment as 1.0. The experiments were repeated three times with biological replicates, and the data were presented as means ± SD.
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Fig. 8. Phenotype of OsWOX4 RNAi transgenic plants. A: transgenic plant phenotype at heading stage; B: flag leaf; C: panicle; D: seeds. Wild type (right) and transgenic plants (left); E: OsWOX4 mRNA level in wild type (left) and RNAi transgenic (right) plants.
expression level of target genes was also normalized with that of actin: 2(Ct of actin − Ct of target). 4.7. Vector construction and rice transformation To construct the RNAi vector, a 389-bp cDNA fragment of OsWOX4 was amplified from the cDNA clone using the primer set (Supplemental Table ) and was inserted into the KpnI and BamHI sites (for forward insert) and the SacI and SpeI sites (for the reverse insert) of the pDS1301 vector (Zhao et al., 2009). Agrobacterium tumefaciens (strains EHA105)-mediated transformation of MH63 plants was conducted. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.gene.2014.08.003. Authors' contributions Yu Zhao designed the research, supervised the experimental design and wrote the manuscript; Saifeng Cheng, Yulan Huang, and Ning Zhu carried out the whole experiments; Yu Zhao, Saifeng Cheng, and Yulan Huang analyzed the data. All authors read and approved the final manuscript. Acknowledgements This research was supported by grants from the National Natural Science Foundation of China (30971661 and 31371468), Program for New Century Excellent Talents in University (NCET-12-0863), and the Fundamental Research Funds for the Central Universities (2012ZYTS057 and 2013PY021).
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Gene journal homepage: www.elsevier.com/locate/gene
The rice WUSCHEL-related homeobox genes are involved in reproductive organ development, hormone signaling and abiotic stress response Saifeng Cheng, Yulan Huang, Ning Zhu, Yu Zhao ⁎ National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan 430070, China
a r t i c l e
i n f o
Article history: Received 2 May 2014 Received in revised form 21 July 2014 Accepted 1 August 2014 Available online 6 August 2014 Keywords: Abiotic stress Development Hormone response Rice WOX genes
a b s t r a c t The WUSCHEL-related homeobox (WOX) genes are important transcription regulators participated in plant development processes. Rice (Oryza sativa L.) genome encodes at least 13 WOX members. In this study, a systematic microarray-based gene expression profiling of eleven WOX genes was performed for the whole life cycle of rice at 16 different tissues/organs of MH63 (rice indica cultivar), which included eight reproductive organs and eight vegetative tissues. The results demonstrated that four genes (OsWUS, OsNS1/OsNS2, OsWOX3 and OsWOX9A) were specifically expressed in panicle and endosperm development, and six genes (OsWOX5, OsWOX9B, OsWOX9D, OsWOX11, OsWOX12A and OsWOX12B) were preferentially expressed in seeds (72 h after imbibitions) during root emergence or growth. In situ hybridization analysis revealed differential transcript levels of OsWOX4, OsWOX5, OsWOX9A and OsWOX12B during panicle development and embryogenesis. Results of qRT-PCR showed that expression of four rice WOX genes (OsWOX5, OsWOX11, OsWOX12B and OsWOX12A) was up- or downregulated by plant hormones (auxin, cytokinin and gibberellin). More interestingly, most WOX genes were responsive to abiotic stress stimuli of drought, salt and cold. The molecular studies presented here will further provide insight in understanding the functions of rice WOX gene family in rice development, hormone signaling, and abiotic stress response. © 2014 Elsevier B.V. All rights reserved.
1. Introduction The WOX (WUSCHEL related homeobox) gene family is one of plant homeobox (HB) transcription factor families, which is characterized by the presence of a short stretch of amino acid residues with the helix– loop–helix–turn–helix structure. It is distinguished by the phylogenetic relatedness of its homeodomains from other HB transcription factor (Gehring et al., 1990). Compared with the animal HOX homeodomain, homology modeling of the plant WOX homeodomain reveals two extended loops between helices 1 and 2 within a generally highly conserved structure (Haecker et al., 2004). In addition to homeodomain, some WOX proteins contain the distinct WUS-box motif that locates carboxy-terminal to the homeodomain (van der Graaff et al., 2009). In Arabidopsis, the WUS-box motif is shown to be essential for WUS function in the shoot stem-cell population maintenance or differentiation, lateral organ formation, floral patterning, and embryogenesis (Haecker et al., 2004; Kamiya et al., 2003; Skylar et al., 2010).
Abbreviations: WOX gene, WUSCHEL homeobox gene; SAM/RAM, shoot/root apical meristem; PAT, polar auxin transport; DAP, day after pollination. ⁎ Corresponding author. E-mail addresses: [email protected] (S. Cheng), [email protected] (Y. Huang), [email protected] (N. Zhu), [email protected] (Y. Zhao).
http://dx.doi.org/10.1016/j.gene.2014.08.003 0378-1119/© 2014 Elsevier B.V. All rights reserved.
Analysis of WOX gene expression, function and evolution in a variety of plant species indicates that they play important roles in regulating key development of plant tissues and organs by determining cell fate, such as shoot/root apical meristem (SAM/RAM) cell maintenance or differentiation, lateral organ formation, and embryogenesis (Chandler et al., 2008; Hedman et al., 2013; Nardmann and Werr, 2006; Ten Hove and Heidstra, 2008; Zhang et al., 2010). For instance, Arabidopsis WUSCHEL (WUS) is the founding member of this family, which is found to be specifically required in maintaining central meristem identity of shoot and floral meristem structural and functional integrity (Haecker et al., 2004; Laux et al., 1996; Mayer et al., 1998). AtWOX5 performs a similar function in the root apical meristem and is specifically expressed in the cells of quiescent center (QC) of root (Gonzali et al., 2005; Sarkar et al., 2007). PRESSED FLOWER1 (PRS1/AtWOX3) is involved in the development of lateral and marginal regions of leaves and leaf orthologs of the flower by recruiting founder cells from all meristem (Matsumoto and Okada, 2001; Shimizu et al., 2009). While another member of Arabidopsis WOX genes, PRETTY FEW SEEDS2/WOX6 affects either ovules patterning by regulating cell proliferation of the maternal integuments or differentiation of megaspore mother cell (MMC) (Park et al., 2005). STIMPY1/WOX9 is required for maintaining cell division and preventing premature differentiation in vegetative shoot apical meristem (SAM) and early embryogenesis in Arabidopsis (Skylar et al., 2010; Wu et al., 2007, 2005). Arabidopsis WOX2 and STIMPY-LIKE1/ WOX8 (STPL1) are important cell fate regulators of early pre-embryo
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and cotyledon boundary formation (Breuninger et al., 2008; Haecker et al., 2004; Lie et al., 2012; Wu et al., 2007). In Arabidopsis and tomato, accumulation of WOX4 mRNA is found in the developing vascular bundle of root and shoot lateral organs, which promotes differentiation and/ or maintains the vascular procambium (Ji et al., 2010). WUS homolog ZmWUS1 is expressed in a few cells underlying the emerging coleoptile and in the seedling SAM after germination, while ZmWUS2 is activated in the P1 leaf primordium in Maize (Nardmann and Werr, 2006). PaWOX2 is found to be regulated by auxin polar transport (PAT), and PaWOX8 and PaWOX9 are involved in zygote and embryo development in conifers (Palovaara and Hakman, 2009; Palovaara et al., 2010). Recent results also show that WOX genes have redundant functions in plant development. For example, WOX4 acts redundantly with WOX14 in regulating vascular cell division (Etchells et al., 2013). WOX11 acts redundantly with its homolog WOX12 to function in the first-step cell fate transition during de novo root organogenesis (Liu et al., 2014). In green algae, bryophytes, lycophytes, fern and gymnosperm, many WOX complete genome sequences provide an unprecedented opportunity to explore the diversity among this class of protein in various organisms and to study their roles in regulating critical developmental processes at the whole genome level. Based on the above-mentioned observations that WOX genes play important roles in regulating plant development in a variety of species, presumably rice WOX gene function would be also important for programming rice development and growth regulation. By far at least 13 WOX genes are found in rice (Oryza sativa L.) genome (Zhang et al., 2010). Among them, WOX5, also named QHB/OsWOX9, which is a homolog of AtWOX5, is involved in specification and maintenance of QC cell in root apical meristem (Cho et al., 2013; Kamiya et al., 2003). WOX11 is shown to be induced by exogenous auxin or cytokinin and involved in activation of crown root growth and development (Zhao et al., 2009). OsWUS is expressed at the abaxial face of the auxiliary bud, not expressed in the organizing center (OC) of the vegetative SAM (Nardmann and Werr, 2006). OsWOX3 (or OsNS2), which is homolog of AtWOX3 (or PRS), is expressed in the leaf and floral organ primordia and its expression is shown to be required for leaf development (Cho et al., 2013; Dai et al., 2007; Ishiwata et al., 2013). Recently, WUSCHELrelated homeobox4 (WOX4), which is associated with cytokinin action, is involved in the maintenance of vegetative and reproductive meristem in rice (Ohmori et al., 2013). DAWAR TILLER, a WUSCHEL-related homeobox transcription factor is required for rice tiller growth (Wang et al., 2014). However, the functions of the other rice WOX genes are not clear. Because plant growth and development are also controlled by external conditions such as abiotic stress and hormones that work through impacting the expression of development-related genes, it would be interesting to know whether WOX gene expression is regulated by abiotic stress and plant hormones. In the present study, an attempt was made to gain insight into the expression pattern of rice WOX genes during seed germination, panicle development, and embryogenesis, responsiveness to plant hormones and abiotic stresses. Utilizing a microarray-based gene expression data, expression profiles of eleven WOX genes were compiled from tissues of the seed, vegetative organs, panicle and endosperm, together with seedlings subjected to plant hormones (GA, NAA and 6-BA) and abiotic stresses including cold, NaCl and drought. These results will be greatly useful for further exploring the developmental and regulatory function aimed at understanding the roles of rice WOX genes. 2. Results 2.1. Expression profiles of rice WOX genes during the entire life cycle To gain insight into the developmental windows during which rice WOX genes are expressed, spatial and temporal expression profiles of these genes were analyzed in different tissues/organs. For this purpose,
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affymetrix microarray data were collected from Rice Expression Profiles database (http://crep.ncpgr.cn), in which the expression profiles of whole rice genome were available for various tissues/organs collected during the entire life cycle of rice. In this study, a total of 16 representative tissues/organs, including eight from vegetative tissues and eight from reproductive organs, were selected for expression level analysis of WOX genes. Eleven WOX genes had corresponding probe sets on the Affymetrix Genechips. Based on the normalized hybridization signals in 16 tissues/organs including the seed (72 h after imbibitions), radicle (48 h after emergence), root, leaf, flag leaf, panicle and endosperm, almost all of the studied WOX genes showed tissue-/organ-specific expression patterns (Fig. 1). According to the analysis, expression patterns of the eleven genes could be classified into two types: reproductive organpreferential and vegetative organ-preferential. OsWUS, OsNS1/OsNS2, OsWOX3, OsWOX4 and OsWOX9A predominantly expressed in reproductive tissues such as young panicles at stages 3–5 and heading stage, spikelet at 3 days after pollination, and endosperm (7, 14 and 21 days after pollination, DAP) (Fig. 1). In reproductive tissues/organs, WOX genes had different expression levels. For example, OsWOX3 was mainly expressed in the spikelet (3 DAP) and endosperm (7 and 14 DAP). Expression pattern of OsWUS was similar to that of OsWOX4, they had higher transcription level in panicle at stages 4 and 5, and heading time. OsNS1/OsNS2 and OsWOX9A were expressed during reproductive organ formation processes except 21-day endosperm after pollination. Additionally, OsWOX4 accumulated more mRNA in flag leaf and OsWOX9A was highly expressed in the seed (72 h after imbibitions) (Fig. 1). Six genes (OsWOX9D, OsWOX12B, OsWOX12A, OsWOX5, OsWOX11 and OsWOX9B) seemed to be preferentially expressed in the seed at 72 h after imbibitions, and radicle (48 h after emergence) and root (seedling with two tillers) in comparison with panicle and endosperm (Fig. 1). The results indicated that expression of WOX genes in rice were specific to tissues/organs. 2.2. Rice WOX genes involved in floral organ development To further validate the microarray data, panicle at different developmental stages were selected and detail tissue expression levels of ten genes were tested by qRT-PCR. The results showed that OsWUS, OsNS1/ OsNS2, OsWOX4, OsWOX5 and OsWOX9B were found to be expressed highly in young panicle at early stage (3–5 and 4–5 cm), while transcripts for OsWOX3, OsWOX9A and OsWOX11 showed maximum accumulation in spikelet (3 DAP), stamen at one day before flowering (Fig. 2). OsWOX4, OsWOX5, OsWOX9A, and OsWOX12B were further confirmed by in situ hybridization. The transcript accumulation patterns for all four genes observed were similar to those observed from microarray analysis (Figs. 1 and 3). OsWOX4, OsWOX9A and OsWOX12B mRNA had more accumulation in floral primordium (Fig. 3A, G, J) and in gynoecium primordium (gp) and developing stamen (st) (Fig. 3B, H and K). OsWOX5 transcripts were only observed in floral primordium (fp) (Fig. 3D). In mature flower, only OsWOX9A had weaker mRNA signal in anther (Fig. 3I). These results suggested that WOX family proteins played different roles in the panicle formation process and might have redundancy functions. 2.3. Expression pattern of rice WOX genes in embryogenesis In order to know the functions of WOX family genes in rice embryogenesis, four WOX gene (OsWOX4, OsWOX5, OsWOX9A and OsWOX12B) transcripts during embryogenesis were detected by in situ hybridization. OsWOX4 and OsWOX12B mRNA had similar distribution, and were transcribed specifically in leaf primordium (lp) and margin of root primordium (rp) in mature embryo (7-DAP) (Fig. 4C, L). Weaker expressions of OsWOX4 and OsWOX12B mRNA were detected in 1-DAP and 3-DAP embryos (Fig. 4A, B, J and K). OsWOX5 was expressed in early embryo development, such as in embryo proper of 1-DAP embryo (Fig. 4D) and in
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Fig. 1. Hierarchical analysis of expression profiles of eleven rice WOX genes in 16 different organs/tissues and developmental stages. The signal value is log10-transformed and subjected to complete linkage hierarchical clustering with Treeview program. The color bar at the bottom represents the z-score values transformed from log2-based expression values with green color representing low level, black indicating medium level, and red signifying high level. The 16 organs/tissues are as follows: S1, seed at 72 h after imbibition; S2, radicle at 48 h after emergence, dark; S3, radicle at 48 h after emergence, light; S4, root (seedling with 2 tillers); S5, leaf from plants with young panicle at stage 3; S6, leaf from young panicle 4–5 cm in length; S7, flag leaf at 5 days before heading; S8, flag leaf at 5 days after heading; S9, young panicle at stage 4; S10, young panicle at stage 5; S11, panicle at heading stage; S12, panicle 4–5 cm in length; S13, spikelet at 3 days after pollination; S14, endosperm at 7 days after pollination; S15, endosperm at 14 days after pollination; S16, endosperm at 21 days after pollination.
Fig. 2. WOX gene expression profiles in floral developmental stages by qRT-PCR. T1, young panicle at stage 3; T2, young panicle at stage 4; T3 young panicle at stage 5; T4, 4–5 cm young panicle; T5, panicle at heading stage; T6, stamen, one day before flowering; T7, spikelet, 3 days after pollination.
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Fig. 3. In situ hybridization detection of OsWOX4, OsWOX5, OsWOX9A, and OsWOX12B gene transcripts in a floral organ primordium and mature flower. OsWOX4 (A–C), OsWOX5 (D–F), OsWOX9A (G–I) and OsWOX12B (J–L) expression was investigated. fp, flower primordium; st, stamen; gy, gynoecium; bar = 50 μm.
coleoptile of 3-DAP embryo (Fig. 4E). In mature embryo (7-DAP), a small amount of OsWOX5 mRNA was detectable in root primordium, shoot apical meristem and leaf primordium (Fig. 4F). OsWOX9A mRNA was distributed evenly in 1-DAP and 3-DAP embryos (Fig. 4G, H). However in 7-DAP embryo, the mRNA was mainly restricted in shoot primordium (sp), leaf primordium (lp), hypocotyl (hy) and root primordium (rp) (Fig. 4I). These results suggested that OsWOX4 and OsWOX12B might have a function in shoot, root and leaf primordium development during embryogenesis. OsWOX5 might play roles in embryo formation and polar construction. OsWOX9A was involved in organ development during embryogenesis. The results indicated that some rice WOX genes might play key roles during embryogenesis including embryo pattern formation and differentiation. 2.4. Response of WOX genes to hormone treatments in seedling stages Plant hormones such as auxin, gibberellin (GA), cytokinin, abscisic acid (ABA) and ethylene have been shown to be involved in the regulation of plant development. Analysis of the promoter regions of the WOX genes by PLACE program or by TOUCAN software showed that all WOX genes included cytokinin response elements (NAGATT) and auxin
Fig. 4. In situ hybridization detection of WOX gene transcripts during embryogenesis. OsWOX4 (A–C), OsWOX5 (D–F), OsWOX9A (G–I), and OsWOX12B (J–L) mRNA were detected in 1-DAP, 3-DAP, and 7-DAP embryos. Antisense line (M–O) was used as control. em, embryo; en, endosperm; lp, leaf primordia; sp, shoot primordium; rp, root primordium; s, suspensor; sc, scutellum; st, stigma; c, coleoptilar. Bar = 50 μm.
response element (TGTATC or GAGACA) in their promoters (Fig. 5, Table 1). ABA response element (MACGYGB) existed in ten WOX gene (OsWOX3, OsNS1/OsNS2, OsWOX4, OsWOX5, OsWOX9A, OsWOX9B, OsWOX9D, OsWOX11, OsWOX12A and OsWOX12B) promoter regions. Ethylene response element existed in six WOX genes (OsWOX3, OsNS1/OsNS2, OsWOX4, OsWOX9A, OsWOX9B and OsWOX12A), while GA response element existed in eight WOX genes (OsWUS, OsWOX2, OsWOX3, OsWOX4, OsWOX5, OsWOX9A, OsWOX11 and OsWOX12A). This suggested that WOX genes in rice might be involved in distinct and overlapping roles in organ development mediated by different plant hormones.
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family genes possibly responded to abiotic stress. Two week-old rice seedlings in the different stress conditions were treated and harvested as samples at different time points. The expression of these genes responding to different stresses such as drought, salt and cold was analyzed by qRT-PCR. We found that the expression of most WOX genes was induced by stress treatment (Fig. 7). For example, eight WOX genes (OsWUS, OsWOX3, OsWOX4, OsWOX5, OsWOX9B, OsWOX11, OsWOX12A, and OsWOX12B) were up-regulated to 4-fold by drought after 3 h induction (Fig. 7). The expression of OsWOX3 and OsWOX5 was quickly increased to 3–8 folds after 1 h treated with 150 mM NaCl, while other WOX genes had no response to NaCl stress (Fig. 7). OsWOX5, OsWOX9B, OsWOX12A and OsWOX12B had a higher transcription level after 6 h cold treatment (Fig. 7). The results indicate that WOX genes might involve in abiotic stresses that are dependent on ABA signal pathway. 3. Discussion
Fig. 5. Analysis of hormone response elements in the promoter of WOX genes. WOX genes possessed a number of cis-elements in their promoter regions. Computational analyses of promoter sequences were performed using analysis tools from the PLACE website (http:// www.dna.affrc.go.jp/htdocs/PLACE/) and manual searches for cis-regulatory elements are performed by TOUCAN software. Elements of TGTATC, ACGTSSC, AGATT, AWTTCAAA, CTCTGG, GAGACA, GCC box, and MACGYGB are shown.
Furthermore, we examined the expression levels of six WOX genes (OsWOX5, OsWOX4, OsWOX9B, OsWOX11, OsWOX12A, OsWOX12B) in 7-day-old seedling treated with cytokinin, auxin and GA by qRT-PCR. The results showed that expression of OsWOX12A and OsWOX12B could be greatly increased by GA after 3 h treatment, consisting with their highest expression level in the seed at 72 h after imbibitions (Figs. 1 and 6). All of these root-specific expressed WOX genes, such as OsWOX5, OsWOX11, OsWOX12A and OsWOX12B could respond rapidly and be induced 2 to 4 times at 1 h treatment by NAA and 6-BA, respectively (Fig. 6). However, OsWOX4 and OsWOX9B did not respond to all of the three hormones (Fig. 6). Collectively, these results indicated that some WOX genes were possibly involved in different hormone-mediated organ development. 2.5. Functional analysis of WOX genes in abiotic stresses Given that ten WOX genes contain ABA response element in their promoter regions (Fig. 5, Table 1), we wanted to know whether WOX
Table 1 Auxin-, ABA-, cytokinin-, and ethylene-regulated cis-elements in promoters of WOX genes. Gene
MACGYGB
NAGATT
GCCGCC
TGTCTC
GAGAC
TAACAAR
WUS OsWOX2 OsWOX3 OsNS1/OsNS2 OsWOX4 OsWOX5 OsWOX9A OsWOX9B OsWOX11 OsWOX12A OsWOX12B
0 0 3 2 2 2 3 3 2 3 1
28 11 23 26 32 25 34 34 23 17 26
0 0 2 3 1 0 6 6 0 2 0
1 1 9 1 0 2 2 2 2 1 0
2 1 0 6 4 7 4 4 5 2 2
4 1 2 0 1 2 0 0 1 1 0
Numbers of cis-elements are listed. ABA-response element: MACGYGB. Cytokininresponse element: NAGATT. Ethylene-response element: GCCGCC. Auxin-response element: TGTATC and GAGAC. GA-response element: TAACAAR.
The expression pattern of a gene is often indicative of its functional relevance. Genome-wide profiling of the WOX family was performed using a whole genome rice microarray for 16 different tissues/organs. Our results showed that three genes (OsWOX4, OsWOX9B and OsWOX9D) were widely expressed at different tissues/organs, suggesting that they might have pleiotropic functions at multiple developmental stages in rice. Ohmori et al. found that WUSCHEL-related homeobox4 (WOX4) promoted the undifferentiated state of the vegetative shoot apex and reproductive meristem in rice and was associated with cytokinin action (Ohmori et al., 2013). Our results also showed that OsWOX4 could be induced by 6-BA after 3 h treatment (Fig. 6), and its RNAi transgenic plants displayed many phenotypes, such as dwarf, narrow and short leaf, abnormal flower branches (Fig. 8), which further confirmed that OsWOX4 was involved in cytokinin-regulated rice development. In contrast to OsWOX4 broad expression, OsWOX12A (also named WOX11 in Zhao et al.), OsWOX5 (also named QHB in Kamyia et al.), OsWOX11 and OsWOX12B were similar and predominantly confined to radicle and root, indicating that they might be implicated in the development of radicle and root regulated by auxin and cytokinin (Figs. 1, 5, 6; Table 1). In the previous reports, OsWOX5 (QHB/OsWOX9) and OsWOX12A (WOX11) were found to be involved in stem cell maintenance in root apical meristem and cell proliferation during crown root development, respectively (Kamiya et al., 2003; Zhao et al., 2009). Zhang et al. also showed that in rice OsWOX5, OsWOX11, OsWOX12A and OsWOX12B were in the same clade (Zhang et al., 2010), further suggesting that they had similar function in rice. However, OsWOX11 and OsWOX12B RNAi transgenic plants did not exhibit any obvious phenotypes especially during root development in the current study (data not shown), indicating that they may have redundant functions in rice root development. In Arabidopsis, the functions of many WOX genes studied so far can be related to either promotion of cell division and/or prevention of premature flower organ development (Ikeda et al., 2009; Lenhard et al., 2001; Lohmann et al., 2001). In this study, microarray data and qRTPCR results revealed that almost all WOX genes were involved in different processes of flower development (Figs. 1, 2 and 3). In situ hybridization results showed that in rice OsWOX4, OsWOX5, OsWOX9A and OsWOX12B were expressed in flower primordia (Fig. 3A, D, G, and J), suggesting that they may stimulate cell division and differentiation in floral organ apical meristem in rice. Accordingly, abnormal panicle development of OsWOX3 over-expression transgenic plants with most florets degenerated, no recognizable anther structure and the absence of ovules were reported (Dai et al., 2007). Similarly, down-regulation of OsWOX4 in rice displayed abnormal spikelets (no second branched panicles formed) (Fig. 8C, Ohmori et al., 2013). These results suggested that WOX genes played key roles in the rice floral organ formation and development. Embryogenesis is the first developmental phase during which morphogenetic events occur to establish fundamental body plan and shoot
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Fig. 6. Kinetics of induction of WOX genes in response to plant hormones NAA, 6-BA and GA3. The transcription levels of OsWOX3, OsWOX4, OsWOX11, OsWOX9B, OsWOX12B and OsWOX12A under treatments of exogenous NAA, 6-BA, and GA3 were examined by qRT-PCR analysis. Ten-day-old seedlings were treated and harvested for analysis. The relative transcription levels were shown by setting the expression of corresponding WOX genes without treatment as 1.0. The experiments were repeated three times with biological replicates, and the data were presented as means ± SD.
and root apical meristem in plants. The complicated regulatory processes are operating during embryogenesis (Dolfini et al., 2007; Ito et al., 2002; Sato et al., 1996). WOX genes play very important roles during embryo formation in Arabidopsis and Confiers (Berleth and Jurgens, 1993; Haecker et al., 2004; Mayer et al., 1993; Palovaara and Hakman, 2009; Palovaara et al., 2010), but the regulatory mechanisms of WOX genes in rice embryogenesis are almost unknown. Our data suggested that OsWOX5 may function in early stage and OsWOX4 and OsWOX12B in mature stage of embryo development, while OsWOX9A may regulate the whole process of embryogenesis. Possibly, rice WOX gene expressions in the embryogenesis might be related to the complexity of cell division or organ formation in this process. So far, there were no studies about WOX gene expression under abiotic stress conditions. Our data showed that expression of WOX family members were responsive to hormones and abiotic stresses (Figs. 6, 7), suggesting that WOX regulated rice developmental processes might be related to plant hormones and stress. It has been shown that WOX gene can be regulated by growth hormones such as auxin, cytokinin and gibberellin (Liu et al., 2014; Ohmori et al., 2013; Wang et al., 2014; Zhao et al., 2009). In addition to the similar observation of WOX gene expression in response to hormone exposure, the present work further noticed that cis-elements of stress hormone abscisic acid (ABA) extensively existed in the promoter region of WOX genes (Table 1; Fig. 5), and ten WOX genes responded to abiotic stress such as drought (Fig. 7). We identified WOX family members by using forward-genetics approaches, but some insertion lines or RNAi transgenic plants of WOX genes did not display obvious phenotypes, possibly because of functional redundancy existed among rice WOX genes. It is also likely that many phenotypes will be apparent only under specific environmental conditions such as biotic or abiotic stress. The expression data we reported here will help define conditions in which WOX functional defects may be expected. Ultimately, these results will provide a foundation for indepth investigation of WOX gene functions. Further investigation of the roles of these proteins in rice development response to hormones/ abiotic stress and molecular mechanisms underlying these activities could extend our knowledge and understanding of the roles of rice WOX genes.
germinated at 28 °C (in light) and 24 °C (in dark) with a 14-h-light/ 10-h-dark cycle and collected panicle, embryo at different developmental stages. 4.2. DNA chip data resources for expression profiling analysis The expression profile data of rice WOX gene family were extracted from the Collection of Rice Expression Profiles (CREP) database established in our laboratory (Wang et al., 2010). 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. 4.3. In situ hybridization analysis The hybridization and immunological detection were performed as described by Dai et al. (2007). Probes for OsWOX4, OsWOX5, OsWOX9A, and OsWOX12B were amplified using the gene-specific primers listed in Supplemental Table 1. Images were photographed using a LEICA MZFL111 microscope with a color CCD camera. 4.4. Cis-element analysis of promoter regions of rice WOX genes Promoter regions of rice WOX genes were analyzed for known consensus cis-regulatory elements using Signal Scan Search (http://www. dna.affrc.go.jp/htdocs/PLACE/). The 3-kb upstream sequences of ATG were extracted and analyzed. In addition, manual searches were performed for the auxin response element (TGTCTC, GAGACA, CTCTGT), ABA response element (MACGYGB), cytokinin response element (AGATT), and ethylene response element (GCC box) by TOUCAN software (http://homes.esat.kuleuven.be/~saerts/software/toucan. php). 4.5. Plant hormone and abiotic treatment
4. Materials and methods
Seeds of MH63 were sowed and germinated on agar medium. After 10 days, the seedlings were transferred to media with or without 10−6 M NAA, 10−5 M 6-BA, or 10−4 M GA3. Total RNA was extracted after 0, 1 and 3 h of treatment and analyzed by qRT-PCR.
4.1. Plant materials and growth conditions
4.6. RNA isolation, reverse transcription and qRT-PCR
The indica rice cultivar MH 63 (O. sativa L. spp. indica) was used to study the expression of WOX genes at different developmental stages by rice Affymetrix microarray data analysis. To further identify tissuespecifically expressed genes, seeds of MH63 were soaked in water and
Total RNA was extracted using TRIzol reagents (Invitrogen, Carlsbad, CA, USA). For Reverse transcription, 4 μg total RNA was treated first with 2 units DNase I (Invitrogen) and then reverse transcribed in a total volume of 20 μL with 0.5 μg oligo (dT)15, 0.75 mM dNTPs, 10 mM
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dithiothreitol, and 200 units SuperScripTMIIIRNase H–reverse transcriptase (Invitrogen). The resulting products were tested by RealTime PCR with gene specific primers (Supplemental Table 1).
qRT-PCR was performed previously by Huang et al. (2007). The rice actin gene was used as the internal control. All primers (Supplemental Table) were annealed at 60 °C and run 42 cycles. The
Fig. 7. Expression of WOX genes under NaCl, cold and drought stresses. The transcription levels of WOX genes under treatments of NaCl, cold and drought stress were examined by qRT-PCR analysis. Ten-day-old seedlings were treated and harvested for analysis. The relative transcription levels were shown by setting the expression of corresponding WOX without treatment as 1.0. The experiments were repeated three times with biological replicates, and the data were presented as means ± SD.
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Fig. 8. Phenotype of OsWOX4 RNAi transgenic plants. A: transgenic plant phenotype at heading stage; B: flag leaf; C: panicle; D: seeds. Wild type (right) and transgenic plants (left); E: OsWOX4 mRNA level in wild type (left) and RNAi transgenic (right) plants.
expression level of target genes was also normalized with that of actin: 2(Ct of actin − Ct of target). 4.7. Vector construction and rice transformation To construct the RNAi vector, a 389-bp cDNA fragment of OsWOX4 was amplified from the cDNA clone using the primer set (Supplemental Table ) and was inserted into the KpnI and BamHI sites (for forward insert) and the SacI and SpeI sites (for the reverse insert) of the pDS1301 vector (Zhao et al., 2009). Agrobacterium tumefaciens (strains EHA105)-mediated transformation of MH63 plants was conducted. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.gene.2014.08.003. Authors' contributions Yu Zhao designed the research, supervised the experimental design and wrote the manuscript; Saifeng Cheng, Yulan Huang, and Ning Zhu carried out the whole experiments; Yu Zhao, Saifeng Cheng, and Yulan Huang analyzed the data. All authors read and approved the final manuscript. Acknowledgements This research was supported by grants from the National Natural Science Foundation of China (30971661 and 31371468), Program for New Century Excellent Talents in University (NCET-12-0863), and the Fundamental Research Funds for the Central Universities (2012ZYTS057 and 2013PY021).
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