The Plant Journal (2014) 80, 1005–1013
doi: 10.1111/tpj.12694
DYT1 directly regulates the expression of TDF1 for tapetum development and pollen wall formation in Arabidopsis Jing-Nan Gu1,†, Jun Zhu1,†, Yu Yu2, Xiao-Dong Teng1, Yue Lou1, Xiao-Feng Xu1, Jia-Li Liu1 and Zhong-Nan Yang1,* 1 Development Center of Plant Germplasm Resources, College of Life and Environment Sciences, Shanghai Normal University, Shanghai, 200234 China, and 2 Shanghai Huangxing School, Shanghai 200093, China Received 11 July 2014; revised 29 September 2014; accepted 30 September 2014; published online 6 October 2014. *For correspondence (e-mail [email protected]). † These authors contributed equally to this work.
SUMMARY The tapetum plays a critical role during the development and maturation of microspores. DYSFUNCTIONAL TAPETUM 1 (DYT1) is essential for early tapetal development. Here, we determined that the promoter region (550 to 463 bp) contains indispensable cis-elements for DYT1 expression. Although DYT1 transcripts can be detected in both meiocytes and tapetal cells, localization of DYT1–GFP demonstrated that DYT1 is strictly located in tapetal cells during microsporogenesis. Chromatin immunoprecipitation (ChIP) analysis revealed that DYT1 directly binds the promoter region of Defective in Tapetal Development and Function 1 (TDF1), a transcription factor essential for tapetum development. When TDF1 driven by the DYT1 promoter is expressed in a dyt1 mutant, the expression of the transcription factors AMS, MS188/MYB80, TEK and MS1 and the pollen wall-related genes are restored. Although the pollen wall is not formed and the microspores are ruptured, DIOC2 staining showed that fatty acids, the precursors of the pollen wall, were synthesized in the transgenic lines. These results indicate that DYT1 regulates the expression of AMS, MS188/MYB80, TEK and MS1 for pollen wall formation, primarily via TDF1. Keywords: Arabidopsis thaliana, transcription factors, tapetum, pollen wall, DYT1.
INTRODUCTION The anther is the male organ that produces pollen for plant reproduction. The mature anther consists of four somatic cell layers, namely the epidermis, endothecium, middle layer, and tapetum enclosing gametophyte cells. The tapetum originates from the L2 layer as one of three germ layers, L1, L2 and L3, in the stamen primordial (Goldberg et al., 1993). As the innermost of the four somatic cell layers surrounding male gametophytes, the tapetum plays an essential role in pollen development (Mariani et al., 1990; Sanders et al., 1999). Tapetal fate determination depends on the signaling pathway triggered by several leucine-rich repeat receptor-like protein kinases (LRR-RLKs) during the early stage of anther development. Dysfunction of these pathways leads to additional microsporocytes lacking the tapetal cells, resulting in complete male sterility (Canales et al., 2002; Zhao et al., 2002; Yang et al., 2003; Albrecht et al., 2005; Colcombet et al., 2005; Ma, 2005; Jia et al., 2008). During anther development, the tapetum undergoes dramatic morphological differentiation to form the binuclear © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd
secretory cell, which is packed with ribosomes, mitochondria, Golgi bodies, endoplasmic reticulum and vesicles (Stevens and Murray, 1981; Bedinger, 1992). During male gametogenesis, the tapetal cells provide the precursors of sporopollenin for pollen exine formation, the callase complex (also termed b-1,3-glucanase) to release microspores from the tetrad, and numerous elaioplasts and cytoplasmic lipid bodies for pollen coat formation (Mascarenhas, 1975; Stieglitz, 1977; Pacini and Juniper, 1979; Hesse and Hess, 1993). In Arabidopsis, several transcription factors regulate the tapetum and its functions. DYT1 encodes a putative basic helix–loop–helix (bHLH) transcription factor, and TDF1 encodes a putative R2R3 MYB transcription factor. Mutations in these two genes cause tapetal hypertrophy extending into the locule and resulting in sporophytic male sterility (Zhang et al., 2006; Zhu et al., 2008). The transcription factor ABORTED MICROSPORES (AMS) encodes a bHLH family protein that plays an important role in tapetum development and pollen wall formation (Sorensen 1005
1006 Jing-Nan Gu et al. et al., 2003; Xu et al., 2010). Another member of the R2R3 MYB family, MS188/MYB80, apparently regulates sexine formation (Zhang et al., 2007; Zhu et al., 2010). MALE STERILITY1 (MS1), a nuclear protein with PHD-finger motifs, is essential for tapetum development at the post-meiotic phase (Wilson et al., 2001; Ito et al., 2007; Yang et al., 2007). These transcription factors form a genetic pathway (DYT1-TDF1-AMS-MS188/MYB80-MS1) for tapetal development and function (Zhu et al., 2011). Cytological evidence indicates that the majority of the precursor materials of the pollen wall originate from the tapetum (Heslop-Harrison, 1962; Dickinson and HeslopHarrison, 1968). The pollen wall consists of two main layers, the outer exine layer and the inner intine layer. The exine is further divided into the sexine and the nexine (Scott, 1994). Recently, MS188/MYB80 and TEK have been reported to control sexine formation and nexine formation, respectively (Zhang et al., 2007; Lou et al., 2014). Both are directly regulated by AMS (Lou et al., 2014). The sexine layer most likely consists of sporopollenin, which is derived from long-chain fatty acids, oxygenated aromatic rings and phenylpropionic acids (Piffanelli et al., 1998; Ariizumi and Toriyama, 2011). Multiple enzymes in the tapetum, including ACOS5, CYP703A2, CYP704B1, MS2, PKSA and PKSB are involved in the biochemical pathways for sexine formation (Morant et al., 2007; de Azevedo Souza et al., 2009; Dobritsa et al., 2009, 2010; Kim et al., 2010; Chen et al., 2011). DYT1 functions during the early stage of tapetum development. Transcriptome analysis showed that many genes in the tapetum are downregulated in the dyt1 mutant (Feng et al., 2012). However, detailed regulation of the tapetum remains unclear. Here, we show that DYT1 directly binds the promoter of TDF1 during tapetum development. Furthermore, a transgenic rescue assay demonstrated that DYT1 regulates the expression of genes for pollen wall formation, primarily via TDF1. RESULTS The promoter region (550 to 463 bp) of DYT1 is essential for its expression Previous studies showed that the 631 bp promoter fragment of DYT1 is sufficient to drive its expression for its function in the anther (Song et al., 2009). This promoter region contains specific cis-acting elements including MYB, WRKY, ARR1, GATABOX, GTGANTG10, ACGTBOX, W-box, and DOF components according to the PLACE database (http://www.dna.affrc.go.jp/PLACE/) (Higo et al., 1999) (Table S1). We created constructs with GUS driven by different lengths of the DYT1 promoter fragments (Figure 1a), and transformed the constructs into wild-type. GUS activity could be detected in transgenic lines with promoter fragments of 631 and 550 bp (Figure 1b,c). In situ hybridization
was used to analyze the expression pattern of GUS driven by the 550 bp fragment. During anther stage 4, the tapetal cells are differentiated when the four-lobed anther pattern is established (Sanders et al., 1999). A weak GUS signal was detected in the anther at this stage (Figure 1i). At stages 5 and 6, when the tapetal layer is clearly present in the anther and the microcytes undergo meiosis, the GUS signal was highest in the tapetum compared to the low level in the meiocytes (Figure 1j,k). At stage 7, when meiosis is complete and the tetrad is formed, the GUS signal was significantly reduced (Figure 1l). The GUS expression pattern driven by the 550 bp fragment was similar to the DYT1 expression in the wild type (Zhang et al., 2006; Zhu et al., 2011). However, no GUS staining was observed in the transgenic plants with the DYT1 promoters of 463, 331, 180 and 91 bp, respectively (Figure 1d–g). These results suggest that the promoter region (550 to 463 bp) contains essential motifs for DYT1 expression. DYT1–GFP is specifically localized in tapetal cells To further understand the function of DYT1 during anther development, we created a DYT1–GFP fusion protein driven by the 631-bp native DYT1 promoter (Figure 2a). After transformed into heterozygous (dyt1-2/+) plants, we identified a transgenic line with a homozygous (dyt1/dyt1) background using closely linked markers (Figure 2c and Table S2). This transgenic line exhibited a fully fertile phenotype (Figure 2b), indicating that the DYT1–GFP fusion protein was enable to rescue the sterile phenotype of the dyt1 mutation. In the wild type, GFP fluorescence was not observed in the anther. In the transgenic line, GFP fluorescence was not detected at stage 4 (Figure 2d). At stage 5, the GFP signal was initially present within the tapetal cells (Figure 2e). At stage 6, a stronger GFP signal was detected in the tapetal cells, which formed a circle within the locule. In meiocytes, the DYT1–GFP fluorescence was not observed (Figure 2f,g). At the tetrad stage (stage 7), the GFP signal was weakly detected in the tapetal cells. No GFP signal was observed in tetrads (Figure 2h,i). At stage 8, when the microspores are released from the tetrads, GFP fluorescence was not detected in the anther (Figure 2j, k). These results indicate that the DYT1 is strictly localized in tapetal cells, which is consistent with its role in tapetal development. ChIP assay demonstrates that DYT1 is associated with the TDF1 promoter TDF1 is a transcriptional regulator downstream of DYT1 and is involved in tapetum development and function (Zhu et al., 2008). In dyt1-2, TDF1 is barely detected (Zhu et al., 2011). Previous studies showed that DYT1 bind to the G-box (TCACGTGA) of target gene promoters (Feng et al., 2012). However, no G-box is presented in the 1000-bp promoter region of TDF1. There are three E-box motifs
© 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd, The Plant Journal, (2014), 80, 1005–1013
DYT1 regulates TDF1 for tapetum and pollen wall formation 1007 Figure 1. Promoter analysis of DYT1. (a) Construction of GUS driven by different length fragments of the DYT1 promoter. (b–g) GUS activity of the transgenic lines with different length fragments of the DYT1 promoter. (b) The anther of proDYT1-630:Gus. (c) The anther of proDYT1-550:Gus. (d) The anther of proDYT1-463:Gus. (e) The anther of proDYT1-331:Gus. (f) The anther of proDYT1-180: Gus. (g) The anther of proDYT1-94:Gus. (h–l) In situ hybridization analysis of transgenic lines with GUS RNA probe during anther development. ProDYT1-550:Gus anthers from stages 3 (h), 4 (i), 5 (j), 6 (k), 7 (l). (m) Stage 6 anther hybridized with the sense probe. Bars = 40 lm.
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(CANNTG) in this region, which is a variant of the G-box. We used chromatin immunoprecipitation (ChIP) with a GFP monoclonal antibody to determine whether DYT1–GFP directly binds to the promoter of TDF1. Four probes were designed in the promoter region of TDF1 (Figure 3a). Quantitative ChIP-PCR (qChIP-PCR) showed no significant enrichment of the TDF1 promoter fragment in the wild type (Figure 3b). By contrast, the pTDF1-3 fragment of the TDF1 promoter was increased when GFP monoclonal antibodies were used (+AB) compared with the AB samples (Figure 3c). No significant increase in the other three TDF1 promoter fragments was observed (Figure 3c). These results suggest that DYT1 binds to the TDF1 promoter in vivo to regulate TDF1 expression. The expression of TDF1 in dyt1-2 can partially rescue the dyt1-2 phenotype To determine whether DYT1 functions through TDF1, we made a construct with the DYT1 promoter (631 bp), the genomic sequence (1143 bp) and 30 untranslated region (257 bp) of TDF1, and introduced this construct into dyt1-2 heterozygous plant (Figure 4a). Of the 23 independent transgenic lines, seven transgenic lines were identified to be dyt1-2 homozygous background using closely linked molecular markers (Table S2). These transgenic lines all showed complete male sterility (Figure 4d). Quantitative RT-PCR showed that the TDF1 transcript was overexpressed in these transgenic lines (Figure 4f). Therefore, the
expression of TDF1 in dyt1-2 does not rescue the fertility. In the transgenic plants with dyt1-2 heterozygous background, the overexpression of TDF1 did not affect their fertility (Figure 4e). Semi-thin sections were analyzed to identify the morphological differences in anther development between the dyt1-2 mutant and the transgenic lines with dyt1-2 background. Although both the mutant and transgenic lines showed a hypertrophic tapetum, the tetrads of the transgenic plants appeared more regular than the dyt1 mutant (Figure 5h,m). The locule space could be observed in transgenic plants at stage 11, whereas it was crushed in the dyt1-2 mutant (Figure 5j,o). This result reveals that tapetum and tetrads in proDYT1::TDF1 transgenic lines are less defective than that in the dyt1-2 plants. We used the proDYT1::TDF1 transgenic line to identify the genes regulated by DYT1 via TDF1. The SPL, EMS1, SERK1, SERK2 and TPD1 have been reported to be important for early anther development and tapetal fate determination. Genetic studies showed that these genes function upstream of DYT1 (Yang et al., 1999; Ma, 2005; Zhang et al., 2006). The qRT-PCR showed that the expressions of these genes were either increased or nearly normal in proDYT1::TDF1 transgenic plants. This result is consistent with previous studies that these genes act upstream of DYT1. Additionally, the GAMYB-like genes, AtMYB33 and AtMYB65 play a redundantly role for tapetum development (Millar and Gubler, 2005). The double mutant of them showed the similar tapetal defect compared with that in
© 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd, The Plant Journal, (2014), 80, 1005–1013
1008 Jing-Nan Gu et al.
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dyt1. These genes likely act at approximately the same stage as DYT1 (Zhang et al., 2006). The expression of these genes was up-regulated in proDYT1::TDF1 transgenic plants (Figure 4h). The transcription factors AMS, MS188/ MYB80, MS1, and TEK are essential for tapetum development and pollen wall formation. They act downstream of DYT1 and TDF1 (Zhu et al., 2008; Lou et al., 2014). The expression of these genes was nearly restored to the level in the wild type (Figure 4g). These results suggest that DYT1 regulates the expression of these transcription factors via TDF1 for tapetum development and pollen wall formation. Genes for pollen wall synthesis act downstream of TDF1 During anther development, tapetal cells are active in the biosynthesis of phenylpropanoids, steryl esters, long-chain alkanes, and flavonoids for exine formation (Hernould et al., 1998; Scott et al., 2004). Although pollen grains are ruptured in the locule of transgenic lines during the late stage (Figure 5o), the expression of the regulators for exine formation, AMS, MS188/MYB80 and TEK, are
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Figure 2. Genetic complement and expression pattern of DYT1–GFP. (a) The proDYT1:DYT1–GFP constructs including 631 bp native promoter, DYT1 genomic fragment and GFP coding region used for genetic complementation assays. (b) The siliques and stained anthers of dyt1-2 and proDYT1:DYT1–GFP line. (c) The molecular identification of the dyt1 mutated site. (d–k) Fluorescence confocal images of the DYT1–GFP fusion protein. The green channel in (d–k) showed the GFP expression (530 nm), red channel showed the chlorophyll autofluorescence (>560 nm). (d) At stage 4, no GFP signal is observed. (e) At stage 5, the GFP is initially detected in the tapetal cells. (f) At stage 6, the GFP is strongly expressed in tapetum. (h) At stage 7, the expression of GFP is dramatically decreased. (j) At stage 8, GFP signal disappeared in anther. The bright-field images of (g), (i) and (k) show that DYT1–GFP is not located in the meiocytes, tetrads and microspores, respectively. T, tapetum; Mc, meiocytes; Td, tetrads; Msp, microspores. Bars = 40 lm.
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recovered in the transgenic line. We determined whether the exine precursors of the pollen wall were synthesized in the tapetal cells of the transgenic plants. The diethyloxadicarbocyanine iodide (DIOC2) and Tinopal were utilized to stain the fatty acid content of exine and cellulose materials in the transgenic line and mutant plant (Regan and Moffatt, 1990; Lou et al., 2014). In the wild type, the red fluorescence of DIOC2 staining was observed in the tapetum and microspores and the blue fluorescence of Tinopal staining showed cell outline in the anther (Figure 6a). In the transgenic plants, the red fluorescence was also detected in the defective tapetum and ruptured pollen, indicating that some exine materials were synthesized in these cells (Figure 6c). However, no red fluorescence was observed in the anther of dyt1-2 mutant (Figure 6b). This result indicated that the materials for the pollen wall could be synthesized when the TDF1 expressed. In Arabidopsis, ACOS5, CYP703A2, CYP704B1, MS2, PKSA, PKSB and ABCG26 are involved in sporopollenin synthesis and transport (Morant et al., 2007; de Azevedo Souza et al., 2009; Dobritsa et al., 2009; Kim et al., 2010;
© 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd, The Plant Journal, (2014), 80, 1005–1013
DYT1 regulates TDF1 for tapetum and pollen wall formation 1009 Quilichini et al., 2010; Chen et al., 2011). The expression of these genes was significantly downregulated in the dyt1-2 mutant. However, the expression levels of these genes in the transgenic plants were restored to the level of the wild type (Figure 6d). These results indicate that the expression of the exine-related genes is restored and that the materials for the pollen wall are synthesized in the transgenic lines. Therefore, the normal expression of TDF1 in dyt1 is sufficient to drive the expression of the exine-related genes directly or indirectly for the synthesis of the pollen wall materials. Figure 3. TDF1 is a direct target of DYT1. (a) The black box indicates the potential DYT1binding sites in the TDF1 promoter region (1 kb). The grey short lines show the fragments amplified in the ChIP-PCR assays. (b, c) The enrichments of TDF1 promoter were confirmed by ChIP–quantitative PCR (qPCR) with the primer sets (pTDF1-1, pTDF1-2, pTDF13, pTDF1-4), using the wild-type (b) and DYT1– GFP (c) samples. Fold of enrichment is calculated from three independent replicates. Error bars represent the standard deviation (n = 3). AB+, presence of antibody; AB, absence of antibody.
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During anther development, SPL/NZZ is a key regulator that controls the early differentiation of primary sporogenous cells into microsporocytes (Schiefthaler et al., 1999; Yang et al., 1999). Tapetum fate determination requires a signaling pathway between reproductive and non-reproductive cells, such as the EMS1/TPD1-dependent pathway (s) (Zhao et al., 2002; Yang et al., 2003). DYT1 is a critical
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DYT1 is required for early tapetum development and function
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Figure 4. The expression of regulatory genes were recovered in proDYT1:TDF1 transgenic line. (a) The proDYT1:TDF1 construct used for genetic complementation assay. (b–d) The main stem of the wild-type (b), dyt1-2 (c), proDYT1:TDF1 with dyt1/dyt1 background (d), and proDYT1:TDF1 with dyt1/+ background (e) plants. (f) Real-time PCR of TDF1 expression in wild-type, dyt1-2, proDYT1:TDF1 with dyt1/dyt1 background and proDYT1:TDF1 with dyt1/+ background plants. (g, h) Expression pattern of selected putative regulatory genes in wild-type, dyt1-2 and proDYT1:TDF1 with dyt1/dyt1 background plants.
© 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd, The Plant Journal, (2014), 80, 1005–1013
1010 Jing-Nan Gu et al.
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Figure 5. TDF1 partially rescues dyt1-2 phenotype. Anther cross-section from wild-type (a–e), dyt-2 mutant (f–j) and proDYT1:TDF1 transgenic lines (k–o). E, epidermis; En, endothecium; ML, middle layer; Ms; microsporocyte; Msp, microspore; T, tapetum; Tds, tetrads. Bar = 20 lm.
transcription factor for early tapetum development acting downstream of SPL, EMS1 and TPD1 (Zhang et al., 2006; Chang et al., 2011). Using promoter-GUS analysis, we determined that the promoter region (550 to 463 bp) of DYT1 contains important cis-elements essential for DYT1 expression (Figure 1). These elements are putative binding sites for MYB, WRKY, and bZIP family proteins. Of the upstream regulators, SPL encodes a putative MADs-Box transcription factor. However, no MADs-Box binding site was found in the essential region (Table S1). It is likely that there are some other factors that can bind to the essential cis-elements of the DYT1 promoter to regulate its expression. The identification of these factors will contribute to understanding the early events of tapetum development. During anther development, the primary sporogenous cells (PSCs) and primary parietal cells (PPCs) are derived from the archesporial cells in the L2 layer of the primordium. PSCs continue dividing, giving rise to a central sporogenous mass, and the PPCs develop to form three concentric parietal layers, including the tapetum (Goldberg et al., 1993). The DYT1 transcript was initially detected both in PPCs and PSCs based on in situ hybridization (Figure 1j; Zhang et al., 2006). The DYT1 transcript levels were highest in the tapetum and low in the meiocytes at stages 5 and 6 (Figure 1j,k). However, DYT1–GFP was only detected in the tapetal cells from stage 5 to stage 7 (Figure 2e–h), indicating that the DYT1 only accumulates in the tapetal cells. The tapetum localization of the DYT1 is consistent with its function in normal tapetal development.
There may exist an unknown mechanism that regulates the translation of DYT1 during early anther development. DYT1 directly regulates TDF1 expression in the tapetum The tdf1 mutant exhibits increased vacuolation and dysfunction of the tapetum, which is similar to the dyt1 mutant (Zhu et al., 2008). RNA in situ hybridization showed that the highest expression of both DYT1 and TDF1 occurred at similar tapetal development stages. Analysis of the dyt1 tdf1 double mutant suggests that TDF1 acts downstream of DYT1 in the tapetal genetic pathway (Zhu et al., 2011). The induction of DYT1 activity in vivo activates TDF1 expression (Feng et al., 2012). In this study, ChIP assays demonstrated that the DYT1 can bind the TDF1 promoter in vivo (Figure 3c). DYT1 binds to the G-box of the target gene promoters. Any mutation in a single base of the G-box sequence affects the direct binding of DYT1 in vitro (Feng et al., 2012). The TDF1 promoter contains several E-box motifs, which is a variant of the G-box. However, the EMSA assay utilizing the DYT1 recombinant protein showed that DYT1 does not bind to the E-box motif on the promoter of TDF1 in vitro. However, the DYT1 could bind to the G-box motif (Figure S1). Recently, CIB1, a bHLH protein, has been reported to form heterodimers with other CIB proteins to bind E-boxes in vitro (Liu et al., 2014). In addition, DYT1 interacts with other bHLH proteins (Feng et al., 2012). It is likely that DYT1 and several other bHLH proteins form a heterodimer to bind to the E-box motif of the TDF1 promoter to regulate its expression.
© 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd, The Plant Journal, (2014), 80, 1005–1013
DYT1 regulates TDF1 for tapetum and pollen wall formation 1011 Figure 6. Pollen wall material synthesis in proDYT1:TDF1. (a–c) Cytochemical staining of semi-thin sections of wild-type, dyt1-2, proDTY1:TDF1 plants at stage 10. The red fluorescence indicates DiOC2 could stain the sporopollenin precursors. The blue fluorescence indicates the Tinopal which binds to the cellulose. (a) Wild-type tapetal cells exhibit red fluorescence. (b) No red fluorescence are detected in the dyt1-2 tapetum. (c) Red fluorescence is observed in proDYT1:TDF1 tapetal cells. The arrows point to tapetal cells. (d) The expression pattern of selected pollen wall material synthesis-related genes in wildtype, dyt1-2 and proDYT1:TDF1 plants. Bar = 40 lm.
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The transcription factors TDF1, AMS, MS188/MYB80, TEK and MS1 are essential for tapetal development and functions (Zhu et al., 2011; Lou et al., 2014). In the dyt1 mutant, the expression of these proteins is very low. Feng et al. (2012) proposed that DYT1 might regulate distinct temporal patterns of gene expression through feed-forward loops. In the present study, we expressed TDF1 in the dyt1-2 mutant using a transgenic method. We found that the expression of AMS, MS188/MYB80, TEK and MS1 was restored in the dyt1 knockout mutant with normal expression of TDF1 (Figure 4g). Therefore, TDF1 is sufficient to activate the expression of these transcriptional regulators during anther development.
wild type. Therefore, the normal tapetal development is important for pollen formation and DYT1 likely regulates several other genes essential for tapetum development and pollen formation. EXPERIMENTAL PROCEDURES Plant materials Both the wild-type and mutant plants are the Landsberg erecta (Ler) ecotype of Arabidopsis. Plants were grown under 16-h light/ 8-h dark conditions at approximately 22°C. The dyt1-2 mutant was isolated from EMS mutation lines as described by Song et al. (2009).
Analyses of the DYT1 promoter DYT1 plays a role during the synthesis of pollen wall materials via TDF1 Previous transcriptome analysis showed that DYT1 integrates multiple biological processes for pollen development (Feng et al., 2012). In the current study, many genes essential for pollen wall material synthesis were recovered in the proDYT1::TDF1 transgenic lines (Figure 6d). The histochemical staining showed the fluorescence of pollen wall materials in the transgenic plants (Figure 6c). This indicated that TDF1 is important for pollen wall material synthesis and transport. Although the material for the pollen wall was synthesized in the tapetum, no mature pollen grain was formed in the proDYT1:TDF1 transgenic lines, indicating that material synthesis is not sufficient for pollen wall formation. Cytological analysis showed that the tapetum development remained abnormal compared to the
The promoter fragments of DYT1 were amplified by PCR with specific primers and ligated into the multiple cloning sites of a modified binary vector pBI121 to construct the DYT1::GUS vectors, which were then introduced into the wild-type plants. The seeds of transformed plants were screened for kanamycin-resistant seedlings, which were transferred to the soil. GUS staining was performed according to the method of Jefferson et al. (1987).
In situ hybridization Non-radioactive RNA in situ hybridization was performed as described in the Digoxigenin (DIG) RNA Labeling Kit (Roche, http://lifescience.roche.com/) and the PCR DIG Probe Synthesis Kit (Roche). The PCR product was cloned into the pSK vector and sequenced. The plasmid DNA was completely digested and prepared for transcription templates with T3 or T7 RNA polymerase (Roche), respectively. The Olympus BX-51 digital camera took the photos.
© 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd, The Plant Journal, (2014), 80, 1005–1013
1012 Jing-Nan Gu et al. ChIP Young flower buds were collected for ChIP assay as described by Haring et al. (2007). The experimental procedure and reagents preparation were as described (Yang et al., 2013). The DNA from immunocomplexes was dissolved with water and used for qChiPPCR analysis. The Ct difference (DCt) between the AB+ and AB– samples were obtained. The fold enrichment was calculated as 2ðDCt Þ . In this experiment, wild type was used as a control.
Microscopy Nikon D7000 camera was used to photograph, and Olympus DP70 camera to take flower images (Nikon, http://www.nikon.com/). Alexander’s solution was used as described (Alexander, 1969). DYT1–GFP in the anther was observed using a Carl Zeiss confocal laser scanning microscope (LSM 5 PASCAL; Zeiss, http://www.zeiss.com). The procedure of semi-thin section and DiOC2 staining were performed as described previously (Zhang et al., 2007; Lou et al., 2014). The sections were photographed by BX51 camera (Olympus, http://www.olympus-global.com/en/).
Quantitative PCR Quantitative PCR analysis was performed as described by Lou et al. (2014). The b-tubulin gene was analysis as a positive control, and each sample had three replicates. The relative expression levels were calculated according to cycle number. The relevant primer sequences we designed are provided in Table S2. Sequence data from this article can be found in the GenBank/ EMBL data libraries under accession numbers DYT1 (AT4G21330), TDF1 (AT3G28470), AMS (AT2G16910), MS188/MYB80 (AT5G 56110), TEK (AT2G42940), MS1 (AT5G22260), SPL (AT4G27330), EMS1 (AT5G07280), TPD1 (AT4G24972), SERK1 (AT1G71830), SERK2 (AT1G34210), MYB33 (AT5G06100), MYB65 (AT3G11440), ACOS5 (AT1G62940), CYP703A2 (AT1G01280), CYP704B1 (AT1G69500), MS2 (AT3G11980), PKSA (AT1G02050), PKSB (AT4G34850), ABCG26 (AT3G13220) and TUB (AT5G23860).
ACKNOWLEDGEMENTS This work was supported by grants from the National Science Foundation of China (31100227) and the Innovation Program of Shanghai Municipal Education Commission (12YZ087).
SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article. Figure S1. DYT1 recombinant protein binds to G-box rather than E-box motif. Table S1. Cis-acting elements are shown in DYT1 promoter region. Table S2. List of primers used in this study.
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Wilson, Z.A., Morroll, S.M., Dawson, J., Swarup, R. and Tighe, P.J. (2001) The Arabidopsis MALE STERILITY1 (MS1) gene is a transcriptional regulator of male gametogenesis, with homology to the PHD-finger family of transcription factors. Plant J. 28, 27–39. Xu, J., Yang, C., Yuan, Z., Zhang, D., Gondwe, M.Y., Ding, Z., Liang, W., Zhang, D. and Wilson, Z.A. (2010) The ABORTED MICROSPORES regulatory network is required for postmeiotic male reproductive development in Arabidopsis thaliana. Plant Cell, 22, 91–107. Yang, W.C., Ye, D., Xu, J. and Sundaresan, V. (1999) The SPOROCYTELESS gene of Arabidopsis is required for initiation of sporogenesis and encodes a novel nuclear protein. Genes Dev. 13, 2108–2117. Yang, S.L., Xie, L.F., Mao, H.Z., Puah, C.S., Yang, W.C., Jiang, L., Sundaresan, V. and Ye, D. (2003) Tapetum determinant1 is required for cell specialization in the Arabidopsis anther. Plant Cell, 15, 2792–2804. Yang, C., Vizcay-Barrena, G., Conner, K. and Wilson, Z.A. (2007) MALE STERILITY1 is required for tapetal development and pollen wall biosynthesis. Plant Cell, 19, 3530–3548. Yang, J., Tian, L., Sun, M.X., Huang, X.Y., Zhu, J., Guan, Y.F., Jia, Q.S. and Yang, Z.N. (2013) AUXIN RESPONSE FACTOR17 is essential for pollen wall pattern formation in Arabidopsis. Plant Physiol. 162, 720–731. Zhang, W., Sun, Y., Timofejeva, L., Chen, C., Grossniklaus, U. and Ma, H. (2006) Regulation of Arabidopsis tapetum development and function by DYSFUNCTIONAL TAPETUM1 (DYT1) encoding a putative bHLH transcription factor. Development, 133, 3085–3095. Zhang, Z.B., Zhu, J., Gao, J.F. et al. (2007) Transcription factor AtMYB103 is required for anther development by regulating tapetum development, callose dissolution and exine formation in Arabidopsis. Plant J. 52, 528– 538. Zhao, D.Z., Wang, G.F., Speal, B. and Ma, H. (2002) The excess microsporocytes1 gene encodes a putative leucine-rich repeat receptor protein kinase that controls somatic and reproductive cell fates in the Arabidopsis anther. Genes Dev. 16, 2021–2031. Zhu, J., Chen, H., Li, H., Gao, J.F., Jiang, H., Wang, C., Guan, Y.F. and Yang, Z.N. (2008) Defective in Tapetal development and function 1 is essential for anther development and tapetal function for microspore maturation in Arabidopsis. Plant J. 55, 266–277. Zhu, J., Zhang, G.Q., Chang, Y.H., Li, X.C., Yang, J., Huang, X.Y., Yu, Q.B., Chen, H., Wu, T.L. and Yang, Z.N. (2010) AtMYB103 is a crucial regulator of several pathways affecting Arabidopsis anther development. Sci. China Life Sci. 53, 1112–1122. Zhu, J., Lou, Y., Xu, X.F. and Yang, Z.N. (2011) A genetic pathway for tapetum development and function in Arabidopsis. J. Integr. Plant Biol. 53, 892–900.
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doi: 10.1111/tpj.12694
DYT1 directly regulates the expression of TDF1 for tapetum development and pollen wall formation in Arabidopsis Jing-Nan Gu1,†, Jun Zhu1,†, Yu Yu2, Xiao-Dong Teng1, Yue Lou1, Xiao-Feng Xu1, Jia-Li Liu1 and Zhong-Nan Yang1,* 1 Development Center of Plant Germplasm Resources, College of Life and Environment Sciences, Shanghai Normal University, Shanghai, 200234 China, and 2 Shanghai Huangxing School, Shanghai 200093, China Received 11 July 2014; revised 29 September 2014; accepted 30 September 2014; published online 6 October 2014. *For correspondence (e-mail [email protected]). † These authors contributed equally to this work.
SUMMARY The tapetum plays a critical role during the development and maturation of microspores. DYSFUNCTIONAL TAPETUM 1 (DYT1) is essential for early tapetal development. Here, we determined that the promoter region (550 to 463 bp) contains indispensable cis-elements for DYT1 expression. Although DYT1 transcripts can be detected in both meiocytes and tapetal cells, localization of DYT1–GFP demonstrated that DYT1 is strictly located in tapetal cells during microsporogenesis. Chromatin immunoprecipitation (ChIP) analysis revealed that DYT1 directly binds the promoter region of Defective in Tapetal Development and Function 1 (TDF1), a transcription factor essential for tapetum development. When TDF1 driven by the DYT1 promoter is expressed in a dyt1 mutant, the expression of the transcription factors AMS, MS188/MYB80, TEK and MS1 and the pollen wall-related genes are restored. Although the pollen wall is not formed and the microspores are ruptured, DIOC2 staining showed that fatty acids, the precursors of the pollen wall, were synthesized in the transgenic lines. These results indicate that DYT1 regulates the expression of AMS, MS188/MYB80, TEK and MS1 for pollen wall formation, primarily via TDF1. Keywords: Arabidopsis thaliana, transcription factors, tapetum, pollen wall, DYT1.
INTRODUCTION The anther is the male organ that produces pollen for plant reproduction. The mature anther consists of four somatic cell layers, namely the epidermis, endothecium, middle layer, and tapetum enclosing gametophyte cells. The tapetum originates from the L2 layer as one of three germ layers, L1, L2 and L3, in the stamen primordial (Goldberg et al., 1993). As the innermost of the four somatic cell layers surrounding male gametophytes, the tapetum plays an essential role in pollen development (Mariani et al., 1990; Sanders et al., 1999). Tapetal fate determination depends on the signaling pathway triggered by several leucine-rich repeat receptor-like protein kinases (LRR-RLKs) during the early stage of anther development. Dysfunction of these pathways leads to additional microsporocytes lacking the tapetal cells, resulting in complete male sterility (Canales et al., 2002; Zhao et al., 2002; Yang et al., 2003; Albrecht et al., 2005; Colcombet et al., 2005; Ma, 2005; Jia et al., 2008). During anther development, the tapetum undergoes dramatic morphological differentiation to form the binuclear © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd
secretory cell, which is packed with ribosomes, mitochondria, Golgi bodies, endoplasmic reticulum and vesicles (Stevens and Murray, 1981; Bedinger, 1992). During male gametogenesis, the tapetal cells provide the precursors of sporopollenin for pollen exine formation, the callase complex (also termed b-1,3-glucanase) to release microspores from the tetrad, and numerous elaioplasts and cytoplasmic lipid bodies for pollen coat formation (Mascarenhas, 1975; Stieglitz, 1977; Pacini and Juniper, 1979; Hesse and Hess, 1993). In Arabidopsis, several transcription factors regulate the tapetum and its functions. DYT1 encodes a putative basic helix–loop–helix (bHLH) transcription factor, and TDF1 encodes a putative R2R3 MYB transcription factor. Mutations in these two genes cause tapetal hypertrophy extending into the locule and resulting in sporophytic male sterility (Zhang et al., 2006; Zhu et al., 2008). The transcription factor ABORTED MICROSPORES (AMS) encodes a bHLH family protein that plays an important role in tapetum development and pollen wall formation (Sorensen 1005
1006 Jing-Nan Gu et al. et al., 2003; Xu et al., 2010). Another member of the R2R3 MYB family, MS188/MYB80, apparently regulates sexine formation (Zhang et al., 2007; Zhu et al., 2010). MALE STERILITY1 (MS1), a nuclear protein with PHD-finger motifs, is essential for tapetum development at the post-meiotic phase (Wilson et al., 2001; Ito et al., 2007; Yang et al., 2007). These transcription factors form a genetic pathway (DYT1-TDF1-AMS-MS188/MYB80-MS1) for tapetal development and function (Zhu et al., 2011). Cytological evidence indicates that the majority of the precursor materials of the pollen wall originate from the tapetum (Heslop-Harrison, 1962; Dickinson and HeslopHarrison, 1968). The pollen wall consists of two main layers, the outer exine layer and the inner intine layer. The exine is further divided into the sexine and the nexine (Scott, 1994). Recently, MS188/MYB80 and TEK have been reported to control sexine formation and nexine formation, respectively (Zhang et al., 2007; Lou et al., 2014). Both are directly regulated by AMS (Lou et al., 2014). The sexine layer most likely consists of sporopollenin, which is derived from long-chain fatty acids, oxygenated aromatic rings and phenylpropionic acids (Piffanelli et al., 1998; Ariizumi and Toriyama, 2011). Multiple enzymes in the tapetum, including ACOS5, CYP703A2, CYP704B1, MS2, PKSA and PKSB are involved in the biochemical pathways for sexine formation (Morant et al., 2007; de Azevedo Souza et al., 2009; Dobritsa et al., 2009, 2010; Kim et al., 2010; Chen et al., 2011). DYT1 functions during the early stage of tapetum development. Transcriptome analysis showed that many genes in the tapetum are downregulated in the dyt1 mutant (Feng et al., 2012). However, detailed regulation of the tapetum remains unclear. Here, we show that DYT1 directly binds the promoter of TDF1 during tapetum development. Furthermore, a transgenic rescue assay demonstrated that DYT1 regulates the expression of genes for pollen wall formation, primarily via TDF1. RESULTS The promoter region (550 to 463 bp) of DYT1 is essential for its expression Previous studies showed that the 631 bp promoter fragment of DYT1 is sufficient to drive its expression for its function in the anther (Song et al., 2009). This promoter region contains specific cis-acting elements including MYB, WRKY, ARR1, GATABOX, GTGANTG10, ACGTBOX, W-box, and DOF components according to the PLACE database (http://www.dna.affrc.go.jp/PLACE/) (Higo et al., 1999) (Table S1). We created constructs with GUS driven by different lengths of the DYT1 promoter fragments (Figure 1a), and transformed the constructs into wild-type. GUS activity could be detected in transgenic lines with promoter fragments of 631 and 550 bp (Figure 1b,c). In situ hybridization
was used to analyze the expression pattern of GUS driven by the 550 bp fragment. During anther stage 4, the tapetal cells are differentiated when the four-lobed anther pattern is established (Sanders et al., 1999). A weak GUS signal was detected in the anther at this stage (Figure 1i). At stages 5 and 6, when the tapetal layer is clearly present in the anther and the microcytes undergo meiosis, the GUS signal was highest in the tapetum compared to the low level in the meiocytes (Figure 1j,k). At stage 7, when meiosis is complete and the tetrad is formed, the GUS signal was significantly reduced (Figure 1l). The GUS expression pattern driven by the 550 bp fragment was similar to the DYT1 expression in the wild type (Zhang et al., 2006; Zhu et al., 2011). However, no GUS staining was observed in the transgenic plants with the DYT1 promoters of 463, 331, 180 and 91 bp, respectively (Figure 1d–g). These results suggest that the promoter region (550 to 463 bp) contains essential motifs for DYT1 expression. DYT1–GFP is specifically localized in tapetal cells To further understand the function of DYT1 during anther development, we created a DYT1–GFP fusion protein driven by the 631-bp native DYT1 promoter (Figure 2a). After transformed into heterozygous (dyt1-2/+) plants, we identified a transgenic line with a homozygous (dyt1/dyt1) background using closely linked markers (Figure 2c and Table S2). This transgenic line exhibited a fully fertile phenotype (Figure 2b), indicating that the DYT1–GFP fusion protein was enable to rescue the sterile phenotype of the dyt1 mutation. In the wild type, GFP fluorescence was not observed in the anther. In the transgenic line, GFP fluorescence was not detected at stage 4 (Figure 2d). At stage 5, the GFP signal was initially present within the tapetal cells (Figure 2e). At stage 6, a stronger GFP signal was detected in the tapetal cells, which formed a circle within the locule. In meiocytes, the DYT1–GFP fluorescence was not observed (Figure 2f,g). At the tetrad stage (stage 7), the GFP signal was weakly detected in the tapetal cells. No GFP signal was observed in tetrads (Figure 2h,i). At stage 8, when the microspores are released from the tetrads, GFP fluorescence was not detected in the anther (Figure 2j, k). These results indicate that the DYT1 is strictly localized in tapetal cells, which is consistent with its role in tapetal development. ChIP assay demonstrates that DYT1 is associated with the TDF1 promoter TDF1 is a transcriptional regulator downstream of DYT1 and is involved in tapetum development and function (Zhu et al., 2008). In dyt1-2, TDF1 is barely detected (Zhu et al., 2011). Previous studies showed that DYT1 bind to the G-box (TCACGTGA) of target gene promoters (Feng et al., 2012). However, no G-box is presented in the 1000-bp promoter region of TDF1. There are three E-box motifs
© 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd, The Plant Journal, (2014), 80, 1005–1013
DYT1 regulates TDF1 for tapetum and pollen wall formation 1007 Figure 1. Promoter analysis of DYT1. (a) Construction of GUS driven by different length fragments of the DYT1 promoter. (b–g) GUS activity of the transgenic lines with different length fragments of the DYT1 promoter. (b) The anther of proDYT1-630:Gus. (c) The anther of proDYT1-550:Gus. (d) The anther of proDYT1-463:Gus. (e) The anther of proDYT1-331:Gus. (f) The anther of proDYT1-180: Gus. (g) The anther of proDYT1-94:Gus. (h–l) In situ hybridization analysis of transgenic lines with GUS RNA probe during anther development. ProDYT1-550:Gus anthers from stages 3 (h), 4 (i), 5 (j), 6 (k), 7 (l). (m) Stage 6 anther hybridized with the sense probe. Bars = 40 lm.
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(CANNTG) in this region, which is a variant of the G-box. We used chromatin immunoprecipitation (ChIP) with a GFP monoclonal antibody to determine whether DYT1–GFP directly binds to the promoter of TDF1. Four probes were designed in the promoter region of TDF1 (Figure 3a). Quantitative ChIP-PCR (qChIP-PCR) showed no significant enrichment of the TDF1 promoter fragment in the wild type (Figure 3b). By contrast, the pTDF1-3 fragment of the TDF1 promoter was increased when GFP monoclonal antibodies were used (+AB) compared with the AB samples (Figure 3c). No significant increase in the other three TDF1 promoter fragments was observed (Figure 3c). These results suggest that DYT1 binds to the TDF1 promoter in vivo to regulate TDF1 expression. The expression of TDF1 in dyt1-2 can partially rescue the dyt1-2 phenotype To determine whether DYT1 functions through TDF1, we made a construct with the DYT1 promoter (631 bp), the genomic sequence (1143 bp) and 30 untranslated region (257 bp) of TDF1, and introduced this construct into dyt1-2 heterozygous plant (Figure 4a). Of the 23 independent transgenic lines, seven transgenic lines were identified to be dyt1-2 homozygous background using closely linked molecular markers (Table S2). These transgenic lines all showed complete male sterility (Figure 4d). Quantitative RT-PCR showed that the TDF1 transcript was overexpressed in these transgenic lines (Figure 4f). Therefore, the
expression of TDF1 in dyt1-2 does not rescue the fertility. In the transgenic plants with dyt1-2 heterozygous background, the overexpression of TDF1 did not affect their fertility (Figure 4e). Semi-thin sections were analyzed to identify the morphological differences in anther development between the dyt1-2 mutant and the transgenic lines with dyt1-2 background. Although both the mutant and transgenic lines showed a hypertrophic tapetum, the tetrads of the transgenic plants appeared more regular than the dyt1 mutant (Figure 5h,m). The locule space could be observed in transgenic plants at stage 11, whereas it was crushed in the dyt1-2 mutant (Figure 5j,o). This result reveals that tapetum and tetrads in proDYT1::TDF1 transgenic lines are less defective than that in the dyt1-2 plants. We used the proDYT1::TDF1 transgenic line to identify the genes regulated by DYT1 via TDF1. The SPL, EMS1, SERK1, SERK2 and TPD1 have been reported to be important for early anther development and tapetal fate determination. Genetic studies showed that these genes function upstream of DYT1 (Yang et al., 1999; Ma, 2005; Zhang et al., 2006). The qRT-PCR showed that the expressions of these genes were either increased or nearly normal in proDYT1::TDF1 transgenic plants. This result is consistent with previous studies that these genes act upstream of DYT1. Additionally, the GAMYB-like genes, AtMYB33 and AtMYB65 play a redundantly role for tapetum development (Millar and Gubler, 2005). The double mutant of them showed the similar tapetal defect compared with that in
© 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd, The Plant Journal, (2014), 80, 1005–1013
1008 Jing-Nan Gu et al.
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dyt1. These genes likely act at approximately the same stage as DYT1 (Zhang et al., 2006). The expression of these genes was up-regulated in proDYT1::TDF1 transgenic plants (Figure 4h). The transcription factors AMS, MS188/ MYB80, MS1, and TEK are essential for tapetum development and pollen wall formation. They act downstream of DYT1 and TDF1 (Zhu et al., 2008; Lou et al., 2014). The expression of these genes was nearly restored to the level in the wild type (Figure 4g). These results suggest that DYT1 regulates the expression of these transcription factors via TDF1 for tapetum development and pollen wall formation. Genes for pollen wall synthesis act downstream of TDF1 During anther development, tapetal cells are active in the biosynthesis of phenylpropanoids, steryl esters, long-chain alkanes, and flavonoids for exine formation (Hernould et al., 1998; Scott et al., 2004). Although pollen grains are ruptured in the locule of transgenic lines during the late stage (Figure 5o), the expression of the regulators for exine formation, AMS, MS188/MYB80 and TEK, are
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Figure 2. Genetic complement and expression pattern of DYT1–GFP. (a) The proDYT1:DYT1–GFP constructs including 631 bp native promoter, DYT1 genomic fragment and GFP coding region used for genetic complementation assays. (b) The siliques and stained anthers of dyt1-2 and proDYT1:DYT1–GFP line. (c) The molecular identification of the dyt1 mutated site. (d–k) Fluorescence confocal images of the DYT1–GFP fusion protein. The green channel in (d–k) showed the GFP expression (530 nm), red channel showed the chlorophyll autofluorescence (>560 nm). (d) At stage 4, no GFP signal is observed. (e) At stage 5, the GFP is initially detected in the tapetal cells. (f) At stage 6, the GFP is strongly expressed in tapetum. (h) At stage 7, the expression of GFP is dramatically decreased. (j) At stage 8, GFP signal disappeared in anther. The bright-field images of (g), (i) and (k) show that DYT1–GFP is not located in the meiocytes, tetrads and microspores, respectively. T, tapetum; Mc, meiocytes; Td, tetrads; Msp, microspores. Bars = 40 lm.
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recovered in the transgenic line. We determined whether the exine precursors of the pollen wall were synthesized in the tapetal cells of the transgenic plants. The diethyloxadicarbocyanine iodide (DIOC2) and Tinopal were utilized to stain the fatty acid content of exine and cellulose materials in the transgenic line and mutant plant (Regan and Moffatt, 1990; Lou et al., 2014). In the wild type, the red fluorescence of DIOC2 staining was observed in the tapetum and microspores and the blue fluorescence of Tinopal staining showed cell outline in the anther (Figure 6a). In the transgenic plants, the red fluorescence was also detected in the defective tapetum and ruptured pollen, indicating that some exine materials were synthesized in these cells (Figure 6c). However, no red fluorescence was observed in the anther of dyt1-2 mutant (Figure 6b). This result indicated that the materials for the pollen wall could be synthesized when the TDF1 expressed. In Arabidopsis, ACOS5, CYP703A2, CYP704B1, MS2, PKSA, PKSB and ABCG26 are involved in sporopollenin synthesis and transport (Morant et al., 2007; de Azevedo Souza et al., 2009; Dobritsa et al., 2009; Kim et al., 2010;
© 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd, The Plant Journal, (2014), 80, 1005–1013
DYT1 regulates TDF1 for tapetum and pollen wall formation 1009 Quilichini et al., 2010; Chen et al., 2011). The expression of these genes was significantly downregulated in the dyt1-2 mutant. However, the expression levels of these genes in the transgenic plants were restored to the level of the wild type (Figure 6d). These results indicate that the expression of the exine-related genes is restored and that the materials for the pollen wall are synthesized in the transgenic lines. Therefore, the normal expression of TDF1 in dyt1 is sufficient to drive the expression of the exine-related genes directly or indirectly for the synthesis of the pollen wall materials. Figure 3. TDF1 is a direct target of DYT1. (a) The black box indicates the potential DYT1binding sites in the TDF1 promoter region (1 kb). The grey short lines show the fragments amplified in the ChIP-PCR assays. (b, c) The enrichments of TDF1 promoter were confirmed by ChIP–quantitative PCR (qPCR) with the primer sets (pTDF1-1, pTDF1-2, pTDF13, pTDF1-4), using the wild-type (b) and DYT1– GFP (c) samples. Fold of enrichment is calculated from three independent replicates. Error bars represent the standard deviation (n = 3). AB+, presence of antibody; AB, absence of antibody.
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During anther development, SPL/NZZ is a key regulator that controls the early differentiation of primary sporogenous cells into microsporocytes (Schiefthaler et al., 1999; Yang et al., 1999). Tapetum fate determination requires a signaling pathway between reproductive and non-reproductive cells, such as the EMS1/TPD1-dependent pathway (s) (Zhao et al., 2002; Yang et al., 2003). DYT1 is a critical
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DYT1 is required for early tapetum development and function
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DISCUSSION
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Figure 4. The expression of regulatory genes were recovered in proDYT1:TDF1 transgenic line. (a) The proDYT1:TDF1 construct used for genetic complementation assay. (b–d) The main stem of the wild-type (b), dyt1-2 (c), proDYT1:TDF1 with dyt1/dyt1 background (d), and proDYT1:TDF1 with dyt1/+ background (e) plants. (f) Real-time PCR of TDF1 expression in wild-type, dyt1-2, proDYT1:TDF1 with dyt1/dyt1 background and proDYT1:TDF1 with dyt1/+ background plants. (g, h) Expression pattern of selected putative regulatory genes in wild-type, dyt1-2 and proDYT1:TDF1 with dyt1/dyt1 background plants.
© 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd, The Plant Journal, (2014), 80, 1005–1013
1010 Jing-Nan Gu et al.
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Figure 5. TDF1 partially rescues dyt1-2 phenotype. Anther cross-section from wild-type (a–e), dyt-2 mutant (f–j) and proDYT1:TDF1 transgenic lines (k–o). E, epidermis; En, endothecium; ML, middle layer; Ms; microsporocyte; Msp, microspore; T, tapetum; Tds, tetrads. Bar = 20 lm.
transcription factor for early tapetum development acting downstream of SPL, EMS1 and TPD1 (Zhang et al., 2006; Chang et al., 2011). Using promoter-GUS analysis, we determined that the promoter region (550 to 463 bp) of DYT1 contains important cis-elements essential for DYT1 expression (Figure 1). These elements are putative binding sites for MYB, WRKY, and bZIP family proteins. Of the upstream regulators, SPL encodes a putative MADs-Box transcription factor. However, no MADs-Box binding site was found in the essential region (Table S1). It is likely that there are some other factors that can bind to the essential cis-elements of the DYT1 promoter to regulate its expression. The identification of these factors will contribute to understanding the early events of tapetum development. During anther development, the primary sporogenous cells (PSCs) and primary parietal cells (PPCs) are derived from the archesporial cells in the L2 layer of the primordium. PSCs continue dividing, giving rise to a central sporogenous mass, and the PPCs develop to form three concentric parietal layers, including the tapetum (Goldberg et al., 1993). The DYT1 transcript was initially detected both in PPCs and PSCs based on in situ hybridization (Figure 1j; Zhang et al., 2006). The DYT1 transcript levels were highest in the tapetum and low in the meiocytes at stages 5 and 6 (Figure 1j,k). However, DYT1–GFP was only detected in the tapetal cells from stage 5 to stage 7 (Figure 2e–h), indicating that the DYT1 only accumulates in the tapetal cells. The tapetum localization of the DYT1 is consistent with its function in normal tapetal development.
There may exist an unknown mechanism that regulates the translation of DYT1 during early anther development. DYT1 directly regulates TDF1 expression in the tapetum The tdf1 mutant exhibits increased vacuolation and dysfunction of the tapetum, which is similar to the dyt1 mutant (Zhu et al., 2008). RNA in situ hybridization showed that the highest expression of both DYT1 and TDF1 occurred at similar tapetal development stages. Analysis of the dyt1 tdf1 double mutant suggests that TDF1 acts downstream of DYT1 in the tapetal genetic pathway (Zhu et al., 2011). The induction of DYT1 activity in vivo activates TDF1 expression (Feng et al., 2012). In this study, ChIP assays demonstrated that the DYT1 can bind the TDF1 promoter in vivo (Figure 3c). DYT1 binds to the G-box of the target gene promoters. Any mutation in a single base of the G-box sequence affects the direct binding of DYT1 in vitro (Feng et al., 2012). The TDF1 promoter contains several E-box motifs, which is a variant of the G-box. However, the EMSA assay utilizing the DYT1 recombinant protein showed that DYT1 does not bind to the E-box motif on the promoter of TDF1 in vitro. However, the DYT1 could bind to the G-box motif (Figure S1). Recently, CIB1, a bHLH protein, has been reported to form heterodimers with other CIB proteins to bind E-boxes in vitro (Liu et al., 2014). In addition, DYT1 interacts with other bHLH proteins (Feng et al., 2012). It is likely that DYT1 and several other bHLH proteins form a heterodimer to bind to the E-box motif of the TDF1 promoter to regulate its expression.
© 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd, The Plant Journal, (2014), 80, 1005–1013
DYT1 regulates TDF1 for tapetum and pollen wall formation 1011 Figure 6. Pollen wall material synthesis in proDYT1:TDF1. (a–c) Cytochemical staining of semi-thin sections of wild-type, dyt1-2, proDTY1:TDF1 plants at stage 10. The red fluorescence indicates DiOC2 could stain the sporopollenin precursors. The blue fluorescence indicates the Tinopal which binds to the cellulose. (a) Wild-type tapetal cells exhibit red fluorescence. (b) No red fluorescence are detected in the dyt1-2 tapetum. (c) Red fluorescence is observed in proDYT1:TDF1 tapetal cells. The arrows point to tapetal cells. (d) The expression pattern of selected pollen wall material synthesis-related genes in wildtype, dyt1-2 and proDYT1:TDF1 plants. Bar = 40 lm.
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The transcription factors TDF1, AMS, MS188/MYB80, TEK and MS1 are essential for tapetal development and functions (Zhu et al., 2011; Lou et al., 2014). In the dyt1 mutant, the expression of these proteins is very low. Feng et al. (2012) proposed that DYT1 might regulate distinct temporal patterns of gene expression through feed-forward loops. In the present study, we expressed TDF1 in the dyt1-2 mutant using a transgenic method. We found that the expression of AMS, MS188/MYB80, TEK and MS1 was restored in the dyt1 knockout mutant with normal expression of TDF1 (Figure 4g). Therefore, TDF1 is sufficient to activate the expression of these transcriptional regulators during anther development.
wild type. Therefore, the normal tapetal development is important for pollen formation and DYT1 likely regulates several other genes essential for tapetum development and pollen formation. EXPERIMENTAL PROCEDURES Plant materials Both the wild-type and mutant plants are the Landsberg erecta (Ler) ecotype of Arabidopsis. Plants were grown under 16-h light/ 8-h dark conditions at approximately 22°C. The dyt1-2 mutant was isolated from EMS mutation lines as described by Song et al. (2009).
Analyses of the DYT1 promoter DYT1 plays a role during the synthesis of pollen wall materials via TDF1 Previous transcriptome analysis showed that DYT1 integrates multiple biological processes for pollen development (Feng et al., 2012). In the current study, many genes essential for pollen wall material synthesis were recovered in the proDYT1::TDF1 transgenic lines (Figure 6d). The histochemical staining showed the fluorescence of pollen wall materials in the transgenic plants (Figure 6c). This indicated that TDF1 is important for pollen wall material synthesis and transport. Although the material for the pollen wall was synthesized in the tapetum, no mature pollen grain was formed in the proDYT1:TDF1 transgenic lines, indicating that material synthesis is not sufficient for pollen wall formation. Cytological analysis showed that the tapetum development remained abnormal compared to the
The promoter fragments of DYT1 were amplified by PCR with specific primers and ligated into the multiple cloning sites of a modified binary vector pBI121 to construct the DYT1::GUS vectors, which were then introduced into the wild-type plants. The seeds of transformed plants were screened for kanamycin-resistant seedlings, which were transferred to the soil. GUS staining was performed according to the method of Jefferson et al. (1987).
In situ hybridization Non-radioactive RNA in situ hybridization was performed as described in the Digoxigenin (DIG) RNA Labeling Kit (Roche, http://lifescience.roche.com/) and the PCR DIG Probe Synthesis Kit (Roche). The PCR product was cloned into the pSK vector and sequenced. The plasmid DNA was completely digested and prepared for transcription templates with T3 or T7 RNA polymerase (Roche), respectively. The Olympus BX-51 digital camera took the photos.
© 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd, The Plant Journal, (2014), 80, 1005–1013
1012 Jing-Nan Gu et al. ChIP Young flower buds were collected for ChIP assay as described by Haring et al. (2007). The experimental procedure and reagents preparation were as described (Yang et al., 2013). The DNA from immunocomplexes was dissolved with water and used for qChiPPCR analysis. The Ct difference (DCt) between the AB+ and AB– samples were obtained. The fold enrichment was calculated as 2ðDCt Þ . In this experiment, wild type was used as a control.
Microscopy Nikon D7000 camera was used to photograph, and Olympus DP70 camera to take flower images (Nikon, http://www.nikon.com/). Alexander’s solution was used as described (Alexander, 1969). DYT1–GFP in the anther was observed using a Carl Zeiss confocal laser scanning microscope (LSM 5 PASCAL; Zeiss, http://www.zeiss.com). The procedure of semi-thin section and DiOC2 staining were performed as described previously (Zhang et al., 2007; Lou et al., 2014). The sections were photographed by BX51 camera (Olympus, http://www.olympus-global.com/en/).
Quantitative PCR Quantitative PCR analysis was performed as described by Lou et al. (2014). The b-tubulin gene was analysis as a positive control, and each sample had three replicates. The relative expression levels were calculated according to cycle number. The relevant primer sequences we designed are provided in Table S2. Sequence data from this article can be found in the GenBank/ EMBL data libraries under accession numbers DYT1 (AT4G21330), TDF1 (AT3G28470), AMS (AT2G16910), MS188/MYB80 (AT5G 56110), TEK (AT2G42940), MS1 (AT5G22260), SPL (AT4G27330), EMS1 (AT5G07280), TPD1 (AT4G24972), SERK1 (AT1G71830), SERK2 (AT1G34210), MYB33 (AT5G06100), MYB65 (AT3G11440), ACOS5 (AT1G62940), CYP703A2 (AT1G01280), CYP704B1 (AT1G69500), MS2 (AT3G11980), PKSA (AT1G02050), PKSB (AT4G34850), ABCG26 (AT3G13220) and TUB (AT5G23860).
ACKNOWLEDGEMENTS This work was supported by grants from the National Science Foundation of China (31100227) and the Innovation Program of Shanghai Municipal Education Commission (12YZ087).
SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article. Figure S1. DYT1 recombinant protein binds to G-box rather than E-box motif. Table S1. Cis-acting elements are shown in DYT1 promoter region. Table S2. List of primers used in this study.
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