Planta DOI 10.1007/s00425-014-2160-9
ORIGINAL ARTICLE
An anther development F-box (ADF) protein regulated by tapetum degeneration retardation (TDR) controls rice anther development Li Li • Yixing Li • Shufeng Song • Huafeng Deng • Na Li • Xiqin Fu • Guanghui Chen • Longping Yuan
Received: 17 June 2014 / Accepted: 20 August 2014 Ó Springer-Verlag Berlin Heidelberg 2014
Abstract Main conclusion In this study, we reported that a F-box protein, OsADF, as one of the direct targets of TDR, plays a critical role in rice tapetum cell development and pollen formation. Abstract The tapetum, the innermost sporophytic tissue of anther, plays an important supportive role in male reproduction in flowering plants. After meiosis, tapetal cells undergo programmed cell death (PCD) and provide nutrients for pollen development. Previously we showed that tapetum degeneration retardation (TDR), a basic helix-loop-helix transcription factor, can trigger tapetal PCD and control pollen wall development during anther development. However, the comprehensive regulatory network of TDR remains to be investigated. In this study, we cloned and characterized a panicle-specific expression F-box protein, anther development F-box (OsADF). By qRT-PCR and RNA in situ hybridization, we further confirmed that OsADF expressed Electronic supplementary material The online version of this article (doi:10.1007/s00425-014-2160-9) contains supplementary material, which is available to authorized users. L. Li S. Song H. Deng N. Li X. Fu L. Yuan (&) State Key Laboratory of Hybrid Rice, Hunan Hybrid Rice Research Centre, Changsha 410125, China e-mail: [email protected] L. Li e-mail: [email protected] L. Li H. Deng G. Chen Collaborative Innovation Center for Modern Production of Multiple Cropping System in Southern Paddy Area, Changsha 410128, China Y. Li G. Chen Hunan Agricultural University, Changsha 410128, China
specially in tapetal cells from stage 9 to stage 12 during anther development. In consistent with this specific expression pattern, the RNAi transgenic lines of OsADF exhibited abnormal tapetal degeneration and aborted microspores development, which eventually grew pollens with reduced fertility. Furthermore, we demonstrated that the TDR, a key regulator in controlling rice anther development, could regulate directly the expression of OsADF by binding to E-box motifs of its promoter. Therefore, this work highlighted the possible regulatory role of TDR, which regulates tapetal cell development and pollen formation via triggering the possible ADF-mediated proteolysis pathway. Keywords OsADF
Rice Male sterility Tapetum TDR PCD
Introduction Rice (Oryza sativa) is the most important agricultural crop feeding more than half of the world’s population, and has became a model monocot crop for both fundamental biology and agricultural traits because of its small genome size, efficient transformation system, available mutant collections, etc. (Jiang et al. 2012; Jung et al. 2005). Hybrid rice technology requires the utilization of male sterile lines due to higher vigor (heterosis) of hybrid plants over the parent’s lines with 20–30 % yield increase (Yuan et al. 2003). Expression analysis revealed that about 29,000 unique transcripts were detectable in rice anther and male reproductive organs, suggesting that the anther development requires the function of various genes. However, relatively little is known about the molecular regulatory network of male reproduction development, particularly in crop rice (Zhang et al. 2011).
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Although rice flower has a distinct structure compared to that of dicot Arabidopsis (Zhang and Yuan 2014; Zhang et al. 2013), the process of rice anther ontology is similar to that of Arabidopsis (Zhang et al. 2011), including the formation of stamen primordium, cell differentiation, and establishment of characteristic tissues. After the morphogenesis, the anther develops four lobes connected to the filament, and each lobe tissue consists of four somatic wall layers, i.e. the epidermis, the endothecium, the middle layer, and the tapetum in addition to the microspore mother cells (MMCs) within the locule (Ma 2005; McCormick 1993; Scott et al. 1991). As a nutritive tissue, tapetal cells directly contact with developing gametophytes and have been assumed to provide materials/signals for microspore formation, release and subsequent pollen maturation (Goldberg et al. 1993; Sanders et al. 2000; Wu and Cheun 2000) via tapetal disintegration promoted by programmed cell death (PCD) after the meiosis (Li et al. 2006). Premature or delayed tapetal PCD always causes abnormal tapetal development, leading to male sterility. Recently, genetic studies have identified that several transcription factors play a relatively conserved role in controlling tapetum identity, differentiation and degradation. Generally, the identity and numbers of the tapetal cells were suggested to be controlled by the species specific cell surface-localized leucine-rich repeat receptor-like kinases (LRR-RLKs) signaling. In rice, MULTIPLE SPOROCYTE 1 (MSP1) expressed in cells neighboring the male and female sporocytes, but not in the sporocytes. While OsTDL1A (TPD1-like 1A)/MICROSPORELESS 2 (MIL2) were mainly detected in inner parietal cells (Hong 2012, #33). Both of them were suggested to play key role in specifying the normal differentiation of primary parietal cells in rice. Then, rice GAMYB (Aya et al. 2009), MYB33/MYB65 (Millar and Gubler 2005), DYSFUNCTIONAL TAPETUM1 (DYT1) (Zhang et al. 2006), DEFECTIVE IN TAPETAL DEVELOPMENT AND FUNCTION1 (TDF1) (Zhu et al. 2008), ABORTED MICROSPORE (AMS) (Xu et al. 2010; Yang et al. 2014), MALE STERILITY1 (Wilson et al. 2001), PERSISTENT TAPETAL CELL 1 (PTC1) (Li et al. 2011), TDR INTERACTING PROTEIN2 (Fu et al. 2014),Undeveloped Tapetum1 (UDT1) (Jung et al. 2005), TAPETUM DEGENERATION RETARDATION (TDR) (Li et al. 2006; Zhang et al. 2008), ETERNAL TAPETUM 1 (EAT1) (Niu et al. 2013)and PERSISTENT TAPETAL CELL 1 (PTC1) (Li et al. 2011) play key roles in regulating tapetal cells development and degeneration, as well as normal microsporogenesis. TDR encodes a putative basic helix–loop–helix (bHLH) transcription factor and mainly expressed in the tapetum (Li et al. 2006). The TDR mutant exhibits delayed tapetal PCD and retarded degeneration with the increased size of
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tapetal cells as well as aborted pollen development, causing complete male sterile (Li et al. 2006). Mechanically, TDR was shown to affect the expression of genes relative to tapetal PCD and pollen wall formation (Zhang et al. 2008). Particularly, TDR can directly regulate the expression of OsCP1, Osc6, and CYP703A3 (Li et al. 2006; Wu and Cheun 2000). OsCP1 encodes a Cys protease which belongs to an enzyme family widely distributed in animals, plants, and microorganisms that play crucial functions in degrading intracellular proteins and promoting PCD. OsC6 encodes a specified lipid transfer protein (LTP) with lipid binding activity, and OsC6 is required for the development of the specified tapetal structures: orbicules (i.e. Ubisch bodies) and outer pollen wall (call exine) (Wu and Cheun 2000). Rice CYP703A3 is a cytochrome P450 hydroxylase catalyzing an in-chain hydroxylation for a specific substrate, lauric acid, and CYP703A3 required for development of anther cuticle and pollen exine (Yang et al. 2014). Furthermore, it was found recently that TDR could interact with two bHLH proteins: TDR INTERACTING PROTEIN2 (TIP2) (Fu et al. 2014) and ETERNAL TAPETUM 1 (EAT1) (Niu et al. 2013), respectively. TDR acts downstream of TIP2 and upstream of EAT1, the three bHLH proteins TIP2, TDR, and EAT1 form a regulatory cascade in controlling differentiation, morphogenesis, and degradation of anther wall layers, and pollen development (Fu et al. 2014). Totally, all these results confirm that TDR is a key regulator in promoting PCD-associated tapetal degeneration and limiting the cell size of tapetal layer. However, the detail regulatory networks of TDR in controlling tapetal PCD process still remains largely unknown. Therefore, we screened a set of panicle-specific expression genes and studied their role in regulating rice tapetum development, as well as its relationship to TDR. In this study, we reported that TDR is able to directly regulate the expression of a F-box protein-encoding gene, OsADF. The RNAi transgenic lines of OsADF grew defective tapetal cell and pollen formation. This finding highlighted the possible regulatory role of TDR in regulating the tapetal PCD via OsADF-mediated proteolysis pathway.
Materials and methods Isolation and sequence analysis The OsADF clone was obtained from a rice panicle cDNA. Nucleotide sequence and putative amino acid sequence were analyzed with the basic local alignment search tool (BLAST) at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov) and the soft Vector NTI AdvanceÒ 11.5 (Invitrogen). OsADF amino acid sequence was examined for the F-box domain using the
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hidden Markov model of SMART tool (http://smart.emblheidelberg.de/). Sequence comparisons were conducted using MUSCLE 3.6.
antisense or sense RNA probe hybridization and immunological detection of the hybridized probes were performed according to the procedure of Kouchi and Hata (1993).
RT-PCR and quantitative real-time PCR assay pHB-OsADF-RNAi vector construction and transfection Rice total RNA from root, shoot, leaf, lemma, palea, and pistil at stage 12 of anther development as well as anthers at various stages (stages 6–12) was extracted using Trizol Reagent kit (Invitrogen, USA) The stages of anthers were classified according to Zhang and Wilson (2009). After treatment with DNase (Promega, USA), 0.3 mg of RNA was used to synthesize oligo (dT)-primed first-strand cDNA using the ReverTra Ace-a-first strand cDNA synthesis kit (TOYOBO, Japan). Two microliters of the reverse transcription product was then used as template for conventional and quantitative RT-PCR analysis. PCR was performed with TaKaRa ExTaq DNA polymerase. RTPCR primers are listed in Supplemental Table 1, with Oryza sativa L. actin as internal control. The primers for real-time PCR were the same as the primers for RT–PCR. Quantitative real-time PCR was performed with a RotorGene RG3000A detection system (Corbett RESEARCH, Australia) using SYBR Green I master mix (Generay Biotech, Co. Ltd, Shanghai).
A 500 bp OsADF cDNA fragment with low similarity to other rice genes after sequence analysis was amplified from the cDNA of wild-type rice using primers RNAi-F and RNAi-R (Supplemental Table 1) and digested with EcoRV/PstI and XbaI/BamHI, respectively. The two fragments were subsequently inserted in opposite directions into the binary RNAi vector pHB (Mao et al. 2005) to generate the pHB-OsADF-RNAi plasmid with a double cauliflower mosaic virus 35S promoter. Rice calli were used for transformation with Agrobacterium EHA105 carrying pHB-OsADF-RNAi and the control plasmid pHB, respectively, as described by Hiei et al. (1994). The transfected plants and flowers were photographed at mature stage with a Nikon E995 digital camera. Flowers were randomly collected from ADF–RNAi lines. Anthers were dissected and immersed in I2-KI solution (1 % I2-KI), crushed, and photographed with a microscope (Leica DM2500). Observation of anther development by semithin sections was done as described by Li et al. (2006).
Expression profile analysis of OsADF Subcellular localization analysis of OsADF The promoter region (2,000 bp upstream of the initiation codon) of OsADF was amplified using Pro-F and Pro-R, which are listed in Supplemental Table 1. The PCR product was cloned into pMD18-T vector (TaKaRa), and the after the sequence was confirmed; a fragment digested with BamHI and NcoI was subcloned into the binary vector pCAMBIA1301 and fused to a beta-glucuronidase (GUS)reporter gene to generate OsADFPro::GUS. The construct was introduced into wild-type rice via Agrobacterium EHA105. GUS activity was visualized by staining the root, stem, leaf, and flowers from spikelets of transgenic lines overnight in X-Gluc (Willemsen et al. 1998) and then cleared in 75 % (v/v) ethanol.
A 1420 bp OsADF cDNA fragment was amplified from the cDNA of wild-type rice with primers YFP-F and YFP-R (Supplemental Table 1). The amplified fragment was digested with XhoI/BamHI and ligated into the XhoI/ BamHI digested pA7-YFP vector to create pA7-OsADFYFP. pA7-YFP was used as positive control vector (provided by Zhang dasheng, Shanghai Jiaotong University, China). The onion epidermis was peeled and bombarded with gold particle-coated plasmids. Cells with YFP fluorescence were observed under a microscope (Leica DM2500). ChIP-assay
In situ hybridization A 482 bp fragment of OsADF cDNA was amplified from the wild-type rice with the In Site Hybridization-F and In Site Hybridization-R (Supplemental Table 1). The PCR product was cloned into pMD18-T vector (TaKaRa), digested with BamHI and XbaI, and subcloned into the vector pBluescript II SK? (Stratagene). Subsequently, the vector was transcribed in vitro under the control of T7 or SP6 promoter with RNA polymerase using the DIG RNA labeling kit (Roche). The digoxigenin-labeled RNA
ChIP and quantitative PCR analysis: The procedure for ChIP of TDR-DNA complexes in rice wild type was modified from Haring et al. (2007). Rice spikelets at stages 9 and 11 were fixed with formaldehyde under vacuum. Chromatin was isolated and sonicated to produce DNA fragments shorter than 500 bp. Some untreated sonicated chromatin was reversely cross-linked and used as the total input DNA control. Immunoprecipitation with TDR-specific immune antiserum and without any serum was performed as described elsewhere (Haring et al. 2007).
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Oligonucleotide primers specific for the upstream of ChIP-F and ChIP-R were added to PCR reactions in which the templates were ChIP populations from immune or control immunoprecipitations. Typically, 34 cycles of PCR
Fig. 1 Schematic representation of the exon and intron organization of OsADF. A schematic representation of the exon and intron organization of OsADF. 50 -ATG indicates the putative starting nucleotide of translation, and the stop codon is TAA-30 . Black boxes indicate exons, and intervening lines indicate introns. The region with gray box indicates the F-box domain
Fig. 2 Protein Phylogeny of the OsADF and related F-box protein. A neighbor-joining analysis was performed using MEGA 3.1 based on the alignment given in Gramene online data of OsADF with the most similar F-box sequences from difference species. Bootstrap values are percentage of 1,000 replicates. Blue branch show the most similar members of OsADF from diverse species
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were performed, and the products were analyzed by agarose gel electrophoresis. EMSA analysis The DNA fragments (P3) containing the E-box binding site 50 -CANNTG-3 regulated by plant bHLH protein were generated using PCR amplification with the primers EMSA-F and EMSA-R. The DNA fragment was cloned into pMD18-T vector (TaKaRa) for sequence confirmation. Then, the fragment was labeled with DIG-labeled kit (DDLK-010) using the specific primers. The DNA binding reactions were performed according to Wang et al. (2002) with the following modifications.
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Fig. 3 Expression pattern of OsADF. a Spatial and temporal expression analyses of OsADF by RT-PCR. Rice Actin1(OsActin1) expression was used as a control. From stage 6 to stage 12, the flowers with different stage of anther development. GDNA, genomic DNA. b Spatial and temporal expression analyses of OsADF by qRT-PCR. L/P: lemma and palea; From stage 6 to stage 12, the flowers with different stage of anther development. c GUS expression (blue staining) patterns in the heterozygous spikelets of the OsADFpro::
GUS transgenic line at various stages. 1 stage 7, 2 stage 8, 3 stage 9, 4 stage 10, 5 stage 11. d, e RNA in situ hybridization of OsADF. Successive section to that shown in (d) and (e), probed with the OsADF sense-probe and antisense-probe respectively. A wild-type anther at the stage 9 showing stronger OsADF expression in tapetal cells. f GUS staining in the flower shown at stage 11 after removal of the palea and lemma
Reaction components were incubated in binding buffer [10 mM Tris–HCl, pH 7.5, 50 mM NaCl, 1 mM EDTA, 5 % glycerol, 0.05 mg mL 21 poly(dI-dC), and 0.1 mg mL21 BSA] at room temperature for 20 min. The entire reaction mixture was analyzed on a 5 % PAGE gel. After drying the gel, DIG-labeled DNA fragments were detected.
family of eukaryotic proteins characterized by an F-box motif (Risseeuw et al. 2003) and they are the substrate specificity initiating part of the Skp, Cullin, F-box (SCF), a multi-protein E3 ubiquitin ligase complex, which ubiquitinates proteins for their subsequent proteasomal degradation (Zheng et al. 2002). These proteins have been shown to be critical for many physiological processes, such as cell-cycle transition, signal transduction, gene transcription, and they are also involved in programmed cell death (Kipreos and Pagano 2000). For this reason we chose this gene for further study. The full length of OsADF cDNA was 3237 bp (Supplemental Fig. 1), OsADF contains twelve exons, and it encodes a predicted protein with 1,078 amino acid residues with two F-box domains (Fig. 1).
Results OsADF encodes a F-Box protein OsADF is one of our selected panicle-specific expression transcription factor (Li 2010), which is a member of the F-box protein family. F-box proteins are an expanding
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conserved function of OsADF during plant male reproductive development (Fig. 2). A neighbor-joining analysis was performed using MEGA 3.1 based on the alignment given in Gramene online data of OsADF with the most similar F-box sequences from difference species. Bootstrap values are percentage of 1000 replicates. Blue branch shows the most similar members of OsADF from diverse species. These observations suggest an essential and conserved function of OsADF during plant male reproductive development. OsADF is mainly expressed in tapetal cells and microspores
Fig. 4 The onion epidermal cell that expressed YFP and OsADFYFP. a A cell that expressed free YFP showing fluorescence in nucleus, cytoplasm, and plasma membrane. b A cell that expressed OsADF-YFP showing fluorescence in the plasma membrane. c A cell that expressed OsADF-YFP showing fluorescence mainly in the cell membrane after plasmolysis
OsADF belongs to F-box family members among terrestrial plants F-box proteins are an expanding family of eukaryotic proteins characterized by an ‘‘F-box’’ motif, which is responsible for substrate specificity in the ubiquitin–proteasome pathway and therefore play a pivotal role in many physiological activities such as cell-cycle progression, transcriptional regulation, programmed cell death and cell signal transduction (Kipreos and Pagano 2000). To gain the information on its potential function of OsADF in the F-box evolutionary tree, we used the OsADF full-length protein sequence as the query to search for its closest relatives form diverse species among terrestrial plants. A total of 33 putative F-box protein and annotated protein sequences that are related to rice OsADF were obtained from 16 different species. OsADF and other five F-box protein, Bra039616.1 (Brassica rapa), POPTR 0007s12980 (Populus trichocarpa), GRMZM2G162086 (Zea mays), EFJ21596 (Selaginella moellendorffii) and AT3G51940.1 (Arabidopsis thaliana) belonged to same branch. Altogether, these observations suggest an essential and
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To understand the function of OsADF, we analyzed the expression pattern of OsADF. We first detected OsADF expression by RT-PCR with total RNA extracted from different organs of rice (root, shoot, leaf, and floral organs) (Fig. 3a). There was no detectable transcription of OsADF in vegetative and floral organs other than the anther. OsADF expression was detectable in anthers starting from stage 10, and was highest at stage 12. Also qRT-PCR analysis showed that OsADF expression was detectable in anthers starting from early stage 9 of development, and was highest at stage 12; it was detectable only marginally in other vegetative organs and not detectable in root, shoot or leaf (Fig. 3b). The results of qRT-PCR are in accordance with RT-PCR and showed that the OsADF gene was mainly expressed in middle-late stage of anther development. To further analyze OsADF temporal and spatial expression characteristics, we used RNA in situ hybridization of the rice anther. The results showed that the expression of OsADF was strong in late stage tapetal cells and microspores (Fig. 3d, e). The OsADFPro::GUS fusion construct (expressing GUS marker protein driven by the 2,000 bp OsADF promoter region) was transformed into wild-type rice. Histochemical5-bromo-4-chloro-3-indolyl glucuronide (X-Gluc) staining of wild-type lines containing pOsADF-GUS showed that GUS activity was detectable at a maximum level at stage 12 (Fig. 3c, f). OsADF Fusion protein was located in the cell membrane The fusion protein of OsADF and yellow fluorescent protein (YFP) was constructed and was transformed into onion epidermal cells through gene gun (Fig. 4). The results showed that the cell expressed free YFP showing fluorescence in nucleus, cytoplasm, and plasma membrane, and the cell expressed OsADF-YFP showing fluorescence in the cell membrane.
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Fig. 5 Comparison of the Wild Type and the tdr Mutant. a Comparison of WT (left) and ADF-RNAi plant (right) after heading. b, c the spikelet of WT (b) and ADF-RNAi plant (c). d, e the spikelet of WT (d) and ADF-RNAi plant (e) after removing the palea and the lemma. f, g anthers and I2-KI–stained pollen grains of WT (f) and ADF-RNAi plant (g), the single anther in right-bottom. h Comparison of male
sterility, high level of phenotype 70–100 %, middle level of phenotype 40–70 % and weak level of phenotype 0–40 %, between WT and ADF-RNAi plant. i Expression level of OsADF between WT and ADF-RNAi plant in anther development stage 11 and stage 12 using qRT-PCR
Fig. 6 Transverse section comparison of the anther development of WT and ADF-RNAi lines. Five stages of anther development were compared. WT sections are shown in a, c, e, g and i, and other panels
show ADF-RNAi sections. a, b stage 8, c, d stage 9, e, f stage 10, g, h stage 11, i, j stage 12. E epidermis, En endothecium, T tapetum; Ms microsporocyte, Tds tetrads, MP mature pollen, Bars = 15 lm
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Fig. 7 Direct binding of TDR to the regulatory regions of OsADF. a Presence of the E-box motifs in the promoters of OsADF (left) and qChIP-PCR results (right).The site of E-box motif: E1 (-749 to -744), E2 (-584 to -579), E3 (-401 to -396), E4 (-151 to -146),
E5 (-141 to -136) and E6 (-17 to -12). b Recombinant TDR binding to the promoter region of OsADF with containing E-box was determined by EMSA. c RT-PCR analysis of OsADF in tdr
observations suggested that silencing of OsADF could alter pollen development in rice. Figure 5i showed that the transcription level of OsADF differed significantly between WT plant and ADF-RNAi plants in anthers and particularly at stage 12 the ADF-RNAi plants lost their OsADF expression. OsADF-RNAi application leads to abnormal anther development
Fig. 8 Gene regulatory network of OsADF for anther development in rice. TDR might function downstream of UDT1, OsCP1 and OsC6 which encoding a cysteine protease and plant lipid transfer protein (LTP), respectively, were regulated by TDR, TDR and GAMYB likely co-regulate OsC6, OsCP1, OsADF for controlling rice anther development
Silencing of OsADF reduces pollen fertility To understand the biological role of OsADF in anther development, we used RNAi. The pHB-OsADF–RNAi construct used a 482 bp OsADF cDNA fragment. Twelve independent T0 generation ADF–RNAi plants were obtained. To further identify the plant is positive, we use the PCR and southern-blot analysis (data not shown). The phenotype of the transgenic plants is not clearly different from the wild-type (WT) plant (Fig. 5a–c). The anthers of ADF-RNAi plants were white and smaller and showed reduced seed setting compared with the WT plant (Fig. 5d, e). We used a I2-KI staining of the pollen of these twelve independent ADF-RNAi lines at stage 13 (Fig. 5f, g). All of the lines showed pollen sterility; among them 81.8 % belonged to a high level of sterility, 67.8 % showed a middle level, and 33 % a weak level of sterility (Fig. 5h). These
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To determine the morphological defects of anthers of the ADF-RNAi lines, transverse sections were examined (Fig. 6). At stage 8, pollen mother cells underwent normal meiosis and formed tetrads. There was no detectable difference between WT plant and ADF-RNAi plant at this stage. At late stage 9 microspores were released from the tetrads and there was still no obvious difference in anther cellular morphology between WT plant and ADF-RNAi plant. At stage 10, the middle layer of the WT plant was hardly visible in the RNAi-control plant, but in contrast, the ADF-RNAi plant’s tapetal cells continued to expand, and the middle layers were still clearly visible. At stage 11, WT tapetal cells differentiated and degenerated. However, the ADF-RNAi middle layer and tapetum became more vacuolated and expanded. At the mature pollen stage 12, WT pollen grains were full of starch, lipids, and other nutrients, and the tapetum was fully degenerated. In contrast, the ADFRNAi microspores were completely degenerated, whereas tapetum cells became abnormally large and extremely vacuolated and the middle layer did not degenerate. OsADF is regulated by TDR The DNA fragments of upstream promoter region of genes regulated by TDR have been screeded. The 151
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downstream genes were shown to be likely direct targets of TDR (Li 2010). To investigate the possible direct regulation of OsADF by TDR, we used quantitative ChIP-PCR in vivo and EMSA in vitro. Our results indicated that OsADF is directly regulated by the TDR in specific binding E-box sites of OsADF upstream promoter region (Fig. 7).
Discussion Tapetum plays an important role in the process of pollen development in flowering plants, because it provides cellular contents supporting pollen wall formation and the subsequent pollen development (Wu and Cheun 2000). On the other hand its degeneration in the late stages of the pollen development is essential for the release of fertile pollen. This degeneration is proposed to be triggered by a programmed cell death (PCD) process during late stages of pollen development (Wu and Cheun 2000). TDR plays a central role for this morphological anther changes. However, the molecular basis regulating tapetum PCD in plants remains poorly understood. In this study, we report about the key role of OsADF, which belongs to the F-Box family in the process of pollen development in rice. OsADF expression is mainly detectable in tapetal cells and microspores from stage 9 to stage 12 of anther development (Fig. 3). Moreover, plants in which OsADF was silenced exhibited abnormal degeneration retardation of the tapetum as well as collapse of microspores and had reduced pollen fertility (Fig. 5). In our experiments, we could identify OsADF as another TDR transcription target, which is decreased expressed in TDR plants (Fig. 7). OsADF contains two F-boxes, one at the N terminus and one at the C terminus (Fig. 1). F-box proteins are also involved in protein degradation and function as adapter for directing substrate proteins to the SCF-complex, which ubiquitinates the substrate then for subsequent selective proteasomal degradation. Taken together our results underline a previous finding, that tapetum degeneration is related to protein degradation and its inhibition leads to changed developmental anther morphology with increased infertile pollen development. Furthermore, the transcriptional regulation of our newly discovered tapetum degeneration related gene OsADF could be determined as anther specific and is depending on TDR, a transcription factor, which is also transcribing other tapetum development related genes like OsC6 and OsCP1 (Zhang et al. 2008) (Fig. 8). The exact role of the F-box containing OsADF gene needs further investigation. This work provides new insights into the role of OsADF in anther development and pollen formation.
Author contribution Conception and design: Li Li. Analysis and interpretation: Li Li, Yixing Li. Data collection: Li Li, Shufeng Song, Guanghui Chen. Writing the article: Li Li, Critical revision of the article: Xiqin Fu, Huafeng Deng. Final approval of the article: Longping Yuan. Statistical analysis: Li Li, Na Li. Overall responsibility: Longping Yuan. Acknowledgments We thank Zong Jie for data analysis, Yi Wenwei for rice transformation, Luo Qingsong for field work. Prof. Zhang Dabing and Prof. Yuan Zheng from Shanghai Jiao Tong University is gratefully acknowledged for his valuable suggestions on the experimental design and manuscript. This study was supported by the National Natural Science Foundation of China (#31201184), Natural Science Foundation of Hunan Province, China (#14JJ2138), the National Key Programs for Transgenic Crops (#2011ZX08001-004) and the Program of Breeding and Application of hybrid Rice with Strong Heterosis (#2011AA10A101). Conflict of interest of interest.
The authors declare that they have no conflict
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ORIGINAL ARTICLE
An anther development F-box (ADF) protein regulated by tapetum degeneration retardation (TDR) controls rice anther development Li Li • Yixing Li • Shufeng Song • Huafeng Deng • Na Li • Xiqin Fu • Guanghui Chen • Longping Yuan
Received: 17 June 2014 / Accepted: 20 August 2014 Ó Springer-Verlag Berlin Heidelberg 2014
Abstract Main conclusion In this study, we reported that a F-box protein, OsADF, as one of the direct targets of TDR, plays a critical role in rice tapetum cell development and pollen formation. Abstract The tapetum, the innermost sporophytic tissue of anther, plays an important supportive role in male reproduction in flowering plants. After meiosis, tapetal cells undergo programmed cell death (PCD) and provide nutrients for pollen development. Previously we showed that tapetum degeneration retardation (TDR), a basic helix-loop-helix transcription factor, can trigger tapetal PCD and control pollen wall development during anther development. However, the comprehensive regulatory network of TDR remains to be investigated. In this study, we cloned and characterized a panicle-specific expression F-box protein, anther development F-box (OsADF). By qRT-PCR and RNA in situ hybridization, we further confirmed that OsADF expressed Electronic supplementary material The online version of this article (doi:10.1007/s00425-014-2160-9) contains supplementary material, which is available to authorized users. L. Li S. Song H. Deng N. Li X. Fu L. Yuan (&) State Key Laboratory of Hybrid Rice, Hunan Hybrid Rice Research Centre, Changsha 410125, China e-mail: [email protected] L. Li e-mail: [email protected] L. Li H. Deng G. Chen Collaborative Innovation Center for Modern Production of Multiple Cropping System in Southern Paddy Area, Changsha 410128, China Y. Li G. Chen Hunan Agricultural University, Changsha 410128, China
specially in tapetal cells from stage 9 to stage 12 during anther development. In consistent with this specific expression pattern, the RNAi transgenic lines of OsADF exhibited abnormal tapetal degeneration and aborted microspores development, which eventually grew pollens with reduced fertility. Furthermore, we demonstrated that the TDR, a key regulator in controlling rice anther development, could regulate directly the expression of OsADF by binding to E-box motifs of its promoter. Therefore, this work highlighted the possible regulatory role of TDR, which regulates tapetal cell development and pollen formation via triggering the possible ADF-mediated proteolysis pathway. Keywords OsADF
Rice Male sterility Tapetum TDR PCD
Introduction Rice (Oryza sativa) is the most important agricultural crop feeding more than half of the world’s population, and has became a model monocot crop for both fundamental biology and agricultural traits because of its small genome size, efficient transformation system, available mutant collections, etc. (Jiang et al. 2012; Jung et al. 2005). Hybrid rice technology requires the utilization of male sterile lines due to higher vigor (heterosis) of hybrid plants over the parent’s lines with 20–30 % yield increase (Yuan et al. 2003). Expression analysis revealed that about 29,000 unique transcripts were detectable in rice anther and male reproductive organs, suggesting that the anther development requires the function of various genes. However, relatively little is known about the molecular regulatory network of male reproduction development, particularly in crop rice (Zhang et al. 2011).
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Although rice flower has a distinct structure compared to that of dicot Arabidopsis (Zhang and Yuan 2014; Zhang et al. 2013), the process of rice anther ontology is similar to that of Arabidopsis (Zhang et al. 2011), including the formation of stamen primordium, cell differentiation, and establishment of characteristic tissues. After the morphogenesis, the anther develops four lobes connected to the filament, and each lobe tissue consists of four somatic wall layers, i.e. the epidermis, the endothecium, the middle layer, and the tapetum in addition to the microspore mother cells (MMCs) within the locule (Ma 2005; McCormick 1993; Scott et al. 1991). As a nutritive tissue, tapetal cells directly contact with developing gametophytes and have been assumed to provide materials/signals for microspore formation, release and subsequent pollen maturation (Goldberg et al. 1993; Sanders et al. 2000; Wu and Cheun 2000) via tapetal disintegration promoted by programmed cell death (PCD) after the meiosis (Li et al. 2006). Premature or delayed tapetal PCD always causes abnormal tapetal development, leading to male sterility. Recently, genetic studies have identified that several transcription factors play a relatively conserved role in controlling tapetum identity, differentiation and degradation. Generally, the identity and numbers of the tapetal cells were suggested to be controlled by the species specific cell surface-localized leucine-rich repeat receptor-like kinases (LRR-RLKs) signaling. In rice, MULTIPLE SPOROCYTE 1 (MSP1) expressed in cells neighboring the male and female sporocytes, but not in the sporocytes. While OsTDL1A (TPD1-like 1A)/MICROSPORELESS 2 (MIL2) were mainly detected in inner parietal cells (Hong 2012, #33). Both of them were suggested to play key role in specifying the normal differentiation of primary parietal cells in rice. Then, rice GAMYB (Aya et al. 2009), MYB33/MYB65 (Millar and Gubler 2005), DYSFUNCTIONAL TAPETUM1 (DYT1) (Zhang et al. 2006), DEFECTIVE IN TAPETAL DEVELOPMENT AND FUNCTION1 (TDF1) (Zhu et al. 2008), ABORTED MICROSPORE (AMS) (Xu et al. 2010; Yang et al. 2014), MALE STERILITY1 (Wilson et al. 2001), PERSISTENT TAPETAL CELL 1 (PTC1) (Li et al. 2011), TDR INTERACTING PROTEIN2 (Fu et al. 2014),Undeveloped Tapetum1 (UDT1) (Jung et al. 2005), TAPETUM DEGENERATION RETARDATION (TDR) (Li et al. 2006; Zhang et al. 2008), ETERNAL TAPETUM 1 (EAT1) (Niu et al. 2013)and PERSISTENT TAPETAL CELL 1 (PTC1) (Li et al. 2011) play key roles in regulating tapetal cells development and degeneration, as well as normal microsporogenesis. TDR encodes a putative basic helix–loop–helix (bHLH) transcription factor and mainly expressed in the tapetum (Li et al. 2006). The TDR mutant exhibits delayed tapetal PCD and retarded degeneration with the increased size of
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tapetal cells as well as aborted pollen development, causing complete male sterile (Li et al. 2006). Mechanically, TDR was shown to affect the expression of genes relative to tapetal PCD and pollen wall formation (Zhang et al. 2008). Particularly, TDR can directly regulate the expression of OsCP1, Osc6, and CYP703A3 (Li et al. 2006; Wu and Cheun 2000). OsCP1 encodes a Cys protease which belongs to an enzyme family widely distributed in animals, plants, and microorganisms that play crucial functions in degrading intracellular proteins and promoting PCD. OsC6 encodes a specified lipid transfer protein (LTP) with lipid binding activity, and OsC6 is required for the development of the specified tapetal structures: orbicules (i.e. Ubisch bodies) and outer pollen wall (call exine) (Wu and Cheun 2000). Rice CYP703A3 is a cytochrome P450 hydroxylase catalyzing an in-chain hydroxylation for a specific substrate, lauric acid, and CYP703A3 required for development of anther cuticle and pollen exine (Yang et al. 2014). Furthermore, it was found recently that TDR could interact with two bHLH proteins: TDR INTERACTING PROTEIN2 (TIP2) (Fu et al. 2014) and ETERNAL TAPETUM 1 (EAT1) (Niu et al. 2013), respectively. TDR acts downstream of TIP2 and upstream of EAT1, the three bHLH proteins TIP2, TDR, and EAT1 form a regulatory cascade in controlling differentiation, morphogenesis, and degradation of anther wall layers, and pollen development (Fu et al. 2014). Totally, all these results confirm that TDR is a key regulator in promoting PCD-associated tapetal degeneration and limiting the cell size of tapetal layer. However, the detail regulatory networks of TDR in controlling tapetal PCD process still remains largely unknown. Therefore, we screened a set of panicle-specific expression genes and studied their role in regulating rice tapetum development, as well as its relationship to TDR. In this study, we reported that TDR is able to directly regulate the expression of a F-box protein-encoding gene, OsADF. The RNAi transgenic lines of OsADF grew defective tapetal cell and pollen formation. This finding highlighted the possible regulatory role of TDR in regulating the tapetal PCD via OsADF-mediated proteolysis pathway.
Materials and methods Isolation and sequence analysis The OsADF clone was obtained from a rice panicle cDNA. Nucleotide sequence and putative amino acid sequence were analyzed with the basic local alignment search tool (BLAST) at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov) and the soft Vector NTI AdvanceÒ 11.5 (Invitrogen). OsADF amino acid sequence was examined for the F-box domain using the
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hidden Markov model of SMART tool (http://smart.emblheidelberg.de/). Sequence comparisons were conducted using MUSCLE 3.6.
antisense or sense RNA probe hybridization and immunological detection of the hybridized probes were performed according to the procedure of Kouchi and Hata (1993).
RT-PCR and quantitative real-time PCR assay pHB-OsADF-RNAi vector construction and transfection Rice total RNA from root, shoot, leaf, lemma, palea, and pistil at stage 12 of anther development as well as anthers at various stages (stages 6–12) was extracted using Trizol Reagent kit (Invitrogen, USA) The stages of anthers were classified according to Zhang and Wilson (2009). After treatment with DNase (Promega, USA), 0.3 mg of RNA was used to synthesize oligo (dT)-primed first-strand cDNA using the ReverTra Ace-a-first strand cDNA synthesis kit (TOYOBO, Japan). Two microliters of the reverse transcription product was then used as template for conventional and quantitative RT-PCR analysis. PCR was performed with TaKaRa ExTaq DNA polymerase. RTPCR primers are listed in Supplemental Table 1, with Oryza sativa L. actin as internal control. The primers for real-time PCR were the same as the primers for RT–PCR. Quantitative real-time PCR was performed with a RotorGene RG3000A detection system (Corbett RESEARCH, Australia) using SYBR Green I master mix (Generay Biotech, Co. Ltd, Shanghai).
A 500 bp OsADF cDNA fragment with low similarity to other rice genes after sequence analysis was amplified from the cDNA of wild-type rice using primers RNAi-F and RNAi-R (Supplemental Table 1) and digested with EcoRV/PstI and XbaI/BamHI, respectively. The two fragments were subsequently inserted in opposite directions into the binary RNAi vector pHB (Mao et al. 2005) to generate the pHB-OsADF-RNAi plasmid with a double cauliflower mosaic virus 35S promoter. Rice calli were used for transformation with Agrobacterium EHA105 carrying pHB-OsADF-RNAi and the control plasmid pHB, respectively, as described by Hiei et al. (1994). The transfected plants and flowers were photographed at mature stage with a Nikon E995 digital camera. Flowers were randomly collected from ADF–RNAi lines. Anthers were dissected and immersed in I2-KI solution (1 % I2-KI), crushed, and photographed with a microscope (Leica DM2500). Observation of anther development by semithin sections was done as described by Li et al. (2006).
Expression profile analysis of OsADF Subcellular localization analysis of OsADF The promoter region (2,000 bp upstream of the initiation codon) of OsADF was amplified using Pro-F and Pro-R, which are listed in Supplemental Table 1. The PCR product was cloned into pMD18-T vector (TaKaRa), and the after the sequence was confirmed; a fragment digested with BamHI and NcoI was subcloned into the binary vector pCAMBIA1301 and fused to a beta-glucuronidase (GUS)reporter gene to generate OsADFPro::GUS. The construct was introduced into wild-type rice via Agrobacterium EHA105. GUS activity was visualized by staining the root, stem, leaf, and flowers from spikelets of transgenic lines overnight in X-Gluc (Willemsen et al. 1998) and then cleared in 75 % (v/v) ethanol.
A 1420 bp OsADF cDNA fragment was amplified from the cDNA of wild-type rice with primers YFP-F and YFP-R (Supplemental Table 1). The amplified fragment was digested with XhoI/BamHI and ligated into the XhoI/ BamHI digested pA7-YFP vector to create pA7-OsADFYFP. pA7-YFP was used as positive control vector (provided by Zhang dasheng, Shanghai Jiaotong University, China). The onion epidermis was peeled and bombarded with gold particle-coated plasmids. Cells with YFP fluorescence were observed under a microscope (Leica DM2500). ChIP-assay
In situ hybridization A 482 bp fragment of OsADF cDNA was amplified from the wild-type rice with the In Site Hybridization-F and In Site Hybridization-R (Supplemental Table 1). The PCR product was cloned into pMD18-T vector (TaKaRa), digested with BamHI and XbaI, and subcloned into the vector pBluescript II SK? (Stratagene). Subsequently, the vector was transcribed in vitro under the control of T7 or SP6 promoter with RNA polymerase using the DIG RNA labeling kit (Roche). The digoxigenin-labeled RNA
ChIP and quantitative PCR analysis: The procedure for ChIP of TDR-DNA complexes in rice wild type was modified from Haring et al. (2007). Rice spikelets at stages 9 and 11 were fixed with formaldehyde under vacuum. Chromatin was isolated and sonicated to produce DNA fragments shorter than 500 bp. Some untreated sonicated chromatin was reversely cross-linked and used as the total input DNA control. Immunoprecipitation with TDR-specific immune antiserum and without any serum was performed as described elsewhere (Haring et al. 2007).
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Oligonucleotide primers specific for the upstream of ChIP-F and ChIP-R were added to PCR reactions in which the templates were ChIP populations from immune or control immunoprecipitations. Typically, 34 cycles of PCR
Fig. 1 Schematic representation of the exon and intron organization of OsADF. A schematic representation of the exon and intron organization of OsADF. 50 -ATG indicates the putative starting nucleotide of translation, and the stop codon is TAA-30 . Black boxes indicate exons, and intervening lines indicate introns. The region with gray box indicates the F-box domain
Fig. 2 Protein Phylogeny of the OsADF and related F-box protein. A neighbor-joining analysis was performed using MEGA 3.1 based on the alignment given in Gramene online data of OsADF with the most similar F-box sequences from difference species. Bootstrap values are percentage of 1,000 replicates. Blue branch show the most similar members of OsADF from diverse species
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were performed, and the products were analyzed by agarose gel electrophoresis. EMSA analysis The DNA fragments (P3) containing the E-box binding site 50 -CANNTG-3 regulated by plant bHLH protein were generated using PCR amplification with the primers EMSA-F and EMSA-R. The DNA fragment was cloned into pMD18-T vector (TaKaRa) for sequence confirmation. Then, the fragment was labeled with DIG-labeled kit (DDLK-010) using the specific primers. The DNA binding reactions were performed according to Wang et al. (2002) with the following modifications.
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Fig. 3 Expression pattern of OsADF. a Spatial and temporal expression analyses of OsADF by RT-PCR. Rice Actin1(OsActin1) expression was used as a control. From stage 6 to stage 12, the flowers with different stage of anther development. GDNA, genomic DNA. b Spatial and temporal expression analyses of OsADF by qRT-PCR. L/P: lemma and palea; From stage 6 to stage 12, the flowers with different stage of anther development. c GUS expression (blue staining) patterns in the heterozygous spikelets of the OsADFpro::
GUS transgenic line at various stages. 1 stage 7, 2 stage 8, 3 stage 9, 4 stage 10, 5 stage 11. d, e RNA in situ hybridization of OsADF. Successive section to that shown in (d) and (e), probed with the OsADF sense-probe and antisense-probe respectively. A wild-type anther at the stage 9 showing stronger OsADF expression in tapetal cells. f GUS staining in the flower shown at stage 11 after removal of the palea and lemma
Reaction components were incubated in binding buffer [10 mM Tris–HCl, pH 7.5, 50 mM NaCl, 1 mM EDTA, 5 % glycerol, 0.05 mg mL 21 poly(dI-dC), and 0.1 mg mL21 BSA] at room temperature for 20 min. The entire reaction mixture was analyzed on a 5 % PAGE gel. After drying the gel, DIG-labeled DNA fragments were detected.
family of eukaryotic proteins characterized by an F-box motif (Risseeuw et al. 2003) and they are the substrate specificity initiating part of the Skp, Cullin, F-box (SCF), a multi-protein E3 ubiquitin ligase complex, which ubiquitinates proteins for their subsequent proteasomal degradation (Zheng et al. 2002). These proteins have been shown to be critical for many physiological processes, such as cell-cycle transition, signal transduction, gene transcription, and they are also involved in programmed cell death (Kipreos and Pagano 2000). For this reason we chose this gene for further study. The full length of OsADF cDNA was 3237 bp (Supplemental Fig. 1), OsADF contains twelve exons, and it encodes a predicted protein with 1,078 amino acid residues with two F-box domains (Fig. 1).
Results OsADF encodes a F-Box protein OsADF is one of our selected panicle-specific expression transcription factor (Li 2010), which is a member of the F-box protein family. F-box proteins are an expanding
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conserved function of OsADF during plant male reproductive development (Fig. 2). A neighbor-joining analysis was performed using MEGA 3.1 based on the alignment given in Gramene online data of OsADF with the most similar F-box sequences from difference species. Bootstrap values are percentage of 1000 replicates. Blue branch shows the most similar members of OsADF from diverse species. These observations suggest an essential and conserved function of OsADF during plant male reproductive development. OsADF is mainly expressed in tapetal cells and microspores
Fig. 4 The onion epidermal cell that expressed YFP and OsADFYFP. a A cell that expressed free YFP showing fluorescence in nucleus, cytoplasm, and plasma membrane. b A cell that expressed OsADF-YFP showing fluorescence in the plasma membrane. c A cell that expressed OsADF-YFP showing fluorescence mainly in the cell membrane after plasmolysis
OsADF belongs to F-box family members among terrestrial plants F-box proteins are an expanding family of eukaryotic proteins characterized by an ‘‘F-box’’ motif, which is responsible for substrate specificity in the ubiquitin–proteasome pathway and therefore play a pivotal role in many physiological activities such as cell-cycle progression, transcriptional regulation, programmed cell death and cell signal transduction (Kipreos and Pagano 2000). To gain the information on its potential function of OsADF in the F-box evolutionary tree, we used the OsADF full-length protein sequence as the query to search for its closest relatives form diverse species among terrestrial plants. A total of 33 putative F-box protein and annotated protein sequences that are related to rice OsADF were obtained from 16 different species. OsADF and other five F-box protein, Bra039616.1 (Brassica rapa), POPTR 0007s12980 (Populus trichocarpa), GRMZM2G162086 (Zea mays), EFJ21596 (Selaginella moellendorffii) and AT3G51940.1 (Arabidopsis thaliana) belonged to same branch. Altogether, these observations suggest an essential and
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To understand the function of OsADF, we analyzed the expression pattern of OsADF. We first detected OsADF expression by RT-PCR with total RNA extracted from different organs of rice (root, shoot, leaf, and floral organs) (Fig. 3a). There was no detectable transcription of OsADF in vegetative and floral organs other than the anther. OsADF expression was detectable in anthers starting from stage 10, and was highest at stage 12. Also qRT-PCR analysis showed that OsADF expression was detectable in anthers starting from early stage 9 of development, and was highest at stage 12; it was detectable only marginally in other vegetative organs and not detectable in root, shoot or leaf (Fig. 3b). The results of qRT-PCR are in accordance with RT-PCR and showed that the OsADF gene was mainly expressed in middle-late stage of anther development. To further analyze OsADF temporal and spatial expression characteristics, we used RNA in situ hybridization of the rice anther. The results showed that the expression of OsADF was strong in late stage tapetal cells and microspores (Fig. 3d, e). The OsADFPro::GUS fusion construct (expressing GUS marker protein driven by the 2,000 bp OsADF promoter region) was transformed into wild-type rice. Histochemical5-bromo-4-chloro-3-indolyl glucuronide (X-Gluc) staining of wild-type lines containing pOsADF-GUS showed that GUS activity was detectable at a maximum level at stage 12 (Fig. 3c, f). OsADF Fusion protein was located in the cell membrane The fusion protein of OsADF and yellow fluorescent protein (YFP) was constructed and was transformed into onion epidermal cells through gene gun (Fig. 4). The results showed that the cell expressed free YFP showing fluorescence in nucleus, cytoplasm, and plasma membrane, and the cell expressed OsADF-YFP showing fluorescence in the cell membrane.
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Fig. 5 Comparison of the Wild Type and the tdr Mutant. a Comparison of WT (left) and ADF-RNAi plant (right) after heading. b, c the spikelet of WT (b) and ADF-RNAi plant (c). d, e the spikelet of WT (d) and ADF-RNAi plant (e) after removing the palea and the lemma. f, g anthers and I2-KI–stained pollen grains of WT (f) and ADF-RNAi plant (g), the single anther in right-bottom. h Comparison of male
sterility, high level of phenotype 70–100 %, middle level of phenotype 40–70 % and weak level of phenotype 0–40 %, between WT and ADF-RNAi plant. i Expression level of OsADF between WT and ADF-RNAi plant in anther development stage 11 and stage 12 using qRT-PCR
Fig. 6 Transverse section comparison of the anther development of WT and ADF-RNAi lines. Five stages of anther development were compared. WT sections are shown in a, c, e, g and i, and other panels
show ADF-RNAi sections. a, b stage 8, c, d stage 9, e, f stage 10, g, h stage 11, i, j stage 12. E epidermis, En endothecium, T tapetum; Ms microsporocyte, Tds tetrads, MP mature pollen, Bars = 15 lm
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Fig. 7 Direct binding of TDR to the regulatory regions of OsADF. a Presence of the E-box motifs in the promoters of OsADF (left) and qChIP-PCR results (right).The site of E-box motif: E1 (-749 to -744), E2 (-584 to -579), E3 (-401 to -396), E4 (-151 to -146),
E5 (-141 to -136) and E6 (-17 to -12). b Recombinant TDR binding to the promoter region of OsADF with containing E-box was determined by EMSA. c RT-PCR analysis of OsADF in tdr
observations suggested that silencing of OsADF could alter pollen development in rice. Figure 5i showed that the transcription level of OsADF differed significantly between WT plant and ADF-RNAi plants in anthers and particularly at stage 12 the ADF-RNAi plants lost their OsADF expression. OsADF-RNAi application leads to abnormal anther development
Fig. 8 Gene regulatory network of OsADF for anther development in rice. TDR might function downstream of UDT1, OsCP1 and OsC6 which encoding a cysteine protease and plant lipid transfer protein (LTP), respectively, were regulated by TDR, TDR and GAMYB likely co-regulate OsC6, OsCP1, OsADF for controlling rice anther development
Silencing of OsADF reduces pollen fertility To understand the biological role of OsADF in anther development, we used RNAi. The pHB-OsADF–RNAi construct used a 482 bp OsADF cDNA fragment. Twelve independent T0 generation ADF–RNAi plants were obtained. To further identify the plant is positive, we use the PCR and southern-blot analysis (data not shown). The phenotype of the transgenic plants is not clearly different from the wild-type (WT) plant (Fig. 5a–c). The anthers of ADF-RNAi plants were white and smaller and showed reduced seed setting compared with the WT plant (Fig. 5d, e). We used a I2-KI staining of the pollen of these twelve independent ADF-RNAi lines at stage 13 (Fig. 5f, g). All of the lines showed pollen sterility; among them 81.8 % belonged to a high level of sterility, 67.8 % showed a middle level, and 33 % a weak level of sterility (Fig. 5h). These
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To determine the morphological defects of anthers of the ADF-RNAi lines, transverse sections were examined (Fig. 6). At stage 8, pollen mother cells underwent normal meiosis and formed tetrads. There was no detectable difference between WT plant and ADF-RNAi plant at this stage. At late stage 9 microspores were released from the tetrads and there was still no obvious difference in anther cellular morphology between WT plant and ADF-RNAi plant. At stage 10, the middle layer of the WT plant was hardly visible in the RNAi-control plant, but in contrast, the ADF-RNAi plant’s tapetal cells continued to expand, and the middle layers were still clearly visible. At stage 11, WT tapetal cells differentiated and degenerated. However, the ADF-RNAi middle layer and tapetum became more vacuolated and expanded. At the mature pollen stage 12, WT pollen grains were full of starch, lipids, and other nutrients, and the tapetum was fully degenerated. In contrast, the ADFRNAi microspores were completely degenerated, whereas tapetum cells became abnormally large and extremely vacuolated and the middle layer did not degenerate. OsADF is regulated by TDR The DNA fragments of upstream promoter region of genes regulated by TDR have been screeded. The 151
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downstream genes were shown to be likely direct targets of TDR (Li 2010). To investigate the possible direct regulation of OsADF by TDR, we used quantitative ChIP-PCR in vivo and EMSA in vitro. Our results indicated that OsADF is directly regulated by the TDR in specific binding E-box sites of OsADF upstream promoter region (Fig. 7).
Discussion Tapetum plays an important role in the process of pollen development in flowering plants, because it provides cellular contents supporting pollen wall formation and the subsequent pollen development (Wu and Cheun 2000). On the other hand its degeneration in the late stages of the pollen development is essential for the release of fertile pollen. This degeneration is proposed to be triggered by a programmed cell death (PCD) process during late stages of pollen development (Wu and Cheun 2000). TDR plays a central role for this morphological anther changes. However, the molecular basis regulating tapetum PCD in plants remains poorly understood. In this study, we report about the key role of OsADF, which belongs to the F-Box family in the process of pollen development in rice. OsADF expression is mainly detectable in tapetal cells and microspores from stage 9 to stage 12 of anther development (Fig. 3). Moreover, plants in which OsADF was silenced exhibited abnormal degeneration retardation of the tapetum as well as collapse of microspores and had reduced pollen fertility (Fig. 5). In our experiments, we could identify OsADF as another TDR transcription target, which is decreased expressed in TDR plants (Fig. 7). OsADF contains two F-boxes, one at the N terminus and one at the C terminus (Fig. 1). F-box proteins are also involved in protein degradation and function as adapter for directing substrate proteins to the SCF-complex, which ubiquitinates the substrate then for subsequent selective proteasomal degradation. Taken together our results underline a previous finding, that tapetum degeneration is related to protein degradation and its inhibition leads to changed developmental anther morphology with increased infertile pollen development. Furthermore, the transcriptional regulation of our newly discovered tapetum degeneration related gene OsADF could be determined as anther specific and is depending on TDR, a transcription factor, which is also transcribing other tapetum development related genes like OsC6 and OsCP1 (Zhang et al. 2008) (Fig. 8). The exact role of the F-box containing OsADF gene needs further investigation. This work provides new insights into the role of OsADF in anther development and pollen formation.
Author contribution Conception and design: Li Li. Analysis and interpretation: Li Li, Yixing Li. Data collection: Li Li, Shufeng Song, Guanghui Chen. Writing the article: Li Li, Critical revision of the article: Xiqin Fu, Huafeng Deng. Final approval of the article: Longping Yuan. Statistical analysis: Li Li, Na Li. Overall responsibility: Longping Yuan. Acknowledgments We thank Zong Jie for data analysis, Yi Wenwei for rice transformation, Luo Qingsong for field work. Prof. Zhang Dabing and Prof. Yuan Zheng from Shanghai Jiao Tong University is gratefully acknowledged for his valuable suggestions on the experimental design and manuscript. This study was supported by the National Natural Science Foundation of China (#31201184), Natural Science Foundation of Hunan Province, China (#14JJ2138), the National Key Programs for Transgenic Crops (#2011ZX08001-004) and the Program of Breeding and Application of hybrid Rice with Strong Heterosis (#2011AA10A101). Conflict of interest of interest.
The authors declare that they have no conflict
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