- American Society of Plant Biologists
Abstract
In flowering plants, tapetum degeneration is proposed to be triggered by a programmed cell death (PCD) process during late stages of pollen development; the PCD is thought to provide cellular contents supporting pollen wall formation and to allow the subsequent pollen release. However, the molecular basis regulating tapetum PCD in plants remains poorly understood. We report the isolation and characterization of a rice (Oryza sativa) male sterile mutant tapetum degeneration retardation (tdr), which exhibits degeneration retardation of the tapetum and middle layer as well as collapse of microspores. The TDR gene is preferentially expressed in the tapetum and encodes a putative basic helix-loop-helix protein, which is likely localized to the nucleus. More importantly, two genes, Os CP1 and Os c6, encoding a Cys protease and a protease inhibitor, respectively, were shown to be the likely direct targets of TDR through chromatin immunoprecipitation analyses and the electrophoretic mobility shift assay. These results indicate that TDR is a key component of the molecular network regulating rice tapetum development and degeneration.
INTRODUCTION
The life cycle of flowering plants alternates between diploid sporophyte and haploid gametophyte generations. Male gametophytes develop in the anther compartment of the stamen within the flower, the sporophytic reproductive structure, and require cooperative functional interactions between gametophytic and sporophytic tissues (Scott et al., 1991; Goldberg et al., 1993; McCormick, 1993; Raghavan, 1997; Ma, 2005). The anther has four lobes that are similar in structure and are attached to a central core with connective and vascular tissues. When anther morphogenesis is complete, the meiotic cells (also called microsporocytes) at the center of each anther lobe are surrounded by four somatic layers, which are, from the surface to interior, the epidermis, endothecium, middle layer, and tapetum (Goldberg et al., 1993). As the innermost of the four sporophytic layers of the anther wall, the tapetum directly contacts with the developing gametophytes and plays a crucial role in the development from microspore to pollen grains (Pacini et al., 1985; Shivanna et al., 1997). As a secretory cell layer, the tapetum provides enzymes for the release of microspores from tetrads and nutrients for pollen development (Goldberg et al., 1993).
It is known that the tapetum undergoes cellular degradation during late stages of pollen development. This degradation process is considered to be a programmed cell death (PCD) event (Papini et al., 1999; Wu and Cheung, 2000). At the structural level, the tapetum PCD is characterized by sequential elimination of the cellular structures. For example, in both Lobivia rauschii and Tillandsia albida, the cytological features of tapetum PCD include cytoplasmic shrinkage, oligonucleosomal cleavage of DNA, vacuole rupture, and swelling of the endoplasmic reticulum (Papini et al., 1999). Tapetal cell differentiation and subsequent disintegration coincides very well with the anther postmeiotic developmental program, and premature or delayed degradation of tapetum is associated with male sterility.
Molecular genetic studies have identified a few genes that control the formation of tapetum (Ma, 2005), including the Arabidopsis thaliana EXCESS MICROSPOROCYTES1 (EMS1)/EXTRA SPOROGENOUS CELLS (EXS) gene encoding a leucine-rich repeat (LRR) receptor-like protein kinase (Canales et al., 2002; Zhao et al., 2002) and the TAPETAL DETERMINANT1 gene encoding a putative small secreted protein (Yang et al., 2003, 2005; Ma, 2005). Recently, two highly similar Arabidopsis LRR receptor-like protein kinases, SOMATIC EMBRYOGENESIS RECEPTOR KINASE1 (SERK1) and SERK2, were found to be required in tapetum formation in a way similar to that of EMS1/EXS (Albrecht et al., 2005; Colcombet et al., 2005). In rice (Oryza sativa), the MULTIPLE SPOROCYTE1 gene encodes an LRR receptor-like protein kinase that is highly similar to EMS1 and has a function resembling that of EMS1/EXS (Nonomura et al., 2003). More recently, the rice Undeveloped Tapetum1 (Udt1) gene was shown to be important for tapetum differentiation and the formation of microspores (Jung et al., 2005).
Other genes have been shown to affect postmeiotic tapetum development and/or function and microspore development. For instance, the Arabidopsis ABORTED MICROSPORE (AMS) gene encoding a basic helix-loop-helix (bHLH)–containing protein plays a crucial role in tapetum and microspore development (Sorensen et al., 2003). In addition, the Arabidopsis MALE STERILITY1 gene encodes a protein with a PHD finger and is important for proper tapetum function and normal microspore development (Wilson et al., 2001). Moreover, At MYB103 is required for the development of tapetum, pollen, and trichome (Higginson et al., 2003). In Penutia hybrida, the tapetum-specific zinc finger gene TAZ1 is important for postmeiotic tapetum development (Kapoor et al., 2002).
On the other hand, little is known about the genetic basis regulating PCD of tapetum during late pollen development. In this report, we describe the isolation and characterization of a rice male sterile tapetum degeneration retardation (tdr) mutant with a mutation in a putative bHLH transcription factor gene. In the tdr anther, the tapetum PCD was retarded and the middle layer cells persisted, accompanied by aborted pollen development and complete male sterility. Furthermore, TDR is expressed highly preferentially in the tapetal cells, suggesting that TDR acts within the tapetum to promote its normal development and postmeiotic degradation. TDR encodes a putative transcription factor with a bHLH domain. Moreover, using chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assay (EMSA), we show that two genes, Os CP1 and Os c6, encoding a Cys protease and a protease inhibitor, respectively, are likely direct targets of TDR. These results provide important insights into the crucial role of TDR in a transcriptional regulatory network for tapetum development and degradation.
RESULTS
Isolation and Phenotypic Analyses of the tdr Mutant
To identify rice genes that are important for the regulation of anther development, we generated a rice mutant library of the japonica subspecies using γ-ray radiation and found the tdr mutant by its complete male sterility. Genetic analysis indicated that a single recessive nuclear locus controlled the mutant phenotype (Liu et al., 2005). The tdr plant was normal in vegetative and floral development but failed to produce any viable pollen (Figures 1A to 1D ). Compared with wild-type anthers, the mutant anthers were small and white, without mature pollen grains (cf. Figures 1E to 1H).
Comparison of the Wild Type and the tdr Mutant.
(A) Comparison of a wild-type plant (left) and a tdr mutant plant (right) after bolting.
(B) Comparison of a wild-type panicle (left) and a tdr mutant panicle at the heading stage.
(C) A wild-type spikelet.
(D) A mutant spikelet.
(E) A wild-type spikelet after removing the lemma and palea.
(F) A mutant spikelet after removing the lemma and palea.
(G) A wild-type yellow anther.
(H) A mutant white and smaller anther.
le, lemma; pa, palea; gl, glume; st, stamen. Bars = 2 mm.
To determine the anther morphological defects in the tdr mutant, anther transverse sections were further examined. Based on the cellular events visible under the light microscope and previous classification of anther development (Feng et al., 2001), we divided rice anther development into eight stages. During the early premeiosis stage, the archesporial cells divided to form primary parietal cells and primary sporogenous cells. The primary sporogenous cells then divided to generate the sporogenous cells, and the primary parietal cells divided to form a layer of endothecial cells and a layer of secondary parietal cells. There was no detectable difference between the wild type and the tdr anthers at this stage (Figures 2A and 2B ). Up to the microspore mother cell (MMC) stage, there was still no obvious difference in anther cellular morphology between the wild type and tdr. Normal epidermis, endothecium, middle layer, tapetum, and microsporocytes were found in both wild-type and the tdr anthers (Figures 2C and 2D).
Transverse Section Comparison of the Anther Development of the Wild Type and the tdr Mutant.
Eight stages of anther development in the wild type and the corresponding stages of development in the tdr mutant were compared. The images are of cross sections through single locules. Wild-type sections are shown in (A), (C), (E), (G), (I), (K), (M), and (O), and other panels show tdr sections. E, epidermis; En, endothecium; ML, middle layer; T, tapetum; Ms, microsporocyte; Tds, tetrads; Msp, microspore; MP, mature pollen; SPC, secondary parietal cell; SC, sporogenous cell. Bars = 15μm.
(A) and (B) The early premeiosis stage.
(C) and (D) The MMC stage.
(E) and (F) The late meiosis stage.
(G) and (H) The tetrad stage.
(I) and (J) The young microspore stage.
(K) and (L) The vacuolated pollen stage.
(M) and (N) The pollen mitosis stage.
(O) and (P) The mature pollen stage.
Subsequently, the tdr mutant anther had detectable morphological abnormalities. During the meiosis stage, wild-type MMCs underwent meiosis to form tetrads of four haploid microspores. The tapetal cells then differentiated and their cytoplasm became deeply stained, while the middle layer cells became very thin and degenerated (Figure 2E). In the tdr anther, microsporocytes seemed normal and could undergo meiosis to form tetrads, as observed using 4′,6-diamidino-2-phenylindole staining (see Supplemental Figure 1 online). However, the cytoplasm of tapetum and middle layer cells in the tdr mutant were not deeply stained (Figure 2F). At the tetrad stage, wild-type meiocytes had formed tetrads, the tapetal cytoplasm continued to agglomerate, and the middle layer assumed a band-like shape (Figure 2G). In the tdr anther, although the tetrads had formed, the tapetum seemed to be vacuolated and the middle layer remained relatively thick (Figure 2H). Subsequently, at the young microspore stage in wild-type anthers, microspores were released from tetrads, tapetal cells had deeply stained cytoplasm but no longer had large vacuoles, and the middle layer was hardly visible (Figure 2I). By contrast, the tdr tapetal cells continued to expand and remained vacuolated, and the middle layers were still clearly visible (Figure 2J). From the vacuolated pollen stage to the pollen mitosis stage, wild-type pollen exine deposition was completed, and the uninucleate pollen developed to trinucleate pollen through two mitotic divisions, while the tapetal cells differentiated and degenerated (Figures 2K and 2M). However, the tdr mutant microspores collapsed following release from tetrads. The middle layer and tapetum became more vacuolated and expanded (Figures 2L and 2N). At the mature pollen stage, wild-type pollen grains were full of starch, lipids, and other nutrients, and the tapetum was fully degenerated (Figure 2O). However, the tdr microspores were completely degenerated with only densely staining remnants at the center of the anther locule, whereas tapetum cells became abnormally large and extremely vacuolated and occupied the majority of locule. The middle layer did not degenerate (Figure 2P).
To gain a more detailed understanding of the abnormalities of the tdr tapetal cells, we used transmission electron microscopy (TEM) to study the mutant anther. At the early meiosis stage, there were no distinct differences between wild-type and the tdr mutant anthers (Figures 3A and 3B ). At the tetrad stage, the tetrad was formed in wild-type anthers through meiosis, and the cytoplasm of tapetal cell was condensed and deeply stained (Figures 3C and 3E). Also, tetrads also could be observed in the tdr mutant anther. However, the cytoplasm of the tdr tapetal cell was not as darkly stained as that of the wild-type tapetum (Figures 3D and 3F). At the young microspore stage, wild-type tapetal cells became collapsed in conjunction with the dissolution of cell walls, as is typical for the secretory tapetum in angiosperms and crucial for supplying nutrition to the developing microspores (Figure 3G). By contrast, the tdr tapetal cells became enlarged with large vacuoles and did not degenerate (Figure 3H). Consistent with previous results (Owen and Makaroff, 1995), the wild-type tapetal cells at this stage had significant dilation of endoplasmic reticulum cisternae consisting of >12 layers. Mitochondria could be recognized as having an electron-dense matrix and enlarged cristae, and the nucleus of the tapetal cell was lobed. All of these signified cellular degeneration in the wild-type tapetal cell during the tetrad stage (Figure 3I). However, in the tdr mutant, the tapetal cytoplasm was full of high electron density matrix, and the nucleus showed no shrinkage. The endoplasmic reticulum dilation in tdr tapetal cells was greatly reduced, unusual patterns of endoplasmic reticulum could be observed occasionally, and the mutant mitochondria lost its normal features with reduced cristae (Figure 3J). Therefore, the wild-type tapetal cells at the young microspore stage show distinct characteristics of cellular degeneration; however, the lack of ultrastructural characteristics of degeneration in the tdr tapetal cells indicates their failure to degrade. Meanwhile, the tdr tapetum cytological abnormalities were accompanied by structural defects in microspores. At the young microspore stage, the spherical wild-type microspores contained mitochondria, plastids, and vacuoles distributed around the endoplasmic reticulum (Figure 3K). The exine of wild-type pollen was established with distinct tectum, bacula, and nexine layers (Figure 3M). However, the tdr microspore was abnormal in shape, with irregularly distributed vacuoles and reduced endoplasmic reticulum (Figure 3L). Particularly, there was no detectable exine (Figure 3N). At the vacuolated pollen stage, further degenerated tapetum and vacuolated microspores were observed in the wild-type anthers (Figure 3O). However, at this stage, the extremely expanded tapetal cell occupied the majority of the locule, and microspores entirely collapsed (Figure 3P). These observations suggest that the tdr mutation resulted in the defects in tapetum degeneration, further affecting microspore development, particularly exine formation.
Transmission Electron Micrographs of the Anthers from the Wild Type and the tdr Mutant.
(A) The early meiosis stage wild-type anther showing microsporocyte.
(B) The tdr mutant anther at early meiosis stage showing microsporocyte.
(C) The tetrad stage wild-type anther showing tetrad.
(D) The tdr mutant anther at the tetrad stage showing tetrad.
(E) A higher magnification of tetrad in (C) showing callose (arrows).
(F) A higher magnification of tetrad in (D) showing callose (arrows).
(G) The young microspore stage wild-type anther showing highly condensed tapetal cytoplasm and spherical microspores.
(H) The young microspore stage of the tdr mutant anther showing the hypertrophy of tapetal cells and abnormal microspores.
(I) Wild-type tapetal cytoplasm showing dilation of the endoplasmic reticulum and lobed nucleus (arrows).
(J) The tdr mutant tapetal cytoplasm showing unusual pattern of the endoplasmic reticulum, abnormal mitochondria, and intact nucleus membrane (arrow).
(K) Wild-type microspore showing the endoplasmic reticulum and developing vacuoles being distributed all around the ER. Arrow shows the exine.
(L) The tdr mutant microspore with abnormal shape and more irregularly distributed vacuoles. Arrow indicates the exine.
(M) Higher-magnification view of the exine in (K) showing tectum, bacula, and nexine (arrows).
(N) The tdr mutant microspore with coarse primexine (arrow).
(O) The vacuolated pollen stage wild-type anther showing degenerated tapetum and vacuolated microspores.
(P) The tdr mutant anther at the vacuolated pollen stage showing overly expanded tapetum.
E, epidermis; En, endothecium; ML, middle layer; T, tapetum; Ms, microsporocyte; Tds, tetrads; Msp, microspore; Mt, mitochondria; ER, endoplasmic reticulum; V, vacuole; N, nucleus; Ex, exine; Tc, tectum; Ba, bacula; Ne, nexine. Bars = 5 μm in (A) to (H), (O), and (P), 1 μm in (K) and (L), and 0.5 μm in (I), (J), (M), and (N).
Loss of TDR Function Causes Aborted Tapetal PCD
The phenotypic analysis described above suggests that the tdr mutation affected the differentiation and degradation of tapetal cells. In plants, the degeneration of tapetum is considered to be the result of PCD, which is characterized by the cleavage of nuclear DNA. To test whether the tdr mutant anthers are defective in PCD, we performed the TUNEL (for terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling) assay in wild-type and the tdr anthers. A range of developmental stages was analyzed, including meiosis stage, tetrad stage, microspore stage, and vacuolated pollen stage from the wild type and the corresponding stages selected from the tdr mutant. During the meiosis stage, both wild-type and the tdr tapetal cells showed TUNEL-negative nuclei (Figures 4A and 4B ), indicating lack of DNA fragmentation at this stage. At the tetrad stage, the TUNEL-positive nuclei were detected in wild-type tapetal cells, suggesting that PCD occurred in the tapetum (Figure 4C). However, little fragmented DNA signal was observed in the tdr mutant anthers at this stage (Figure 4D). Wild-type anthers at the young microspore stage showed more strong TUNEL-positive signals in tapetal cells. Simultaneously, the TUNEL signals were also observed in the outer cell layers of the anther (endothecium and middle layer) and vascular bundle cells (Figure 4E). However, TUNEL fluorescence signal was still not detected in the tdr tapetum at the young microspore stage (Figure 4F). At the vacuolated pollen stage, TUNEL-positive nuclei were present in degenerating tapetal cells and stomium cells in wild-type anthers (Figure 4G), and a weak signal could be observed in the expanded tapetal cells and collapsed microspores in the tdr mutant (Figure 4H). These observations demonstrate that the PCD of tapetum commences at the tetrad stage in wild-type anther, and the retardation of PCD in tapetum possibly results in the failure of tapetum degeneration in the tdr mutant.
DNA Fragmentation in Wild-Type and tdr Mutant Anthers.
The anthers of the four developmental stages in the wild type and the tdr mutant were compared for nuclear DNA fragmentation using the TUNEL assay. Nuclei have been stained with propidium iodide indicated by red fluorescence, while yellow to green fluorescence is TUNEL-positive nuclei staining. T, tapetum; Ms, microsporocyte; Tds, tetrads; Msp, microspore. Bars = 50 μm.
(A) The wild type at meiosis stage.
(B) The tdr mutant at meiosis stage.
(C) Wild-type anther at tetrad stage showing TUNEL-positive signal in tapetal cells (arrow).
(D) The tdr mutant at tetrad stage.
(E) The wild type at young micropsore stage. TUNEL-positive signal is detected in the tapetum, outer cell layers, and vascular bundle cells (arrows).
(F) The tdr mutant at young microspore stage.
(G) Wild-type anther at the vacuolated pollen stage showing TUNEL-positive signal in the tapetal cells and stomium area (arrows).
(H) The tdr mutant at vacuolated pollen stage. TUNEL-positive signal is detected in expanded tapetal cells and collapsed microspores (arrows).
To quantify the results from the TUNEL assay, we compared DNA damage levels between the wild-type and the tdr mutant anthers using the comet assay (Wang and Liu, 2006). The tdr mutant anthers at the MMC stage exhibited similar levels of DNA damage to those of the wild type. From the tetrad stage to vacuolated pollen stage, wild-type anthers exhibited significant increases in DNA damage. However, the tdr mutant anthers exhibited lower than normal levels of DNA damage from the tetrad stage to the vacuolated pollen stage (Figure 5 ). The result of the comet assay also confirmed the retardation of PCD in the tdr anthers.
Analyses of DNA Damage in the Wild Type and the tdr Mutant Anther Tissues.
Comet assay used to assess the relative amount of DNA damage in the wild type and the tdr mutant anther at different stages. The tdr mutant anthers at the MMC stage exhibited similar levels of DNA damage to those of the wild type. The DNA damage level in the wild type was increased from the tetrad stage and reached the maximum at the vacuolated pollen stage. The tdr mutant exhibited an increased level of DNA damage just from the vacuolated pollen stage. The extent of DNA damage in each nucleus is indicated by the units 0, 1, 2, or 3. An increased unit correlated with a higher DNA percentage in tail, as illustrated in the inset. This assignation was described by Wang and Liu (2006). The DNA damage units were obtained by summing the units from 100 nuclei on each slide. Bars indicate sd.
Isolation of the TDR Gene
The TDR locus was previously mapped to the short arm of rice chromosome 2 between the two InDel molecular markers LHS10 and LHS6 with a physical distance of 133 kb (Liu et al., 2005). To further map the TDR gene, we generated a large F2 mapping population, and 2450 segregants showing the tdr mutant phenotype were analyzed. The TDR gene was located between two newly developed InDel markers LHS12 and LHS3 defining a region of 52 kb (Figure 6A ). Through repeated sequencing, we confirmed a single nucleotide deletion in the seventh exon of an annotated bHLH gene (Os02g02820), which caused a frame shift and premature translational termination (Figure 6B). The Os02g02820 gene was confirmed to be TDR by a functional complementation experiment. A binary plasmid carrying a 6.4-kb wild-type BamHI-SalI genomic fragment from the BAC clone AP005851 was able to rescue the sterile phenotype of the tdr homozygous plants (see Supplemental Figure 2 online).
Molecular Identification of TDR.
(A) Fine mapping of the TDR gene on chromosome 2. Names and positions of the molecular markers are indicated. AP005851 and AP004078 are genomic DNA accession numbers. The TDR locus is mapped to a 52-kb region between two molecular markers (LHS12 and LHS3). cM, centimorgan.
(B) A schematic representation of the exon and intron organization of TDR. The mutant sequence has one base deletion in the seventh exon. +1 indicates the starting nucleotide of translation, and the stop codon (TAG) is +1659. Black boxes indicate exons, intervening lines indicate introns, the gray box indicates the 3′-untranslated region, and the white box indicates the bHLH domain.
(C) The TDR protein sequence. Putative bHLH domain is underlined. Putative nuclear localization signal is boxed.
(D) A cell that expressed free GFP showing fluorescence in nucleus, cytoplasm, and plasma membrane.
(E) A cell that expressed TDR-GFP showing fluorescence in the nucleus.
(F) Nuclear-localizing AS2 is used as a positive control, and AS2-GFP is exclusively detected in the nucleus. Bars = 20 μm in (D) to (F).
The TDR open reading frame encodes a putative bHLH protein of 552 amino acids with a bHLH domain between the 280th and 341st amino acids (Figure 6C). The presence of a potential nuclear localization signal (RKRRKK, amino acids 290 to 296) in the TDR protein suggests that TDR protein is possibly targeted to the nucleus (Figure 6C). To determine the subcellular localization of TDR, we constructed a translation fusion between the cDNA for the green fluorescent protein (GFP) and the full-length TDR coding region. The TDR-GFP fusion construct and the GFP alone control, both driven by the 35S promoter, were introduced into onion epidermal cells by particle bombardment. As expected, the free GFP was found in the nucleoplasm and in the cytoplasm (Figure 6D). By contrast, the TDR-GFP fusion protein was observed exclusively in the nucleus (Figure 6E). Nuclear-localizing AS2 was used as a positive control, and AS2-GFP fusion protein was observed specifically in the nucleus (Figure 6F). These results suggest that TDR is localized to the nucleus.
Sequence Analysis of TDR and Related Proteins
To gain additional insights into the phylogenetic relationship between TDR and its close homologs, we searched public databases using BLAST with the TDR sequence as a query. The full-length amino acid sequences of TDR and its 12 closest homologs were used for phylogenetic analysis. Our result revealed that TDR and Arabidopsis AMS (Sorensen et al., 2003) were supported as an orthologous pair (Figure 7A ). Furthermore, sequence comparison indicated that TDR shares 32% overall identity with Arabidopsis AMS (Figure 7B). In Arabidopsis, the ams mutation results in abnormal tapetum expansion and persistence of middle layer, which is similar to the phenotype of the tdr mutant. However, PCD was not analyzed in the ams mutant.
Phylogenetic Analysis of TDR-Related Proteins and Comparison of the Amino Acid Sequences of TDR with AMS and Udt1.
(A) Bootstrap neighbor-joining phylogenetic tree was constructed using MEGA and 1000 replicates. The proteins are named according to their gene names or National Center for Biotechnology Information accession numbers. Zm IN1 is defined as an outgroup. The length of the branches refers to the amino acid variation rates. The alignment on which the tree was constructed is shown in Supplemental Figure 3 online.
(B) The deduced amino acid sequence of TDR is compared with the sequences of AMS and Udt1. bHLH domains are boxed. Black boxes indicate identical residues, and gray boxes indicate similar residues.
Additionally, the rice Udt1 gene also encodes a bHLH protein, which plays a major role in maintaining rice tapetum development at the early meiosis stage (Jung et al., 2005). Comparative analysis revealed that the full-length amino acid sequences between the TDR and Udt1 have only 12% identity (Figure 7B). In the udt1 mutant, the transcript of TDR is reduced (Jung et al., 2005), while the tdr mutation has no obvious effect on the expression of Udt1 (see Supplemental Figure 4 online). So, the Udt1 gene probably acts upstream of TDR.
TDR Is Preferentially Expressed in the Tapetum
The tdr mutation affected the tapetum postmeiotic degeneration and the morphology of other anther wall cells and microspores but had little effect on rice vegetative growth and other flower organ development. To test whether TDR acts within the anther or from a distant tissue, we analyzed the TDR expression pattern.
We first detected TDR expression by RT-PCR with total RNA extracted from vegetative and reproductive organs (Figure 8A ). There was no detectable expression of TDR in vegetative and floral organs other than the anther. By contrast, TDR expression was clearly detected at relatively early stages of anther development, starting at the meiosis stage and reaching the maximum level at the young microspore stage. When the young microspores developed into the vacuolated pollen stage, the transcript level of TDR was greatly reduced. At the heading stage, the TDR transcripts were hardly detectable.
TDR Expression Pattern.
(A) Spatial and temporal expression analyses of TDR by RT-PCR. M, meiosis; Y, young microspore; V, vacuolated pollen; H, heading stage; GDNA, genomic DNA.
(B) to (G) In situ analyses of TDR.
(B) A wild-type anther at the early premeiosis stage showing no TDR expression.
(C) Successive section to that shown in (B), probed with the TDR sense probe.
(D) A meiosis stage wild-type anther showing TDR expression in tapetum, middle layer, and endothecium.
(E) A wild-type anther at the meiosis stage with sense probe.
(F) A wild-type anther at the tetrad stage showing stronger TDR expression in tapetal cells.
(G) Successive section to that shown in (F), probed with the TDR sense probe.
SPC, secondary parietal cell; SC, sporogenous cell; Ms, microsporocytes; T, tapetum; Tds, tetrads. Bars = 25 μm.
To more precisely determine the spatial and temporal patterns of TDR expression, we performed RNA in situ hybridization with wild-type floral sections (Figures 8B to 8G). At the early premeiosis stage, the TDR transcript was hardly detectable (Figure 8B). The TDR transcripts were initially detected in the tapetal, middle layer, and endothecium of the meiosis stage anthers (Figure 8D). At the tetrad stage, the TDR gene was more strongly expressed in the tapetum (Figure 8F). Only background levels of signal were detected with the sense probe (Figures 8C, 8E, and 8G). Endo et al. (2004) showed that TDR (Os02g02820) was mainly expressed in the tapetum at the young microspore stage through in situ hybridization probed with another DNA fragment of TDR. Therefore, TDR expression is associated with the differentiation of tapetal cells during rice anther development.
TDR Interacts with Os CP1 and Os c6
TDR is a putative bHLH transcription factor expected to regulate gene expression by binding to an E-box (CANNTG) (Bouchard et al., 1998; Chinnusamy et al., 2003). To identify the regulatory target genes of TDR, we performed transcriptional analysis of the wild type and the tdr mutant anther at the meiosis/young microspore stages using the Affymetrix rice chips (data not shown). From the preliminary data, we identified two genes, Os CP1 and Os c6, for further testing of direct in vivo binding with TDR (Figure 9 ). Cys proteases (CPs) belong to a family of enzymes found in animals, plants, and microorganisms that play important roles in intracellular protein degradation and are PCD hallmarks (Solomon et al., 1999). Os CP1 is a rice Cys protease gene, and a loss-of-function mutation in Os CP1 results in the collapse of microspore after release from tetrads (Lee et al., 2004). The Os c6 gene encodes a putative protease inhibitor and shows tapetum-specific expression (Tsuchiya et al., 1992, 1994). Our RT-PCR analysis indicated that Os CP1 and Os c6 were expressed in wild-type anthers at the early stages, and their transcripts were greatly reduced as the anther developed into the vacuolated and mature pollen stages. However, reduced Os CP1 transcript and no expression of Os c6 were detected in the tdr anthers, suggesting that they are possible downstream genes of TDR (Figure 9A). Six and four predicted E-box sequences (CANNTG) were also found in the promoter regions of Os CP1 and Os c6, respectively. As a control, three predicted E-box sequences were found in the promoter regions of Actin1, which have sequence variation with those of predicted E-box sequences (CANNTG) and were also found in the promoter regions of Os CP1 and Os c6 (Figure 9B). Both 514-bp Os CP1 and 514-bp Os c6 upstream DNA fragments were specifically enriched when the affinity-purified TDR antibodies were used. However, no enrichment of the 307-bp upstream DNA fragment of Actin1 was observed using the affinity-purified TDR antibodies (Figure 9C).
Direct Binding of TDR to the Regulatory Regions of Os CP1 and Os c6.
(A) RT-PCR analyses of Os CP1 and Os c6 in wild-type and the tdr anthers. M, meiosis; Y, young microspore; V, vacuolated pollen; H, heading stage.
(B) Predicted E-boxes of Os CP1, Os c6, and Actin1 within 1-kb upstream regions starting from ATG. The bent arrow denotes the translational start site. The black boxes indicate canonical binding sites for bHLH proteins of the form CANNTG (i.e., E-box). Arrows indicate oligonucleotide primers used in enrichment assays.
(C) Enrichment of the Os CP1 and Os c6 regulatory fragments required for the anti-TDR antibody. Enrichment of the Os CP1 and Os c6 fragments was only observed when affinity-purified TDR antibody was used, and no Actin1 fragment was enriched.
(D) TDR was assayed for binding to the regulatory regions of Os CP1 and Os c6 in EMSA. The length of DNA probes for Os CP1 (P-Os CP1) and Os c6 (P-Os c6) are 161 and 170 bp, respectively. Dashes represent the omitted nucleotides. The underlined nucleotides indicate E-box. TDR protein was mixed with 32P-labeled Os CP1 probe (lane 2) and Os c6 probe (lane 7), and 1-, 100-, and 10,000-fold molar excess of unlabeled Os CP1 probe (lanes 3 to 5) and Os c6 probe (lanes 8 to 10) were added as competitor to the EMSA reaction. As a control, translated pET32a control plasmid was added (cont. lanes 1 and 6). An arrow marks free probe. An arrowhead marks supershift band.
To further confirm that TDR has the ability to bind to the promoter regions of Os CP1 and Os c6, EMSA was employed. We observed that TDR could bind the 161-bp DNA fragment (−673 ∼ −513) of the Os CP1 promoter region and the 170-bp DNA fragment (−881 ∼ −712) of Os c6 (data not shown). The DNA binding activities were further tested in the competition experiments by addition of unlabeled DNA fragments as competitors. As shown in Figure 9D, the addition of excess Os CP1 and Os c6 competitor DNAs reduced the formation of the complex in a concentration-dependant manner. These results support the hypothesis that TDR directly regulates Os CP1 and Os c6 and may regulate tapetum degeneration by upregulating their expression.
DISCUSSION
A Mutation in TDR Impairs Rice Anther Development
We report here the characterization of the TDR gene in rice. Based on morphological studies, formation of four anther wall cell layers and MMC appeared to be normal. The mutant MMCs entered meiosis and progressed through to the tetrad stage. However, the postmeiotic development of both tapetum and middle layer was disrupted in tdr anthers, as indicated by the appearance of large vacuoles in expanded cells. In addition, microspores collapsed after released from tetrads. Therefore, TDR is critical for normal tapetum development and function, which are essential for normal pollen development.
Production of functional pollen grains relies significantly on the timely death and degeneration of the tapetum. Through their degeneration and release of cellular contents, the tapetal cells contribute to the completion of the extracellular sculpting of the pollen grains, providing them with adhesive and signaling molecules of proteinaceous and lipoidal nature that are critical for pollination (Wu et al., 1997; Piffanelli et al., 1998). Using genetic cell ablation, Kawanabe et al. (2006) showed that the PCD signal commences at the tetrad stage, but these authors did not provide cytological evidence of tapetum PCD. In this article, our TUNEL analysis provides direct evidence that the PCD of tapetal cells starts at the tetrad stage. Simultaneously, we also found that the tapetum PCD is retarded in the tdr mutant. We propose that the TDR gene encodes a key positive regulator of rice tapetum PCD. Combined with TEM observation, we believe that the absence of PCD in the tdr mutant tapetum caused a failure to provide critical materials and signals for proper microspore development, resulting in collapse of microspores and male sterility. According to Varnier et al. (2005), anther PCD in Lilium is a progressive process that is initiated in the tapetum and then expanded to other anther cell layers. This type of PCD progression can also explain why the lack of PCD in the tdr mutant tapetum may further impact the other somatic anther cell layers, such as the middle layer.
How does the tdr mutation abolish rice tapetal PCD? In animal systems, the Bcl-2 family proteins are key regulators of both cell survival and cell death (Gross et al., 1999). One member of this family, the mammalian Bax gene, has been shown to induce plant PCD that is similar to the PCD associated with the hypersensitive response induced by mosaic virus (Lacomme and Cruz, 1999; Kawai-Yamada et al., 2001). Overexpression of an Arabidopsis homolog of human Bax inhibitor-1 (At BI-1) has been shown to block the tapetum PCD, resulting in male sterility (Kawanabe et al., 2006). Although the tdr mutation has no effect on the expression of the Os BI-1 gene (see Supplemental Figure 5 online), our data reveal that the expression of Os CP1 and Os c6, encoding a Cys protease and a proteases inhibitor, respectively, are highly reduced in the tdr anthers. Cys proteases and their inhibitors are known to be associated with PCD in various stress responses and during leaf senescence (Minami and Fukuda, 1995; Solomon et al., 1999; Xu and Chye, 1999). Therefore, such Cys proteases likely play crucial roles in tapetum degeneration. Further support for this idea comes from the evidence for a direct regulation of Os CP1 and Os c6 by TDR from ChIP and EMSA analyses. Therefore, our data strongly suggest that TDR positively regulates tapetum PCD by controlling tissue-specific effector genes important for PCD.
The TDR Gene Is Involved in a Crucial Regulation Network Controlling Postmeiotic Anther Development
The bHLH proteins belong to a superfamily of transcription factors that are important regulatory components in controlling various processes, from cell proliferation to cell lineage establishments. These proteins usually consist of two domains (i.e., a basic domain necessary for DNA binding and a HLH domain for dimerization) (Chinnusamy et al., 2003; Baudry et al., 2004). In plants, bHLH proteins have been reported to regulate a number of biochemical and developmental processes (Murre et al., 1989; Goodrich et al., 1992; Ferre-D'Amare et al., 1994; Kawagoe and Murai, 1996; Abe et al., 1997; Ni et al., 1998; Fairchild et al., 2000; Grandori et al., 2000; Massari and Murre, 2000; Spelt et al., 2000; Heisler et al., 2001; Chinnusamy et al., 2003).
Phylogenetic and BLAST analyses indicate that TDR is most similar to the Arabidopsis AMS protein, which is required for male fertility (Sorensen et al., 2003). Additionally, Li et al. (2006) performed phylogenic analysis of 167 rice bHLH genes and 147 Arabidopsis bHLH genes, and TDR and AMS also were grouped in the same subfamily. The AMS expression starts at a low level premeiotically and increases in postmeiotic anthers. The microspores in the ams anther collapse after release from the tetrad, and the tapetum becomes abnormally large and vacuolated at the late stage of anther development, while the middle layer persists (Sorensen et al., 2003). Our results further indicate that the TDR protein is likely localized to the nucleus and has a function similar to that of AMS, suggesting the TDR is likely the rice ortholog of AMS.
Additionally, it is notable that the rice Udt1 gene encoding a bHLH protein plays a major role in maintaining rice tapetum development at the early meiosis stage. The udt1 mutation results in the vacuolation and expansion of the tapetal cells from the meiosis stage; the udt1 meiocytes degenerate at the tetrad stage, and the degeneration of the middle layer is inhibited (Jung et al., 2005). However, the full-length protein sequences of TDR and Udt1 have only 12% identity. In the udt1 mutant, the transcript of TDR is reduced, while the tdr mutation has no impact on the expression of Udt1. Therefore, Udt1 is probably upstream of TDR. Furthermore, though the expanded tapetum is observed in both tdr and udt1 mutants, the microspore formation occurs only in the tdr mutant but not in the udt1 mutant, which further supports the possibility that TDR acts downstream of Udt1.
In summary, we show here that TDR promotes anther development by positively regulating the tapetum PCD in rice. Also, we further identify two downstream target genes, Os CP1 and Os c6, directly regulated by TDR. Our studies have opened a window into the genetic control of postmeiotic tapetum degeneration and development. Recently, it was shown that the Arabidopsis DYT1 gene encoding a bHLH protein is required for tapetum development and normal levels of AMS expression, suggesting that DYT1 acts upstream of AMS (Zhang et al., 2006). Phylogenetic analysis supports the orthology of the Arabidopsis DYT1 and rice Udt1 genes (Zhang et al., 2006). Therefore, it is likely that the rice Udt1-TDR genes and the Arabidopsis DYT1-AMS genes represent an evolutionarily conserved regulatory module critical for normal anther development.
METHODS
Mutant Material and Growth Conditions
The F2 mapping population was generated from a cross between the tdr mutant (japonica) and LongTeFu B (indica). In the F2 population, male sterile plants were selected for gene mapping. All plants were grown in the paddy field of Shanghai Academy of Agriculture Sciences.
Characterization of Mutant Phenotype
Plants or flowers were photographed with a Nikon E995 digital camera and a Motic K400 dissecting microscope. Anther sections were stained by 0.2% 4,6-diamino-2-phenylindole dihydrochloride n-hydrate to stain the nuclei. Observation of anther development was performed on standard plastic sections as described by Hong et al. (1995). Spikelets of different developmental stages were collected, based on the length of spikelet, and fixed with 3% (w/v) paraformaldehyde and 0.25% glutaraldehyde in 0.2 n sodium phosphate buffer, pH 7.0, for 20 h at 4°C, rinsed with 0.1 M phosphate buffer, pH 7.0, and dehydrated in an ethanol series. The samples were embedded in Technovit 7100 resin (Hereaus Kulzer), polymerized at 45°C. Transverse sections of 2 μm were cut using an Ultratome III ultramicrotome (LKB) stained with 0.25% toluidine blue O (Chroma Gesellshaft Shaud) and photographed using a Nikon E600 microscope and a Nikon DXM1200 digital camera.
For TEM, spikelets at various stages of development were fixed in 3% (w/v) paraformaldehyde and 0.25% glutaraldehyde in 0.2 n sodium phosphate buffer, pH 7.0, and were postfixed in 2% OsO4 in PBS, pH 7.2. Following ethanol dehydration, samples were embedded in acrylic resin (London Resin Company). Ultra-thin sections (50 to 70 nm) were double stained with 2% (w/v) uranyl acetate and 2.6% (w/v) lead citrate aqueous solution and examined with a JEM-1230 transmission electron microscope (JEOL) at 80 kV.
Molecular Cloning of TDR
For fine-mapping of the TDR locus, we developed InDel molecular markers based on the sequence difference between japonica variety Nipponbare and indica variety 9311 (Jander et al., 2002). The primer sequences of InDel markers were as follows: LHS3 (5′-CACTACTCCCTCTATCGCACG-3′and 5′-ATTATCATTGGATTGACATTTG-3′), LHS6 (5′-AGGTTAGTGCTTCGGAGTGG-3′ and 5′-ACAGACAGAACAGCGGTCAA-3′), LHS10 (5′-CCTTTCAAAGCGCCACAG-3′ and 5′-AGCAGCCGACGTTCCTAA-3′), and LHS12 (5′-CTTGGGGTCTCGCAGCATA-3′ and 5′-GAAGAAGCGGATGAATGGG-3′). PCR amplification and polyacrylamide gel electrophoresis analysis were performed as described previously (Liu et al., 2005).
RT-PCR
Total RNA was isolated using Trizol reagent (Invitrogen) as described by the supplier from rice (Oryza sativa) tissues: root shoot, leaf, glume, lemma, palea, and anthers at different stages. The stages of anthers were classified into the following categories according to spikelet length (Feng et al., 2001): meiosis stage anthers within 1- to 3-mm spikelets, young microspore stage anthers within 3- to 5-mm spikelets, vacuolated pollen stage anthers within 5- to 7-mm spikelets, and mature pollen stage anthers within 7- to 8-mm spikelets. After treatment with DNase (Promega), 0.3 μg RNA was used to synthesize the oligo(dT) primed first-strand cDNA using the ReverTra Ace-α-First Strand cDNA synthesis kit (TOYOBO). Three microliters of the reverse transcription products were subsequently used as template in a PCR reaction. All the primers for RT-PCR are listed in Supplemental Table 1 online.
In Situ Hybridization
Wild-type spikelets of different developmental stages were fixed in 5% acetic acid, 50% ethanol, and 3.7% formaldehyde in water for 16 h at 4°C. They were dehydrated through ethanol series, embedded in Paraplast Plus (Oxford Labware), and sectioned at 8 μm using an YL3-A rotary microtome (Shanghai Instrument Factory). The full-length TDR cDNA fragment was digested from the TDR cDNA clone vector pCMVFL3 (Rice Genome Resource Center–National Institute of Agrobiological Sciences [RGRC-NIAS]; http://www.rgrc.dna.affrc.go.jp/stock.html) with EcoRI or BamHI and was transcribed in vitro under T7 or SP6 promoter with RNA polymerase using the DIG RNA labeling kit (Roche). This mixture was prepared for the DIG-labeled RNA antisense or sense probe. RNA hybridization and immunological detection of the hybridized probes were performed according to the protocol of Kouchi and Hata (1993).
Complementation of the tdr Mutant
For functional complementation, an ∼6.4-kb genomic DNA fragment containing the entire TDR coding region, a 2430-bp upstream sequence, and a 1857-bp downstream sequence was digested from BAC clone AP005851 with BamHI and SalI and subcloned into the binary vector pCAMBIA1301 (CAMBIA) carrying a hygromycin resistance marker to generate p1301TDR construct. We induced the calli using the homogenous tdr young panicles, which mainly included palea and lemma, and then the calli were used for transformation with Agrobacterium tumefaciens EHA105 carrying the p1301TDR plasmid and the control plasmid p1301.
TDR Nuclear Localization Analysis
The GFP cDNA was amplified from pBSK-GFP vector with the following primers: 5′-aaaaAGATCTATGGGTAAAGGAGAAGAACTTTTCACTG-3′ and 5′-aaaaCACGTGTTAT TTGTATAGTTCATCCATGCCATGTG-3′ (attached restriction site is underlined). The PCR product was cloned into pMD18-T vector (TaKaRa), released by BglII-SmaI digestion, and subcloned into the BglII-PmacI–digested pCAMBIA1301 vector (CAMBIA) containing 35S promoter to generate p1301-GFP. The TDR cDNA was amplified from the cDNA clone vector pCMVFL3 (RGRC-NIAS) with primers 5′-AGTCACGACGTTGTA-3′ and 5′-aaaaAGATCTATCAAACGCGAGGTAATGCAGGTGCT-3′. The amplified fragment was digested with NcoI and BglII and ligated with the same enzyme-digested p1301-GFP to create p1301-TDR-GFP. pAS2-GFP was used as positive control vector (provided by A.-W. Dong, Fudan University, China). The onion epidermis was peeled and bombarded with golden particle–coated plasmids. Cells with GFP fluorescence were observed under a fluorescence confocal microscope (Zeiss LSM 510).
TUNEL
Sections were washed in PBS (160 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, and 1.5 mM KH2PO4) for 5 min and incubated in 20 μg/mL proteinase K in 100 mM Tris-HCl, pH 8.0, and 50 mM Na2EDTA (100 μL per slide in a humid chamber). The sections were washed in PBS for 5 min and fixed in 4% (w/v) paraformaldehyde in PBS for 10 min, and then the PBS washing was repeated. In situ nick-end labeling of nuclear DNA fragmentation was performed in a humid chamber for 1 h in the dark at 37°C with a TUNEL apoptosis detection kit (DeadEnd Fluorometric TUNEL system; Promega) according to the supplier's instructions. Samples were analyzed under a fluorescence confocal scanner microscope (Zeiss Axioplan).
Comet Assay for DNA Damage
The comet assay kit from Trevigen was used with minor modifications. Anthers of different developmental stages were chopped with a razor in 1.5-mL centrifuge tubes containing 500 μL of ice-cold 1× PBS plus 20 mM EDTA. The resulting mixture was filtered through a 100-μm nylon net filter (Millipore). Thirty microliters of nuclei was mixed with 300 μL of 1% low-melting-point agarose (prewarmed at 37°C) and pipetted onto Trevigen-precoated slides. After incubating in lysis solution at 4°C for 1 h, the slides were dipped in alkaline solution (0.3 n NaOH and 1 mM EDTA) for 30 min and then washed with 1× TBE (Tris-borate/EDTA) three times. The slides were run at 1 V/cm for 10 min in 1× TBE and then dipped in 70% ethanol for 5 min. After air-drying, the slides were stained with a 1:10,000 dilution of SYBR green and then were examined with a Nikon E600 microscope. The percentage of DNA in each comet tail (T DNA%) was evaluated with CometScore software (http://www.autocomet.com). The extent of DNA damage in different samples was derived according to the method described by Wang and Liu (2006).
TDR-Specific Polyclonal Antibody Preparation and Purification
A TDR-specific fragment was amplified from cDNA clone vector pCMVFL3 (RGRC-NIAS) using the primer pairs N12F (5′-aaaGAATTCAACAGCCACCAACAGCAGC-3′) and N12R (5′-aaaAAGCTTTCATCAATCAAACGCGAGGTAATGC-3′). The PCR product was cloned into the EcoRI and HindIII sites of pET-32a vector (Novagen) to produce p32-TDR. The fusion protein expression and purification were performed according to the manual of pET-32a from Novagen, and antibody preparation was performed as described by Huang et al. (2003).
ChIP and Enrichment Test for in Vivo Binding of TDR
The procedure for ChIP of TDR-DNA complexes in rice wild-type anther was modified from Bowler et al. (2004). Briefly, rice anthers at the meiosis stage were treated with formaldehyde, and the chromatin solution was sonicated on an Ultrasonic Crasher Noise Isolating Chamber (SCIENTZ). The soluble chromatin fragments were obtained from isolated nuclei. Preadsorption with sheared salmon sperm DNA/protein A agarose mix (Sigma-Aldrich) was performed to remove nonspecific binding DNA. Immunoprecipitation with TDR-specific immune antiserum and preadsorption with protein A-Sepharose beads and without any serum were performed as in the reference above.
Oligonucleotide primers specific for the upstream of Os CP1 (5′-CCTAAAGAAGCAGTTGCCAT-3′ and 5′-GATTGGGGATTGAGGTGTTA-3′), Os c6 (5′-CAACGAACGGCATTTGTTTT-3′ and 5′-GACTTTTTAGCACATGTTTA-3′), and Actin1 (5′-GTTCCTAAAGCCCAAAGTGC-3′ and 5′-TGTATATAGTGGCGATGGGG-3′) were added to PCR reactions in which the templates were ChIP populations from immune or control immunoprecipitations. Typically, 34 cycles of PCR were performed, and the products were analyzed by agarose gel electrophoresis.
EMSA
The DNA fragments containing the E-box of the Os CP1 and Os c6 regulatory region were generated using PCR amplification with the following specific primers: P-Os CP1 (5′-TTCCCCTTCCACCAAAAACG-3′ and 5′-AAATCTAGGCGAGACTCCAC-3′) and P-Os c6 (5′-CAACGAACGGCATTTGTTTT-3′ and 5′-GCAGGAACTAATTAAATAAGTTTGT-3′). The two DNA fragments were cloned into pMD18-T vector (TaKaRa) for sequence confirmation. The specific primers (25 pmol/μL) were incubated with polynucleotide kinase (Promega), 10× polynucleotide kinase buffer, and 32P-dATP at 37°C for 30 min and labeled with 32P. The two inserts were labeled with 32P through PCR with 32P-labeled specific primers and purified on an 8% PAGE gel.
Full-length TDR was amplified from cDNA clone vector pCMVFL3 (RGRC-NIAS) using the primer pairs 32a-F (5′-GAATTCATGGGAAGAGGAGACCACC-3′) and 32a-R (5′-GTCGACTGGTAAAATCAATCATCATC-3′). The PCR product was cloned into the pMD18-T vector (TaKaRa) and released by EcoRI and SalI digestion. This fragment was then ligated with the same enzyme-digested pET32a (Novagen). The fusion protein was expressed in Escherichia coli as described by Huang et al. (2003).
The DNA binding reactions were performed according to Wang et al. (2002) with the following modifications. Reaction components were incubated in 1× binding buffer [10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM EDTA, 5% glycerol, 0.05 μg μL−1 poly(dI-dC), and 0.1 μg μL−1 BSA] at room temperature for 20 min. The entire reaction mix was analyzed on a 3.5% PAGE gel. After drying the gel, 32P-labeled DNA fragments were detected using an autoradiograph.
Computational and Database Analysis
A phylogenetic tree was constructed with the aligned plant bHLH protein sequences using MEGA software (version 3.0) (http://www.megasoftware.net/index.html) (Kumar et al., 2004) based on the neighbor-joining method with the following parameters: p-distance model, pairwise deletion, and bootstrap (1000 replicates; random seed). The multiple alignments were performed using ClustalW (http://www.ebi.ac.uk/clustalw/).
Accession Numbers
Sequence data from this article for the cDNA and genomic DNA of TDR can be found in the GenBank/EMBL data libraries under accession numbers XM_463907 and AP004078, respectively.
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Table 1. List of the Primers Used for RT-PCR Analyses.
Supplemental Figure 1. Results of DAPI Staining to Detect Nuclei.
Supplemental Figure 2. Complementation of the tdr Mutant by TDR Genomic DNA.
Supplemental Figure 3. Sequence Alignment of TDR and 12 TDR-Related bHLH Proteins.
Supplemental Figure 4. Expression Analysis of Udt1.
Supplemental Figure 5. Expression Analysis of Os BI-1.
Acknowledgments
We thank B. Han (Rice Genome Resource Center) and A.-W. Dong for providing the BAC clone, cDNA clone, and pAS2-GFP plasmid. We thank Z.-J. Luo and M.-J. Chen for mutant screening and generation of F2 populations for the mapping, C.-M. Zhang for rice transformation, Y.-J. Lu for plastic sections, X.-S. Gao and K.-Y. Chen for confocal microscope imaging, and Q. Song for TEM observation. Z.-C. Liu is gratefully acknowledged for her valuable suggestions regarding ChIP and comet assay experiments and helpful comments on the manuscript. We are also grateful to H. Yu for editing this manuscript. This work was supported by funds from the National Key Basic Research Developments Program of the Ministry of Science and Technology, People's Republic of China (2006CB101700 and 2005CB120802), the National “863” High-Tech Project (2005AA2710330), the Program for New Century Excellent Talents in University (NCET-04-0403), the Shuguang Scholarship (04SG15), and the Shanghai Institutes of Biological Sciences (Reproductive Development Project).
Footnotes
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The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Da-Bing Zhang (zhangdb{at}sjtu.edu.cn).
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↵1 These authors contributed equally to this work.
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↵[W] Online version contains Web-only data.
- Received May 13, 2006.
- Revised October 11, 2006.
- Accepted November 2, 2006.
- Published November 30, 2006.
References
