Molecular Plant • Volume 6 • Number 5 • Pages 1715–1718 • September 2013
LETTER TO THE EDITOR
A Novel Rice bHLH Transcription Factor, DTD, Acts Coordinately with TDR in Controlling Tapetum Function and Pollen Development Dear Editor, Male reproductive development is an essential biological process for flowering plants and crucial for crop seed production. Formation of the male reproductive organ, the anther, involves a number of developmental events, including stamen meristem specification, generation of sporogenous cells and their differentiation into microspore mother cells (MMCs), meiosis, microspore (pollen) maturation, and pollination (Ma, 2005). The formation of microspores and their development into mature pollen grains require cooperative interactions between gametophytic (microspores) and sporophytic (anther wall) cells, with the innermost cell layer, the tapetum, playing the most crucial role (Ma, 2005). Tapetal cells undergo degeneration by programmed cell death (PCD). The tapetal PCD and the consequent cellular degradation of the tapetum must occur during the proper anther developmental stages for the normal tapetum function and pollen development; premature, suppressed, or delayed tapetal PCD and cellular degeneration cause male sterility (Papini et al., 1999; Kawanabe et al., 2006; Li et al., 2006a; Luo et al., 2013). In rice, several genes are known to be involved in the differentiation, degeneration, and function of the tapetum and microspore development, including TAPETUM1 (UDT1), OsGAMYB, TAPETUM DEGENERATION RETARDATION (TDR), OsCP1, OsC6, PERSISTENT TAPETAL CELL 1 (PTC1), RICE TAPETUM-SPECIFIC GENE (RTS), WAX-DEFICIENT ANTHER 1 (WDA1), Defective Pollen Wall (DPW), MADS3, CYP703A3, and CYP704B2 (reviewed by Zhang et al., 2011 and Guo and Liu, 2012). For example, TDR is preferentially expressed in the tapetum; in the tdr mutant, the tapetal PCD was retarded, accompanied by abortive pollen development and complete male sterility (Li et al., 2006a). To better understand the molecular genetic network of anther development in rice, we identified a number of malesterile mutants from a rice mutant library created by γ-ray mutagenesis of a japonica rice cultivar Nipponbare (unpublished results). One of the mutants exhibited abnormal anther morphology, lacking pollen (Figure 1A). Observation of anther transverse sections showed that the MMCs in the mutant anthers could undergo meiosis to produce microspores, but the microspore cells were eventually degraded before they could develop into mature pollen grains (Supplemental Figure 1). In wild-type rice anthers, the tapetum degenerated gradually from the microspore stage (stage
9) and disappeared completely at the bicellular pollen stage (stage 11) (Zhang et al., 2011; Luo et al., 2013). However, in the mutant anthers, tapetum degeneration was largely delayed and was not completed even at the last stage (stage 12) (Supplemental Figure 1). To examine tapetal PCD in the mutant anthers, we performed TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) assay, which detects nuclear DNA fragmentation to indicate PCD process. In wild-type anthers, tapetal PCD started at the dyad stage (stage 8a) and finished at the middle microspore stage (stage 9); however, in the mutant anthers, tapetal PCD started at stage 9 and had strongest TUNEL signals at the late microspore stage (stage 10) (Figure 1B). Since its tapetal PCD and cellular degeneration were delayed, we named this mutant delayed tapetum degeneration (dtd). Genetic analysis with a segregating population from a cross between dtd and an indica rice variety showed that, of 293 F2 individuals investigated, 226 were male-fertile and 67 malesterile (χ2 = 0.71, P > 0.05), indicating a genetically sporophytic male-sterile phenotype controlled by a recessive locus. Using the mapping population from this cross (about 10 000 F2 individuals), we fine-mapped the locus to an 11-kb region on chromosome 4, where only one gene (Os04g0599300 by http://rapdb.dna.affrc.go.jp/; or LOC_Os04g51070 by www.gramene.org/) was present (Supplemental Figure 1A). Sequencing analysis uncovered a 7-bp deletion in the third exon of this gene, which resulted in a premature stop codon (Figure 1C). To test its function, we transferred a genomic fragment containing this gene of wild-type into the progeny of a heterozygous (DTD/dtd) plant. Seven transgenic plants that were homozygous for dtd were obtained, and they showed a recovered male fertility phenotype (Supplemental Figure 2B and 2C), confirming that the mutation in Os04g0599300 conferred the dtd phenotype. DTD encodes a 464-aa (amino acid) basic helix-loop-helix (bHLH) transcription factor, OsbHLH141 (Li et al., 2006b). Expression analysis by quantitative reverse transcription polymerase chain reaction (qRT–PCR) showed that DTD was expressed specifically in © The Author 2013. Published by the Molecular Plant Shanghai Editorial Office in association with Oxford University Press on behalf of CSPB and IPPE, SIBS, CAS. doi: 10.1093/mp/sst046, Advance Access publication 21 March 2013 Received 8 January 2013; accepted 3 March 2013
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Letter to the Editor
Figure 1. DTD Acts Coordinately with TDR in Normal Tapetum Function and Male Fertility. (A) Phenotypes of the anthers in wild-type (WT) rice and the male-sterile mutant. The lower panels show that anthers of WT and the mutant (stained with 1% potassium iodide) had pollen grains and no pollen, respectively. Bars = 1 mm. (B) Detection of tapetal PCD by TUNEL in the developing anthers of the WT and mutant plants. Yellow signal indicates TUNEL-positive nuclei of PCD cells. T, tapetum; Msp, microspore. Bars = 20 μm. (C) The structure of the DTD gene and the nucleotide deletion in dtd. Premature stop codon (TAA) is indicated in red. (D) Expression analysis of DTD and dtd by qRT–PCR. The relative expression levels indicate the ratios of DTD/OsActin1. S6 to S12 indicate stages 6–12 of anther development, as in Zhang et al. (2011). Error bars show SD (n = 3). (E) P35S::DTD–EGFP and P35S::TDR–RFP constructs were co-transformed into tobacco protoplasts to confirm that the fusion protein targeted to the nucleus. TDR–RFP marked the nucleus. Scale bars = 20 μm. (F) BiFC assays showing the interaction of DTD with TDR. Scale bars = 20 μm. (G) Relative expression changes of OsC4, OsC6, and RTS in WT and dtd plants, measured by qRT–PCR. The relative expression levels indicate the ratio of the target gene/OsActin1. Error bars show SD (n = 3). (H) A proposed model for the molecular function of DTD, coordinately with TDR, in controlling proper tapetal PCD and pollen maturation by activating OsC4, OsC6, and RTS. The dysfunction of the tapetum caused by the defective dtd gene may induce a feedback regulation to some tapetum function-related genes.
the anthers, mainly from stage 8a to stage 10 (Figure 1D). The levels of the dtd transcript at the anther stages were substantially decreased, suggesting that the 7-nt (nucleotide) deletion resulted in instability of the mRNA. Consistently with its putative function as a transcription factor, DTD was localized to the nucleus (Figure 1E).
It is known that TDR, another rice bHLH transcription factor with low amino acid sequence similarity (54%) to DTD only in the bHLH domains, functions in promotion of tapetal PCD; the tdr mutant is defective in proper tapetal PCD, leading to the suppression of tapetum degradation and male sterility (Li et al., 2006a). Because dtd also
Letter to the Editor
affected tapetum degradation, we hypothesized that these two bHLH proteins may co-act to control tapetum function. Indeed, a yeast two-hybrid test showed that DTD interacted with TDR (Supplemental Figure 3), and the in vivo interaction in the nucleus of tobacco protoplasts was confirmed by a bimolecular fluorescence complementation assay (Figure 1F). TDR positively regulates the expression of two tapetumspecific genes, OsCP1 and OsC6 (Li et al., 2006a). OsCP1 encodes a member of the Cys protease family; these proteases play important roles in protein degradation and PCD (Lee et al., 2004). OsC6 and OsC4 encode lipid transfer proteins that are involved in synthesis of lipidic orbicules and pollen exine during anther development (Tsuchiya et al., 1994; Zhang et al., 2010). To know whether the dtd mutation affects known genes that function in tapetal degeneration and function, we measured the expression of UDT1, OsGAMYB, TDR, OsCP1, OsC4, OsC6, PTC1, RTS, WDA1, CYP703A3, and CYP704B2. In the dtd anthers, OsC4, OsC6, and RTS expression was completely suppressed (Figure 1G). In wild-type plants, the expression of these tapetum-specific genes terminates at stage 10, when the tapetum is almost completely degraded. However, in the dtd mutant, tapetal PCD and cellular degeneration were delayed (Figure 1B and Supplemental Figure 1), leading to the continued expression of UDT1, OsGAMYB, TDR, OsCP1, PTC1, WDA1, CYP703A3, and CYP704B2 until stage 11 or stage 12, most of them (OsGAMYB,TDR, OsCP1, WDA, CYP703A3) even having higher expression levels than their top levels in wild-type anthers (Supplemental Figure 4). It is notable that, although TDR interacts with DTD and is required for the OsCP1 expression (Li et al., 2006a), the defective dtd did not suppress the OsCP1 expression, suggesting that OsCP1 is not the target gene of DTD. Based on these results, we propose that DTD functions coordinately with TDR to control tapetal PCD and degeneration in developmental anthers (Figure 1H). It is notable that the defective phenotype of the tapetal PCD and cellular degeneration of dtd was relatively weaker as compared to that in the tdr mutant (Li et al., 2006a). This suggests that dtd may still have weak function; probably due to the fact that the mutation in this gene does not affect the encoding bHLH domain (Figure 1C), and that dtd was expressed, although at low levels (Figure 1D). DTD also is required for the expression of OsC4, OsC6, and RTS, which function in microspore development. The dysfunction of the tapetum by the defective dtd may induce a positive feedback regulation to some tapetum function-related regulators, including TDR. However, due to the absence of the key components (OsC4, OsC6, and RTS) downstream of DTD, the up-regulated expression of these genes by the feedback regulation eventually cannot recover male fertility. Recently, Niu et al. (2013) reported three allelic mutants of DTD, which is named ETERNAL TAPETUM 1 (EAT1), demonstrating that EAT1 interacts with TDR and promote tapetal cell
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death by regulating expression of two aspartic protease genes. Therefore, DTD (EAT1) is an important regulator of male reproductive development in rice, and a similar regulatory network may be present in other plants such as Arabidopsis.
SUPPLEMENTARY DATA Supplementary Data are available at Molecular Plant Online.
FUNDING This work was supported by grants from the Ministry of Science and Technology of China (2012AA10A303, 2011CB100203).
Acknowledgments We thank Dr Letian Chen for critical reading of the manuscript. No conflict of interest declared.
Chonghui Ji2, Heying Li2, Libin Chen, Min Xie, Fengping Wang, Yuanling Chen and Yao-Guang Liu1 State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Key Laboratory of Plant Functional Genomics and Biotechnology of Guangdong Provincial Higher Education Institutions, College of Life Sciences, South China Agricultural University, Guangzhou 510642, China 1 To whom correspondence should be addressed. E-mail
[email protected], tel. +86-20-85281908. 2 These authors equally contributed to this work.
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