Key message
Several QTLs and genes responsible for seed dormancy were detected and SNP candidates were shown to cause changes in seed germination.
Seed dormancy is a key agricultural trait to prevent ...pre-harvest sprouting in crop plants such as rice (
Oryza sativa
L.), wheat (
Triticum aestivum
), and barley (
Hordeum vulgare
L.). However, our knowledge of seed dormancy is hampered by the complexities of studying a trait that changes over time after seed harvest, and is complicated by interactions between phytohormones, seed coat components and the environment. Here, we have conducted a genome-wide association study using a panel of 311 natural accessions of cultivated rice, examining a total of 519,158 single nucleotide polymorphisms (SNPs). Eight quantitative trait loci (QTLs) were found to associate with seed dormancy in the whole panel and five in the
Japonica
and
Indica
subpanel; expression of candidate genes within 100 kb of each QTL was examined in two published, germination-specific transcriptomic datasets. Ten candidate genes, differentially expressed within the first four days post-imbibition, were identified. Five of these genes had previously been associated with awn length, heading date, yield, and spikelet length phenotypes. Two candidates were validated using Quantitative Reverse Transcription (qRT)-PCR. In addition, previously identified genes involved in hormone signaling during germination were found to be differentially expressed between a
japonica
and an
indica
line; SNPs in the promoter of
Os9BGlu33
were associated with germination index, with qRT-PCR validation. Collectively, our results are useful for future characterization of seed dormancy mechanism and crop improvement, and suggest haplotypes for further analysis that may be of use to boost PHS resistance in rice.
Dissection of the genetic pathways and mechanisms by which anther development occurs in grasses is crucial for both a basic understanding of plant development and for examining traits of agronomic ...importance such as male sterility. In rice, MULTIPLE SPOROCYTES1 (MSP1), a leucine-rich-repeat receptor kinase, plays an important role in anther development by limiting the number of sporocytes. OsTDL1a (a TPD1-like gene in rice) encodes a small protein that acts as a cofactor of MSP1 in the same regulatory pathway. In this study, we analyzed small RNA and mRNA changes in different stages of spikelets from wild-type rice, and from msp1 and ostdl1a mutants. Analysis of the small RNA data identified miRNAs demonstrating differential abundances. miR2275 was depleted in the two rice mutants; this miRNA is specifically enriched in anthers and functions to trigger the production of 24-nt phased secondary siRNAs (phasiRNAs) from PHAS loci. We observed that the 24-nt phasiRNAs as well as their precursor PHAS mRNAs were also depleted in the two mutants. An analysis of co-expression identified three Argonaute-encoding genes (OsAGO1d, OsAGO2b, and OsAGO18) that accumulate transcripts coordinately with phasiRNAs, suggesting a functional relationship. By mRNA in situ analysis, we demonstrated a strong correlation between the spatiotemporal pattern of these OsAGO transcripts and phasiRNA accumulations.
Programmed cell death is essential for the development of multicellular organisms, yet pathways of plant programmed cell death and its regulation remain elusive. Here we report that ETERNAL TAPETUM ...1, a basic helix-loop-helix transcription factor conserved in land plants, positively regulates programmed cell death in tapetal cells in rice anthers. eat1 exhibits delayed tapetal cell death and aborted pollen formation. ETERNAL TAPETUM 1 directly regulates the expression of OsAP25 and OsAP37, which encode aspartic proteases that induce programmed cell death in both yeast and plants. Expression and genetic analyses revealed that ETERNAL TAPETUM 1 acts downstream of TAPETUM DEGENERATION RETARDATION, another positive regulator of tapetal programmed cell death, and that ETERNAL TAPETUM 1 can also interact with the TAPETUM DEGENERATION RETARDATION protein. This study demonstrates that ETERNAL TAPETUM 1 promotes aspartic proteases triggering plant programmed cell death, and reveals a dynamic regulatory cascade in male reproductive development in rice.
Synthesis of lipidic components in anthers, including of the pollen exine, is essential for plant male reproductive development. Plant lipid transfer proteins (LTPs) are small, abundant lipid-binding ...proteins that have the ability to exchange lipids between membranes in vitro. However, their biological role in male reproductive development remains less understood. Here, we report the crucial role of OsC6 in regulating postmeiotic anther development in rice (Oryza sativa). Found in monocots, OsC6 belongs to a distinct clade from previously identified LTP1 and LTP2 family members found in both dicots and monocots. OsC6 expression is mainly detectable in tapetal cells and weakly in microspores from stage 9 to stage 11 of anther development. Immunological assays indicated that OsC6 is widely distributed in anther tissues such as the tapetal cytoplasm, the extracellular space between the tapetum and middle layer, and the anther locule and anther cuticle. Biochemical assays indicated that recombinant OsC6 has lipid binding activity. Moreover, plants in which OsC6 was silenced had defective development of orbicules (i.e. Ubisch bodies) and pollen exine and had reduced pollen fertility. Furthermore, additional evidence is provided that the expression of OsC6 is positively regulated by a basic helix-loop-helix transcription factor, Tapetum Degeneration Retardation (TDR). Extra granule-like structures were observed on the inner surface of the tdr tapetal layer when the expression of OsC6 was driven by the TDR promoter compared with the tdr mutant. These data suggest that OsC6 plays a crucial role in the development of lipidic orbicules and pollen exine during anther development in rice.
In male reproductive development in plants, meristemoid precursor cells possessing transient, stem cell-like features undergo cell divisions and differentiation to produce the anther, the male ...reproductive organ. The anther contains centrally positioned microsporocytes surrounded by four distinct layers of wall: the epidermis, endothecium, middle layer, and tapetum. Here, we report that the rice (Oryza sativa) basic helix-loop-helix (bHLH) protein TDR INTERACTING PROTEIN2 (TIP2) functions as a crucial switch in the meristemoid transition and differentiation during early anther development. The tip2 mutants display undifferentiated inner three anther wall layers and abort tapetai programmed cell death, causing complete male sterility. TIP2 has two paralogs in rice, TDR and EAT1, which are key regulators of tapetai programmed cell death. We revealed that TIP2 acts upstream of TDR and EAT1 and directly regulates the expression of TDR and EAT1. In addition, TIP2 can interact with TDR, indicating a role of TIP2 in later anther development. Our findings suggest that the bHLH proteins TIP2, TDR, and EAT1 play a central role in regulating differentiation, morphogenesis, and degradation of anther somatic cell layers, highlighting the role of paralogous bHLH proteins in regulating distinct steps of plant cell-type determination.
Anther cuticle and pollen exine act as protective envelopes for the male gametophyte or pollen grain, but the mechanism underlying the synthesis of these lipidic polymers remains unclear. Previously, ...a tapetum‐expressed CYP703A3, a putative cytochrome P450 fatty acid hydroxylase, was shown to be essential for male fertility in rice (Oryza sativa L.). However, the biochemical and biological roles of CYP703A3 has not been characterized. Here, we observed that cyp703a3‐2 caused by one base insertion in CYP703A3 displays defective pollen exine and anther epicuticular layer, which differs from Arabidopsis cyp703a2 in which only defective pollen exine occurs. Consistently, chemical composition assay showed that levels of cutin monomers and wax components were dramatically reduced in cyp703a3‐2 anthers. Unlike the wide range of substrates of Arabidopsis CYP703A2, CYP703A3 functions as an in‐chain hydroxylase only for a specific substrate, lauric acid, preferably generating 7‐hydroxylated lauric acid. Moreover, chromatin immunoprecipitation and expression analyses revealed that the expression of CYP703A3 is directly regulated by Tapetum Degeneration Retardation, a known regulator of tapetum PCD and pollen exine formation. Collectively, our results suggest that CYP703A3 represents a conserved and diversified biochemical pathway for in‐chain hydroxylation of lauric acid required for the development of male organ in higher plants.
In higher plants, timely degradation of tapetai cells, the innermost sporophytic cells of the anther wall layer, is a prerequisite for the development of viable pollen grains. However, relatively ...little is known about the mechanism underlying programmed tapetai cell development and degradation. Here, we report a key regulator in monocot rice (Oryza sauva), PERSISTANT TAPETAL CELLI (PTCl), which controls programmed tapetal development and functional pollen formation. The evolutionary significance of PTCl was revealed by partial genetic complementation of the homologous mutation MALE STERILITY1 (MSI) in the dicot Arabidopsis (Arabidopsis thaliana). PTCl encodes a PHD-finger (for plant homeodomain) protein, which is expressed specifically in tapetai cells and microspores during anther development in stages 8 and 9, when the wild-type tapetai cells initiate a typical apoptosis-like cell death. Even though ptcl mutants show phenotypic similarity to msl in a lack of tapetai DNA fragmentation, delayed tapetai degeneration, as well as abnormal pollen wall formation and aborted microspore development, the ptcl mutant displays a previously unreported phenotype of uncontrolled tapetai proliferation and subsequent commencement of necrosis-like tapetai death. Microarray analysis indicated that 2,417 tapetum-and microspore-expressed genes, which are principally associated with tapetai development, degeneration, and pollen wall formation, had changed expression in ptcl anthers. Moreover, the regulatory role of PTCl in anther development was revealed by comparison with MS2 and other rice anther developmental regulators. These findings suggest a diversified and conserved switch of PTCl /MSI in regulating programmed male reproductive development in both dicots and monocots, which provides new insights in plant anther development.
Acyl-CoA Synthetase (ACOS) is one of the enzymes activating fatty acids for various metabolic functions in plants. Here, we show that OsACOS12, an orthologue of Arabidopsis ACOS5 in rice, is crucial ...for rice fertility. Similar to acos5, osaocs12 mutant had no mature pollen. But unlike acos5, osaocs12 produced defective anthers lacking cutin and Ubisch bodies on the epidermal and inner surfaces, respectively, and delayed programmed cell death (PCD)-induced tapetum degradation. Those phenotypic changes were evident at stage 10, during which OsACOS12 had its maximum expression in tapetal cells and microspores. Chemical analysis revealed that the levels of anther cuticular lipid components (wax and cutin monomers) were significantly reduced in osaocs12, while the expression levels of three known lipid biosynthetic genes were unchanged. Recombinant OsACOS12 enzyme was shown to catalyze the conversion of C18:1 fatty acid to C18:1 CoA in vitro. Phylogenetic analysis indicated that OsACOS12 is an ancient and conserved enzyme associated with the plant’s colonization to earth. Collectively, our study suggests that OsACOS12 is an ancient enzyme participating in a conserved metabolic pathway for diversified biochemical functions to secure male reproduction in plants.
The rice (Oryza sativa) floral homeotic C-class gene, MADS3, was previously shown to be required for stamen identity determination during early flower development. Here, we describe a role for MADS3 ...in regulating late anther development and pollen formation. Consistent with this role, MADS3 is highly expressed in the tapetum and microspores during late anther development, and a newly identified MADS3 mutant alíele, mads3-4, displays defective anther walls, aborted microspores, and complete male sterility. During late anther development, mads3-4 exhibits oxidative stress-related phenotypes. Microarray analysis revealed expression level changes in many genes in mads3-4 anthers. Some of these genes encode proteins involved in reactive oxygen species (ROS) homeostasis; among them is MT-1-4b, which encodes a type 1 small Cys-rich and metal binding protein. In vivo and in vitro assays showed that MADS3 is associated with the promoter of MT-1-4b, and recombinant MT-1-4b has Superoxide anión and hydroxyl radical scavenging activity. Reducing the expression of MT-1-4b causes decreased pollen fertility and an increased level of superoxide anion in transgenic plants. Our findings suggest that MADS3 is a key transcriptional regulator that functions in rice male reproductive development, at least in part, by modulating ROS levels through MT-1-4b.
In flowering plants, formation of the haploid male gametophytes in anthers requires the interaction between reproductive cells and the neighboring somatic cells, yet the underlying mechanism remains ...poorly understood. Here, we reveal the crucial role of a fasciclin glycoprotein, MICROSPORE AND TAPETUM REGULATOR1 (MTR1), in controlling the development of sporophytic and reproductive cells in rice (Oryza sativa). MTR1 is specifically expressed in the male reproductive cells, yet its mutant exhibits defects in both tapetum and microspore development, causing complete male sterility. We also demonstrate that the fasciclin domains, N-glycolation, and N-terminal signal peptide-mediated plasma membrane localization of MTR1 are required for normal anther development and pollen fertility. Our findings show that rice male reproductive cells secrete the MTR1 protein to control the development of reproductive cells and their adjacent somatic cells, thus providing novel insights into the mechanism of plant male reproductive development.
Display omitted
► The fasciclin glycoprotein MTR1 regulates male reproductive development in rice ► Rice male reproductive cells secrete MTR1 to control somatic tapetum development
Tan et al. identify a plasma-membrane-localized fasciclin glycoprotein, MTR1, which is required for normal anther development and pollen fertility in rice. MTR1 is secreted by male reproductive cells and coordinates the development of reproductive cells and their adjacent somatic cells.
PDF
