Earlier Degraded Tapetum1 (EDT1) Encodes an ATP-Citrate Lyase Required for Tapetum Programmed Cell Death.

In flowering plants, the tapetum cells in anthers undergo programmed cell death (PCD) at the late meiotic stage, providing nutrients for further development of microspores, including the formation of the pollen wall. However, the molecular basis of tapetum PCD remains elusive. Here we report a tapetum PCD-related mutant in rice (Oryza sativa), earlier degraded tapetum 1 (edt1), that shows complete pollen abortion associated with earlier-than-programmed tapetum cell death. EDT1 encodes a subunit of ATP-citrate lyase (ACL), and is specifically expressed in the tapetum of anthers. EDT1 localized in both the nucleus and the cytoplasm as observed in rice protoplast transient assays. We demonstrated that the A and B subunits of ACL interacted with each other and might function as a heteromultimer in the cytoplasm. EDT1 catalyzes the critical steps in cytosolic acetyl-CoA synthesis. Our data indicated a decrease in ATP level, energy charge, and fatty acid content in mutant edt1 anthers. In addition, the genes encoding secretory proteases or lipid transporters, and the transcription factors known to regulate PCD, were downregulated. Our results demonstrate that the timing of tapetum PCD must be tightly regulated for successful pollen development, and that EDT1 is involved in the tapetum PCD process. This study furthers our understanding of the molecular basis of pollen fertility and fecundity in rice and may also be relevant to other flowering plants.

A GARP transcription factor anther dehiscence defected 1 (OsADD1) regulates rice anther dehiscence.

Anther dehiscence, one of the essential steps in pollination and double fertilization, is regulated by a complex signaling pathway encompassing hormones and environmental factors. However, key components underlying the signaling pathway that regulate anther dehiscence remain largely elusive. Here, we isolated a rice mutant anther dehiscence defected 1 (Osadd1) that exhibited defects in anther dehiscence and glume open. Map-based cloning revealed that OsADD1 encoded a GARP (Golden2, ARR-B and Psr1) transcription factor. Sequence analysis showed that a single base deletion in Osadd1 mutant resulted in pre-termination of the GARP domain. OsADD1 was constitutively expressed in various tissues, with more abundance in the panicles. The major genes associated with anther dehiscence were affected in the Osadd1 mutant, and the expression level of the cellulose synthase-like D sub-family 4 (OsCSLD4) was significantly decreased. We demonstrate that OsADD1 regulated the expression of OsCSLD4 by binding to its promoter, and affects rice anther dehiscence.

OsSRF8 interacts with OsINP1 and OsDAF1 to regulate pollen aperture formation in rice.

In higher plants, mature male gametophytes have distinct apertures. After pollination, pollen grains germinate, and a pollen tube grows from the aperture to deliver sperm cells to the embryo sac, completing fertilization. In rice, the pollen aperture has a single-pore structure with a collar-like annulus and a plug-like operculum. A crucial step in aperture development is the formation of aperture plasma membrane protrusion (APMP) at the distal polar region of the microspore during the late tetrad stage. Previous studies identified OsINP1 and OsDAF1 as essential regulators of APMP and pollen aperture formation in rice, but their precise molecular mechanisms remain unclear. We demonstrate that the Poaceae-specific OsSRF8 gene, encoding a STRUBBELIG-receptor family 8 protein, is essential for pollen aperture formation in Oryza sativa. Mutants lacking functional OsSRF8 exhibit defects in APMP and pollen aperture formation, like loss-of-function OsINP1 mutants. OsSRF8 is specifically expressed during early anther development and initially diffusely distributed in the microsporocytes. At the tetrad stage, OsSRF8 is recruited by OsINP1 to the pre-aperture region through direct protein-protein interaction, promoting APMP formation. The OsSRF8-OsINP1 complex then recruits OsDAF1 to the APMP site to co-regulate annulus formation. Our findings provide insights into the mechanisms controlling pollen aperture formation in cereal species.

A toxin-antidote system contributes to interspecific reproductive isolation in rice.

Breakdown of reproductive isolation facilitates flow of useful trait genes into crop plants from their wild relatives. Hybrid sterility, a major form of reproductive isolation exists between cultivated rice (Oryza sativa) and wild rice (O. meridionalis, Mer). Here, we report the cloning of qHMS1, a quantitative trait locus controlling hybrid male sterility between these two species. Like qHMS7, another locus we cloned previously, qHMS1 encodes a toxin-antidote system, but differs in the encoded proteins, their evolutionary origin, and action time point during pollen development. In plants heterozygous at qHMS1, ~ 50% of pollens carrying qHMS1-D (an allele from cultivated rice) are selectively killed. In plants heterozygous at both qHMS1 and qHMS7, ~ 75% pollens without co-presence of qHMS1-Mer and qHMS7-D are selectively killed, indicating that the antidotes function in a toxin-dependent manner. Our results indicate that different toxin-antidote systems provide stacked reproductive isolation for maintaining species identity and shed light on breakdown of hybrid male sterility.

OsPHS1 is required for both male and female gamete development in rice.

Meiosis plays an essential role in the production of male and female gametes. Extensive studies have elucidated that homologous chromosome association and pairing are essential for crossing-over and recombination of chromosomal segments. However, the molecular mechanism of chromosome recognition and pairing remains elusive. Here, we identified a rice male-female sterility mutant plant. Cytological observations showed that the development of both pollen and embryo sacs of the mutant were abnormal due to defects in homologous chromosome recognition and pairing during prophase I. Map-based cloning revealed that Os06g0473000 encoding a poor homologous synapsis 1 (PHS1) protein is the candidate target gene, which was confirmed by knockout using CRISPR/Cas9 technology. Sequence analysis revealed a single base mutation (G > A) involving the junction of the fourth exon and intron of OsPHS1, which is predicted to alter splicing, resulting in an Osphs1 mutant. Expression pattern analysis indicated that OsPHS1 expression levels were mainly expressed in panicles at the beginning of meiosis. Subcellular localization analysis demonstrated that the OsPHS1 protein is situated in the nucleus and cytoplasm. Taken together, our results suggest an important role for OsPHS1 in homologous chromosome pairing in both male and female gametogenesis in rice.

OsMFS1/OsHOP2 Complex Participates in Rice Male and Female Development.

Meiosis plays an essential role in the production of gametes and genetic diversity of posterities. The normal double-strand break (DSB) repair is vital to homologous recombination (HR) and occurrence of DNA fragment exchange, but the underlying molecular mechanism remain elusive. Here, we characterized a completely sterile Osmfs1 (male and female sterility 1) mutant which has its pollen and embryo sacs both aborted at the reproductive stage due to severe chromosome defection. Map-based cloning revealed that the OsMFS1 encodes a meiotic coiled-coil protein, and it is responsible for DSB repairing that acts as an important cofactor to stimulate the single strand invasion. Expression pattern analyses showed the OsMFS1 was preferentially expressed in meiosis stage. Subcellular localization analysis of OsMFS1 revealed its association with the nucleus exclusively. In addition, a yeast two-hybrid (Y2H) and pull-down assay showed that OsMFS1 could physically interact with OsHOP2 protein to form a stable complex to ensure faithful homologous recombination. Taken together, our results indicated that OsMFS1 is indispensable to the normal development of anther and embryo sacs in rice.

Small Grain and Dwarf 2, encoding an HD-Zip II family transcription factor, regulates plant development by modulating gibberellin biosynthesis in rice.

Homeodomain leucine zipper (HD-Zip) proteins are transcription factors that regulate plant development. Bioactive gibberellin (GA) is a key endogenous hormone that participates in plant growth. However, the relationship between HD-Zip genes and modulation of GA biosynthesis in rice remains elusive. Here, we identified a rice mutant, designated as small grain and dwarf 2 (sgd2), which had reduced height and grain size compared with the wild type. Cytological observations indicated that the defective phenotype was mainly due to decreased cell length. Map-based cloning and complementation tests demonstrated that a 9 bp deletion in a homeodomain leucine zipper (HD-Zip) II family transcription factor was responsible for the sgd2 mutant phenotype. Expression of SGD2 was pronounced in developing panicles, and its protein was localized in nucleus. Luciferase reporter system and transactivation assays in yeast suggested that SGD2 functioned as a transcriptional repressor. High performance liquid chromatography assays showed that the endogenous GA1 level in the sgd2 mutant was dramatically decreased, and exogenous GA3 recovered the second leaf sheath to normal length. Results of qRT-PCR showed that the expression levels of genes positively regulating GA-biosynthesis were mostly down-regulated in the mutant. Our data identified the role of an HD-Zip transcription factor that affects rice plant development by modulating gibberellin biosynthesis.