The IPGA1-ANGUSTIFOLIA module regulates microtubule organisation and pavement cell shape in Arabidopsis.

Plant cells continuously experience mechanical stress resulting from the cell wall that bears internal turgor pressure. Cortical microtubules align with the predicted maximal tensile stress direction to guide cellulose biosynthesis and therefore results in cell wall reinforcement. We have previously identified Increased Petal Growth Anisotropy (IPGA1) as a putative microtubule-associated protein in Arabidopsis, but the function of IPGA1 remains unclear. Here, using the Arabidopsis cotyledon pavement cell as a model, we demonstrated that IPGA1 forms protein granules and interacts with ANGUSTIFOLIA (AN) to cooperatively regulate microtubule organisation in response to stress. Application of mechanical perturbations, such as cell ablation, led to microtubule reorganisation into aligned arrays in wild-type cells. This microtubule response to stress was enhanced in the IPGA1 loss-of-function mutant. Mechanical perturbations promoted the formation of IPGA1 granules on microtubules. We further showed that IPGA1 physically interacted with AN both in vitro and on microtubules. The ipga1 mutant alleles exhibited reduced interdigitated growth of pavement cells, with smooth shape. IPGA1 and AN had a genetic interaction in regulating pavement cell shape. Furthermore, IPGA1 genetically and physically interacted with the microtubule-severing enzyme KATANIN. We propose that the IPGA1-AN module regulates microtubule organisation and pavement cell shape.

Auxin regulates source-sink carbohydrate partitioning and reproductive organ development in rice.

Carbohydrate partitioning between the source and sink tissues plays an important role in regulating plant growth and development. However, the molecular mechanisms regulating this process remain poorly understood. In this study, we show that elevated auxin levels in the rice dao mutant cause increased accumulation of sucrose in the photosynthetic leaves but reduced sucrose content in the reproductive organs (particularly in the lodicules, anthers, and ovaries), leading to closed spikelets, indehiscent anthers, and parthenocarpic seeds. RNA sequencing analysis revealed that the expression of AUXIN RESPONSE FACTOR 18 (OsARF18) and OsARF2 is significantly up- and down-regulated, respectively, in the lodicule of dao mutant. Overexpression of OsARF18 or knocking out of OsARF2 phenocopies the dao mutant. We demonstrate that OsARF2 regulates the expression of OsSUT1 through direct binding to the sugar-responsive elements (SuREs) in the OsSUT1 promoter and that OsARF18 represses the expression of OsARF2 and OsSUT1 via direct binding to the auxin-responsive element (AuxRE) or SuRE in their promoters, respectively. Furthermore, overexpression of OsSUT1 in the dao and Osarf2 mutant backgrounds could largely rescue the spikelets' opening and seed-setting defects. Collectively, our results reveal an auxin signaling cascade regulating source-sink carbohydrate partitioning and reproductive organ development 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.

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.

Rice albino 1, encoding a glycyl-tRNA synthetase, is involved in chloroplast development and establishment of the plastidic ribosome system in rice.

The chloroplast is an important organelle that performs photosynthesis as well as biosynthesis and storage of many metabolites. Aminoacyl-tRNA synthetases (aaRSs) are key enzymes in protein synthesis. However, the relationship between chloroplast development and aaRSs still remains unclear. In this study, we isolated a rice albino 1 (ra1) mutant through methane sulfonate (EMS) mutagenesis of rice japonica cultivar Ningjing 4 (Oryza sativa L.), which displayed albinic leaves in seedling stage due to abnormal chloroplast development. Compared with wild type (WT), ra1 showed significantly decreased levels of chlorophylls (Chl) and carotenoids (Car) in 2-week-old seedlings, which also showed obvious plastidic structural defects including abnormal thylakoid membrane structures and more osmiophilic particles. These defects caused albino phenotypes in seedlings. Map-based cloning revealed that RA1 gene encodes a glycyl-tRNA synthetase (GlyRS), which was confirmed by genetic complementation and knockout by Crispr/Cas9 technology. Sequence analysis showed that a single base mutation (T to A) occurred in the sixth exon of RA1 and resulted in a change from Isoleucine (Ile) to Lysine (Lys). Real-time PCR analyses showed that RA1 expression levels were constitutive in most tissues, but most abundant in the leaves and stems. By transient expression in Nicotiana benthamiana, we found that RA1 protein was localized in the chloroplast. Expression levels of chlorophyll biosynthesis and plastid development related genes were disordered in the ra1 mutant. RNA analysis revealed biogenesis of chloroplast rRNAs was abnormal in ra1. Meanwhile, western blotting showed that synthesis of proteins associated with plastid development was significantly repressed. These results suggest that RA1 is involved in early chloroplast development and establishment of the plastidic ribosome system in rice.

A selfish genetic element confers non-Mendelian inheritance in rice.

Selfish genetic elements are pervasive in eukaryote genomes, but their role remains controversial. We show that qHMS7, a major quantitative genetic locus for hybrid male sterility between wild rice (Oryza meridionalis) and Asian cultivated rice (O. sativa), contains two tightly linked genes [Open Reading Frame 2 (ORF2) and ORF3]. ORF2 encodes a toxic genetic element that aborts pollen in a sporophytic manner, whereas ORF3 encodes an antidote that protects pollen in a gametophytic manner. Pollens lacking ORF3 are selectively eliminated, leading to segregation distortion in the progeny. Analysis of the genetic sequence suggests that ORF3 arose first, followed by gradual functionalization of ORF2 Furthermore, this toxin-antidote system may have promoted the differentiation and/or maintained the genome stability of wild and cultivated rice.