Regulation by distinct MYB transcription factors defines the roles of OsCYP86A9 in anther development and root suberin deposition.

Cytochrome P450 proteins (CYPs) play critical roles in plant development and adaptation to fluctuating environments. Previous reports have shown that CYP86A proteins are involved in the biosynthesis of suberin and cutin in Arabidopsis. However, the functions of these proteins in rice remain obscure. In this study, a rice mutant with incomplete male sterility was identified. Cytological analyses revealed that this mutant was defective in anther development. Cloning of the mutant gene indicated that the responsible mutation was on OsCYP86A9. OsMYB80 is a core transcription factor in the regulation of rice anther development. The expression of OsCYP86A9 was abolished in the anther of osmyb80 mutant. In vivo and in vitro experiments showed that OsMYB80 binds to the MYB-binding motifs in OsCYP86A9 promoter region and regulates its expression. Furthermore, the oscyp86a9 mutant exhibited an impaired suberin deposition in the root, and was more susceptible to drought stress. Interestingly, genetic and biochemical analyses revealed that OsCYP86A9 expression was regulated in the root by certain MYB transcription factors other than OsMYB80. Moreover, mutations in the MYB genes that regulate OsCYP86A9 expression in the root did not impair the male fertility of the plant. Taken together, these findings revealed the critical roles of OsCYP86A9 in plant development and proposed that OsCYP86A9 functions in anther development and root suberin formation via two distinct tissue-specific regulatory pathways.

Ornithine δ-aminotransferase OsOAT is critical for male fertility and cold tolerance during rice plant development.

Cold stress is a major factor limiting the production and geographical distribution of rice (Oryza sativa) varieties. However, the molecular mechanisms underlying cold tolerance remain to be elucidated. Here, we report that ornithine δ-aminotransferase (OsOAT) contributes to cold tolerance during the vegetative and reproductive development of rice. osoat mutant was identified as a temperature-sensitive male sterile mutant with deformed floral organs and seedlings sensitive to cold stress. Comparative transcriptome analysis showed that OsOAT mutation and cold treatment of the wild-type plant led to similar changes in the global gene expression profiles in anthers. OsOAT genes in indica rice Huanghuazhan (HHZ) and japonica rice Wuyungeng (WYG) are different in gene structure and response to cold. OsOAT is cold-inducible in WYG but cold-irresponsive in HHZ. Further studies showed that indica varieties carry both WYG-type and HHZ-type OsOAT, whereas japonica varieties mostly carry WYG-type OsOAT. Cultivars carrying HHZ-type OsOAT are mainly distributed in low-latitude regions, whereas varieties carrying WYG-type OsOAT are distributed in both low- and high-latitude regions. Moreover, indica varieties carrying WYG-type OsOAT generally have higher seed-setting rates than those carrying HHZ-type OsOAT under cold stress at reproductive stage, highlighting the favorable selection for WYG-type OsOAT during domestication and breeding to cope with low temperatures.

The ribosomal protein P0A is required for embryo development in rice.

BACKGROUND: The P-stalk is a conserved and vital structural element of ribosome. The eukaryotic P-stalk exists as a P0-(P1-P2)2 pentameric complex, in which P0 function as a base structure for incorporating the stalk onto 60S pre-ribosome. Prior studies have suggested that P0 genes are indispensable for survival in yeast and animals. However, the functions of P0 genes in plants remain elusive. RESULTS: In the present study, we show that rice has three P0 genes predicted to encode highly conserved proteins OsP0A, OsP0B and OsP0C. All of these P0 proteins were localized both in cytoplasm and nucleus, and all interacted with OsP1. Intriguingly, the transcripts of OsP0A presented more than 90% of the total P0 transcripts. Moreover, knockout of OsP0A led to embryo lethality, while single or double knockout of OsP0B and OsP0C did not show any visible defects in rice. The genomic DNA of OsP0A could well complement the lethal phenotypes of osp0a mutant. Finally, sequence and syntenic analyses revealed that OsP0C evolved from OsP0A, and that duplication of genomic fragment harboring OsP0C further gave birth to OsP0B, and both of these duplication events might happen prior to the differentiation of indica and japonica subspecies in rice ancestor. CONCLUSION: These data suggested that OsP0A functions as the predominant P0 gene, playing an essential role in embryo development in rice. Our findings highlighted the importance of P0 genes in plant development.

OsMYB80 Regulates Anther Development and Pollen Fertility by Targeting Multiple Biological Pathways.

Pollen development is critical to the reproductive success of flowering plants, but how it is regulated is not well understood. Here, we isolated two allelic male-sterile mutants of OsMYB80 and investigated how OsMYB80 regulates male fertility in rice. OsMYB80 was barely expressed in tissues other than anthers, where it initiated the expression during meiosis, reached the peak at the tetrad-releasing stage and then quickly declined afterward. The osmyb80 mutants exhibited premature tapetum cell death, lack of Ubisch bodies, no exine and microspore degeneration. To understand how OsMYB80 regulates anther development, RNA-seq analysis was conducted to identify genes differentially regulated by OsMYB80 in rice anthers. In addition, DNA affinity purification sequencing (DAP-seq) analysis was performed to identify DNA fragments interacting with OsMYB80 in vitro. Overlap of the genes identified by RNA-seq and DAP-seq revealed 188 genes that were differentially regulated by OsMYB80 and also carried an OsMYB80-interacting DNA element in the promoter. Ten of these promoter elements were randomly selected for gel shift assay and yeast one-hybrid assay, and all showed OsMYB80 binding. The 10 promoters also showed OsMYB80-dependent induction when co-expressed in rice protoplast. Functional annotation of the 188 genes suggested that OsMYB80 regulates male fertility by directly targeting multiple biological processes. The identification of these genes significantly enriched the gene networks governing anther development and provided much new information for the understanding of pollen development and male fertility.

The plant-specific ABERRANT GAMETOGENESIS 1 gene is essential for meiosis in rice.

Meiotic recombination plays a central role in maintaining genome stability and increasing genetic diversity. Although meiotic progression and core components are widely conserved across kingdoms, significant differences remain among species. Here we identify a rice gene ABERRANT GAMETOGENESIS 1 (AGG1) that controls both male and female gametogenesis. Cytological and immunostaining analysis showed that in the osagg1 mutant the early recombination processes and synapsis occurred normally, but the chiasma number was dramatically reduced. Moreover, OsAGG1 was found to interact with ZMM proteins OsHEI10, OsZIP4, and OsMSH5. These results suggested that OsAGG1 plays an important role in crossover formation. Phylogenetic analysis showed that OsAGG1 is a plant-specific protein with a highly conserved N-terminal region. Further genetic and protein interaction analyses revealed that the conserved N-terminus was essential for the function of the OsAGG1 protein. Overall, our work demonstrates that OsAGG1 is a novel and critical component in rice meiotic crossover formation, expanding our understanding of meiotic progression. This study identified a plant-specific gene ABERRANT GAMETOGENESIS 1 that is required for meiotic crossover formation in rice. The conserved N-terminus of the AGG1 protein was found to be essential for its function.

GDSL esterase/lipases OsGELP34 and OsGELP110/OsGELP115 are essential for rice pollen development.

Pollen exine contains complex biopolymers of aliphatic lipids and phenolics. Abnormal development of pollen exine often leads to plant sterility. Molecular mechanisms regulating exine formation have been studied extensively but remain ambiguous. Here we report the analyses of three GDSL esterase/lipase protein genes, OsGELP34, OsGELP110, and OsGELP115, for rice exine formation. OsGELP34 was identified by cloning of a male sterile mutant gene. OsGELP34 encodes an endoplasmic reticulum protein and was mainly expressed in anthers during pollen exine formation. osgelp34 mutant displayed abnormal exine and altered expression of a number of key genes required for pollen development. OsGELP110 was previously identified as a gene differentially expressed in meiotic anthers. OsGELP110 was most homologous to OsGELP115, and the two genes showed similar gene expression patterns. Both OsGELP110 and OsGELP115 proteins were localized in peroxisomes. Individual knockout of OsGELP110 and OsGELP115 did not affect the plant fertility, but double knockout of both genes altered the exine structure and rendered the plant male sterile. OsGELP34 is distant from OsGELP110 and OsGELP115 in sequence, and osgelp34 and osgelp110/osgelp115 mutants were different in anther morphology despite both were male sterile. These results suggested that OsGELP34 and OsGELP110/OsGELP115 catalyze different compounds for pollen exine development.

Lectin receptor kinase OsLecRK-S.7 is required for pollen development and male fertility.

Pollen grains are covered by exine that protects the pollen from stress and facilitates pollination. Here we isolated a male sterile mutant s13283 in rice exhibiting aborted pollen with abnormal exine and defective aperture. The mutant gene encodes a novel plasma membrane-localized legume-lectin receptor kinase that we named OsLecRK-S.7. OsLecRK-S.7 was expressed at different levels in all tested tissues and throughout anther development. In vitro kinase assay showed OsLecRK-S.7 capable of autophosporylation. Mutation in s13283 (E560K) and mutation of the conserved ATP binding site (K418E) both knocked out the kinase activity. Mass spectrometry showed Thr376 , Ser378 , Thr386 , Thr403 , and Thr657 to be the autophosphorylation sites. Mutation of individual autophosphorylation site affected the in vitro kinase activity to different degrees, but did not abolish the gene function in fertility complementation. oslecrk-s.7 mutant plant overexpressing OsLecRK-S.7 recovered male fertility but showed severe growth retardation with reduced number of tillers, and these phenotypes were abolished by E560K or K418E mutation. The results indicated that OsLecRK-S.7 was a key regulator of pollen development.

Construction of a male sterility system for hybrid rice breeding and seed production using a nuclear male sterility gene.

The breeding and large-scale adoption of hybrid seeds is an important achievement in agriculture. Rice hybrid seed production uses cytoplasmic male sterile lines or photoperiod/thermo-sensitive genic male sterile lines (PTGMS) as female parent. Cytoplasmic male sterile lines are propagated via cross-pollination by corresponding maintainer lines, whereas PTGMS lines are propagated via self-pollination under environmental conditions restoring male fertility. Despite huge successes, both systems have their intrinsic drawbacks. Here, we constructed a rice male sterility system using a nuclear gene named Oryza sativa No Pollen 1 (OsNP1). OsNP1 encodes a putative glucose-methanol-choline oxidoreductase regulating tapetum degeneration and pollen exine formation; it is specifically expressed in the tapetum and miscrospores. The osnp1 mutant plant displays normal vegetative growth but complete male sterility insensitive to environmental conditions. OsNP1 was coupled with an α-amylase gene to devitalize transgenic pollen and the red fluorescence protein (DsRed) gene to mark transgenic seed and transformed into the osnp1 mutant. Self-pollination of the transgenic plant carrying a single hemizygous transgene produced nontransgenic male sterile and transgenic fertile seeds in 1:1 ratio that can be sorted out based on the red fluorescence coded by DsRed Cross-pollination of the fertile transgenic plants to the nontransgenic male sterile plants propagated the male sterile seeds of high purity. The male sterile line was crossed with ∼1,200 individual rice germplasms available. Approximately 85% of the F1s outperformed their parents in per plant yield, and 10% out-yielded the best local cultivars, indicating that the technology is promising in hybrid rice breeding and production.

The Arabidopsis ceramidase AtACER functions in disease resistance and salt tolerance.

Ceramidases hydrolyze ceramide into sphingosine and fatty acids. In mammals, ceramidases function as key regulators of sphingolipid homeostasis, but little is known about their roles in plants. Here we characterize the Arabidopsis ceramidase AtACER, a homolog of human alkaline ceramidases. The acer-1 T-DNA insertion mutant has pleiotropic phenotypes, including reduction of leaf size, dwarfing and an irregular wax layer, compared with wild-type plants. Quantitative sphingolipid profiling showed that acer-1 mutants and the artificial microRNA-mediated silenced line amiR-ACER-1 have high ceramide levels and decreased long chain bases. AtACER localizes predominantly to the endoplasmic reticulum, and partially to the Golgi complex. Furthermore, we found that acer-1 mutants and AtACER RNAi lines showed increased sensitivity to salt stress, and lines overexpressing AtACER showed increased tolerance to salt stress. Reduction of AtACER also increased plant susceptibility to Pseudomonas syringae. Our data highlight the key biological functions of ceramidases in biotic and abiotic stresses in plants.

Dual roles for CND1 in maintenance of nuclear and chloroplast genome stability in plants.

The coordination of chloroplast and nuclear genome status is critical for plant cell function. Here, we report that Arabidopsis CHLOROPLAST AND NUCLEUS DUAL-LOCALIZED PROTEIN 1 (CND1) maintains genome stability in the chloroplast and the nucleus. CND1 localizes to both compartments, and complete loss of CND1 results in embryo lethality. Partial loss of CND1 disturbs nuclear cell-cycle progression and photosynthetic activity. CND1 binds to nuclear pre-replication complexes and DNA replication origins and regulates nuclear genome stability. In chloroplasts, CND1 interacts with and facilitates binding of the regulator of chloroplast genome stability WHY1 to chloroplast DNA. The defects in nuclear cell-cycle progression and photosynthesis of cnd1 mutants are respectively rescued by compartment-restricted CND1 localization. Light promotes the association of CND1 with HSP90 and its import into chloroplasts. This study provides a paradigm of the convergence of genome status across organelles to coordinately regulate cell cycle to control plant growth and development.

N6-methyladenosine RNA modification regulates photosynthesis during photodamage in plants.

N6-methyladenosine (m6A) modification of mRNAs affects many biological processes. However, the function of m6A in plant photosynthesis remains unknown. Here, we demonstrate that m6A modification is crucial for photosynthesis during photodamage caused by high light stress in plants. The m6A modification levels of numerous photosynthesis-related transcripts are changed after high light stress. We determine that the Arabidopsis m6A writer VIRILIZER (VIR) positively regulates photosynthesis, as its genetic inactivation drastically lowers photosynthetic activity and photosystem protein abundance under high light conditions. The m6A levels of numerous photosynthesis-related transcripts decrease in vir mutants, extensively reducing their transcript and translation levels, as revealed by multi-omics analyses. We demonstrate that VIR associates with the transcripts of genes encoding proteins with functions related to photoprotection (such as HHL1, MPH1, and STN8) and their regulatory proteins (such as regulators of transcript stability and translation), promoting their m6A modification and maintaining their stability and translation efficiency. This study thus reveals an important mechanism for m6A-dependent maintenance of photosynthetic efficiency in plants under high light stress conditions.

Ceramide-Induced Cell Death Depends on Calcium and Caspase-Like Activity in Rice.

Ceramide sphingolipids are major components of membranes. C2 and C6 ceramides induce programmed cell death (PCD) in animals and plants, and we previously showed that C2 and C6 ceramides induce PCD in rice (Oryza sativa) protoplasts. However, the mechanistic link between sphingolipids and PCD in rice remains unclear. Here, we observed that calcium levels increased rapidly after ceramide treatment. Moreover, the calcium channel inhibitor LaCl3 and the intracellular calcium chelator acetoxymethyl-1, 2-bis (2-aminophenoxy) ethic acid (BAPTA-AM) inhibited this calcium increase and prevented ceramide-induced PCD. Moreover, caspase-3-like protease activity increased significantly in C6 ceramide-treated protoplasts, and a caspase-specific inhibitor prevented C6 ceramide-induced cell death. We also detected the other typical PCD events including ATP loss. DIDS (4, 49-diisothiocyanatostilbene- 2, 29-disulfonic acid), an inhibitor of voltage-dependent anion channels (VDACs), decreased C6 ceramide-induced cell death. Together, this evidence suggests that mitochondria played an important role in C6 ceramide-induced PCD.

The ATP-binding cassette (ABC) transporter OsABCG3 is essential for pollen development in rice.

BACKGROUND: The pollen wall, which protects male gametophyte against various stresses and facilitates pollination, is essential for successful reproduction in flowering plants. The pollen wall consists of gametophyte-derived intine and sporophyte-derived exine. From outside to inside of exine are tectum, bacula, nexine I and nexine II layers. How these structural layers are formed has been under extensive studies, but the molecular mechanisms remain obscure. RESULTS: Here we identified two osabcg3 allelic mutants and demonstrated that OsABCG3 was required for pollen development in rice. OsABCG3 encodes a half-size ABCG transporter localized on the plasma membrane. It was mainly expressed in anther when exine started to form. Loss-function of OsABCG3 caused abnormal degradation of the tapetum. The mutant pollen lacked the nexine II and intine layers, and shriveled without cytoplasm. The expression of some genes required for pollen wall formation was examined in osabcg3 mutants. The mutation did not alter the expression of the regulatory genes and lipid metabolism genes, but altered the expression of lipid transport genes. CONCLUSIONS: Base on the genetic and cytological analyses, OsABCG3 was proposed to transport the tapetum-produced materials essential for pollen wall formation. This study provided a new perspective to the genetic regulation of pollen wall development.

An ABC transporter, OsABCG26, is required for anther cuticle and pollen exine formation and pollen-pistil interactions in rice.

Wax, cutin and sporopollenin are essential components for the formation of the anther cuticle and the pollen exine, respectively. Their lipid precursors are synthesized by secretory tapetal cells and transported to the anther and microspore surface for deposition. However, the molecular mechanisms involved in the formation of the anther cuticle and pollen exine are poorly understood in rice. Here, we characterized a rice male sterile mutant osabcg26. Molecular cloning and sequence analysis revealed a point mutation in the gene encoding an ATP binding cassette transporter G26 (OsABCG26). OsABCG26 was specifically expressed in the anther and pistil. Cytological analysis revealed defects in tapetal cells, lipidic Ubisch bodies, pollen exine, and anther cuticle in the osabcg26 mutant. Expression of some key genes involved in lipid metabolism and transport, such as UDT1, WDA1, CYP704B2, OsABCG15, OsC4 and OsC6, was significantly altered in osabcg26 anther, possibly due to a disturbance in the homeostasis of anther lipid metabolism and transport. Additionally, wild-type pollen tubes showed a growth defect in osabcg26 pistils, leading to low seed setting in osabcg26 cross-pollinated with the wild-type pollen. These results indicated that OsABCG26 plays an important role in anther cuticle and pollen exine formation and pollen-pistil interactions in rice.