Controlling diurnal flower-opening time by manipulating the jasmonate pathway accelerates development of indica-japonica hybrid rice breeding.

Inter-subspecific indica-japonica hybrid rice (Oryza sativa) has the potential for increased yields over traditional indica intra-subspecies hybrid rice, but limited yield of F1 hybrid seed production (FHSP) hinders the development of indica-japonica hybrid rice breeding. Diurnal flower-opening time (DFOT) divergence between indica and japonica rice has been a major contributing factor to this issue, but few DFOT genes have been cloned. Here, we found that manipulating the expression of jasmonate (JA) pathway genes can effectively modulate DFOT to improve the yield of FHSP in rice. Treating japonica cultivar Zhonghua 11 (ZH11) with methyl jasmonate (MeJA) substantially advanced DFOT. Furthermore, overexpressing the JA biosynthesis gene OPDA REDUCTASE 7 (OsOPR7) and knocking out the JA inactivation gene CHILLING TOLERANCE 1 (OsHAN1) in ZH11 advanced DFOT by 1- and 2-h respectively; and knockout of the JA signal suppressor genes JASMONATE ZIM-DOMAIN PROTEIN 7 (OsJAZ7) and OsJAZ9 resulted in 50-min and 1.5-h earlier DFOT respectively. The yields of FHSP using japonica male-sterile lines GAZS with manipulated JA pathway genes were significantly higher than that of GAZS wildtype. Transcriptome analysis, cytological observations, measurements of elastic modulus and determination of cell wall components indicated that the JA pathway could affect the loosening of the lodicule cell walls by regulating their composition through controlling sugar metabolism, which in turn influences DFOT. This research has vital implications for breeding japonica rice cultivars with early DFOT to facilitate indica-japonica hybrid rice breeding.

An E3 ubiquitin ligase CSIT2 controls critical sterility-inducing temperature of thermo-sensitive genic male sterile rice.

Thermo-sensitive genic male sterile (TGMS) lines are the core of two-line hybrid rice (Oryza sativa). However, elevated or unstable critical sterility-inducing temperatures (CSITs) of TGMS lines are bottlenecks that restrict the development of two-line hybrid rice. However, the genes and molecular mechanisms controlling CSIT remain unknown. Here, we report the CRITICAL STERILITY-INDUCING TEMPERATURE 2 (CSIT2) that encodes a really interesting new gene (RING) type E3 ligase, controlling the CSIT of thermo-sensitive male sterility 5 (tms5)-based TGMS lines through ribosome-associated protein quality control (RQC). CSIT2 binds to the large and small ribosomal subunits and ubiquitinates 80S ribosomes for dissociation, and may also ubiquitinate misfolded proteins for degradation. Mutation of CSIT2 inhibits the possible damage to ubiquitin system and protein translation, which allows more proteins such as catalases to accumulate for anther development and inhibits abnormal accumulation of reactive oxygen species (ROS) and premature programmed cell death (PCD) in anthers, partly rescuing male sterility and raised the CSIT of tms5-based TGMS lines. These findings reveal a mechanism controlling CSIT and provide a strategy for solving the elevated or unstable CSITs of tms5-based TGMS lines in two-line hybrid rice.

SET DOMAIN GROUP 711-mediated H3K27me3 methylation of cytokinin metabolism genes regulates organ size in rice.

Organ size shapes plant architecture during rice (Oryza sativa) growth and development, affecting key factors influencing yield, such as plant height, leaf size, and seed size. Here, we report that the rice Enhancer of zeste [E(z)] homolog SET DOMAIN GROUP 711 (OsSDG711) regulates organ size in rice. Knockout of OsSDG711 produced shorter plants with smaller leaves, thinner stems, and smaller grains. We demonstrate that OsSDG711 affects organ size by reducing cell length and width and increasing cell number in leaves, stems, and grains. The result of chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) using an anti-trimethylation of histone H3 lysine 27 (H3K27me3) antibody showed that the levels of H3K27me3 associated with cytokinin oxidase/dehydrogenase genes (OsCKXs) were lower in the OsSDG711 knockout line Ossdg711. ChIP-qPCR assays indicated that OsSDG711 regulates the expression of OsCKX genes through H3K27me3 histone modification. Importantly, we show that OsSDG711 directly binds to the promoters of these OsCKX genes. Furthermore, we measured significantly lower cytokinin contents in Ossdg711 plants than in wild-type plants. Overall, our results reveal an epigenetic mechanism based on OsSDG711-mediated modulation of H3K27me3 levels to regulate the expression of genes involved in the cytokinin metabolism pathway and control organ development in rice. OsSDG711 may be an untapped epigenetic resource for ideal plant type improvement.

The E3 ubiquitin ligase CSIT1 regulates critical sterility-inducing temperature by ribosome-associated quality control to safeguard two-line hybrid breeding in rice.

Two-line hybrid breeding can fully utilize heterosis in crops. In thermo-sensitive genic male sterile (TGMS) lines, low critical sterility-inducing temperature (CSIT) is vital to safeguard the production of two-line hybrid seeds in rice (Oryza sativa), but the molecular mechanism determining CSIT is unclear. Here, we report the cloning of CSIT1, which encodes an E3 ubiquitin ligase, and show that CSIT1 modulates the CSIT of thermo-sensitive genic male sterility 5 (tms5)-based TGMS lines through ribosome-associated quality control (RQC). Biochemical assays demonstrated that CSIT1 binds to the 80S ribosomes and ubiquitinates abnormal nascent polypeptides for degradation in the RQC process. Loss of CSIT1 function inhibits the possible damage of tms5 to the ubiquitination system and protein translation, resulting in enhanced accumulation of anther-related proteins such as catalase to suppress abnormal accumulation of reactive oxygen species and premature programmed cell death in the tapetum, thereby leading to a much higher CSIT in the tms5-based TGMS lines. Taken together, our findings reveal a regulatory mechanism of CSIT, providing new insights into RQC and potential targets for future two-line hybrid breeding.

Advances on the Study of Diurnal Flower-Opening Times of Rice.

The principal goal of rice (Oryza sativa L.) breeding is to increase the yield. In the past, hybrid rice was mainly indica intra-subspecies hybrids, but its yield has been difficult to improve. The hybridization between the indica and japonica subspecies has stronger heterosis; the utilization of inter-subspecies heterosis is important for long-term improvement of rice yields. However, the different diurnal flower-opening times (DFOTs) between the indica and japonica subspecies seriously reduce the efficiency of cross-pollination and yield and increase the cost of indica-japonica hybrid rice seeds, which has become one of the main constraints for the development of indica-japonica hybrid rice breeding. The DFOT of plants is adapted to their growing environment and is also closely related to species stability and evolution. Herein, we review the structure and physiological basis of rice flower opening, the factors that affect DFOT, and the progress of cloning and characterization of DFOT genes in rice. We also analyze the problems in the study of DFOT and provide corresponding suggestions.

BOTRYOID POLLEN 1 regulates ROS-triggered PCD and pollen wall development by controlling UDP-sugar homeostasis in rice.

Uridine diphosphate (UDP)-sugars are important metabolites involved in the biosynthesis of polysaccharides and may be important signaling molecules. UDP-glucose 4-epimerase (UGE) catalyzes the interconversion between UDP-Glc and UDP-Gal, whose biological function in rice (Oryza sativa) fertility is poorly understood. Here, we identify and characterize the botryoid pollen 1 (bp1) mutant and show that BP1 encodes a UGE that regulates UDP-sugar homeostasis, thereby controlling the development of rice anthers. The loss of BP1 function led to massive accumulation of UDP-Glc and imbalance of other UDP-sugars. We determined that the higher levels of UDP-Glc and its derivatives in bp1 may induce the expression of NADPH oxidase genes, resulting in a premature accumulation of reactive oxygen species (ROS), thereby advancing programmed cell death (PCD) of anther walls but delaying the end of tapetal degradation. The accumulation of UDP-Glc as metabolites resulted in an abnormal degradation of callose, producing an adhesive microspore. Furthermore, the UDP-sugar metabolism pathway is not only involved in the formation of intine but also in the formation of the initial framework for extine. Our results reveal how UDP-sugars regulate anther development and provide new clues for cellular ROS accumulation and PCD triggered by UDP-Glc as a signaling molecule.

Environment-sensitive genic male sterility in rice and other plants.

Environment-sensitive genic male sterility is a type of male sterility that is affected by both genetic and environmental factors. Environment-sensitive genic male sterile lines are not only used in two-line hybrid breeding but are also good materials for studying plant-environment interactions. In this study we review the research progress on environment-sensitive genic male sterility in rice from the perspectives of epigenetic, transcriptional, posttranscriptional, posttranslational and metabolic mechanisms as well as signal transduction processes. While significant progress has been made in the genetics, gene cloning and understanding of the molecular mechanisms of environment-sensitive genic male sterility in recent years, the relevant regulatory network is still poorly understood in rice. We therefore also review studies of environment-sensitive genic male sterility in Arabidopsis and other crops, hoping to promote research in this field in rice. Finally, we analyse the challenges posed by environment-sensitive genic male sterility and provide corresponding suggestions. This review will contribute towards an understanding the molecular genetics of environment-sensitive genic male sterility and its application in two-line hybrid breeding in rice and other species.

Methylesterification of cell-wall pectin controls the diurnal flower-opening times in rice.

Flowers are the core reproductive organ of plants, and flowering is essential for cross-pollination. Diurnal flower-opening time is thus a key trait influencing reproductive isolation, hybrid breeding, and thermostability in plants. However, the molecular mechanisms controlling this trait remain unknown. Here, we report that rice Diurnal Flower Opening Time 1 (DFOT1) modulates pectin methylesterase (PME) activity to regulate pectin methylesterification levels of the lodicule cell walls, which affect lodicule swelling to control diurnal flower-opening time. DFOT1 is specifically expressed in the lodicules, and its expression gradually increases with the approach to flowering but decreases with flowering. Importantly, a knockout of DFOT1 showed earlier diurnal flower opening. We demonstrate that DFOT1 interacts directly with multiple PMEs to promote their activity. Knockout of PME40 also resulted in early diurnal flower opening, whereas overexpression of PME42 delayed diurnal flower opening. Lower PME activity was observed to be associated with higher levels of pectin methylesterification and the softening of cell walls in lodicules, which contribute to the absorption of water by lodicules and cause them to swell, thus promoting early diurnal flower opening. Higher PME activity had the opposite effect. Collectively, our work uncovers a molecular mechanism underlying the regulation of diurnal flower-opening time in rice, which would help reduce the costs of hybrid breeding and improve the heat tolerance of flowering plants by avoiding higher temperatures at anthesis.

Heterologous Expression of Jatropha curcas Fatty Acyl-ACP Thioesterase A (JcFATA) and B (JcFATB) Affects Fatty Acid Accumulation and Promotes Plant Growth and Development in Arabidopsis.

Plant fatty acyl-acyl carrier protein (ACP) thioesterases terminate the process of de novo fatty acid biosynthesis in plastids by hydrolyzing the acyl-ACP intermediates, and determine the chain length and levels of free fatty acids. They are of interest due to their roles in fatty acid synthesis and their potential to modify plant seed oils through biotechnology. Fatty acyl-ACP thioesterases (FAT) are divided into two families, i.e., FATA and FATB, according to their amino acid sequence and substrate specificity. The high oil content in Jatropha curcas L. seed has attracted global attention due to its potential for the production of biodiesel. However, the detailed effects of JcFATA and JcFATB on fatty acid biosynthesis and plant growth and development are still unclear. In this study, we found that JcFATB transcripts were detected in all tissues and organs examined, with especially high accumulation in the roots, leaves, flowers, and some stages of developing seeds, and JcFATA showed a very similar expression pattern. Subcellular localization of the JcFATA-GFP and JcFATB-GFP fusion protein in Arabidopsis leaf protoplasts showed that both JcFATA and JcFATB localized in chloroplasts. Heterologous expression of JcFATA and JcFATB in Arabidopsis thaliana individually generated transgenic plants with longer roots, stems and siliques, larger rosette leaves, and bigger seeds compared with those of the wild type, indicating the overall promotion effects of JcFATA and JcFATB on plant growth and development while JcFATB had a larger impact. Compositional analysis of seed oil revealed that all fatty acids except 22:0 were significantly increased in the mature seeds of JcFATA-transgenic Arabidopsis lines, especially unsaturated fatty acids, such as the predominant fatty acids of seed oil, 18:1, 18:2, and 18:3. In the mature seeds of the JcFATB-transgenic Arabidopsis lines, most fatty acids were increased compared with those in wild type too, especially saturated fatty acids, such as 16:0, 18:0, 20:0, and 22:0. Our results demonstrated the promotion effect of JcFATA and JcFATB on plant growth and development, and their possible utilization to modify the seed oil composition and content in higher plants.

A cytosolic pentatricopeptide repeat protein is essential for tapetal plastid development by regulating OsGLK1 transcript levels in rice.

Most plant pentatricopeptide repeat (PPR) proteins localize to and function inside plastids and mitochondria. However, the function of PPRs that only localize to the cytoplasm remains unknown. Here, we demonstrated that the rice (Oryza sativa) PPR protein CYTOPLASM-LOCALIZED PPR1 (OsCPPR1) contributes to pollen development and localizes to the cytoplasm. Knocking down OsCPPR1 led to abnormal plastid development in tapetal cells, prolonged tapetal programmed cell death (PCD) and tapetum degradation, and significantly reduced pollen fertility. Transcriptome analysis revealed that the transcript level of OsGOLDEN-LIKE1 (OsGLK1), which encodes a transcription factor that regulates plastid development and maintenance, was significantly higher in the OsCPPR1 knockdown plants compared to wild-type plants. We further determined that OsCPPR1 downregulates OsGLK1 transcription by directly binding to the single-stranded regions of OsGLK1 mRNAs. Overexpression of OsGLK1 resulted in abnormal tapetum and plastid development, similar to that seen in OsCPPR1 knockdown plants, and suppression of OsGLK1 partially restored pollen fertility in the OsCPPR1 knockdown plants. We therefore conclude that OsCPPR1 suppresses OsGLK1 in the regulation of plastid development and PCD in the tapetum. Our work revealed novel functions for a cytosolic PPR, demonstrating the diverse roles of PPRs in plants and identifying a new regulatory mechanism for regulating pollen development in rice.

Hexokinase gene OsHXK1 positively regulates leaf senescence in rice.

BACKGROUND: Leaf senescence is a highly complex and meticulous regulatory process, and the disruption of any factor involved in leaf senescence might lead to premature or delayed leaf senescence and thus result in reduced or increased crop yields. Despite sincere efforts by scientists, there remain many unsolved problems related to the regulatory factors and molecular mechanisms of leaf senescence. RESULTS: This study successfully revealed that OsHXK1 was highly expressed in senescent leaves of rice. The upregulation of OsHXK1 led to premature senescence of rice leaves, a decreased level of chlorophyll, and damage to the chloroplast structure. The overexpression of OsHXK1 resulted in increases in glucose and ROS levels and produced programmed cell death (PCD) signals earlier at the booting stage. Further analysis showed that expression level of the respiratory burst oxidase homolog (RBOH) genes and OsGLO1 were increased in OsHXK1-overexpressing plants at the booting stage. CONCLUSIONS: Overall, the outcomes of this study suggested that OsHXK1 could act as a positive regulator of rice leaf senescence by mediating glucose accumulation and inducing an increase in ROS.

Initiation and Execution of Programmed Cell Death and Regulation of Reactive Oxygen Species in Plants.

Programmed cell death (PCD) plays crucial roles in plant development and defence response. Reactive oxygen species (ROS) are produced during normal plant growth, and high ROS concentrations can change the antioxidant status of cells, leading to spontaneous cell death. In addition, ROS function as signalling molecules to improve plant stress tolerance, and they induce PCD under different conditions. This review describes the mechanisms underlying plant PCD, the key functions of mitochondria and chloroplasts in PCD, and the relationship between mitochondria and chloroplasts during PCD. Additionally, the review discusses the factors that regulate PCD. Most importantly, in this review, we summarise the sites of production of ROS and discuss the roles of ROS that not only trigger multiple signalling pathways leading to PCD but also participate in the execution of PCD, highlighting the importance of ROS in PCD.

OsCER1 regulates humidity-sensitive genic male sterility through very-long-chain (VLC) alkane metabolism of tryphine in rice.

Humidity-sensitive genic male sterility (HGMS) is a novel type of environment-sensitive male sterility (EGMS) which plants are male sterile at low humidity and male fertile at high humidity. Previous studies have revealed that OsCER1 contributes to very-long-chain (VLC) alkanes biosynthesis in rice (Oryza sativa L.). Here, applying the CRISPR/Cas9 technique, we obtained two independent OsCER1 knockout lines (OsCER1Cas). Both OsCER1Cas lines exhibited HGMS. Mutant pollen showed defects in adhesion and germination on stigmas at low humidity, whereas high humidity enhanced the pollen germination rate. Transmission electron microscopy (TEM) observations of mutant pollen revealed abnormal tryphine structure, potentially representing the basis of HGMS. Furthermore, co-pollination with mixed OsCER1Cas mutant and maize (Zea mays L.) pollen could rescue the fertility of the mutant, thereby establishing the key role of tryphine in germination on stigmas. OsCER1 knockout might affect VLC alkane metabolism and therefore alter the lipid composition of tryphine. It could lead to the defects in pollen grain adhesion, hydration and germination, resulting in HGMS. This work identified the mechanism of HGMS induced by VLC alkanes in rice and the generality of tryphine in different species of Gramineae.

The structural characteristics and the substrate recognition properties of RNase ZS1.

TMS5 encodes an RNase ZS1 protein that can process ubiquitin-60S ribosomal protein L40 family (UbL40) mRNAs to regulate thermo-sensitive genic male sterility in rice. Despite the importance of this protein, the structural characteristics and substrate recognition properties of RNase ZS1 remain unclear. Here, we found that the variations in several conservative amino acids alter the activation of RNase ZS1, and its recognition of RNA substrates depends on the structure of RNA. RNase ZS1 acts as a homodimer. The conserved amino acids in or adjacent to enzyme center play a critical role in the enzyme activity of RNase ZS1 and the conserved amino acids that far from active center have little impact on its enzyme activity. The cleavage efficiency of RNase ZS1 for pre-tRNA-MetCAU35 and UbL401 mRNA with cloverleaf-like structure was higher than that of pre-tRNA-AspAUC9 and UbL404 mRNA with imperfect cloverleaf-like structure. This difference implies that the enzyme activity of RNase ZS1 depends on the cloverleaf-like structure of the RNA. Furthermore, the RNase ZS1 activity was not inhibited by the 5' leader sequence and 3' CCA motif of pre-tRNA. These findings provide new insights for studying the cleavage characteristics and substrate recognition properties of RNase ZS.

An effective method for Agrobacterium tumefaciens-mediated transformation of Jatropha curcas L. using cotyledon explants.

Jatropha curcas is one of oilseed crops and has been considered as an energy crop. In the present study, efficient plant regeneration protocol and transformation method were developed for J. curcas. Because the regeneration efficiency of adventitious bud from cotyledon explants of J. curcas induced by traditional methods is low, and it takes a long time to get complete plants. It is necessary to establish a new regeneration system to improve regeneration efficiency. Cotyledon explants were dipped into TDZ solution at different concentrations respectively for various times to obtain higher efficiency of adventitious bud regeneration. This new regeneration method was then applied to genetic transformation of J. curcas. Cotyledon explants were precultured for 1 day after treated with high concentration of Thidiazuron (TDZ) solution (20 mg/L for 40 min), followed by Agrobacterium tumefaciens infection. After co-cultured for 2 days, the explants were placed on the induction hormone-free media for bud regeneration and resistant screening. After 30 days, selected shoot buds were transferred onto elongation medium for 15 days. Young leaf sections of the regenerated shoots were used for PCR (Polymerase chain reaction) detection of the transgenic shoots. The PCR positive shoots were isolated and used for in vitro grafting. The intact plants were obtained within 20 days. GUS (ß-Glucosidase) staining and Southern analysis confirmed the transformation events. Briefly, a transformation efficiency of 34.32% was achieved and an intact transgenic plant could be obtained within 65 days.

Molecular Control and Application of Male Fertility for Two-Line Hybrid Rice Breeding.

The significance of the climate change may involve enhancement of plant growth as well as utilization of the environmental alterations in male fertility (MF) regulation via male sterility (MS) systems. We described that MS systems provide a fundamental platform for improvement in agriculture production and have been explicated for creating bulk germplasm of the two-line hybrids (EGMS) in rice as compared to the three-line, to gain production sustainability and exploit its immense potential. Environmental alterations such as photoperiod and/or temperature and humidity regulate MS in EGMS lines via genetic and epigenetic changes, regulation of the noncoding RNAs, and RNA-metabolism including the transcriptional factors (TFs) implication. Herein, this article enlightens a deep understanding of the molecular control of MF in EGMS lines and exploring the regulatory driving forces that function efficiently during plant adaption under a changing environment. We highlighted a possible solution in obtaining more stable hybrids through apomixis (single-line system) for seed production.

HMS1 interacts with HMS1I to regulate very-long-chain fatty acid biosynthesis and the humidity-sensitive genic male sterility in rice (Oryza sativa).

Environment-sensitive genic male sterility (EGMS) lines are used widely in two-line hybrid breeding in rice (Oryza sativa). At present, photoperiod-sensitive genic male sterility (PGMS) lines and thermo-sensitive genic male sterility (TGMS) lines are predominantly used in two-line hybrid rice, with humidity-sensitive genic male sterility (HGMS) lines rarely being reported. Here, it is shown that HUMIDITY-SENSITIVE GENIC MALE STERILITY 1 (HMS1), encoding a ß-ketoacyl-CoA synthase, plays key roles in the biosynthesis of very-long-chain fatty acids (VLCFAs) and HGMS in rice. The hms1 mutant displayed decreased seed setting under low humidity, but normal seed setting under high humidity. HMS1 catalyzed the biosynthesis of the C26 and C28 VLCFAs, contributing to the formation of bacula and tryphine in the pollen wall, which protect the pollen from dehydration. Under low-humidity conditions, hms1 pollen showed poor adhesion and reduced germination on the stigmas, which could be rescued by increasing humidity. HMS1-INTERACTING PROTEIN (HMS1I) interacted with HMS1 to coregulate HGMS. Furthermore, both japonica and indica rice varieties with defective HMS1 exhibited HGMS, suggesting that hms1 potentially could be used in hybrid breeding. The results herein reveal the novel mechanism of VLCFA-mediated pollen wall formation, which protects pollen from low-humidity stress in rice, and has a potential use in hybrid crop breeding.

Overexpression of OsAGO1b Induces Adaxially Rolled Leaves by Affecting Leaf Abaxial Sclerenchymatous Cell Development in Rice.

BACKGROUND: ARGONAUTE 1 (AGO1) proteins can recruit small RNAs to regulate gene expression, involving several growth and development processes in Arabidopsis. Rice genome contains four AGO1 genes, OsAGO1a to OsAGO1d. However, the regulatory functions to rice growth and development of each AGO1 gene are still unknown. RESULTS: We obtained overexpression and RNAi transgenic lines of each OsAGO1 gene. However, only up- and down-regulation of OsAGO1b caused multiple abnormal phenotypic changes in rice, indicating that OsAGO1b is the key player in rice growth and organ development compared with other three OsAGO1s. qRT-PCR assays showed that OsAGO1b was almost unanimously expressed in leaves at different developmental stages, and strongly expressed in spikelets at S1 to S3 stages. OsAGO1b is a typical AGO protein, and co-localized in both the nucleus and cytoplasm simultaneously. Overexpression of OsAGO1b caused adaxially rolled leaves and a series of abnormal phenotypes, such as the reduced tiller number and plant height. Knockdown lines of OsAGO1b showed almost normal leaves, but the seed setting percentage was significantly reduced accompanied by the disturbed anther patterning and reduced pollen fertility. Further anatomical observation revealed that OsAGO1b overexpression plants showed the partially defective development of sclerenchymatous cells on the abaxial side of leaves. In situ hybridization showed OsAGO1b mRNA was uniformly accumulated in P1 to P3 primordia without polarity property, suggesting OsAGO1b did not regulate the adaxial-abaxial polarity development directly. The expression levels of several genes related to leaf polarity development and vascular bundle differentiation were observably changed. Notably, the accumulation of miR166 and TAS3-siRNA was decreased, and their targeted OSHBs and OsARFs were significantly up-regulated. The mRNA distribution patterns of OSHB3 and OsARF4 in leaves remained almost unchanged between ZH11 and OsAGO1b overexpression lines, but their expression levels were enhanced at the regions of vascular bundles and sclerenchymatous cell differentiation. CONCLUSIONS: In summary, we demonstrated OsAGO1b is the leading player among four OsAGO1s in rice growth and development. We propose that OsAGO1b may regulate the abaxial sclerenchymatous cell differentiation by affecting the expression of OSHBs in rice.

OsAGO2 controls ROS production and the initiation of tapetal PCD by epigenetically regulating OsHXK1 expression in rice anthers.

Proteins of the ARGONAUTE (AGO) family function in the epigenetic regulation of gene expression. Although the rice (Oryza sativa) genome encodes 19 predicted AGO proteins, few of their functions have thus far been characterized. Here, we show that the AGO protein OsAGO2 regulates anther development in rice. OsAGO2 was highly expressed in anthers. Knockdown of OsAGO2 led to the overaccumulation of reactive oxygen species (ROS) and abnormal anther development, causing premature initiation of tapetal programmed cell death (PCD) and pollen abortion. The expression level of Hexokinase 1 (OsHXK1) increased significantly, and the methylation levels of its promoter decreased, in plants with knocked-down OsAGO2 expression. Overexpression of OsHXK1 also resulted in the overaccumulation of ROS, premature initiation of PCD, and pollen abortion. Moreover, knockdown of OsHXK1 restored pollen fertility in OsAGO2 knockdown plants. Chromatin immunoprecipitation assays demonstrated that OsAGO2 binds directly to the OsHXK1 promoter region, suggesting that OsHXK1 is a target gene of OsAGO2. These results indicate that OsHXK1 controls the appropriate production of ROS and the proper timing of tapetal PCD and is directly regulated by OsAGO2 through epigenetic regulation.

OsCER1 Plays a Pivotal Role in Very-Long-Chain Alkane Biosynthesis and Affects Plastid Development and Programmed Cell Death of Tapetum in Rice (Oryza sativa L.).

Cuticle waxes, which are primarily comprised of very-long-chain (VLC) alkanes, play an important role in plant reproductive development. ECERIFERUM1 (CER1) is recognized as the core element for VLC alkane biosynthesis in Arabidopsis (Arabidopsis thaliana). However, genes involved in the VLC alkane biosynthesis in rice remain unclear, and the alkane-form pathway in rice has still to be further explored. Here, we show that OsCER1, a homology of CER1, functions in VLC alkanes biosynthesis, which also could regulate anther development and plastids differentiation in rice. OsCER1 was highly expressed in the tapetum (stage 10) and bicellular pollen cells (stage 11). The decreased content of VLC alkanes (C25 and C27) in the OsCER1 knocked down plants as well as the increased content of C27 alkanes in the OsCER1 overexpression plants indicates that OsCER1 participates in VLC alkane biosynthesis. Downregulation of OsCER1 in rice led to sterility, and fewer amyloplasts within the mature pollen grains. In addition, the downregulation of OsCER1 in rice caused delayed tapetal programmed cell death and abnormal development of plastids in the tapetal cells. Furthermore, significantly altered levels of expression of genes involved in the pollen development were exhibited in the OsCER1 knocked down plants. These results indicate that OsCER1 is critical for VLC alkanes biosynthesis, plastids differentiation, and pollen development. This work provides insights into the VLC alkanes biosynthesis in anther development in rice.