Genome-Wide Identification and Analysis of Collar Region-Preferential Genes in Rice.

The collar region plays a crucial role in leaf angle formation and plant architecture, which is important for improving crop yield given the challenges of diminishing arable land and changing environmental conditions. To determine collar region-preferential genes (CRPGs) affecting plant architecture and crop yield, we conducted genome-wide transcriptomic analysis. By integrating our RNA sequencing data with public rice anatomical expression data, we identified 657 CRPGs. Verification involved testing six randomly selected CRPGs, all of which exhibited collar-preferential expression. The functional significance of CRPGs was assessed via Gene Ontology enrichment analysis, utilizing MapMan and KEGG, and literature analysis provided additional information for characterized CRPGs. Our findings revealed links between manipulating leaf angle and phytohormone-related pathways and stress responses. Moreover, based on the CRPGs, five transcription factors downstream of the liguleless 1 (LG1) gene were identified. Overall, the identified CRPGs provide potential targets for further research and breeding applications aimed at improving crop productivity by manipulating leaf architecture.

Genomic basis of multiphase evolution driving divergent selection of zinc-finger homeodomain genes.

Gene families divergently evolve and become adapted as different genes with specific structures and functions in living organisms. We performed comprehensive structural and functional analyses of Zinc-finger homeodomain genes (ZF-HDs), including Mini zinc-finger genes (MIFs) and Zinc-finger with homeodomain genes (ZHDs), displaying competitive functions each other. Intensive annotation updates for 90 plant genomes verified that most MIFs (MIF-Is) exhibited distinct motif compositions from ZHDs, although some MIFs (MIF-Zs) contained ZHD-specific motifs. Phylogenetic analyses suggested that MIF-Zs and ZHDs originated from the same ancestral gene, whereas MIF-Is emerged from a distinct progenitor. We used a gene-editing system to identify a novel function of MIF-Is in rice: regulating the surface material patterns in anthers and pollen through transcriptional regulation by interacting ZHDs. Kingdom-wide investigations determined that (i) ancestral MIFs diverged into MIF-Is and MIF-Zs in the last universal common ancestor, (ii) integration of HD into the C-terminal of MIF-Zs created ZHDs after emergence of green plants and (iii) MIF-Is and ZHDs subsequently expanded independently into specific plant lineages, with additional formation of MIF-Zs from ZHDs. Our comprehensive analysis provides genomic evidence for multiphase evolution driving divergent selection of ZF-HDs.

Reinterpretation of anthocyanins biosynthesis in developing black rice seeds through gene expression analysis.

The biosynthesis of anthocyanins is still questionable in regulating the quantities of anthocyanins biosynthesized in rice seeds and the expression levels of transcription factors and the structural genes involved in the biosynthetic pathway of anthocyanins. We herein investigated the relationship between the accumulated anthocyanin contents and the expression levels of genes related to the biosynthesis of anthocyanins in rice seeds. Liquid chromatography/mass spectrometry-mass spectrometry analysis of cyanidin 3-glucoside (C3G) in rice seeds showed no accumulation of C3G in white and red rice cultivars, and the differential accumulation of C3G among black rice cultivars. RNA-seq analysis in rice seeds, including white, red, and black rice cultivars, at twenty days after heading (DAH) further exhibited that the genes involved in the biosynthesis of anthocyanins were differentially upregulated in developing seeds of black rice. We further verified these RNA-seq results through gene expression analysis by a quantitative real-time polymerase chain reaction in developing seeds of white, red, and black rice cultivars at 20 DAH. Of these genes related to the biosynthesis of anthocyanins, bHLHs, MYBs, and WD40, which are regulators, and the structural genes, including chalcone synthase (CHS), flavanone 3-hydroxylase (F3H), flavonoid 3´-hydroxylase (F3´H), dihydroflavonol 4-reductase (DFR), and anthocyanidin synthase (ANS), were differentially upregulated in black rice seeds. The correlation analysis revealed that the quantities of C3G biosynthesized in black rice seeds were positively correlated to the expression levels of bHLHs, MYBs and WD40, CHS, F3H, F3´H, DFR, and ANS. In addition, we present bHLH2 (LOC_Os04g47040) and MYBs (LOC_Os01g49160, LOC_Os01g74410, and LOC_Os03g29614) as new putative transcription factor genes for the biosynthesis of anthocyanins in black rice seeds. It is expected that this study will help to improve the understanding of the molecular levels involved in the biosynthesis of anthocyanins in black rice seeds.

Rice pollen-specific OsRALF17 and OsRALF19 are essential for pollen tube growth.

Pollen tube growth is essential for successful double fertilization, which is critical for grain yield in crop plants. Rapid alkalinization factors (RALFs) function as ligands for signal transduction during fertilization. However, functional studies on RALF in monocot plants are lacking. Herein, we functionally characterized two pollen-specific RALFs in rice (Oryza sativa) using multiple clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9-induced loss-of-function mutants, peptide treatment, expression analyses, and tag reporter lines. Among the 41 RALF members in rice, OsRALF17 was specifically expressed at the highest level in pollen and pollen tubes. Exogenously applied OsRALF17 or OsRALF19 peptide inhibited pollen tube germination and elongation at high concentrations but enhanced tube elongation at low concentrations, indicating growth regulation. Double mutants of OsRALF17 and OsRALF19 (ralf17/19) exhibited almost full male sterility with defects in pollen hydration, germination, and tube elongation, which was partially recovered by exogenous treatment with OsRALF17 peptide. This study revealed that two partially functionally redundant OsRALF17 and OsRALF19 bind to Oryza sativa male-gene transfer defective 2 (OsMTD2) and transmit reactive oxygen species signals for pollen tube germination and integrity maintenance in rice. Transcriptomic analysis confirmed their common downstream genes, in osmtd2 and ralf17/19. This study provides new insights into the role of RALF, expanding our knowledge of the biological role of RALF in regulating rice fertilization.

OsSNDP3 Functions for the Polar Tip Growth in Rice Pollen Together with OsSNDP2, a Paralog of OsSNDP3.

Understanding pollen tube growth is critical for crop yield maintenance. The pollen tube provides a path for sperm cells for fertilization with egg cells. Cells must be subdivided into functionally and structurally distinct compartments for polar tip growth, and phosphoinositides are thought to be one of the facilitators for polarization during pollen tube growth. OsSNDP3 encodes Sec14-nodulin domain-containing protein and localizes in the nucleus and the microdomains of the plasma membrane in tobacco leaf epidermis cells. OsSNDP3 is thought to bind with phosphatidylinositol 4,5-bisphosphate based on the data including the information of basic amino acids in the C-terminal and colocalization with 2X Pleckstrin homology domain of Phospholipase C delta-1. OsSNDP3 interacts with a protein that contains a class I nodulin domain. We discovered that OsSNDP3 plays a significant role in pollen tube germination using CRISPR/Cas9 systems, whereas another pollen-preferential Sec14-nodulin domain-containing protein, OsSNDP2, additively functions with OsSNDP3 during pollen tube germination. Gene Ontology analysis using downregulated genes in ossndp3 indicated that the expression of genes involved in the phosphatidylinositol metabolic process and tip growth was significantly altered in ossndp3. OsSNDP3 aids pollen polar tip growth by binding with phosphatidylinositol 4,5-bisphosphate. We can better understand the roles of phosphoinositides during pollen tube growth by studying the functions of OsSNDP3 and OsSNDP2. And downregulated genes in ossndp3 might be useful targets for future research on polar tip growth.

A myosin XI adaptor, TAPE, is essential for pollen tube elongation in rice.

Pollen tube (PT) elongation is important for double fertilization in angiosperms and affects the seed-setting rate and, therefore, crop productivity. Compared to Arabidopsis (Arabidopsis thaliana L.), information on PT elongation in rice (Oryza sativa L.) is limited by the difficulty in obtaining homozygous mutants. In a screen of T-DNA insertional mutants, we identified a mutant in the Tethering protein of actomyosin transport in pollen tube elongation (TAPE) gene with an unusual segregation ratio by genotyping analysis. A CRISPR/Cas9 knockout mutant of TAPE that produced a short PT was sterile, and TAPE was expressed specifically in pollen grains. TAPE is a homolog of a myosin XI adaptor in Arabidopsis with three tetratricopeptide repeat and Phox and Bem1 protein domains. TAPE showed latrunculin B-sensitive, actin-dependent localization to the endoplasmic reticulum. Yeast two-hybrid screening and transcriptome analysis revealed that TAPE interacted with pollen-specific LIM protein 2b and elongation factor 1-alpha. Loss of TAPE affected transcription of 1,259 genes, especially genes related to cell organization, which were downregulated. In summary, TAPE encodes a myosin XI adaptor essential for rice PT elongation.

Cytokinin increases vegetative growth period by suppressing florigen expression in rice and maize.

Increasing the vegetative growth period of crops can increase biomass and grain yield. In rice (Oryza sativa), the concentration of trans -zeatin, an active cytokinin, was high in the leaves during vegetative growth and decreased rapidly upon induction of florigen expression, suggesting that this hormone is involved in the regulation of the vegetative phase. To elucidate whether exogenous cytokinin application influences the length of the vegetative phase, we applied 6-benzylaminopurine (BAP) to rice plants at various developmental stages. Our treatment delayed flowering time by 8-9 days when compared with mock-treated rice plants, but only at the transition stage when the flowering signals were produced. Our observations also showed that flowering in the paddy field is delayed by thidiazuron, a stable chemical that mimics the effects of cytokinin. The transcript levels of florigen genes Heading date 3a (Hd3a) and Rice Flowering locus T1 (RFT1) were significantly reduced by the treatment, but the expression of Early heading date 1 (Ehd1), a gene found directly upstream of the florigen genes, was not altered. In maize (Zea mays), similarly, BAP treatment increased the vegetative phage by inhibiting the expression of ZCN8, an ortholog of Hd3a. We showed that cytokinin treatment induced the expression of two type-A response regulators (OsRR1 and OsRR2) which interacted with Ehd1, a type-B response regulator. We also observed that cytokinin did not affect flowering time in ehd1 knockout mutants. Our study indicates that cytokinin application increases the duration of the vegetative phase by delaying the expression of florigen genes in rice and maize by inhibiting Ehd1.

Comparative transcriptome analysis of pollen and anther wall reveals novel insights into the regulatory mechanisms underlying anther wall development and its dehiscence in rice.

To further understand the regulatory mechanism for anther dehiscence in rice, we carried out transcriptome analysis for the following two tissues: the anther wall and pollen at the anthesis stage. With the anatomical meta-expression data, in addition to these tissues, the differentially expressed genes (DEGs) between the two tissues were further refined to identify 1,717 pollen-preferred genes and 534 anther wall-preferred genes. A GUS transgenic line and RT-qPCR analysis for anther wall-preferred genes supported the fidelity of our gene candidates for further analysis. The refined DEGs were functionally classified through Gene Ontology (GO) enrichment and MapMan analyses. Through the analysis of cis-acting elements and alternative splicing variants, we also suggest the feature of regulatory sequences in promoter regions for anther wall-preferred expression and provide information of the unique splicing variants in anther wall. Subsequently, it was found that hormone signaling and the resulting transcriptional regulation pathways may play an important role in anther dehiscence and anther wall development. Our results could provide useful insights into future research to broaden the molecular mechanism of anther dehiscence or anther wall development in rice.

Transcriptional Changes in the Developing Rice Seeds Under Salt Stress Suggest Targets for Manipulating Seed Quality.

Global sea-level rise, the effect of climate change, poses a serious threat to rice production owing to saltwater intrusion and the accompanying increase in salt concentration. The reclaimed lands, comprising 22.1% of rice production in Korea, now face the crisis of global sea-level rise and a continuous increase in salt concentration. Here, we investigated the relationship between the decrease in seed quality and the transcriptional changes that occur in the developing rice seeds under salt stress. Compared to cultivation on normal land, the japonica rice cultivar, Samgwang, grown on reclaimed land showed a greatly increased accumulation of minerals, including sodium, magnesium, potassium, and sulfur, in seeds and a reduced yield, delayed heading, decreased thousand grain weight, and decreased palatability and amylose content. Samgwang showed phenotypical sensitivity to salt stress in the developing seeds. Using RNA-seq technology, we therefore carried out a comparative transcriptome analysis of the developing seeds grown on reclaimed and normal lands. In the biological process category, gene ontology enrichment analysis revealed that the upregulated genes were closely associated with the metabolism of biomolecules, including amino acids, carboxylic acid, lignin, trehalose, polysaccharide, and chitin, and to stress responses. MapMan analysis revealed the involvement of upregulated genes in the biosynthetic pathways of abscisic acid and melatonin and the relationship of trehalose, raffinose, and maltose with osmotic stress. Interestingly, many seed storage protein genes encoding glutelins and prolamins were upregulated in the developing seeds under salt stress, indicating the negative effect of the increase of storage proteins on palatability. Transcription factors upregulated in the developing seeds under salt stress included, in particular, bHLH, MYB, zinc finger, and heat shock factor, which could act as potential targets for the manipulation of seed quality under salt stress. Our study aims to develop a useful reference for elucidating the relationship between seed response mechanisms and decreased seed quality under salt stress, providing potential strategies for the improvement of seed quality under salt stress.

Recurrent mutations promote widespread structural and functional divergence of MULE-derived genes in plants.

Transposable element (TE)-derived genes are increasingly recognized as major sources conferring essential traits in agriculturally important crops but underlying evolutionary mechanisms remain obscure. We updated previous annotations and constructed 18,744 FAR-RED IMPAIRED RESPONSE1 (FAR1) genes, a transcription factor family derived from Mutator-like elements (MULEs), from 80 plant species, including 15,546 genes omitted in previous annotations. In-depth sequence comparison of the updated gene repertoire revealed that FAR1 genes underwent continuous structural divergence via frameshift and nonsense mutations that caused premature translation termination or specific domain truncations. CRISPR/Cas9-based genome editing and transcriptome analysis determined a novel gene involved in fertility-regulating transcription of rice pollen, denoting the functional capacity of our re-annotated gene models especially in monocots which had the highest copy numbers. Genomic evidence showed that the functional gene adapted by obtaining a shortened form through a frameshift mutation caused by a tandem duplication of a 79-bp sequence resulting in premature translation termination. Our findings provide improved resources for comprehensive studies of FAR1 genes with beneficial agricultural traits and unveil novel evolutionary mechanisms generating structural divergence and subsequent adaptation of TE-derived genes in plants.

Optimization of Protein Isolation and Label-Free Quantitative Proteomic Analysis in Four Different Tissues of Korean Ginseng.

Korean ginseng is one of the most valuable medicinal plants worldwide. However, our understanding of ginseng proteomics is largely limited due to difficulties in the extraction and resolution of ginseng proteins because of the presence of natural contaminants such as polysaccharides, phenols, and glycosides. Here, we compared four different protein extraction methods, namely, TCA/acetone, TCA/acetone-MeOH/chloroform, phenol-TCA/acetone, and phenol-MeOH/chloroform methods. The TCA/acetone-MeOH/chloroform method displayed the highest extraction efficiency, and thus it was used for the comparative proteome profiling of leaf, root, shoot, and fruit by a label-free quantitative proteomics approach. This approach led to the identification of 2604 significantly modulated proteins among four tissues. We could pinpoint differential pathways and proteins associated with ginsenoside biosynthesis, including the methylerythritol 4-phosphate (MEP) pathway, the mevalonate (MVA) pathway, UDP-glycosyltransferases (UGTs), and oxidoreductases (CYP450s). The current study reports an efficient and reproducible method for the isolation of proteins from a wide range of ginseng tissues and provides a detailed organ-based proteome map and a more comprehensive view of enzymatic alterations in ginsenoside biosynthesis.

A Systemic View of Carbohydrate Metabolism in Rice to Facilitate Productivity.

Carbohydrate metabolism is an important biochemical process related to developmental growth and yield-related traits. Due to global climate change and rapid population growth, increasing rice yield has become vital. To understand whole carbohydrate metabolism pathways and find related clues for enhancing yield, genes in whole carbohydrate metabolism pathways were systemically dissected using meta-transcriptome data. This study identified 866 carbohydrate genes from the MapMan toolkit and the Kyoto Encyclopedia of Genes and Genomes database split into 11 clusters of different anatomical expression profiles. Analysis of functionally characterized carbohydrate genes revealed that source activity and eating quality are the most well-known functions, and they each have a strong correlation with tissue-preferred clusters. To verify the transcriptomic dissection, three pollen-preferred cluster genes were used and found downregulated in the gori mutant. Finally, we summarized carbohydrate metabolism as a conceptual model in gene clusters associated with morphological traits. This systemic analysis not only provided new insights to improve rice yield but also proposed novel tissue-preferred carbohydrate genes for future research.

OsMTD2-mediated reactive oxygen species (ROS) balance is essential for intact pollen-tube elongation in rice.

The highly specialized haploid male gametophyte-pollen consist of two sperm cells and a large vegetative cell. Successful fertilization requires proper growth timing and rupture of the pollen tube until it delivers sperm cells, which occur immediately after a pollen grain hydrates. Although a tight regulation on polar cell-wall expansion of the pollen tube is fundamentally important, the underlying molecular mechanism remains largely unknown, especially in crop plants. Here, we characterized the function of male-gene transfer defective 2 (OsMTD2) gene in rice (Oryza sativa), which belongs to the plant-specific receptor-like kinase, the CrRLK1L family. We demonstrated that OsMTD2 is an essential male factor participating in pollen-tube elongation based on genetic evidence and physiological observations. Because of unavailability of homozygous mutant via conventional methods, we used CRISPR-Cas9 system to obtain homozygous knockout mutant of OsMTD2. We were able to identify phenotypic changes including male sterility due to early pollen-tube rupture in the mutant. We observed that the production of reactive oxygen species (ROS) was dramatically reduced in mutants of OsMTD2 pollen grain and tubes with defective pectin distribution. Transcriptome analysis of osmtd2-2 versus wild-type anthers revealed that genes involved in defense responses, metabolic alteration, transcriptional and protein modification were highly upregulated in the osmtd2-2 mutant. Through yeast-two-hybrid screening, we found that OsMTD2 kinase interacts with E3 ligase SPL11. Taken together, we propose that OsMTD2 has crucial functions in promoting pollen-tube elongation through cell-wall modification, possibly by modulating ROS homeostasis during pollen-tube growth.

Interaction of OsRopGEF3 Protein With OsRac3 to Regulate Root Hair Elongation and Reactive Oxygen Species Formation in Rice (Oryza sativa).

Root hairs are tip-growing cells that emerge from the root epidermis and play a role in water and nutrient uptake. One of the key signaling steps for polar cell elongation is the formation of Rho-GTP by accelerating the intrinsic exchange activity of the Rho-of-plant (ROP) or the Rac GTPase protein; this step is activated through the interaction with the plant Rho guanine nucleotide exchange factor (RopGEFs). The molecular players involved in root hair growth in rice are largely unknown. Here, we performed the functional analysis of OsRopGEF3, which is highly expressed in the root hair tissues among the OsRopGEF family genes in rice. To reveal the role of OsRopGEF3, we analyzed the phenotype of loss-of-function mutants of OsRopGEF3, which were generated using the CRISPR-Cas9 system. The mutants had reduced root hair length and increased root hair width. In addition, we confirmed that reactive oxygen species (ROS) were highly reduced in the root hairs of the osropgef3 mutant. The pairwise yeast two-hybrid experiments between OsRopGEF3 and OsROP/Rac proteins in rice revealed that the OsRopGEF3 protein interacts with OsRac3. This interaction and colocalization at the same subcellular organelles were again verified in tobacco leaf cells and rice root protoplasts via bimolecular functional complementation (BiFC) assay. Furthermore, among the three respiratory burst oxidase homolog (OsRBOH) genes that are highly expressed in rice root hair cells, we found that OsRBOH5 can interact with OsRac3. Our results demonstrate an interaction network model wherein OsRopGEF3 converts the GDP of OsRac3 into GTP, and OsRac3-GTP then interacts with the N-terminal of OsRBOH5 to produce ROS, thereby suggesting OsRopGEF3 as a key regulating factor in rice root hair growth.

CTP synthase is essential for early endosperm development by regulating nuclei spacing.

Cereal grain endosperms are an important source of human nutrition. Nuclear division in early endosperm development plays a major role in determining seed size; however, this development is not well understood. We identified the rice mutant endospermless 2 (enl2), which shows defects in the early stages of endosperm development. These phenotypes arise from mutations in OsCTPS1 that encodes a cytidine triphosphate synthase (CTPS). Both wild-type and mutant endosperms were normal at 8 h after pollination (HAP). In contrast, at 24 HAP, enl2 endosperm had approximately 10-16 clumped nuclei while wild-type nuclei had increased in number and migrated to the endosperm periphery. Staining of microtubules in endosperm at 24 HAP revealed that wild-type nuclei were evenly distributed by microtubules while the enl2-2 nuclei were tightly packed due to their reduction in microtubule association. In addition, OsCTPS1 interacts with tubulins; thus, these observations suggest that OsCTPS1 may be involved in microtubule formation. OsCTPS1 transiently formed macromolecular structures in the endosperm during early developmental stages, further supporting the idea that OsCTPS1 may function as a structural component during endosperm development. Finally, overexpression of OsCTPS1 increased seed weight by promoting endosperm nuclear division, suggesting that this trait could be used to increase grain yield.

Global Identification of ANTH Genes Involved in Rice Pollen Germination and Functional Characterization of a Key Member, OsANTH3.

Pollen in angiosperms plays a critical role in double fertilization by germinating and elongating pollen tubes rapidly in one direction to deliver sperm. In this process, the secretory vesicles deliver cell wall and plasma membrane materials, and excessive materials are sequestered via endocytosis. However, endocytosis in plants is poorly understood. AP180 N-terminal homology (ANTH) domain-containing proteins function as adaptive regulators for clathrin-mediated endocytosis in eukaryotic systems. Here, we identified 17 ANTH domain-containing proteins from rice based on a genome-wide investigation. Motif and phylogenomic analyses revealed seven asparagine-proline-phenylalanine (NPF)-rich and 10 NPF-less subgroups of these proteins, as well as various clathrin-mediated endocytosis-related motifs in their C-terminals. To investigate their roles in pollen germination, we performed meta-expression analysis of all genes encoding ANTH domain-containing proteins in Oryza sativa (OsANTH genes) in anatomical samples, including pollen, and identified five mature pollen-preferred OsANTH genes. The subcellular localization of four OsANTH proteins that were preferentially expressed in mature pollen can be consistent with their role in endocytosis in the plasma membrane. Of them, OsANTH3 represented the highest expression in mature pollen. Functional characterization of OsANTH3 using T-DNA insertional knockout and gene-edited mutants revealed that a mutation in OsANTH3 decreased seed fertility by reducing the pollen germination percentage in rice. Thus, our study suggests OsANTH3-mediated endocytosis is important for rice pollen germination.

Arachis hypogaea resveratrol synthase 3 alters the expression pattern of UDP-glycosyltransferase genes in developing rice seeds.

The resveratrol-producing rice (Oryza sativa L.) inbred lines, Iksan 515 (I.515) and Iksan 526 (I.526), developed by the expression of the groundnut (Arachis hypogaea) resveratrol synthase 3 (AhRS3) gene in the japonica rice cultivar Dongjin, accumulated both resveratrol and its glucoside, piceid, in seeds. Here, we investigated the effect of the AhRS3 transgene on the expression of endogenous piceid biosynthesis genes (UGTs) in the developing seeds of the resveratrol-producing rice inbred lines. Ultra-performance liquid chromatography (UPLC) analysis revealed that I.526 accumulates significantly higher resveratrol and piceid in seeds than those in I.515 seeds and, in I.526 seeds, the biosynthesis of resveratrol and piceid reached peak levels at 41 days after heading (DAH) and 20 DAH, respectively. Furthermore, RNA-seq analysis showed that the expression patterns of UGT genes differed significantly between the 20 DAH seeds of I.526 and those of Dongjin. Quantitative real-time PCR (RT-qPCR) analyses confirmed the data from RNA-seq analysis in seeds of Dongjin, I.515 and I.526, respectively, at 9 DAH, and in seeds of Dongjin and I.526, respectively, at 20 DAH. A total of 245 UGTs, classified into 31 UGT families, showed differential expression between Dongjin and I.526 seeds at 20 DAH. Of these, 43 UGTs showed more than 2-fold higher expression in I.526 seeds than in Dongjin seeds. In addition, the expression of resveratrol biosynthesis genes (PAL, C4H and 4CL) was also differentially expressed between Dongjin and I.526 developing seeds. Collectively, these data suggest that AhRS3 altered the expression pattern of UGT genes, and PAL, C4H and 4CL in developing rice seeds.

OsPP2C09 Is a Bifunctional Regulator in Both ABA-Dependent and Independent Abiotic Stress Signaling Pathways.

Clade A Type 2C protein phosphatases (PP2CAs) negatively regulate abscisic acid (ABA) signaling and have diverse functions in plant development and in response to various stresses. In this study, we showed that overexpression of the rice ABA receptor OsPYL/RCAR3 reduces the growth retardation observed in plants exposed to osmotic stress. By contrast, overexpression of the OsPYL/RCAR3-interacting protein OsPP2C09 rendered plant growth more sensitive to osmotic stress. We tested whether OsPP2CAs activate an ABA-independent signaling cascade by transfecting rice protoplasts with luciferase reporters containing the drought-responsive element (DRE) or ABA-responsive element (ABRE). We observed that OsPP2CAs activated gene expression via the cis-acting drought-responsive element. In agreement with this observation, transcriptome analysis of plants overexpressing OsPP2C09 indicated that OsPP2C09 induces the expression of genes whose promoters contain DREs. Further analysis showed that OsPP2C09 interacts with DRE-binding (DREB) transcription factors and activates reporters containing DRE. We conclude that, through activating DRE-containing promoters, OsPP2C09 positively regulates the drought response regulon and activates an ABA-independent signaling pathway.

Transcriptome Analysis of Triple Mutant for OsMADS62, OsMADS63, and OsMADS68 Reveals the Downstream Regulatory Mechanism for Pollen Germination in Rice (Oryza sativa).

The MADS (MCM1-AGAMOUS-DEFFICIENS-SRF) gene family has a preserved domain called MADS-box that regulates downstream gene expression as a transcriptional factor. Reports have revealed three MADS genes in rice, OsMADS62, OsMADS63, and OsMADS68, which exhibits preferential expression in mature rice pollen grains. To better understand the transcriptional regulation of pollen germination and tube growth in rice, we generated the loss-of-function homozygous mutant of these three OsMADS genes using the CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR associated protein 9) system in wild-type backgrounds. Results showed that the triple knockout (KO) mutant showed a complete sterile phenotype without pollen germination. Next, to determine downstream candidate genes that are transcriptionally regulated by the three OsMADS genes during pollen development, we proceeded with RNA-seq analysis by sampling the mature anther of the mutant and wild-type. Two hundred and seventy-four upregulated and 658 downregulated genes with preferential expressions in the anthers were selected. Furthermore, downregulated genes possessed cell wall modification, clathrin coat assembly, and cellular cell wall organization features. We also selected downregulated genes predicted to be directly regulated by three OsMADS genes through the analyses for promoter sequences. Thus, this study provides a molecular background for understanding pollen germination and tube growth mediated by OsMADS62, OsMADS63, and OsMADS68 with mature pollen preferred expression.