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Understanding the intricacies of pollen tube growth in cereal crops, such as rice, is crucial for understanding crossbreeding, seed formation, and crop productivity. In this study, we investigated the molecular mechanisms underlying pollen tube germination and elongation in rice, focusing on the interaction between OsPUB14 and OsMTD2 and its impact on reactive oxygen species (ROS) regulation. Expression studies revealed that OsPUB14 was highly expressed in pollen and anther tissues, indicating its involvement in pollen function. We demonstrated that OsPUB14 belonging to group II U-box domain proteins, interacts with the kinase domain of OsMTD2 (a pollen-specific CrRLK1L member) and degrades it. This interaction subsequently reduces OsMTD2-mediated ROS generation. Moreover, the overexpression of OsPUB14 resulted in decreased ROS levels and reduced fertility in rice plants, emphasizing its role in reproductive processes. Yeast two-hybrid screening identified OsCRK10P and OsNET2D as potential interactors of OsPUB14, further expanding our understanding of the regulatory networks associated with pollen development. This study provides insight into the intricate interplay between pollen-specific plant U-box domain proteins (PUBs), demonstrating their roles in regulating ROS levels and ultimately influencing plant fertility. [ABSTRACT FROM AUTHOR]

Flooding stress caused by climate change is a serious threat to crop productivity. To enhance our understanding of flooding stress in soybean, we analyzed the transcriptome of the roots of soybean plants after waterlogging treatment for 10 days at the V2 growth stage. Through RNA sequencing analysis, 870 upregulated and 1129 downregulated differentially expressed genes (DEGs) were identified and characterized using Gene Ontology (GO) and MapMan software (version 3.6.0RC1). In the functional classification analysis, "alcohol biosynthetic process" was the most significantly enriched GO term in downregulated DEGs, and phytohormone-related genes such as ABA, cytokinin, and gibberellin were upregulated. Among the transcription factors (TFs) in DEGs, AP2/ERFs were the most abundant. Furthermore, our DEGs encompassed eight soybean orthologs from Arabidopsis and rice, such as 1-aminocyclopropane-1-carboxylate oxidase. Along with a co-functional network consisting of the TF and orthologs, the expression changes of those genes were tested in a waterlogging-resistant cultivar, PI567343. These findings contribute to the identification of candidate genes for waterlogging tolerance in soybean, which can enhance our understanding of waterlogging tolerance. [ABSTRACT FROM AUTHOR]

The transfer of sperm cells to the egg cell during pollen germination and pollen tube (PT) growth is an essential process for successful reproduction in higher plants. In this process, proper cell wall assembly and remodeling is important for the normal growth of PTs. The functions of members of the Beta(β)-Expansin (EXPB) family, which encode proteins that loosen cell walls, remain poorly understood. Here, we performed a meta-expression analysis of all OsEXPBs sourced from anatomical samples comprising 22 tissues and/or organs. We identified five pollen-preferred OsEXPBs (i.e., OsEXPB1a, OsEXPB1b, OsEXPB9, OsEXPB10, and OsEXPB13). We also identified gene duplication events that specifically involved pollen-preferred OsEXPBs. Subcellular localization of the OsEXPB proteins was found to match well with their roles as cell wall loosening factors, and these were also visible when in transit through the secretory pathway. Further functional characterization of OsEXPBs using a gene editing system for all five targets after removing probable redundancy revealed that a quintuple expb1a;1b;9;10;13 mutant was sterile due to defects in PT elongation. Taken together, the results of our study suggest that the role of five pollen-preferred OsEXPBs that share common expression patterns is important for normal PT growth in rice. [ABSTRACT FROM AUTHOR]

Female gametogenesis has been rarely studied due to gametophyte lethality and the unavailability of related genetic resources. In this study, we identified a rice ATP‐binding cassette transporter, OsABCB24, whose null function displayed a significantly reduced seed setting rate by as much as 94%–100% compared with that of the wild type (WT). The reciprocal cross of WT and mutant plants demonstrated that the female reproductive organs in mutants were functionally impaired. Confocal microscopy observations revealed that, although megasporogenesis remained unaffected in CRISPR/Cas9 osabcb24 mutants, the formation of female gametophytes was interrupted. Additionally, the structure of the syncytial nucleus was impaired during the initial stages of endosperm formation. Histochemical analysis showed that OsABCB24 was preferentially expressed at the conjunction of receptacle and ovary, spanning from the functional megaspore stage to the two‐nucleate embryo sac stage. Further, OsABCB24 was identified as an endoplasmic reticulum membrane‐localized protein. Notably, the overexpression of OsABCB24 triggered a 1.5‐ to 2‐fold increase in grain production compared to the WT. Our findings showed that OsABCB24 plays a key role in both female gametophyte development and the early development of seeds. [ABSTRACT FROM AUTHOR]

Plants, being sessile organisms, are exposed to a diverse range of environmental challenge. Over time, they have evolved various mechanisms to withstand these harsh conditions, often acquiring functional redundancy in genes with similar sequence. This genetic redundancy, while advantageous for the plant's adaptability, poses challenges in functional genomics research. To address this issue, we developed CAFRI-Arabidopsis (https://cafri.khu.ac.kr/arabidopsis/), a web resource designed to intuitively explore functional redundancy in Arabidopsis, a model plant that has significantly contributed to understanding the morphological and physiological traits. Our tool combines transcriptome data with Pfam-based phylogenetic data across 33 tissues/organs to visualize and quantify functional redundancy. Its effectiveness has been validated through a comprehensive literature review. User-friendly in design, CAFRI-Arabidopsis allows users to quickly generate results by inputting the desired locus. We anticipate that this tool will be invaluable in uncovering new insights in plant developmental and physiological studies that have been previously obscured due to functional redundancy. [ABSTRACT FROM AUTHOR]

Inorganic phosphate (Pi) homeostasis plays an important role in plant growth and abiotic stress tolerance. Several MYB-CC transcription factors involved in Pi homeostasis have been identified in rice (Oryza sativa). PHOSPHATE STARVATION RESPONSE-LIKE 7 (PHL7) is a class II MYC-CC protein, in which the MYC-CC domain is located at the N terminus. In this study, we established that OsPHL7 is localized to the nucleus and that the encoding gene is induced by Pi deficiency. The Pi-responsive genes and Pi transporter genes are positively regulated by OsPHL7. The overexpression of OsPHL7 enhanced the tolerance of rice plants to Pi starvation, whereas the RNA interference-based knockdown of this gene resulted in increased sensitivity to Pi deficiency. Transgenic rice plants overexpressing OsPHL7 produced more roots than wild-type plants under both Pi-sufficient and Pi-deficient conditions and accumulated more Pi in the shoots and roots. In addition, the overexpression of OsPHL7 enhanced rice tolerance to salt stress. Together, these results demonstrate that OsPHL7 is involved in the maintenance of Pi homeostasis and enhances tolerance to Pi deficiency and salt stress in rice. [ABSTRACT FROM AUTHOR]

Rice is an important cereal crop worldwide, the growth of which is affected by rice blast disease, caused by the fungal pathogen Magnaporthe oryzae. As climate change increases the diversity of pathogens, the disease resistance genes (R genes) in plants must be identified. The major blast-resistance genes have been identified in indica rice varieties; therefore, japonica rice varieties with R genes now need to be identified. Because leucine-rich repeat (LRR) domain proteins possess R-gene properties, we used bioinformatics analysis to identify the rice candidate LRR domain receptor-like proteins (OsLRR-RLPs). OsLRR-RLP2, which contains six LRR domains, showed differences in the DNA sequence, containing 43 single-nucleotide polymorphisms (SNPs) in indica and japonica subpopulations. The results of the M. oryzae inoculation analysis indicated that indica varieties with partial deletion of OsLRR-RLP2 showed susceptibility, whereas japonica varieties with intact OsLRR-RLP2 showed resistance. The oslrr-rlp2 mutant, generated using clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), showed increased pathogen susceptibility, whereas plants overexpressing this gene showed pathogen resistance. These results indicate that OsLRR-RLP2 confers resistance to rice, and OsLRR-RLP2 may be useful for breeding resistant cultivars. [ABSTRACT FROM AUTHOR]

Phosphate (Pi) starvation is a critical factor limiting crop growth, development, and productivity. Rice (Oryza sativa) R2R3-MYB transcription factors function in the transcriptional regulation of plant responses to various abiotic stresses and micronutrient deprivation, but little is known about their roles in Pi starvation signaling and Pi homeostasis. Here, we identified the R2R3-MYB transcription factor gene OsMYB58, which shares high sequence similarity with AtMYB58. OsMYB58 expression was induced more strongly by Pi starvation than by other micronutrient deficiencies. Overexpressing OsMYB58 in Arabidopsis thaliana and rice inhibited plant growth and development under Pi-deficient conditions. In addition, the overexpression of OsMYB58 in plants exposed to Pi deficiency strongly affected root development, including seminal root, lateral root, and root hair formation. Overexpressing OsMYB58 strongly decreased the expression of the rice microRNAs OsmiR399a and OsmiR399j. By contrast, overexpressing OsMYB58 strongly increased the expression of rice PHOSPHATE 2 (OsPHO2), whose expression is repressed by miR399 during Pi starvation signaling. OsMYB58 functions as a transcriptional repressor of the expression of its target genes, as determined by a transcriptional activity assay. These results demonstrate that OsMYB58 negatively regulates OsmiR399-dependent Pi starvation signaling by enhancing OsmiR399s expression. [ABSTRACT FROM AUTHOR]

Root hairs are extensions of epidermal cells on the root tips that increase the root contract surface area with the soil. For polar tip growth, newly synthesized proteins and other materials must be incorporated into the tips of root hairs. Here, we report the characterization of PRX102, a root hair preferential endoplasmic reticulum peroxidase. During root hair growth, PRX102 has a polar localization pattern within the tip regions of root hairs but it loses this polarity after growth termination. Moreover, PRX102 participates in root hair outgrowth by regulating dense cytoplasmic streaming toward the tip. This role is distinct from those of other peroxidases playing roles in the root hairs and regulating reactive oxygen species homeostasis. RNA-seq analysis using prx102 root hairs revealed that 87 genes including glutathione S-transferase were downregulated. Our results therefore suggest a new function of peroxidase as a player in the delivery of substances to the tips of growing root hairs. [ABSTRACT FROM AUTHOR]

Exogenous or endogenous caffeine application confers resistance to diverse biotic stresses in plants. In this study, we demonstrate that endogenous caffeine in caffeine-producing rice (CPR) increases tolerance even to abiotic stresses such as water deficit. Caffeine produced by CPR plants influences the cytosolic Ca2+ ion concentration gradient. We focused on examining the expression of Ca2+-dependent protein kinase genes, a subset of the numerous proteins engaged in abiotic stress signaling. Under normal conditions, CPR plants exhibited increased expressions of seven OsCPKs (OsCPK10, OsCPK12, OsCPK21, OsCPK25, OsCPK26, OsCPK30, and OsCPK31) and biochemical modifications, including antioxidant enzyme (superoxide dismutase, catalase, peroxidase, and ascorbate peroxidase) activity and non-enzymatic antioxidant (ascorbic acid) content. CPR plants exhibited more pronounced gene expression changes and biochemical alterations in response to water-deficit stress. CPR plants revealed increased expressions of 16 OsCPKs (OsCPK1, OsCPK2, OsCPK3, OsCPK4, OsCPK5, OsCPK6, OsCPK9, OsCPK10, OsCPK11, OsCPK12, OsCPK14, OsCPK16, OsCPK18, OsCPK22, OsCPK24, and OsCPK25) and 8 genes (OsbZIP72, OsLEA25, OsNHX1, OsRab16d, OsDREB2B, OsNAC45, OsP5CS, and OsRSUS1) encoding factors related to abiotic stress tolerance. The activity of antioxidant enzymes increased, and non-enzymatic antioxidants accumulated. In addition, a decrease in reactive oxygen species, an accumulation of malondialdehyde, and physiological alterations such as the inhibition of chlorophyll degradation and the protection of photosynthetic machinery were observed. Our results suggest that caffeine is a natural chemical that increases the potential ability of rice to cope with water-deficit stress and provides robust resistance by activating a rapid and comprehensive resistance mechanism in the case of water-deficit stress. The discovery, furthermore, presents a new approach for enhancing crop tolerance to abiotic stress, including water deficit, via the utilization of a specific natural agent. [ABSTRACT FROM AUTHOR]

Soil salinity has a negative effect on crop yield. Therefore, plants have evolved many strategies to overcome decreases in yield under saline conditions. Among these, E3‐ubiquitin ligase regulates salt tolerance. We characterized Oryza sativa Really Interesting New Gene (RING) Finger C3HC4‐type E3 ligase (OsRFPHC‐4), which plays a positive role in improving salt tolerance. The expression of OsRFPHC‐4 was downregulated by high NaCl concentrations and induced by abscisic acid (ABA) treatment. GFP‐fused OsRFPHC‐4 was localized to the plasma membrane of rice protoplasts. OsRFPHC‐4 encodes a cellular protein with a C3HC4‐RING domain with E3 ligase activity. However, its variant OsRFPHC‐4C161A does not possess this activity. OsRFPHC‐4‐overexpressing plants showed enhanced salt tolerance due to low accumulation of Na+ in both roots and leaves, low Na+ transport in the xylem sap, high accumulation of proline and soluble sugars, high activity of reactive oxygen species (ROS) scavenging enzymes, and differential regulation of Na+/K+ transporter expression compared to wild‐type (WT) and osrfphc‐4 plants. In addition, OsRFPHC‐4‐overexpressing plants showed higher ABA sensitivity under exogenous ABA treatment than WT and osrfphc‐4 plants. Overall, these results suggest that OsRFPHC‐4 contributes to the improvement of salt tolerance and Na+/K+ homeostasis via the regulation of changes in Na+/K+ transporters. [ABSTRACT FROM AUTHOR]

For a successful reproduction in angiosperms, pollen germination and pollen tube growth must proceed normally. The processes are regulated by transcriptional regulation and cellular activities, in which signaling proteins are the key. Ras homolog (Rho)-type GTPases (Rops), which are known as RACs, are a group of Rho small GTP-binding proteins which evolved in plants. However, the molecular mechanisms of the rice Rac family (OsRac) genes in pollen germination are not yet understood. In this investigation, the OsRac6 gene, which is only expressed only during the pollen development stage, was identified. A functional characterization of OsRac6 using gene-edited mutants exhibits male sterility due to the disruption of pollen germination. Then, to determine downstream candidate genes modulated by the OsRac6 genes during pollen development, we performed RNA-seq analysis by comparing osrac6-1 and wild-type (WT) anthers. Subsequently, we identified 652 genes with pollen-preferential expression which were upregulated compared with the WT, and 608 genes which were downregulated. Furthermore, gene ontology (GO) enrichment and MapMan analyses show that downregulated genes are closely associated with transcription, protein ubiquitination, proteolysis, cell wall modification, cell organization, cell cycle, and calcium regulation. The results of this study will help in elucidating the molecular mechanisms of pollen germination in rice mediated by OsRac6. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR/Cas9) system has widespread usage in the process of gene knockout. Due to the phenomenon of gene redundancy in higher organisms, there has been an increasing need for CRISPR/Cas9 vectors capable of editing multiple sites in the genome. In rice, a CRISPR/Cas9 vector that utilized a tandem-arrayed tRNA–gRNA structure for editing multiple sites in the genome demonstrated a remarkably efficient multiplex editing capability. To construct a CRISPR/Cas9 vector that utilizes a tandem-arrayed tRNA–gRNA structure, the Golden Gate assembly method has been used, which needs multi-step manipulations. Here, we designed a simple method to construct CRISPR/Cas9 vector with tandem-arrayed tRNA–gRNA structure for knocking out multiple sites in rice genome. This method enables easy construction of tandem-arrayed tRNA–gRNA structure via simple PCR and ligation steps. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Necrotrophic pathogen Cochliobolus miyabeanus (C. miyabeanus) causes rice brown leaf spot disease and drastically affects the yield and quality of rice grains. However, the molecular mechanism of rice-C. miyabeanus remains poorly understood due to the limited research conducted on this pathosystem. To elucidate the molecular mechanism of rice-C. miyabeanus, a transcriptome analysis was conducted from in vitro and in planta grown C. miyabeanus. This analysis led to the identification of a total of 24,060 genes of which 426 in vitro and 57 in planta expressed genes were predicted to encode for secretory proteins. As these 57 genes were specifically expressed in planta and were predicted to be secretory in nature, these were consider as putative effectors, highlighting their possible roles in the fungal pathogenicity. Notably, among these putative effectors, CmXyn1 which encodes a glycosyl hydrolase 11 displayed the highest expression level under in planta conditions and was thus selected for further functional characterization. Interestingly, the extracellular expression of CmXyn1 transiently induced cell death in Nicotiana benthamiana leaves, while intracellular expression was comparatively lesser effective. In addition, transcriptome analysis on rice leaves during C. miyabeanus infection and comparing it to the rice leaf transcriptome data obtained during hemibiotrophic pathogen Magnaporthe oryzae infection led to the discovery of 18 receptors/receptor-like kinases that were commonly expressed in response to both pathogens, indicating their key roles in rice defense response. Taken together, our findings provide new insights into rice-C. miyabeanus interaction as well as the unique and common defense responses of rice against hemibiotroph and necrotroph model systems. [ABSTRACT FROM AUTHOR]

Rice is an important staple food crop for over half of the world's population. However, abiotic stresses seriously threaten rice yield improvement and sustainable production. Breeding and planting rice varieties with high environmental stress tolerance are the most cost-effective, safe, healthy, and environmentally friendly strategies. In-depth research on the molecular mechanism of rice plants in response to different stresses can provide an important theoretical basis for breeding rice varieties with higher stress resistance. This review presents the molecular mechanisms and the effects of various abiotic stresses on rice growth and development and explains the signal perception mode and transduction pathways. Meanwhile, the regulatory mechanisms of critical transcription factors in regulating gene expression and important downstream factors in coordinating stress tolerance are outlined. Finally, the utilization of omics approaches to retrieve hub genes and an outlook on future research are prospected, focusing on the regulatory mechanisms of multi-signaling network modules and sustainable rice production. [ABSTRACT FROM AUTHOR]

The Rice Transporter Database (https://ricephylogenomics.ucdavis.edu/transporter/tree.php) integrates host functional genomic information for 1228 putative rice transporters. Here, when we analyzed diverse transcriptome data, we found that 270 transporter genes were preferentially expressed in tissue- and organ-specific patterns. Specifically, transcription of 205 genes was upregulated in response to abiotic stressors, such as salt, drought, cold, and heat; 231 genes were upregulated in response to hormones, such as ABA, gibberellic acid, cytokines, auxin, and jasmonic acid; and 60 genes were upregulated in response to pathogens, such as fungi, bacteria, viruses, and insects. Among them, the functions of 49 genes were characterized using the information acquired from the OGRO database. The association between the transporter families and featured expression patterns were also assessed. The ABC transporter family was found to be associated with root-preferential expression, the responses of M. oryzae and Xoo to infection, and treatment with the hormone JA. On the other hand, the TAT transporter family was found to be significantly enriched in the categories of heat stress and preferential shoot/leaf-expression. POT was highly enriched when coupled with M. oryzae/Xoo infection and tZ hormone treatment. The application to identify the function of predicted ABA responded OsABCC7 transporter genes in rice revealed that the expression of OsABCC7 was triggered by exogenous ABA and that the CRISPR–Cas9 mutant was sensitive to ABA. These predicted results can serve as a premise for more specific studies in the future and contribute to the foundation of transporter research in rice. [ABSTRACT FROM AUTHOR]

We reported in our recent studies that the combined treatment of Paenibacillus yonginensis DCY84T (DCY84T) and Silicon (Si) promotes initial plant growth and increases resistance to biotic and abiotic stress. To understand the molecular background of these phenotypes, Liquid Chromatography Mass Spectrometry (LC–MS) analysis was performed, and it was confirmed that unsaturated fatty acid metabolites such as oleic acid and linoleic acid decreased in response to the combined treatment of DCY84T and Si. The stearoyl-acyl carrier protein desaturase (SACPD) introduces the cis double bond into the acyl-ACPs at C9, resulting in the production of unsaturated fatty acid. We identified OsSSI2 encoding SACPD in rice and found that the expression of OsSSI2 was reduced under DCY84T and Si treatment. Furthermore, qRT-PCR analysis revealed that the expression of OsWRKY45, which is downstream of OsSSI2, was upregulated in response to DCY84T and Si treatment. These results enable the speculation that activation of the salicylic acid (SA)-responsive gene, OsWRKY45, may contribute to enhancing biological stress resistance. Based on this, we propose a probable model for the rice defense pathway following DCY84T and Si treatment. This model retains a WRKY45-dependent but NH1(NPR1)-independent SA signaling pathway. [ABSTRACT FROM AUTHOR]

Glycoside hydrolase family 18 (GH18) genes are known as chitinase, which catalyzes the ß-1,4-glycosidic bond in amino polysaccharides. GH18 genes have been found in diverse organisms with multiple physiological roles. Despite their vital role in plant defense, the role of the GH18 family in ginseng (Panax ginseng) has not been investigated to date. In this study, we identified 26 members of the GH18 family from the ginseng genome and cloned a Class III member, PgGH18. Expression of PgGH18 was highly up-regulated in response to salt stress in ginseng. In subcellular localization analysis, PgGH18 protein was predominantly identified in the cytoplasm, and signals in the plasma membrane were connected with apoplast by plasmolysis. Overexpression of PgGH18 in Arabidopsis exhibited a higher germination ratio and maintained longer root growth than the wild type (WT) under salt stress. When treated with salt stress, it was observed that reactive oxygen species (ROS) accumulated more in transformants than it did in the WT. Our study suggests that a Class III member of the GH18 gene family, PgGH18, plays a role in abiotic stress defense in ginseng rather than fungal response. PgGH18 functions as a positive regulator of salinity tolerance, representing an important target for developing salt-tolerant ginseng cultivars via genetic engineering or breeding marker. [ABSTRACT FROM AUTHOR]

In the angiosperm, pollen germinates and rapidly expands the pollen tube toward the ovule. This process is important for plant double fertilization and seed setting. It is well known that the tip-focused calcium gradient is essential for pollen germination and pollen tube growth. However, little is known about the Ca2+ channels that play a role in rice pollen germination and tube growth. Here, we divided the 16 cyclic nucleotide-gated channel (CNGC) genes from rice into five subgroups and found two subgroups (clades II and III) have pollen-preferential genes. Then, we performed a meta-expression analysis of all OsCNGC genes in anatomical samples and identified three pollen-preferred OsCNGCs (OsCNGC4, OsCNGC5, and OsCNGC8). The subcellular localization of these OsCNGC proteins is matched with their roles as ion channels on the plasma membrane. Unlike other OsCNGCs, these genes have a unique cis-acting element in the promoter. OsCNGC4 can act by forming a homomeric complex or a heteromeric complex with OsCNGC5 or OsCNGC8. In addition, it was suggested that they can form a multi-complex with Mildew Resistance Locus O (MLO) protein or other types of ion transporters, and that their expression can be modulated by Ruptured Pollen tube (RUPO) encoding receptor-like kinase. These results shed light on understanding the regulatory mechanisms of pollen germination and pollen tube growth through calcium channels in rice. [ABSTRACT FROM AUTHOR]

SUMMARY: Grass xylan, the major hemicellulose in both primary and secondary cell walls, is heavily decorated with α‐1,3‐linked arabinofuranosyl (Araf) residues that may be further substituted at O‐2 with xylosyl (Xyl) or Araf residues. Although xylan 3‐O‐arabinosyltransferases (XATs) catalyzing 3‐O‐Araf addition onto xylan have been characterized, glycosyltransferases responsible for the transfer of 2‐O‐Xyl or 2‐O‐Araf onto 3‐O‐Araf residues of xylan to produce the Xyl‐Araf and Araf‐Araf disaccharide side chains remain to be identified. In this report, we showed that a rice GT61 member, named OsXAXT1 (xylan arabinosyl 2‐O‐xylosyltransferase 1) herein, was able to mediate the addition of Xyl‐Araf disaccharide side chains onto xylan when heterologously co‐expressed with OsXAT2 in the Arabidopsis gux1/2/3 (glucuronic acid substitution of xylan 1/2/3) triple mutant that lacks any glycosyl substitutions. Recombinant OsXAXT1 protein expressed in human embryonic kidney 293 cells exhibited a xylosyltransferase activity catalyzing the addition of Xyl from UDP‐Xyl onto arabinosylated xylooligomers. Consistent with its function as a xylan arabinosyl 2‐O‐xylosyltransferase, CRISPR‐Cas9‐mediated mutations of the OsXAXT1 gene in transgenic rice plants resulted in a reduction in the level of Xyl‐Araf disaccharide side chains in xylan. Furthermore, we revealed that XAXT1 close homologs from several other grass species, including switchgrass, maize, and Brachypodium, possessed the same functions as OsXAXT1, indicating functional conservation of XAXTs in grass species. Together, our findings establish that grass XAXTs are xylosyltransferases catalyzing Xyl transfer onto O‐2 of Araf residues of xylan to form the Xyl‐Araf disaccharide side chains, which furthers our understanding of genes involved in xylan biosynthesis. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

SUMMARY: 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. Significance Statement: Cytokinins mediate various aspects of plant development including flowering time. Here we show that exogenous cytokinin application prolongs the duration of the vegetative phase by suppressing florigen expression in rice (Oryza sativa) and maize (Zea mays). Further experiments elucidated the genetic and molecular mechanisms influencing the transition from vegetative growth to the reproductive phase by the hormone treatment. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Epitranscriptomics has added a new layer of regulatory machinery to eukaryotes, and the advancement of sequencing technology has revealed more than 170 post-transcriptional modifications in various types of RNAs, including messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), and long non-coding RNA (lncRNA). Among these, N6-methyladenosine (m6A) and N5-methylcytidine (m5C) are the most prevalent internal mRNA modifications. These regulate various aspects of RNA metabolism, mainly mRNA degradation and translation. Recent advances have shown that regulation of RNA fate mediated by these epitranscriptomic marks has pervasive effects on a plant's development and responses to various biotic and abiotic stresses. Recently, it was demonstrated that the removal of human-FTO-mediated m6A from transcripts in transgenic rice and potatoes caused a dramatic increase in their yield, and that the m6A reader protein mediates stress responses in wheat and apple, indicating that regulation of m6A levels could be an efficient strategy for crop improvement. However, changing the overall m6A levels might have unpredictable effects; therefore, the identification of precise m6A levels at a single-base resolution is essential. In this review, we emphasize the roles of epitranscriptomic modifications in modulating molecular, physiological, and stress responses in plants, and provide an outlook on epitranscriptome engineering as a promising tool to ensure food security by editing specific m6A and m5C sites through robust genome-editing technology. [ABSTRACT FROM AUTHOR]

Floral organ development and regulation of flowering time in rice is closely associated with MADS-box transcriptional factors (TFs). MADS-box proteins contain a conserved 60 amino acid MADS-box motif for a sequence-specific DNA-binding activity that also functions in dimerization and accessory factor binding. A systematic understanding of this MADS-box TF subfamily is needed to explain the functional complexity among the gene family members. In total, the function of 33 out of 75 MADS-box TFs in rice has been characterized. Meta-anatomical expression data based on a large collection of spatiotemporal microarray data integrated to the phylogenetic tree context was very effective at explaining the functional complexity or specificity among a large gene family. Subsequently, a cluster of MADS-box TFs consisting of 15 gene members showed preferred expression in floral organs, and their expression patterns are well correlated with previously identified functions in floral organ identity. In addition, the hierarchical structure of a group of MADS-box TFs, based on the expression patterns, was well supported by the genetic studies. OsMADS1 functions in the earliest step of floral organ development and affects the overall floral organ development by identifying downregulation of nine members in the same clade in osmads1 mutant and observed phenotypic changes. The combination of hierarchical structure and possible hetero-dimerization among MADS-box TFs allows for understanding the molecular and genetic regulatory model for floral organ development in rice. This work provides a comprehensive understanding of the functions of MADS-box TFs and will be a useful guide for further studies. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Summary: 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. [ABSTRACT FROM AUTHOR]

The preparation of microsomal membrane proteins (MPs) is critically important to microsomal proteomics. To date most research studies have utilized an ultracentrifugation-based approach for the isolation and solubilization of plant MPs. However, these approaches are labor-intensive, time-consuming, and unaffordable in certain cases. Furthermore, the use of sodium dodecyl sulfate (SDS) and its removal prior to a mass spectrometry (MS) analysis through multiple washing steps result in the loss of proteins. To address these limitations, this study introduced a simple micro-centrifugation-based MP extraction (MME) method from rice leaves, with the efficacy of this approach being compared with a commercially available plasma membrane extraction kit (PME). Moreover, this study assessed the subsequent solubilization of isolated MPs in an MS-compatible surfactant, namely, 4-hexylphenylazosulfonate (Azo) and SDS using a label-free proteomic approach. The results validated the effectiveness of the MME method, specifically in the enrichment of plasma membrane proteins as compared with the PME method. Furthermore, the findings showed that Azo demonstrated several advantages over SDS in solubilizing the MPs, which was reflected through a label-free quantitative proteome analysis. Altogether, this study provided a relatively simple and rapid workflow for the efficient extraction of MPs with an Azo-integrated MME approach for bottom-up proteomics. [ABSTRACT FROM AUTHOR]

SUMMARY: 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. Significance Statement: In rice, OsMTD2 interacts with SPL11 and regulates ROS generation in pollen grain and tube, which is critical for cell‐wall modification, thus an intact tube elongation. [ABSTRACT FROM AUTHOR]

Pre-harvest sprouting (PHS) is one of the primary problems associated with seed dormancy in rice (Oryza sativa L.). It causes yield loss and reduces grain quality under unpredictable humid conditions at the ripening stage, thus affecting the economic value of the rice crop. To resolve this issue, understanding the molecular mechanism underlying seed dormancy in rice is important. Recent studies have shown that seed dormancy is affected by a large number of genes associated with plant hormone regulation. However, understanding regarding the effect of heat stress on seed dormancy and plant hormones is limited. This study compared the transcriptome and small RNAome of the seed embryo and endosperm of two contrasting japonica rice accessions, PHS susceptible (with low seed dormancy) and PHS resistant (with high seed dormancy), at three different maturation stages. We found that 9,068 genes and 35 microRNAs (miRNAs) were differentially expressed in the embryo, whereas 360 genes were differentially expressed in the endosperm. Furthermore, we identified and verified the candidate genes associated with seed dormancy and heat stress-related responses in rice using quantitative real-time PCR. We newly discovered eight hormone-related genes, four heat shock protein-related genes, and two miRNAs potentially involved in PHS. These findings provide a strong foundation for understanding the dynamics of transcriptome and small RNAome of hormone- and heat stress-related genes, which affect PHS during seed maturation. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Summary: Successful delivery of sperm cells to the embryo sac in higher plants is mediated by pollen tube growth. The molecular mechanisms underlying pollen germination and tube growth in crop plants remain rather unclear, although these mechanisms are crucial to plant reproduction and seed formation. By screening pollen‐specific gene mutants in rice (Oryza sativa), we identified a T‐DNA insertional mutant of Germinating modulator of rice pollen (GORI) that showed a one‐to‐one segregation ratio for wild type (WT) to heterozygous. GORI encodes a seven‐WD40‐motif protein that is homologous to JINGUBANG/REN4 in Arabidopsis. GORI is specifically expressed in rice pollen, and its protein is localized in the nucleus, cytosol and plasma membrane. Furthermore, a homozygous mutant, gori‐2, created through CRISPR‐Cas9 clearly exhibited male sterility with disruption of pollen tube germination and elongation. The germinated pollen tube of gori‐2 exhibited decreased actin filaments and altered pectin distribution. Transcriptome analysis revealed that 852 pollen‐specific genes were downregulated in gori‐2 compared with the WT, and Gene Ontology enrichment analysis indicated that these genes are strongly associated with cell wall modification and clathrin coat assembly. Based on the molecular features of GORI, phenotypical observation of the gori mutant and its interaction with endocytic proteins and Rac GTPase, we propose that GORI plays key roles in forming endo‐/exocytosis complexes that could mediate pollen tube growth in rice. Significance Statement: Pollen tubes deliver sperm cells for fertilization in flowering plants, producing seeds. We found that Germinating modulator of rice pollen (GORI), a rice gene encoding a seven‐WD40‐domain protein, is essential for initiating pollen germination and tube elongation. Using CRISPR‐Cas9 gene editing, we created a homozygous mutant that exhibited male sterility through a pollen tube growth defect owing to altered pectin and actin distribution and imbalance between endocytosis and exocytosis. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Oryza sativa (rice) is an economically important crop, which is valued as a staple food source and as genetic model for cereals. Rice production needs to increase substantially to meet the growing global demand. Genetic studies have indicated that anther and pollen development genes can be used as targets to improve yield. Although several genes related to male organ developmental pathways have been characterized, the complete signaling network remains unclear. We used publicly available anther and pollen development transcriptome datasets to identify candidates for functional genomics studies. In addition, we newly generated RNA-Seq data for anther, anther wall and pollen samples with leaf sheath as control to provide the expression data for genes that are not covered by microarray technologies in rice. We constructed a platform, the rice male gamete expression database (RMEDB), comprising microarray and RNA-Seq based 188 transcriptome samples across diverse rice tissues and developmental stages of O. sativa japonica and O. sativa indica. Meta-expression analysis identified genes expressed at specific stages of anther or pollen developments. The functionalities in RMEDB database (http://ricephylogenomics-khu.org/RMEDB/home.php) can serve to identify candidates in male organ development for functional genomic studies of rice. [ABSTRACT FROM AUTHOR]

Sheath blight disease (ShB) severely affects rice production; however, the details of defense against ShB remain unclear. To understand the rice defense mechanism against ShB, an RNA sequencing analysis was performed using Rhizoctonia solani inoculated rice leaves after 48 h of inoculation. Among them, 3417 genes were upregulated and 2532 were downregulated when compared with the control group (> twofold or < 1/2). In addition, the differentially expressed genes were classified via Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and MapMan analyses. Fifty-nine GO terms and seven KEGG pathways were significantly enriched. A MapMan analysis demonstrated that the phytohormone and metabolic pathways were significantly altered. Interestingly, the expression levels of 359 transcription factors, including WRKY, MYB, and NAC family members, as well as 239 transporter genes, including ABC, MFS, and SWEET, were significantly changed in response to R. solani AG1-IA inoculation. Additionally, OsWRKY53 and OsAKT1 negatively regulate the defense response in rice against R. solani via gain of function study for OsWRKY53 and loss of function study for OsAKT1, respectively. Furthermore, several differentially expressed genes contain R. solani-responsive cis acting regulatory elements in their promoter regions. Taken together, our analyses provide valuable information for the additional study of the defense mechanisms against ShB, and the candidate genes identified in this study will be useful resource for future breeding to enhance resistance against ShB. [ABSTRACT FROM AUTHOR]

SUMMARY: Rice (Oryza sativa L.) is a staple crop with agricultural traits that have been intensively investigated. However, despite the variety of mutant population and multi‐omics data that have been generated, rice functional genomic research has been bottlenecked due to the functional redundancy in the genome. This phenomenon has masked the phenotypes of knockout mutants by functional compensation and redundancy. Here, we present an intuitive tool, CRISPR applicable functional redundancy inspector to accelerate functional genomics in rice (CAFRI‐Rice; cafri‐rice.khu.ac.kr). To create this tool, we generated a phylogenetic heatmap that can estimate the similarity between protein sequences and expression patterns, based on 2,617 phylogenetic trees and eight tissue RNA‐sequencing datasets. In this study, 33,483 genes were sorted into 2,617 families, and about 24,980 genes were tested for functional redundancy using a phylogenetic heatmap approach. It was predicted that 7,075 genes would have functional redundancy, according to the threshold value validated by an analysis of 111 known genes functionally characterized using knockout mutants and 5,170 duplicated genes. In addition, our analysis demonstrated that an anther/pollen‐preferred gene cluster has more functional redundancy than other clusters. Finally, we showed the usefulness of the CAFRI‐Rice‐based approach by overcoming the functional redundancy between two root‐preferred genes via loss‐of‐function analyses as well as confirming the functional dominancy of three genes through a literature search. This CAFRI‐Rice‐based target selection for CRISPR/Cas9‐mediated mutagenesis will not only accelerate functional genomic studies in rice but can also be straightforwardly expanded to other plant species. Significance Statement: Although a huge number of gene‐indexed mutant population and multi‐omics data are available, functional genomic studies in rice, which is a representative crop model, are still not making rapid progress. The main reason for this is the redundancy of gene function in the genome; to overcome this limitation, the best candidate gene(s) for gene editing technology is (are) proposed through the CAFRI‐Rice tool, which combines phylogenetic tree and transcriptome analyses. [ABSTRACT FROM AUTHOR]

The AMT1 family comprises major ammonium transporters in rice roots. In this study, we utilized AMT1 RNAi mutants (amt1) to explore how AMT1 affects NH4+- and NO3–-mediated morphological development and NH4+-responsive gene expression in roots. In the presence of NH4+, amt1 showed inhibition of NO3–- dependent lateral root development. The inhibitory action of NH4+ on lateral root growth was independent of the NO3– concentrations supplied to amt1 roots. The results of split root assays indicated that NH4+ exerts systemic action in inhibiting NO3–-dependent lateral root development in amt1. Further study with NAA and NOA, a potent auxin flux inhibitor, suggested that perturbation of membrane dynamics might not be the primary cause of the inhibitory action of NH4+ on NO3–-mediated lateral root growth in amt1 mutants. RNA-seq analysis of NH4+-responsive genes showed that approximately half of DEGs observed in wild-type roots were not detected in the DEGs of amt1 roots. Gene ontology enrichment analysis suggested that the expression of specific functional gene groups were affected by amt1 during the early response to NH4+. Auxin-responsive gene expression and root gravity responses were altered in amt1. This study demonstrated that AMT1 affects the interactions not only between ammonium and nitrate in lateral root growth but also between auxin and NH4+ in rice roots. [ABSTRACT FROM AUTHOR]

Root meristem activity is the most critical process influencing root development. Although several factors that regulate meristem activity have been identified in rice, studies on the enhancement of meristem activity in roots are limited. We identified a T-DNA activation tagging line of a zinc-finger homeobox gene, OsZHD2 , which has longer seminal and lateral roots due to increased meristem activity. The phenotypes were confirmed in transgenic plants overexpressing OsZHD2. In addition, the overexpressing plants showed enhanced grain yield under low nutrient and paddy field conditions. OsZHD2 was preferentially expressed in the shoot apical meristem and root tips. Transcriptome analyses and quantitative real-time PCR experiments on roots from the activation tagging line and the wild type showed that genes for ethylene biosynthesis were up-regulated in the activation line. Ethylene levels were higher in the activation lines compared with the wild type. ChIP assay results suggested that OsZHD2 induces ethylene biosynthesis by controlling ACS5 directly. Treatment with ACC (1-aminocyclopropane-1-carboxylic acid), an ethylene precursor, induced the expression of the DR5 reporter at the root tip and stele, whereas treatment with an ethylene biosynthesis inhibitor, AVG (aminoethoxyvinylglycine), decreased that expression in both the wild type and the OsZHD2 overexpression line. These observations suggest that OsZHD2 enhances root meristem activity by influencing ethylene biosynthesis and, in turn, auxin. [ABSTRACT FROM AUTHOR]

Main conclusion: A new imaging platform was constructed to analyze drought-tolerant traits of rice. Rice was used to quantify drought phenotypes through image-based parameters and analyzing tools. Climate change has increased the frequency and severity of drought, which limits crop production worldwide. Developing new cultivars with increased drought tolerance and short breeding cycles is critical. However, achieving this goal requires phenotyping a large number of breeding populations in a short time and in an accurate manner. Novel cutting-edge technologies such as those based on remote sensors are being applied to solve this problem. In this study, new technologies were applied to obtain and analyze imaging data and establish efficient screening platforms for drought tolerance in rice using the drought-tolerant mutant osphyb. Red–Green–Blue images were used to predict plant area, color, and compactness. Near-infrared imaging was used to determine the water content of rice, infrared was used to assess plant temperature, and fluorescence was used to examine photosynthesis efficiency. DroughtSpotter technology was used to determine water use efficiency, plant water loss rate, and transpiration rate. The results indicate that these methods can detect the difference between tolerant and susceptible plants, suggesting their value as high-throughput phenotyping methods for short breeding cycles as well as for functional genetic studies of tolerance to drought stress. [ABSTRACT FROM AUTHOR]

The tapetum plays a crucial role in pollen development by nursing and releasing the microspore. The tapetum undergoes programmed cell death (PCD), and appropriate timing of PCD is essential for microsporogenesis. Undeveloped Tapetum1 (UDT1) is known to function in anther development, but the regulation mechanism of tapetum differentiation and degeneration by UDT1 is largely unknown. Through comparative transcriptome analysis, we selected 100 genes as udt1 downregulated genes. The biological process related to the negative regulation of translation is the most enriched in udt1 downregulated genes. It is attributed by ribosome inactivating proteins (RIPs), but the role of RIPs is not well defined, and they are assumed to participate in tapetal PCD. Lipid transport is another overrepresented Gene Ontology (GO) term in udt1 downregulated genes, indicating the functional loss of the tapetum as the nursing cell. Using comparative analysis and real-time PCR, we infer that UDT1 can trigger tapetal PCD by controlling the expression of RIPs and three aspartyl proteases (AP) grouped with OsAP37, which is well known as being involved in tapetum degeneration. [ABSTRACT FROM AUTHOR]

Rice (Oryza sativa. L) has been intensively studied to ensure a stable global supply of this commodity in the face of rapid global climate change. A critical factor that decreases crop yield is drought, which has been analyzed in various ways through many researches. Microbiome-based studies of rice investigate the symbiosis between rice and bacteria, which has been proposed as a way to overcome problems caused by drought. Several rice-associated metagenomic profiles obtained under drought conditions have been reported since the advent of next generation sequencing (NGS) technology. To elucidate the future diversity of plants and microorganisms and to promote sustainable agriculture, we reanalyzed 64 of the publicly available 16S amplicon sequencing data produced under drought condition. In the process of integrating data sets, however, we found an inconsistency that serves as a bottleneck for microbiome-based sustainability research. While this report provides clues about the composition of the microbiome under the drought conditions, the results are affected by differences in the location of the experiments, sampling conditions, and analysis protocols. Re-analysis of amplicon sequencing data of the soil microbiome in rice fields suggests that microbial composition shifts in response to drought condition and the presence of plants. Among the bacteria involved, the phylum Proteobacteria appears to play the most important role in the survival of rice under drought condition. [ABSTRACT FROM AUTHOR]

The mutant allele of rice calmodulin-binding transcription activator OsCBT, oscbt-1, exhibits broad-spectrum resistance against rice pathogens. Previously, we reported that the strong resistance of the oscbt-1 mutant to pathogens was conferred by a constitutive upregulation of defense-related genes even under pathogen-free conditions. We also found strong induction of the hypersensitive response as a reaction to pathogen invasion. The results suggest that OsCBT acts as a negative regulator of basal resistance to pathogen attack. To identify the transcriptional network regulated by OsCBT, we compared global gene expression profiles between wild-type (WT) and oscbt-1 rice plants grown under pathogen-free conditions. The results of a 3′ tiling microarray revealed that in oscbt-1 plants, 81 genes are upregulated and 200 genes are downregulated when compared with the WT. A gene ontology analysis showed that differentially regulated genes in oscbt-1 were very closely associated with "death" GO term in a biological process category, and "catalytic activity" and "binding" GO terms in a molecular function category. A MapMan analysis indicated that the functions of these genes were associated with plant responses to biotic stress. Moreover, the results from quantitative real-time PCR in the oscbt-1 mutant showed a significant effect on the gene expression patterns of the fungal pathogen response. Our results suggested that the OsCBT regulates a rice defense response by modulating the expressions of various defense-related genes. [ABSTRACT FROM AUTHOR]