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Panax ginseng is a major medicinal crop with pharmaceutical efficacy derived from ginsenoside metabolites. Despite its genome information, the inefficiency of ginseng transformation hinders the study of the molecular mechanism of ginseng plant metabolism. Thus, this protocol aimed to develop an easy and efficient system for ginseng transformation. We established a transformation system using ginseng callus cultured in a liquid medium, which has a higher ratio compared with cotyledon explant. In addition, Agrobacterium tumefaciens has been used for plant transformation. Compared with the LBA4404 strain, C58C1 was inappropriate for ginseng transformation using ginseng callus. We induced and maintained calli in liquid medium and cut them into small pieces before infection. After infection, we selected calli that survived from the antibiotic medium until identification of newly growing cells. In β-glucuronidase (GUS) gene assay, the expression of the GUS gene was observed in cells that were newly generated from explants. We treated calli with 0.05 M of MgSO4 before infection. After MgSO4 pre-treatment, the transformation efficiency of growing cells around infected callus was increased. Moreover, we constructed and introduced a visible reporter RUBY system to easily identify transformed cells. Using this system, we could identify the cells by a red color with naked eyes. Based on our transformation protocol, the success rate has increased to 77.27% in surviving lines during selection culture. This stable ginseng transformation could facilitate the overexpression and knockout of ginseng lines for functional or synthetic biological studies.

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.

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.

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.

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.

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.

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.

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

The regulatory factor ZHD2 promotes root meristem activity and affects grain yield in rice by inducing ethylene biosynthesis., 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.

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.

Rice is an important food staple that is consumed by half of the human population. Therefore, understanding the regulatory mechanism of male fertility in rice can improve production by enhancing the efficiency of hybrid seed production. However, information on the control mechanism of male fertility by anther dehiscence or wall development in rice is very limited. To further understand the regulatory mechanism for anther dehiscence in rice, we carried out transcriptome analysis for 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 walls. 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 result could provide useful insight for future research to broaden the molecular mechanism of anther dehiscence or anther wall development in rice.

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.

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.

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.

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.

Plant homologs of mammalian NADPH oxidase, respiratory burst oxidase homologs (RBOH), mainly oxidize NADPH to NADP+ and transfer electrons to water. The membrane residing RBOHs thus produce reactive oxygen species (ROS), which allows plants to withstand abiotic and biotic environmental stresses. To understand the spatial and temporal function of rice (Oryza sativa) RBOH genes, we performed expression analysis of nine RBOH genes using qRT-PCR and microarray data. The expression profiling data suggest that RBOH genes have diverse roles in various tissues and organs as well as responses to hormonal treatments. Among them, OsRBOH2, OsRBOH3, and OsRBOH5 are preferentially expressed in root hairs. Exogenous auxin upregulates the expression of OsRBOH1, OsRBOH2, OsRBOH3, OsRBOH4, and OsRBOH8 in root hairs, while the expression of OsRBOH7 and OsRBOH9 is downregulated. In roots, treatment with an RBOH inhibitor, diphenyleneiodonium (DPI), suppressed the accumulation of ROS in trichoblast cells which initiate root hairs, suggesting that RBOH-mediated ROS could play an important role in root hair initiation in the trichoblast cells of rice roots. Promoter analysis revealed that OsRBOH3 and OsRBOH5 contain many known cis-acting regulatory elements (CREs) associated with root hair development such as the root hair cis-acting element, RHE. OsRBOH2 and OsRBOH3, which are upregulated after treatment with indole-3-acetic acid (IAA) with significant expression in root hairs, might be key players in root hair elongation via an auxin-dependent pathway in rice.

Root hairs are tubular shaped protuberances of root epidermal cells and are found in nearly all vascular plants. Co-ordinate expression of a number of root hair morphogenesis genes involved in cytoskeleton reorganization, changes in homeostasis and distribution of ion gradients, and cell wall reassembly are required during root hair cell elongation. In this report, we have characterized a root hairspecific putative cellulase gene in rice, OsRHC. OsRHC is specifically expressed in elongating root hairs and OsRHC is targeted to the plasma membrane. The mutation of the OsRHC gene by a T-DNA knock-out and CRISPR-Cas9 system causes a severe reduction in root hair length. Bimolecular fluorescence complementation analysis demonstrated that the OsRHC protein interacts with a root hair-specific cellulose synthase protein (OsCSLD1) in the plasma membrane. Furthermore, we observed a moderate reduction of cellulose content in the osrhc mutant. Our results suggest that the plasma membrane-localized OsRHC plays a critical role in cell wall remodeling during root hair extension.

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.

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.

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.

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.

Rice (Oryza sativa L.), a staple crop plant that is a major source of calories for approximately 50% of the human population, exhibits various physiological responses against temperature stress. These responses are known mechanisms of flexible adaptation through crosstalk with the intrinsic circadian clock. However, the molecular regulatory network underlining this crosstalk remains poorly understood. Therefore, we performed systematic transcriptome data analyses to identify the genes involved in both cold stress responses and diurnal rhythmic patterns. Here, we first identified cold-regulated genes and then identified diurnal rhythmic genes from those (119 cold-upregulated and 346 cold-downregulated genes). We defined cold-responsive diurnal rhythmic genes as CD genes. We further analyzed the functional features of these CD genes through Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses and performed a literature search to identify functionally characterized CD genes. Subsequently, we found that light-harvesting complex proteins involved in photosynthesis strongly associate with the crosstalk. Furthermore, we constructed a protein&ndash, protein interaction network encompassing four hub genes and analyzed the roles of the Stay-Green (SGR) gene in regulating crosstalk with sgr mutants. We predict that these findings will provide new insights in understanding the environmental stress response of crop plants against climate change.

Additional file 7: Table S1. All amplicon sequence data collected for re-analysis.

Additional file 1:. Materials and Methods.

Additional file 9: Table S2. OsRopGEF isogene-specific primers for qPCR and cloning.

Additional file 6: Figure S5. Bar plot of the integrated abundance the 16 most abundant phyla from sorghum under drought conditions. Ctrl, xxx, and DR refer to Control, well-watered, and drought state, respectively. Samples were grown under well-watered condition for 4 weeks to establish roots and then water was drained at 5 weeks after planting. Drought-stressed plants were sampled every week starting from 5 weeks after planting. Control samples were collected from a well-watered bathtub.

Additional file 4: Figure S3. Bar plot of the integrated abundance of the 16 most dominant phyla from endosphere data collected under drought stress. Each bar represents the mean value under each condition. The 16 most abundant phyla in each treatment are integrated, resulting in the identification of 16 phyla, including unassigned phyla. Only groups within the Proteobacteria phylum are presented as classes. Phyla other than those classified are clustered as “Others.” Detailed information on each sample is presented in Table S11.

Additional file 2: Figure S2. Meta-expression analysis of entire AtRopGEF genes. The heatmap was prepared using the Genevestigator. We chose five representative tissues, including pollen. It revealed that five Arabidopsis RopGEFs were highly expressed in pollen. The dark red color of the heatmap indicated the highest expression; white color, lowest expression.

Additional file 3: Figure S3. Protein sequence alignment domain analysis and conserved phosphorylated amino acid residues of C-termini of RopGEF genes. Every OsRopGEF and AtRopGEF protein sequence was collected and aligned, followed by the PRONE domain (C1, C2, C3) and the WW-motif. At the end part of the sequence, we found some conserved regions. According to previous studies, S510 in the C-terminus of AtRopGEF12 is involved in the C-terminal inhibition of GEF activity. As in AtRopGEF12, the serine residue is conserved in each of the OsRopGEF2, OsRopGEF3, and OsRopGEF8 genes but not in OsRopGEF6. However, K (Lysine) can also be phosphorylated.

Additional file 5: Figure S4. Bar plot of the 16 most abundant phyla from rhizosphere and rhizoplane data collected under drought stress. Each bar represents the mean value under each condition. The 16 most abundant phyla in each treatment are integrated, resulting in the identification of 16 phyla, including unassigned phyla. Only groups within the Proteobacteria are presented as classes. Phyla other than those classified are clustered as “Others.” Detailed information on each sample is presented in Table S11.

Additional file 3: Figure S2. Bar plot of the integrated abundance of the 16 most dominant phyla in each environment. Each bar represents the mean value under each condition. Phyla that are not included in the integrated set of 16 phyla are classified as “others.”

Additional file 6: Figure S6. Single mutant assay using CRISPR-Cas9 system. (a-b, d-e) In-vitro pollen germination test of wild-type (a, d), ropgef2 (b, c), and ropgef3 (e-f) single mutants on solid pollen media, Bar = 50 μm; (c,f) enlarged pollen pictures, Bar = 20 μm. Each control and mutant pollen in-vitro assays were performed in the same environment on the same day. (g) The ratio of pollen germination. Black bar indicates wild type; grey bar indicates ropgef2; white bar indicates ropgef3 mutant pollen. Error bars are standard deviation of three technical repeats. (h) Fertility ratio comparison between wild-type and ropgef2 single mutant plants. Error bars are standard deviation of more than three panicles in the plants.

Additional file 1: Figure S1. Meta-expression analysis and genome-wide identification of RopGEF in rice. The expressions from various rice tissues were examined using Microarray database. Yellow color in the heatmap indicates high level of expression; dark blue, low level of expression. Numeric values indicate the average of the normalized log2 intensity of microarray data.

Additional file 7: Figure S7. Meta-expression analysis and genome-wide identification of the seven OsRac and ten AtRop genes. (a) Heatmap expression analysis of OsRac gene. (b) Heatmap of AtRop using Genevestigator. (c) Phylogenetic tree constructs including every OsRac and AtRop.