OsMYB58 Negatively Regulates Plant Growth and Development by Regulating Phosphate Homeostasis.

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.

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

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

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

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

GORI, encoding the WD40 domain protein, is required for pollen tube germination and elongation in rice.

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.

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

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

CAFRI-Rice: CRISPR applicable functional redundancy inspector to accelerate functional genomics in rice.

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.

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

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

A Multiprotein Complex Regulates Interference-Sensitive Crossover Formation in Rice.

In most eukaryotes, a set of conserved proteins that are collectively termed ZMM proteins (named for molecular zipper 1 [ZIP1], ZIP2, ZIP3, and ZIP4, MutS homologue 4 [MSH4] and MSH5, meiotic recombination 3, and sporulation 16 [SPO16] in yeast [Saccharomyces cerevisiae]) are essential for the formation of the majority of meiotic crossovers (COs). Recent reports indicated that ZIP2 acts together with SPO16 and ZIP4 to control CO formation through recognizing and stabilizing early recombination intermediates in budding yeast. However, whether this mechanism is conserved in plants is not clear. Here, we characterized the functions of SHORTAGE OF CHIASMATA 1 (OsSHOC1; ZIP2 ortholog) and PARTING DANCERS (OsPTD; SPO16 ortholog) and their interactions with other ZMM proteins in rice (Oryza sativa). We demonstrated that disruption of OsSHOC1 caused a reduction of CO numbers to ∼83% of wild-type CO numbers, whereas synapsis and early meiotic recombination steps were not affected. Furthermore, OsSHOC1 interacts with OsPTD, which is responsible for the same set of CO formations as OsSHOC1. In addition, OsSHOC1 and OsPTD are required for the normal loading of other ZMM proteins, and conversely, the localizations of OsSHOC1 and OsPTD were also affected by the absence of OsZIP4 and human enhancer of invasion 10 in rice (OsHEI10). OsSHOC1 interacts with OsZIP4 and OsMSH5, and OsPTD interacts with OsHEI10. Furthermore, bimolecular fluorescence complementation and yeast-three hybrid assays demonstrated that OsSHOC1, OsPTD, OsHEI10, and OsZIP4 were able to form various combinations of heterotrimers. Moreover, statistical and genetic analysis indicated that OsSHOC1 and OsPTD are epistatic to OsHEI10 and OsZIP4 in meiotic CO formation. Taken together, we propose that OsSHOC1, OsPTD, OsHEI10, and OsZIP4 form multiple protein complexes that have conserved functions in promoting class I CO formation.

RSL Class II Transcription Factors Guide the Nuclear Localization of RHL1 to Regulate Root Hair Development.

Root hairs are important for absorption of nutrients and water from the rhizosphere. The Root Hair Defective-Six Like (RSL) Class II family of transcription factors is expressed preferentially in root hairs and has a conserved role in root hair development in land plants. We functionally characterized the seven members of the RSL Class II subfamily in the rice (Oryza sativa) genome. In root hairs, six of these genes were preferentially expressed and four were strongly expressed. Phenotypic analysis of each mutant revealed that Os07g39940 plays a major role in root hair formation, based on observations of a short root hair phenotype in those mutants. Overexpression (OX) for each of four family members in rice resulted in an increase in the density and length of root hairs. These four members contain a transcription activation domain and are targeted to the nucleus. They interact with rice Root Hairless1 (OsRHL1), a key regulator of root hair development. When heterologously expressed in epidermal cells of Nicotiana benthamiana leaves, OsRHL1 was predominantly localized to the cytoplasm. When coexpressed with each of the four RSL Class II members, however, OsRLH1 was translocated to the nucleus. Transcriptome analysis using Os07g39940-OX plants revealed that 86 genes, including Class III peroxidases, were highly up-regulated. Furthermore, reactive oxygen species levels in the root hairs were increased in Os07g39940-OX plants but were drastically reduced in the os07g39940 and rhl1 mutants. Our results demonstrate that RSL Class II members function as essential regulators of root hair development in rice.

Deficiency of rice hexokinase HXK5 impairs synthesis and utilization of starch in pollen grains and causes male sterility.

There is little known about the function of rice hexokinases (HXKs) in planta. We characterized hxk5-1, a Tos17 mutant of OsHXK5 that is up-regulated in maturing pollen, a stage when starch accumulates. Progeny analysis of self-pollinated heterozygotes of hxk5-1 and reciprocal crosses between the wild-type and heterozygotes revealed that loss of HXK5 causes male sterility. Homozygous hxk5-1, produced via anther culture, and additional homozygous hxk5-2, hxk5-3 and hxk5-4 lines created by CRISPR/Cas9 confirmed the male-sterile phenotype. In vitro pollen germination ability and in vivo pollen tube growth rate were significantly reduced in the hxk5 mutant pollen. Biochemical analysis of anthers with the mutant pollen revealed significantly reduced hexokinase activity and starch content, although they were sufficient to produce some viable seed. However, the mutant pollen was unable to compete successfully against wild-type pollen. Expression of the catalytically inactive OsHXK5-G113D did not rescue the hxk5 male-sterile phenotype, indicating that its catalytic function was responsible for pollen fertility, rather than its role in sugar sensing and signaling. Our results demonstrate that OsHXK5 contributes to a large portion of the hexokinase activity necessary for the starch utilization pathway during pollen germination and tube growth, as well as for starch biosynthesis during pollen maturation.

Meta-expression analysis of unannotated genes in rice and approaches for network construction to suggest the probable roles.

KEY MESSAGE: This work suggests 2020 potential candidates in rice for the functional annotation of unannotated genes using meta-analysis of anatomical samples derived from microarray and RNA-seq technologies and this information will be useful to identify novel morphological agronomic traits. Although the genome of rice (Oryza sativa) has been sequenced, 14,365 genes are considered unannotated because they lack putative annotation information. According to the Rice Genome Annotation Project Database ( http://rice.plantbiology.msu.edu/ ), the proportion of functionally characterized unannotated genes (0.35%) is quite limited when compared with the approximately 3.9% of annotated genes with assigned putative functions. Researchers require additional information to help them investigate the molecular mechanisms associated with those unannotated genes. To determine which of them might regulate morphological or physiological traits in the rice genome, we conducted a meta-analysis of expression data that covered a wide range of tissue/organ samples. Overall, 2020 genes showed cultivar-, tissue-, or organ-preferential patterns of expression. Representative candidates from featured groups were validated by RT-PCR, and the GUS reporter system was used to validate the expression of genes that were clustered according to their leaf or root preference. Taking a molecular and genetics approach, we examined meta-expression data and found that 127 genes were differentially expressed between japonica and indica rice cultivars. This is potentially significant for future agronomic applications. We also used a T-DNA insertional mutant and performed a co-expression network analysis of Sword shape dwarf1 (SSD1), a gene that regulates cell division. This network was refined via RT-PCR analysis. Our results suggested that SSD1 represses the expression of four genes related to the processes of DNA replication or cell division and provides insight into possible molecular mechanisms. Together, these strategies present a valuable tool for in-depth characterization of currently unannotated genes.

Defective Tapetum Cell Death 1 (DTC1) Regulates ROS Levels by Binding to Metallothionein during Tapetum Degeneration.

After meiosis, tapetal cells in the innermost anther wall layer undergo program cell death (PCD)-triggered degradation. This step is essential for microspore development and pollen wall maturation. We identified a key gene, Defective Tapetum Cell Death 1 (DTC1), that controls this degeneration by modulating the dynamics of reactive oxygen species (ROS) during rice male reproduction. Mutants defective in DTC1 exhibit phenotypes of an enlarged tapetum and middle layer with delayed degeneration, causing male sterility. The gene is preferentially expressed in the tapetal cells during early anther development. In dtc1 anthers, expression of genes encoding secretory proteases or lipid transporters is significantly reduced, while transcripts of PCD regulatory genes, e.g. UDT1, TDR1, and EAT1/DTD, are not altered. Moreover, levels of DTC1 transcripts are diminished in udt1, tdr, and eat1 anthers. These results suggest that DTC1 functions downstream of those transcription factor genes and upstream of the genes encoding secretory proteins. DTC1 protein interacts with OsMT2b, a ROS scavenger. Whereas wild-type plants accumulate large amounts of ROS in their anthers at Stage 9 of development, those levels remain low during all stages of development in dtc1 anthers. These findings indicate that DTC1 is a key regulator for tapetum PCD by inhibiting ROS-scavenging activity.

Genome-wide identification and analysis of rice genes preferentially expressed in pollen at an early developmental stage.

Microspore production using endogenous developmental programs has not been well studied. The main limitation is the difficulty in identifying genes preferentially expressed in pollen grains at early stages. To overcome this limitation, we collected transcriptome data from anthers and microspore/pollen and performed meta-expression analysis. Subsequently, we identified 410 genes showing preferential expression patterns in early developing pollen samples of both japonica and indica cultivars. The expression patterns of these genes are distinguishable from genes showing pollen mother cell or tapetum-preferred expression patterns. Gene Ontology enrichment and MapMan analyses indicated that microspores in rice are closely linked with protein degradation, nucleotide metabolism, and DNA biosynthesis and regulation, while the pollen mother cell or tapetum are strongly associated with cell wall metabolism, lipid metabolism, secondary metabolism, and RNA biosynthesis and regulation. We also generated transgenic lines under the control of the promoters of eight microspore-preferred genes and confirmed the preferred expression patterns in plants using the GUS reporting system. Furthermore, cis-regulatory element analysis revealed that pollen specific elements such as POLLEN1LELAT52, and 5659BOXLELAT5659 were commonly identified in the promoter regions of eight rice genes with more frequency than estimation. Our study will provide new sights on early pollen development in rice, a model crop plant.

Rice glycosyltransferase1 encodes a glycosyltransferase essential for pollen wall formation.

The pollen wall consists of an exine and an intine. The mechanism underlying its formation is not well understood. Glycosyltransferases catalyze the modification of biological molecules by attaching a single or multiple sugars and play key roles in a wide range of biological processes. We examined the role of GLYCOSYLTRANSFERASE1 (OsGT1) in pollen wall development in rice (Oryza sativa). This gene is highly expressed in mature pollen, and plants containing alleles caused by transfer DNA insertion do not produce homozygous progeny. Reciprocal crosses between OsGT1/osgt1 and the wild type indicated that the mutation leads to a male gametophyte defect. Microscopic analyses revealed that osgt1 pollen developed normally to the pollen mitosis stage but failed to produce mature grains. In osgt1 pollen, intine structure was disrupted. In addition, starch and protein levels were much lower in the mutant grains. Recombinant OsGT1 transferred glucose from UDP-glucose to the third and seventh positions of quercetin, a universal substrate of glycosyltransferases. Consistent with the role of OsGT1, an OsGT1-green fluorescent protein fusion protein was localized to the Golgi apparatus. Taken together, our results suggest that OsGT1 is a Golgi-localized glycosyltransferase essential for intine construction and pollen maturation, providing new insight into male reproductive development.

The MYB-CC Transcription Factor PHOSPHATE STARVATION RESPONSE-LIKE 7 (PHL7) Functions in Phosphate Homeostasis and Affects Salt Stress Tolerance in Rice.

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.

The rice gene DEFECTIVE TAPETUM AND MEIOCYTES 1 (DTM1) is required for early tapetum development and meiosis.

Tapetum development and meiosis play crucial roles in anther development. Here we identified a rice gene, DEFECTIVE TAPETUM AND MEIOCYTES 1 (DTM1), which controls the early stages of that development. This gene encodes for an endoplasmic reticulum (ER) membrane protein that is present only in cereals. Our T-DNA insertion mutations gave rise to abnormal tapetal formation. Cellular organelles, especially the ER, were underdeveloped, which led to hampered differentiation and degeneration of the tapetum. In addition, the development of pollen mother cells was arrested at the early stages of meiotic prophase I. RNA in-situ hybridization analyses showed that DTM1 transcripts were most abundant in tapetal cells at stages 6 and 7, and moderately in the pollen mother cells and meiocytes. Transcripts of UDT1, which functions in tapetum development during early meiosis, were reduced in dtm1 anthers, as were those of PAIR1, which is involved in chromosome pairing and synapsis during meiosis. However, expression of MSP1 and MEL1, which function in anther wall specification and germ cell division, respectively, was not altered in the dtm1 mutant. Moreover, transcripts of DTM1 were reduced in msp1 mutant anthers, but not in udt1 and pair1 mutants. These results, together with their mutant phenotypes, suggest that DTM1 plays important roles in the ER membrane during early tapetum development, functioning after MSP1 and before UDT1, and also in meiocyte development, after MEL1 and before PAIR1.

Genome-wide expression analysis of rice aquaporin genes and development of a functional gene network mediated by aquaporin expression in roots.

The world population continually faces challenges of water scarcity for agriculture. A common strategy called water-balance control has evolved to adapt plant growth to these challenges. Aquaporins are a family of integral membrane proteins that play a central role in water-balance control. In this study, we identified 34 members of the rice aquaporin gene family, adding a novel member to the previous list. A combination of phylogenetic tree and anatomical meta-expression profiling data consisting of 983 Affymetrix arrays and 209 Agilent 44 K arrays was used to identify tissue-preferred aquaporin genes and evaluate functional redundancy among aquaporin family members. Eight aquaporins showed root-preferred expression in the vegetative growth stage, while 4 showed leaf/shoot-preferred expression. Integrating stress-induced expression patterns into phylogenetic tree and semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) analyses revealed that 3 rice aquaporin genes were markedly downregulated and 4 were upregulated by water deficiency in the root, suggesting that these candidate genes are key regulators of water uptake from the soil. Finally, we constructed a functional network of genes mediated by water stress and refined the network by confirming the differential expression using RT-PCR and real-time PCR. Our data will be useful to elucidate the molecular mechanism of water-balance control in rice root.

PRX102 Participates in Root Hairs Tip Growth of Rice.

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.

Tissue-specific enhancement of OsRNS1 with root-preferred expression is required for the increase of crop yield.

INTRODUCTION: Root development is a fundamental process that supports plant survival and crop productivity. One of the essential factors to consider when developing biotechnology crops is the selection of a promoter that can optimize the spatial-temporal expression of introduced genes. However, there are insufficient cases of suitable promoters in crop plants, including rice. OBJECTIVES: This study aimed to verify the usefulness of a new rice root-preferred promoter to optimize the function of a target gene with root-preferred expression in rice. METHODS: osrns1 mutant had defects in root development based on T-DNA insertional mutant screening and CRISPR technology. To optimize the function of OsRNS1, we generated OsRNS1-overexpression plants under two different promoters: a whole-plant expression promoter and a novel root-preferred expression promoter. Root growth, yield-related agronomic traits, RNA-seq, and reactive oxygen species (ROS) accumulation were analyzed for comparison. RESULTS: OsRNS1 was found to be involved in root development through T-DNA insertional mutant analysis and gene editing mutant analysis. To understand the gain of function of OsRNS1, pUbi1::OsRNS1 was generated for the whole-plant expression, and both root growth defects and overall growth defects were found. To overcome this problem, a root-preferential overexpression line using Os1-CysPrxB promoter (Per) was generated and showed an increase in root length, plant height, and grain yield compared to wild-type (WT). RNA-seq analysis revealed that the response to oxidative stress-related genes was significantly up-regulated in both overexpression lines but was more obvious in pPer::OsRNS1. Furthermore, ROS levels in the roots were drastically decreased in pPer::OsRNS1 but were increased in the osrns1 mutants compared to WT. CONCLUSION: The results demonstrated that the use of a root-preferred promoter effectively optimizes the function of OsRNS1 and is a useful strategy for improving root-related agronomic traits as well as ROS regulation.

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

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