OsBBP1, a newly identified protein containing DUF630 and DUF632 domains confers drought tolerance in rice.

Domain of unknown function (DUF) protein families, which are uncharacterized and numerous within the Pfam database. Recently, studies have demonstrated that DUFs played crucial roles in plant development, but whether, or how, they function in drought resistance remain unclear. In this study, we identified the Os03g0321500 gene, encoding OsbZIP72 binding protein 1 (OsBBP1), as a target of OsbZIP72 using chromatin immunoprecipitation sequencing in rice. OsBBP1 is a novel member of DUFs, which localize both in the nuclei and cytoplasm of rice protoplasts. Furthermore, yeast one-hybrid and electrophoretic mobility shift assays confirmed the specific binding between OsbZIP72 and OsBBP1. Additionally, a luciferase reporter analysis illustrated that OsbZIP72 activated the expression of OsBBP1. Drought tolerance experiments demonstrate that the OsBBP1 CRISPER-CAS9 transgenic mutants were sensitive to drought stress, but the transgenic OsBBP1 over-expressing rice plants showed enhanced drought resistance. Moreover, drought tolerance experiments in a paddy field suggested that OsBBP1 contributed to less yield or yield-related losses under drought conditions. Mechanistically, OsBBP1 might confer drought resistance by inducing more efficient reactive oxygen species (ROS) scavenging. Several ROS scavenging-related genes showed increased expression levels in OsBBP1 overexpression lines and decreased expression levels in OsBBP1 CRISPER-CAS9 mutants under drought conditions. Thus, OsBBP1, acting downstream of OsbZIP72, contributes to drought resistance and causes less yield or yield-related losses under drought conditions.

Multiplex-genome-editing based rapid directional improvement of complex traits in rice.

Although thousands of genes have been identified or cloned in rice (Oryza sativa) in the last two decades, the majority of them have only been separately characterized in specific varieties or single-gene modified backgrounds, thus limiting their practical application. We developed an optimized multiplex genome editing (MGE) toolbox that can efficiently assemble and stably express up to twelve sgRNA targets in a single plant expression vector. In this study, we established the MGE-based Rapid Directional Improvement (MRDI) strategy for directional improvement of complex agronomic traits in one small-scale rice transformation. This approach provides a rapid and practical procedure, encompassing sgRNA assembly, transgene-free screening and the creation of promising germplasm, by combining the precision of gene editing with phenotype-based field breeding. The MRDI strategy was used to generate the full diversity of twelve main agronomic genes in rice cultivar FXZ for the directional improvement of its growth duration and plant architecture. After applying the MRDI to FXZ, ideal plants with the desired traits of early heading date reduced plant height, and more effective panicles were generated without compromising yield, blast resistance and grain quality. Furthermore, the results of whole-genome sequencing (WGS), including the analysis of structural variations (SVs) and single nucleotide variations (SNVs) in the MGE plants, confirmed the high specificity and low frequency of unwanted mutations associated with this strategy. The MRDI breeding strategy would be a robust approach for exploring and applying crucial agronomic genes, as well as for generating novel elite germplasm in the future.

The lipoxygenase OsLOX10 affects seed longevity and resistance to saline-alkaline stress during rice seedlings.

Prolonged storage of rice seeds can lead to a decrease in seed vigor and seedling quality. The Lipoxygenase (LOX) gene family is widely distributed in plants, and LOX activity is closely related to seed viability and stress tolerance. In this study, the lipoxygenase OsLOX10 gene from the 9-lipoxygenase metabolic pathway was cloned from rice, and its roles in determining seed longevity and tolerance to saline-alkaline stress caused by Na2CO3 in rice seedlings were mainly investigated. CRISPR/Cas9 knockout of OsLOX10 increased seed longevity compared with the wild-type and OsLOX10 overexpression lines in response to artificial aging. The expression levels of other 9-lipoxygenase metabolic pathway related genes, such as LOX1, LOX2 and LOX3, were increased in the LOX10 overexpression lines. Quantitative real-time PCR and histochemical staining analysis showed that the expression of LOX10 was highest in seed hulls, anthers and the early germinating seeds. KI-I2 staining of starch showed that LOX10 could catalyze the degradation of linoleic acid. Furthermore, we found that the transgenic lines overexpressing LOX10 showed better tolerance to saline-alkaline stress than the wild-type and knockout mutant lines. Overall, our study demonstrated that the knockout LOX10 mutant increased seed longevity, whereas overexpression of LOX10 enhanced tolerance to saline-alkaline stress in rice seedlings.

Overexpression of a GIPC glycosyltransferase gene, OsGMT1, suppresses plant immunity and delays heading time in rice.

Glycosylinositol phosphorylceramides (GIPCs) are the major sphingolipids in the plant plasma membrane. In Arabidopsis, mutations of genes involved in the synthesis of GIPCs affect many physiological aspects of plants, including growth, pollen fertility, defense, and stress signaling. Loss of function of the GIPC MANNOSYL-TRANSFERASE1 (AtGMT1) results in GIPC misglycosylation and induces plant immune responses accompanied by a severely dwarfed phenotype, thus indicating that GIPCs play important roles in plant immunity. Here, we investigated the enzymatic activity and phenotypes of transgenic lines of OsGMT1, the ortholog of AtGMT1. Sphingolipidomic analysis indicated that OsGMT1 retained the enzymatic activity of GIPC hexose (Hex) glycosylation, but the knockout lines did not accumulate H2O2. In contrast, the OsGMT1 overexpression lines showed significant down-regulation of several defense-associated or cell wall synthesis-associated genes, and enhanced sensitivity to rice blast. Furthermore, we first demonstrated the sensitivity of rice cells to MoNLP1 protein through calcein AM release assays using rice protoplasts, thus legitimizing the presence of MoNLPs in rice blast fungus. In addition, yeast two-hybrid screens using OsGMT1 as bait revealed that OsGMT1 may regulate heading time through the OsHAP5C signaling pathway. Together, our findings suggested clear physiological functional differentiation of GMT1 orthologs between rice and Arabidopsis.

Analysis of co-expression and gene regulatory networks associated with sterile lemma development in rice.

BACKGROUND: The sterile lemma is a unique organ of the rice (Oryza sativa L.) spikelet. However, the characteristics and origin of the rice sterile lemma have not been determined unequivocally, so it is important to elucidate the molecular mechanism of the development of the sterile lemma. RESULTS: In the paper, we outline the regulatory mechanism of sterile lemma development by LONG STERILE LEMMA1 (G1), which has been identified as the gene controlling sterile lemma development. Based on the comprehensive analyses of transcriptome dynamics during sterile lemma development with G1 alleles between wild-type (WT) and mutant (MT) in rice, we obtained co-expression data and regulatory networks related to sterile lemma development. Co-transfection assays of rice protoplasts confirmed that G1 affects the expression of various phytohormone-related genes by regulating a number of critical transcription factors, such as OsLBD37 and OSH1. The hormone levels in sterile lemmas from WT and MT of rice supports the hypotheses that lower auxin, lower gibberellin, and higher cytokinin concentrations are required to maintain a normal phenotype of sterile lemmas. CONCLUSION: The regulatory networks have considerable reference value, and some of the regulatory relationships exhibiting strong correlations are worthy of further study. Taken together, these work provided a detailed guide for further studies into the molecular mechanism of sterile lemma development.

Gradual daylength sensing coupled with optimum cropping modes enhances multi-latitude adaptation of rice and maize.

To expand crop planting areas, reestablishment of crop latitude adaptation based on genetic variation in photoperiodic genes can be performed, but it is quite time consuming. By contrast, a crop variety that already exhibits multi-latitude adaptation has the potential to increase its planting areas to be more widely and quickly available. However, the importance and potential of multi-latitude adaptation of crop varieties have not been systematically described. Here, combining daylength-sensing data with the cropping system of elite rice and maize varieties, we found that varieties with gradual daylength sensing coupled with optimum cropping modes have an enhanced capacity for multi-latitude adaptation in China. Furthermore, this multi-latitude adaptation expanded their planting areas and indirectly improved China's nationwide rice and maize unit yield. Thus, coupling the daylength-sensing process with optimum cropping modes to enhance latitude adaptability of excellent varieties represents an exciting approach for deploying crop varieties with the potential to expand their planting areas and quickly improve nationwide crop unit yield in developing countries.

SH3P2, an SH3 domain-containing protein that interacts with both Pib and AvrPib, suppresses effector-triggered, Pib-mediated immunity in rice.

Plants usually keep resistance (R) proteins in a static state under normal conditions to avoid autoimmunity and save energy for growth, but R proteins can be rapidly activated upon perceiving pathogen invasion. Pib, the first cloned blast disease R gene in rice, encoding a nucleotide-binding leucine-rich repeat (NLR) protein, mediates resistance to the blast fungal (Magnaporthe oryzae) isolates carrying the avirulence gene AvrPib. However, the molecular mechanisms about how Pib recognizes AvrPib and how it is inactivated and activated remain largely unclear. In this study, through map-based cloning and CRISPR-Cas9 gene editing, we proved that Pib contributes to the blast disease resistance of rice cultivar Yunyin (YY). Furthermore, an SH3 domain-containing protein, SH3P2, was found to associate with Pib mainly at clathrin-coated vesicles in rice cells, via direct binding with the coiled-coil (CC) domain of Pib. Interestingly, overexpression of SH3P2 in YY compromised Pib-mediated resistance to M. oryzae isolates carrying AvrPib and Pib-AvrPib recognition-induced cell death. SH3P2 competitively inhibits the self-association of the Pib CC domain in vitro, suggesting that binding of SH3P2 with Pib undermines its homodimerization. Moreover, SH3P2 can also interact with AvrPib and displays higher affinity to AvrPib than to Pib, which leads to dissociation of SH3P2 from Pib in the presence of AvrPib. Taken together, our results suggest that SH3P2 functions as a "protector" to keep Pib in a static state by direct interaction during normal growth but could be triggered off by the invasion of AvrPib-carrying M. oryzae isolates. Our study reveals a new mechanism about how an NLR protein is inactivated under normal conditions but is activated upon pathogen infection.

Transcriptome Analyses Indicate Significant Association of Increased Non-Additive and Allele-Specific Gene Expression with Hybrid Weakness in Rice (Oryza sativa L.).

The heterosis in hybrid rice is highly affected by the environment and hybrid weakness occurs frequently depending on the genotypes of the hybrid and its parents. Hybrid weakness was also observed in our field experiments on nine rice hybrids produced by 3 × 3 incomplete diallel crosses. Among the nine hybrids, five displayed mid-parent heterosis (MPH) for grain yield per plant, while four showed mid-parent hybrid weakness (MPHW). A sequencing analysis of transcriptomes in panicles at the seed-filling stage revealed a significant association between enhanced non-additive gene expression (NAE) and allele-specific gene expression (ASE) with hybrid weakness. High proportions of ASE genes, with most being of mono-allele expression, were detected in the four MPHW hybrids, ranging from 22.65% to 45.97%; whereas only 4.80% to 5.69% of ASE genes were found in the five MPH hybrids. Moreover, an independence test indicated that the enhancements of NAE and ASE in the MPHW hybrids were significantly correlated. Based on the results of our study, we speculated that an unfavorable environment might cause hybrid weakness by enhancing ASE and NAE at the transcriptome level.

Characterization and expression analysis of the glycosyltransferase 64 family in rice (Oryza sativa).

The glycosyltransferase 64 (GT64) family is widely conserved in many species, including animals and plants. The functions of GT64 family genes in animals have been well characterized in the biosynthesis of extracellular heparan sulfate, whereas two GT64 members in Arabidopsis thaliana are involved in the glycosylation of plasma membrane glycosylinositol phosphorylceramides (GIPCs). GIPCs are the main components of plant sphingolipids and serve as important signal molecules in various developmental processes and stress responses. Rice (Oryza sativa), a model monocot plant, contains four GT64 members in its genome. Using phylogenetic analysis, 73 GT64s from 19 plant species were divided into three main groups. Each group can be represented by the three members in Arabidopsis and show a trend of monocot-eudicot divergence. A promoter and genomic variation analysis of GT64s in rice showed that various stress-related regulatory elements exist in their promoters, and many sequence variations were found between the two main rice subspecies, japonica and indica. Additionally, transmembrane domain and subcellular localization analyses revealed that these genes all encode membrane-bound glycosyltransferases and are localized to the Golgi apparatus. Finally, expression analysis of the four GT64 genes in rice, as assessed by quantitative real-time PCR, showed that they have distinct tissue-specific expression patterns and respond to different hormone treatments or abiotic stresses. Our results indicated that this family of genes may play a role in different stress responses and hormone signaling pathways in rice, which will provide fundamental information for further investigation of their functions in future.

Ubiquitylomes and proteomes analyses provide a new interpretation of the molecular mechanisms of rice leaf senescence.

MAIN CONCLUSION: We identified a typical rice premature senescence leaf mutant 86 (psl86) and exhibited the first global ubiquitination data during rice leaf senescence. Premature leaf senescence affects the yield and quality of rice, causing irreparable agricultural economic losses. In this study, we reported a rice premature senescence leaf mutant 86 (psl86) in the population lines of rice (Oryza sativa) japonica cultivar 'Yunyin' (YY) mutagenized using ethyl methane sulfonate (EMS) treatment. Immunoblotting analysis revealed that a higher ubiquitination level in the psl86 mutant compared with YY. Thus, we performed the proteome and ubiquitylome analyses to identify the differential abundance proteins and ubiquitinated proteins (sites) related to leaf senescence. Among 885 quantified lysine ubiquitination (Kub) sites in 492 proteins, 116 sites in 94 proteins were classified as up-regulated targets and seven sites in six proteins were classified as down-regulated targets at a threshold of 1.5. Proteins with up-regulated Kub sites were mainly enriched in the carbon fixation in photosynthetic organisms, glycolysis/gluconeogenesis and the pentose phosphate pathway. Notably, 14 up-regulated Kub sites in 11 proteins were enriched in the carbon fixation in photosynthetic organism pathway, and seven proteins (rbcL, PGK, GAPA, FBA5, ALDP, CFBP1 and GGAT) were down-regulated, indicating this pathway is tightly regulated by ubiquitination during leaf senescence. To our knowledge, we present the first global data on ubiquitination during rice leaf senescence.

SDR7-6, a short-chain alcohol dehydrogenase/reductase family protein, regulates light-dependent cell death and defence responses in rice.

Lesion mimic mutants resembling the hypersensitive response without pathogen attack are an ideal material to understand programmed cell death, the defence response, and the cross-talk between defence response and development in plants. In this study, mic, a lesion mimic mutant from cultivar Yunyin treated with ethyl methanesulphonate (EMS), was screened. By map-based cloning, a short-chain alcohol dehydrogenase/reductase with an atypical active site HxxxK was isolated and designated as SDR7-6. It functions as a homomultimer in rice and is localized at the endoplasmic reticulum. The lesion mimic phenotype of the mutant is light-dependent. The mutant displayed an increased resistance response to bacterial blight, but reduced resistance to rice blast disease. The mutant and knockout lines showed increased reactive oxygen species, jasmonic acid content, antioxidant enzyme activity, and expression of pathogenicity-related genes, while chlorophyll content was significantly reduced. The knockout lines showed significant reduction in grain size, seed setting rate, 1000-grain weight, grain weight per plant, panicle length, and plant height. SDR7-6 is a new lesion mimic gene that encodes a short-chain alcohol dehydrogenase with atypical catalytic site. Disruption of SDR7-6 led to cell death and had adverse effects on multiple agricultural characters. SDR7-6 may act at the interface of the two defence pathways of bacterial blight and rice blast disease in rice.

RNA-seq profiling of primary calli induced by different media and photoperiods for japonica rice 'Yunyin'.

The induction of embryogenic calli plays a vital role in the genetic transformation and regeneration of rice (Oryza sativa L.). Despite progress in rice tissue culture, the molecular mechanisms of embryogenic callus induction remain unknown. In this study, gene expression profiles associated with calli were comprehensively analyzed during callus induction of japonica rice 'Yunyin'. We first confirmed that NMB medium with 24 h of light and 0 h of dark (NMB-L) was the optimal condition for 'Yunyin' callus induction, while J3 medium with 0 h of light and 24 h of dark (J3-D) was the worst condition. After transcriptome analysis, 33,597 unigenes were assembled, among which we identified 6,063 DEGs (Differentially Expressed Genes) related to media and seven DEGs related to photoperiod. Phenylpropanoid biosynthesis, plant hormone signal, and starch and sucrose metabolism were the top three pathways affected by media, while the circadian rhythm-plant pathway was associated with photoperiod. Furthermore, we identified two candidate genes, Os01g0965900 and Os12g0555200, affected by both medium and photoperiod. Statistical analysis of RNA-seq libraries showed that the expression levels of these two genes in J3-D calli were over 2.5 times higher than those in NMB-L calli, which was further proved by RT-qPCR analysis. Based on FPKM (Fragments Per Kilobase of transcript Per Million mapped reads), unigenes belonging to the NMB-L group were mainly assigned to ribosome, carbon metabolism, biosynthesis of amino acids, protein processing in endoplasmic reticulum, and plant hormone signal transduction pathways. We transformed Os12g0555200Nip and Os12g05552009311 into 'Nipponbare' calli and observed their effects on the growth and development process of rice calli using TEM (Transmission Electron Microscopy) and SEM (Scanning Electron Microscopy). Observations showed that Os12g05552009311 was more disadvantageous to rice callus growth than Os12g0555200Nip. Our results reveal that the Os12g0555200, identified from transcriptomic profiles, has a negative influence during 'Yunyin' callus induction. Supplementary information: The online version contains supplementary material available at 10.1007/s11032-022-01283-y.

From Green Super Rice to green agriculture: Reaping the promise of functional genomics research.

Producing sufficient food with finite resources to feed the growing global population while having a smaller impact on the environment has always been a great challenge. Here, we review the concept and practices of Green Super Rice (GSR) that have led to a paradigm shift in goals for crop genetic improvement and models of food production for promoting sustainable agriculture. The momentous achievements and global deliveries of GSR have been fueled by the integration of abundant genetic resources, functional gene discoveries, and innovative breeding techniques with precise gene and whole-genome selection and efficient agronomic management to promote resource-saving, environmentally friendly crop production systems. We also provide perspectives on new horizons in genomic breeding technologies geared toward delivering green and nutritious crop varieties to further enhance the development of green agriculture and better nourish the world population.

PEPC of sugarcane regulated glutathione S-transferase and altered carbon-nitrogen metabolism under different N source concentrations in Oryza sativa.

BACKGROUND: Phosphoenolpyruvate carboxylase (PEPC) plays an important role in the primary metabolism of higher plants. Several studies have revealed the critical importance of PEPC in the interaction of carbon and nitrogen metabolism. However, the function mechanism of PEPC in nitrogen metabolism is unclear and needs further investigation. RESULTS: This study indicates that transgenic rice expressing the sugarcane C4-PEPC gene displayed shorter primary roots and fewer crown roots at the seedling stage. However, total nitrogen content was significantly higher in transgenic rice than in wild type (WT) plants. Proteomic analysis revealed that there were more differentially expressed proteins (DEPs) responding to nitrogen changes in transgenic rice. In particular, the most enriched pathway "glutathione (GSH) metabolism", which mainly contains GSH S-transferase (GST), was identified in transgenic rice. The expression of endogenous PEPC, GST and several genes involved in the TCA cycle, glycolysis and nitrogen assimilation changed in transgenic rice. Correspondingly, the activity of enzymes including GST, citrate synthase, 6-phosphofructokinase, pyruvate kinase and ferredoxin-dependent glutamate synthase significantly changed. In addition, the levels of organic acids in the TCA cycle and carbohydrates including sucrose, starch and soluble sugar altered in transgenic rice under different nitrogen source concentrations. GSH that the substrate of GST and its components including glutamic acid, cysteine and glycine accumulated in transgenic rice. Moreover, the levels of phytohormones including indoleacetic acid (IAA), zeatin (ZT) and isopentenyladenosine (2ip) were lower in the roots of transgenic rice under total nutrients. Taken together, the phenotype, physiological and biochemical characteristics of transgenic rice expressing C4-PEPC were different from WT under different nitrogen levels. CONCLUSIONS: Our results revealed the possibility that PEPC affects nitrogen metabolism through regulating GST, which provide a new direction and concepts for the further study of the PEPC functional mechanism in nitrogen metabolism.

Genome-Wide Association Study Reveals the QTLs for Seed Storability in World Rice Core Collections.

Seed storability is a main agronomically important trait to assure storage safety of grain and seeds in rice. Although many quantitative trait loci (QTLs) and associated genes for rice seed storability have been identified, the detailed genetic mechanisms of seed storability remain unclear in rice. In this study, a genome-wide association study (GWAS) was performed in 456 diverse rice core collections from the 3K rice genome. We discovered the new nine QTLs designated as qSS1-1, qSS1-2, qSS2-1, qSS3-1, qSS5-1, qSS5-2, qSS7-1, qSS8-1, and qSS11-1. According to the analysis of the new nine QTLs, our results could well explain the reason why seed storability of indica subspecies was superior to japonica subspecies in rice. Among them, qSS1-2 and qSS8-1 were potentially co-localized with a known associated qSS1/OsGH3-2 and OsPIMT1, respectively. Our results also suggest that pyramiding breeding of superior alleles of these associated genes will lead to new varieties with improved seed storability in the future.

Fine-mapping of qTGW2, a quantitative trait locus for grain weight in rice (Oryza sativa L.).

BACKGROUND: Grain weight is a grain yield component, which is an integrated index of grain length, width and thickness. They are controlled by a large number of quantitative trait loci (QTLs). Besides major QTLs, minor QTLs play an essential role. In our previous studies, QTL analysis for grain length and width was performed using a recombinant inbred line population derived from rice cross TQ/IRBB lines. Two major QTLs were detected, which were located in proximity to GS3 and GW5 that have been cloned. In the present study, QTLs for grain weight and shape were identified using rice populations that were homozygous at GS3 and GW5. METHOD: Nine populations derived from the indica rice cross TQ/IRBB52 were used. An F10:11population named W1, consisting of 250 families and covering 16 segregating regions, was developed from one residual heterozygote (RH) in the F7generation of Teqing/IRBB52. Three near isogenic line (NIL)-F2 populations, ZH1, ZH2 and ZH3 that comprised 205, 239 and 234 plants, respectively, were derived from three RHs in F10:11. They segregated the target QTL region in an isogenic background. Two NIL populations, HY2 and HY3, were respectively produced from homozygous progeny of the ZH2 and ZH3 populations. Three other NIL-F2 populations, Z1, Z2 and Z3, were established using three RHs having smaller heterozygous segments. QTL analysis for 1000-grain weight (TGW), grain length (GL), grain width (GW), and length/width ratio (LWR) was conducted using QTL IciMapping and SAS procedure with GLM model. RESULT: A total of 27 QTLs distributed on 12 chromosomes were identified. One QTL cluster, qTGW2/qGL2/qGW2 located in the terminal region of chromosome 2, were selected for further analysis. Two linked QTLs were separated in region Tw31911-RM266. qGL2 was located in Tw31911-Tw32437 and mainly controlled GL and GW. The effects were larger on GL than on GW and the allelic directions were opposite. qTGW2 was located in Tw35293-RM266 and affected TGW, GL and GW with the same allelic direction. Finally, qTGW2 was delimited within a 103-kb region flanked by Tw35293 and Tw35395. CONCLUSION: qTGW2 with significant effects on TGW, GL and GW was validated and fine-mapped using NIL and NIL-F2 populations. These results provide a basis for map-based cloning of qTGW2 and utilization of qTGW2 in the breeding of high-yielding rice varieties.

Brassinosteroid signaling may regulate the germination of axillary buds in ratoon rice.

BACKGROUND: Rice ratooning has traditionally been an important component of the rice cropping system in China. However, compared with the rice of the first harvest, few studies on factors effecting ratoon rice yield have been conducted. Because ratoon rice is a one-season rice cultivated using axillary buds that germinate on rice stakes and generate panicles after the first crop's harvest, its production is mainly affected by the growth of axillary buds. The objectives of this study were to evaluate the sprouting mechanism of axillary buds to improve the ratoon rice yield. RESULTS: First, we observed the differentiation and growth dynamics of axillary buds at different nodes of Shanyou 63, and found that they differentiated from bottom to top before the heading of the mother stem, and that they developed very slowly. After heading they differentiated from top to bottom, and the ones on the top, especially the top 2nd node, developed much faster than those at the other nodes. The average length and dry weight of the axillary buds were significantly greater than those at other nodes by the yellow ripe stage, and they differentiated into pistils and stamens by 6 d after the yellow ripe stage. The morphology of vegetative organs from regenerated tillers of Shanyou 63 also suggested the superior growth of the upper buds, which was regulated by hormones, in ratoon rice. Furthermore, a comprehensive proteome map of the rice axillary buds at the top 2nd node before and after the yellow ripe stage was established, and some proteins involved in steroid biosynthesis were significantly increased. Of these, four took part in brassinosteroid (BR) biosynthesis. Thus, BR signaling may play a role in the germination of axillary buds of ratoon rice. CONCLUSIONS: The data provide insights into the molecular mechanisms underlying BR signaling, and may allow researchers to explore further the biological functions of endogenous BRs in the germination of axillary buds of ratoon rice.

Single-Cell RNA Sequencing Efficiently Predicts Transcription Factor Targets in Plants.

Discovering transcription factor (TF) targets is necessary for the study of regulatory pathways, but it is hampered in plants by the lack of highly efficient predictive technology. This study is the first to establish a simple system for predicting TF targets in rice (Oryza sativa) leaf cells based on 10 × Genomics' single-cell RNA sequencing method. We effectively utilized the transient expression system to create the differential expression of a TF (OsNAC78) in each cell and sequenced all single cell transcriptomes. In total, 35 candidate targets having strong correlations with OsNAC78 expression were captured using expression profiles. Likewise, 78 potential differentially expressed genes were identified between clusters having the lowest and highest expression levels of OsNAC78. A gene overlapping analysis identified 19 genes as final candidate targets, and various assays indicated that Os01g0934800 and Os01g0949900 were OsNAC78 targets. Additionally, the cell profiles showed extremely similar expression trajectories between OsNAC78 and the two targets. The data presented here provide a high-resolution insight into predicting TF targets and offer a new application for single-cell RNA sequencing in plants.

OsGL6, a conserved AP2 domain protein, promotes leaf trichome initiation in rice.

Trichomes are specialized epidermal cells that play crucial roles in resisting environmental stress and enhancing plant development. In Arabidopsis thaliana, the main genes controlling trichome formation have been consecutively identified. However, few genes like this were reported in rice. In this study, we identified the hairy phenotype of indica variety 75-1-127. This was used to construct a segregation population with a cross of hairless variety Minghui63 (MH63) to fine map the trichome formation genes. Genetic analysis indicated that hairy phenotype was controlled by a pair of dominant genes on chromosome 6, which was designated as GLABRA6 (OsGL6). OsGL6 was an allele of HL6 gene whose sequences containing rich variations in genomes. Compared to wild type, the overexpressing transgenic lines revealed that OsGL6 promoted trichome initiation. We found that OsGL6 interacted with serine/threonine protein kinase OSK3 (OSK3) or COP9 signalosome complex subunit 5a (CSN5) in yeast. These results provide potential information for understanding the molecular mechanism of trichome formation in rice.

Dissecting a heterotic gene through GradedPool-Seq mapping informs a rice-improvement strategy.

Hybrid rice breeding for exploiting hybrid vigor, heterosis, has greatly increased grain yield. However, the heterosis-related genes associated with rice grain production remain largely unknown, partly because comprehensive mapping of heterosis-related traits is still labor-intensive and time-consuming. Here, we present a quantitative trait locus (QTL) mapping method, GradedPool-Seq, for rapidly mapping QTLs by whole-genome sequencing of graded-pool samples from F2 progeny via bulked-segregant analysis. We implement this method and map-based cloning to dissect the heterotic QTL GW3p6 from the female line. We then generate the near isogenic line NIL-FH676::GW3p6 by introgressing the GW3p6 allele from the female line Guangzhan63-4S into the male inbred line Fuhui676. The NIL-FH676::GW3p6 exhibits grain yield highly increased compared to Fuhui676. This study demonstrates that it may be possible to achieve a high level of grain production in inbred rice lines without the need to construct hybrids.