The RAC/ROP GTPase activator OsRopGEF10 functions in crown root development by regulating cytokinin signaling in rice.

RAC/Rho of plant (ROP) GTPases are major molecular switches that control diverse signaling cascades for plant growth, development, and defense. Here, we discovered a signaling node that connects RAC/ROPs to cytokinins. Rice (Oryza sativa) plants develop a fibrous root system mainly composed of crown roots. Cytokinin signaling via a phosphorelay system is critical for crown root development. We show that OsRopGEF10, which activates RAC/ROPs, acts upstream of the cytoplasmic-nuclear shuttling phosphotransfer proteins AHPs of the cytokinin signaling pathway to promote crown root development. Mutations of OsRopGEF10 induced hypersensitivity to cytokinin, whereas overexpressing this gene reduced the cytokinin response. Loss of OsRopGEF10 function reduced the expression of the response regulator gene OsRR6, a repressor of cytokinin signaling, and impaired crown root development. Mutations in OsAHP1/2 led to increased crown root production and rescued the crown root defect of Osropgef10. Furthermore, auxin activates the ROP GTPase OsRAC3, which attenuates cytokinin signaling for crown root initiation. Molecular interactions between OsRopGEF10, OsRAC3, and OsAHP1/2 implicate a mechanism whereby OsRopGEF10-activated OsRAC3 recruits OsAHP1/2 to the cortical cytoplasm, sequestering them from their phosphorelay function in the nucleus. Together, our findings uncover the OsRopGEF10-OsRAC3-OsAHP1/2 signaling module, establish a link between RAC/ROPs and cytokinin, and reveal molecular crosstalk between auxin and cytokinin during crown root development.

Improvement of Salinity Tolerance in Water-Saving and Drought-Resistance Rice (WDR).

Rice is one of the most economically important staple food crops in the world. Soil salinization and drought seriously restrict sustainable rice production. Drought aggravates the degree of soil salinization, and, at the same time, increased soil salinity also inhibits water absorption, resulting in physiological drought stress. Salt tolerance in rice is a complex quantitative trait controlled by multiple genes. This review presents and discusses the recent research developments on salt stress impact on rice growth, rice salt tolerance mechanisms, the identification and selection of salt-tolerant rice resources, and strategies to improve rice salt tolerance. In recent years, the increased cultivation of water-saving and drought-resistance rice (WDR) has shown great application potential in alleviating the water resource crisis and ensuring food and ecological security. Here, we present an innovative germplasm selection strategy of salt-tolerant WDR, using a population that is developed by recurrent selection based on dominant genic male sterility. We aim to provide a reference for efficient genetic improvement and germplasm innovation of complex traits (drought and salt tolerance) that can be translated into breeding all economically important cereal crops.

Breeding an early maturing, blast resistance water-saving and drought-resistance rice (WDR) cultivar using marker-assisted selection coupled with rapid generation advance.

The japonica water-saving and drought-resistance rice (Oryza sativa L.) (WDR) cultivar Huhan 9 harbors genes for resistance to rice blast (Magnaporthe oryzae), including Pi-ta and Pi-b. The early maturing japonica rice cultivar Suhuxiangjing and the high-yield WDR cultivars Huhan 3 and Huhan 11 were used as the parents to conduct single cross breeding and composite hybridization breeding. Strict drought resistance screening was conducted in the segregating generations, the genotypes of which were determined using functional markers of Pi-ta and Pi-b genes. By combining the rapid generation advance of the industrialized breeding system and multi-site shuttle identification, the new WDR cultivar Huhan 106 with early maturity, blast resistance, high yield, and high quality was bred, and it was certified by the Agricultural Crop Variety Certification Commission of Shanghai in 2020. Molecular marker-assisted selection coupled with rapid generation advance and multi-site shuttle identification is a rapid and efficient breeding method for the value-added improvement of crop varieties. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-022-01319-3.

Cystathionine beta-lyase is crucial for embryo patterning and the maintenance of root stem cell niche in Arabidopsis.

The growth and development of roots in plants depends on the specification and maintenance of the root apical meristem. Here, we report the identification of CBL, a gene required for embryo and root development in Arabidopsis, and encodes cystathionine beta-lyase (CBL), which catalyzes the penultimate step in methionine (Met) biosynthesis, and which also led to the discovery of a previous unknown, but crucial, metabolic contribution by the Met biosynthesis pathway. CBL is expressed in embryos and shows quiescent center (QC)-enriched expression pattern in the root. cbl mutant has impaired embryo patterning, defective root stem cell niche, stunted root growth, and reduces accumulation of the root master regulators PLETHORA1 (PLT1) and PLT2. Furthermore, mutation in CBL severely decreases abundance of several PIN-FORMED (PIN) proteins and impairs auxin-responsive gene expression in the root tip. cbl seedlings also exhibit global reduction in histone H3 Lys-4 trimethylation (H3K4me3) and DNA methylation. Importantly, mutation in CBL reduces the abundance of H3K4me3 modification in PLT1/2 genes and downregulates their expression. Overexpression of PLT2 partially rescues cbl root meristem defect, suggesting that CBL acts in part through PLT1/2. Moreover, exogenous supplementation of Met also restores the impaired QC activity and the root growth defects of cbl. Taken together, our results highlight the unique role of CBL to maintain the root stem cell niche by cooperative actions between Met biosynthesis and epigenetic modification of key developmental regulators.

Development and validation of a PCR-based functional marker system for identifying the low amylose content-associated gene Wx hp in rice.

Low amylose content (AC) is a desirable trait for rice (Oryza sativa L.) cooking quality and is selected in soft rice breeding. The Wx hp allele was derived from a Yunnan rice landrace in China, Haopi, with low AC. To efficiently and rapidly utilize the low amylose content-associated gene Wx hp in rice molecular breeding programs, we developed a tetra-primer amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) method, according to the single-nucleotide variation of the Wx hp . Four Wx hp -specific primers were used to perform PCR assays using genomic DNA extracted from several rice varieties. Based on the band pattern of the amplified products after electrophoresis, this method can accurately distinguish three Wx hp -related genotypes (i.e., Wx hp homozygotes, Wx hp heterozygotes, and wild-type), and the genotypes completely correspond to the appearance of mature endosperm. This method represents a novel approach that is both inexpensive and highly efficient and can be widely used for genotyping Wx hp alleles in rice germplasm collections and may aid breeding programs with marker-assisted selection (MAS).

RopGEF1 Plays a Critical Role in Polar Auxin Transport in Early Development.

Polar auxin transport, facilitated by the combined activities of auxin influx and efflux carriers to maintain asymmetric auxin distribution, is essential for plant growth and development. Here, we show that Arabidopsis (Arabidopsis thaliana) RopGEF1, a guanine nucleotide exchange factor and activator of Rho GTPases of plants (ROPs), is critically involved in polar distribution of auxin influx carrier AUX1 and differential accumulation of efflux carriers PIN7 and PIN2 and is important for embryo and early seedling development when RopGEF1 is prevalently expressed. Knockdown or knockout of RopGEF1 induces embryo defects, cotyledon vein breaks, and delayed root gravity responses. Altered expression from the auxin response reporter DR5rev:GFP in the root pole of RopGEF1-deficient embryos and loss of asymmetric distribution of DR5rev:GFP in their gravistimulated root tips suggest that auxin distribution is affected in ropgef1 mutants. This is reflected by the polarity of AUX1 being altered in ropgef1 embryos and roots, shifting from the normal apical membrane location to a basal location in embryo central vascular and root protophloem cells and also reduced PIN7 accumulation at embryos and altered PIN2 distribution in gravistimulated roots of mutant seedlings. In establishing that RopGEF1 is critical for AUX1 localization and PIN differential accumulation, our results reveal a role for RopGEF1 in cell polarity and polar auxin transport whereby it imapcts auxin-mediated plant growth and development.

ROP3 GTPase contributes to polar auxin transport and auxin responses and is important for embryogenesis and seedling growth in Arabidopsis.

ROP GTPases are crucial for the establishment of cell polarity and for controlling responses to hormones and environmental signals in plants. In this work, we show that ROP3 plays important roles in embryo development and auxin-dependent plant growth. Loss-of-function and dominant-negative (DN) mutations in ROP3 induced a spectrum of similar defects starting with altered cell division patterning during early embryogenesis to postembryonic auxin-regulated growth and developmental responses. These resulted in distorted embryo development, defective organ formation, retarded root gravitropism, and reduced auxin-dependent hypocotyl elongation. Our results showed that the expression of AUXIN RESPONSE FACTOR5/MONOPTEROS and root master regulators PLETHORA1 (PLT1) and PLT2 was reduced in DN-rop3 mutant embryos, accounting for some of the observed patterning defects. ROP3 mutations also altered polar localization of auxin efflux proteins (PINs) at the plasma membrane (PM), thus disrupting auxin maxima in the root. Notably, ROP3 is induced by auxin and prominently detected in root stele cells, an expression pattern similar to those of several stele-enriched PINs. Our results demonstrate that ROP3 is important for maintaining the polarity of PIN proteins at the PM, which in turn ensures polar auxin transport and distribution, thereby controlling plant patterning and auxin-regulated responses.

Effect of life cycle and venation pattern on the coordination between stomatal and vein densities of herbs.

Life cycle (annual vs perennial) and leaf venation pattern (parallel and reticular) are known to be related to water use strategies in herb species and critical adaptation to certain climatic conditions. However, the effect of these two traits and how they influence the coordination between vein density (vein length per area, VLA) and stomatal density (SD) remains unclear. In this study, we examined the leaves of 53 herb species from a subtropical botanical garden in Guangdong Province, China, including herbs with different life cycles and leaf venation patterns. We assessed 21 leaf water-related functional traits for all species, including leaf area (LA), major and minor VLA, major and minor vein diameter (VD), SD and stomatal length (SL). The results showed no significant differences in mean SD and SL between either functional group (parallel venation vs reticular venation and annual vs perennial). However, parallel vein herbs and perennial herbs displayed a significantly higher mean LA and minor VD, and lower minor VLA compared to reticular vein herbs and annual herbs, respectively. There was a linear correlation between total VLA and SD in perennial and reticular vein herbs, but this kind of correlation was not found in annual and parallel vein herbs. The major VLA and minor VD were significantly affected by the interaction between life cycle and leaf venation pattern. Our findings suggested that VLA, rather than SD, may serve as a more adaptable structure regulated by herbaceous plants to support the coordination between leaf water supply and demand in the context of different life cycles and leaf venation patterns. The results of the present study provide mechanistic understandings of functional advantages of different leaf types, which may involve in species fitness in community assembly and divergent responses to climate changes.

Effect of leaf phenology and morphology on the coordination between stomatal and minor vein densities.

Leaf phenology (evergreen vs. deciduous) and morphology (simple vs. compound) are known to be related to water use strategies in tree species and critical adaptation to certain climatic conditions. However, the effect of these two traits and their interactions on the coordination between minor vein density (MVD) and stomatal density (SD) remains unclear. In this study, we examined the leaves of 108 tree species from plots in a primary subtropical forest in southern China, including tree species with different leaf morphologies and phenologies. We assessed nine leaf water-related functional traits for all species, including MVD, SD, leaf area (LA), minor vein thickness (MVT), and stomatal length (SL). The results showed no significant differences in mean LA and SD between either functional group (simple vs. compound and evergreen vs. deciduous). However, deciduous trees displayed a significantly higher mean MVD compared to evergreen trees. Similarly, compound-leaved trees have a higher (marginally significant) MVD than simple-leaved trees. Furthermore, we found that leaf morphology and phenology have significantly interactive effects on SL, and the compound-leafed deciduous trees exhibited the largest average SL among the four groups. There were significant correlations between the MVD and SD in all different tree groups; however, the slopes and interceptions differed within both morphology and phenology. Our results indicate that MVD, rather than SD, may be the more flexible structure for supporting the coordination between leaf water supply and demand in different leaf morphologies and phenologies. The results of the present study provide mechanistic understandings of the functional advantages of different leaf types, which may involve species fitness in community assembly and divergent responses to climate changes.

Patterns of compound-leaf form and deciduous-leaf habit across forests in China: Their association and key climatic factors.

Leaf form (compound vs. simple) and habit (evergreen vs. deciduous) are key functional traits of trees to adapt to various climates and are vital in determining plant response to climate change. However, their association and climatic determinants remain uncertain, especially in East Asian forests in the largest monsoon region on earth. To fill these knowledge gaps, we compiled a dataset comprising 42 intact forests and over 2200 angiosperm tree species across China (spanning 30 latitudes and 47 longitudes). The geographical and climatic patterns of leaf form and habit were analyzed. The association between compound leaf and deciduousness was tested for tropical, subtropical and temperate climatic zones. We found that both the percentage of compound leaf (CT%) and deciduous tree species (DT%) increased with latitude and decreased with mean annual precipitation (MAP). For all forests, DT% was negatively related to mean annual temperature (MAT), whereas CT% was not. Nevertheless, both DT% and CT% increased with increasing MAT in the tropics, possibly owing to the high vapor pressure deficits (VPD) and canopy water deficits associated with high temperatures. A positive linear relationship between CT% and DT% was found across all forests and within different climatic zones except for temperate, and the intercept of the regression line was significantly higher in the tropics than in the subtropics. Overall, as supported by principal component analysis, deciduousness was negatively associated with both temperature and precipitation, while CT negatively with precipitation only across zones and positively with temperature in the tropics. Different relationships in different climatic zones suggest potentially different selective forces. Our findings provide novel insights into the linkage between leaf form and habit, as well as how climate shapes the landscape of broadleaf forests, which has important implications regarding the response of forest composition to climate change.

Molecular Breeding of Water-Saving and Drought-Resistant Rice for Blast and Bacterial Blight Resistance.

Rice production is often affected by biotic and abiotic stressors. The breeding of resistant cultivars is a cost-cutting and environmentally friendly strategy to maintain a sustainable high production level. An elite water-saving and drought-resistant rice (WDR), Hanhui3, is susceptible to blast and bacterial blight (BB). This study was conducted to introgress three resistance genes (Pi2, xa5, and Xa23) for blast and BB into Hanhui3, using marker-assisted selection (MAS) for the foreground selection and a whole-genome single-nucleotide polymorphism (SNP) array for the background selection. As revealed by the whole-genome SNP array, the recurrent parent genome (RPG) recovery of the improved NIL was 94.2%. The resistance levels to blast and BB of the improved NIL and its derived hybrids were higher than that of the controls. In addition, the improved NIL and its derived hybrids retained the desired agronomic traits from Hanhui3, such as yield. The improved NIL could be useful to enhance resistance against biotic stressors and produce stable grain yields in Oryza sativa subspecies indica rice breeding programs.

Linkage mapping and association analysis to identify a reliable QTL for stigma exsertion rate in rice.

The commercialization of hybrid rice has greatly contributed to the increase in rice yield, with the improvement of its seed production capacity having played an important role. The stigma exsertion rate (SER) is a key factor for improving the outcrossing of the sterile line and the hybrid rice seed production. We used the Zhenshan 97B × IRAT109 recombinant inbred population comprising 163 lines and a natural population of 138 accessions to decipher the genetic foundation of SER over 2 years in three environments. Additionally, we detected eight QTLs for SER on chromosomes 1, 2, and 8 via linkage mapping. We also identified seven and 19 significant associations for SER using genome-wide association study in 2016 and 2017, respectively. Interestingly, we located two lead SNPs (sf0803343504 and sf083344610) on chromosome 8 in the qTSE8 QTL region that were significantly associated with total SER. After transcriptomic analysis, quantitative real-time PCR, and haplotype analysis, we found 13 genes within this reliable region as important candidate genes. Our study results will be beneficial to molecular marker-assisted selection of rice lines with high outcrossing rate, thereby improving the efficiency of hybrid seed production.

eIF4E1 Regulates Arabidopsis Embryo Development and Root Growth by Interacting With RopGEF7.

Eukaryotic translation initiation factor 4E1 (eIF4E1) is required for the initiation of protein synthesis. The biological function of eIF4E1 in plant-potyvirus interactions has been extensively studied. However, the role of eIF4E1 in Arabidopsis development remains unclear. In this study, we show that eIF4E1 is highly expressed in the embryo and root apical meristem. In addition, eIF4E1 expression is induced by auxin. eIF4E1 mutants show embryonic cell division defects and short primary roots, a result of reduced cell divisions. Furthermore, our results show that mutation in eIF4E1 severely reduces the accumulation of PIN-FORMED (PIN) proteins and decreases auxin-responsive gene expression at the root tip. Yeast two-hybrid assays identified that eIF4E1 interacts with an RAC/ROP GTPase activator, RopGEF7, which has been previously reported to be involved in the maintenance of the root apical meristem. The interaction between eIF4E1 and RopGEF7 is confirmed by protein pull-down and bimolecular fluorescent complementation assays in plant cells. Taken together, our results demonstrated that eIF4E1 is important for auxin-regulated embryo development and root growth. The eIF4E1-RopGEF7 interaction suggests that eIF4E1 may act through ROP signaling to regulate auxin transport, thus regulating auxin-dependent patterning.

Molecular Breeding of a Novel PTGMS Line of WDR for Broad-Spectrum Resistance to Blast Using Pi9, Pi5, and Pi54 Genes.

BACKGROUND: The two-line method based on the photoperiod and thermo-sensitive genic male sterile (PTGMS) lines is more cost-effective, simple, and efficient than the three-line system based on cytoplasmic male-sterility. Blast and drought are the most prevalent biotic and abiotic stress factors hampering rice production. Molecular techniques demonstrate higher efficacy in the pyramiding of disease resistance genes, providing green performance under the background of water-saving and drought-resistance rice. RESULTS: This study employed molecular marker-assisted selection, conventional hybridization, and high-intensity stress screening to integrate three broad-spectrum blast resistance genes Pi9, Pi5, and Pi54 into Huhan 1S. Subsequently, a novel water-saving and drought-resistance rice (WDR) PTGMS line Huhan 74S was developed. The drought resistance of the new PTGMS line Huhan 74S was comparable to that of Huhan 1S. Pathogenicity assays involving the inoculation of 14 blast prevalent isolates in the glasshouse showed that the blast resistance frequency of Huhan 74S was 85.7%. Further evaluation under natural blast epidemic field conditions showed that Huhan 74S and its hybrids were resistant to leaf and neck blast. The critical temperature point of fertility-sterility alteration of Huhan 74S was 23 °C daily mean temperature. The complete male sterility under natural growth conditions in 2017 at Shanghai lasted for 67 days. Also, both the agronomic and grain quality traits met the requirement for two-line hybrid rice production. CONCLUSION: These results indicate that the newly bred PTGMS line Huhan 74S can be used to breed high-yielding, good-quality, disease-resistant two-line hybrid water-saving and drought-resistance rice (WDR), hence promoting sustainable rice production in China.

Difference between emergent aquatic and terrestrial monocotyledonous herbs in relation to the coordination of leaf stomata with vein traits.

Emergent aquatic plants mostly occur in shallow waters and root in bottom substrates, but their leaves emerge from the water surface and are thus exposed to air, similar to the leaves of terrestrial plants. Previous studies have found coordination between leaf water supply and demand in terrestrial plants; however, whether such a coordination exists in emergent aquatic plants remains unknown. In this study, we analysed leaf veins and stomatal characteristics of 14 emergent aquatic and 13 terrestrial monocotyledonous herb species (EMH and TMH), with 5 EMH and 8 TMH belonging to Poaceae. We found that EMH had significantly higher mean leaf area, leaf thickness, stomatal density, stomatal number per vein length and major vein diameter, but lower mean major vein length per area (VLA) and total VLA than TMH. There was no significant difference in stomatal length, minor VLA and minor vein diameter between the two groups. Stomatal density and total VLA were positively correlated among the EMH, TMH, as well as the 8 Poaceae TMH species, but this correlation became non-significant when data from both the groups were pooled. Our results showed that the differences in water supply between emergent aquatic and terrestrial plants modify the coordination of their leaf veins and stomatal traits.

Transcriptomic divergence between upland and lowland ecotypes contributes to rice adaptation to a drought-prone agroecosystem.

INTRODUCTION: Transcriptomic divergence drives plant ecological adaptation. Upland rice is differentiated in drought tolerance from lowland rice during its adaptation to the drought-prone environment. They provide a good system to learn the evolution of drought tolerance in rice. METHODS AND RESULTS: We estimate morphological differences between the two rice ecotypes under well-watered and drought conditions, as well as their genetic and transcriptomic divergences by the high-throughput sequencing. Upland rice possesses higher expression diversity than lowland rice does. Thousands of genes exhibit expression divergences between the two rice ecotypes, which contributes to their morphological differences in drought tolerance. These transcriptomic divergences contribute to drought adaptation of upland rice during its domestication. Mutations in transcriptional regulatory regions, which cause presence and absence of cis-elements, are the cause of expression divergence. About 15.3% transcriptionally selected genes also receive sequence-based selection in upland or lowland ecotype. Some highly differentiated genes promote the transcriptomic divergence between rice ecotypes via gene co-expression network. In addition, we also detected transcriptomic trade-offs between drought tolerance and productivity. DISCUSSION: Many key genes, which promote transcriptomic adaptation to drought in upland rice, have great prospective in breeding water-saving and drought-resistant rice. Meanwhile, appropriate strategies are required in breeding to overcome the potential transcriptomic trade-off.

Correlation and quantitative trait loci analyses of total chlorophyll content and photosynthetic rate of rice (Oryza sativa) under water stress and well-watered conditions.

In order to explore the relevant molecular genetic mechanisms of photosynthetic rate (PR) and chlorophyll content (CC) in rice (Oryza sativa L.), we conducted a series of related experiments using a population of recombinant inbred lines (Zhenshan97B x IRAT109). We found a significant correlation between CC and PR (R= 0.19**) in well-watered conditions, but no significant correlation during water stress (r= 0.08). We detected 13 main quantitative trait loci (QTLs) located on chromosomes 1, 2, 3, 4, 5, 6, and 10, which were associated with CC, including six QTLs located on chromosomes 1, 2, 3, 4, and 5 during water stress, and seven QTLs located on chromosomes 2, 3, 4, 6, and 10 in well-watered conditions. These QTLs explained 47.39% of phenotypic variation during water stress and 56.19% in well-watered conditions. We detected four main QTLs associated with PR; three of them (qPR2, qPR10, qPR11) were located on chromosomes 2, 10, and 11 during water stress, and one (qPR10) was located on chromosome 10 in well-watered conditions. These QTLs explained 34.37% and 18.41% of the phenotypic variation in water stress and well-watered conditions, respectively. In total, CC was largely controlled by main QTLs, and PR was mainly controlled by epistatic QTL pairs.

Effect of moisture gradient on rice yields and greenhouse gas emissions from rice paddies.

Fluxes of methane (CH4) and nitrous oxide (N2O) from two rice varieties, Huayou 14 and Hanyou 8, were monitored using closed chamber/gas chromatography method. Huayou 14 is a commonly grown variety of rice whereas Hanyou 8 is a water-saving and drought-resistant rice (WDR) variety. Low soil volumetric water content (VWC) existed in the treatments on the slope (W5 < W4 < W3 < W2). On the slope, rice yields of Hanyou 8 decreased by 12-39%, and Huayou 14 by 11-46% as compared to the plots on the flat. The total compatible solutes in Hanyou 8 had a greater variational range than Huayou 14. Compared to W1, CH4 emissions from W2-W5 decreased by 58-86% in Hanyou 8 and 38-86% in Huayou 14, whereas those of N2O increased by 26-121% in Hanyou 8 and 49-189% in Huayou 14 across both two seasons, which was mainly because the VWC varied in W2-W5 treatment. Under the treatments in the slope (W2, W3, W4, and W5), the global warming potential (GWP) was dominated by N2O emissions, which accounted for 69-90% of the GWP. Hanyou 8 had greater tolerance for water stress than Huayou 14 did, as evident from the smaller reductions in rice yield and greater variational range of total compatible solutes content. Water stress could reduce CH4 emissions but decrease N2O emissions for both rice varieties. This results suggest that planting WDR varieties under water shortage irrigation (such as W4, W5) will be able to maintain rice yields and reduce the GWP with less water.

Fine mapping a quantitative trait locus, qSER-7, that controls stigma exsertion rate in rice (Oryza sativa L.).

BACKGROUND: Stigma exsertion rate (SER) is a key determinant of outcrossing in hybrid rice seed production. A quantitative trait locus (QTL) for stigma exsertion rate in rice, qSER-7, has previously been detected on chromosome 7 by using a F2 population derived from two indica cytoplasmic male sterility (CMS) maintainers, Huhan 1B and II-32B. RESULTS: The chromosomal location of qSER-7 was precisely delimited by fine-scale mapping. Near-isogenic lines (NILs) were established, one of which isolated the locus in the qSER-7II-32B line, which contains an introgressed segment of II-32B in the Huhan 1B genetic background, and exhibits a significantly higher stigma exsertion rate than that of the recurrent parent. Using 3192 individuals from the BC4F2 segregation population, the QTL qSER-7 was narrowed down to a 28.4-kb region between the markers RM3859 and Indel4373 on chromosome 7. According to the rice genome annotation database, three genes were predicted within the target region. Real-time PCR analysis showed significantly higher expression levels of LOC_Os07g15370 and LOC_Os07g15390 in II-32B than in Huhan 1B. LOC_Os07g15370(OsNRAMP5) was a previously reported gene for Mn and Cd transporter. The stigma exertion rates of OsNRAMP5-overexpressing plants were significantly higher than that of wild type plants, in contrast, a T-DNA insertion mutant osnramp5 showed a lower stigma exertion rate. CONCLUSIONS: In the present study, the QTL qSER-7 was isolated to a region between the markers RM3859 and Indel4373. Two candidate genes were selected based on the expression difference between the two parents, which can facilitate the further cloning of the gene underlying the quantitative trait associated with qSER-7 as well as the marker-assisted transfer of desirable genes for stigma exsertion rate improvement in rice.

Enhanced rice salinity tolerance via CRISPR/Cas9-targeted mutagenesis of the OsRR22 gene.

Salinity is one of the most important abiotic stress affecting the world rice production. The cultivation of salinity-tolerant cultivars is the most cost-effective and environmentally friendly approach for salinity control. In recent years, CRISPR/Cas9 systems have been widely used for target-site genome editing; however, their application for the improvement of elite rice cultivars has rarely been reported. Here, we report the improvement of the rice salinity tolerance by engineering a Cas9-OsRR22-gRNA expressing vector, targeting the OsRR22 gene in rice. Nine mutant plants were identified from 14 T0 transgenic plants. Sequencing showed that these plants had six mutation types at the target site, all of which were successfully transmitted to the next generations. Mutant plants without transferred DNA (T-DNA) were obtained via segregation in the T1 generations. Two T2 homozygous mutant lines were further examined for their salinity tolerance and agronomic traits. The results showed that, at the seedling stage, the salinity tolerance of T2 homozygous mutant lines was significantly enhanced compared to wild-type plants. Furthermore, no significantly different agronomic traits were found between T2 homozygous mutant lines and wild-type plants. Our results indicate CRISPR/ Cas9 as a useful approach to enhance the salinity tolerance of rice.