The superior allele LEA12OR in wild rice enhances salt tolerance and yield.

Soil salinity has negative impacts on food security and sustainable agriculture. Ion homeostasis, osmotic adjustment and reactive oxygen species scavenging are the main approaches utilized by rice to resist salt stress. Breeding rice cultivars with high salt tolerance (ST) and yield is a significant challenge due to the lack of elite alleles conferring ST. Here, we report that the elite allele LEA12OR, which encodes a late embryogenesis abundant (LEA) protein from the wild rice Oryza rufipogon Griff., improves osmotic adjustment and increases yield under salt stress. Mechanistically, LEA12OR, as the early regulator of the LEA12OR-OsSAPK10-OsbZIP86-OsNCED3 functional module, maintains the kinase stability of OsSAPK10 under salt stress, thereby conferring ST by promoting abscisic acid biosynthesis and accumulation in rice. The superior allele LEA12OR provides a new avenue for improving ST and yield via the application of LEA12OR in current rice through molecular breeding and genome editing.

Alternative splicing of OsGS1;1 affects nitrogen-use efficiency, grain development, and amylose content in rice.

Excessive nitrogen fertilizer application is harmful to the environment and reduces the quality of cereal crops. Maintaining crop yields under low nitrogen (LN) conditions and improving quality are important goals for cereal crop breeding. Although the effects of nitrogen assimilation on crop nitrogen-use efficiency (NUE) have been intensively studied, natural variations of the key assimilation genes underlying grain development and quality are largely unclear. Here, we identified an NUE-associated gene, OsGS1;1, encoding glutamine synthase, through genome-wide association analysis, followed by validation experiments and functional analysis. Fifteen single-nucleotide polymorphisms in the OsGS1;1 region led to alternative splicing that generated two functional transcripts: OsGS1;1a and OsGS1;1b. The elite haplotype of OsGS1;1 showed high OsGS1;1b activity, which improved NUE, affected grain development, and reduced amylose content. The results show that OsGS1;1, which is induced under LN conditions, affects grain formation by regulating sugar metabolism and may provide a new avenue for the breeding of high-yield and high-quality rice (Oryza sativa).

OsTUB1 confers salt insensitivity by interacting with Kinesin13A to stabilize microtubules and ion transporters in rice.

Salt stress is one of the major environmental factors limiting plant growth and development. Although microtubule (MT) organization is known to be involved in response to salt stress, few tubulin genes have been identified that confer salt insensitivity in plants. In this study, we identified a MT encoding gene, OsTUB1, that increased the survival rate of rice plants under salt stress by stabilizing MT organization and ion transporters. We found that OsTUB1 interacted with Kinesin13A protein, which was essential for OsTUB1-regulated MT organization under salt stress. Further molecular evidence revealed that a OsTUB1-Kinesin13A complex protected rice from salt stress by sustaining membrane-localized Na+ transporter OsHKT1;5, a key regulator of ionic homeostasis. Our results shed light on the function of tubulin and kinesin in regulating MT organization and stabilizing Na+ transporters and Na+ flux at the plasma membrane in rice. The identification of the OsTUB1-Kinesin13A complex provides novel genes for salt insensitivity rice breeding in areas with high soil salinity.

Enhanced OsNLP4-OsNiR cascade confers nitrogen use efficiency by promoting tiller number in rice.

Increased use of nitrogen fertilizers has deleterious impact on the environment. Increase in yield potential at low nitrogen supply is regarded as a cereal breeding goal for future agricultural sustainability. Although natural variations of nitrogen transporters have been investigated, key genes associated with assimilation remain largely unexplored for nitrogen use efficiency (NUE) enhancement. Here, we identified a NIN-like protein NLP4 associated with NUE through a GWAS in rice. We found that OsNLP4 transactivated OsNiR encoding nitrite reductase that was critical in nitrogen assimilation in rice. We further constructed quadrupling NREs (Nitrate-responsive cis-elements) in the promoter of OsNiR (p4xNRE:OsNiR) and enhanced nitrogen assimilation significantly. We demonstrated that OsNLP4-OsNiR increased tiller number and yield through enhancing nitrogen assimilation and NUE. Our discovery highlights the genetic modulation of OsNLP4-OsNiR signalling cascade as a strategy for high NUE and yield breeding in rice.

Heterosis-associated genes confer high yield in super hybrid rice.

KEY MESSAGE: Heterosis QTLs, including qSS7 and qHD8, with dominance effects were identified through GBS and large-scale phenotyping of CSSLs and hybrid F1 populations in a paddy field. Heterosis has contributed immensely to agricultural production, but its genetic basis is unclear. We evaluated dominance effects by creating two hybrid populations: a B-homo set with a homozygous background and heterozygous chromosomal segments and a B-heter set with a heterozygous background and homozygous segments. This was achieved by crossing a set of 156 backcrossed-derived chromosome segment substitution lines (CSSLs) with their recurrent parent (9311), the male parent of the first super-high-yield hybrid Liangyoupei9 (LYP9), and with the female parent (PA64s) of the hybrid. The CSSLs were subjected to a genotyping-by-sequencing analysis to develop a genetic map of segments introduced from the PA64s. We evaluated the heterotic effects on eight yield-related traits in the hybrid variety and F1 populations in large-scale field experiments over 2 years. Using a linkage map consisting of high-density SNPs, we identified heterosis-associated genes in LYP9. Five candidate genes contributed to the high yield of LYP9, with qSS7 and qHD8 repeatedly detected in both B-hybrid populations. The heterozygous segments harboring qSS7 and qHD8 showed dominance effects that contributed to the heterosis of yield components in the hybrid rice variety Liangyoupei9.

OsPPR6, a pentatricopeptide repeat protein involved in editing and splicing chloroplast RNA, is required for chloroplast biogenesis in rice.

KEY MESSAGE: OsPPR6, a pentatricopeptide repeat protein involved in editing and splicing chloroplast RNA, is required for chloroplast biogenesis in rice. The chloroplast has its own genetic material and genetic system, but it is also regulated by nuclear-encoded genes. However, little is known about nuclear-plastid regulatory mechanisms underlying early chloroplast biogenesis in rice. In this study, we isolated and characterized a mutant, osppr6, that showed early chloroplast developmental defects leading to albino leaves and seedling death. We found that the osppr6 mutant failed to form thylakoid membranes. Using map-based cloning and complementation tests, we determined that OsPPR6 encoded a new Pentatricopeptide Repeat (PPR) protein localized in plastids. In the osppr6 mutants, mRNA levels of plastidic genes transcribed by the plastid-encoded RNA polymerase decreased, while those of genes transcribed by the nuclear-encoded RNA polymerase increased. Western blot analyses validated these expression results. We further investigated plastidic RNA editing and splicing in the osppr6 mutants and found that the ndhB transcript was mis-edited and the ycf3 transcript was mis-spliced. Therefore, we demonstrate that OsPPR6, a PPR protein, regulates early chloroplast biogenesis and participates in editing of ndhB and splicing of ycf3 transcripts in rice.

TSV, a putative plastidic oxidoreductase, protects rice chloroplasts from cold stress during development by interacting with plastidic thioredoxin Z.

Rice is vulnerable to cold stress. Seedlings are very sensitive to cold stress and this harms global rice production. The effects of cold on chloroplast development are well known, but little is known about the underlying molecular mechanisms. Here, we isolated a temperature-sensitive virescent (tsv) mutant that is extremely sensitive to cold stress. It displayed defective chloroplasts, decreased chlorophyll and zero survivorship under cold stress. We isolated and identified TSV by map-based cloning and rescue experiments, combined with genetic, cytological and molecular biological analyses. We found that TSV, a putative plastidic oxidoreductase, is a new type of virescent protein. A mutation in tsv causes premature termination of the gene product. The activity of plastid-encoded RNA polymerase (PEP) and the expression of genes participating in chlorophyll synthesis were severely reduced in the tsv mutant under cold stress, but not at normal temperatures. TSV expression was induced by low temperatures. Strikingly, TSV interacted with OsTrxZ (a subunit of PEP in chloroplasts) and enhanced OsTrxZ stability under low temperatures. We demonstrated that TSV protects rice chloroplasts from cold stress by interacting with OsTrxZ. These results provide novel insights into ways in which rice chloroplast development and chlorophyll synthesis are protected by TSV under cold stress.

Association mapping and genetic dissection of nitrogen use efficiency-related traits in rice (Oryza sativa L.).

The increases in the usage of nitrogen fertilizer result in deleterious impacts on the environment; thus, there is an urgent need to improve nitrogen use efficiency (NUE) in crops including rice (Oryza sativa L.). Attentions have focused on quantitative trait loci (QTL) mapping of NUE-related traits using single experimental population, but to date, very few studies have taken advantage of association mapping to examine hundreds of lines for identifying potentially novel QTLs in rice. Here, we conducted association analysis on NUE-related traits using a population containing 184 varieties, which were genotyped with 157 genome-wide simple sequence repeat (SSR) markers. We detected eight statistically significant marker loci associating with NUE-related traits, of which two QTLs at RM5639 and RM3628 harbored known NUE-related genes GS1;2 and AspAt3, respectively. At a novel NUE-related locus RM5748, we developed Kompetitive Allele Specific PCR (KASP) single nucleotide polymorphism (SNP) markers and searched for putative NUE-related genes which are close to the associated SNP marker. Based on a transcriptional map of N stress responses constructed by our lab, we evaluated expressions of the NUE-related genes in this region and validated their effect on NUE. Meanwhile, we analyzed NUE-related alleles of the eight loci that could be utilized in marker-assisted selection. Moreover, we estimated breeding values of all the varieties through genomic prediction approach that could be beneficial for rice NUE enhancement.

SNP-based analysis of genetic diversity reveals important alleles associated with seed size in rice.

BACKGROUND: Single-nucleotide polymorphisms (SNPs) have become the genetic markers of choice in various genetic, ecological, and evolutionary studies. Genotyping-by-sequencing (GBS) is a next-generation-sequencing based method that takes advantage of reduced representation to enable high-throughput genotyping using a large number of SNP markers. RESULTS: In the present study, the distribution of non-redundant SNPs in the parents of 12 rice recombination line populations was evaluated through GBS. A total of 45 Gigabites of nucleotide sequences conservatively provided satisfactory genotyping of rice SNPs. By assembling to the genomes of reference genomes of japonica Nipponbare, we detected 22,682 polymorphic SNPs that may be utilized for QTL/gene mapping with the Recombinant Inbred Lines (RIL) populations derived from these parental lines. Meanwhile, we identified polymorphic SNPs with large effects on protein-coding and miRNA genes. To validate the effect of the polymorphic SNPs, we further investigated a SNP (chr4:28,894,757) at the miRNA binding site in the 3'-UTR region of the locus Os4g48460, which is associated with rice seed size. Os4g48460 encodes a putative cytochrome P450, CYP704A3. Direct degradation of the 3'-UTR of the CYP704A3 gene by a miRNA (osa-miRf10422-akr) was validated by in planta mRNA degradation assay. We also showed that rice seeds of longer lengths may be produced by downregulating CYP704A3 via RNAi. CONCLUSIONS: Our study has identified the genome-wide SNPs by GBS of the parental varieties of RIL populations and identified CYP704A3, a miRNA-regulated gene that is responsible for rice seed length.

A Novel Allele in the Promoter of Wx Decreases Gene Expression and Confers Lower Apparent Amylose Contents in Japonica Rice (Oryza sativa L.).

Wx is the key gene that controls amylose content (AC), and various alleles have been found in rice populations. Wxb is the major allele in japonica and produces moderate AC (15~18%). It was recently found that editing the promoter of Wx could produce a series of alleles that have different Wx activities. Although some studies have edited the promoter, few studies have focused on the natural variations in Wx. Here, we used the Rice3K database to investigate variations in the Wx promoter and found that the allele Wx1764178 (A/G) has a higher LD (linkage disequilibrium) with the two key SNPs (1765751, T/G; 1768006, A/C), which could produce different Wx alleles and influence AC, as reported previously. Further study showed that the Wx1764178 allele (A/G) is functional and influences the expression of Wx positively. Editing the A allele using CRISPR‒Cas9 produced 36 and 3 bp deletions and caused a decrease in the expression of Wx. The apparent amylose content (AAC) in the edited lines was decreased by 7.09% and 11.50% compared with that of the wild type, which was the japonica variety Nipponbare with Wxb and the A allele at 1764178, while a complementary line with the G allele showed a lower AAC than the A allele with no effect on other agronomic traits. The AAC of the edited lines showed a higher increase than that of the wild type (Nipponbare, Wxb) in low-nitrogen conditions relative to high-nitrogen conditions. We also developed a dCAPS marker to identify the allele and found that the G allele has widely been used (82.95%) in japonica-bred varieties from Jiangsu Province, China. Overall, we found a functional allele (Wx1764178, A/G) in the Wx promoter that could affect AAC in japonica cultivars and be developed as markers for quality improvement in rice breeding programs.

The elite haplotype OsGATA8-H coordinates nitrogen uptake and productive tiller formation in rice.

Excessive nitrogen promotes the formation of nonproductive tillers in rice, which decreases nitrogen use efficiency (NUE). Developing high-NUE rice cultivars through balancing nitrogen uptake and the formation of productive tillers remains a long-standing challenge, yet how these two processes are coordinated in rice remains elusive. Here we identify the transcription factor OsGATA8 as a key coordinator of nitrogen uptake and tiller formation in rice. OsGATA8 negatively regulates nitrogen uptake by repressing transcription of the ammonium transporter gene OsAMT3.2. Meanwhile, it promotes tiller formation by repressing the transcription of OsTCP19, a negative modulator of tillering. We identify OsGATA8-H as a high-NUE haplotype with enhanced nitrogen uptake and a higher proportion of productive tillers. The geographical distribution of OsGATA8-H and its frequency change in historical accessions suggest its adaption to the fertile soil. Overall, this study provides molecular and evolutionary insights into the regulation of NUE and facilitates the breeding of rice cultivars with higher NUE.

Identification of risk factors for air traffic controllers' unsafe acts based on online reviews.

Online reviews may influence unsafe acts and are significant in the context of big data. This study acquired online reviews related to air traffic control from social media websites. The word frequency statistics and coding of negative comments were taken to mine risk factors. Combined with the human factors analysis and classification system (HFACS), a conceptual model of the risk factors associated with the unsafe acts of air traffic controllers (ATCers) was constructed. The results indicate that the frequency of risk factors in online reviews, ranked from high to low, is organizational influences, ATCers' adverse states, environmental factors and unsafe supervision. Organizational influences, environmental factors and unsafe supervision indirectly affect the unsafe acts through the ATCers' adverse states. It is demonstrated that the combination of HFACS and online reviews to identify risk factors enables the identification of problems in the air traffic control industry and demands of ATCers.

Genome-wide association studies identify OsWRKY53 as a key regulator of salt tolerance in rice.

Salinity stress progressively reduces plant growth and productivity, while plant has developed complex signaling pathways to confront salt stress. However, only a few genetic variants have been identified to mediate salt tolerance in the major crop rice, and the molecular mechanism remains poorly understood. Here, we identify ten candidate genes associated with salt-tolerance (ST) traits by performing a genome-wide association analysis in rice landraces. We characterize two ST-related genes, encoding transcriptional factor OsWRKY53 and Mitogen-activated protein Kinase Kinase OsMKK10.2, that mediate root Na+ flux and Na+ homeostasis. We further find that OsWRKY53 acts as a negative modulator regulating expression of OsMKK10.2 in promoting ion homeostasis. Furthermore, OsWRKY53 trans-represses OsHKT1;5 (high-affinity K+ transporter 1;5), encoding a sodium transport protein in roots. We show that the OsWRKY53-OsMKK10.2 and OsWRKY53-OsHKT1;5 module coordinate defenses against ionic stress. The results shed light on the regulatory mechanisms underlying plant salt tolerance.

Genome-wide associated study identifies NAC42-activated nitrate transporter conferring high nitrogen use efficiency in rice.

Over-application of nitrogen fertilizer in fields has had a negative impact on both environment and human health. Domesticated rice varieties with high nitrogen use efficiency (NUE) reduce fertilizer for sustainable agriculture. Here, we perform genome-wide association analysis of a diverse rice population displaying extreme nitrogen-related phenotypes over three successive years in the field, and identify an elite haplotype of nitrate transporter OsNPF6.1HapB that enhances nitrate uptake and confers high NUE by increasing yield under low nitrogen supply. OsNPF6.1HapB differs in both the protein and promoter element with natural variations, which are differentially trans-activated by OsNAC42, a NUE-related transcription factor. The rare natural allele OsNPF6.1HapB, derived from variation in wild rice and selected for enhancing both NUE and yield, has been lost in 90.3% of rice varieties due to the increased application of fertilizer. Our discovery highlights this NAC42-NPF6.1 signaling cascade as a strategy for high NUE and yield breeding in rice.