Investigation of a UPR-Related Gene Signature Identifies the Pro-Fibrotic Effects of Thrombospondin-1 by Activating CD47/ROS/Endoplasmic Reticulum Stress Pathway in Lung Fibroblasts.

Unfolded protein response (UPR) signaling and endoplasmic reticulum (ER) stress have been linked to pulmonary fibrosis. However, the relationship between UPR status and pulmonary function and prognosis in idiopathic pulmonary fibrosis (IPF) patients remains largely unknown. Through a series of bioinformatics analyses, we established a correlation between UPR status and pulmonary function in IPF patients. Furthermore, thrombospondin-1 (TSP-1) was identified as a potential biomarker for prognostic evaluation in IPF patients. By utilizing both bulk RNA profiling and single-cell RNA sequencing data, we demonstrated the upregulation of TSP-1 in lung fibroblasts during pulmonary fibrosis. Gene set enrichment analysis (GSEA) results indicated a positive association between TSP-1 expression and gene sets related to the reactive oxygen species (ROS) pathway in lung fibroblasts. TSP-1 overexpression alone induced mild ER stress and pulmonary fibrosis, and it even exacerbated bleomycin-induced ER stress and pulmonary fibrosis. Mechanistically, TSP-1 promoted ER stress and fibroblast activation through CD47-dependent ROS production. Treatment with either TSP-1 inhibitor or CD47 inhibitor significantly attenuated BLM-induced ER stress and pulmonary fibrosis. Collectively, these findings suggest that the elevation of TSP-1 during pulmonary fibrosis is not merely a biomarker but likely plays a pathogenic role in the fibrotic changes in the lung.

Fibroblast Upregulation of Vitamin D Receptor Represents a Self-Protective Response to Limit Fibroblast Proliferation and Activation during Pulmonary Fibrosis.

Dysregulation of vitamin D receptor (VDR) is implicated in chronic obstructive pulmonary disease. However, whether VDR dysregulation contributes to the development of pulmonary fibrosis remains largely unknown. Analysis of bulk and single-cell RNA profiling datasets revealed VDR upregulation in lung fibroblasts from patients with pulmonary fibrosis or fibrotic mice, which was validated in lung fibroblasts from bleomycin-exposed mice and bleomycin-treated fibroblasts. Stable VDR knockdown promoted, whereas the VDR agonist paricalcitol suppressed lung fibroblast proliferation and activation. Gene set enrichment analysis (GSEA) showed that the JAK/STAT pathway and unfolded protein response (UPR), a process related to endoplasmic reticulum (ER) stress, were enriched in lung fibroblasts of fibrotic lungs. Stable VDR knockdown stimulated, but paricalcitol suppressed ER stress and JAK1/STAT3 activation in lung fibroblasts. The STAT3 inhibitor blocked bleomycin- or stable VDR knockdown-induced ER stress. Paricalcitol inhibited the bleomycin-induced enrichment of STAT3 to the ATF6 promoter, thereby suppressing ATF6 expression in fibroblasts. Paricalcitol or intrapulmonary VDR overexpression inactivated JAK1/STAT3 and suppressed ER stress in bleomycin-treated mice, thus resulting in the inhibition of fibroblast proliferation and activation. Collectively, this study suggests that fibroblast VDR upregulation may be a self-protective response to limit fibroblast proliferation and activation during pulmonary fibrosis by suppressing the JAK1/STAT3/ER stress pathway.

Natural variation of OsML1, a mitochondrial transcription termination factor, contributes to mesocotyl length variation in rice.

Mesocotyl length (ML) is a crucial factor in determining the establishment and yield of rice planted through dry direct seeding, a practice that is increasingly popular in rice production worldwide. ML is determined by the endogenous and external environments, and inherits as a complex trait. To date, only a few genes have been cloned, and the mechanisms underlying mesocotyl elongation remain largely unknown. Here, through a genome-wide association study using sequenced germplasm, we reveal that natural allelic variations in a mitochondrial transcription termination factor, OsML1, predominantly determined the natural variation of ML in rice. Natural variants in the coding regions of OsML1 resulted in five major haplotypes with a clear differentiation between subspecies and subpopulations in cultivated rice. The much-reduced genetic diversity of cultivated rice compared to the common wild rice suggested that OsML1 underwent selection during domestication. Transgenic experiments and molecular analysis demonstrated that OsML1 contributes to ML by influencing cell elongation primarily determined by H2 O2 homeostasis. Overexpression of OsML1 promoted mesocotyl elongation and thus improved the emergence rate under deep direct seeding. Taken together, our results suggested that OsML1 is a key positive regulator of ML, and is useful in developing varieties for deep direct seeding by conventional and transgenic approaches.

OsNAC15 Regulates Tolerance to Zinc Deficiency and Cadmium by Binding to OsZIP7 and OsZIP10 in Rice.

Zinc (Zn) deficiency and cadmium (Cd) stress are severe threats to the growth and development of plants. Increasing Zn content and/or decreasing Cd content in grain are also important objectives of rice breeding. However, the molecular mechanisms of Zn deficiency tolerance (ZDT) and Cd stress tolerance (CDT) are largely unknown in rice. Here, we report that a NAM/CUC2-like transcription factor, OsNAC15, contributes to ZDT and CDT in rice. Knockout of OsNAC15 reduced ZDT and CDT at the vegetative stage. OsNAC15 expresses in all tissues of different developmental stages, and is repressed by Zn deficiency and induced by Cd stress. OsNAC15 is a functional transcription factor with transactivation and DNA binding activities. Expression analysis of rice ZIP family genes suggested that the knockout of OsNAC15 activates or inhibits their transcriptions under Zn deficiency or Cd stress conditions. The yeast one-hybrid assay, transient transcriptional activity assay using the dual-luciferase reporter system and electrophoretic mobility shift assay demonstrated that OsNAC15 directly binds to the zinc deficiency-responsive element motifs in the promoters of OsZIP7 and OsZIP10 to repress their transcriptions. The OsNAC15-OsZIP7/10 module is an essential foundation for further study on the regulatory mechanisms of ZDT and CDT in rice.

Multi-Trait Genomic Prediction Models Enhance the Predictive Ability of Grain Trace Elements in Rice.

Multi-trait (MT) genomic prediction models enable breeders to save phenotyping resources and increase the prediction accuracy of unobserved target traits by exploiting available information from non-target or auxiliary traits. Our study evaluated different MT models using 250 rice accessions from Asian countries genotyped and phenotyped for grain content of zinc (Zn), iron (Fe), copper (Cu), manganese (Mn), and cadmium (Cd). The predictive performance of MT models compared to a traditional single trait (ST) model was assessed by 1) applying different cross-validation strategies (CV1, CV2, and CV3) inferring varied phenotyping patterns and budgets; 2) accounting for local epistatic effects along with the main additive effect in MT models; and 3) using a selective marker panel composed of trait-associated SNPs in MT models. MT models were not statistically significantly (p < 0.05) superior to ST model under CV1, where no phenotypic information was available for the accessions in the test set. After including phenotypes from auxiliary traits in both training and test sets (MT-CV2) or simply in the test set (MT-CV3), MT models significantly (p < 0.05) outperformed ST model for all the traits. The highest increases in the predictive ability of MT models relative to ST models were 11.1% (Mn), 11.5 (Cd), 33.3% (Fe), 95.2% (Cu) and 126% (Zn). Accounting for the local epistatic effects using a haplotype-based model further improved the predictive ability of MT models by 4.6% (Cu), 3.8% (Zn), and 3.5% (Cd) relative to MT models with only additive effects. The predictive ability of the haplotype-based model was not improved after optimizing the marker panel by only considering the markers associated with the traits. This study first assessed the local epistatic effects and marker optimization strategies in the MT genomic prediction framework and then illustrated the power of the MT model in predicting trace element traits in rice for the effective use of genetic resources to improve the nutritional quality of rice grain.

Validation of Genes Affecting Rice Grain Zinc Content Through Candidate Gene-Based Association Analysis.

Several key genes governing Zn homeostasis and grain zinc content (GZC) have been functionally characterized. However, the effects of these genes in diverse breeding populations have not been evaluated; thus, their availability in breeding is unclear. In this study, the effects of 65 genes related to rice zinc responses on GZC were evaluated using two panels of breeding lines, and the superior haplotypes were identified. One panel consisted of mega varieties from the International Rice Research Institute (IRRI), South Asia, and Southeast Asia (SEA), and the other panel is breeding lines/varieties from South China (SC). In addition, a multiparent advanced generation intercross (MAGIC) population, named as DC1, was also employed. Three analytical methods, single-locus mixed linear model (SL-MLM), multilocus random-SNP-effect mixed linear model (mrMLM), and haplotype-based association analysis (Hap-AA), were applied. OsIDEF1 (which explained 12.3% of the phenotypic variance) and OsZIFL7 (8.3-9.1%), OsZIP7 (18.9%), and OsIRT1 (17.9%) were identified by SL-MLM in SEA and SC, respectively, whereas no gene was significantly associated with GZC in DC1. In total, five (OsNRAMP6, OsYSL15, OsIRT1, OsIDEF1, and OsZIFL7, 7.70-15.39%), three (OsFRDL1, OsIRT1, and OsZIP7, 11.87-17.99%), and two (OsYSL7 and OsZIP7, 9.85-10.57%) genes were detected to be significantly associated with GZC in SEA, SC, and DC1 by mrMLM, respectively. Hap-AA indicated that Hap1-OsNRAMP5, Hap5-OsZIP4, Hap1-OsIRT1, Hap3-OsNRAMP6, Hap6-OsMTP1, and Hap6-OsYSL15 had the largest effects for GZC in SEA, whereas Hap3-OsOPT7, Hap4-OsIRT2, Hap4-OsZIP7, Hap5-OsIRT1, and Hap5-OsSAMS1 were the most significant in the SC population. Besides, superior alleles were also identified for the significant genes. The genes significantly associated with GZC and their superior haplotypes identified in different panels could be used in enhancing GZC through molecular breeding, which could further address the problem of Zn malnutrition among rice consumers.

Mesocotyl elongation, an essential trait for dry-seeded rice (Oryza sativa L.): a review of physiological and genetic basis.

MAIN CONCLUSIONS: (1) Mesocotyl elongation is responsive to abiotic stresses, such as deep sowing drought, submergence, chilling, and salinity. (2) Humus soil culture with a burial depth of 6 cm and at the temperature of 30 °C could be the optimum method for mesocotyl length phenotyping, The frequently colocalized quantitative trait loci (QTL) controlling mesocotyl elongation were located on chromosome (3) 1 (RM562-RG146), chromosome 2 (RZ288-RM145), and chromosome 3 (RM426-RM520). Dry direct-seeding is becoming a popular rice cultivation technology in many countries, which reduces water use and labor costs enormously. Meanwhile, direct-seeding rice is also facing the problems of low seedling emergence rate, poor seedling establishment, weed infestation, and high crop lodging rate. To take the full advantages of direct-seeding, both agronomic and genetic solutions are needed. Varieties with optimum mesocotyl length are desired for improving rice seedling emergence rate, particularly under deep sowing and submergence, which is adopted to reduce lodging and increase tolerance to abiotic stresses. In this review, we summarized the physiological and genetic mechanisms of mesocotyl elongation in rice. The elongation of mesocotyl is affected by light, temperature, and water, and, as a result, is responsive to sowing depth, water content, and soil salinity. Plant hormones such as abscisic acid (ABA), brassinosteroid (BR), strigolactones (SLs), cytokinin (CTK), ethylene (ETH), jasmonic acid (JA), gibberellin (GA), and indole-3-acetic acid (IAA) play important roles in regulating mesocotyl elongation. A humus soil culture protocol developed by our team was shown to be a better high-throughput method for measuring mesocotyl length in large scale. Sixty-seven QTL controlling mesocotyl length were reported, which are distributed on all the 12 chromosomes. Twelve chromosomal regions were repeatedly found to have QTL using various mapping populations and methods. These regions should be targeted in future studies to isolate genes and develop markers for molecular breeding. Two genes with very different molecular functions have been cloned, highlighting the genetic complexity of mesocotyl elongation.

Genome-wide Association Study (GWAS) for Mesocotyl Elongation in Rice (Oryza sativa L.) under Multiple Culture Conditions.

Mesocotyl is a crucial organ for pushing buds out of soil, which plays a vital role in seedling emergence and establishment in dry direct-seeded rice. However, the genetic mechanisms of mesocotyl elongation remains unclear. In our study, 208 rice accessions were used to identify the SNPs significantly associated with mesocotyl length under various culture conditions, including sand, water and soil. The mesocotyl length ranges from 0 to 4.88 cm, 0 to 3.99 cm and 0 to 4.51 cm in sand, water and soil covering, respectively. A total of 2,338,336 SNPs were discovered by re-sequencing of 208 rice accessions. Genome-wide association study (GWAS) based on mixed linear model (MLM) was conducted and 16 unique loci were identified on chromosomes 1, 2 (2), 3, 4, 5 (2), 6 (2), 7, 8, 9 (2) and 12 (3), respectively, explaining phenotypic variations ranging from 6.3 to 15.9%. Among these loci, 12 were stable across two or more environments. Ten out of the sixteen loci coincided with known genes or quantitative trait locus (QTL), whereas the other six were potentially novel loci. Furthermore, five high-confidence candidate genes related to mesocotyl elongation were identified on chromosomes 1, 3, 5, 9 and 12. Moreover, qRT-PCR analysis showed that all the five genes showed significant expression difference between short-mesocotyl accessions and long-mesocotyl accessions. This study provides new insights into the genetic architecture of rice mesocotyl, the associated SNPs and germplasms with long mesocotyl could be useful in the breeding of mechanized dry direct-seeded rice.