Leaf chlorophyll content is an important physiological indicator of plant growth, metabolism and nutritional status, and it is highly correlated with leaf nitrogen content and photosynthesis. In this study, we report the cloning and identification of a xylan glucuronosyltransferase gene (OsGUX1) that affects relative chlorophyll content in rice leaf. Using a set of chromosomal segment substitution lines derived from a cross of wild rice accession ACC10 and indica variety Zhenshan 97 (ZS97), we identified numerous quantitative trait loci for relative chlorophyll content. One major locus of them for relative chlorophyll content was mapped to a 10.3-kb region that contains OsGUX1. The allele OsGUX1AC from ACC10 significantly decreases nitrogen content and chlorophyll content of leaf compared with OsGUX1ZS from ZS97. The overexpression of OsGUX1 reduced chlorophyll content, and the suppression of this gene increased chlorophyll content of rice leaf. OsGUX1 is located in Golgi apparatus, and highly expressed in seedling leaf and the tissues in which primary cell wall synthesis occurring. Our experimental data indicate that OsGUX1 is responsible for addition of glucuronic acid residues onto xylan and participates in accumulation of cellulose and hemicellulose in the cell wall deposition, thus thickening the primary cell wall of mesophyll cells, which might lead to reduced chlorophyll content in rice leaf. These findings provide insights into the association of cell wall components with leaf nitrogen content in rice.

Rice is highly sensitive to salinity stress during the seedling establishment phase. Salt stress is widely occurring in cultivated areas and severely affects seed germination ability and seedling establishment, which may result in a complete crop failure. The objective of the present study is to identify quantitative trait loci (QTLs) related to salt tolerance of the germination and seedling stages in a rice backcross inbred line (BIL) population that was derived from a backcross of an Africa rice ACC9 as donor and indica cultivar Zhenshan97 (ZS97) as the recurrent parent. Under salt stress, ACC9 exhibited a higher germination percentage, but more repressed seedling growth than ZS97. Using the BIL population, 23 loci for germination parameters were detected at the germination stage and 46 loci were identified for several morphological and physiological parameters at the seedling stage. Among them, nine and 33 loci with the ACC9 alleles increased salt tolerance at the germination and seedling stages, respectively. Moreover, several major QTLs were found to be co-localized in the same or overlapping regions of previously reported genes for salt stress. These major loci will facilitate improving salt-tolerance rice in genome-breeding programs.

Grain chalkiness is an important grain quality related to starch granules in the endosperm. A high percentage of grain chalkiness is a major problem because it diminishes grain quality in rice. Here, we report quantitative trait loci identification for grain chalkiness using high-throughput single nucleotide polymorphism genotyping of a chromosomal segment substitution line population in which each line carried one or a few introduced japonica cultivar Nipponbare segments in the genetic background of the indica cultivar ZS97. Ten quantitative trait loci regions were commonly identified for the percentage of grain chalkiness and the degree of endosperm chalkiness. The allelic effects at nine of these quantitative trait loci reduced grain chalkiness. Furthermore, a quantitative trait locus (qPGC8-2) on chromosome 8 was validated in a chromosomal segment substitution line-derived segregation population, and had a stable effect on chalkiness in a multiple-environment evaluation of the near-isogenic lines. Residing on the qPGC8-2 region, the isoamylase gene (ISA1) was preferentially expressed in the endosperm and revealed some nucleotide polymorphisms between two varieties, Nipponbare and ZS97. Transgenic lines with suppression of ISA1 by RNA interference produced grains with 20% more chalkiness than the control. The results support that the gene may underlie qPGC8-2 for grain chalkiness. The multiple-environment trials of the near-isogenic lines also show that combination of the favorable alleles such as the ISA1 gene for low chalkiness and the GS3 gene for long grains considerably improved grain quality of ZS97, which proves useful for grain quality improvement in rice breeding programs.

Seed storability largely determines the vigor of seeds during storage and is significant in agriculture and ecology. However, the underlying genetic basis remains unclear. In the present study, we report the cloning and characterization of the rice (Oryza sativa) indole-3-acetic acid (IAA)-amido synthetase gene GRETCHEN HAGEN3-2 (OsGH3-2) associated with seed storability. OsGH3-2 was identified by performing a genome-wide association study in rice germplasms with linkage mapping in chromosome substitution segment lines, contributing to the wide variation of seed viability in the populations after long periods of storage and artificial ageing. OsGH3-2 was dominantly expressed in the developing seeds and catalyzed IAA conjugation to amino acids, forming inactive auxin. Transgenic overexpression, knockout, and knockdown experiments demonstrated that OsGH3-2 affected seed storability by regulating the accumulation level of abscisic acid (ABA). Overexpression of OsGH3-2 significantly decreased seed storability, while knockout or knockdown of the gene enhanced seed storability compared with the wild-type. OsGH3-2 acted as a negative regulator of seed storability by modulating many genes related to the ABA pathway and probably subsequently late embryogenesis-abundant proteins at the transcription level. These findings shed light on the molecular mechanisms underlying seed storability and will facilitate the improvement of seed vigor by genomic breeding and gene-editing approaches in rice.

Variations in the gene promoter play critical roles in the evolution of important adaptive traits in crops, but direct links of the regulatory mutation to the adaptive change are not well understood. Here, we examine the nucleotide variations in the promoter region of a transcription factor (Ghd8) that control grain number, plant height and heading date in rice. We find that a dominant promoter type of subspecies japonica displayed a high activity for Ghd8 expression in comparison with the one in indica. Transgenic analyses revealed that higher expression levels of Ghd8 delayed heading date and enhanced cold tolerance in rice. Furthermore, a single-nucleotide polymorphism (T1279G) at the position -1279 bp that locates on the potential GA-responsive motif in the Ghd8 promoter affected the expression of this gene. The 1279 T variant has elevated expression of Ghd8, thus conferring increased cold tolerance of rice seedlings. Nucleotide diversity analysis revealed that the approximately 25-kb genomic region surrounding Ghd8 in the subspecies japonica was under significant selection pressure. Our findings demonstrate that the join effects of the regulatory and coding variants largely contribute to the divergence of japonica and indica and increase the adaptability of japonica to the cold environment.

Seed dormancy, a quality characteristic that plays a role in seed germination, seedling establishment and grain yield, is affected by multiple genes and environmental factors. The genetic and molecular mechanisms underlying seed dormancy in rice remain largely unknown.

Seed deterioration during storage poses a significant challenge to rice production, leading to a drastic decline in both edible quality and viability, thereby impacting overall crop yield. This study aimed to address this issue by further investigating candidate genes associated with two previously identified QTLs for seed storability through genome association analysis. Among the screened genes, two superoxide dismutase (SOD) genes, OsCSD2 (Copper/zinc Superoxide Dismutase 2) and OsCSD3, were selected for further study. The generation of overexpression and CRISPR/Cas9 mutant transgenic lines revealed that OsCSD2 and OsCSD3 play a positive regulatory role in enhancing rice seed storability. Subsequent exploration of the physiological mechanisms demonstrated that overexpression lines exhibited lower relative electrical conductivity, indicative of reduced cell membrane damage, while knockout lines displayed the opposite trend. Furthermore, the overexpression lines of OsCSD2 and OsCSD3 showed significant increases not only in SOD but also in CAT and POD activities, highlighting an augmented antioxidant system in the transgenic seeds. Additionally, hormone profiling indicated that ABA contributed to the improved seed storability observed in these lines. In summary, these findings provide valuable insights into the regulatory mechanisms of OsCSDs in rice storability, with potential applications for mitigating grain loss and enhancing global food security.

Ammonium is one of the major forms in which nitrogen is available for plant growth. OsAMT1;1 is a high-affinity ammonium transporter in rice (Oryza sativa L.), responsible for ammonium uptake at low nitrogen concentration. The expression pattern of the gene has been reported. However, variations in its nucleotides and the evolutionary pathway of its descent from wild progenitors are yet to be elucidated. In this study, nucleotide diversity of the gene OsAMT1;1 and the diversity pattern of seven gene fragments spanning a genomic region approximately 150 kb long surrounding the gene were surveyed by sequencing a panel of 216 rice accessions including both cultivated rice and wild relatives.

Timing of germination determines whether a new plant life cycle can be initiated; therefore, appropriate dormancy and rapid germination under diverse environmental conditions are the most important features for a seed. However, the genetic architecture of seed dormancy and germination behavior remains largely elusive. In the present study, a linkage analysis for seed dormancy and germination behavior was conducted using a set of 146 chromosome segment substitution lines (CSSLs), of which each carries a single or a few chromosomal segments of Nipponbare (NIP) in the background of Zhenshan 97 (ZS97). A total of 36 quantitative trait loci (QTLs) for six germination parameters were identified. Among them, qDOM3.1 was validated as a major QTL for seed dormancy in a segregation population derived from the qDOM3.1 near-isogenic line, and further delimited into a genomic region of 90 kb on chromosome 3. Based on genetic analysis and gene expression profiles, the candidate genes were restricted to eight genes, of which four were responsive to the addition of abscisic acid (ABA). Among them, LOC_Os03g01540 was involved in the ABA signaling pathway to regulate seed dormancy. The results will facilitate cloning the major QTLs and understanding the genetic architecture for seed dormancy and germination in rice and other crops.

Trichome formation has been extensively studied as a mechanistic model for epidermal cell differentiation and cell morphogenesis in plants. However, the genetic and molecular mechanisms underlying trichome formation (i.e., initiation and elongation) in rice remain largely unclear. Here, we report an AP2/ERF transcription factor, Hairy Leaf 6 (HL6), which controls trichome formation in rice. Functional analyses revealed that HL6 transcriptionally regulates trichome elongation in rice, which is dependent on functional OsWOX3B, a homeodomain-containing protein that acts as a key regulator in trichome initiation. Biochemical and molecular genetic analyses demonstrated that HL6 physically interacts with OsWOX3B, and both of them regulate the expression of some auxin-related genes during trichome formation, in which OsWOX3B likely enhances the binding ability of HL6 with one of its direct target gene, OsYUCCA5. Population genetic analysis indicated that HL6 was under negative selection during rice domestication. Taken together, our findings provide new insights into the molecular regulatory network of trichome formation in rice.