Summary: Yellow‐seed is widely accepted as a good‐quality trait in Brassica crops. Previous studies have shown that the flavonoid biosynthesis pathway is essential for the development of seed colour, but its function in Brassica napus, an important oil crop, is poorly understood. To systematically explore the gene functions of the flavonoid biosynthesis pathway in rapeseed, several representative TRANSPARENT TESTA (TT) genes, including three structural genes (BnaTT7, BnaTT18, BnaTT10), two regulatory genes (BnaTT1, BnaTT2) and a transporter (BnaTT12), were selected for targeted mutation by CRISPR/Cas9 in the present study. Seed coat colour, lignin content, seed quality and yield‐related traits were investigated in these Bnatt mutants together with Bnatt8 generated previously. These Bnatt mutants produced seeds with an elevated seed oil content and decreased pigment and lignin accumulation in the seed coat without any serious defects in the yield‐related traits. In addition, the fatty acid (FA) composition was also altered to different degrees, i.e., decreased oleic acid and increased linoleic acid and α‐linolenic acid, in all Bnatt mutants except Bnatt18. Furthermore, gene expression analysis revealed that most of BnaTT mutations resulted in the down‐regulation of key genes related to flavonoid and lignin synthesis, and the up‐regulation of key genes related to lipid synthesis and oil body formation, which may contribute to the phenotype. Collectively, our study generated valuable resources for breeding programs, and more importantly demonstrated the functional divergence and overlap of flavonoid biosynthesis pathway genes in seed coat colour, oil content and FA composition of rapeseed.

Seed storability, defined as the ability to remain alive during storage, is an important agronomic and physiological characteristic, but the underlying genetic mechanism remains largely unclear. Here, we report quantitative trait loci (QTLs) analyses for seed storability using a high-density single nucleotide polymorphism linkage map in the backcross recombinant inbred lines that was derived from a cross of a japonica cultivar, Nipponbare, and an indica cultivar, 9311. Seven putative QTLs were identified for seed storability under natural storage, each explaining 3.6–9.0% of the phenotypic variation in this population. Among these QTLs, qSS1 with the 9311 alleles promoting seed storability was further validated in near-isogenic line and its derived-F₂ population. The other locus (qSS3.1) for seed storability colocalized with a locus for germination ability under hydrogen peroxide, which is recognized as an oxidant molecule that causes lipid damage. Transgenic experiments validated that a candidate gene (OsFAH2) resides the qSS3.1 region controlling seed storability and antioxidant capability. Overexpression of OsFAH2 that encodes a fatty acid hydroxylase reduced lipid preoxidation and increased seed storability. These findings provide new insights into the genetic and physiological bases of seed storability and will be useful for the improvement of seed storability in rice.

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.

Non-dormant seeds are continuously aging and deteriorating during storage, leading to declining seed vigor, which is a challenge for the rice seed industry. Improving the storability of seeds is of great significance to ensure the quality of rice and national food security. Through a set of chromosome segment substitution lines population constructed using japonica rice NIP as donor parent and indica rice ZS97 as recurrent parent, we performed seed storability QTL analysis and selected four non-storable NILs to further investigate the storability regulatory mechanisms underlying it. The seeds were divided into four tissues, which were the embryo, endosperm, aleurone layer, and hull, and tissue-specific transcriptome and metabolome analyses were performed on them. By exploring the common differentially expressed genes and differentially accumulated metabolites, as well as the KEGG pathway of the four non-storable NILs, we revealed that the phenylpropanoid biosynthesis pathway and diterpenoid biosynthesis pathway played a central role in regulating seed storability. Integrated analysis pinpointed 12 candidate genes that may take part in seed storability. The comprehensive analysis disclosed the divergent and synergistic effect of different seed tissues in the regulation of rice storability.