The E3 ligase BnNLA1 modulates seed coat color in <i>Brassica napus</i> through ubiquitination of BnC07MYB3a

In rapeseed (Brassica napus L.), excessive accumulation of flavonoid pigments (anthocyanins/proanthocyanidins) compromises the nutrition, flavor, and commercial value. Therefore, reducing the accumulation of seed coat pigments is one of the main objectives in B. napus breeding. To elucidate the mechanisms affecting the genetic architecture of seed coat color, a genome-wide association study (GWAS) of seed coat color was conducted with a diverse group of 393 B. napus cultivars. NITROGEN LIMITATION ADAPTATION1 (NLA1) was identified as a previously unrecognized regulator that controls seed coat color and participates in flavonoid biosynthesis and accumulation in B. napus. Increasing evidence suggests that BnNLA1 (BnA09NLA1/BnC08NLA1) interacts with and ubiquitinates BnC07MYB3a, an R2R3-MYB-type transcription factor and a candidate regulator of the seed coat color in B. napus, and the lysine residue K164 of BnC07MYB3a is the key ubiquitination. Both the BnA09NLA1/BnC08NLA1 knock-out (KO) mutants and overexpressing BnC07MYB3a lines in B. napus exhibit lighter seed coat color indicating lower anthocyanin and proanthocyanidin accumulation compared with the wild-type plants. BnC07MYB3a also directly binds to the promoter of the TRANSPARENT TESTA 6 (BnTT6) and BANYULS (BnBAN), and represses their expression in B. napus. As expected, the expression levels of BnTT6 and BnBAN are significantly reduced in the BnA09NLA1/BnC08NLA1 KO mutants compared to the wild-type plants. Our findings reveal a novel regulatory framework mediated by the BnNLA1-BnC07MYB3a module for controlling seed coat color in B. napus and provide a new strategy for breeding high-quality B. napus cultivars.