Creating a Superior Wx Allele with Temperature-Responsive Amylose Regulation and a Novel Transcriptional Pattern in Rice via CRISPR/Cas9-Mediated Promoter Editing

High quality stands as a pivotal competitive edge in the rice industry. Optimizing amylose content (AC) and the physicochemical properties of endosperm starch by regulating the Wx gene is crucial for enhancing rice grain quality. In this study, we created a novel Wxb-d25 allele by deleting a 25 bp segment (−26 to −2) within the Wx core promoter using CRISPR/Cas9. Compared with the wild type and the previously reported Wxb-i1, Wxb-d25 exhibited no significant changes in agronomic traits. However, its grains displayed temperature-dependent variations in AC and altered transparency and viscosity characteristics, holding the potential to synergistically improve both the eating and cooking quality (ECQ) and appearance quality (AQ) of rice. Further studies demonstrated that this promoter modification, by partially disrupting the transcription initiator, significantly downregulated the original Wx-01 transcript and generated a novel Wx transcript (ONT.7395.1) in Wxb-d25 grains. Despite its low expression abundance, the ONT.7395.1 transcript could be completely processed into mature Wx mRNA. The combined effects of the dual transcripts resulted in significantly increased Wx gene expression and AC in Wxb-d25 grains under conventional cultivation conditions. These findings provide a genetic resource and a theoretical foundation for utilizing the Wxb-d25 allele to improve rice grain quality.