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 <i>Wx</i> gene is crucial for enhancing rice grain quality. In this study, we created a novel <i>Wx^b-d25</i> allele by deleting a 25 bp segment (-26 to -2) within the <i>Wx</i> core promoter using CRISPR/Cas9. Compared with the wild type and the previously reported <i>Wx^b-i1</i>, <i>Wx^b-d25</i> 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 <i>Wx</i>-01 transcript and generated a novel <i>Wx</i> transcript (ONT.7395.1) in <i>Wx^b-d25</i> grains. Despite its low expression abundance, the ONT.7395.1 transcript could be completely processed into mature <i>Wx</i> mRNA. The combined effects of the dual transcripts resulted in significantly increased <i>Wx</i> gene expression and AC in <i>Wx^b-d25</i> grains under conventional cultivation conditions. These findings provide a genetic resource and a theoretical foundation for utilizing the <i>Wx^b-d25</i> allele to improve rice grain quality.