Endogenous Anthocyanins Are Associated with Improved Starch Quality Stability in Black Rice Under Accelerated Aging: Evidence from an OsKala4 Knockout Model

Black rice is rich in anthocyanins with potential antioxidant benefits, but their specific role in storage stability remains unclear due to confounding genetic backgrounds in previous studies. In this study, we used CRISPR/Cas9-mediated gene editing to generate OsKala4 knockout lines in the black rice cultivar Heizhen (HZ), creating an isogenic system to test whether endogenous anthocyanins contribute to storage-related quality stability. Knockout lines showed blocked anthocyanin biosynthesis (0.5–0.6 vs. 155.6 mg/100 g, p < 0.001) and altered grain composition. Under accelerated aging (45 °C, 90% RH, 2 weeks), HZ maintained higher antioxidant capacity (p < 0.05) and exhibited less pronounced starch aging than the representative knockout line KO2. Apparent amylose content increased less in HZ than in KO2 (16.7% vs. 28.1%, p < 0.05). HZ also showed smaller changes in pasting, thermal, and structural properties. XRD and FTIR analyses further suggested better maintenance of starch crystallinity and molecular order in HZ under accelerated aging conditions. These results suggest that endogenous anthocyanins were associated with storage-related quality stability in black rice. However, direct mechanistic evidence and validation under natural storage conditions are still needed.