CsbHLH122/CsMYBS3-CsSUT2 contributes to the rapid accumulation of sugar in the ripening stage of sweet orange (Citrus sinensis).

The sugar content serves as the fundamental metabolic component that determines both the flavor quality and the nutritional value of fruits. Nevertheless, the regulatory mechanism underlying the rapid accumulation of sugars during citrus fruit maturation remains elusive. In this study, we demonstrated that the expression level of sucrose transporter CsSUT2 is increased during citrus fruit ripening and sugar accumulation. Functional assays confirmed that CsSUT2 is localized in the plasma membrane and exhibits sucrose transporter activity. Homologous and heterologous overexpression of CsSUT2 in citrus juice sacs, calli, and tomato resulted in an increase in sugar content. Conversely, virus-induced gene silencing and RNAi-mediated silencing of CsSUT2 led to a decrease in sugar levels in transgenic citrus tissues. We further identified CsMYBS3 as an upstream transcription factor that positively regulates the expression of CsSUT2. Transgenic evidence supported that the induction of sugar accumulation by CsMYBS3 depends on the transcript level of CsSUT2. Additionally, we found that CsbHLH122 physically interacts with CsMYBS3 to form a transcription factor complex, enhancing promoter transcriptional activity of CsSUT2. This study expands our understanding of the function and regulatory mechanism of sugar transporter in citrus, providing valuable insights for regulating sugar accumulation and quality control in citrus fruit.