The role of Calvin cycle enzymes <scp>AcFBA2</scp> and <scp>AcRBCS1</scp> in regulating de‐greening of kiwifruit

Chlorophyll is central to the capture of light energy but also contributes to pigmentation of plant products such as fruit. Bagging treatments are widely used to enhance chlorophyll degradation and to improve the visual quality of fruit such as apples, pears, and peaches. In this study, a bagging treatment of kiwifruit Actinidia chinensis (cultivar Jinshi; "JS") accelerated flesh chlorophyll degradation. RNA-seq analysis indicated that AcFBA2, which encodes a fructose-1,6-bisphosphate aldolase (FBA) enzyme, and AcRBCS1, encoding Rubisco small subunit (RBCS), both in the Calvin cycle, were significantly downregulated. Knockout of either gene resulted in leaf yellowing in kiwifruit, accompanied by severe damage to chloroplast structure. Further experiments indicated that AcFBA2 and AcRBCS1 could form a protein complex, which also occurred in orthologous Arabidopsis enzymes. Additionally, we identified two HSF transcription factors, AcHSFB2a and AcHSFA7a, which bind to the promoters of AcFBA2 and AcRBCS1, respectively, and repress their expression, thereby promoting yellowing of kiwifruit flesh. Transient overexpression of AcHSFB2a or AcHSFA7a could reduce chlorophyll content in tobacco leaves. In summary, an unexpected physical interaction between two key Calvin cycle enzymes (FBA and RBCS) was shown in kiwifruit and Arabidopsis, and downregulation of these two genes by bagging treatment and genome editing could lead to de-greening. Transcriptional regulation by AcHSFB2a and AcHSFA7a suggests a potential regulatory network linking HSF transcription factors with photosynthesis and chlorophyll degradation.