Mutagenesis of AcSQBP9 in kiwifruit results in reduction of malate via alteration of the expression of a plastidial malate dehydrogenase

Organic acids are major contributors to the flavor of fleshy fruits. In kiwifruit, the Al-ACTIVATED MALATE TRANSPORTER gene (AcALMT1) is key to the accumulation of citrate, while factors driving malate metabolism remain largely unknown. During kiwifruit (Actinidia chinensis cv "Hongyang") development, a rapid decline of malate content was observed between 6 and 12 weeks after full bloom (WAFB), which was studied using RNA-seq analysis. Co-expression network analysis indicated that expression of the chloroplast localized AcPNAD-MDH1 (Plastid-Localized NAD-Dependent Malate Dehydrogenase) negatively correlated with malate content. Overexpression of AcPNAD-MDH1 in kiwifruit resulted lower malate and citrate content in leaves. Among 15 transcription factors that are highly correlated with the expression of AcPNAD-MDH1, AcSQBP9 (SQUAMOSA PROMOTER-BINDING PROTEIN) was shown to directly bind the promoter of AcPNAD-MDH1 to repress transcriptional activity. Moreover, targeted CRISPR-Cas9-induced mutagenesis of AcSQBP9 in kiwifruit produced a significant decrease in malate and citrate, accompanied by an increase in AcPNAD-MDH1 expression. Both PNAD-MDH and SQBP have not been widely studied in fruit metabolism, so the present omics-oriented study provides insights for both kiwifruit and general plant organic acid metabolism.