CitFBA6 regulates soluble sugars and malate accumulation during fruit ripening in Citrus (Citrus reticulata Blanco)

Fructose-bisphosphate aldolase (FBA), a crucial enzyme in glycolysis/gluconeogenesis, plays a significant role in sugar accumulation. However, the information available on the FBA family in citrus is very limited. In this study, we performed the first functional characterization of the FBA gene family in citrus, identifying four CitFBA genes which were classified into chloroplastic and cytosolic subfamilies based on phylogenetic and structural analyses. Comparative genomic analysis revealed significant protein homology among CitFBAs, while gene structure analysis highlighted variability in exon-intron organization. Protein motif analysis showed conserved glycolytic domains across the CitFBA gene family. Promoter analysis revealed complex regulation of CitFBA genes by environmental and hormonal signals, with light-responsive elements being the most prevalent. RNA-seq data elucidated CitFBA expression patterns during fruit development and, in response to postharvest phytohormone applications, showed differential patterns, with CitFBA6 exhibiting high expression, suggesting its positive role in fruit ripening. Virus-induced gene silencing (VIGS) of CitFBA6 led to reduced fructose and glucose levels, increased sucrose content, enhanced malate accumulation and altered the activity and expression of other important enzymes in gluconeogenesis. These results highlight the pivotal role of CitFBA6 in regulating soluble sugar and organic acid accumulation during citrus fruit ripening. Together, our study provides comprehensive information about the FBA family in citrus and can potentially enhance citrus fruit flavor and postharvest quality through targeted manipulation of FBA enzymes.