Transcriptome analysis reveals the common and specific pathways of citric acid accumulation in different citrus species

Citric acid is an important contributor to fruit flavor. The extremely significant differences in citric acid levels are observed among citrus species and varieties. The molecular basis of citric acid accumulation remains largely unknown on the inter-species level. Here, we performed transcriptome analysis of five pairs of acidic and acidless variety fruits from kumquat (Citrus crassifolia), lemon (C. limon), pummelo (C. maxima), mandarin (C. reticulata), and sweet orange (C. sinensis) to investigate the common and specific genes related to citric acid accumulation in these citrus species. Transcript profiles of the citrate metabolism genes revealed that the mechanism of citric acid accumulation in kumquat differed from that in the other four species. The comparative analysis and weighted gene co-expression network analysis identified 61 candidate genes from lemon, pummelo, mandarin, and sweet orange. Vacuolar acidification regulated by CitPH4-CitAN1-CitPH5 was a common mechanism underlying citric acid accumulation in these four species. In addition, gene network analysis indicated that CitPH4 was a central regulator, exhibiting a strong correlation with the genes involved in vacuolar trafficking and potassium (K⁺) transport. The co-expression and functional enrichment analyses showed that active sugar metabolism and increased citrate synthesis were mainly responsible for the accumulation of citric acid in kumquat. Twenty-four transcription factors were identified as candidate regulators, of which CitbHLH28 was found to regulate acidity level via transactivating CWINV2 and PK3 expressions. Overall, this study provides an insight into the mechanism of citric acid accumulation in citrus fruit and provides useful references for fruit quality improvement through acidity control in different citrus species.