Molecular elucidation for the variance of organic acid profile between citrus top and bottom canopy fruits

Organic acids are key metabolites for diverse fruit flavoring and vital for fruit quality measures. Their contents are significantly varied regarding the canopy position. However, the underlying mechanism is still unclear. In this study by using Citrus raticulata cv. Bendizao as research material, a significant 2-fold increase in citrate content and a 2-fold decrease in malate content were noticed in bottom-canopy fruits subjected to less light and low temperature compared to top-canopy fruits. Moreover, transcript levels of genes like citrate synthase (CS1), aconitate hydratase genes (ACO1 and ACO2), NAD-dependent malic enzyme gene (NAD-ME) and malate dehydrogenase genes (MDH1 and MDH2) were significantly higher, while transcript levels of other genes such as citrate utilization genes (ACL2, ACL3 and ACO3), malate synthase gene (MS) and cytosolic NAD-dependent malate dehydrogenase gene (cytNAD-MDH) were significantly lower in bottom-canopy fruits compared to top-canopy fruits. Additionally, transcript levels of proton pump genes (CsPHs and VHA), two transporter genes (MATE and ALMT9), and four transcription factor genes (MYB73, Noemi, MYC-N, and bHLH) were significantly higher, while mRNA levels of two transcription factor genes (MYB10 and WRKY14) and transporter genes (CsCit1 and ALMT9-like) were significantly lower in bottom-canopy fruits than those in top-canopy fruits. Taken together, the variance of organic acid profiles in the canopy could be attributed to the microclimate change, which comprehensively influences the organic acid accumulation-related genes’ transcript levels; reduced malate accumulation in bottom-canopy fruits is mainly due to the minimal expression of MS and cytNAD-MDH, and high expression of malate utilization genes (MDH and NAD-ME), while the higher citrate content in the bottom-canopy fruits is probably due to the enhanced vacuolar storage through increasing vacuolar proton pump genes and MATE transcript levels, and the minimal citrate degradation and efflux through decreasing ACL2, ACL3, ACO3 and CsCit1 transcript levels.