Table_2_The Interaction Between CitMYB52 and CitbHLH2 Negatively Regulates Citrate Accumulation by Activating CitALMT in Citrus Fruit.DOCX

Citric acid plays significant roles in numerous physiological processes in plants, including carbon metabolism, signal transduction, and tolerance to environmental stress. For fruits, it has a major effect on fruit organoleptic quality by directly influencing consumer taste. Citric acid in citrus is mainly regulated by the balance between synthesis, degradation, and vacuolar storage. The genetic and molecular regulations of citric acid synthesis and degradation have been comprehensively elucidated. However, the transporters for citric acid in fruits are less well understood. Here, an aluminum-activated malate transporter, CitALMT, was characterized. Transient overexpression and stable transformation of CitALMT significantly reduced citrate concentration in citrus fruits and transgenic callus. Correspondingly, transient RNA interference-induced silencing of CitALMT and increased citrate significantly, indicating that CitALMT plays an important role in regulating citrate concentration in citrus fruits. In addition, dual-luciferase assays indicated that CitMYB52 and CitbHLH2 could trans-activate the promoter of CitALMT. EMSA analysis showed that CitbHLH2 could physically interact with the E-box motif in the CitALMT promoter. Bimolecular fluorescence complementation assays, yeast two-hybrid, coimmunoprecipitation and transient overexpression, and RNAi assay indicated that the interaction between CitMYB52 and CitbHLH2 could synergistically trans-activate CitALMT to negatively regulate citrate accumulation.

柠檬酸在植物的诸多生理过程中发挥关键作用，涵盖碳代谢、信号转导以及环境胁迫耐受调控。对于果实而言，柠檬酸可通过直接影响消费者的味觉体验，对果实的感官品质产生决定性影响。柑橘中的柠檬酸含量主要由合成、降解与液泡储存之间的动态平衡所调控。目前，柠檬酸合成与降解的遗传及分子调控机制已得到全面阐明，但果实中负责柠檬酸转运的转运蛋白相关研究仍较为匮乏。本研究对铝激活苹果酸转运蛋白（aluminum-activated malate transporter, ALMT）家族成员CitALMT进行了功能表征。将CitALMT进行瞬时过表达与稳定转化后，均可显著降低柑橘果实及转基因愈伤组织中的柠檬酸浓度。与之相对，通过瞬时RNA干扰（RNA interference, RNAi）沉默CitALMT时，果实内柠檬酸含量显著升高，这表明CitALMT在调控柑橘果实柠檬酸浓度过程中发挥重要功能。此外，双荧光素酶测定（dual-luciferase assays）结果显示，CitMYB52与CitbHLH2可反式激活CitALMT的启动子区域。电泳迁移率变动分析（electrophoretic mobility shift assay, EMSA）证实，CitbHLH2可与CitALMT启动子中的E-box基序发生特异性物理结合。双分子荧光互补（bimolecular fluorescence complementation, BiFC）、酵母双杂交（yeast two-hybrid, Y2H）、免疫共沉淀（coimmunoprecipitation, CoIP）以及瞬时过表达与RNAi实验结果均表明，CitMYB52与CitbHLH2之间的相互作用可协同反式激活CitALMT，进而负向调控柠檬酸的积累。