The Interaction Between CitMYB52 and CitbHLH2 Negatively Regulates Citrate Accumulation by Activating CitALMT in Citrus Fruit

Abstract

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.

Keywords: MYB-bHLH protein complex; aluminum-activated malate transporter; citric acid; citrus fruit; transcriptional regulation.

Figure 1

Identification of CitALMT. (A) The amino acid sequences analysis of CitALMT compared with some ALMTs reported previously. (B) Subcellular localization of CitALMT in tobacco leaves stably transformed with a red nuclear localization marker and agroinfiltrated with CitALMT-GFP and tonoplast-RFP marker. White arrows highlight co-localization of CitALMT with tonoplast marker. The empty vector control is on the top without tonoplast-RFP marker. The fluorescence was measured at 488 nm with a LSM780 microscope and photographed. Bars = 20 μm. (C) The citrate concentration and expression of the CitALMT genes in flesh of “Ponkan” fruits during the late stage of fruit development. DAFB, days after full blossom.

Figure 2

Gene function analysis of CitALMT. The transcript level of CitALMT (left) and organic acid concentration (right) were analyzed in citrus fruits and citrus callus. (A) Transient overexpression of CitALMT in citrus fruits. SK represents empty vector. (B) Transient RNAi of CitALMT in citrus fruits. pHB represents empty vector. (C) The analysis of transgenic CitALMT callus. Error bars indicate SE from three biological replicates. ^*Significant differences (p < 0.05), ^***Significant differences (p < 0.001).

Figure 3

Interaction between transcription factors and CitALMT’s promoter. (A) In vivo interaction of transcription factors with the promoter of the CitALMT gene from “Ponkan” fruit. In vivo associations of the transcription factors and promoter were obtained by transient expression assays in tobacco leaves. The ratio of LUC/REN of the empty vector (SK) plus promoter was used as calibrator (set as 1). Error bars indicate SE from at least five replicates. ^***Significant differences (p < 0.001). (B) Effect of the combination of CitMYB52 and CitbHLH2 on the CitALMT’ promoter. The ratio of LUC/REN of the empty vector (SK) plus promoter was used as calibrator (set as 1). Error bars indicate SEs from five biological replicates. Different letters above the columns represent significant differences (the combination effects were compared to two individual effects, p < 0.05). (C) Electrophoretic mobility shift assay (EMSA) of CitbHLH2 binding to the CitALMT promoter. Purified CitbHLH2 proteins and biotin-labeled DNA probe were mixed and analyzed on 6% native polyacrylamide gels. The presence (+) or absence (−) of specific probes is indicated. The concentration of the cold probe is shown; the biotinylated probe concentration was 1 nM.

Figure 4

Characterization of CitMYB52 and CitbHLH2. (A) Subcellular localization analysis was performed in N. benthamiana leaves stably transformed with a red nuclear localization marker. GFP fluorescence of CitMYB52-GFP and CitbHLH2-GFP are indicated. The empty vector control is at the top. Bars = 25 μm. (B) Expression of the CitMYB52 and CitbHLH2 genes in the flesh of “Ponkan” fruits during the late stage of fruit development, DAFB, days after full blossom. Error bars represent SE (n = 3).

Figure 5

Protein–protein interaction between CitMYB52 and CitbHLH2. (A) In vivo interaction between CitMYB52 and CitbHLH2, determined using BiFC. N- and C-terminal fragments of YFP (indicated on the figure as YFPN and YFPC) were fused to the C terminus of CitbHLH2 and CitMYB52, respectively. The pairs of fusion proteins tested were CitbHLH2-YFPN+CitMYB52-YFPC. The other combinations were negative controls. Fluorescence of YFP represents protein–protein interaction. Bars = 50 μm. (B) Interaction between CitMYB52 and CitbHLH2 in yeast two-hybrid assays. Liquid cultures of double transformants were plated at OD600 = 0.01 dilutions on synthetic dropout selective media: (1) SD medium lacking Trp and Leu; (2) SD medium lacking Trp, Leu, His, and Ade; and (3) SD medium lacking Trp, Leu, His, and Ade, and supplemented with AbA. pGBKT7-p53 and pGADT7-T were used as positive controls, while pGBKT7-Lam and pGADT7-T were used as negative controls. (C) Interactions between CitMYB52 and CitbHLH2 measured by CoIP. Total protein extracts (Input) and protein complexes immunoprecipitated with anti-HA agarose (IP) were separated on gels and blotted. Anti-HA and anti-myc antibodies were used in Western blotting.

Figure 6

Effects on the citrate concentration of transient overexpression or RNAi inhibition of CitMYB52 and CitbHLH2. The transcript level of CitALMT (left) and citrate concentration (right) were analyzed in citrus fruits. (A) Transient overexpression of CitMYB52 and CitbHLH2 in citrus fruits. SK represents empty vector. (B) Transient RNAi of CitMYB52 and CitbHLH2 in citrus fruits. pHB represents empty vector. Error bars indicate SE from three biological replicates. Different letters above the columns represent significant differences (p < 0.05).