Calcyclin-binding protein-promoted degradation of MdFRUCTOKINASE2 regulates sugar homeostasis in apple

Abstract

Fructokinase (FRK) activates fructose through phosphorylation, which sends the activated fructose into primary metabolism and regulates fructose signaling capabilities in plants. The apple (Malus × domestica) FRK gene MdFRK2 shows especially high affinity to fructose, and its overexpression decreases fructose levels in the leaves of young plants. However, in the current study of mature plants, fruits of transgenic apple trees overexpressing MdFRK2 accumulated a higher level of fructose than wild-type (WT) fruits (at both young and mature stages). Transgenic apple trees with high mRNA MdFRK2 expression showed no significant differences in MdFRK2 protein abundance or FRK enzyme activity compared to WT in mature leaves, young fruits, and mature fruits. Immunoprecipitation-mass spectrometry analysis identified an skp1, cullin, F-box (SCF) E3 ubiquitin ligase, calcyclin-binding protein (CacyBP), that interacted with MdFRK2. RNA-sequencing analysis provided evidence for ubiquitin-mediated post-transcriptional regulation of MdFRK2 protein for the maintenance of fructose homeostasis in mature leaves and fruits. Further analyses suggested an MdCacyBP-MdFRK2 regulatory module, in which MdCacyBP interacts with and ubiquitinates MdFRK2 to facilitate its degradation by the 26S proteasome, thus decreasing the FRK enzyme activity to elevate fructose concentration in transgenic apple trees. This result uncovered an important mechanism underlying plant fructose homeostasis in different organs through regulating the MdFRK2 protein level via ubiquitination and degradation. Our study provides usable data for the future improvement of apple flavor and expands our understanding of the molecular mechanisms underlying plant fructose content and signaling regulation.

Figure 1

Levels of fructose, MdFRK2 transcript, FRK enzyme activity, and MdFRK2 protein in the YL, ML, YF (40 days after bloom), and MF (130 days after bloom) of WT and transgenic apple overexpressing MdFRK2 (OE-4 and OE-9). (A) Concentration of fructose in the YL, ML, YF, and MF of the WT and transgenic lines. (B) MdFRK2 expression levels in the YL, ML, YF, and MF of WT and transgenic apple trees were measured via RT-qPCR. The transcript levels were normalized to those of MdActin. The relative expression level for MdFRK2 was obtained via the 2^−ΔΔCT method, setting its expression in ML-WT as 1. (C) Activity of FRK enzyme in the YL, ML, YF, and MF of WT and MdFRK2-OE lines. (D) The abundance of MdFRK2 protein in the YL, ML, YF, and MF of WT and transgenic OE lines. The MdFRK2 protein level was detected by western blotting using anti-MdFRK2 antibodies. The number below each protein band indicates the relative protein abundance measured using density scanning, with that from WT of YL set as 1. M: protein marker. β-Actin was used as the reference protein. Bars represent the mean value ± Se (n ≥ 3). The asterisks in A, B, and C indicate significant differences based on independent t tests (*P < 0.05).

Figure 2

Identification of MdFRK2-interacting proteins by IP–MS. (A) M, marker. P₁, total proteins were extracted from ML of MdFRK2-OE transgenic apple plants and incubated with purified MdFRK2-GST protein for 2 h. The protein complexes were immunoprecipitated with anti-GST agarose beads. The immunoprecipitated proteins (red box) were analyzed by liquid chromatography–tandem mass spectrometry (LC–MS/MS). (B) Functional category enrichment of the MdFRK2-interacting proteins. After annotation, 602 candidate-interacting proteins were found to relate to protein post-transcriptional modification (yellow box). PS, photosynthesis; CHO metabolism, carbohydrate metabolism. (C) Venn diagram of co-upregulated DEGs involved in the MdFRK2 post-transcriptional regulation in ML and YL of WT and MdFRK2-OE lines. Total RNA from ML and YL was extracted and subjected to RNA-sequencing (RNA-seq). For this purpose, we used FPKM and identified genes with Fold Change (∣log₂ (FC)∣≥ 1.5) and FDR <0.05. Four pairwise comparisons (YL-OE-4 versus YL-WT, YL-OE-9 versus YL-WT, ML-OE-4 versus ML-WT, and ML-OE-4 versus ML-WT) from different tissues indicated a considerable number of DEGs. (D) Relative expression levels of MdFBS2 (MD05G1325400) and MdCacyBP (MD05G1283600) were measured using RT-qPCR. For RT-qPCR, the transcript levels were normalized to those of MdActin. The relative expression levels for MdFBS2 and MdCacyBP were obtained via the 2^−ΔΔCT method, setting their expression in YL-WT as 1. Bars represent the mean value ± Se (n ≥ 3). Different letters indicate significant differences at P < 0.05 based on one-way ANOVA.

Figure 3

MdCacyBP interacted with MdFRK2. (A) Interaction between MdCacyBP and MdFRK2 as determined by the Y2H assay. The pGBKT7-p53 and pGADT7-T pair were used as positive controls. Pairs of BD-MdFRK2 and AD-pGADT7, and BD-pGBKT7 and AD-MdCacyBP were used as negative controls. Each colony was dissolved in 5 μL sterile NaCl and then diluted to 10⁻¹–10⁻³. Yeasts were grown on SD/-Leu/-Trp, SD/-Leu/-Trp/-His/-Ade, and SD/-Leu/-Trp/-His/-Ade + X-α-gal media. (B) MdCacyBP interacted with MdFRK2 in a BiFC assay. MdFRK2 was fused to the C-terminus of yellow fluorescent protein (YFP^C), and MdCacyBP was fused to the N-terminus of YFP^N. Pairs of MdFRK2-YFP^C + YFP^N and YFP^C + MdCacyBP-YFP^N were used as negative controls. Different pairs of constructs were co-expressed in N. benthamiana. After 3 days, YFP fluorescence was detected by confocal microscopy. Scale bars = 20 μm. (C) Detection of the interaction between MdCacyBP and MdFRK2 proteins by Co-IP assays. MdFRK2-GFP and MdCacyBP-Flag were transiently expressed in N. benthamiana leaves. MdFRK2-GFP protein was immunoprecipitated using an anti-GFP antibody. The eluted solution was examined using an anti-Flag antibody.

Figure 4

MdCacyBP mediates the ubiquitination and degradation of MdFRK2. (A, B) MdCacyBP ubiquitinated MdFRK2 in N. benthamiana leaves. MdFRK2-GFP was expressed alone or co-expressed with MdCacyBP-Flag in N. benthamiana, and proteins were extracted from N. benthamiana leaves. Immunoprecipitation was performed using anti-GFP antibody, proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), and the proteins were detected by western blotting using anti-GFP and anti-ubiquitin antibodies. IP, immunoprecipitate; IB, immunoblot; Ubi, ubiquitin. (C, D) Cell-free degradation assay shows that MdCacyBP promotes the degradation of MdFRK2 by the 26S proteasome. Total protein was extracted from WT and MdFRK2-OE apple leaves and incubated with MdCacyBP-His protein with 50 μM MG132 (proteasome inhibitor) or its solvent DMSO for 0, 0.5, 1, or 3 h at 22°C. The number below each protein band indicates the relative protein abundance measured using density scanning, with that from 0 h of MdFRK2-OE set as 1. M, protein marker. MdFRK2 protein level was detected by western blotting using anti-MdFRK2 antibody. β-Actin was used as the reference protein.

Figure 5

MdFRK2 degradation was mediated via the 26S proteasome pathway. (A) Agroinfiltration of apple leaves. Black arrows point to sites of infiltration of MG132-containing MES buffer into subcultured ML of WT and MdFRK2-overexpressing transgenic apple, with DMSO as a control. (B) Protein levels of MdFRK2 assayed by western blotting after agroinfiltration. Three days after injection, total protein was extracted from the leaves and MdFRK2 protein level was detected by western blotting with a specific MdFRK2 antibody. β-Actin was used as the reference protein. (C) FRK enzyme activity and (D) fructose concentration were also detected from the collected samples after injection. Bars represent the mean value ± Se (n ≥ 3). Different asterisks in (C) and (D) indicate significant differences based on independent t tests (*P < 0.05).

Figure 6

Virus-induced silencing of MdCacyBP decreased the fructose level in apple leaves. (A) MdCacyBP expression was investigated by RT-qPCR. The transcript levels were normalized to those of MdActin. The relative expression level for MdCacyBP was obtained via the 2^−ΔΔCT method, setting its expression in WT leaves infiltrated with the empty vector pTRV2 as 1. (B) Abundances of MdCacyBP and MdFRK2 proteins in ML of the WT and MdFRK2-OE lines inside the injection areas 3 days after VIGS treatments. The leaf tissues at the injection region were used for protein detection. Proteins were extracted and probed with anti-MdCacyBP and anti-MdFRK2 antibodies, respectively. β-Actin was used as the reference protein. (C) FRK enzyme activity and fructose (D) measurements using three biological replicates. The empty pTRV2 vector was used as a control. Bars represent the mean value ± Se (n ≥ 3). The asterisks in A, C, and D indicate significant differences based on independent t tests (*P < 0.05).

Figure 7

Virus-induced silencing of MdCacyBP decreased the fructose level in apple flesh. (A) Black arrows show the injection sites of Agrobacterium solution into the flesh of the WT and MdFRK2 overexpression transgenic apple at 40 days after bloom (DAB). Scale bars = 20 µm. (B) The RT-qPCR analysis of the relative expression levels of MdCacyBP around the injection sites. The transcript levels were normalized to those of MdActin. The relative expression level for MdCacyBP was obtained via the 2^−ΔΔCT method, setting its expression in WT infiltrated with the empty vector pTRV2 as 1. The flesh infiltrated with an empty vector pTRV2 was used as a control. (C) Abundance of the MdCacyBP and MdFRK2 proteins in WT and MdFRK2-OE transgenic apple flesh inside the injection areas 3 days after VIGS treatments. The flesh at each injection region was used for protein detection. Proteins were extracted and probed with anti-MdCacyBP and anti-MdFRK2 antibodies, respectively. β-Actin was used as the reference protein. (D) FRK enzyme activity and (E) fructose measurements using three biological replicates. The empty pTRV2 vector was used as a control. Bars represent the mean value ± Se (n ≥ 3). The asterisks in B, D, and E indicate significant differences based on independent t tests (*P < 0.05).

Figure 8

A dynamic regulatory module for the MdCacyBP–MdFRK2 regulation of fructose homeostasis in apple plants. MdCacyBP interacts with MdFRK2, promoting its degradation via the ubiquitin–proteasome pathway, which reduces the FRK enzyme activity to elevate fructose concentration and functioning as a braking mechanism for the regulation of fructose homeostasis in apple plants. U, Ubiquitin. Green ellipses indicate the MdFRK2 protein. Gray ellipses indicate the MdCacyBP protein. Different colored circles indicate the levels of fructose in seven different apple tissues. ST, shoot tips. YL, young leaves. ML, mature leaves. Ph, phloem; R, roots. YF, young fruits. MF, mature fruits. Black arrows indicate positive regulation.