MdERDL6-mediated glucose efflux to the cytosol promotes sugar accumulation in the vacuole through up-regulating TSTs in apple and tomato

Abstract

Sugar transport across tonoplasts is essential for maintaining cellular sugar homeostasis and metabolic balance in plant cells. It remains unclear, however, how this process is regulated among different classes of sugar transporters. Here, we identified a tonoplast H⁺/glucose symporter, MdERDL6-1, from apples, which was highly expressed in fruits and exhibited expression patterns similar to those of the tonoplast H⁺/sugar antiporters MdTST1 and MdTST2. Overexpression of MdERDL6-1 unexpectedly increased not only glucose (Glc) concentration but also that of fructose (Fru) and sucrose (Suc) in transgenic apple and tomato leaves and fruits. RNA sequencing (RNA-seq) and expression analyses showed an up-regulation of TST1 and TST2 in the transgenic apple and tomato lines overexpressing MdERDL6-1 Further studies established that the increased sugar concentration in the transgenic lines correlated with up-regulation of TST1 and TST2 expression. Suppression or knockout of SlTST1 and SlTST2 in the MdERDL6-1-overexpressed tomato background reduced or abolished the positive effect of MdERDL6-1 on sugar accumulation, respectively. The findings demonstrate a regulation of TST1 and TST2 by MdERDL6-1, in which Glc exported by MdERDL6-1 from vacuole up-regulates TST1 and TST2 to import sugars from cytosol to vacuole for accumulation to high concentrations. The results provide insight into the regulatory mechanism of sugar accumulation in vacuoles mediated by the coordinated action of two classes of tonoplast sugar transporters.

Keywords: H+/glucose symporter; H+/sugar antiporter; cytosolic sugar signaling; intracellular communication; sugar transport.

Fig. 1.

The expression profiles of vacuolar sugar transporter genes, including MdERDL6s, MdTSTs, and MdvGTs, in apples. (A) A heat map of gene expression levels based on RNA-seq and sugar concentrations in different tissues including developing fruit. RPKM values measured with RNA-seq are shown in Dataset S1A. Fold difference is designated as a log2 value, with the data in young fruit at 16 DAB set as 1. (B) Relative transcript levels of MdERDL6-1, MdTST1, and MdTST2 in different tissues, including developing fruits based on qRT-PCR. For each sample, transcript levels were normalized with those of MdActin. Relative expression levels for each gene were obtained via the ddCT method, setting its expression in young fruit at 15 DAB as 1. The bars represent the mean value ± SD (n ≥ 3).

Fig. 2.

Tonoplast localization of MdERDL6-1 in tobacco leaves and apple calli and its transport prosperities. (A and B) Tonoplast localization of the MdERDL6-1-GFP fusion protein in the isolated vacuoles of protoplasts from tobacco leaves (A) and the protoplasts from apple calli (B). The green fluorescence represents MdERDL6-1-GFP fusion proteins. Note the chloroplasts showing red auto fluorescence are located outside the ring of GFP fluorescence (A). For the apple calli protoplast (B), the MdERDL6-1-GFP fluorescence exhibited a ring-like pattern inside the plasma membrane (black arrows), indicating that MdERDL6-1 is localized to the tonoplast. (C) The localization of MdERDL6-1 to plasma membrane in yeast, based on MdERDL6-1-GFP fusion expression, in comparison with that of pYST II-GFP control in the yeast mutant strain EBY.VW4000. (Scale bars = 2 μm.) (D) Growth of the yeast mutant strain EBY.VW4000 expressing MdERDL6-1 on culture medium with 2% of different sugars. The deficient yeast mutant carrying the empty pYST II vector (Control 1) or the MdERDL6-1-antisense recombinant plasmid (Control 2) could not grow normally on each monosaccharide culture medium, whereas the yeast mutant transformed with MdERDL6-1-sense construct grew normally on glucose but slowly on Fru, Gal, and Xyl. (E) The transport capacity of [¹⁴C] glucose was measured in yeast cells expressing MdERDL6-1 cDNA in the sense orientation (strain EBY.VW4000; blue circles) or the empty vector on 100 mM of glucose at pH 5.5. (Inset) The uptake rates with increasing concentrations of [¹⁴C] glucose were determined 2 min after substrate addition and used to calculate the K_m value. The plot of a typical K_m determination is presented with five independent measurements. Here, a K_m value of 21.7 mM and a maximum uptake rate (V max) of 1.214 mmol ⋅ h⁻¹ ⋅ ml⁻¹ packed cells were determined for Glc uptake, driven by the MdERDL6-1 transporter. (F) Relative uptake rates of sugars at an initial concentration of 100 mM and the influence on glucose uptake by 50 μM uncoupler CCCP in MdERDL6-1–expressing yeast cells. Data represent average values of five independent transport tests (mean ± SD).

Fig. 3.

The impact of altering MdERDL6-1 expression on sugar (Glc, Fru, Suc, and Sor) concentrations in apple fruit calli. (A) MdERDL6-1 expression levels in the overexpressing (OL3 and OL6) and silencing (SL1 and SL3) calli lines were measured by using qRT-PCR. The transcript levels were normalized to those of MdActin. The relative expression level for MdERDL6-1 was obtained via the ddCT method, setting its expression in WT as 1. (B) The growth of the transformed calli on 3% Suc or 3% Glc medium. (C and D) The sugar concentrations of the transformed calli lines cultured on 3% Suc (C) or 3% Glc (D) medium. The bars represent the mean value ± SD (n ≥ 4). *P ≤ 0.05, a significant difference from the WT.

Fig. 4.

The carbohydrate levels of the transgenic apple (OEm-1, OEm-3, and OEm-4) leaves and tomato (OEs-1 and OEs-2) fruits overexpressing MdERDL6-1. (A) Glc, Fru, Suc, Sor, and starch levels in mature leaves of transgenic apple. (B) The carbohydrate levels and soluble solids content in ripening fruits of transgenic tomato and longitudinal sections of the fruits. The bars represent the mean value ± SD (n ≥ 4). *P ≤ 0.05, a significant difference from the WT.

Fig. 5.

The expression of genes related to carbohydrate metabolism and transport in the transgenic apple or tomato lines overexpressing MdERDL6-1. (A) A heat map illustrating different expression genes related to carbohydrate metabolism and transport in the leaves of transgenic apple lines (OEm-1 and OEm-3) based on RNA-seq. APL, ADP-glucose pyrophosphorylase; SS, starch synthase; SBE, starch branching enzyme; AMY, α-amylase; BMY, β-amylase; PHS, α-glucan phosphorylase; CWINV, cell wall invertase; NINV, neutral invertase; vAINV, vacuolar acid invertase; SUSY, sucrose synthase; FRK, fructokinse; SUC, sucrose transporter; SOT, sorbitol transporter; INT, inositol transporter; HT, hexose transporter; TST, tonoplast sugar transporter; SWEET, sugars will eventually be exported transporter; ERDL6, early response to dehydration like six. (B) A heat map illustrating the expression of marker genes involved in sugar response and other members of MdERDL6 family in the leaves of transgenic lines based on RNA-seq. RPKM values measured with RNA-seq are shown in Dataset S2 B and C, respectively. The fold difference is designated as a log2 value, while the data in WT was set as 1 for each gene. ACT, actin; CAB, chlorophyll a/b binding protein; GPT, glucose-6-phosphate transporter. (C) Expression levels of MdTST1 and MdTST2 mRNAs in the transgenic apple calli overexpressing MdERDL6-1 (OL3, 6) in comparison with the WT and two silencing lines (SL1, 3). (D and E) The expression levels of MdTST1 and MdTST2 mRNAs (D) and proteins (E) in the transgenic apple (OEm-1, OEm-3, and OEm-4) leaves overexpressing MdERDL6-1. (F and G) The expression levels of SlTST1 and SlTST2 mRNAs (F) and proteins (G) in the ripening fruits of transgenic tomato (OEs-1 and OEs-2) overexpressing MdERDL6-1. For qRT-PCR, the transcript levels were normalized to those of MdActin (D) and SlActin (F), respectively. Relative expression levels for each gene were obtained via the ddCT method, with its expression in WT set as 1. The bars represent the mean value ± SD (n ≥ 3). *P ≤ 0.05, a significant difference from the WT.

Fig. 6.

The influence of sugar feeding and coexpression of 35S:MdERDL6-1 on the promoter activities of MdTST1 (MdTST1P) and MdTST2 (MdTST2P). (A and B) The impact of sugar feeding on MdTST1 (A) and MdTST2 (B) promoter activities. Tobacco leaves were fed for 24 h with 2% different exogenous sugar, starting from 48 h after infiltrating with Agrobacterium harboring MdTST1P or MdTST2P plasmids. The treated and control leaves were harvested for GUS activity assay after 24 h feeding. (C) The impact of 35S:MdERDL6-1 coexpression on MdTST1 and MdTST2 promoter activities. The 35S:GUS infiltration was used as a positive control. (D) The relative GUS activities of different combinations of infiltration. The treated leaves were harvested for GUS activity assay at 2 d after infiltration. “+” and “−” represent the presence or absence, respectively, of the Agrobacterium containing corresponding plasmids in the mixture for infiltration. The bars represent the mean value ± SD (n ≥ 4). *P ≤ 0.05, a significant difference from control.

Fig. 7.

CRISPR-Cas9–mediated knockout of SlTST1 and SlTST2 abolished the positive effect on sugar levels exerted by MdERDL6-1 overexpression in tomato fruits. (A) The DNA sequence of the target region. SlTST1 and SlTST2 target sequences were chosen from CRISPR direct for gene editing. Sequences were aligned using DNAMAN. The dark region is the target sequence, and the other colored region is the difference in sequence among the lines indicated; the target sequences of WT and OEs-1 (transgenic line overexpressing MdERDL6-1) were the same as the control. The edited lines tst1/2-4 had two bases missing for SlTST1 and one base inserted for SlTST2, while the tst1/2-15 had two bases missing for SlTST1 and two bases missing for SlTST2. The OEs-1-tst1/2-1 had two bases missing for SlTST1 and one base inserted for SlTST2, whereas the OEs-1-tst1/2-4 had one base missing for each of SlTST1 and SlTST2. (B) The Glc, Fru, and Suc concentrations and soluble solids content in ripening fruit. The bars represent the mean value ± SD (n ≥ 4). Different letters indicate significant differences at P ≤ 0.05.

Fig. 8.

A model on how MdERDL6 could modulate sugar accumulation in vacuole through regulating MdTST1/2 or their orthologous genes. In apple fruits, sugars are unloaded from the phloem into the parenchyma cells via transporters MdSUT, MdHT, and MdSOT for sucrose, hexose, and sorbitol, respectively. Upon meeting the requirements for energy and carbon skeleton production, excessive soluble sugars are imported into the vacuoles for storage, mediated by a set of tonoplast sugar transporters including the H⁺/sugar antiporters, MdTST1 and MdTST2. On the other hand, MdERDL6-1 acts as an H⁺/sugar symporter to export glucose from the vacuole to cytosol. Findings from our study indicate that the MdERDL6-mediated glucose efflux to the cytosol activates or enhances the expression of MdTST1 and MdTST2 to import sugars into the vacuole, leading to the accumulation of high concentration of sugars. Our data also indicate that the model is likely applicable to other systems such as tomato fruits (see article text for details). G/Glc, glucose; F/Fru, fructose; S/Suc, sucrose.