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The SnRK2.3-AREB1-TST1/2 cascade activated by cytosolic glucose regulates sugar accumulation across tonoplasts in apple and tomato

Nature Plants volume 9, pages 951–964 (2023)Cite this article

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Abstract

Soluble sugars are the core components of fruit quality, and the degree of sugar accumulation is largely determined by tonoplast-localized sugar transporters. We previously showed that two classes of tonoplast sugar transporters, MdERDL6 and MdTST1/2, coordinately regulate sugar accumulation in vacuoles. However, the mechanism underlying this coordination remains unknown. Here we discovered that two transcription factors, MdAREB1.1/1.2, regulate MdTST1/2 expression by binding their promoters in apple. The enhanced MdAREB1.1/1.2 expression in MdERDL6-1-overexpression plants resulted in an increase in MdTST1/2 expression and sugar concentration. Further studies established that MdSnRK2.3, whose expression could be regulated by expressing MdERDL6-1, could interact with and phosphorylate MdAREB1.1/1.2, thereby promoting the MdAREB1.1/1.2-mediated transcriptional activation of MdTST1/2. Finally, the orthologous SlAREB1.2 and SlSnRK2.3 exhibited similar functions in tomato fruit as in their apple counterparts. Together, our findings provide insights into the regulatory mechanism of tonoplast sugar transport exerted by SnRK2.3-AREB1-TST1/2 for fruit sugar accumulation.

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Fig. 1: The expression profiles of MdAREB1.1/1.2.
Fig. 2: MdAREB1.1/1.2 regulate the expression of MdTST1/2 by binding to their promoters.
Fig. 3: Impacts of altering MdAREB1.1/1.2 expression on MdTST1/2 expression levels and sugar concentrations in fruit of ‘Fuji’ apple and leaves of MdERDL6-1-overexpressing apple lines.
Fig. 4: MdSnRK2.3 interacts with both MdAREB1.1 and MdAREB1.2.
Fig. 5: MdSnRK2.3 phosphorylates and suppresses MdAREB1.1/1.2 protein degradation in vitro.
Fig. 6: The effects of co-expression and sugar feeding on the promoter activities of MdTST1/2 and MdSnRK2.3 in apple calli.
Fig. 7: Impacts of altering MdSnRK2.3 expression on MdAREB1.1/1.2 phosphorylation levels and sugar concentrations in fruit of ‘Fuji’ apple and leaves of MdERDL6-1-overexpressing apple lines.
Fig. 8: A model of the ‘MdERDL6-Glc-SnRK2.3-AREB1-TST1/2’ pathway promoting sugar accumulation in the vacuole.

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Data availability

All data supporting the findings of this study are available in the main text or the Supplementary Information. Additional data related to this study are available from the corresponding author upon request. All biological materials used in this study are available from the corresponding author on reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank J. Zhang and J. Zhao (Horticulture Science Research Center, Northwest A&F University, Yangling, China) for providing professional technical assistance with GC–MS analysis; H. Zhao and F. Yuan (State Key Laboratory of Crop Stress Biology for Arid Areas, Northwest A&F University, Yangling, China) and Y. Yuan and R. Chen (Horticulture Science Research Center, Northwest A&F University, Yangling, China) for providing confocal microscopy experimental assistance. This work was supported by the Program for the National Natural Science Foundation of China (31872043) to M.L., the Shaanxi Science and Technology Innovation Team Project (2022TD-18) to M.L., the Australian Research Council (DP180103834) to Y.-L.R., and the Earmarked Fund for the China Agriculture Research System (CARS-27) to F.M.

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  1. These authors contributed equally: Lingcheng Zhu, Yanzhen Li.

Authors and Affiliations

  1. State Key Laboratory of Crop Stress Biology in Arid Areas/Shaanxi Key Laboratory of Apple, College of Horticulture, Northwest A&F University, Xianyang, China

    Lingcheng Zhu, Yanzhen Li, Chengcheng Wang, Zhiqi Wang, Wenjing Cao, Jing Su, Yunjing Peng, Baiyun Li, Baiquan Ma, Fengwang Ma, Yong-Ling Ruan & Mingjun Li

  2. College of Life Science, Northwest A&F University, Xianyang, China

    Lingcheng Zhu

  3. Division of Plant Sciences, Research School of Biology, The Australian National University, Canberra, Australian Capital Territory, Australia

    Yong-Ling Ruan

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Contributions

L.Z., M.L., F.M. and Y.-L.R. designed this research. L.Z., Y.L., C.W., Z.W., J.S., Y.P., B.L., W.C. and B.M. performed the experiments. L.Z., J.S., Y.L., M.L. and Y.-L.R. analysed the data. L.Z., M.L. and Y.-L.R. wrote the paper. M.L. and F.M. supervised the study. All authors read and approved the final paper.

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Correspondence to Fengwang Ma, Yong-Ling Ruan or Mingjun Li.

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Nature Plants thanks Jintao Cheng, Li-Qing Chen and H. Ekkehard Neuhaus for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Relative distribution of compartment-specific marker enzyme activities and subcellular sugar concentration in MdERDL6-1 overexpressed plants using NAF method.

a, b, Relative distribution of compartment-specific markers in MdERDL6-1 overexpressed apple leaves (a) and tomato fruits (b). Tissues were fractionated using a NAF procedure and the activities of marker enzymes in the five fractions were determined. Data are expressed as percentage activities in each fraction. AGPase, UGPase, and ACP were used as plastidic, cytosolic, and vacuolar markers, respectively. c, d, Subcellular concentrations of Glc, Fru, and Suc in plastid, cytosol, and vacuole fractions of MdERDL6-1 overexpressed apple leaves (c) and tomato fruits (d). nd, no data. The bars represent the mean value ± SD (n = 3 independent biological replicates). The asterisks in (c,d) indicate significant differences as assessed by Student’s t-test (two-sided). ***P < 0.001, **P < 0.01, *P < 0.05, ns, not significant.

Source data

Extended Data Fig. 2 The screening of candidate transcription factors.

a, Differentially expressed transcription factors in the RNA-seq of MdERDL6-1 transgenic apples. The threshold was set as: RPKM > 1, fold change > 1.5. b, c, Predicted active transcription factors regulate MdTST1 (b) and MdTST2 (c) expression based on ATAC-seq in apple fruit. d, The ABRE element analysis on MdTST1/2 promoters. e, qRT-PCR validation of three differential transcription factors in the RNA-seq of MdERDL6-1 transgenic apples. The transcript levels were normalized with those of MdActin in transgenic apple. Relative expression levels for each gene were obtained via ddCT method, with the expression in wild type (WT) set as ‘1’.The bars represent the mean value ± SD (n = 3 seedlings). The asterisks in (e) indicate significant differences as assessed by Student’s t-test (two-sided). ***P < 0.001, **P < 0.01, ns, not significant.

Source data

Extended Data Fig. 3 The gene expression levels and promoter activities of MdSnRK2.3 and MdAREB1.1/1.2 after sugars feeding in apple calli.

a, b, The impacts of sugars feeding on MdSnRK2.3 and MdAREB1.1/1.2 expression levels. Apple calli was cultured on MS liquid medium with 2% different exogenous sugars (Water was used as control) and different concentrations of Glc for 24 h, and the gene expression levels were detected by qRT-PCR. The transcript levels were normalized with those of MdActin. Relative expression level was obtained via ddCT method, setting the expression level in water feeding as ‘1’. c, d, The impacts of sugars feeding on MdAREB1.1/1.2 promoter activities. Apple calli was cultured on MS liquid medium with different exogenous sugars and different concentrations of Glc for 24 h after infiltrating with Agrobacterium harboring MdAREB1.1/1.2pro-GUS plasmids. The treated and control apple calli were harvested for GUS activity assay after 24 h feeding. The 1924bp and 1937bp promoters of MdAREB1.1 and MdAREB1.2 were used in the GUS experiments. Man: mannose; Glc: glucose; Fru: fructose; Suc: sucrose; and Sor: sorbitol. Bars represent the mean value ± SD (n = 3 independent biological replicates). Different letters indicate significant differences as assessed by one-way ANOVA (Tukey’s test), respectively (P < 0.05).

Source data

Extended Data Fig. 4 Phylogenetic analysis, subcellular localization, self-activation assay and expression profiles of MdAREB1.1/1.2.

a, Phylogenetic tree of MdAREBs from apple and the orthologous genes from Arabidopsis and tomato. The AREB orthologous sequences were collected to build an un-rooted phylogenetic tree using the neighbour-joining method of the MEGA7 software. A 1000 trial of bootstrap analysis was used to provide confident estimates for phylogenetic tree topology. The colored circles are studied genes in this research. The scale bar corresponds to 0.05. b, Nuclear localization of MdAREB1.1/1.2. 35Spro:MdAREB1.1/1.2-GFP were transiently expressed in protoplasts of Arabidopsis leaves. DAPI served as a nuclear dye. The GFP signals of MdAREB1.1/1.2 overlapped with DAPI, indicating that MdAREB1.1/1.2 localized in the nuclear. Scale bars, 20 μm. c, The transcriptional self-activation assay of MdAREB1.1/1.2. The pGBKT7-MdAREB1.1/1.2 and empty pGBKT7 vector (negative control) were introduced into the yeast strain Y2H respectively, and the transformed cells were spotted on SD-Trp and SD-Trp-His-Leu+x-α-Gal medium. The plates were incubated at 30 °C for 3 d. d, The heat map of MdAREBs expression levels based on RNA-seq in mature leaves (ML) and five stages of developing fruits (S1-S5). Fold difference is designated as a log2 value, with the data in S1 set as 1. The experiments in (b, c) were repeated independently at least three times, with similar results.

Source data

Extended Data Fig. 5 Impacts of altering MdAREB1.1 or MdAREB1.2 expression on MdTST1/2 expression levels and sugar concentrations in ‘Orin’ apple calli.

a, The complete coding sequences of MdAREB1.1 or MdAREB1.2 were inserted into the pMDC83 vector for gene stable overexpression (OE-MdAREB1.1#1, #2; OE-MdAREB1.2#1, #2), while the specific cDNA fragments were cloned into pK7GWIWG2 vector for gene stable silencing (PK7-MdAREB1.1#1, #2; PK7-MdAREB1.2#1, #2). The empty vectors pMDC83 and PK7 served as controls respectively. b, The mRNA relative expression levels of MdAREB1.1/1.2 and MdTST1/2 in transgenic apple calli, setting that from pMDC83 as ‘1’. c, The sugar concentrations (Fru, fructose; Glc, glucose; Suc, sucrose) in transgenic apple calli. The bars represent the mean value ± SD (n = 3 independent biological replicates). Different letters indicate significant differences as assessed by one-way ANOVA (Tukey’s test) (P < 0.05).

Source data

Extended Data Fig. 6 Phylogenetic tree, expression profiles, and subcellular localization analysis of MdSnRK2s.

a, Phylogenetic analysis of MdSnRK2s from apple and the orthologous genes from Arabidopsis and tomato. The SnRK2s orthologous sequences were collected to build an un-rooted phylogenetic tree using the neighbour-joining method of the MEGA7 software, which was obviously clustered into three subclasses. A 1000 trial of bootstrap analysis was used to provide confident estimates for phylogenetic tree topology. The colored circles are studied genes in this research. The scale bar corresponds to 0.05. b, The heat map of MdSnRK2s expression levels based on RNA-seq in MdERDL6-1 overexpressing apple lines. Fold difference is designated as a log2 value, with the data in WT set as 1. c, The protein abundances (quantified proteins) of MdSnRK2.1/2.3/2.5 are extracted from tandem mass tags (TMT) quantitative proteomics of five developing stages of apple fruits. The bars represent the mean value ± SD (n = 3 independent biological replicates). d, Subcellular localization of MdSnRK2.3-GFP fusion protein in the protoplasts from Arabidopsis leaves. GFP, green fluorescent protein. DAPI (4’,6-diamidino-2-phenylindole) was used to stain the nucleus. Auto, the red auto fluorescence of chloroplasts. Bar = 20 μm. The experiments in (d) were repeated independently at least three times, with similar results.

Source data

Extended Data Fig. 7 Y1H and dual-luciferase assay analysis of MdAREB1.1/1.2 and SlAREB1.2 binding to their own promoters.

a, b, Yeast one-hybrid assays showed that MdAREB1.1/1.2 and SlAREB1.2 bound to their own promoters containing ABRE cis-elements. Schematic diagram of P1 and P2 truncation promoters with or without ABRE cis-elements, the vertical lines represent ABRE cis-elements (a). The positive controls were pAbAi-P53 and pGADT7-53. The screening concentration of AbA were 150 ng/mL, 180 ng/mL, and 200 ng/mL of MdAREB1.1, MdAREB1.2, and SlAREB1.2 promoters respectively. Each colony was dissolved in 5 μL sterile NaCl and then diluted to 10−1 to 10−3. c, Dual-luciferase assays in tobacco (N. benthamiana) leaves revealed that MdAREB1.1/1.2 and SlAREB1.2 binding to their own promoters. The left are the dual-luciferase signal imagings, and the right are dual-luciferase activity assays. The 1924bp and 1937bp promoters of MdAREB1.1 and MdAREB1.2, 1911bp promoter of SlAREB1.2 were used in this assay. The positive controls are pGreenII 0800-LUC-P53 and pGreenII 62-SK-53. The experiments were repeated independently at least three times, with similar results.

Extended Data Fig. 8 Impacts of altering SlAREB1.2 expression on SlTST1/2 expression levels and sugar concentrations in tomato fruits.

a, The fruits of transgenic tomato lines (overexpression lines: OE#2, OE#5; silence lines: PK7#1, PK7#3). b, c, The relative expression levels of SlAREB1.2 and SlTST1/2 in the transgenic tomato measured using qRT-PCR and normalized to those of SlActin, setting that from WT as ‘1’. d, e, The soluble solids contents and carbohydrate levels in ripening fruits of transgenic tomato. Glc, glucose; Fru, fructose; Suc, sucrose. The bars represent the mean value ± SD (n = 3 independent biological replicates). The asterisks indicate significant differences as assessed by Student’s t-test (two-sided). ***P < 0.001, **P < 0.01, *P < 0.05.

Source data

Extended Data Fig. 9 Impacts of VIGS-induced gene silencing of SlAREB1.2 on SlTST1/2 expression levels and sugar concentrations in MdERDL6-1 overexpressing tomato fruits.

a, The Agrobacterium containing the resulting vectors pTRV2-SlAREB1.2 and pTRV1 were mixed in equal proportions, which were used for injection in two MdERDL6-1 overexpressing tomato lines (OE1-pTRV#SlAREB1.2 and OE2-pTRV#SlAREB1.2), with empty pTRV2 and pTRV1 mixture for injection as controls (WT, OE-1, and OE-2). The arrows indicate the injection sites in fruits. b, The mRNA relative expression levels of SlAREB1.2 and SlTST1/2 in transgenic tomato fruits of wild-type and MdERDL6-1 overexpressed background, setting that from WT as ‘1’. c, The sugar concentrations (Fru, fructose; Glc, glucose; Suc, sucrose) in transgenic tomato fruits of wild-type and MdERDL6-1 overexpressed background. The bars represent the mean value ± SD (n = 3 independent biological replicates). Different letters indicate significant differences as assessed by one-way ANOVA (Tukey’s test) (P < 0.05).

Source data

Extended Data Fig. 10 Impacts of VIGS-induced gene silencing of SlSnRK2.3 on SlTST1/2 expression levels and sugar concentrations in MdERDL6-1 overexpressing tomato fruits.

a, The Agrobacterium containing the resulting vectors pTRV2-SlSnRK2.3 and pTRV1 were mixed in equal proportions, which were used for injection in two MdERDL6-1 overexpressing tomato lines (OE1-pTRV#SlSnRK2.3 and OE2-pTRV#SlSnRK2.3), with empty pTRV2 and pTRV1 mixture for injection as controls (WT, OE-1, and OE-2). The arrows indicate the injection sites in fruits. b, The mRNA relative expression levels of SlSnRK2.3, SlAREB1.2 and SlTST1/2 in transgenic tomato fruits of wild-type and MdERDL6-1 overexpressed background, setting that from WT as ‘1’. c, The sugar concentrations (Fru, fructose; Glc, glucose; Suc, sucrose) in transgenic tomato fruits of wild-type and MdERDL6-1 overexpressed background. The bars represent the mean value ± SD (n = 3 independent biological replicates). Different letters indicate significant differences as assessed by one-way ANOVA (Tukey’s test) (P < 0.05).

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Zhu, L., Li, Y., Wang, C. et al. The SnRK2.3-AREB1-TST1/2 cascade activated by cytosolic glucose regulates sugar accumulation across tonoplasts in apple and tomato. Nat. Plants 9, 951–964 (2023). https://doi.org/10.1038/s41477-023-01443-8

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