A Tonoplast Sugar Transporter Underlies a Sugar Accumulation QTL in Watermelon

Abstract

How sugar transporters regulate sugar accumulation in fruits is poorly understood and particularly so for species storing high-concentration Suc. Accumulation of soluble sugars in watermelon (Citrullus lanatus) fruit, a major quality trait, had been selected during domestication. Still, the molecular mechanisms controlling this quantitative trait are unknown. We resequenced 96 recombinant inbred lines, derived from crossing sweet and unsweet accessions, to narrow down the size of a previously described sugar content quantitative trait locus, which contains a putative Tonoplast Sugar Transporter gene (ClTST2). Molecular and biochemical analyses indicated that ClTST2 encodes a vacuolar membrane protein, whose expression is associated with tonoplast uptake and accumulation of sugars in watermelon fruit flesh cells. We measured fruit sugar content and resequenced the genomic region surrounding ClTST2 in 400 watermelon accessions and associated the most sugar-related significant single-nucleotide polymorphisms (SNPs) to the ClTST2 promoter. Large-scale population analyses strongly suggest increased expression of ClTST2 as a major molecular event in watermelon domestication associated with a selection sweep around the ClTST2 promoter. Further molecular analyses explored the binding of a sugar-induced transcription factor (SUSIWM1) to a sugar-responsive cis-element within the ClTST2 promoter, which contains the quantitative trait locus (QTL) causal SNP. The functional characterization of ClTST2 and its expression regulation by SUSIWM1 provide novel tools to increase sugar sink potency in watermelon and possibly in other vegetable and fruit crops.

Figure 1.

Mapping, transmembrane domain modeling, and phylogenetic analysis of the ClTST2 gene. A, SNP bin map of the sugar content QTL (QBRX2-1). B, Mapping of QBRX2-1 to a physical region of 797.4 kb on chromosome 2 between nucleotides 17,657,266 and 18,454,759 in the watermelon genome. C, Five expressed genes (Supplemental Table S2) localized in the region, one of which is ClTST2 (Cla000264). D, Association study of sugar content, based on 21,249 SNPs identified in ClTST2 region (Chr2 16–19 Mb) by genomic resequencing of this region in 400 watermelon accessions. The most significant SNPs, marked with red dots, are located in the promoter of ClTST2. E, Transmembrane helix modeling for the ClTST2 protein showing 12 transmembrane domains. F, A neighbor-joining phylogenetic tree of Arabidopsis (At), sugar beet (Bv, B. vulgaris), and watermelon (Cl, C. lanatus) ClTST1, ClTST2, and ClTST3.

Figure 2.

High expression of ClTST2 increases sugar accumulation. ClTST2-specific mRNA relative levels determined by qRT-PCR in the mature fruit of 96 RILs (A) and 50 core germplasm accessions (B) selected from Supplemental Table S4. Basta resistance in seedling stage (shown in red circles) and fruit phenotype at 30 DAP for transgenic control (C) and representative ClTST2 overexpression T1 lines (D and E). In lines ClTST2 OE-2 and ClTST2 OE-5 with higher levels of ClTST2 mRNA, the fruit sugar contents increased significantly compared with the control (F).

Figure 3.

Transport function of ClTST2. Incubation with 50 mm Suc (A), Fru (B), and Glc (C) solutions induced inward current responses (gray bars) in HEK293T cells expressing the ClTST2. HEK293T cells only did not exhibit a significant change in current in response to incubation of 50-mm Suc/Glc solution (D). Tracer efflux assay in Xenopus oocytes at pH 5.5. [¹⁴C] Suc (E), [¹⁴C] Glc (F), and [¹⁴C] Fru (G) efflux by ClTST2 in Xenopus oocytes in 3- and 6-mm [¹⁴C] sugars. As negative controls, we used oocytes injected with water to efflux [¹⁴C] sugars (n = 9).

Figure 4.

ClTST2 increases sugar accumulation in fruits. A to C, The parental lines 97103, G35, F₁, and BC₁ fruits for NIL population construction. D, Sugar content and relative expression levels of ClTST2 in (from left to right) 97103 and G35 parental lines, F₁, BC₁ plants, and 50 segregating (BC₂F₂) NILs. E, Suc injection induces early maturity in strawberry fruits; red arrows point at the injection site. F to H, ClTST2 overexpressing strawberry fruit sections (OE), coloration compared to the empty vector control. I, Accumulation of sugars in ClTST2 overexpressing strawberry fruit sections (OE) and in the empty vector control. J, Semiquantitative RT-PCR expression analysis of FaACT and ClTST2 in the empty vector controls 1 to 3 and in ClTST2 overexpressing strawberry fruit sections (ClTST2-1-3 OE). The same sample order as in I for FaACT and ClTST2 expression analyses, respectively.

Figure 5.

SUSIWM1 regulates expression of ClTST2. A, Association of sugar content with ClTST2 promoter haplotype in cultivated, semiwild, and wild watermelons. B, XP-CLR estimation of the ClTST2 genome region. ClTST2 is localized in the 40-kb regions surrounded by red frame, which resides in the genome region that went through selective sweep during watermelon domestication. C, Y1H assays showing that SUSIWM1 activated AUR1-C expression when both the 97103 (97) and PI296341-FR (PI) sequence motifs were used in the presence of 200 to 600 ng/mL AbA. No activation was detected for the empty vector. D, EMSA of GST-SUSIWM1 fusion protein binding to the SURE cis-elements in ClTST2 from 97103 (97) and PI296341-FR (PI). FP assay showing probed SUSIWM1 dose-dependent binding activity to SURE cis-elements in ClTST2 from 97 and PI. E, LUC assay of strawberry fruits cotransformed with p35S::SUSIWM1::tNOS as the effector or with p35S::MYC::tNOS as the control, with the −1,500 bp promoter region of the sweet and unsweet lines fused to LUC. Relative fluorescence results are presented as histograms, representing the means of five replicates. F, Digital expression (in RPKM) of SUSIWM1 in high-sugar-content fruit line 97103, in low-sugar fruit (PI296341-FR) and low-sugar tissue (97103 rind).

Figure 6.

Regulation of ClTST2 and SUSIWM1. A, ChIP analysis of SUSIWM1 recruitment to the ClTST2 promoter W box3, which contains the −1,368 SNP (A/C) in a SURE element. The results are expressed as means ± sd. B, Changes in fruit sugar content (Brix) and color 3 d after injection of 1 mL 10% Suc into 10 DAP immature watermelon fruit. C, Relative level of SUSIWM1 mRNA after treatment with Suc. D, Proposed model for regulation of ClTST2 by SUSIWM1 based on RNA-seq transcriptome data. Suc induces the expression of SUSIWM1, which up-regulates ClTST2 in cultivar sweet 97103 fruit flesh, while transcription of ClTST2 is repressed during all stages of fruit maturation in low-sugar-content tissues such as PI296341-FR fruit and 97103 fruit rind.