Spatiotemporal Expression and Substrate Specificity Analysis of the Cucumber SWEET Gene Family

Abstract

The functions of SWEET (Sugar Will Eventually be Exported Transporter) proteins have been studied in a number of crops, but little is known about their roles in cucumber (Cucumis sativus L.), a model plant for studying stachyose metabolism and phloem function. Here, we identified 17 cucumber SWEET genes (CsSWEETs), located on chromosomes 1-6, and classified them into four clades. Two genes from each clade were selected for spatiotemporal expression, subcellular localization, and substrate specificity analyses. Clade I and II proteins were all hexose transporters and targeted to the plasma membrane, while clade III proteins also localized to the plasma membrane, but used sucrose as a substrate. Clade IV SWEET proteins were localized to the tonoplast, and used hexose as a substrate. The eight tested CsSWEET genes were most highly expressed in flower, which represents a large sink in plants. However, each gene also showed specific expression patterns: three of the eight tested genes were highly expressed in mature leaves, two in roots, two in fruit, two in stems, and one was detected in all tested organs. The likely biological roles of each are discussed based on the above results.

Keywords: SWEET transporters; cucumber; phylogeny; spatiotemporal expression; subcellular localization; substrate specificity; sugar.

FIGURE 1

Phylogenetic tree of cucumber (Cucumis sativus), Arabidopsis thaliana, and tomato (Solanum lycopersicum) SWEET proteins. AtSWEET is A. thaliana (yellow), CsSWEET is Cucumis sativus (red), and SlSWEET is Solanum lycopersicum (green). The evolutionary history was inferred using the neighbor joining method with 1,000 replicates. The evolutionary distances were computed using the Poisson correction distance model and are in the units of the number of amino acid substitutions per site. Evolutionary analyses were conducted using MEGA5.0 software. Accessions are listed in Supplementary Table S2.

FIGURE 2

The transmembrance domains of CsSWEET proteins. The position of N- and C-terminal domains of the protein are indicated by blue or pink lines. Table on the right bottom shows the location of each protein in this figure. The website used for predictions was TMHMM.

FIGURE 3

Heterologous expression of eight CsSWEET genes in yeast. Yeast strains with recombinant vectors or the empty pDR196 vector (as a negative control) were grown on SD (synthetic deficient)-ura medium supplemented with 2% maltose or different sugars (listed above) as the sole carbon source for 5 days. Medium with maltose as sole carbon source is a positive control for strain EBY.WV4000, while with glucose as sole carbon source is a positive control for strain SUSY7/ura. Yeast cell suspensions were diluted (×10, ×10², ×10³) for serial dilutions assay. The other results of the experiment are in Supplementary Figure S2.

FIGURE 4

The expression patterns of eight CsSWEET genes. (A–H) Relative expression of CsSWEETs. R, root; S, stem; YL, young leaf; ML, mature leaf; MF, male flower; FF, female flower; 0DAA, ovary/fruit on the day of anthesis, 9DAA, fruit on the ninth day after anthesis. (I) Schematic model of the areas of CsSWEET gene expression in cucumber. Letters in (I) match the expression pattern in (A–H). Error bars represent the SE for three technical replicates of three biological replicates.

FIGURE 5

The subcellular localization of CsSWEET1, -7b, -12c, -17a green fluorescent protein (GFP) fusions in A. thaliana protoplasts. The white arrows indicate the tonoplast. Scale bars = 10 μm.