Functional Characterization of CsSWEET5a, a Cucumber Hexose Transporter That Mediates the Hexose Supply for Pollen Development and Rescues Male Fertility in Arabidopsis

Abstract

Pollen cells require large amounts of sugars from the anther to support their development, which is critical for plant sexual reproduction and crop yield. Sugars Will Eventually be Exported Transporters (SWEETs) have been shown to play an important role in the apoplasmic unloading of sugars from anther tissues into symplasmically isolated developing pollen cells and thereby affect the sugar supply for pollen development. However, among the 17 CsSWEET genes identified in the cucumber (Cucumis sativus L.) genome, the CsSWEET gene involved in this process has not been identified. Here, a member of the SWEET gene family, CsSWEET5a, was identified and characterized. The quantitative real-time PCR and β-glucuronidase expression analysis revealed that CsSWEET5a is highly expressed in the anthers and pollen cells of male cucumber flowers from the microsporocyte stage (stage 9) to the mature pollen stage (stage 12). Its subcellular localization indicated that the CsSWEET5a protein is localized to the plasma membrane. The heterologous expression assays in yeast demonstrated that CsSWEET5a encodes a hexose transporter that can complement both glucose and fructose transport deficiencies. CsSWEET5a can significantly rescue the pollen viability and fertility of atsweet8 mutant Arabidopsis plants. The possible role of CsSWEET5a in supplying hexose to developing pollen cells via the apoplast is also discussed.

Keywords: SWEET; cucumber; hexose transporter; plasma membrane; pollen development.

Figure 1

Sequence analysis of CsSWEET5a. (A) Multiple sequence alignment of SWEET5 proteins from Cucumis sativus (CsSWEET5a), Arabidopsis thaliana (AtSWEET5), Oryza sativa (OsSWEET5), Solanum lycopersicum (SlSWEET5b), Cucumis melo (CmeSWEET5), Cucurbita pepo (CpSWEET5), Cucurbita moschata (CmoSWEET5), Cucurbita maxima (CmaSWEET5), Benincasa hispida (BhSWEET2), Momordica charantia (McSWEET5), Rosa chinensis (RcSWEET5), Ziziphus jujuba (ZjSWEET5), and Punica granatum (PgSWEET5). The seven transmembrane domains (TMs) are outlined. The identical amino acids are denoted by white characters on a red background, and the conserved amino acids are indicated by a yellow background. (B) Phylogenetic analysis of SWEET proteins from cucumber (CsSWEET5a) and Arabidopsis (AtSWEET1 to AtSWEET17). A phylogenetic tree was constructed using the neighbor joining method and the p-distance model via MEGA 7.0 software. The scale bar represents the evolutionary distance of the number of amino acid differences per site. Bootstrapping was performed with 1000 replicates, and the values on the branches are shown as %. The amino acid sequences of the SWEET proteins used for the analysis are listed in Table S3.

Figure 2

Expression of CsSWEET genes in cucumber organs. (A) Expression analysis of CsSWEET5a in different cucumber organs according to the transcriptome data. Raw data were obtained from the Cucurbit Expression Atlas (http://cucurbitgenomics.org/rnaseq/home (accessed on 2 December 2022)) under the project PRJNA80169. RPKM, reads per kilobase per million mapped reads. (B) Expression analysis of CsSWEET5a in different cucumber organs by quantitative real-time PCR (qRT–PCR). (C) Images of whole male flowers at various developmental stages. The stage division of male cucumber flowers was performed essentially as described previously [40], with slight modifications. The main features of each developmental stage were clarified in Table S4. Scale bars: 5 mm. (D) qRT–PCR analysis of CsSWEET5a in whole male flowers at various developmental stages. (E) Expression analysis of CsSWEET5a in various organs of male flowers at stages 11, 12, and 13. (F,G) Expression analysis of 17 CsSWEET genes in anthers (F) or pollen cells (G) that were isolated from male flowers at stage 11. A monoecious cucumber inbred line, C49, was used in (B–G). Error bars (B,D–G) represent the SEs from three biological replicates. FF, whole female flower at the time of opening; L, leaf; MF, whole male flower at the time of opening; O, ovary; OF, ovary_fertilized; OU, ovary_unfertilized; R, root; S, stem; T, tendril; TB, tendril base; 3 DAA, fruit at 3 days after anthesis; 6 DAA, fruit at 6 days after anthesis.

Figure 3

Histochemical staining of β-glucuronidase (GUS) activity in flower buds of cucumber and Arabidopsis plants. (A–L) Transient expression of pCsSWEET5a::GUS in male cucumber flowers from stages 9 to 12. Predominant GUS staining was observed in the anthers (indicated by arrows) at every stage of male flower development but not in the sepals or petals (E–H). Strong GUS activity was also observed in microsporocytes (I), tetrad microspores (J), uninuclear microspores (K), and mature pollen (L). (A–H) show the same male cucumber flower buds before and after GUS staining, respectively. (A,E) stage 9; (B,F) stage 10; (C,G), stage 11; (D,H) stage 12. (M–X) GUS staining of flower buds from T₂ pCsSWEET5a::GUS transgenic Arabidopsis plants at different developmental stages. Dominant GUS expression was detected in anthers but not in sepals, petals, pistils, filaments, or peduncles (Q–T). To better visualize GUS expression in anthers, the sepals and petals in which GUS staining was not observed (Supplementary Figure S2) were removed from (Q–T). GUS staining was also strongly detected in the tetrad microspores (U), uninuclear microspores (V), bicellular pollen (W), and tricellular pollen (X) of the pCsSWEET5a::GUS transgenic Arabidopsis plants. (M–T) show the same Arabidopsis flower buds before and after GUS staining, respectively. Scale bars: 5 mm (A–H); 20 µM (I–L,U–X); 1 mm (M–T).

Figure 4

Subcellular location of the CsSWEET5a-YFP fusion protein in tobacco (Nicotiana benthamiana) leaf epidermal cells (A–H) and Arabidopsis mesophyll protoplasts (I–P). An empty vector expressing untargeted YFP was used as a control (A–D,I–L). The white arrowheads in (O,P) indicate chloroplasts. An mCherry-labeled marker (CD3-1007) was used to mark the plasma membrane position. The merged image shows YFP (green), chlorophyll (blue), and plasma membrane marker (red) fluorescence. The bright-field images are also presented. These images demonstrated that CsSWEET5a-YFP-derived fluorescence colocalized with the fluorescence of plasma membrane markers in a lining outside the chloroplast, indicating localization to the plasma membrane. Scale bars: 50 μm (A–D); 30 μm (E–H); 10 µM (I–P).

Figure 5

Analysis of the transport activity of CsSWEET5a in yeast. (A) Transport activity of CsSWEET5a in the yeast mutant EBY.VW4000. Yeast cells expressing an empty vector (negative control), CsSWEET5a, or AtSWEET1 (positive control) were serially diluted (10-fold) and cultured on solid synthetic deficient media without uracil (SD-Ura) supplemented with 2% (w/v) maltose, 2% (w/v) glucose, or 2% (w/v) fructose as the sole carbon source. CsSWEET5a and AtSWEET1 complemented the glucose and fructose uptake deficiency of EBY.VM4000, but the empty vector did not. (B) Transport activity of CsSWEET5a in the yeast mutant SUSY7/ura3. Yeast cells expressing an empty vector (negative control), CsSWEET5a or AtSWEET12 (positive control) were serially diluted (10-fold) and cultured on solid SD-Ura media supplemented with 2% (w/v) glucose or 2% (w/v) sucrose as the sole carbon source. AtSWEET12 complemented the sucrose uptake deficiency of SUSY7/ura3, but the empty vector and CsSWEET5a did not. Images were captured after incubation at 30 °C for 3–5 days.

Figure 6

Characterization of atsweet8 mutant Arabidopsis plants overexpressing CsSWEET5a. (A) Inflorescences of the primary stems of atsweet8, CsSWEET5a/atsweet8-overexpressing (CsSWEET5a/atsweet8-OE) transgenic lines, and the wild type (WT). (B) The position and corresponding seed number of siliques generated from the primary stem of the atsweet8, CsSWEET5a/atsweet8-OE, and WT plants. Five primary stems were randomly selected for presentation. (C,D) Total length of siliques (C) or total number of seeds (D) per primary stem of the atsweet8, CsSWEET5a/atsweet8-OE, and WT plants. The data are presented as the means ± SDs of ten primary stems. The different letters above the bars indicate significant differences (p < 0.01) determined by Duncan’s test. The siliques generated from the 1st to 40th flowers on the primary stem of the atsweet8, CsSWEET5a/atsweet8-OE, and WT plants were statistically analyzed, and the results are shown in (B–D). (E) Morphological features of the flowers of the atsweet8, CsSWEET5a/atsweet8-OE, and WT plants. Note that many more pollen grains were observed on the stigmas of the CsSWEET5a/atsweet8-OE lines than on those of the atsweet8 mutant plants. (F) Triphenyl tetrazolium chloride (TTC) staining of anthers from the atsweet8, CsSWEET5a/atsweet8-OE, and WT lines. The red-stained pollen grains are viable and fertile. The flowers from the 10th to 20th on the primary stem were used to obtain the results shown in (E,F). Scale bars: 1 cm (A); 1 mm (E); 100 µm (F).

Figure 7

Hypothetical model of the role of CsSWEET5a in sugar transport from anther tissues into developing cucumber pollen at stages 9–12. CWINV, cell wall invertase.