Down-regulation of the Sucrose Transporter CsSUT1 Causes Male Sterility by Altering Carbohydrate Supply

Abstract

In plants, male sterility is an important agronomic trait, especially in hybrid crop production. Many factors are known to affect crop male sterility, but it remains unclear whether Suc transporters (SUTs) participate directly in this process. Here, we identified and functionally characterized the cucumber (Cucumis sativus) CsSUT1, a typical plasma membrane-localized energy-dependent high-affinity Suc-H⁺ symporter. CsSUT1 is expressed in male flowers and encodes a protein that is localized primarily in the tapetum, pollen, and companion cells of the phloem of sepals, petals, filaments, and pedicel. The male flowers of CsSUT1-RNA interference (RNAi) lines exhibited a decrease in Suc, hexose, and starch content, relative to those of the wild type, during the later stages of male flower development, a finding that was highly associated with male sterility. Transcriptomic analysis revealed that numerous genes associated with sugar metabolism, transport, and signaling, as well as with auxin signaling, were down-regulated, whereas most myeloblastosis (MYB) transcription factor genes were up-regulated in these CsSUT1-RNAi lines relative to wild type. Our findings demonstrate that male sterility can be induced by RNAi-mediated down-regulation of CsSUT1 expression, through the resultant perturbation in carbohydrate delivery and subsequent alteration in sugar and hormone signaling and up-regulation of specific MYB transcription factors. This knowledge provides a new approach for bioengineering male sterility in crop plants.

Figure 1.

Spatio-temporal expression of CsSUT1 in cucumber. A, RT-qPCR analysis of CsSUT1 in different plant organs. B, RT-qPCR analysis of CsSUT1 at various stages of male flower development. Images in (B) were obtained from the male flower at various developmental stages, illustrated by different floral bud lengths. C, Expression analysis of three cucumber SUTs in sepals, petals, anthers, and pollen of stage 12 male flowers. Error bars represent ± sem (n = 3). Abbreviations: R, root; S, stem; YL, young leaf; ML, mature leaf; MF, male flower; FF, female flower; F, fruit.

Figure 2.

Histological and cellular localization of CsSUT1. A–N, Immunolocal detection of CsSUT1 in stage 10 male flowers using a CsSUT1-antiserum. A, C, E, G, I, K, L, N, Immunolabel detected in the tapetum and microspores in anthers, and phloem cells in sepals, petals, filaments, and the pedicel. B, D, F, H, J, M, Control sections incubated with preimmune serum were devoid of label. (G), (H), (K), and (N) are transverse sections, and the others are longitudinal sections. (L), (M), and (N), are the close-ups of (I), (J), and (K), respectively. O, Plasma membrane localization of CsSUT1-GFP fusion protein in protoplasts derived from cucumber cotyledons. GFP served as a control. Abbreviations: T, tapetum; M, microspore; Ph, phloem; Xy, xylem; SE, sieve element; PM, plasma membrane; Auto, chlorophyll autofluorescence. Scale bars = 50 μm in (A–N) and 5 μm in (O).

Figure 3.

Morphological and structural characterization of CsSUT1-RNAi male flowers. A, Schematic illustration of the CsSUT1-RNAi expression vector used in this study. B, RT-qPCR analysis of CsSUT1 transcripts present in stage 12 male flowers of wild-type (WT) and CsSUT1-RNAi lines. Error bars = ± sem (n = 3). Two lines were selected for further research (red arrows). C, Morphological features of male flowers at different developmental stages in wild type and CsSUT1-RNAi lines. D, Anther development in wild type and CsSUT1-RNAi male flowers. E, Transverse sections illustrating anther and pollen development in wild type and CsSUT1-RNAi lines. F, Transmission electron micrographs of developing pollen from wild type and CsSUT1-RNAi 14-1 plants. G, Phenotype of male flowers at anthesis stage (a–c), and triphenyltetrazolium chloride stained pollen grains (d–f, white arrows), with percentage viability (%) of wild type and CsSUT1-RNAi lines. Red arrow in (a) indicates abundant pollen. H, Scanning electron micrographs of anthers (a–c) and pollen grains (d–f) from stage 12 male flowers of wild type and CsSUT1-RNAi lines. Abbreviations: M, microspore; MP, mature pollen; Po, pollen; T, tapetum. Scale bars = 1 cm in (C), 200 μm in (D) and (G), and 50 μm in (E) and (H).

Figure 4.

Carbohydrate levels analysis in male flowers from wild-type (WT) and CsSUT1-RNAi lines. Data presented are means ± sem (n = 3) with units of µg/g fresh weight (FW). An ANOVA was undertaken using Tukey Honestly Significant Difference test, n = 3; the letters above the bars indicate significant differences (P < 0.05). Inset: Starch levels in male flowers and pollen of wild-type and CsSUT1-RNAi lines, detected by I₂-KI staining. Abbreviations: Sta, stachyose; Raf, raffinose; TSS, total soluble sugar. Scale bars = 1 mm in inset figure.

Figure 5.

Identification of genes related to carbohydrate metabolism in the cucumber male flower. A, Volcano plot of down-regulated (Down) and up-regulated (Up) DEGs developed based on RNA-Seq data from CsSUT1-RNAi lines versus wild-type (WT) lines. B, Functional categories of DEGs in RNA-Seq data identified by Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis; the top ten categories are shown. ‘Frequency’ stands for the percentage of genes in a certain category to the total DEGs. C, The DEGs involved in carbohydrate metabolism, transport, and signaling. D, Expression of several genes related to carbohydrate metabolism in (C), verified by RT-qPCR. α-Tubulin (AJ715498) was used as the normalization control. Error bars = ± sem (n = 3). Student’s t test, *P < 0.05; **P < 0.01; ***P < 0.001. Abbreviations: FC, fold change; SWEET, sugar will eventually be exported transporter; PFK, phosphofructokinase; UGP, UTP-Glc-1-phosphate uridylyltransferase; HXK1, hexokinase 1; FK2, fructokinase 2; SnRK1, SNF1-related protein kinase; FBP, Fru-1,6-bisphosphatase; TPS, alpha-trehalose-phosphate synthase; PG, polygalacturonase; PEL, pectate lyase; PE/PEI, pectinesterase/pectinesterase inhibitor; FPKM, fragments per kilobase of transcript per million fragments mapped; Min, minimum; Max, maximum.

Figure 6.

Identification of auxin signaling and MYB transcription factor genes whose expression patterns were changed in flowers of CsSUT1-RNAi compared to wild-type (WT) plants. A, Heat map of DEGs involved in auxin metabolic pathways, including auxin biosynthesis, transport, and response. B, Heat map showing normalized FRKM values of MYB transcription factors; five genes that changed significantly are highlighted in red. C, Phylogenetic analysis of MYB transcription factors. The five cucumber proteins highlighted in red font shared high sequence similarity with Arabidopsis AtMYB21, 24, 57, 108 (green font), and cotton GhMYB24 (purple font). The genes presented in (A) and (B) were selected with an FPKM value ≥ 10 before down-regulation or after up-regulation. Abbreviations: FC, fold change; PIN3, PIN-formed 3; PIF5, phytochrome-interacting factor 5; AUX1, auxin transporter-like protein; FPKM, fragments per kilobase of transcript per million fragments mapped.