SUT2, a putative sucrose sensor in sieve elements

Abstract

In leaves, sucrose uptake kinetics involve high- and low-affinity components. A family of low- and high-affinity sucrose transporters (SUT) was identified. SUT1 serves as a high-affinity transporter essential for phloem loading and long-distance transport in solanaceous species. SUT4 is a low-affinity transporter with an expression pattern overlapping that of SUT1. Both SUT1 and SUT4 localize to enucleate sieve elements of tomato. New sucrose transporter-like proteins, named SUT2, from tomato and Arabidopsis contain extended cytoplasmic domains, thus structurally resembling the yeast sugar sensors SNF3 and RGT2. Features common to these sensors are low codon bias, environment of the start codon, low expression, and lack of detectable transport activity. In contrast to LeSUT1, which is induced during the sink-to-source transition of leaves, SUT2 is more highly expressed in sink than in source leaves and is inducible by sucrose. LeSUT2 protein colocalizes with the low- and high-affinity sucrose transporters in sieve elements of tomato petioles, indicating that multiple SUT mRNAs or proteins travel from companion cells to enucleate sieve elements. The SUT2 gene maps on chromosome V of potato and is linked to a major quantitative trait locus for tuber starch content and yield. Thus, the putative sugar sensor identified colocalizes with two other sucrose transporters, differs from them in kinetic properties, and potentially regulates the relative activity of low- and high-affinity sucrose transport into sieve elements.

Figure 1.

Sucrose Transporter Sequence Analysis. (A) Structural comparison of SUT1 and SUT2. Hydrophobicity plots (Kyte and Doolittle, 1982) (window of 11 amino acids) of LeSUT1 and LeSUT2 were overlayed. Both contain 12 putative transmembrane domains (labeled with roman numerals I to XII), but SUT2 contains several hydrophilic domains not present in SUT1. (B) Predicted topology of LeSUT2. All amino acids conserved in both SUT2 orthologs are shown in black. The central loop region contains two highly conserved sequence motifs: central conserved box (CCB) 1 and 2, which are present only in SUT2 orthologs (underlined). (C) Homology of sucrose transporters. The tree is based on maximum parsimony analysis (Swofford, 1998) for protein sequences of aligned sucrose transporters from tobacco (NtSUT1 [Bürkle et al., 1998] and NtSUT3 [Lemoine et al., 1999]), potato (StSUT1 [Riesmeier et al., 1993]), tomato (LeSUT1, LeSUT2, and LeSUT4), Arabidopsis (AtSUC1, AtSUC2 [Sauer and Stolz, 1994], AtSUT2, and AtSUT4), and Schizosaccharomyces pombe (SpSUT1 [CAB16264]). Percentage bootstrap values of 1000 replicates are given at each branch point. Branch lengths (drawn in the horizontal dimension only) are proportional to phylogenetic distance.

Figure 2.

Genomic Structure of the SUT Loci. (A) Comparison of the genomic structure of LeSUT2 versus AtSUT2, showing the high conservation of the number of exons (exons 1–14). (B) DNA gel blot analysis of the LeSUTs under high stringency. Genomic DNA from tomato was digested with restriction enzymes, resolved on a 1% agarose gel (20 μg/lane), and blotted to Hybond N⁺. The gel at left shows hybridization with a ³²P-labeled 1.3-kb fragment of LeSUT1. The gel at right shows hybridization with a ³²P-labeled LeSUT2 cDNA.

Figure 3.

Expression of LeSUT1 and LeSUT2 in Yeast. (A) Expression of LeSUT1 and LeSUT2 in SUSY7/ura3. The dish at left shows that LeSUT1 functionally complements the yeast mutant, SUSY7/ura3 on sucrose (0.5%), whereas LeSUT2 reduced growth below that of the empty expression vector. At right, growth on glucose (2%). (B) Protein gel blot analysis of LeSUT1 and LeSUT2. At left is shown purified tomato source-leaf membranes (50 μg) resolved by electrophoresis. LeSUT1 was detected by using an affinity-purified antiserum against StSUT1. MM, microsomal membranes; IM, intracellular membranes; PM, plasma membranes. At right, LeSUT2 was expressed in SUSY7/ura3, and 50 μg of the total membrane protein was resolved by electrophoresis. LeSUT2 was detected as a doublet at 66 kD and was absent in yeast transformed with the empty expression vector.

Figure 4.

Specificity of the LeSUT2 Antiserum Shown by Yeast Immunofluorescence. LeSUT1, LeSUT2, or the empty vector pDR195 was expressed in SUSY7/ura3, the cells were fixed, and SUT proteins were detected with affinity-purified antisera. No cross-reactivity of the SUT1 antiserum on SUT2-expressing yeast or SUT2 antiserum on SUT1-expressing yeast was detected. No signals were detected on yeast expressing pDR195.

Figure 5.

Expression of SUT2. (A) Organ-specific expression of LeSUT1 and LeSUT2 (20 μg/lane) in stringent hybridization conditions. Probes were the full-length cDNA of LeSUT1 and a 1.3-kb fragment of LeSUT2. (B) Comparison of SUT1, SUT2, and patatin expression under different treatments in potato. SUT2 was specifically induced after treatment with sucrose (100 mM), whereas SUT1 expression remained unchanged. Both sucrose and to a lesser extent glucose (100 mM) induced patatin expression. Sorbitol also was used as an osmotic control at a concentration of 100 mM.

Figure 6.

Immunocytochemical Localization of SUT Proteins. (A) Immunofluorescent detection of SUT1 in enucleate sieve elements of tomato stems, colocalized with the aniline blue–stained callose in the sieve plate. DAPI-stained nuclei are visible in neighboring phloem cells. (B) SUT2 colocalized with aniline blue–stained sieve plate callose in enucleate sieve elements of stem. (C) and (D) Loss of LeSUT2 signal after incubation of SUT2 antiserum with its corresponding peptide (C). No reduction in signal after incubation of SUT2 antiserum with a control peptide (D). (E) No signals detected with preimmune serum; aniline blue–stained sieve plate callose indicates presence of sieve element. (F) No immunofluorescence labeling detectable in source-leaf minor veins of tomato plants treated with anti-LeSUT2 antiserum. (G) Bright-field view of the same section as in (F), showing minor vein anatomy. (H) LeSUT1 detectable in source-leaf minor veins on a serial section as in (F). (I) Source leaf of a transgenic Arabidopsis plant transformed with the 1.2-kb AtSUT2 promoter–GUS fusion construct. GUS staining is detectable in first-, second-, and third-order veins but not in minor veins. (J) Immunogold labeling of the sieve element plasma membrane in a petiole cross-section by using anti-LeSUT2 antibodies. (K) Control treated with IgG-enriched preimmune serum on a serial section as in (J). (H) ; (K) . n, nucleus; pp, phloem parenchyma cell; se, sieve element; sp, sieve plate; w, cell wall.

Figure 7.

Mapping of SUT Loci. cDNA clones StSUT1, LeSUT4, and LeSUT2 were used as RFLP markers in mapping population BC916² and identified SUT1 (StSUT1) on linkage group XI, SUT4 (LeSUT4) on linkage group IV, and SUT2 (LeSUT2) on linkage group V. Centimorgan distances between SUT loci and anchor RFLP markers (Gebhardt et al., 1999) are indicated to the left of the linkage groups. GP and CP markers are anonymous potato genomic and cDNA fragments, respectively. Small letters in parentheses indicate that more than one locus was identified by the marker probe. Functional gene markers (Gebhardt et al., 1999) are as follows: BE, branching enzyme; MBF, mas binding factor; StPto, Solanum tuberosum homolog of tomato resistance gene Pto; UGPase, UDP–glucose pyrophosphorylase. Positions of QTLs for tuber starch content [ts(i), ts(k), ts(n), ts(a)] and tuber yield [yi(a)], shown to the left of the linkage groups, are derived from Schäfer-Pregl et al. (1998), based on anchor RFLP markers with known genetic distance to QTL and SUT loci.