XB-ART-54872
Front Plant Sci
2018 Mar 26;9:430. doi: 10.3389/fpls.2018.00430.
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Protoplast-Esculin Assay as a New Method to Assay Plant Sucrose Transporters: Characterization of AtSUC6 and AtSUC7 Sucrose Uptake Activity in Arabidopsis Col-0 Ecotype.
Rottmann TM
,
Fritz C
,
Lauter A
,
Schneider S
,
Fischer C
,
Danzberger N
,
Dietrich P
,
Sauer N
,
Stadler R
.
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The best characterized function of sucrose transporters of the SUC family in plants is the uptake of sucrose into the phloem for long-distance transport of photoassimilates. This important step is usually performed by one specific SUC in every species. However, plants possess small families of several different SUCs which are less well understood. Here, we report on the characterization of AtSUC6 and AtSUC7, two members of the SUC family in Arabidopsis thaliana. Heterologous expression in yeast (Saccharomyces cerevisiae) revealed that AtSUC6Col-0 is a high-affinity H+-symporter that mediates the uptake of sucrose and maltose across the plasma membrane at exceptionally low pH values. Reporter gene analyses revealed a strong expression of AtSUC6Col-0 in reproductive tissues, where the protein product might contribute to sugar uptake into pollen tubes and synergid cells. A knockout of AtSUC6 did not interfere with vegetative development or reproduction, which points toward physiological redundancy of AtSUC6Col-0 with other sugar transporters. Reporter gene analyses showed that AtSUC7Col-0 is expressed in roots and pollen tubes and that this sink specific expression of AtSUC7Col-0 is regulated by intragenic regions. Transport activity of AtSUC7Col-0 could not be analyzed in baker's yeast or Xenopus oocytes because the protein was not correctly targeted to the plasma membrane in both heterologous expression systems. Therefore, a novel approach to analyze sucrose transporters in planta was developed. Plasma membrane localized SUCs including AtSUC6Col-0 and also sucrose specific SWEETs were able to mediate transport of the fluorescent sucrose analog esculin in transformed mesophyll protoplasts. In contrast, AtSUC7Col-0 is not able to mediate esculin transport across the plasma membrane which implicates that AtSUC7Col-0 might be a non-functional pseudogene. The novel protoplast assay provides a useful tool for the quick and quantitative analysis of sucrose transporters in an in planta expression system.
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FIGURE 1. RT-PCR based expression analysis of AtSUC6Col-0 and AtSUC7Col-0 in pollen tubes. Comparison of AtSUC6Col-0 and AtSUC7Col-0 expression in in vitro germinated pollen tubes, pollen tubes grown through the stigma (semi-in vivo) and virgin stigmata with gene specific primers (Supplementary Table 1). Arrows indicate the size of PCR products derived from reverse-transcribed mRNA (white) and genomic DNA (black). The presence of cDNA in each sample was confirmed with ACTIN2 specific primers (Supplementary Table 1). | |
FIGURE 2. Analysis of pAtSUC6:AtSUC6g-reporter plants and subcellular localization of AtSUC6Col-0. (A–L) Histochemical detection of β-glucuronidase activity in Arabidopsis Col-0 expressing AtSUC6g-GUS under the control of the native AtSUC6Col-0 promoter. (A) Two-week-old seedling with GUS staining in the vascular tissue of roots, hypocotyl and leaves. (B) Lateral root tips of a 2-week-old seedling. (C) Root tip with GUS activity in the protophloem at higher magnification. (D) Rosette leaf. (E) GUS staining in the vasculature of the root differentiation zone. (F) Patchy GUS pattern in the vasculature of a source leaf. (G) Unpollinated flower in early stage 13 (all flower stages according to Smyth et al., 1990) with GUS signal in the ovules. (H) Pollinated flower of stage 14. (I) Peeled ovary of a stage-14 flower. (J) Pollen tubes grown semi-in vivo through a WT stigma and a part of the transmitting tract. (K) Pollen tubes germinated semi-in vivo on a WT stigma. (L) Pollen tubes at higher magnification. (M–R) Detection of GFP fluorescence (green) in pAtSUC6:AtSUC6g-GFP reporter plants. Chlorophyll autofluorescence is given in red. (M) Peeled ovary with GFP fluorescence in pollen tubes. (N) Pollen tubes growing in the transmitting tract of a peeled ovary. (O) Tip of a pollen tube grown semi-in vivo. (P) Bright field image of (O). (Q) Maximum projection of an excised ovule stained with propidium iodide (red) with GFP fluorescence in the synergids. (R) Synergid cells at higher magnification. (S,T) Single optical section (S) and maximum projection (T) of a protoplast expressing AtSUC6c-GFP under the control of the 35S promoter. Scale bars: 2.5 mm in (A,D); 100 μm in (B,M); 50 μm in (C,E,I,J,K,N,Q); 1 mm in (F); 500 μm in (G,H); 10 μm in (L,O,P,R); 5 μm in (S,T). | |
FIGURE 3. Analysis of pAtSUC7:AtSUC7g-reporter plants and subcellular localization of AtSUC7Col-0. (A–N) Histochemical detection of β-glucuronidase activity in Col-0 expressing pAtSUC7:AtSUC7g-GUS. (A) Five-day-old seedling with GUS staining in the main root (arrowhead). (B) Two-week-old seedling with GUS activity in roots and stipules (arrowhead). (C) Main root with lateral root primordium. (D) Tip of the main root at higher magnification. (E) Root cross sections in the differentiation (left) and elongation zone (right). (F) Stipules of a 2-week old seedling. (G) Inflorescence with flowers of different developmental stages. (H) Unpollinated flower. (I) Pollinated flower. (J,K) Peeled ovary of unpollinated (J) or pollinated (K) flower. (L,M) Pollen tubes grown semi-in vivo through a WT stigma with (L) or without (M) a part of the ovary. (N) Pollen tubes grown semi-in vivo at higher magnification. (O) GFP fluorescence (green) of pollen tubes in a peeled ovary of a pAtSUC7:AtSUC7g-GFP reporter plant. Chlorophyll autofluorescence is given in red. (P,Q) Single optical section (P) and maximum projection (Q) of an Arabidopsis mesophyll protoplast expressing GFP-AtSUC7 under the control of the 35S promoter. Scale bars: 1 mm in (A,B,D); 50 μm in (C,D,E,L,M,O); 100 μm in (F,J,K); 500 μm in (G–I); 20 μm in (N); 5 μm in (P,Q). | |
FIGURE 4. Analysis of AtSUC7Col-0 and AtSUC6Col-0 transport properties in transgenic baker’s yeast. (A) Uptake analysis of 14C-sucrose into yeast strains TRY1002 (black triangles) or TRY1001 (gray circles) expressing AtSUC7cCol-0 in sense or antisense (as) orientation, respectively, per ml packed cells (p.c.) at an initial outside concentration of 100 μM sucrose at pH 5.5. Yeast strain SEY2102 expressing the sucrose transporter Srt1 was used as a positive control for sucrose uptake (black circles). (B) Uptake of 14C-sucrose into AtSUC6Col-0 strain TRY1039 (circles) and AtSUC6Col-0-antisense (as) control strain TRY1040 (triangles) per ml packed cells (p.c.) at an initial outside concentration of 100 μM sucrose at pH 5.5. (C) For the calculation of the KM value of AtSUC6Col-0 for sucrose uptake according to Lineweaver–Burk uptake rates of TRY1039 for increasing concentrations of 14C-sucrose were determined. The plot represents mean values ± standard deviations of three biological replicates for each sucrose concentration. (D) Uptake rates of AtSUC6Col-0 for 14C-sucrose at different pH values at an initial outside concentration of 100 μM sucrose. (E) Analysis of AtSUC6Col-0 substrate specificity and sensitivity to uncouplers. Binding capacity of AtSUC6Col-0 for different sugars was determined by competitive inhibition of 14C-sucrose uptake (100 μM initial outside concentration) in the presence of non-radioactive sugars in 10-fold excess at pH 5.5. Addition of 1-mM cold sucrose was used as a control. CCCP was added to a final concentration of 50 μM. Means ± standard errors (SEs) of three independent biological replicates are shown. ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001 by Student’s t-test. | |
FIGURE 5. Control experiments for the establishment of a protoplast esculin assay. (A–H,J) Protoplasts expressing different GFP-fusion constructs were incubated with 1 mM esculin in W5 buffer (pH5.6) for 40 min. GFP is given in green, esculin fluorescence in cyan and chlorophyll autofluorescence in red. (A) Overview image of Col-0 protoplasts transformed with p35S:AtSUC2c-GFP. (B) Bright field to (A). (C) Col-0 protoplast expressing p35S:GFP-STP10c. (D) Individual protoplast expressing p35S:AtSUC2c-GFP with esculin in the vacuole at higher magnification. (E) Protoplast of a Attmt1/tmt2 knockout-plant transformed with p35S:AtSUC2c-GFP. (F) Col-0 protoplasts with p35S:AtSUC2c-GFP. The arrowhead indicates a small non-transformed protoplast showing esculin uptake. (G) Companion cell protoplast of a pEPS1 line (stably transformed with pAtSUC2:GFP), labeled by cytosolic GFP. (H) GFP-labeled companion cell protoplast of pMH5a (stably transformed with a pAtSUC2:erGFP construct). (I) Leaf epidermal peel of Col-0 incubated with 1 mM esculin in W5 for 1 h. Overlay of bright field, esculin and chlorophyll fluorescence. The arrowhead points to a guard cell, the asterisk marks a neighboring subsidiary cell with esculin fluorescence. (J) WT protoplasts expressing p35S:AtSUC2c-GFP at different levels. (K) Correlation of GFP fluorescence in the plasma membrane and esculin fluorescence intensity in the vacuole of protoplasts transformed with p35S:AtSUC2c-GFP. Fluorescence intensities were normalized to the radius of the respective protoplast under the assumption of a spherical shape (n = 115). Scale bars: 50 μm in (A,B), 10 μm in (C–J). | |
FIGURE 6. Esculin protoplast assay for SWEET transporters. Confocal images of protoplasts transformed with p35S:SWEET10c-GFP (A) or p35S:SWEET4c-GFP (B) and incubated with 1 mM esculin in W5 buffer (pH5.6) for 40 min. Left to right: overview image, single section and maximum projection without esculin detection channel of individual protoplasts. GFP is given in green, esculin fluorescence in cyan and chlorophyll autofluorescence in red. Scale bars: 20 μm in overview images, else: 10 μm. | |
FIGURE 7. Esculin uptake into protoplasts via different AtSUCs. (A–H) Confocal images of Arabidopsis mesophyll protoplasts expressing GFP fusions of different AtSUCs under the control of the 35S promoter as indicated. Protoplasts were incubated with 1-mM esculin for 40 min. Upper part: overview image. Bottom part: single optical sections of individual protoplasts. GFP is given green, esculin fluorescence in cyan and chlorophyll autofluorescence in red. Scale bars: 25 μm in overview images, 5 μm in single protoplast images. | |
FIGURE 8. Protoplast esculin uptake assay for AtSUC sequence variants. (A–J) Confocal images of protoplasts transformed with GFP fusion constructs of AtSUC7 wild type sequences of ecotypes Col-0 (A) or Ws (F), or point mutated sequences of AtSUC7Col-0 (B–E), AtSUC5Col-0 (G,H) or AtSUC2Col-0 (I,J) as indicated. GFP is given in green, esculin in cyan and chlorophyll autofluorescence in red. (E,H,J) Maximum projections without esculin detection channel. Scale bars: 10 μm. (K,L) Correlation of GFP fluorescence in the plasma membrane and esculin fluorescence intensity in the vacuole of protoplasts transformed with p35S:GFP-AtSUC9 (K) or p35S:GFP-AtSUC7P67S/R436G (L). Protoplasts were incubated for 30 min with 1 mM esculin only (blue) or with 1 mM esculin in the presence of sucrose in 10-fold excess (orange). Fluorescence intensities were normalized to the radius of the respective protoplast under the assumption of a spherical shape (n > 19 protoplasts for each measurement). | |
FIGURE 9. Identification and characterization of Atsuc6 T-DNA insertion lines. (A) Genomic organization of AtSUC6. Introns and untranslated regions are shown as black lines; exon regions containing coding sequences are represented by numbered gray bars. Arrows indicate the primers used for PCRs shown in (B,C). The positions of the T-DNA insertions Atsuc6.1–Atsuc6.4 are marked. LB, left border; RB, right border. (B) PCR products obtained from genomic DNA preparations of homozygous Atsuc6.3 and WT plants with primer combinations for the detection of wild type (WT) and the mutant allele (m). For primer combinations see Supplementary Table 2. (C) RT-PCR analyses of RNA obtained from flowers of a homozygous Atsuc6.3 mutant plant and a WT plant with primers amplifying either the AtSUC6 sequence traversing, upstream of or downstream of the insertion (Supplementary Table 3). WT genomic DNA was used as control for contaminations with genomic DNA. (D) Length of main roots of 14-day-old Atsuc6.3 and WT seedlings on MS-0 or MS medium supplemented with 2% (w/v) sucrose. Means of three biological replicates ± SD are shown. n > 30 for each genotype. (E) Average number of seeds/silique ± SD of Atsuc6.3 and WT plants after self-pollination. n > 50 siliques/genotype. (F) Genotypes of F1 descendants of a cross-pollination experiment with heterozygous Atsuc6.3/AtSUC6 pollen and pistils from a WT plant. Bars represent mean values (±SE) of WT and heterozygous plants in the F1 generation resulting from eight independent crossings (n = 76 in total). | |
FIGURE 10. Identification and characterization of Atsuc7 T-DNA insertion lines. (A) Genomic organization of AtSUC7. Introns and untranslated regions are shown as black lines; exon regions containing coding sequences are represented by numbered gray bars. Arrows indicate the primers used for PCRs shown in (B,C). The positions of the insertion in the T-DNA lines are marked. LB, left border; RB, right border. (B) PCR products obtained from genomic DNA preparations of homozygous Atsuc7.3 and WT plants. Primer combinations for the detection of wild type (WT) and mutant allele (m) are listed in Supplementary Table 2. (C) RT-PCR analyses of pollen tube RNA obtained from a homozygous Atsuc7.3 mutant and a WT plant with primers (Supplementary Table 3) amplifying either the AtSUC7 sequence traversing, upstream of or downstream of the insertion. WT genomic DNA was used a control for genomic contaminations. (D) Length of main roots of 12-day-old Atsuc7.3 and WT seedlings on MS-0 or MS medium supplemented with 2% (w/v) sucrose. Means of five biological replicates ± SE are shown. n > 50 for each genotype. (E) Average number of seeds/silique ± SD of Atsuc7.3 and WT plants after self-pollination. n > 55 siliques/genotype. (F) Pollen tube lengths of WT and Atsuc7.3 in vitro. Pollen were grown on medium with different sucrose concentrations for 8 h. Bars represent mean values of three biological replicates ± SE (n > 500 in total per sucrose concentration for each genotype). (G) Genotypes of F1 descendants of a cross-pollination experiment with heterozygous Atsuc7.3/AtSUC7 pollen and pistils from a WT plant. Bars represent mean values (±SE) of WT and heterozygous plants in the F1 generation resulting from seven independent crossings (n = 89 in total). |
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