Sucrose (JrSUT1) and hexose (JrHT1 and JrHT2 ... - Tree Physiology

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218 DECOURTEIX ET AL. Fifteen-µm sections were rinsed in PBS (pH 7.2) and bathed for 15 min in PBS containing 0.1% (v/v) Triton X-100 and 0.2% (w/v) glycine (pH 7.2). After saturation of nonspecific sites, sections were incubated overnight with either 1/100 diluted antibody against JrSUT1, 1/5 diluted antibody against JrHT1 and JrHT2 or the same antibodies saturated with the KLH-coupled synthetic peptide. After washing, sections were incubated for 3 h with goat anti-rabbit antibodies conjugated to alkaline phosphatase (Sigma) diluted in blocking solution. Tissue sections were washed with Tris buffered saline and incubated with the chromogenic substrates nitro-blue tetrazolium chloride and 5-bromo-4-chloro-3′-indolyphosphate p-toluidine salt (BioRad). Color development was carried out at room temperature and was stopped by washing in H2O. Statistical analysis Means and standard errors were calculated from replicates. Date and sugar effects were evaluated by analysis of variance (ANOVA) and by rank and Duncan tests (at 5%). Results Starch and sugar in xylem parenchyma cells From March to May, mean starch concentration was 1.8- to 2.5-fold higher in apical stem segments than in basal stem segments of 1-year-old shoots, but there was no difference in June (Figure 1). The seasonal pattern in starch concentration was similar in the apical and basal stem segments. Starch concentration increased by a factor of two in early spring (March– April), reached a maximum in April and decreased to a minimum in June. Starch concentration was around ten- and fourfold higher in April than in June, in the apical and basal stem segments, respectively. Except in June, starch concentrations were higher than soluble sugar concentrations. In March and April, mean soluble sugar concentration in xylem parenchyma cells was at a maximum, and was 1.7-fold higher in apical stem segments than in basal stem segments (Figure 1). Xylem sap sugar concentrations and uptake at bud break Mean total concentrations of sucrose and hexose in the xylem sap were 2.5-fold higher in apical stem segments than in basal stem segments of the 1-year-old shoots (2.3 versus 0.9 mg ml –1 , respectively; P < 0.05) (Figure 2A). Mean sucrose concentration was about 4-fold higher in apical stem segments than in basal stem segments, whereas hexose concentrations were similar in both (Figure 2A). Active sucrose uptake was estimated at +15 °C by subtracting passive sugar uptake (in the presence of HgCl2 ) from total sugar uptake (in the absence of HgCl2 ). At the start of bud break, mean active sucrose uptake in apical stem segments was 4.3-fold higher than in basal stem segments (9.5 versus 2.2 nmol g DM –1 h –1 ; P < 0.001) (Figure 2B). In contrast to sucrose, there was little active glucose uptake under the same experimental conditions. TREE PHYSIOLOGY VOLUME 28, 2008 Figure 1. Changes in starch and soluble sugar (GFS = glucose + fructose + sucrose) concentrations in xylem parenchyma cells of the apical and basal segments of 1-year-old walnut shoots. Values are means ± SE of at least three replicates. JrHT1 and JrHT2: putative hexose transporters To obtain putative hexose transporter cDNA from xylem tissue, conserved domains of known hexose transporter sequences from different species were used to design degenerate primers for RT-PCR. An amplified cDNA fragment of about 460 bp was sequenced and found to be homologous to hexose Figure 2. (A) Xylem sap sucrose (filled bars) and hexose (glucose + fructose; open bars) concentrations in apical and basal segments of 1-year-old walnut shoots at the end of April (onset of bud break). Values are means ± SE of five replicates. (B) Active sucrose uptake by parenchyma cells in the apical and basal segments of 1-year-old walnut shoots estimated by subtracting passive uptake (in the presence of HgCl2 ) from total uptake (in the absence of HgCl2 ) of sucrose. Values are means + SE of at least three replicates. Downloaded from http://treephys.oxfordjournals.org/ by guest on December 27, 2012
SUCROSE AND HEXOSE TRANSPORTERS IN WALNUT XYLEM PARENCHYMA 219 transporters from other species. Thus, it has been used as a homologous probe to screen a xylem-tissue-derived cDNA library (Sakr et al. 2003). Two full-length cDNA clones encoding putative hexose transporters, JrHT1 and JrHT2, were isolated. The complete nucleotide sequences of the clones JrHT1 and JrHT2 contained an open reading frame of 1563 and 1524 bp, respectively, and had 98% sequence similarity. JrHT1 and JrHT2 encode 521 and 508 amino acid polypeptides, respectively. The putative amino acid sequence of JrHT1 was aligned with the deduced protein sequences of known hexose transporters (Figure 3A). The identity between JrHT1 and other hexose transporters ranges from 51 to 85%, and the closest identity was found with the RcSCP from Ricinus communis L. (Weig et al. 1994). Expression analysis of these putative hexose transporters in different organs was carried out by a semi-quantitative RT- PCR approach, with RNAs extracted from leaf, fine root, xylem, bark and flowers (male and female). Because the primer pair (Hex11/Hex12) is common to JrHT1 and JrHT2, the amplified band was named JrHT1 and JrHT2. JrHT1 and JrHT2 transcripts were detected in all of the tested organs, including the xylem tissue (Figure 3B), contrasting with the sink-specific expression of a cDNA encoding a putative sucrose transporter isolated by Decourteix et al. (2006) from walnut xylem tissue (JrSUTs; Figure 3B). No significant difference in transcript abundance of JrHT1 and JrHT2 was found between source leaf and sink organs (xylem, fine root and bark). The lowest abundance of JrHT1 and JrHT2 was in male flowers. Transcript patterns of JrSUT1 and JrHT1 and JrHT2 during the growing season The relative transcript abundance of JrHT1 and JrHT2 and a putative sucrose transporter JrSUT1 previously isolated from walnut xylem tissue by Decourteix et al. (2006) was examined by semi-quantitative RT-PCR as previously applied to walnut xylem tissue (Sakr et al. 2003, Decourteix et al. 2006). As shown in Figure 4A, the apical and basal stem segments exhibited a contrasting pattern of JrSUT1 transcript abundance. In the apical segment, JrSUT1 transcripts were abundant in March and April, whereas they were undetected in May and June. In the basal segment, the transcript level of JrSUT1 was high in April and June and low in March and May. The pattern of JrHT1 and JrHT2 transcripts differed from that of JrSUT1 and between the apical and basal stem segments (Figure 4B). In the apical segment, JrHT1 and JrHT2 transcripts were slightly more abundant in April and May than in March and June. In the basal segment, JrHT1 and JrHT2 transcripts increased from March to May, but decreased in June. Accumulation and localization of sugar transporters during the growing season The affinity-purified anti-JrSUT1 antibody labeled a single band of 54 kDa on immunoblots, which is consistent with the molecular mass of several sucrose transporters (Lemoine 2000). In the apical segment, band intensity was highest in TREE PHYSIOLOGY ONLINE at http://heronpublishing.com Figure 3. (A) Comparison of the deduced amino acid sequence of JrHT1 with those of other hexose transporters from mono- and dicotyledonous plants. The dendrogram was generated by comparison of the sequences by the CLUSTAL W 1.74 software (Higgins et al. 1994). VvHT2 (Accession no. AAT77693); AtSTP1 (CAA39037); AtSTP4 (BAB01308); VfSTP1 (CAB07812); AtSTP9 (NP175449); AtSTP5 (CAC69071); AtSTP6 (CAC69073); AtSTP11 (CAC- 69075); tMST2.1 (CAG27605); tMST2.2 (CAG27609); tMST3.1 (CAG27606); ZmMST1 (AAT90503); PaMST1 (CAB06079); PMT1 (AAC61852); LeHT2 (CAB52689); VvHT1 (CAA04511); RcSTC (AAA79761); OsMST3 (BAB19864). Percentage identity is noted on the right. (B) Expression patterns of JrHTs and JrSUTs in various organs of walnut tree. Semi-quantitative reverse transcriptase polymerase chain reactions (RT-PCRs) were performed with either Sut1A/Sut1B for JrSUTs, Hex11/Hex12 for JrHTs or Act11/Act12 for JrAct. At least four RT-PCRs were conducted on each of the two biological replicates. All yielded similar results to those shown. March (early spring) and April (beginning of bud break), whereas no labeled band was found in May and June (Figure 5A). In the basal segment, the band increased in intensity from March to April and disappeared in May and June. In March and April, there was more JrSUT1 protein in xylem parenchyma cells of the apical than of the basal stem segments (Figure 5A). Downloaded from http://treephys.oxfordjournals.org/ by guest on December 27, 2012
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