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

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220 DECOURTEIX ET AL. Figure 4. Expression patterns of JrSUT1 and JrHT1 and JrHT2 in xylem parenchyma cells in apical and basal segments of walnut stems harvested from March to June. Semi-quantitative reverse transcriptase polymerase chain reactions (RT-PCRs) were performed with either (A) Sut11/Sut12 for JrSUT1 or (B) Hex11/Hex12 for JrHT1 and JrHT2. (C) JrEF1 was used as control. At least four RT-PCRs were conducted on each of two biological replicates, yielding similar results to those presented here. The affinity-purified anti-JrHT1 and JrHT2 antibody yielded only a single band of 52 kDa, which is comparable to the molecular mass of other hexose transporters (Williams et al. 2000, Vignault et al. 2005). The protein pattern of JrHT1 and JrHT2 contrasted with that of JrSUT1 (Figure 5B). In the apical xylem parenchyma cells, JrHT1 and JrHT2 were barely detectable in March, and only a faint signals were observed in April and June. The greatest amount of JrHT1 and JrHT2 was found in May, coinciding with a period of intense radial growth in walnut (Daudet et al. 2005). In the basal xylem parenchyma cells, no significant signal was found, except in May, when JrHT1 and JrHT2 were abundant. In May, JrHT1 and JrHT2 were more abundant in the basal than in the apical stem segment. Immunolocalization analysis of the hexose transporters was performed in sections harvested from apical and basal stem segments of 1-year-old stems at the end of April (beginning of bud break). No signal was detected when the immunolocalization was performed either with the saturated primary antibody (data not shown) or in the absence of the primary antibody (Figures 6B, 6D, 6F and 6H). For JrSUT1, immunolabeling was limited to the living cells of the xylem in both apical (Figure 6A) and basal (Figure 6C) stem segments. It was strongly detected in VACs, which are highly specialized in nutrient transports, and in both axial and ray parenchyma cells (Figures 6A and 6C). Similar patterns were found for JrHT1 and JrHT2. The immunolabelling was detected in living cells including axial and ray parenchyma cells and VACs (Figures 6E and 6G). Total transport of sucrose and glucose from xylem vessels to bud cells The sucrose transporter JrSUT1 accumulated preferentially in the apical segment of the walnut shoot and was more highly expressed than the glucose transporters JrHT1 and JrHT2 (Figures 4 and 5) at the beginning of bud break in April. TREE PHYSIOLOGY VOLUME 28, 2008 Figure 5. Immunoblot analyses of microsomal fractions of xylem parenchyma cells in the apical and basal segments of walnut stems harvested from March to June and incubated with either (A) the affinity-purified anti-JrSUT1 antibody or (B) the affinity–purified anti-JrHT1 and anti-JrHT2 antibody. Four independent experiments yielded similar results to those presented here. Therefore, we checked whether the accumulation pattern of sugar transporters in xylem tissues and the import of sugar into vegetative buds was coordinated. At the beginning of bud break, sucrose influx was more than 14-fold higher into buds of the apical stem segment than the basal stem segment (1453 and 104 nmol g DM –1 h –1 , respectively) and was more than 70-fold higher than glucose influx for both locations (18.7 and 1.39 in buds of the apical and basal stem segments, respectively; Table 1). Discussion In temperate woody species, bud break marks the beginning of vegetative growth, which is characterized by intense physiological activity that creates a high demand for soluble carbohydrates (Maurel et al. 2004). Based on a double-labeling experiment with walnut trees, Lacointe et al. (2004) reported that the greatest consumption of labeled reserves occurred in spring between the onset of bud break (end of April) and the acquisition of autotrophy for carbon by new shoots (mid-June). In keeping with these findings, our data show that starch stored in stem xylem parenchyma cells was hydrolyzed during the same period (Figure 1). In poplar, a strong and rapid mobilization of starch reserves has been reported during bud break and has been related to activation of amylases (Witt and Sauter 1994). In parallel with the breakdown of starch, soluble sugar concentrations (sucrose, glucose and fructose) in xylem parenchyma cells declined significantly (Figure 1). These findings differ from those reported for the rest period (Sauter and Kloth 1987, Sauter 1988, Améglio et al. 2000) and are consistent with increased consumption of starch-derived carbohydrates during early vegetative growth. Recently, JrSUT1, a putative stem-specific sucrose transporter, has been characterized in walnut xylem parenchyma cells, and has been implicated in sucrose retrieval from xylem sap during winter (Decourteix et al. 2006). To identify hexose transporters expressed in the xylem tissue, we screened a xylem cDNA library with a homologous probe and isolated two putative hexose transporters designated JrHT1 and JrHT2 (Juglans regia hexose transporters 1 and 2). These transporters cluster only with plant hexose or monosaccharide transporters Downloaded from http://treephys.oxfordjournals.org/ by guest on December 27, 2012
SUCROSE AND HEXOSE TRANSPORTERS IN WALNUT XYLEM PARENCHYMA 221 (Figure 3A), and the highest identity (85%) was with RcSCP (Weig et al. 1994). JrHT1 and JrHT2 had all the characteristics of monosaccharide transporters including 12 transmembrane-spanning domains and the conserved V/ LPETK or Table 1. Capacities of buds of apical and basal stem segments of 1-year-old walnut stems to import sucrose and glucose during bud break. Values are means ± SE of at least five replicates. Stem segment Sucrose import Glucose import (nmol g DM –1 h –1 ) (nmol g DM –1 h –1 ) Apical 1453.5 ± 217.8 18.7 ± 8.1 Basal 103.7 ± 23.9 1.4 ± 0.4 TREE PHYSIOLOGY ONLINE at http://heronpublishing.com Figure 6. Transverse sections showing the general distribution of JrSUT1 and JrHT1 and JrHT2 in the xylem parenchyma cells of walnut stem. Immunolabeling of JrSUT1 in living cells of the (A) apical and (C) basal stem segments and immunolabeling of JrHT1 and JrHT2 in living cells of the (E) apical and (G) basal stem segments. No labeling was detected in the controls (B, D, F, H). Abbreviations: A, axial parenchyma cell; F, fiber; R, ray parenchyma cell; V, vessel; and VAC, vessel associated cell. Scale bars equal 25 µm. (R/K)XGR(R/K) motifs, and JrHT1 and JrHT2 transcripts were found in both source and sink organs, as is the case for other hexose or monosaccharide transporters (e.g., LeHT2, Gear et al. 2000; AtSTP4, Trüernit et al. 1996; or PaMST1, Nehls et al. 2000). During early spring, walnut xylem sap is considered the primary route by which soluble sugars are transported from exporting organs to recipient sinks, mainly the buds and possibly the cambium (Lacointe et al. 2001, 2004). This was verified by our experiments, which showed a large amount of radiolabeled sugar, perfused into xylem vessels, moved to vegetative bud cells (Table 1). At bud burst, bud vascular tissue includes only protoxylem, so that, to reach the meristematic zone, sugars must pass by the VACs, which control nutrient exchanges between xylem vessels and parenchyma cells (Fromard et al. Downloaded from http://treephys.oxfordjournals.org/ by guest on December 27, 2012
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Sucrose (JrSUT1) and hexose (JrHT1 and JrHT2 ... - Tree Physiology

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