Tree Physiology 28, 215–224
© 2008 Heron Publishing—Victoria, Canada
Sucrose (JrSUT1) and hexose (JrHT1 and JrHT2) transporters in
walnut xylem parenchyma cells: their potential role in early events of
growth resumption
MÉLANIE DECOURTEIX,1 GEORGES ALVES,1 MARC BONHOMME,2 MEDERIC PEUCH,2
KHAOULA BEN BAAZIZ,1 NICOLE BRUNEL,1 AGNÈS GUILLIOT,1 RÉMY RAGEAU,2
THIERRY AMÉGLIO,2 GILLES PÉTEL1 and SOULAÏMAN SAKR1,3
1
UMR 547-PIAF, site des Cézeaux, Université Blaise Pascal, 24 avenue des Landais, 63170 Aubière, France
2
UMR 547-PIAF, site INRA de Crouelle, 234 avenue du Brézet, 63100 Clermont-Ferrand, France
3
Corresponding author ([email protected])
Received July 12, 2006; accepted April 23, 2007; published online December 3, 2007
Summary In temperate woody species, the vegetative
growth period is characterized by active physiological events
(e.g., bud break), which require an adequate supply of soluble
sugars imported in the xylem sap stream. One-year-old shoots
of walnut (Juglans regia L. cv. ‘Franquette’) trees, which have
an acrotonic branching pattern (only apical and distal vegetative buds burst), were used to study the regulation of xylem
sugar transporters in relation to bud break. At the end of April
(beginning of bud break), a higher xylem sap sucrose concentration and a higher active sucrose uptake by xylem parenchyma cells were found in the apical portion (bearing buds able
to burst) than in the basal portion (bearing buds unable to burst)
of the sample shoots. At the same time, xylem parenchyma
cells of the apical portion of the shoots exhibited greater
amounts of both transcripts and proteins of JrSUT1 (Juglans
regia putative sucrose transporter 1) than those of the basal
stem segment. Conversely, no pronounced difference was
found for putative hexose transporters JrHT1 and JrHT2 (Juglans regia hexose transporters 1 and 2). These findings demonstrate the high capacity of bursting vegetative buds to import
sucrose. Immunological analysis revealed that sucrose transporters were localized in all parenchyma cells of the xylem, including vessel-associated cells, which are highly specialized in
nutrient exchange. Taken together, our results indicate that xylem parenchyma sucrose transporters make a greater contribution than hexose transporters to the imported carbon supply of
bursting vegetative buds.
Keywords: branching, bud break, cambium, influx, Juglans
regia, vessel-associated cells, walnut tree.
Introduction
The bursting vegetative bud is a photosynthetically inactive
sink, so it must import soluble sugars to meet its carbon and
energy demands (Quinlan 1969, Kelner et al. 1993, Maurel et
al. 2004). Before the formation of an efficient photosynthetic
apparatus in spring, the vegetative bud grows mainly at the expense of carbohydrate reserves. Loescher et al. (1990) proposed that xylem sap is the main route by which reserve-derived soluble sugars are transported to different sink organs
during the leafless period. This implies: (1) mobilization of
carbohydrate reserves in different storage compartments of the
tree (Loescher et al. 1990, Witt and Sauter 1994, Lacointe et
al. 2004); (2) loading and long-distance transport of soluble
sugars into xylem vessels (Bollard 1960, Sauter 1980, Lacointe et al. 2001); and (3) unloading into the parenchyma
cells near the recipient sink. Hence, soluble sugars must be actively absorbed from xylem sap by vessel-associated cells
(VACs) before delivery to recipient organs. The presence of
VACs with sugar transporters can, thus, determine the intensity of carbon supply to sink organs.
Despite the need for soluble sugars imported from the xylem sap stream for early events of bud break (Sauter 1980,
1981, Sauter and Ambrosius 1986, Lacointe et al. 2001,
Maurel et al. 2004), direct information about soluble sugar
transporters in xylem tissue is lacking. In Robinia pseudoacacia L., study of the physiological characteristics of sugar
influx in xylem parenchyma cells indicated the involvement of
an H+/sucrose co-transporter during the resumption of vegetative growth (Fromard 1990). In contrast, in the xylem parenchyma cells of Populus, the operation of an H+/hexose cotransporter has been proposed (Himpkamp 1988). Few sugar
transporters have been cloned from woody species to date.
Some, such as BpSUC1 from birch root (Betula pendula Roth;
Wright et al. 2000), SUT1 and 2 from citrus root (Citrus sp.; Li
et al. 2003) and VvSUTs from berry grape (Vitis vinifera L.;
Davies et al. 1999, Ageorges et al. 2000), share characteristics
with those identified in herbaceous species. Others, such as
VvHT1 from berry grape (Fillion et al. 1999, Vignaut et al.
2005) and BpHEX1 and BpHEX2 from birch root (Wright et
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DECOURTEIX ET AL.
al. 2000), are highly homologous with the already isolated
hexose transporters.
To date, the only sugar co-transporter gene identified from
xylem tissue of a woody species is the Juglans regia L. putative sucrose transporter (JrSUT1), which encodes a putative
stem-specific sucrose transporter (Decourteix et al. 2006). Its
role has been investigated only in the autumn–winter period,
when it appears to mediate the influx of sucrose released into
the xylem sap after freeze/thaw events (Decourteix et al.
2006). Although the physiological significance of some sugar
transporters has been investigated, their involvement in vegetative bud burst has not been studied. However, because the
plasma membrane H+-ATPase (PM-H+-ATPase) energizes a
large set of secondary transporters, such as sugar transporters,
studies have focused mainly on this protein (Fromard et al.
1995, Arend et al. 2002, 2004, Alves et al. 2004). Plasma
membrane H+-ATPase is reported to be seasonally regulated in
walnut xylem tissue, because its amount and activity are
higher when bud break occurs at the end of April, than in February (Alves et al. 2004, 2007). These findings support the
idea that PM-H+-ATPase contributes to the early events of bud
break by permitting sugar uptake from xylem sap, presumably
via H+ /sugar co-transporters.
Our study focused on the characterization of putative sugar
transporters in xylem tissues during the first stages of the
growing season, with particular attention to the early events of
bud break. Walnut (J. regia) cv. ‘Franquette’ was chosen as the
study species because it exhibits an acrotonic branching pattern (the apical part of the shoot bears buds that are able to
grow, whereas the basal part of the shoot bears buds that do not
normally grow). Because sucrose and hexoses (glucose and
fructose) comprise 95% of the total soluble sugars in walnut
xylem sap during bud break (Alves 2003), the possible involvement of their respective transporters was investigated
based on their patterns of activity and accumulation during the
study period.
Materials and methods
Plant material
Thirteen-year-old walnut trees (J. regia) cv. ‘Franquette’
grown in an INRA orchard near Clermont-Ferrand (south-central France, 45° N, 3° E) were selected for study. One-year-old
shoots were collected at the end of each month, from March
(early spring) to June (acquisition of autotrophy for carbon by
new shoots). Segments were sampled 10 cm below the apical
vegetative bud (apical stem segment) and 10 cm above the
lowermost portion of the shoot (basal stem segment), and the
xylem tissue collected as described by Alves et al. (2007).
Starch and soluble sugar in xylem parenchyma cells
Soluble sugars were extracted from the xylem tissue with
80:20 (v/v) hot (80 °C) ethanol:water and purified as described by Moing and Gaudillière (1992). Glucose, fructose
and sucrose concentrations were determined spectrophotometrically at 340 nm after enzymatic assays (Boehringer
1984). Starch concentration was determined by a hexokinasecatalyzed glucose-6-phosphate linked assay (Kunst et al.
1984) after hydrolysis of the sample with amyloglucosidase
(Boehringer 1984).
Xylem sap sugar concentration
Immediately after stem excision, xylem sap was collected separately from the apical and basal portions of the excised shoots
by vacuum extraction, according to the method of Bollard
(1953). To avoid contamination of extracted xylem sap by
phloem sap, the bark was scraped to a distance of about 3 cm
from each end of the shoots. Following extraction, the xylem
sap was heated to 80 °C for 20 min, and its sugar content determined as described by Maurel et al. (2004).
Uptake of sucrose and glucose by xylem parenchyma cells
Twenty-cm-long segments were cut from the apical and basal
ends of 1-year-old stems preconditioned at 15 °C for 48 h in a
temperature-controlled chamber (Liebherr 650l). Tissues outside the cambium were scraped from the ends of the segments
to prevent phloem constituents entering the xylem at the ends
of the stem segments. Each segment was perfused with 3 ml of
the control solution (0.1 mM MgCl2, 0.1 mM KCl, 0.1 mM
CaCl2 and 10 mM MES adjusted to pH 5.5 with HCl) under
moderate pressure (0.1 MPa), and then perfused with 2 ml of a
radioactive solution (control solution supplemented with
1 mM 14C-labeled sucrose (70 kBeq ml – 1) or with 1 mM
14
C-labeled glucose (70 kBeq ml – 1) (both from NEN Life Science) in the presence and absence of 1 mM HgCl2 to dissipate
the electrochemical potential difference. After a 1-h incubation at 15 °C, xylem vessels were rinsed with 6 ml of control
solution and the shoot segments immediately frozen and
stored at –20 °C until lyophilized, ground and the sugar extracted and radioactivity determined.
Soluble sugar flux from xylem to vegetative buds
During vegetative bud break (April), total fluxes of soluble
sugars from xylem vessels to buds were determined by a
method based on that of Valentin et al. (2001). Five-cm-long
segments were cut from the apical and basal ends of 1-year-old
shoots preconditioned at 15 °C for 48 h. Each segment was
perfused first with 3 ml of control solution (1 mM KCl and
NaCl and 0.2 mM CaCl2 adjusted to pH 6 with HCl) under
moderate pressure (0.1 MPa) of nitrogen gas, and then with
0.5 ml of a radioactive-sugar solution (control solution supplemented with 10 mM 14C-labeled sucrose (70 kBeq ml – 1), or
10 mM 14C-labeled glucose (70 kBeq ml – 1), or 200 mM 3H-labeled mannitol (400 kBeq ml – 1) (Sigma). Because a mannitol
transporter has not been described in walnut, and mannitol has
never been found in the xylem sap of this species, mannitol
was assumed to be a non-permeating osmoticum.
A mannitol concentration of 200 mM was chosen to match
the osmolarity of walnut xylem sap. After a 30-min incubation
at 15 °C, xylem vessels were perfused (0.1 MPa) with 3 ml of
unlabeled-sugar solution (10 mM sucrose or 10 mM glucose,
200 mM mannitol). After 8 h, the solution was expelled from
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the perfused vessels with nitrogen gas. The solution was collected for measurement of 14C / 3H ratio at equilibrium and the
bud immediately frozen in liquid nitrogen and stored at –20 °C
until lyophilized, ground and the sugar extracted and its radioactivity determined.
The sugar content of cells of vegetative buds was obtained
by correcting the total 14C radioactivity in the bud by the
apoplasmic 14C radioactivity (14C cells = total 14C – 14C apoplasm, where 14C apoplasm = (14C / 3H)( 3H in vegetative bud)).
Total RNA extraction, and construction and screening of a
xylem-derived cDNA library
Total RNA was extracted from xylem, bark, source leaf, fine
root and flowers (male and female) as described by Gévaudant
et al. (1999). The RNA was quantified spectrophotometrically
and its quality assessed by gel electrophoresis.
A xylem hexose transporter probe was obtained by reverse
transcription polymerase chain reaction (RT-PCR). Reverse
transcription was carried out with 1 µg of total RNA extracted
from xylem tissue in May and the Ready To Go T-primed
First-Strand kit (Amersham). For the PCR, the following degenerate primers were used: Hex1, 5′-G(C/T)(T/G/C)AT(T/C)ATGTT(C/T)TA(C/T)GC(A/C/G)CCTGT-3′; and
Hex2, 5′-CCA(T/A)ACTT(C/G)A(T/A)(A/C/G)CT(G/C)(A/C)CA(C/A)(C/A)AGCAT-3′; which were deduced from
conserved regions of plant hexose transporter cDNAs (EMBL
data library).
Amplification was carried out in a thermal cycler (Gene
Amp PCR system, Perkin Elmer) according to standard protocols. The amplified fragment (460 bp) was sequenced and
used as a hexose transporter homologous probe to screen a xylem cDNA library (Sakr et al. 2003) under low stringency conditions. Two clones were obtained, completely sequenced and
named JrHT1 and JrHT2 for Juglans regia hexose transporter
1 and 2, respectively. The sequences of JrHT1 and JrHT2 have
EMBL/Genbank/DDBJ Accession nos. DQ026508 and
DQ026509, respectively.
Semi-quantitative RT-PCR
One µg of total RNA, extracted from xylem tissues as described above, was reverse transcribed. Three µl of the synthesized first-strand cDNA was amplified by PCR. Amplification
of JrHT1 and JrHT2, JrSUT1 and JrAct (J. regia actin) was
performed with the following specific primers: Hex11 (sense),
5′-AGGAGATGAACAGAGTATGGAAGACC-3′ and Hex12
(antisense), 5′-AGGHAAAACTAAAGAGCAAGGTCCC-3′
for the amplification of JrHT1 and JrHT2; Sut11 (sense),
5′-AGATGCAATATCCGCAGTTCCC-3′ and Sut12 (antisense), 5′-GCATGGHCTTTTGTAGCATTGGG-3′, for the
amplification of JrSUT1; Act11 (sense), 5′-GATGAAGCCCAATCAAAGAGGGGT-3′ and Act12 (antisense) and 5′-TGTCCATCACCAGAAATCCAGCAC-3′ for the amplification of
JrAct; JrEF1A (sense), 5′-TGG(A/T/C)GG(A/T)ATTGA(T/C)AAGCGTG-3′ and JrEF1B (antisense), 5′-CAAT(T/C)TTGTA(A/G)ACATCCTGAAG-3′ for the amplification of JrEF1 (J. regia Elongation Factor 1 alpha). Each PCR
217
was carried out as follows: 2 min at 94 °C for denaturation,
20 s at 94 °C, 20 s at 59 °C and 45 s at 72 °C for 35 (JrHT1,
JrHT2 and JrSUT1), and 25 cycles (JrAct) followed by 10 min
at 72 °C for the final extension. The amplification of JrEF1
was carried out as follows: 2 min at 94 °C for denaturation,
20 s at 94 °C, 30 s at 57 °C and 45 s at 72 °C, for 35 cycles. The
optimal numbers of PCR cycles were established to generate
unsaturated (linear phase) but detectable signals. All PCRs
were performed in parallel on the same cDNA under the same
conditions.
Preparation of the affinity-purified anti-JrHTs antibody
Anti-JrHT1 and anti-JrHT2 antiserum was raised in rabbits
(Eurogentec, Herstal, Belgium) against a synthetic peptide
(PKVEMGKGGRAIKNV) coupled to a carrier protein
(KLH). This sequence was derived from the C-terminal region
of JrHT1 and JrHT2, but is not conserved in all hexose transporters from other species. Antibodies were affinity-purified
against the synthetic peptide and checked for cross-reaction
against the C-terminal region of JrHT1. The proteins cross-reacting with anti-JrHT1 and anti-JrHT2 antibodies are called
JrHT1 and JrHT2.
The affinity-purified anti-JrSUT1 antibody was raised
against a synthetic peptide (MEVESANKDMRAAQR) chosen from the N-terminal region of JrSUT1 (Decourteix et al.
2006). As in Decourteix et al. (2006), the proteins cross-reacting with anti-JrSUT1 antibody are called JrSUT1.
Microsomal fraction and analysis by SDS-PAGE and
Western blot
Xylem microsomal fractions from the apical and basal segments of the sample shoots were isolated as described by
Alves et al. (2004). Protein samples (30 µg) were subjected to
sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE) according to Laemmli (1970). The gel system
consisted of a 5% stacking gel and a 10% resolving gel. After
electrophoresis, polypeptides were transferred to a nitrocellulose membrane (Transfer Membrane, BioTrace NT) by
electroblotting. Membranes were probed with either antiJrHT1 and anti-JrHT2 (1/500 dilution) or anti-JrSUT1
(1/3000 dilution) primary antibodies and anti-rabbit IgG
(H + L) (P.A.R.I.S., Compiègne, France) peroxidase-labeled
secondary antibody (1/10,000 dilution). The protein–antibody complex was detected with the ECL Western blotting
analysis system (Amersham Pharmacia Biotech).
JrSUT1, JrHT1 and JrHT2 immunolocalization in xylem
parenchyma cells
Immunolocalization of JrSUT1 and JrHT1 and JrHT2 was
performed on apical and basal segments of 1-year old stems as
described by Decourteix et al. (2006). Briefly, each stem segment of about 4 mm in length was fixed for 45 min at 4 °C in
1.5% (v/v) formaldehyde and 0.5% (v/v) glutaraldehyde in
0.1 M phosphate buffer (pH 7.4) containing 0.5 % (v/v) dimethylsulfoxide and then washed in 0.1 M phosphate buffer
(pH 7.4) followed by phosphate buffered saline (PBS, pH 7.2).
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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%).
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
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.
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.
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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 RTPCR 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
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 (CAC69075); 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).
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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.
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
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SUCROSE AND HEXOSE TRANSPORTERS IN WALNUT XYLEM PARENCHYMA
221
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.
(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
(nmol g DM –1 h –1)
Glucose import
(nmol g DM – 1 h – 1)
Apical
Basal
1453.5 ± 217.8
103.7 ± 23.9
18.7 ± 8.1
1.4 ± 0.4
(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.
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222
DECOURTEIX ET AL.
1995, Sakr et al. 2003, Decourteix et al. 2006). Sucrose and
hexose co-transporters were immunolocalized in VACs at the
onset of bud break, indicating that active sugar absorption
from xylem vessels can be mediated by VAC-localized transporters.
Assays of respiratory and phosphatase enzymes have shown
that VACs are metabolically highly active during the growth
period (Alves et al. 2001). However, JrSUT1 and JrHT1 and
JrHT2 are not confined to VACs, but are located in axial and
ray xylem parenchyma cells (Figure 6). This differs from the
distribution observed in winter, when JrSUT1 is located
mainly in VACs (Decourteix et al. 2006). It is possible that parenchyma-localized transporters participate in the transport of
soluble sugars after xylem uptake, either by accelerating the
transfer of soluble sugars from VACs to vegetative buds or by
retrieving sugars leaked to the apoplasm during their symplasmic passage from VACs to bud.
In herbaceous species, many studies have shown that the expression of sucrose and hexose transporters in sink organs is
developmentally regulated in a way that is often related to sink
carbon demand (Weber et al. 1997, Ylstra et al. 1998, Trüernit
et al. 1999, Weschke et al. 2000, Scholz-Starke et al. 2003,
Schneidereit et al. 2003). For instance, the accumulation of
VfSUT1 (Vicia faba L. sucrose transporter), which is limited
to the sites of sugar exchange between maternal and filial tissues, coincides with storage activity of the endosperm parenchyma in developing cotyledons (Weber et al. 1997). We
found that the contribution of JrSUT1 to the supply of soluble
sugars to bursting vegetative buds can be deduced from comparisons of both activity and accumulation of sugar transporters between apical and basal stem segments of 1-year-old walnut shoots. At the end of April (bud break), parenchyma cells
in apical segments exhibited high sucrose concentrations (Figure 2A), rapid sucrose absorption (Figure 2B) and elevated
quantities of JrSUT1 transcripts (Figure 4A) and proteins
(Figure 5A) compared with basal segments. This spatial upregulation of JrSUT1, together with its localization in xylem
parenchyma cells (Figure 6) before and at the time of bud
break, indicate that JrSUT1 can mediate active absorption and
radial transport of xylem-imported sucrose from xylem vessels to vegetative bud cells when cambial growth is low
(Améglio et al. 2002). In summary, JrSUT1 participates in the
supply of sucrose to bursting vegetative apical buds, which exhibit a high capacity for sucrose import (Table 1).
Up-regulation of JrSUT1 in the apical xylem parenchyma
cells started before bud break (in March). Because the bursting
bud is a net importer of sugar and acts as a strong sink by competing for nutrients with other sink organs (Maurel et al.
2004), carbon supply to the breaking bud can be governed by
the relative ability of xylem parenchyma cells to transport xylem-imported sugars. In this context, enrichment of xylem parenchyma cells of upper stem segments with JrSUT1 in early
spring indicates the importance of sucrose to support of physiological activities preceding bud break in walnut trees.
Compared with JrSUT1, JrHT1 and JrHT2 may make a limited contribution to bud carbon supply at the time of bud break.
Xylem parenchyma cells were poorly enriched in both tran-
scripts and proteins of JrHT1 and JrHT2 at bud break. Even if
JrHT1 and JrHT2 were localized in VACs (Figure 6), the
scarcity of both transcripts (Figure 4B) and proteins (Figure 5B) of these transporters precluded detectable uptake of
glucose under our experimental conditions. The limited role of
hexoses at the start of bud break in walnut accords with the
limited capacity of buds to import glucose (Table 1). Differential regulation of sucrose and hexose transporters suggests that
the role of sucrose cannot be extended to all xylem sap-imported soluble sugars, and that hexoses contribute less to the
carbon supply at bud break than sucrose. This finding contrasts with our earlier observations indicating the possible importance of glucose in early bud break in peach tree vegetative
buds (Maurel et al. 2004). This apparent discrepancy may reflect differences in carbon metabolism between peach and
walnut. For example, peach is a polyol-producing plant,
whereas walnut is not.
The greatest quantities of JrHT1 and JrHT2 transcripts and
proteins were found in May (Figures 4B and 5B), coinciding
with intense radial growth (Daudet et al. 2005). It is possible
that these hexose transporters are involved in the carbon demand required for radial growth of walnut stems.
We have demonstrated spatial and temporal regulation of
putative sugar transporters in xylem parenchyma cells during
the period of vegetative growth. These findings are comparable with those previously obtained for fava bean (Vicia faba L.) seeds in showing that sucrose and hexose co-transporters have different temporal and spatial patterns (Weber et al.
1997). Our results suggest that sucrose transporter(s)
(JrSUT1) contributes to the carbon supply of bursting vegetative buds, thereby participating with other physiological factors in the acrotonic branching pattern of walnut.
Acknowledgments
This work was supported by the Ministère de la Recherche et de
l’Education Nationale and the INRA (Environment and Agronomy
department). We thank Brigitte Saint-Joanis, Sylvaine Labernia and
Marc Vandame for contributions, and Professor Alan Schulman (Institute of Biotechnology, Helsinki) for comments on the manuscript.
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