Seasonal and cell type specific expression of sulfate transportersin the phloem of Populus reveals tree specific characteristicsfor SO4

22 storage and mobilization

Jasmin Durr • Heike Bucking • Susanne Mult •

Henning Wildhagen • Klaus Palme • Heinz Rennenberg •

Franck Ditengou • Cornelia Herschbach

Received: 5 August 2009 / Accepted: 2 December 2009

� Springer Science+Business Media B.V. 2010

Abstract The storage and mobilization of nutrients in

wood and bark tissues is a typical feature of trees. Sulfur can

be stored as sulfate, which is transported from source to sink

tissues through the phloem. In the present study two tran-

scripts encoding sulfate transporters (SULTR) were identi-

fied in the phloem of grey poplar (Populus tremula 9

P. alba). Their cell-specific expression was analyzed

throughout poplar in source tissues, such as mature leaves,

and in sink tissues, such as the wood and bark of the stem,

roots and the shoot apex. PtaSULTR1;1 mRNA was detected

in companion cells of the transport phloem, in the phloem of

high-order leaf veins and in fine roots. PtaSULTR3;3a

mRNA was found exclusively in the transport phloem and

here in both, companion cells and sieve elements. Both

sulfate transporter transcripts were located in xylem

parenchyma cells indicating a role for PtaSULTR1;1 and

PtaSULTR3;3a in xylem unloading. Changes in mRNA

abundance of these and of the sulfate transporters

PtaSULTR4;1 and PtaSULTR4;2 were analyzed over an

entire growing season. The expression of PtaSULTR3;3a

and of the putative vacuolar efflux transporter PtaSULTR4;2

correlated negatively with the sulfate content in the bark.

Furthermore, the expression pattern of both PtaSULTR3;3a

and PtaSULTR4;2 correlated significantly with temperature

and day length. Thus both SULTRs seem to be involved in

mobilization of sulfate during spring: PtaSULTR4;2 medi-

ating efflux from the vacuole and PtaSULTR3;3a mediating

loading into the transport phloem. In contrast, the abundance

of PtaSULTR1;1 and PtaSULTR4;1 transcripts was not

affected by environmental changes throughout the whole

season. The transcript abundance of all tested sulfate

transporters in leaves was independent of weather condi-

tions. However, PtaSULTR1;1 abundance correlated nega-

tively with sulfate content in leaves, supporting its function

in phloem loading. Taken together, these findings indicate a

transcriptional regulation of sulfate distribution in poplar

trees.

Keywords Sulfate transporter � Phloem loading �Poplar � In situ gene expression �Microautoradiographics �Seasonal changes � Real-time RT-PCR

Introduction

Sulfur is one of the six macronutrients that plants require for

growth and development. It is available to plants mostly in

Electronic supplementary material The online version of thisarticle (doi:10.1007/s11103-009-9587-6) contains supplementarymaterial, which is available to authorized users.

J. Durr � S. Mult � H. Wildhagen � H. Rennenberg �C. Herschbach (&)

Chair of Tree Physiology, Institute of Forest Botany and Tree

Physiology, Albert-Ludwigs-University Freiburg, Georges-

Kohler-Allee 053/054, 79110 Freiburg, Germany

e-mail: cornelia.herschbach@ctp.uni-freiburg.de

H. Bucking

Biology and Microbiology Department, South Dakota State

University, Northern Plains Biostress (SNP, 251B), Brookings,

SD 57007, USA

K. Palme � F. Ditengou

Institute of Biology II, Faculty of Biology, Albert-Ludwigs-

Universiy Freiburg, Schanzlestrasse 1, 79104 Freiburg, Germany

K. Palme � F. Ditengou

Centre for Biological Signaling Studies, Albert-Ludwigs-

University Freiburg, Albertstrasse 19, 79104 Freiburg, Germany

K. Palme

Freiburg Institute of Advanced Sciences, Albert-Ludwigs-

University Freiburg, Albertstrasse 19, 79104 Freiburg, Germany

123

Plant Mol Biol

DOI 10.1007/s11103-009-9587-6

the form of sulfate in the soil. Sulfate is taken up by the

roots and distributed within the plant by short- and long-

distance transport (Herschbach and Rennenberg 2001a, b).

Short-distance sulfate transport over membranes is medi-

ated by sulfate transporters (SULTR) (Buchner et al. 2004a).

The Arabidopsis thaliana and Oryza sativa genomes both

encode 14 putative transporters (Buchner et al. 2004a)

which cluster into five sub-groups (Hawkesford 2003).

Sulfate uptake into root cells is mediated by high affinity

transporters of group 1 (Smith et al. 1997; Vidmar et al.

1999, 2000; Yoshimoto et al. 2002; Shibagaki et al. 2002)

and has been described to operate through a sulfate/proton

co-transport mechanism (Lass and Ullrich-Eberius 1984;

Hawkesford et al. 1993). In roots, sulfate is transported out

of xylem parenchyma cells into the xylem, where it is

transported to the shoot. Some sulfate transporter sequences

of group 2 are transcribed in vascular tissues: for example

AtSULTR2;1 in the xylem parenchyma, in root pericycle

cells and in the phloem of leaves (Takahashi et al. 2000).

Recent studies have showed co-expression of AtSULTR3;5

and AtSULTR2;1 in xylem parenchyma and root pericycle

cells. It has been demonstrated that AtSULTR3;5 is

important for sulfate loading into the xylem during sulfur

starvation (Kataoka et al. 2004a), but its involvement in

sulfate efflux has not been shown.

GFP fusion proteins have demonstrated the localization

of AtSULTR4;1 and AtSULTR4;2 sulfate transporters in

the tonoplast (Kataoka et al. 2004b). Furthermore, double

knockout mutants showed that both transporters contribute

to xylem loading by releasing sulfate from the vacuole.

These transporters are, thus, involved in controlling the

storage of sulfate in the vacuole (Kataoka et al. 2004b).

Sulfate reaching the shoot via the transpiration stream can

be transported into mesophyll cells and further into plastids

for sulfate assimilation (Saito 2004), or into the vacuoles

for storage (Bell et al. 1994). Alternatively, sulfate can be

loaded into the phloem and transported back to the roots

(Smith and Lang 1988; Adiputra and Anderson 1995).

In phloem exudates of deciduous trees, sulfate is the

predominant sulfur compound (Herschbach and Rennen-

berg 2001b). The loading of sulfate into the phloem of leaf

veins and sulfate transport to trunk and root tissues has

been demonstrated in beech (Herschbach and Rennenberg

1996), oak (Schulte et al. 1998) and poplar (Hartmann et al.

2000). For example, 35S-sulfate fed to young mature and

mature poplar leaves has been detected in positions basal to

the fed leaf in wood and bark tissues and in phloem exu-

dates (Hartmann et al. 2000). Sulfate has to be taken up

into the sieve element/companion cell complex for trans-

port via the phloem by mass flow. In Arabidopsis, one

sulfate transporter AtSULTR1;3, was detected in the sieve

element/companion cell complex of cotyledons and roots

(Yoshimoto et al. 2003), but leaves from vegetative plants

were not investigated. During seedling development

AtSULTR1;3 is important for the loading of remobilized

sulfate into the phloem of the cotyledons. Although

AtSULTR2;2 is expressed in the root phloem and in the

vascular bundle sheath of leaves (Takahashi et al. 2000) its

contribution to phloem loading has not been demonstrated.

One important feature of trees is their seasonal growth

pattern. This requires storage and mobilization of reserve

metabolites including sulfate (Herschbach and Rennenberg

1996; Hartmann et al. 2000; Rennenberg et al. 2007).

During spring, sulfate is mobilized and transported into the

developing buds and leaves (Herschbach and Rennenberg

1996). Hence, sulfate has to be transported from paren-

chyma cells into the xylem and/or phloem. This can be

achieved by symplastic translocation or by sulfate release

into the apoplast followed by subsequent uptake into the

sieve element/companion cell complex.

Many studies have revealed that the abundance of sev-

eral sulfate transporter transcripts change as a response to

the sulfur demand. For example, increasing expression

levels were found under sulfur limitation when the sulfate

uptake capacity increased (Smith et al. 1997; Vidmar et al.

2000; Buchner et al. 2004b; Hopkins et al. 2004; Kataoka

et al. 2004b; Rouached et al. 2008; Koralewska et al.

2009). Moreover, reduced or increased expression was also

observed during heavy metal application (Heiss et al. 1999;

Nocito et al. 2006). Transcriptome profiling of dormant and

active cambial meristem revealed a significant reduction in

complexity of the cambial transcriptome during the dor-

mant state (Schrader et al. 2004). All these findings support

the idea that sulfate distribution within plants, particularly

seasonal changes in phloem loading and unloading, is

regulated at the transcriptional level. Therefore, the present

study aimed (1) to identify sequences encoding phloem-

specific sulfate transporters from the genome of Populus

tremula 9 P. alba; (2) to characterize the cell type-specific

expression pattern of transcripts by in situ hybridization in

source and sink tissues; and (3) to profile seasonal changes

in transcript abundance of phloem-specific, and of putative

vacuolar, sulfate transporters.

Materials and methods

Plant material

Poplars of the hybrid Populus tremula 9 Populus alba,

clone 717 1B4 (Institute National de la Recherche

Agronomique, INRA, France) were micropropagated as

described by Strohm et al. (1995) and Noctor et al. (1996).

Four-week old cuttings were transferred into a soil mixture

containing commercial soil, silica sand, and perlite (1:1:1)

and further grown in a greenhouse (26 ± 5�C) under long

Plant Mol Biol

123

day conditions (Hartmann et al. 2000). After 10 weeks, the

poplar seedlings were harvested and separated into (1) apex

with two visible leaves, (2) the 10th and 11th leaf counted

from the apex (separated into the main leaf vein including

the petiole and the leaf lamina), (3) bark and wood from the

corresponding stem section, main roots and, (4) fine roots

with a diameter up to 1 mm.

For seasonal analyses, poplar cuttings were grown from

February 2005 in the greenhouse, trimmed in autumn 2005

and transferred into the field for acclimation during winter.

The seedlings were planted in a field containing humus in

April 2006 near the institute. They were fertilized at the

beginning once with 120 g of a commercial long-time

fertilizer (Basa cote Plus 12 M, COMPO, Austria). During

drought periods in summer, trees were watered with tap

water. From the end of August 2006 until mid September

2007 leaf and bark samples were harvested between 10 am

and 11 am (sampling dates see Supplemental Table S1).

Two leaves between the seventh and ninth leaf counted

from the twig apex as well as the bark between the fifth and

15th leaf from one twig were rotationally harvested from

three of 16 trees at each sampling date. Dormant buds were

always removed from the twigs. Plant material was frozen

in liquid N2 and stored at -80�C for further analyses.

Preparation of RNA and cDNA

Tissue samples were first homogenized in liquid nitrogen.

Total RNA was then either extracted with an extraction kit

(RNeasy Plant Mini Kit; QIAGEN) according to the

instructions of the manufacturer or, alternatively from

1.2 g homogenized tissue as described by Kolosova et al.

(2004) for northern-blot analyses, or from 120 mg

homogenized tissue for the real-time PCR analyses.

For real-time PCR analyses of bark samples, genomic

DNA was digested with DNase I (Fermentas) prior RNA

transcription. In leaf samples genomic DNA was digested

using the On-Column RNAase-Free DNase Set (Qiagen)

during RNA extraction. The amount and purity of RNA were

determined with a nano-spectrophotometer (NanoDrop�

ND-1000, Peqlab). First strand cDNA was synthesized from

1 or 2 lg RNA using a final concentration of 500 nM random

primer R12 [50-NNN NNN NNN NNN-30] and Superscript II

Reverse Transcriptase (Invitrogen) according to the manu-

facturer’s instructions. No RT was used as a control reaction

and means the omission of the transcriptase enzyme.

Cloning of putative sulfate transporters

Based on in silico research of the Populus trichocarpa

genome database (http://genome.jgi-psf.org/Poptr1/Poptr1.

home.html), 18 putative sulfate transporter (SULTR)

sequences were selected and parts of their open reading

frames were cloned from the poplar hybrid Populus tremula 9

P. alba. The amplification reaction was performed using Taq-

DNA Polymerase (Promega) following the standard protocol

of the manufacturer with cDNA of different tissues. With the

exception of three sequences, all SULTR sequences were

cloned by nested PCR. Primers for PCR were designed based

on the sequence information from Populus trichocarpa

(Supplemental Table S2). The first PCR included the specific

forward 1 primer and PolyT-mix as reverse primer. The sec-

ond PCR contained 1 ll of the first PCR solution as template

and the sequence specific forward 2 and reverse 2 primers.

Amplified DNA segments were separated on a 0.8% agarose

gel, extracted with the QIAquick gel extraction kit (Qiagen),

incorporated into the pCR2.1 vector (Invitrogen) and trans-

formed into competent E. coli cells (INVF0a, Invitrogen).

Segments of the putative SULTR genes were verified by

sequencing (MWG Biotech AG, Martinsried, Germany) and

subsequent multiple sequence alignment using ClustalW

(SDSC Biology WorkBench, http://workbench.sdsc.edu/,

Thompson et al. 1994).

Characterization of putative sulfate transporters

The homology of the selected SULTR amino acid sequences

to the SULTR sequences from Arabidopsis thaliana and

Oryza sativa was determined by neighbor-joining tree

analyses (MEGA 4; Tamura et al. 2007) with gap open

penalty of 50 and gap extension penalty of 0.2 with the score

matrix BLOSUM62. Poisson correction was used as substi-

tution model. Identity and similarity between Arabidopsis or

Oryza and Populus tremula 9 P. alba genes were deter-

mined by local alignment (Tatusova and Madden 1999; http://

www.ncbi.nlm.nih.gov/blast/bl2seq/wblast2.cgi). Full length

amino acid sequences of PtaSULTR1;1, PtaSULTR3;3a and of

the group 4 proteins PtaSULTR4;1, PtaSULTR4;2 were

aligned with the Arabidopsis AtSULTR1;1 sequence using

ClustalW2 (http://www.ebi.ac.uk/Tools/clustalw2/index.html).

A combined analysis with MEMSAT (http://saier-144-37.

ucsd.edu/memsat.html) (Jones et al. 1994) and TMAP (http://

bioinfo4.limbo.ifm.liu.se/tmap/index.html) (Persson and Argos

1994) was used to determine the locations of probable membrane

spanning domains (MSDs). Predicted transit peptide regions

were calculated using the ChloroP 1.1 (Emanuelsson et al. 1999)

program (http://www.cbs.dtu.dk/services/ChloroP/).

Northern-blot analyses

For each tissue, 15 lg of total RNA was loaded per gel slot

and separated on 1% formaldehyde-agarose gels at 100 V

for 80 min by electrophoresis. RNA was transferred onto

Hybond-XL nylon membranes (Amersham) with 109 SSC

Plant Mol Biol

123

and fixed at 80�C for 2 h. Prehybridization with

40 lg ml-1 herring sperm DNA was performed at 65�C in

a 7% SDS-FSB-buffer (FSB: 0.05 M NaP2O7, 0.115 M

NaH2PO4, 0.5 M EDTA). Specific cDNA probes between

150 and 360 bp long including 30UTR parts were prepared

for each isoform. Primers and the lengths of the fragments

are given in Supplemental Table S3. Hybridization with32P-labelled cDNA probes containing 32P-dATP insertion

was performed in 7% SDS-FSB-buffer over night using the

Strip-EZTM DNA kit (Ambion). Northern blots treated with

5.8S rRNA (Acc. No. AY781281) probe were used as

loading controls. After hybridization the membranes were

washed three times under high stringency conditions with

1% SDS-FSB-buffer and examined by a Phosphor Imager

(Molecular Imager FX, Bio-Rad Laboratories).

In situ hybridization

Approximately 3 mm sections of different tissues were

harvested from 10-week old Populus tremula 9 P. alba

trees. These were: (1) the apex, (2) the 11th leaf counted

from the apex, (3) young stem parts with young developing

leaves (fourth and fifth leaf counted from the apex), stems

with young mature leaves (tenth and 11th leaf) and older

stem parts with mature leaves (25th and 27th leaf), (4) the

main root and, (5) fine roots. Tissues were fixed in 4%

paraformaldehyde phosphate buffer (PBS: 140 mM NaCl,

10 mM KCl, 6.4 mM Na2HPO4, 2 mM NaH2PO4, pH 7)

containing 0.01% Triton x-100 overnight at 4�C after

vacuum infiltration according to Cnops et al. (2006).

Samples were dehydrated in a graded ethanol series and

subsequently infiltrated in a Roticlear (Carl Roth) series

described by Deeken et al. (2008). The samples were

embedded in paraplast (Sigma–Aldrich) at 60�C by

changing the solution four times and subsequent vacuum

infiltration with a self-made infiltration device according to

Deeken et al. (2008). Eight to 10 lm sections were cut with

a RM2245 microtome (Leica) and were transferred onto

charged glass slides (SuperFrost� Plus, Menzel, Germany).

Paraplast was removed with a series of HistoClear and

ethanol (Deeken et al. 2008). The proteins were digested by

treating the sections with 125 lg ml-1 Pronase (Sigma–

Aldrich) in PBS-buffer for 10 min at 37�C. Subsequently,

the samples were post-fixed with 4% paraformaldehyde

in PBS.

Gene specific probes were generated by PCR for both

PtaSULTR1;1 and PtaSULTR3;3a (primers and probe length

see Supplemental Table S3). The forward primer carried

the T3 [50-AATTAACCCTCACTAAAGGGAGA-30] and

the reverse primer the T7 RNA polymerase binding site

[50-GCTTCTAATACGACTCACTATAGGGAGA-30] at

the 50 end. Labeling with digoxigenin (DIG) was performed

by in vitro transcription using DIG RNA labeling mix

(Roche Diagnostics) with either T3 or T7 RNA polymerase

(Fermentas) according to the instruction of the manufac-

turer. Labeled probes were dissolved in 100 ll water and the

quality was tested by electrophoresis on a 1%-agarose-TBE-

buffer gel. The RNA concentration was determined spec-

trophotometrically at 260 nm.

Each slide was incubated for 1 h at 50�C in a humid

chamber with 100 ll prehybridization buffer [50% form-

amide and 29 SSC buffer (209 SSC was 3 M NaCl, 0.3 M

sodium citrate) and 40 U ml-1 RNase inhibitor (Fermen-

tas)]. Hybridization with equal concentrations (20–30

lg ml-1) of antisense or sense probe was performed on

slides with 100 ll hybridization buffer [50% formamide,

49 SSC buffer, 109 Denhardt’s solution, 0.5 mg/ml yeast

t-RNA, 10% dextran sulfate and 40 U ml-1 RNase inhib-

itor (Fermentas) in water] overnight at 50�C in a humid

chamber. The following wash steps were performed in

glass boxes at 50�C each for 30 min: 39 SSC, 39 SSC,

1.59 SSC, 0.759 SSC and two further wash steps with

0.39 SSC and 0.19 SSC for 20 min each. After the second

wash step, the sections were treated with RNase (RNase A

20 lg ml-1, Carl Roth) for 20 min at 37�C in TE-buffer

(pH 8.0). Non-specific binding sites were blocked with

BSA (bovine serum albumin) in blocking buffer (100 mM

Tris–HCl, pH 7.5, 150 mM NaCl, 0.5% BSA, 0.02%

Tween 20) for 1 h at room temperature. Incubation with

anti-DIG-UTP-alkaline phosphatase FAB fragments

(1:2,000, Roche Diagnostics) and staining with nitroblue

tetrazolium chloride and 5-bromo-4-chloro-3-indolyl

phosphate (Roche Diagnostics) was carried out according

to Cnops et al. (2006). The slides were mounted in 50%

glycerol and inspected with an inverted microscope

(Axiovert 200 M and Stemi SV 11 Apo, Zeiss). Images

were taken with a digital camera (Zeiss) and processed

with AxioVision 4.6 software (Zeiss).

Microautoradiographic studies

Mature leaves from 8 to 10-week old poplar plants were

fed with 150 lCi carrier free [35S]-SO42- via flap-feeding

(Biddulph 1956) as previously described (Hartmann et al.

2000) to allow a direct penetration of the feeding solution

into the phloem. The stem sections below the fed leaf were

harvested after 24 h. The stem sections were cryofixed by

plunging into melting nitrogen and freeze-dried under low

temperature and high vacuum conditions (CFD, Leica,

Bensheim, Germany). To prevent a re-crystallization of

water in the plant tissues (*-80�C), the samples were first

freeze-dried for 7 days at -100�C, and then for 2 days

each at -90, -80, and -60�C and then slowly warmed up

to room temperature. The samples were then pressure infil-

trated according to the method described by Fritz (1980) in

an epoxy resin (Spurr 1969) using dried diethyl ether as an

Plant Mol Biol

123

inter-medium. After polymerization, dry sections of 1 lm

were cut with Teflon-coated glass knifes and mounted on

filmed microscope slides. The sections were overlaid in the

dark room with a thin layer of the nuclear research emulsion

L4 (Ilford, Dreieich, Germany) with a crystal size of

0.11 lm. The thickness of the film layer was approximately

5 lm. After exposure in the darkness at 4�C for several

weeks (dependent on the radioactivity of the samples), the

film was developed with the fine grain developer D19 A/S

(Sanderson 1981), rinsed in water and than fixed with a

commercial b/w fixer (Tetanol, Norderstedt, Germany). As

controls, slides without sections were overlaid with film and

processed in the same way as described above. The micro-

graphs were analyzed using Nomarski DIC microscopy

(Olympus, Hamburg, Germany).

Quantitative real-time RT PCR

Quantitative PCR measurements of PtaSULTR1;1,

PtaSULTR3;3a, of the putative vacuole localized sulfate

transporters PtaSULTR4;1, PtaSULTR4;2 and of the refer-

ence gene, the elongation factor 1-beta (EF1beta; Acc.

No.: FJ372570), were performed using the following gene-

specific forward and reversed primers: (50-TTTATAACCC

GTGCAGATAAGGAC-3) and (50-CCTTTTAGCAAATG

GTCACCAC-30) for PtaSULTR1;1, (50-GCCCCTCTTGT

GTCTGTGATC-30) and (50-TCCACGAGGGAGGATT-

TAGC-30) for PtaSULTR3;3a, (50-GGCACTGCGTATA-

TATGATATCTGTC-30) and (50-AAACCTTACGACAAG

TATTGCATTG-30) for PtaSULTR4;1, (50-GAGGCAGGG

CGTAGATTG-30) and (50-GGAAGCAAGCCTTACAAT

GC-30) for PtaSULTR4;2 and, (50-TGAGGATCTCTGGT

GTCGAAG-30) and (50-GTCTCAGCAGATGGAGGAGT

G-30) for EF1beta. The resulting PCR fragments were

113 bp (PtaSULTR1;1), 115 bp (PtaSULTR3;3a), 114 bp

(PtaSULTR4;1), 132 bp (PtaSULTR4;2) and 100 bp

(EF1beta). The specificity of the primer pairs was tested by

RT-PCR and amplified segments were controlled by

sequencing after purification from agarose gels. mRNA

abundance in leaf and bark tissues was determined by real

time RT PCR using the LightCycler 480 SYBR Green I

Master mix (Roche, Germany) and the Multiwell Plate 384

(Roche, Germany). The 10 ll reaction volume contained

5 ll 29 SYBR Green I Master mix, 2.5 ll (25 ng) cDNA

and a final primer concentration of 0.8 or 1.2 lM. After a

hot start for 2 min at 95�C, 45 PCR cycles were performed

with a 15 s melting step at 95�C, 15 s annealing time at

55�C, and 15 s extension time at 72�C on a LightCycler�

480 system (Roche, Germany). Transcripts per lg RNA

were calculated based on standard curves generated with

serial dilutions of linearized plasmids containing an insert

of the gene of interest.

Determination of sulfate contents

Sulfate concentration was determined by anion exchange

chromatography from 50 mg powdered bark and leaf tissue

as described by Herschbach et al. (2000).

Statistical analyses

Bivariate data correlations of the seasonal sulfate trans-

porter expression were performed with transformed (ln)

values of the transcript means and the sulfate means from

particular sampling dates using the Pearson linear corre-

lation function. The data transformation resulted in nor-

mality and homogeneity of variances of data. The weather

data (maximum, minimum and mean temperature, relative

humidity and sun shine duration) were available from a

climate station in close vicinity (Station 10803 of the

Deutscher Wetterdienst, Freiburg Airport, Germany).

Meteorological data were mean values from 24 h prior to

the sampling time. Statistical analyses were performed

using the statistical software package (SPSS� GmbH

software, version 16, Munich, Germany). Significant dif-

ferences in transcript abundances and sulfate contents

among sampling dates were tested by analysis of variance

(ANOVA) using a significance level of P \ 0.05. Post-hoc

tests (Turkey test and Games–Howell-test) were used to

determine significant differences between adjacent sam-

pling dates.

Accession numbers

Sequences can be found in the NCBI database (http://

www.ncbi.nlm.nih.gov) under the following accession

numbers for Populus tremula 9 P. alba: DQ906929

(PtaSULTR1;1), DQ174472 (PtaSULTR1;2), DQ906931

(PtaSULTR2;1a), DQ906933 (PtaSULTR2;1b), DQ174473

(PtaSULTR2;2), DQ174470 (PtaSULTR3;1a), DQ906928

(PtaSULTR3;1b), DQ174469 (PtaSULTR3;2a), DQ906934

(PtaSULTR3;2b), DQ906924 (PtaSULTR3;3a), DQ906926

(PtaSULTR3;3b), DQ174467 (PtaSULTR3;4a), DQ174466

(PtaSULTR3;4b), DQ906927 (PtaSULTR3;5), DQ906930

(PtaSULTR4;1), DQ906935 (PtaSULTR4;2), DQ174477

(PtaSULTR5;2), DQ174475 (PtaSULTR5;1), AY781281

(5.8 S rRNA) and for Arabidopsis: AB018695 (AtSULTR1;1),

AB042322 (AtSULTR1;2), AB049624 (AtSULTR1;3),

AB003591 (AtSULTR2;1), D85416 (AtSULTR2;2),

D89631 (AtSULTR3;1), AB004060 (AtSULTR3;2),

AB023423 (AtSULTR3;3), AB054645 (AtSULTR3;4),

AB061739 (AtSULTR3;5), AB008782 (AtSULTR4;1),

AB052775 (AtSULTR4;2), NP_178147 (AtSULTR5;1),

NP_180139 (AtSULTR5;2) and for Oryza: AF493790

(OsSULTR1;1), XP_470587 (OsSULTR1;2), AF493790

(OsSULTR1;3), AAN59769 (OsSULTR2;1), AAN59770

Plant Mol Biol

123

(OsSULTR2;2), NP_921514 (OsSULTR3;1), AAN06871

(OsSULTR3;2), AK104831 (OsSULTR3;3), BAD68396

(OsSULTR3;4), NM_192602 (OsSULTR3;5), NM_191791

(OsSULTR3;6), AF493793 (OsSULTR4;1), BAC05530

(OsSULTR5;1), BAD03554 (OsSULTR5;2).

Results

Identification and expression analyses of SULTR genes

Phylogenetic analysis of the 18 putative SULTR (sulfate

transporter) gene sequences from Populus tremula 9

P. alba revealed that they can be classified into the five sub-

groups proposed by Hawkesford (2003) (Fig. 1a). Sequence

similarity to the homologous Arabidopsis sequences was

between 80 and 90% (Supplemental Table S4). All putative

SULTR sequences showed a tissue-specific expression pat-

tern (Supplemental Fig. S1). Two sulfate transporter tran-

scripts, PtaSULTR1;1 of group 1 and PtaSULTR3;3a of

group 3, were abundant in the bark of the stem, in the main

root and in leaf veins. Small amounts of PtaSULTR1;1

mRNA were also found in the stem wood (Fig. 1c). As the

bark includes the phloem we further focused on these two

transporters. We included the two sequences of group 4,

PtaSULTR4;1 and PtaSULTR4;2 in our analyses as members

of group 4 are putative vacuolar sulfate transporters. Both;

PtaSULTR4;1 and PtaSULTR4;2, were expressed ubiqui-

tously throughout all tissues (Fig. 1c). A combined analysis

with MEMSAT and TMAP showed that the open reading

frames of PtaSULTR1;1, PtaSULTR3;3a, PtaSULTR4;1,

and PtaSULTR4;2 each encode 12 predicted membrane

spanning domains (MSDs) (Fig. 1b). At the carboxyl-ter-

minus, a conserved STAS (sulfate transporter anti sigma

factor antagonist) domain was identified in the PtaSULTR

sequences from groups 1, 2, 3, and 4 by comparing these

sequences with the Arabidopsis SULTR1;1 sequence pre-

viously examined by Shibagaki and Grossman (2004)

(Fig. 1b).

Cell type-specific expression of PtaSULTR1;1

and PtaSULTR3;3a analyzed by in situ hybridization

As the phloem connects mature leaves with different sink

tissues such as young developing leaves, roots and storage

tissues of the trunk, we used in situ hybridization to detect

the expression of both transporters (1) in transverse sec-

tions of leaves and leaf veins, (2) in longitudinal sections of

the apex, (3) in transverse sections of stems at different

developmental stages, of roots with secondary growth, and

(4) in fine roots. The sections were analyzed with antisense

probes. Sense probes used as controls showed no blue

staining (Figs. 2a, g, 3a, g, 4a, 5a, 6a, c). Brown spots

visible in the apex (developing leaves) and in roots repre-

sent the staining of phenolic compounds (green arrows in

Figs. 3e, 6c, d). Thus the staining observed with the anti-

sense probes clearly shows the mRNA abundance of

PtaSULTR1;1 or PtaSULTR3;3a.

Transverse sections of leaves and leaf veins

PtaSULTR1;1 expression was found in major leaf veins,

especially in companion cells of the phloem and in xylem

parenchyma cells (Fig. 2c, d). In minor leaf veins, the entire

phloem was labeled (Fig. 2e, f). Palisade parenchyma,

spongy mesophyll cells, and upper epidermis cells

were weakly stained (Fig. 2e, f). The expression of

PtaSULTR3;3a differed from that of PtaSULTR1;1 (Fig. 2h–m).

The phloem and xylem parenchyma cells of major leaf veins

showed PtaSULTR3;3a expression. Both, sieve elements and

companion cells were labeled with comparable intensity

(Fig. 2i). PtaSULTR3;3a transcripts were not detectable in

minor veins and also not in other leaf cells (Fig. 2l, m).

Longitudinal sections of the apex

Both PtaSULTR1;1 and PtaSULTR3;3a transcripts were

detected in the shoot apex but with clear differences in their

expression patterns (Fig. 3). Whereas a high expression

of PtaSULTR1;1 was found in leaf primordia (Fig. 3b, c) and

in meristem cells of the leaf margin (LMM; Fig. 3d, e),

PtaSULTR3;3a was only weakly detected in the shoot apical

meristem (SAM, Fig. 3h). Both genes were expressed in the

provascular strands (Fig. 3b, h).

Transverse sections of stems

Stem sections with mature leaves (tenth to 11th) (Fig. 4)

showed PtaSULTR1;1 expression in several cell types. Most

significant is the expression in companion cells of the

phloem (Fig. 4e, f) beside a strong mRNA abundance in ray

initials of the vascular cambium and, to a lower extent, in

fusiform cells (Fig. 4b, c, e). PtaSULTR3;3a was expressed

in companion cells but, staining with a comparable intensity

was also found in sieve elements (Fig. 5e, f). Irrespective of

the stem age PtaSULTR3;3a transcripts were not detected in

the vascular cambium (Fig. 5e, Supplemental Fig. S3).

Both, PtaSULTR1;1 and PtaSULTR3;3a were expressed

in developing ray pith cells (Figs. 4c, e, 5c, e), in ray cells

connecting xylem vessels in the mature wood (Figs. 4e, g,

5e, g) and in parenchyma cells of the primary xylem

(Figs. 4h, 5h).

Furthermore, PtaSULTR1;1 transcripts were visible in

cells just below the epidermis (Fig. 4d) while transverse

sections of young trunks (fourth and fifth leaf counted from

the apex) revealed strong PtaSULTR1;1 expression in the

Plant Mol Biol

123

(C)

(A) (B)

PtaSULTR1;1

PtaSULTR3;3a

5.8S rRNA

5.8S rRNAPtaSULTR4;2

PtaSULTR4;1

5.8S rRNA

5.8S rRNA

PtaSULTR1.1

PtaSULTR1.2

AtSULTR1.2

AtSULTR1.3

AtSULTR1.1

OsSULTR1.3

OsSULTR1.1

OsSULTR1.2

PtaSULTR2.1a

PtaSULTR2.1b

AtSULTR2.1

PtaSULTR2.2

AtSULTR2.2

OsSULTR2.1

OsSULTR2.2

PtaSULTR3.1a

PtaSULTR3.1b

PtaSULTR3.2a

PtaSULTR3.2b

AtSULTR3.1

AtSULTR3.2

OsSULTR3.1

OsSULTR3.2

PtaSULTR3.5

AtSULTR3.5

OsSULTR3.5

OsSULTR3.6

PtaSULTR3.3a

PtaSULTR3.3b

AtSULTR3.3

PtaSULTR3.4a

PtaSULTR3.4b

AtSULTR3.4

OsSULTR3.4

PtaSULTR4.1

PtaSULTR4.2

AtSULTR4.1

AtSULTR4.2

OsSULTR4.1

PtaSULTR5.1

PtaSULTR5.2

AtSULTR5.2

OsSULTR5.2

AtSULTR5.1

OsSULTR5.1

94

69

98

95

100

100

99

97

100

100

100

100

100

100

100

100

100

100

100

98

88

96

100

100

98

64

42

100

98

100

97

93

97

98

92

82

100

89

100

98

53

98

0.2 substitutions per site

Fig. 1 Characteristics of the sulfate transporter gene family from

Populus tremula 9 P. alba. a Phylogenetic analysis of predicted

sulfate transporter amino acid sequences from Populus tremula 9

P. alba. The bootstrap values, expressed as percentage, were obtained

from 1,000 replicate trees. b Full length amino acid sequences of

PtaSULTR1;1, PtaSULTR3;3a, PtaSULTR4;1, and PtaSULTR4;2

were aligned with the Arabidopsis AtSULTR1;1 sequence. Predicted

12 membrane-spanning domains (MSDs) were marked with blackbars. Consensus amino acids are highlighted in yellow while those

with similar properties are highlighted in grey. A conserved basic

amino acid residue (Arg) between MSD 9 and 10 is indicated by a

solid circle. The conserved STAS (sulfate transporter anti sigma

factor antagonist) domain is indicated by a red box. Predicted transit

peptide regions are marked with blue bold letters. c Northern blots

from PtaSULTR sequences. A 15 lg total RNA was extracted from

different tissues from 10-week old poplars as indicated: the apex, the

10th and 11th leaf counted from the apex separated into major leaf

vein including the petiole and the remaining leaf lamina, the

corresponding stem section separated into bark and wood, the main

root separated into bark, wood, and fine roots. Northern blots

were hybridized with isoform-specific, [32P] labeled probes for

PtaSULTR1;1, PtaSULTR3;3a, PtaSULTR4;1, and PtaSULTR4;2sequences. 5.8 S rRNA probe was used as a loading control. Data

presented are from pooled samples from three independent plants

from one of two experiments with comparable results

Plant Mol Biol

123

epidermis and the cells around lenticels (Supplemental

Figs. S2, S4). The cortex cells that are located between

sclerenchymatous fibers and the epidermis showed weak

PtaSULTR1;1 expression (Fig. 4b) while only periderm

cells revealed a slight PtaSULTR3;3a staining (Fig. 5c, d).

The expression was also analyzed along the entire stem

to identify age dependent differences. But, the expression

patterns observed in stem sections bearing young mature

leaves (tenth to 11th counted from the apex) was repre-

sentative for the entire stem (Supplemental Figs. S2, S3).

However, whereas PtaSULTR1;1 mRNA was expressed

in roots with secondary growth (Supplemental Fig. S2),

PtaSULTR3;3a was not (data not shown).

Fine roots

PtaSULTR3;3a transcripts were not visible in fine roots

(Supplemental Fig. S5) but PtaSULTR1;1 mRNA was here

expressed in the phloem (Fig. 6d, f). This was also found

when lateral root appear (Fig. 6e). The phloem was stained

in diarch, i.e., near the root tip, as well as in tetrarch

vascular root bundles (Fig. 6f). In longitudinal sections,

PtaSULTR1;1 transcripts were detected in the stele and in

the root meristem of the emerging lateral root (Fig. 6g, h).

Microautoradiographic 35S localization

To test whether transcript abundance of SULTR genes

correlates with sulfate transport activity, a mature leaf was

fed with 35S-sulfate. The radioactivity was examined at the

cellular level by microautoradiographic analysis in stem

sections basal to the fed leaf. 35S distribution is visualized

as silver grains in the photographs. Accumulated 35S was

found in ray initials of the vascular cambium (Fig. 7c),

companion cells of the phloem (Fig. 7b) and pith ray cells

of wood (Fig. 7d). These results clearly demonstrate the

transport of 35S from source leaves to more basal regions of

the stem, and thus the unloading of sulfate from the

phloem, and further transport into ray pith cells. The latter

is indicated by an enrichment of silver grains in pith ray

cells (Fig. 7d).

Seasonal changes in sulfate content and of SULTR

expression in bark

The seasonal expression of PtaSULTR1;1, PtaSULTR3;3a,

and the putative vacuolar sulfate transporters PtaSULTR4;1

and PtaSULTR4;2 was analyzed in leaves and bark

throughout an annual growth cycle (Fig. 8). An elongation

Fig. 2 In situ localization of PtaSULTR1;1 and PtaSULTR3;3a in

leaves. Transverse sections of major leaf veins (a–d, g–k) and of leaf

lamina with minor leaf veins (e, f and l, m) were hybridized with the

sense (a) or antisense (b–f) probe for PtaSULTR1;1 as well as with the

sense (g) or antisense probe (h–m) for PtaSULTR3;3a. Enlarged parts

are indicated by squares. Bars are 200 lm for a, b, g and h and

20 lm for all other. Abbreviations: cc companion cell, le lower

epidermis, p phloem, pp palisade parenchyma, px protoxylem,

se sieve element, sm spongy mesophyll, ue upper epidermis, x xylem,

xp xylem parenchyma, xv xylem vessel

Plant Mol Biol

123

factor gene family sequence (elongation factor 1-beta;

EF1beta) was used as a reference gene (Brunner et al.

2004; Nicot et al. 2005). In bark, the expression of EF1beta

in autumn and spring was twice as abundant as during

dormancy (Fig. 8f). The expression of PtaSULTR1;1 in the

bark decreased in autumn when tree growth switched to

dormancy (mean temperatures below 5�C) (Fig. 8b). Dur-

ing bud break the expression of PtaSULTR1;1 increased

significantly: twofold week-on-week between 3rd April

and 18th April 2007 (P \ 0.05; 3rd April \ 11th

April \ 18th April). At the same time, the sulfate that had

accumulated in the bark during autumn 2006 disappeared

(Fig. 8a). The expression level of PtaSULTR3;3a was

comparable to that of PtaSULTR1;1 during autumn 2006

and spring 2007 and increased significantly during bud

swelling (P \ 0.05; 12th March \ 3rd April, P \ 0.068;

3rd April \ 18th April) (Fig. 8c). During dormancy,

PtaSULTR3;3a mRNA was not detected but the transcript

level increased again at the end of March and remained

high throughout leaf expansion and early leaf development.

The expression of PtaSULTR4;2 decreased during autumn

but, in contrast to PtaSULTR3;3a, low expression levels

were still detected during dormancy (Fig. 8c, e). In spring,

mRNA abundance of PtaSULTR4;2 increased, starting

shortly before bud break (P \ 0.05; 12th March \ 27th

March). PtaSULTR4;1 expression increased significantly

during winter with a maximum on 21st December

(P \ 0.05; 25th October \ 22nd November \ 21st

December [ 22nd January). Subsequently, it remained

constant until bud break, which occurred between 3rd April

and 11th April (Fig. 8d). The expression of all genes tested

was high in the bark of young twigs developed in 2007 and

declined thereafter.

Bivariate Pearson-correlation analyses were used to

calculate the correlation between the expression of differ-

ent sulfate transporters, the sulfate content and meteoro-

logical parameters (maximum, minimum and mean

temperature, day length, relative humidity, wind, rain, and

hours of sunshine). PtaSULTR3;3a and PtaSULTR4;2

expression was positively correlated in bark tissue. Both

genes were negatively correlated to the sulfate content

(Table 1). PtaSULTR3;3a and PtaSULTR4;2 were posi-

tively and the sulfate content negatively correlated to mean

temperature, maximum temperature, minimum temperature

and day lengths (Table 1, see also Supplemental Fig. S6).

The expression of PtaSULTR1;1 and PtaSULTR4;1 corre-

lated positively, but neither showed any correlation to

meteorological parameters.

Fig. 3 In situ localization of PtaSULTR1;1 and PtaSULTR3;3a in the

shoot apex. Longitudinal sections from the shoot apex were hybrid-

ized with the sense (a) or antisense (b–e) probe for PtaSULTR1;1 and,

with the sense (g) or antisense (h) probe for PtaSULTR3;3a. a, b, g,

and h longitudinal sections from the apex; c shoot apical meristem

(SAM); d a lateral bud with leaf primordium; e leaf margin meristem

(LMM). A green arrow in e indicates phenolic compounds. Bars are

200 lm for a, b, g, and h and 50 lm for c, d, and e. Abbreviations:

lp leaf primordium, ps provascular strand

Plant Mol Biol

123

Seasonal changes in sulfate content and of SULTR

expression in leaves

In leaves, the average expression of EF1beta was tenfold

higher than in bark. One peak of EF1beta transcript abun-

dance was detected just after bud break when leaves started

to expand (Fig. 8f). In comparison, the mean expression

level of PtaSULTR1;1 was 100-fold lower, but was compa-

rable in bark and leaves (Fig. 8b). A peak value of

PtaSULTR1;1 and PtaSULTR4;1 mRNA was observed in

autumn 2006 before leaf fall. During leaf expansion, i.e.,

after bud break in spring 2007, expression of PtaSULTR1;1

and PtaSULTR4;2 increased until the leaves were fully

expanded (in May) and decreased thereafter (P \ 0.05; 18th

April \ 18th May [ 14th August, 28th August, 13th Sep-

tember). In contrast, mRNA abundance of PtaSULTR3;3a,

was low during early leaf expansion, increased in June 2007

when the leaves were fully developed and decreased later in

the season (P \ 0.05; 18th May \ 29th June [ 13th Sep-

tember). The expression of PtaSULTR4;1 was constant in

spring and early summer but decreased in late summer. The

sulfate content increased in the developing leaves until late

summer 2007 to a comparable amount to that measured in the

previous year (Aug 2006) (Fig. 8a). Sulfate decreased during

late summer in the leaves developed in 2006 and remained

low during autumn.

The bivariate analyses showed a linear negative corre-

lation between the transcript levels of PtaSULTR1;1 and

the sulfate content in the leaf tissue (Table 2). The

expression levels of PtaSULTR1;1, PtaSULTR4;1, and

PtaSULTR4;2 were positively correlated (Table 2) but only

mRNA values of PtaSULTR4;2 correlated negatively to

maximum temperature.

Discussion

Characteristics of SULTR sequences

In the present study, the sulfate transporter-encoding gene

family of Populus tremula 9 P. alba was investigated. The

overall topology of the SULTR sequences PtaSULTR1;1,

PtaSULTR3;3a, PtaSULTR4;1, and PtaSULTR4;2 fits with

the proposed 12 membrane spanning domain (MSD) model

(Smith et al. 1995; Hawkesford 2003). One Arg (R-391/390/

363/361) residue located between MSD 9 and 10, is con-

served among all sulfate transporter sequences from poplar

(this study), Arabidopsis thaliana, Stylosanthes hamata, and

Hordeum vulgare (Takahashi et al. 1997). As previously

proposed after work in other species this residue may be

involved in the binding of sulfate (Smith et al. 1995). In

Arabidopsis, the STAS domain, which extends into the

Fig. 4 In situ localization of

PtaSULTR1;1 in stem sections

where the 10th to 11th leaves

(counted from the apex) are

attached. Transverse stem

sections were hybridized with

the sense (a) or antisense (b–h)

probe for PtaSULTR1;1. a, bOverview of the transverse

section; c bark and cambial

zone; d epidermis and periderm

of the bark; e cambium and

phloem; f phloem; g primary

wood with pith rays; h primary

xylem at the pith. Enlarged parts

are indicated by squares. Barsare 200 lm for a–c and e and

20 lm for d, f–h.

Abbreviations: br phloem ray

cells, c cambium, cc companion

cell, co cortex, cu cuticle,

ep epidermis, f fusiform cell,

p phloem, ph periderm, pi pith,

pr pith ray cells, px primary

xylem, ri ray initials, se sieve

element, sf sclerenchymatous

fibres, sp sieve plate, x xylem,

xp xylem parenchyma, xv xylem

vessel

Plant Mol Biol

123

cytoplasm (Aravind and Koonin 2000), is necessary for the

correct localization of SULTR proteins within the mem-

brane; it also determines the kinetic characteristics of sulfate

transport (Rouached et al. 2005; Shibagaki and Grossman

2006). All putative poplar sulfate transporters of group 1, 2,

3, and 4 contain this STAS domain and are illustrated

for PtaSULTR1;1, PtaSULTR3;3a, PtaSULTR4;1, and

PtaSULTR4;2 (Fig. 1b) which exhibited significant identi-

ties (E \ 0.01 Pearson 2000) with genes encoding sulfate

transporters in Arabidopsis thaliana (http://blast.ncbi.nlm.

nih.gov/Blast.cgi). The poplar sequence PtaSULTR1;1

showed highest homology to the Arabidopsis sequence

AtSULTR1;3 of group 1, but a lower identity to the

AtSULTR1;1 and AtSULTR1;2 sequences. AtSULTR1;3 is

specifically expressed in the phloem of cotyledons and has

been functionally characterized (Yoshimoto et al. 2003).

PtaSULTR4;1 and PtaSULTR4;2; amino acid sequences

revealed highest identity with, and shared many character-

istics with, AtSULTR4;1 including a transit peptide

sequence (Fig. 1b). As AtSULTR4;1 is located in the tono-

plast and generates sulfate efflux (Kataoka et al. 2004b) it is

assumed that both SULTRs of group 4 from poplar also

functions as a sulfate efflux transporter in the tonoplast. The

two poplar sequences with highest similarity to Arabidopsis

AtSULTR5;2 lack characteristics of sulfate transporters such

as the STAS domain (Aravind and Koonin 2000). This is not

surprising since recently AtSULTR5;2 was identified as a

molybdate transporter (Tomatsu et al. 2007).

In terms of genome evolution, analyses of Arabidopsis

thaliana and poplar ESTs have revealed a whole genome

duplication event in poplar (salicoid duplication) (Tuskan

et al. 2006). Thus, a single-copy gene in Arabidopsis tha-

liana is typically represented by two copies in Populus

species (Jansson and Douglas 2007). This could be the

reason for the observation that two highly similar

PtaSULTR sequences (a and b; Fig. 1) showed significant

identity with only one sulfate transporter in Arabidopsis

thaliana. The possibility that closely related genes are

derived from the two combined genomes of the used hybrid

Populus tremula 9 P. alba can be exclude because corre-

sponding sequences are also represented in the Populus

trichocarpa genome.

Expression of sulfate transporters relevant for sulfate

loading into the phloem of leaves

Sulfate transporters are dependent on the proton motive

force and function as proton-symport carrier (Lass and

Ullrich-Eberius 1984; Hawkesford et al. 1993; Kataoka

et al. 2004b). During active growth, sulfate reaches the

leaves via xylem transport. Therefore, the expression of

PtaSULTR1;1 in leaf mesophyll cells may indicate that

Fig. 5 In situ localization of

PtaSULTR3;3a in stem sections

with the 10th to 11th leaves

(counted from the apex)

connected. Transverse sections

were hybridized with the sense

(a) or antisense (b–h) probe for

PtaSULTR3;3a. a, b Overview

of a transverse section;

c overview of the bark with

cambial zone; d epidermis and

phellem; e phloem and

cambium; f phloem; g primary

wood with pith ray cells;

h primary xylem and xylem

parenchyma at the pith.

Enlarged parts are indicated by

squares. Bars are 200 lm for

a–c and e and 20 lm for d, f–h.

Abbreviations: br phloem ray

cells, c cambium, cc companion

cell, co cortex, cu cuticle,

ep epidermis, f fusiform cell,

p phloem, ph periderm, pi pith,

pr pith ray cells, px primary

xylem, ri ray initials, se sieve

element, sf sclerenchymatous

fibres, x xylem, xp xylem

parenchyma, xv xylem vessel

Plant Mol Biol

123

PtaSULTR1;1 transports sulfate from the apoplast to the

symplast. Mature poplar leaves are sources of carbohy-

drates (Dickson 1991) and sulfate (Hartmann et al. 2000).

Minor leaf veins are associated with the collection phloem

and major leaf veins contribute to the transport phloem

(van Bel 2003). Thus the abundance of PtaSULTR1,1

mRNA in the phloem of minor leaf veins indicates the

importance of PtaSULTR1,1 in loading sulfate into the

phloem. A comparable function can be assumed for

PtaSULTR1;1 and PtaSULTR3;3a in major leaf veins, since

both transcripts were detected in the phloem. Accumulation

of sulfate in the bark starts in late autumn when the

expression of PtaSULTR1;1 in senescent leaves is high.

This indicates an increase in phloem loading of sulfate in

the leaves for sulfate storage in the stem (Fig. 9a). This was

supported by the observation, that the sulfate content in

leaves negatively correlated with the PtaSULTR1;1 mRNA

level. Moreover, the sulfate content in senescent leaves was

significantly lower compared to late summer. However,

whether other sulfur sources, for example thiols or proteins,

are mobilized and converted to sulfate still needs to be

investigated.

An interesting point is that, in leaves, the expression of

sulfate transporters is independent of weather parameters.

Only PtaSULTR4;2 showed a negative correlation with

maximum temperature (Table 2). Hence SULTR gene

expression seems mainly determined by a developmental

program in leaves. Environmental factors may become

more important 1 month after bud break and later in

summer (Wissel et al. 2003). Accumulation of sulfate, and

of PtaSULTR1;1 and PtaSULTR3;3a mRNAs in the leaves

was not constant throughout leaf development. After bud

break, sulfate decreased in expanding leaves, probably due

to a dilution effect caused by leaf expansion or by high

rates of sulfate assimilation. Simultaneously, PtaSULTR1;1

expression increased and reached a maximum when leaves

were fully expanded (Fig. 9b, c). Sulfate uptake into leaf

mesophyll cells, probably mediated by PtaSULTR1;1, may

remain high during leaf development until leaves are fully

expanded. The sulfate content in mature leaves increased

during early summer when the expression of PtaSULTR4;2

and PtaSULTR1;1 decreased. It can be assumed that the

decline in PtaSULTR4;2 and PtaSULTR1;1 mRNA restricts

both sulfate efflux from the vacuole and sulfate loading

into the phloem that enables sulfate accumulation in mature

leaves. Expression of the second phloem-specific sulfate

transporter, PtaSULTR3;3a, which is only expressed in

major leaf veins, increased in early summer when the

expression of PtaSULTR1;1 was low. Hence, phloem

re-loading gets relevant during the summer.

Fig. 6 In situ localization of

PtaSULTR1;1 in fine roots.

Transverse sections of fine roots

were hybridized with the sense

(a, c) or antisense (b, d–h)

probe for PtaSULTR1;1. a, bOverview of a transverse

section of fine roots; c, d cross

section of the stele with a

tetrarch xylem; e transverse

section of a tetrarch stele with

side root; f fine root whose stele

contains a diarch xylem. g, hlongitudinal sections of fine root

with developing side root.

Enlarged parts are indicated by

squares. A green arrow in c, dindicates phenolic compounds.

Bars are 200 lm for a and b and

50 lm for c–h. Abbreviations:

co cortex, ed endodermis,

ep epidermis, mx metaxylem,

p phloem, px protoxylem,

sr side root, st stele

Plant Mol Biol

123

Expression of sulfate transporters in stem tissues

relevant for sulfate storage and mobilization

PtaSULTR3;3a mRNA was detected in sieve elements and

companion cells along the entire stem. Since mature sieve

elements do not possess translation machinery (van Bel et al.

2002), the presence of PtaSULTR3;3a mRNA in sieve ele-

ments seems surprising. However, transcripts of several

cellular proteins including a sucrose transporter have been

found in sieve elements by in situ hybridization (Kuhn et al.

1997) or in phloem exudates (Doering-Saad et al. 2006;

Lough and Lucas 2006; Omid et al. 2007; Le Hir et al. 2008;

Kehr and Buhtz 2008). Leakage of PtaSULTR3;3a mRNA

from the companion cells into the sieve element for move-

ment by phloem mass flow may therefore possible. Such a

movement has been demonstrated by grafting experiments

for several sequences (referred in Kehr and Buhtz 2008).

PtaSULTR1;1 transcripts were strongly detected in

companion cells and ray initials throughout the stem. Fur-

thermore, 35S accumulation was observed in these cells after35S-sulfate was fed to mature leaves (Fig. 7). The accumu-

lation of 35S-sulfate in companion cells below the fed leaf

indicates sieve element unloading. van Bel and Kempers

(1990) found that the sieve element/companion cell com-

plex is symplastically isolated from the surrounding paren-

chyma in Salix alba and Ricinus. If this is also true for

poplar, sulfate must be transported out of the complex into

the apoplast. This unloading could be mediated by voltage

dependent anion channels (Frachisse et al. 1999; Roberts

2006; Fig. 9a). Phloem unloading via PtaSULTR1;1 seems

unlikely because sulfate transport into cells via the plasma

membrane depends on a proton motive force in plants (Lass

and Ullrich-Eberius 1984; Hawkesford et al. 1993) and, a

high Em of the sieve element/companion cell complex (van

Bel and Kempers 1990) probably facilitates sulfate uptake

from the apoplast. Correspondingly, in Populus tricho-

carpa, companion cells but not sieve elements possess

plasma membrane ATPases (Arend et al. 2002). Thus

PtaSULTR1;1 and PtaSULTR3;3a transporters might be

involved in the retrieval of sulfate leaking out of the phloem.

A comparable function has been postulated for sucrose

transporters similarly expressed along the transport phloem

(Williams et al. 2000; van Bel 2003; Carpaneto et al. 2005).

However, an accumulation of sulfate, which comes from

sieve elements, in companion cells cannot be achieved by

diffusion process through plasmodesmata between the two

cell types. This discrepancy can only be investigated by

further studies. For instance, by creating poplar plants

deficient in PtaSULTR1;1 and/or PtaSULTR3;3a expression.

However, a possible complementation of sulfate transport

into the phloem by other sulfate transporters of the gene

family has to be taken into account. This effect is evident

from double and triple sultr knockout mutants of Arabid-

opsis (Lydiate and Higgins, personal communication).

Fig. 7 Microautoradiographic

detection of 35S in stem sections

basal from a mature leaf to

which 35SO42- was fed into the

phloem via flap feeding.

a Overview of a transverse

section of the stem basal to the

fed leaf (209). b Phloem with

strong staining in companion

cells; c cambium with strong

staining in ray initials; d young

wood with pith rays. c to d were

taken at 50-fold extension. The

black grains indicate 35S sulfur.

Bars are 50 lm for a and 20 lm

for b–d. Abbreviations: cccompanion cell, f fusiform cell,

pr pith ray, ri ray initials, sesieve element, x xylem, xvxylem vessel

Plant Mol Biol

123

The phloem-specific sulfate transporter PtaSULTR3;3a

and the putative vacuolar efflux transporter PtaSULTR4;2

are positively correlated and both are negatively correlated

with the sulfate content in bark. The sulfate level in the bark

reached maximum values at the end of December, when

both PtaSULTR3;3a and PtaSULTR4;2 showed nearly no

expression. Since PtaSULTR4;2 is involved in sulfate

mobilization from the vacuole (Kataoka et al. 2004b), and

PtaSULTR3;3a is relevant for phloem loading (see above)

these observations are consistent (Fig. 9a). During bud

break, sulfur was mobilized from soluble and insoluble

storage pools in bark and wood tissues of beech (Herschbach

and Rennenberg 1996). Phloem transport enables swelling

buds to be supplied with metabolites during insufficient

xylem sap flow. The increased level of PtaSULTR4;2

mRNA, encoding a putative vacuolar sulfate transporter,

during bud swelling and early leaf development indicates

sulfate mobilization from the vacuole of bark cells (Fig. 9b).

Table 1 Correlation matrix of sulfate transporter transcript levels, sulfate content of bark and weather parameters

PtaSULTR1;1 PtaSULTR3;3a PtaSULTR4;1 PtaSULTR4;2 Sulfate

PtaSULTR3;3a 0.301

PtaSULTR4;1 0.546** 0.013

PtaSULTR4;2 0.363 0.881** 0.238

Sulfate -0.010 -0.621** 0.375 -0.521**

T mean -0.068 0.749** -0.454 0.596** -0.682**

T max -0.100 0.703** -0.421 0.583** -0.529**

T min 0.008 0.691** -0.399 0.491** -0.750**

Day length -0.067 0.814** -0.138 0.866** -0.639**

Values presented are Pearson correlation coefficients. Two asterisks represent significant positive or negative correlation at P \ 0.01

Fig. 8 Quantitative analyses of SULTR expression and sulfate

contents in bark (left column) and leaf (right column) tissue during

seasonal growth. a Sulfate content of leaves and bark from twigs

which developed in 2006 (light gray squares) and from twigs

which developed in 2007 (dark gray circles). PtaSULTR1;1

(b), PtaSULTR3;3a (c), PtaSULTR4;1 (d), PtaSULTR4;2 (e) and

expression of the elongation factor 1-beta (f) was analyzed in leaves

and bark from twigs developed in 2006 (light gray bars) and in 2007

(dark gray bars). Expression of mRNA copies was related to total

RNA

Plant Mol Biol

123

The expression of PtaSULTR1;1 and PtaSULTR3;3a

increased when sulfate in the bark declined. This strongly

supports the assumption that the sulfate transporters

PtaSULTR1;1 and PtaSULTR3;3a are involved in sulfate

uptake into the phloem during spring. ATPase protein also

increased in the cambium and phloem of Populus tricho-

carpa after cambium reactivation (Arend et al. 2002). If the

active sulfate uptake via SULTR requires a proton motive

force, built by plasma membrane H?-ATPases, the positive

correlation between the appearance of an ATPase protein

and PtaSULTR1;1 as well as PtaSULTR3;3a mRNA

supports the assumption that sulfate uptake into the phloem

is mediated by PtaSULTR1;1 and PtaSULTR3;3a. Sulfate

supply from storage vacuoles of the bark is no longer nec-

essary when leaves are maturing and thus the transcript level

of PtaSULTR4;2 decreased. Also PtaSULTR3;3a mRNA

abundance declined because sulfate loading into the trans-

port phloem gets less important. Interestingly, these effects

are strongly correlated to temperature and day length: strong

parameters for seasonality.

Table 2 Correlation matrix of sulfate transporter transcript levels, sulfate content of leaves and weather parameters

PtaSULTR1;1 PtaSULTR3;3a PtaSULTR4;1 PtaSULTR4;2 Sulfate

PtaSULTR3;3a -0.002

PtaSULTR4;1 0.587** 0.303

PtaSULTR4;2 0.725** 0.076 0.589**

Sulfate -0.645** 0.160 -0.275 -0.529

T mean -0.296 -0.057 -0.224 -0.506 0.383

T max -0.381 -0.057 -0.259 -0.638** 0.328

T min -0.266 -0.194 -0.328 -0.204 0.301

Day length 0.112 0.299 0.185 -0.187 -0.021

Values presented are Pearson correlation coefficients. Two asterisks represent significant positive or negative correlation at P \ 0.01

SULTR4;1SULTR1;1 SULTR3;3a SULTR4;2 anion channel

autumn early spring late spring(A) (B) (C)

Fig. 9 Hypothetical functions of SULTRs in leaves and bark during

autumn (a), early spring (b) and late spring (c). The black (phloem)

and grey (xylem) arrows indicate the flow of sulfate. Putative

functions were developed from the expression pattern of the phloem

localized SULTRs PtaSULTR1;1 (pink) and PtaSULTR3;3a (yellow)

and of the putative vacuole localized SULTRs, PtaSULTR4;1 (darkblue) and PtaSULTR4;2 (light blue). Green squares indicate the

hypothesized anion channel that allows sulfate efflux from companion

cells. The different abundance of sulfate transporter symbols between

each, early spring, late spring and autumn, indicates function. One

important assumption is that SULTR mRNA abundance correlates

with the protein abundance. Abbreviations: se sieve element,

cc companion cells, x xylem, v vacuole

Plant Mol Biol

123

After 35S-sulfate feeding to a mature leaf, 35S became

enriched in ray initials basal to the fed leaf. This indicates the

origin of stored sulfate by sulfate uptake into ray cells.

Hence, the high expression of PtaSULTR1;1 supports sulfate

uptake from the apoplast into ray initials. Further transport

within the ray pith could occur by cell to cell transport via

plasmodesmata. Sulfate storage along the stem in pith ray

cells is also possible after its removal from the xylem. In a

similar manner as the putative sucrose transporter JrSUT1

(Decourteix et al. 2006) PtaSULTR1;1 and PtaSULTR3;3a

encoding mRNAs are detected in pith cells that are con-

nected to xylem vessels. A prerequisite for sucrose and sul-

fate uptake into these cells again requires the presence of a

proton motive force. ATPase was detected in ray cells con-

nected with xylem vessels of Populus trichocarpa twigs after

cambial reactivation during spring (Arend et al. 2002). Both,

the expression of PtaSULTR1;1 and PtaSULTR3;3a as well

as the presence of a plasma membrane H?-ATPase support

an unloading of sulfate from the xylem into vessel-associated

ray cells. The same process could also be relevant for both

transporters in major leaf veins. However, several further

sulfate transporters were found in wood of poplar (see Sup-

plemental Fig. S1) that may contribute to xylem unloading of

sulfate during vegetative growth.

Expression of sulfate transporters in developing tissues

(apex and lateral roots)

Previous investigations showed that sulfate is transported

from leaves to the shoot apex and fine roots (Hartmann

et al. 2000). Whereas PtaSULTR3;3a transcripts were only

visible in the apical meristem of the shoot (SAM),

PtaSULTR1,1 transcripts were highly abundant in leaf

primordia, leaf margin meristems (LMM) and provascular

strands. Both sulfate transporters could be responsible for

sulfate uptake into dividing cells providing sulfate for

assimilation. The relevance of sulfate assimilation in het-

erotrophic and growing tissues is still unknown. Hartmann

et al. (2000) concluded that sulfate assimilation in the apex

is possible and has been demonstrated by 35S-sulfate

feeding to a separated apex (Herschbach 2003).

In fine roots from maize, highest APS reductase activity,

the regulatory step of sulfate reduction (Kopriva and

Koprivova 2004; Martin et al. 2005), has been detected in

the root tips (Kopriva et al. 2001). High PtaSULTR1;1

transcript levels in the root meristem of developing lateral

roots are consistent with these findings. Sulfate uptake via

PtaSULTR1;1 into dividing and growing cells supplies

sulfate for the sulfate reduction and assimilation pathway

relevant in poplar roots (Scheerer et al. 2009). The abun-

dance of PtaSULTR1;1 mRNA in the protophloem of fine

roots supports the idea of loading sulfate into the phloem

for further allocation to the root tip.

Conclusion

Our results provide novel evidence for the seasonal regu-

lation of sulfate storage, mobilization and distribution via

the phloem at the transcription level of sulfate transporters.

In the perennial model species Populus two sulfate trans-

porters, PtaSULTR3;3a and PtaSULTR1;1, are expressed in

the phloem of leaf veins and along the stem. Important

processes of deciduous trees such as poplar are sulfate

storage during winter and sulfate mobilization during

spring (Herschbach and Rennenberg 1996; Rennenberg

et al. 2007). These processes cannot be investigated in

Arabidopsis. The mRNA of PtaSULTR3;3a and of the

putative vacuolar located sulfate efflux transporter

PtaSULTR4;2 in the bark increased in spring during bud

break (summarized in Fig. 9) and correlated with the day

length, temperature and, most importantly, inversely with

sulfate content. In Arabidopsis, one function of the sulfate

transporters of group 4 seems to be the delivery of sulfate

from the vacuole for further loading into the xylem (Kat-

aoka et al. 2004b). Here, we present evidence for the

seasonal function of PtaSULTR4;2 and for its role in sul-

fate mobilization from the vacuole, and of PtaSULTR3;3a

and PtaSULTR1;1 for sulfate loading into the phloem.

Moreover, the clear correlation with temperature and day

length which are characteristic for seasonal changes

revealed environmental control for the expression of

PtaSULTR4;2 and PtaSULTR3;3a in the bark. In contrast,

the expression of sulfate transporters seems mainly under

developmental control in leaves. Therefore, the regulatory

signals, inducing changes in gene expression, may vary

among tissues and different sulfate transporters.

Acknowledgments This work was financial supported by the Deut-

sche Forschungsgemeinschaft (DFG) under the contract numbers HE

3003/2 & 3. The Excellence Initiative of the German Federal and State

Governments (EXC 294), SFB 592 and the Landesstiftung are grate-

fully acknowledged. The authors thank Simone Sikora and Katja Rapp

for technical assistance and Dr. William Teale to improve language.

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