www.elsevier.com/locate/plantsci

Plant Science 169 (2005) 798–804

Genes coding for a putative cell-wall invertase and two putative

monosaccharide/H+ transporters are expressed in roots

of etiolated Glycine max seedlings§

Maria Dimou a, Emmanouil Flemetakis a, Costas Delis a, Georgios Aivalakis a,Karoline G. Spyropoulos b, Panagiotis Katinakis a,*

a Department of Agricultural Biotechnology, Agricultural University of Athens, Iera Odos 75, 11855 Botanikos, Athens, Greeceb Department of Botany, Faculty of Biology, School of Sciences, National and Kapodistrian University of Athens,

Panepistimiopolis, Zografou, 15771 Athens, Greece

Received 8 February 2005; received in revised form 26 May 2005

Available online 22 June 2005

Abstract

Sink tissues depend on the supply of sugars produced by source tissues. Cell-wall invertases (EC 3.2.1.26) are considered to have a pivotal

role in supplying sink tissues with carbohydrates via an apoplasmic pathway, while associated monosaccharide/H+ transporters take up the

produced hexoses. In this study, we characterized genes coding for a putative cell-wall invertase (GmCWINV1) and two putative

monosaccharide/H+ transporters (GmMST1 and GmMST2) of Glycine max. Semi-quantitative RT-PCR analysis revealed that GmMST1

and GmMST2 are expressed in various plant organs. In situ hybridization revealed that they are expressed in different root tissues. These

results propose that different monosaccharide/H+ transporters may play different roles in source and sink organs. In addition, the temporal and

spatial expression of GmCWINV1, as was determined by semi-quantitative RT-PCR and in situ hybridization analyses, was detected in tissues

where GmMST1 and GmMST2 were also expressed, indicating that, at least part of the sucrose unloaded from soybean phloem may be

hydrolyzed into hexoses before being transported from the apoplasmic space into the respective sink cells.

# 2005 Elsevier Ireland Ltd. All rights reserved.

Keywords: Cell-wall invertase; Glycine max; In situ hybridization; Monosaccharide/H+ transporter; Root; Phloem unloading

1. Introduction

The assimilated carbon is exported from the source

tissues and transported to the non-photosynthetic sink

tissues via phloem, mainly in the form of sucrose. Phloem

unloading to the sink tissues may take place either

symplasmically via plasmodesmata or apoplasmically

across the plasma membrane [1]. In the latter case, sucrose

Abbreviations: BCIP, 5-bromo-4-chloro-3-indolyl-phosphate; BSA,

bovine serum albumin; EDTA, ethylenediaminetetraacetic acid; EST,

expressed sequence tag; NBT, nitro blue tetrazolium

Nucleotide sequence data are available in the EMBL database under

accession numbers AJ563366 (GmMST1), AJ563365 (GmMST2),

AJ563368 (GmCWINV1).

* Corresponding author. Tel.: +30 210 529 4342; fax: +30 210 529 4314.

E-mail address: bmbi2kap@aua.gr (P. Katinakis).

0168-9452/$ – see front matter # 2005 Elsevier Ireland Ltd. All rights reserved

doi:10.1016/j.plantsci.2005.05.037

may be taken up by sink cells either intact via sink specific

sucrose transporters or after extracellular hydrolysis by cell-

wall invertases (EC 3.2.1.26), to glucose and fructose, via

monosaccharide/H+ transporters, referred as sugar transport

proteins (STP) [1,2].

Genes and cDNA clones encoding cell-wall invertases

have been identified and characterized from a variety of

plants, and are often found as multigene families that show

sink tissue-enhanced expression pattern [3,4]. It has been

suggested that they may play a role in photoassimilate

partitioning and source/sink regulation [5,6]. However, the

importance of cell-wall invertases came from the analysis of

the miniature1 mutant of maize, which due to the

abolishment of an endosperm specific cell-wall invertase

shows a small seed phenotype [7]. Furthermore, antisense

inhibition of cell-wall invertase in carrot resulted in the

.

M. Dimou et al. / Plant Science 169 (2005) 798–804 799

arrest of taproot formation [8]. In both cases, provision of

apoplasmic hexoses is apparently required for normal

development.

Hexoses liberated by cell-wall invertases are taken up by

associated monosaccharide/H+ transporters and a functional

coupling is supported by a temporal and spatial association

of gene expression [2,9–12]. Genes encoding plant

monosaccharide/H+ transporters are grouped in multigene

families with different members being up-regulated in

various sinks [1,13,14]. The observations that different

Arabidopsis thaliana sugar transport proteins (AtSTPs) have

been shown to catalyze the uptake of exogenous sugars in

heterologous systems [15], and that a knock-out mutation in

the AtSTP1 gene resulted in a decrease in uptake of

exogenous monosaccharides by A. thaliana seedlings [16],

are consistent with AtSTPs functioning in the plasma

membrane to import monosaccharides from the apoplasmic

space. It remains to be determined whether all 14 putative

AtSTPs catalyze monosccaharide transport, and what

physiological functions these and other related proteins

have [15,16].

In the present work, we report the expression of a putative

cell-wall invertase and two putative monosaccharide/H+

transporter genes (designated as GmCWINV1, GmMST1 and

GmMST2, respectively) during primary and lateral root

development of etiolated soybean seedlings. Based on the

temporal and spatial accumulation of transcripts, possible

functions of putative cell-wall invertase and monosacchar-

ide/H+ transporter isoforms are discussed.

2. Materials and methods

2.1. Plant material and growth conditions

Soybean (Glycine max cv. Williams) seeds were pre-

germinated on two sheets of moist paper, in Petri dishes, in

darkness, at 26 8C for 2 days. After germination, the

seedlings were transferred in a modified 0.5 � Hoagland

solution, under a 16-h light/8-h dark cycle, at 26 8C. Young

leaves (YL), mature leaves (ML) and stems (STEMS) of 30

days old plants were used to isolate total RNA. In addition,

some seedlings were left in Petri dishes, in darkness, at

26 8C for 6 more days, and total RNA was isolated from

hypocotyls (HYP), cotyledons (COT) and different parts of

roots, representing the region of cell division (RT), the root

region of elongation (MRI) and the region of laterals

emergence (MRII). For in situ hybridization, the roots from

the 8 days old seedlings were used.

2.2. Characterization of cDNA clones coding for

GmMST1, GmMST2 and GmCWINV1

By performing BLAST searches [17] among the

databases of the American Public Soybean Expressed

Sequence Tag Project, two cDNA clones coding for putative

monosaccharide/H+ transporters, and one cDNA clone

coding for a putative cell-wall invertase were identified

and obtained from ResGen, Invitrogen Corp. Their

nucleotide sequences were determined and as parts of their

50 ends were missing, total RNAwas extracted from the roots

of 8 days old seedlings [18] and a 50 RACE-PCR was

performed with the SMART 50 RACE kit from Clontech

(Westburg, NL). The amplified fragments were cloned into

the pBlueScriptKS+ plasmid vector (STRATAGENE) and

fully sequenced. The full-length cDNA clones were

designated as GmMST1, GmMST2 and GmCWINV1. The

clustal procedure was used for both analysis of the sequences

data and determination of the relatedness of the deduced

polypeptides to related sequences from other species.

2.3. RT-PCR analysis of GmMST1, GmMST2 and

GmCWINV1

Semi-quantitative RT-PCR analysis was performed using

total RNA extracted from the tissues of interest as described

above and quantified by spectrophotometry and agarose gel

electrophoresis. All RNA samples were treated with DNase I

(PROMEGA) according to the manufacturer’s instructions to

eliminate DNA contamination. In each case, the RT-PCR

reactions were performed, according to the manufacturer’s

directions (QIAGEN) using gene-specific oligonucleotides

as primers (GmCWINV1F: 50-CTGCTTGGCTAGGCAA-

AG-30, GmCWINV1R: 50-ACCATTCACCGACGTGTC-30,GmMST1F: 50-CTGAGACACCAAACAGCC-30, GmMS-

T1R: 50-CACAGCCAAGACTGCACC-30, GmMST2F: 50-AGGCTGTAGTTGCAGCTG-30 and GmMST2R: 50-TAAT-

CATCATGCTCCACG-30). RNase inhibitor was obtained

from PROMEGA. For normalization of the different RNA

preparations, a 260 bp fragment of GmUBQ was amplified,

using two gene-specific oligonucleotides as primers

(GmUBQF: 50-GGGTTTTAAGCTCGTTGT-30 and GmU-

BQR: 50-GGACACATTGAGTTCAAC-30). The reverse

transcriptase reactions were performed in a thermal cycler

at 50 8C for 30 min, followed by PCR amplification of 25–30

cycles at 94 8C for 1 min, 54 8C for 1 min and 72 8C for 1 min.

A complete final extension for the PCR products was

performed at 72 8C for 10 min. Amplified products were

separated on 1.5% agarose gels, blotted on to nylon

membranes and hybridized with the respective digoxi-

genin-11-rUTP labeled cDNA probes.

2.4. In situ mRNA hybridization

Root segments were fixed in 4% (w/v) paraformaldehyde

supplemented with 0.25% (v/v) glutaraldehyde in 10 mM

sodium phosphate buffer (pH 7.4), for 1 h, in a vacuum

aspirator, dehydrated with ethanol, and then exchanged with

xylene before embedding in paraffin. Sections (8 mm) were

mounted on poly-L-lysine slides, digested with proteinase K

for 30 min at 37 8C, treated with acetic anhydride, dried in

ethanol, and then hybridized with appropriate gene specific

M. Dimou et al. / Plant Science 169 (2005) 798–804800

digoxigenin-labeled probes overnight, at 42 8C. After

washing with 4 � SSC containing 5 mM dithiothreitol,

the slides were treated with RNase A for 30 min, at 37 8C,

washed again at 37 8C with RNase A buffer (500 mM NaCl,

1 mM EDTA, 10 mM Tris–HCl, pH 7.5) containing 5 mM

dithiothreitol, and then processed for revealing the

digoxigenin antigen. This involved blocking with blocking

reagent and BSA, followed by incubation with an anti-

digoxigenin antibody conjugated to alkaline phosphatase,

and washing with blocking reagent. The color was revealed

by incubation in NBT and BCIP and the reactions were

stopped with water, slides dehydrated, air-dried, and then

mounted in DPX before viewing. Antisense and sense

probes labeled with digoxigenin-11-rUTP (ROCHE) were

used in parallel hybridizations. The sections were examined

using a ZEISS Axiolab (Carl Zeiss, Jena, Germany)

microscope and pictures were taken with SONY DSC-

S75 38P/45 (SONY Corporation, Japan) digital camera

system.

3. Results

3.1. Characterization of G. max cDNA clones coding

for putative monosaccharide/H+ transporters and a

putative cell-wall invertase

Systematic BLAST searches among the public databases

revealed the presence of a number of soybean ESTs derived

from root cDNA libraries showing high homology to

monosaccharide/H+ transporter genes from different plant

species. Several of these cDNA clones were obtained, and

Fig. 1. Relatedness of GmMST1, GmMST2 and GmCWINV1 to protein sequ

transporters and invertases, respectively, which were identified in databanks and a

alignments, using Clustal method with PAM250 residue weight table, are shown

numbers are: (A) Arabidopsis thaliana, AtSTP1 (CAA39037); Ricinus communis, R

Nicotiana tabacum, NtMST1 (CAA47324); Picea abies, PaMst-1 (CAB0607

(CAD91335). (B) Vigna radiata, VrINVA (BAA01107); Vicia faba, VfINVA (

carota, DcInv4 (CAA77268); Zea mays, ZmIVR1 (AAA83439); Lycopersicon escu

Daucus carota, DcINV1 (AAA03516); Daucus carota, DcINV2 (CAA55189); Vici

Zea mays, Incw4 (AAD02264); Zea mays, Incw1 (AAA64487); Zea mays, Incw3 (A

Vulgare, HvCWINV1 (CAD58960); Daucus carota, DcNI (CAA76145) and Gly

their nucleotide sequences were determined. Alignment of

these sequences allowed the identification of two distinct

cDNA clones encoding putative monosaccharide/H+ trans-

porters. Since the two cDNA clones were partial, the missing

50 ends were amplified by a 50-rapid amplification of cDNA

ends (50-RACE) PCR. Both clones were fully sequenced and

designated as GmMST1 (AJ563366) and GmMST2

(AJ563365). In public databases, there are approximately

9 soybean ESTs related to GmMST1 and 35 soybean ESTs

related to GmMST2. Hydropathy analyses of the deduced

aminoacid sequences indicated the existence of a six

transmembrane spanning domain followed by a central

hydrophilic region and another six transmembrane spanning

domain structure, a structure common to plant sugar porter

family members of the Major Facilitator Superfamily [19]

(data not shown). A search for related sequences in the

public databases, at the amino acid level, revealed high

similarities with monosaccharide/H+ transporters from

various plant species. To investigate the relatedness of the

GmMST1 and GmMST2 to protein sequences from other

plant sugar transporters, a dendrogram was constructed

(Fig. 1A). GmMST1 clusters with PaMst-1 from Picea abies

while GmMST2 is related with a number of transporters that

appear to be expressed preferentially in sink tissues.

A similar approach was employed to identify ESTs

encoding for cell-wall invertases. A cDNA from a soybean

root library that encodes a cell-wall invertase was identified,

and a full-length clone was generated by a 50-rapid

amplification of cDNA ends PCR. Its nucleotide sequence

was determined and the clone was designated as

GmCWINV1 (AJ563368). In public databases, there are

approximately six soybean ESTs related to GmCWINV1.

ences from other plant species encoding full-length monosaccharide/H+

ligned using the ClustalW procedure. The dendrograms derived from these

. The sequences used for the analyses and their corresponding accession

cSTC (AAA79761); Vicia faba, VfSTP1 (T12199); MtMST1 (AAB06594);

9); Glycine max, GmMST1 (CAD91336) and Glycine max, GmMST2

CAA89992); Solanum tuberosum, POTBFRUASE (AAA50305); Daucus

lentum, LeLin6 (AAM28823); Nicotiana tabacum, NtNIN88 (AAK57505);

a faba, VfCWINV2 (T12095); Chenopodium rubrum, CrCIN1 (CAA57389);

AD02263); Triticum aestivum, TaIVR1 (T06163); Hordeum vulgare subsp.

cine max, GmCWINV1 (CAD91338).

M. Dimou et al. / Plant Science 169 (2005) 798–804 801

A search for related sequences in the public databases

revealed strong similarities with cell-wall invertases. An

alignment of the deduced GmCWINV1 cell-wall invertases

from other plant species revealed a conserved b-fructosidase

(Asn54, Asp55, Pro56, Asn57, Gly58) and a catalytic cysteine

motif (Met233, Trp234, Glu235, Cys236, Pro237). The fifth

residue in the cysteine motif is a proline that is specific to

cell-wall invertases [4]. A basic pI of 8.84 was calculated

using the ExPasy sequence analysis tool, suggesting that in a

cell-wall environment, where the pH is acidic, the

GmCWINV1 will be ionically bound with cell-wall compo-

nents. A dendrogram was constructed in order to investigate

the relatedness of GmCWINV1 to protein sequences from

other plant invertases (Fig. 1B). The sequences were divided

into three different classes representing neutral, vacuolar and

cell-wall invertases. GmCWINV1 clusters with the group of

other cell-wall invertases.

3.2. Accumulation of GmMST1, GmMST2 and

GmCWINV1 transcripts in various G. max organs

The accumulation of GmMST1, GmMST2 and

GmCWINV1 gene transcripts in various organs of 30 days

old soybean plants (young leaves, mature leaves and stems)

and 8 days old etiolated soybean seedlings (hypocotyls,

cotyledons, the root region of cell division, elongation and

laterals emergence) was investigated using a semi-quanti-

tative reverse transcription-PCR approach. A housekeeping

gene coding for ubiquitin was used as an internal control. As

shown in Fig. 2, expression of GmCWINV1 gene was

observed in all organs examined. The lower levels of

expression were found in mature leaves of 30 days old plants

and in cotyledons of 8 days old etiolated seedlings. The

highest levels of GmMST1 transcript accumulation were

observed in the root tip region (RT) and in hypocotyls of

Fig. 2. Semi-quantitative reverse transcription-polymerase chain reaction

analysis of the expression profiles of GmMST1, GmMST2 and GmCWINV1

genes. Total RNA from young leaves (YL), mature leaves (ML) and stems

(STEMS) of 30 days old plants, and hypocotyls (HYP), cotyledons (COT),

and different parts of roots, representing the region of cell division (RT), the

root region of elongation (MRI) and the region of laterals emergence (MRII)

of 8 days old etiolated seedlings was subjected to reverse transcription-

polymerase chain reaction analysis. The RT-PCR product of the GmUBQ

was used as an internal control. Amplified products were separated on 1.5%

agarose gels, blotted on nylon membranes and hybridized with the respec-

tive digoxigenin-11-rUTP labeled cDNA probes.

8 days old etiolated seedlings and in stems and young leaves

of 30 days old plants. Relatively lower levels were found in

cotyledons of 8 days old etiolated seedlings and in the root

region of elongation (MRI) and the region of laterals

emergence (MRII) of 8 days old etiolated seedlings. On the

contrary, the GmMST2 transcripts were detected in all

organs examined and particularly in the root tip region of

8 days old etiolated seedlings.

3.3. Spatial expression of GmMST1, GmMST2 and

GmCWINV1 transcripts in primary and lateral roots of

etiolated G. max seedlings

Roots of 8 days old etiolated seedlings are depleted of

their own reserves, as has been judged from starch, lipid and

protein staining of root sections (data not shown), and

therefore are typical sink tissues. So we further studied the

expression of GmMST1, GmMST2 and GmCWINV1 during

root development, using the in situ RNA–RNA hybridization

method. Longitudinal and transverse sections of primary and

lateral roots of 8 days old etiolated soybean seedlings were

hybridized with 11-digoxigenin-rUTP-labeled RNA probes

transcribed from GmMST1, GmMST2 and GmCWINV1

cDNA clones (Fig. 3). In longitudinal sections, GmCWINV1

and GmMST2 expression was detected in the root tip region

and especially in the cortex parenchyma of root tip and in the

central root cap region (Fig. 3N and I). Levels of both

transcripts, in the phloem region and in a few cell layers

neighbouring the central tissue, gradually increased from the

region of cell division to the region of elongation and then

decreased in the lateral root emergence region. As the cells

differentiated, GmCWINV1 and GmMST2 expression was

confined in the epidermis, some underlying cell layers, and

the vascular cylinder (Fig. 3N and I).

In order to identify cell types in which GmCWINV1 and

GmMST2 are expressed, we carried out in situ hybridizations

in cross-sections. On the basis of the anatomical features of

root regions we chose sections representing the zone of cell

division, elongation zone as well as the region of lateral root

emergence. As it can be observed in Fig. 3K and F, the cells

in the root cap as well as the cells in other layers in the region

of cell division were strongly stained. In the region of

elongation, the transcripts were strongly detected in cells of

the epidermis and vascular cylinder (Fig. 3L and G).

Hybridization signal was detectable in the epidermis of the

region of maturation and the cortex parenchyma (Fig. 3M

and H).

When similar sections from the same root tissues were

hybridized using an antisense probe of GmMST1, hybridiza-

tion signal was detectable in all different cell types (Fig. 3A–

D). However, root cap enlarging cells as well as enlarging

cortical parenchyma cells give higher signal.

Cross-sections of the primary root containing the lateral

root primordia also revealed that all genes were mainly

expressed in the apical region of the emerging lateral root

and in the phloem of the main root (Fig. 3E, J and O). In

M. Dimou et al. / Plant Science 169 (2005) 798–804802

Fig. 3. In situ localization of GmMST1, GmMST2 and GmCWINV1 transcripts in primary and lateral roots of 8 days old etiolated soybean seedlings.

Hybridization signal is visible as blue–purple precipitate. (A–E) Sections hybridized with the antisense probe for the GmMST1 gene. (A) Cross-section at about

170 mm from the root tip. (B) Cross-section at about 550 mm from the root tip. (C) Cross-sections at about 1.1 mm from the root tip. (D) Longitudinal section

from the root tip region. (E) Cross-section from the lateral root initiation zone of the primary root (approximately 4.5 cm from the primary root tip). (F–J)

Sections hybridized with the antisense probe for the GmMST2 gene. (F) Cross-section at about 170 mm from the root tip. (G) Cross-section at about 550 mm

from the root tip. (H) Cross-section at about 1.1 mm from the root tip. (I) Longitudinal section from the root tip region. (J) Cross-section from the lateral root

initiation zone of the primary root (approximately 4.5 cm from the primary root tip). (K–O) Sections hybridized with the antisense probe for the GmCWINV1

gene. (K) Cross-section at about 170 mm from the root tip. (L) Cross-section at about 550 mm from the root tip. (M) Cross-sections at about 1.1 mm from the

root tip. (N) Longitudinal section from the root tip region. (O) Cross-section from the lateral root initiation zone of the primary root (approximately 4.5 cm from

the primary root tip). (P) Cross-section from the lateral root initiation zone of the primary root hybridized with the sense probe for the GmCWINV1 gene. rc, root

cap; e, epidermis; v, vascular cylinder; m, meristem; c, cortex. Scale bars: 250 mm.

developing lateral roots, strong signal was observed in

epidermis, developing cortex parenchyma, developing

vascular bundles and emerging root cap (Fig. 3E, J and

O). As a negative control, sections were hybridized to sense

RNA probes transcribed from the corresponding cDNAs. In

that case, no significant signal was detected (Fig. 3P).

4. Discussion

The unloading of carbohydrates from the phloem into

various sink tissues, and the post-phloem transport within a

sink organ or to its adjacent sink, can occur symplasmically

via plasmodesmata or apoplasmically across the plasma

membrane [1]. In the latter case, sucrose unloaded from the

phloem may be taken up either directly by sink specific

sucrose transporters or after extracellular hydrolysis by cell-

wall invertases [1,2,20].

Expression of the GmCWINV1 gene, as was determined

by RT-PCR, was detected in all organs examined (Fig. 2).

The localization of GmCWINV1 mRNA (Fig. 3K–O) in the

phloem as well as in epidermal, cortical and root cap cells of

the primary root is in accordance with data from in situ RT-

PCR hybridization studies in maize root tips, where it has

been shown that Incw3 was expressed mainly in the root cap,

the phloem, and the epidermis [21]. Association of invertase

gene expression with the phloem has been noted previously

[22,23], and the physiological role of this may be the

M. Dimou et al. / Plant Science 169 (2005) 798–804 803

maintenance of a steep sucrose concentration gradient

between source and sink regions of the plant and/or the

supply of hexoses, and thus energy, for the metabolism of

companion cells.

The expression of GmCWINV1 in the cell division area of

the root, at a stage when mitotic activity proceeds in the root

tip, might be critical for the supply of fast dividing cells with

hexoses, which in turn may act as a developmental signal.

The importance of the high ratio of hexoses to sucrose in

promoting mitotic activity has been indicated [10,24]. In the

root tip region, GmCWINV1 may, also, double the osmotic

contribution of unloaded sucrose to apoplasmic osmotic

pressures that affect sieve element turgor, and hence rates of

photoassimilate import, irrespective of the primary pathway

of unloading [5,25]. Furthermore, biochemical and physio-

logical studies provide evidence that cell-wall invertases are

most active at sites of sugar transport within rapidly

importing apoplasmic sinks [4,5].

Sink tissues can acquire the products of extracellular

hydrolysis of sucrose, via monosaccharide transport

proteins. Our in situ hybridization experiments have

indicated that the GmMST1 gene was mainly expressed in

the regions of cell division and elongation of the primary

root (Fig. 3A–E). The cells expressing the GmMST1 gene

were generally located in the vascular cylinder as well as in

the epidermis and underlying cell layers of the region of

elongation of the primary root while lower levels of

expression were detected in the cortex parenchyma.

Furthermore, expression of the GmMST1 gene was strongly

up-regulated at the lateral root primordia emerging from the

primary root.

Expression of GmMST2 was observed both in sink and

source organs, as was determined by RT-PCR analysis

(Fig. 2). In situ hybridization analysis revealed that

GmMST2 was predominantly expressed in the phloem of

the primary root as well as in most of the cells of the lateral

root primordia (Fig. 3J). In general, plant monosaccharide/

H+ transporters are thought to function in hexose import into

sink tissues [15,26], although expression associated with

source tissues has been reported [15,27], where they may

play a role in retrieval of monosaccharides lost to the

apoplasmic space by passive leakage [15].

The present study strongly indicates that the unloaded

sucrose in the apoplasmic space may be hydrolyzed into

hexoses by a cell-wall invertase, while the distribution of

the GmCWINV1 mRNA in the epidermis, cortex and root

cap cells suggests that the post-phloem long-distance

transport of sucrose may also be partly apoplasmic. There is

evidence that monosaccharide/H+ transporter gene expres-

sion is coordinately induced with that of cell-wall

invertases. This regulatory mechanism results in higher

tissue uptake of sucrose, due to the apoplasmic invertase

activity, and of hexoses, due to the monosaccharide/H+

transporter activity [9–12,28]. However, such a regulatory

mechanism in etiolated soybean roots remains to be

confirmed.

In conclusion, we have characterized three genes from G.

max that code for two putative monosaccharide/H+

transporters, GmMST1 and GmMST2, and one putative

cell-wall invertase, GmCWINV1. Our temporal and spatial

gene expression studies revealed that GmMST1 and

GmMST2 are expressed in the same organs at the same

developmental stage, albeit at different tissues, suggesting

that different monosaccharide/H+ transporters may play

different roles in both source and sink organs and in various

tissues during root development. In accordance to that, it

would be of great interest to compare the two proteins in

terms of substrate specificity and kinetic properties of

transport activity. Furthermore, these expression patterns are

similar enough with the expression pattern of the

GmCWINV1 gene, suggesting, that, at least part of the

unloaded sucrose in soybean root may be hydrolyzed into

hexoses before being transported from the apoplasmic space

into the respective sink cells.

References

[1] L.E. Williams, R. Lemoine, N. Sauer, Sugar transporters in higher

plants—a diversity of roles and complex regulation, Trends Plant Sci.

5 (2000) 283–290.

[2] W. Eschrich, Free space invertase, its possible role in phloem unload-

ing, Ber. Dtsch. Bot. Ges. 93 (1980) 363–378.

[3] D.E. Godt, T. Roitsch, Regulation and tissue specific distribution of

mRNAs for three extracellular invertase isoenzymes of tomato sug-

gests an important function in establishing and maintaining sink

metabolism, Plant Physiol. 115 (1997) 273–282.

[4] Z. Tymowska-Lalanne, M. Kreis, Expression of the Arabidopsis

thaliana invertase gene family, Planta 207 (1998) 259–265.

[5] T. Roitsch, R. Ehneb, M. Goetz, B. Hause, M. Hofmann, A. Krishna

Sinha, Regulation and function of extracellular invertases from higher

plants in relation to assimilate partitioning, stress responses and sugar

signaling, Aust. J. Plant Physiol. 27 (2000) 815–825.

[6] A. Sturm, Invertases. Primary structures, functions, and roles in

plant development and sucrose partitioning, Plant Physiol. 121

(1999) 1–7.

[7] W.H. Cheng, E.W. Talliercio, P.S. Chourey, The miniature1 seed locus

of maize encodes a cell wall invertase required for normal develop-

ment of endosperm and maternal cells in the pedicel, Plant Cell 8

(1996) 971–983.

[8] G.Q. Tang, M. Luscher, A. Sturm, Antisense repression of vacuolar

and cell wall invertase in transgenic carrot alters early plant devel-

opment and sucrose partitioning, Plant Cell 11 (1999) 177–189.

[9] V. Fotopoulos, J.K. Pittman, A.C. Marver, A.J. Buckana, N. Sauer, J.L.

Hall, L.E. Williams, The monosaccharide transporter gene, AtSTP4,

and the cell-wall invertase, Atbetafruct1, are induced in Arabidopsis

during infection with the fungal biotroph Erysiphe cichoracearum,

Plant Physiol. 132 (2003) 821–829.

[10] H. Weber, L. Borisjuk, U. Heim, P. Buchner, U. Wobus, Seed coat-

associated invertases of fava bean control both unloading and storage

functions: cloning of cDNAs and cell type-specific expression, Plant

Cell 7 (1995) 1835–1846.

[11] H. Weber, L. Borisjuk, U. Heim, N. Sauer, U. Wobus, A role for sugar

transporters during seed development: molecular characterization of a

hexose and a sucrose carrier in fava bean seeds, Plant Cell 9 (1997)

895–908.

[12] W. Weschke, R. Panitz, S. Gubatz, Q. Wang, R. Radchuk, H. Weber, U.

Wobus, The role of invertases and hexose transporters in controlling

M. Dimou et al. / Plant Science 169 (2005) 798–804804

sugar ratios in maternal and filial tissues of barley caryopses during

early development, Plant J. 33 (2003) 395–411.

[13] A. Schneidereit, J. Scholz-Starke, M. Buttner, Functional character-

ization and expression analyses of the glucose-specific AtSTP9 mono-

saccharide transporter in pollen of Arabidopsis, Plant Physiol. 133

(2003) 182–190.

[14] J. Scholz-Starke, M. Buttner, N. Sauer, AtSTP6, a new pollen-specific

H+-monosaccharide symporter from Arabidopsis, Plant Physiol. 131

(2003) 70–77.

[15] M. Buttner, N. Sauer, Monosaccharide transporters in plants: structure,

function and physiology, Biochim. Biophys. Acta 1465 (2000)

263–274.

[16] S.M. Sherson, H.L. Alford, S.M. Forbes, G. Wallace, S.M. Smith,

Roles of cell-wall invertases and monosaccharide transporters in the

growth and development of Arabidopsis, J. Exp. Bot. 54 (2003) 525–

582.

[17] S.F. Altschul, T.L. Madden, A.A. Schaffer, J. Zhang, Z. Zhang, W.

Miller, D.J. Lipman, Gapped BLAST and PSI-BLAST: a new gen-

eration of protein database search programs, Nucleic Acids Res. 25

(1997) 3389–3402.

[18] J.A. Brusslan, E.M. Tobin, Light-independent developmental regula-

tion of cab gene expression in Arabidopsis thaliana seedlings, Proc.

Natl. Acad. Sci. U.S.A. 89 (1992) 7791–7795.

[19] R. Lemoine, Sucrose transporters in plants: update on function and

structure, Biochim. Biophys. Acta 1465 (2000) 246–262.

[20] N. Sauer, K. Baier, M. Gahrtz, R. Stadler, J. Stolz, E. Truernit, Sugar

transport across the plasma membranes oh higher plants, Plant Mol.

Biol. 26 (1994) 1671–1679.

[21] J.Y. Kim, A. Mahe, S. Guy, J. Brangeon, O. Roche, P.S. Chourey, J.L.

Prioul, Characterization of two members of the maize gene family,

Incw3 and Incw4, encoding cell-wall invertases, Gene 245 (2000)

89–102.

[22] P.E. Hedley, A.L. Maddison, D. Davidson, G.C. Machray, Differential

expression of invertase genes in internal and external phloem tissues of

potato (Solanum tuberosum L.), J. Exp. Bot. 51 (2000) 817–821.

[23] L. Zhang, N.S. Cohn, J.P. Mitchell, Induction of a pea cell-wall

invertase by wounding and its localized expression in phloem, Plant

Physiol. 112 (1996) 1111–1117.

[24] H. Weber, P. Buchner, L. Borisjuk, U. Wobus, Sucrose metabolism

during cotyledon development of Vicia faba L. is controlled by the

concerted action of both sucrose-phosphate synthase and sucrose

synthase: expression patterns, metabolic regulation and implications

for seed development, Plant J. 9 (1996) 841–850.

[25] S. Lalonde, M. Tegeder, M. Throne-Holst, W.B. Frommer, J.W.

Patrick, Phloem loading and unloading of sugars and amino acids,

Plant Cell Environ. 26 (2003) 37–56.

[26] S. Lalonde, E. Boles, H. Hellmann, L. Barker, J.W. Patrick, W.B.

Frommer, The dual function of sugar carriers: transport and sugar

sensing, Plant Cell 11 (1999) 707–726.

[27] A. Weig, J. Franz, N. Sauer, E. Komor, Isolation of a family of cDNA

clones from Ricinus communis L. with close homology to the hexose

carriers, J. Plant Physiol. 143 (1994) 178–183.

[28] R. Ehness, M. Ecker, E. Godt, T. Roitsch, Glucose and stress inde-

pendently regulate source and sink metabolism and defense mechan-

isms via signal transduction pathways involving protein

phosphorylation, Plant Cell 9 (1997) 1825–1841.