Plant Cell Physiol. 49(10): 1607–1613 (2008)
doi:10.1093/pcp/pcn134, available online at www.pcp.oxfordjournals.org
ß The Author 2008. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.
All rights reserved. For permissions, please email: [email protected]
A Maltose Transporter from Apple is Expressed in Source and Sink Tissues
and Complements the Arabidopsis Maltose Export-Defective Mutant
Edwin J. Reidel
1, 2, 3
, Robert Turgeon
2
and Lailiang Cheng
1,
*
1
Department of Horticulture, Cornell University, Ithaca, NY 14853, USA
2
Department of Plant Biology, Cornell University, Ithaca, NY 14853, USA
Prior to the cytosolic synthesis of transport sugars during
transitory starch utilization, intermediate products of starch
breakdown, such as maltose, must be exported from chloroplasts. Recent work in Arabidopsis indicates that a novel
transporter mediates maltose transfer across the chloroplast
inner envelope membrane. We cloned a gene from an apple
cDNA library that is highly homologous with the Arabidopsis
maltose transporter, MEX1. Expression levels of MdMEX
determined by real-time PCR were low in the tips of growing
shoots, higher in expanding leaves and maximal in mature
leaves. Expression was also detected in fruits and roots,
indicating a role for MdMEX in starch mobilization in sink
tissues. The cDNA from apple was subcloned into an
expression cassette between the cauliflower mosaic virus 35S
promoter and the sGFP (green fluorescent protein) coding
sequence. Plants of the Arabidopsis maltose excess1-1 mutant,
which is homozygous for a defective MEX1 allele, were
transformed with the 35S:MdMEX:GFP construct.
Fluorescence of GFP was localized to chloroplasts, indicating
that Arabidopsis recognized the predicted 55 amino acid
chloroplast transit peptide in the apple protein. The phenotypes of several independently transformed lines were analyzed. The complemented plants were relieved of the severe
stunting and chlorosis characteristic of mex1-1 plants.
Furthermore, starch levels and concentrations of soluble
sugars, leaf chlorophyll content and maximum quantum efficiency of PSII were restored to wild-type levels. MdMEX
(Malus domestica maltose transporter) is the second member
of the unique maltose transporter gene family.
Keywords: Apple — Maltose transporter — Malus domestica — MEX.
Abbreviations: CaMV, cauliflower mosaic virus; EST,
expressed sequence tag; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein;
LSCM, laser scanning confocal microscopy; MdMEX,
Malus domestica maltose transporter; MEX1, Arabidopsis
maltose transporter; ORF, open reading frame; qRT-PCR,
quantitative real-time PCR; RCP1, root cap protein; TSU,
transitory starch utilization; TPT, triose phosphate/phosphate translocator.
The nucleotide sequence reported in this paper has
been submitted to GenBank under accession number
DQ648082.
Introduction
Starch that is synthesized in chloroplasts during the
day is remobilized at night to fuel plant function in the
absence of photosynthesis. The remobilized starch is
known as ‘transitory starch’, reflecting its nature as a
temporary storage pool and not a true photosynthetic
‘sink’ (Zeeman et al. 2004). Transitory starch utilization
(TSU) allows plants to maintain near steady-state rates
of carbon export diurnally (Chatterton and Silvius
1979).
The understanding of the pathways of starch
breakdown, the products generated and the means of
their transport has recently undergone a dramatic
change. For a long time it was believed that phosphorolytic starch breakdown and the subsequent export of
triose phosphates or 3-phosphoglycerate occurred primarily via triose phosphate/phosphate translocators
(TPTs; Flügge 1999). However, plants with reduced
TPT capacity, even though they produce more starch
during the day, are able to increase starch utilization at
night while maintaining near wild-type growth (Riesmeier
et al. 1993, Heineke et al. 1994). In addition, nuclear
magnetic resonance studies showed that most of the
sucrose synthesized at night in leaves of tomato and
bean derives from products of hydrolytic starch breakdown (Schleucher et al. 1998). Thus, the current view is
that the hydrolytic pathway of starch breakdown and
the export of neutral compounds dominates in leaves
(Zeeman et al. 2004).
Both glucose and maltose are formed during hydrolytic
starch breakdown (Peavey et al. 1977). It was subsequently
demonstrated that both sugars are transported across the
inner membrane of isolated chloroplasts (Herold et al.
1981). Further work established that the glucose and
3
Present address: Nunhems USA, Inc., Brooks, OR 97305, USA
*Corresponding author: E-mail, [email protected]; Fax, þ1-607-255-0599.
1607
1608
Apple maltose transporter
maltose transport pathways do not interact; therefore,
separate mechanisms facilitate movement of the sugars
(Rost et al. 1996).
Perhaps because of the higher glucose concentrations
found in chloroplasts, or because it was conceptually
simpler to envisage a pathway for glucose incorporation
into transport sugars, the role of maltose in TSU was
initially neglected. Research instead was concentrated on a
glucose translocator. Trethewey and ap Rees (1994) isolated
a chloroplast glucose transport mutant lacking a 40 kDa
protein postulated to be the glucose transporter. It was later
shown that this mutant is actually defective in starch:water
dikinase R1 (Zeeman et al. 2004). Another effort resulted in
the cloning of members of a putative glucose transporter
family from several species (Weber et al. 2000). However,
transgenically down-regulated and knockout plants lack a
phenotype suggestive of impaired TSU (Weber 2004). Thus
the mechanism of starch mobilization based on glucose
remains elusive.
More recently the focus has shifted to maltose, which
is found at significantly lower concentrations in chloroplasts. Niittylä et al. (2004) identified two mutants
with altered malto-oligosaccharide levels in Arabidopsis.
The mutation was mapped to the MEX locus (At5g17520)
and characterized as a maltose transporter essential
for starch degradation (Niittylä et al. 2004). Concurrently,
Weise et al. (2004) demonstrated that maltose is the
major export product of hydrolytic starch breakdown at night. Cytosolic maltose then enters the sucrose
synthesis pathway in species which utilize that sugar as
a primary export product (Chia et al. 2004, Lu and
Sharkey 2004).
Sorbitol (D-glucitol) is the predominant end-product of
photosynthesis in apple (Malus domestica B orkh.) and
other woody Rosaceae—particularly the subfamilies
Maloideae (e.g. apple, pear) and Amygdaloideae (e.g.
cherry, peach, almond) (Bieleski and Redgwell 1977).
Under normal conditions, 510% of the newly fixed
carbon is partitioned to starch in apple (Cheng et al.
2005). Compared with sucrose-synthesizing species such
as spinach, pea and Arabidopsis, this is a much lower level
of partitioning to starch. However, the capacity for high
rates of starch synthesis exists in apple. In transgenic
apple trees with decreased sorbitol synthesis, starch
synthesis was concomitantly up-regulated; higher levels of
starch accumulated during the day and more starch was
remobilized at night (Cheng et al. 2005). However, the
regulation of starch mobilization in apple source leaves is
largely unknown. As a first step to understand starch
mobilization in sorbitol-synthesizing species, we cloned an
M. domestica maltose transporter (MdMEX) gene and
studied its function in an Arabidopsis maltose exportdefective mutant.
Results
Molecular cloning of the MdMEX cDNA
The MdMEX cDNA is 1,531 bp and encodes an
1,275 bp open reading frame (ORF). At 425 amino acid
residues, the predicted MdMEX protein is 10 residues
longer than the Arabidopsis ortholog (Fig. 1). The
chloroplast transit peptide prediction server, ChloroP
(Emanuelsson et al. 1999), predicts that the first 55 amino
residues of MdMEX are a chloroplast transit peptide. The
mature protein is 68% identical to the Arabidopsis ortholog
(Fig. 1). MdMEX is predicted to encode eight transmembrane helices (Fig. 1).
Quantitative real-time PCR analysis of MdMEX expression
patterns
Transcript levels of MdMEX were measured in leaves
at various developmental stages, fruits and roots via
quantitative real-time PCR (qRT-PCR) using SYBR
Green I (Fig. 2). As expected, relative transcript levels
were highest in mature leaves. Transcript levels measured in
expanding leaves were similar to those in mature leaves,
while levels in the shoot tip, which includes the apical
meristem and all folded leaves, were 4-fold lower. Transcript
levels were also low in roots and expanding fruits.
Complementing the Arabidopsis mex1-1 mutant with the
apple ortholog, MdMEX
Transformation of the Arabidopsis mex1-1 mutant with
MdMEX as a green fluorescent protein (GFP) fusion
protein resulted in targeting of the mature protein to the
chloroplast envelope (Fig. 3). In addition, GFP fluorescence
was detected on the envelope of plastids in roots (data not
shown). MdMEX restored the phenotype of mutant plants
so that they were nearly indistinguishable empirically from
wild-type plants. Plant size was normal in many transgenic
lines (Fig. 4). Phenology of leaf development and flowering
was also restored, unlike mutant plants which have delayed
development.
Carbohydrate levels in the complemented plants
MdMEX:mex1-1 plants were statistically indistinguishable from the wild type in terms of soluble carbohydrate
levels at the end of both the day and night periods (Fig. 5).
Starch levels, which were significantly higher at both the
end of the day and the end of the night in mutants, were
normal at the end of the day and only slightly higher at the
end of the night in the complemented lines. Maltose levels—
which are 20-fold higher in mutant plants at the end of the
day and 50-fold higher at the end of the night—were
restored to wild-type levels in the complemented lines.
Levels of hexose sugars in mutant plants and the
Apple maltose transporter
MdMEX
AtMEX
MdMEX
AtMEX
1609
1 MAESLMVVPCSSYTPGRPPHPRLQQHHLLKSFPASSSSSSSSIQHNTNSNTLSVS
1 -----MEAKAIATSLGGD-RVLIFPCSPRSSFVFTSRLSSLPLKRASIGGAVSCS
56
50
*
SSAGQGQCFLFKSYHPLPLRLPLQRRSTLSALDSDVPHPLHQGSVKSSSSKTSF
GVNGLTRWNSIVSTRRL---VPVRSINSESDSDSDFPHENQQGNPGLGKFK-EY
MdMEX
AtMEX
-------------110 EQWNSWTAKFSGASNIPFLLLQMPQIYLNAQNLLAGNKAALLAVPWMGMFTGLLG
100 QEWDSWTAKFSGGANIPFLMLQLPQIILNTQNLLAGNNTALSAVPWLGMLTGLLG
MdMEX
AtMEX
------------------------------------165 NLSLLSYFAKKRENEAIVVQTLGVISLYVVFAQLSMADAMPLPYFVITSIVVATG
155 NLSLLSYFAKKREKEAAVVQTLGVVSTHIVLAQLTMAEAMPIQYFVATSAVVTIG
MdMEX
AtMEX
--------------------------220 LVLNFLNYFGWLNAGLWRFWEDFITVGGLSVLPQIMWSTFVPYLPNSILPGALAF
210 LIVNCLYYFGKLSKTVWQLWEDVITIGGLSVLPQIMWSTFVPLVPNSILPGTTAF
MdMEX
AtMEX
----------------------------275 VVAVVAVVMARMGKLSEGGIKFVGAISGWTATLLFMWMPVSQMWTNFLNPDNIKG
265 GIAVAAIIMARTGKLSEKGVRFVGSLSGWTATLMFMWMPVSQMWTNFLNPDNIKG
MdMEX
AtMEX
--------------------------------------------330 LSASSMLLAMIGNGLMIPRALFIRDLMWFTGSTWASFFYGYGNIVCLYWFNSISK
320 LSSITMLLSMMGNGLMIPRALFIRDLMWLTGSLWATLFYGYGNILCLYLVNCTSQ
MdMEX
AtMEX
---------------------385 EFFLAATTGLFLWIGMAVWRDAVVNEYDSPFTSLKELVSGL 425
375 SFFVAATIGLISWIGLALWRDAVAYGHNSPFRSLKELVFGP 415
Fig. 1 Alignment of the translated nucleotide sequence of MdMEX with the Arabidopsis maltose export protein, MEX1 (At5g17520).
Identical residues are white text on a black background; conserved residues have a gray background. The first residue of the predicted
mature protein is denoted with an asterisk; all upstream residues are the predicted chloroplast transit peptide. The eight predicted
transmembrane helices in MdMEX are denoted by bars above the corresponding residues.
complemented lines were not statistically different from the
wild type (Fig. 5).
Chlorophyll levels and chlorophyll fluorescence
The mex1-1 mutant had significantly lower leaf
chlorophyll concentration and maximum quantum efficiency of PSII (Fv/Fm) compared with the wild type
(Table 1), indicating that photosynthetic capacity and
electron transport efficiency are compromised as a result
of blockage of maltose export from the chloroplast.
Complementation of the mex1-1 mutant with MdMEX
restored both chlorophyll concentration and Fv/Fm to wildtype levels.
Discussion
MdMEX is the second member of the unique plant
maltose transporter family. MdMEX fully complemented
the Arabidopsis maltose excess mutant, mex1-1. Levels of
maltose and chlorophyll, and PSII quantum efficiency were
restored to wild-type levels (Table 1; Fig. 5). The phenotype
of complemented plants was generally indistinguishable
from that of the wild type (Fig. 4).
The expression pattern of MdMEX determined by
qRT-PCR suggests that its physiological role is not limited
to starch mobilization as maltose from mature leaves
(Fig. 2). Rather, the data indicate that MdMEX plays a
role in both mature and immature leaves, as well as in roots
and expanding fruits. Transcript levels in expanding leaves
undergoing the sink–source transition were nearly as high as
the levels in mature leaves. Thus, our data support a role for
maltose transporters in starch mobilization in a variety of
tissues.
Our results demonstrate that not only is the function of
the mature protein conserved, but it was targeted to the
chloroplast inner envelope membrane. Presuming that
MdMEX is chloroplast-localized in apple, the amino acid
constitution and the peptide cleavage site motifs are
conserved between different plant orders, i.e. Rosales and
Brassicales (Fig. 1). Our experiments also provide horizontal validation of the Arabidopsis-trained ChloroP algorithm
(Emanuelsson et al. 1999).
Studies of maltose transport in isolated chloroplasts
showed that the stromal sugar concentration equilibrates
with the concentration in the external medium (Rost et al.
1996). Therefore, the transport mechanism does not involve
active transport. Accordingly, there is no information in the
1610
Apple maltose transporter
peptide sequence of MdMEX to suggest that it encodes
anything other than a transmembrane transporter (Fig. 1).
It is not a member of the major facilitator superfamily
of sugar symporters, which have 12–14 transmembrane
domains and operate via sugar:proton symport
1.4
Expression level (relative to Leaf 10)
1.2
1.0
0.8
0.6
0.4
0.2
0.0
SHOOT TIP
LEAF3
LEAF10
FRUIT
ROOT
Fig. 2 MdMEX expression in apple leaves, fruits and roots. Data
were normalized to glyceraldehyde-3-phosphate dehydrogenase,
and mature leaves (‘Leaf 10’) were designated as the calibrator for
calculating relative expression levels. Error bars indicate the
standard deviation of three technical replicates from an RNA
pool representing the same sample collected from multiple plants.
(Marger and Saier 1993). Furthermore, there is no similarity
to ATP-binding cassette proteins, and an ATP-binding
domain is a characteristic of primary active transporters.
Therefore, it is very likely that maltose transporters
facilitate the downhill transport of maltose in the presence
of a concentration gradient between the chloroplast stroma
and cytosol. Indeed, Weise et al. (2005) found that the
subcellular concentration of b-maltose, the metabolically
active anomer of maltose during transitory starch degradation, is higher in the chloroplast than in the cytosol at night.
MEX1 was originally identified from Arabidopsis root
tips as root cap protein (RCP1) (Tsugeki and Fedoroff
1999). Thus, it is expressed in both source and sink tissues,
the latter tissues lacking chloroplasts. Likewise, MdMEX
was targeted to both leaves and roots in Arabidopsis (Fig. 3;
root data not shown); expressed sequence tags (ESTs) have
also been sequenced from apple fruits and seeds. Therefore,
maltose transporters may have a role in sink tissues in
addition to sources; they may function in multiple plastid
types, such as amyloplasts in roots.
In summary, we have identified the second member of
a unique plant gene family that facilitates the transport of
maltose across the chloroplast inner envelope membrane.
Maltose transporters are essential for transitory starch
utilization in leaves and may play a role in generalized
starch utilization in sink tissues such as fruits and roots.
Fig. 3 Localization of the MdMEX:GFP fusion protein to the chloroplast inner envelope membrane. (A) GFP fluorescence in a mutant
plant transformed with 35S:MdMEX:GFP; (B) chlorophyll fluorescence in the same guard cell; (C) merged images. Scale bar, 10 mm.
Fig. 4 Growth of mex1-1 mutants and plants complemented with 35S:MdMEX:GFP. Plants were grown under a 12 h day/night cycle.
(A) Wild-type Arabidopsis Col-0; (B) the mex1-1 mutant is much smaller, chlorotic and developmentally delayed; (C) a representative
mex1-1 plant complemented with 35S:MdMEX:GFP.
Apple maltose transporter
1611
*
A
*
12
*
B
2.0
*
10
1.0
8
6
0.1
4
2
0.0
0
C
Sucrose
(mg g−1 fresh wt)
1.5
D
1.5
1.0
1.0
0.5
0.5
0.0
0.0
E
1.5
Fructose
mg g−1 fresh wt
Maltose
mg g−1 fresh wt
14
Glucose
mg g−1 fresh wt
Starch
mg glucose g−1 fresh wt
16
mex1-1
MdMEX-1
1.0
MdMEX-2
MdMEX-3
0.5
WT Col-0
0.0
End of day
End of night
Fig. 5 Concentrations of starch (A), maltose (B), sucrose (C), glucose (D) and fructose (E) from leaves of mex1-1 mutants, plants
complemented with 35S:MdMEX:GFP and wild-type Arabidopsis col-0. Plants were grown under a 12 h day/night cycle. Five individual
plants were sampled at the end of the light and dark periods. Data presented are the means with standard error. For each grouping, the
leftmost bar represents the mutants, the next three bars are data from independently transformed lines, and the rightmost bar represents the
wild type. Comparisons significantly different from the wild-type controls at the P50.05 level are indicated by an asterisk.
Table 1 Leaf chlorophyll concentration and maximum
quantum efficiency of PSII (Fv/Fm)
Genotype
mex1-1
MdMEX:mex1-1 Line 1
MdMEX:mex1-1 Line 2
MdMEX:mex1-1 Line 3
wt Col-0
Chlorophyll
(mg cm–2)
Fv/Fm
12.2 0.24
21.1 0.65
21.9 0.42
22.7 0.69
21.6 0.67
0.64 0.008
0.83 0.001
0.83 0.002
0.83 0.002
0.84 0.001
Values are the means of five measurements made on individual
plants. Means that are significantly different from the control
(wt Col-0) at P50.05 are denoted with an asterisk.
MdMEX relieved the high maltose phenotype of Arabidopsis
maltose export mutants (Fig. 5). Levels of chlorophyll and
photosynthetic capacity were also restored (Table 1). The
peptide encoded by the apple gene was targeted to
chloroplasts in Arabidopsis, suggesting high conservation
of transit peptide motifs between plant orders. Based on the
amino acid sequence, we project that the proteins form
transmembrane transporters that catalyze maltose transport
down a concentration gradient. There is no evidence for an
active transport mechanism and we argue why such a
mechanism is unnecessary.
Materials and Methods
Plant material
Leaf and fruit samples were collected from field-grown
‘Gala’/M.26 apple (M. domestica Borkh.) trees at Cornell
Orchards in Ithaca, New York at approximately 35 d after
bloom. Leaves were sampled acropetally from various positions
on actively growing shoots. Roots were collected from 2-year-old
potted ‘Gala’/M.26 trees at the same time as leaf and fruit samples.
All the samples were frozen in liquid nitrogen and stored at 808C
until use.
1612
Apple maltose transporter
Plants of Arabidopsis thaliana ecotype Col-0 were grown in a
growth chamber with incandescent-supplemented fluorescent
lighting (150 mmol m–2 s–1) under a 12 h/12 h (day/night) cycle.
The temperature was controlled to 208C/188C.
Identification of a maltose transporter ortholog from apple
The tblastn algorithm was used to align the Arabidopsis
maltose transporter (MEX1; At5g17520), with apple ESTs curated
by NCBI. An EST (gi:48276225) with high similarity to MEX1 was
identified, and the corresponding full-length clone was kindly
sequenced by HortResearch (New Zealand). The gene was named
M. domestica maltose transporter (MdMEX).
Quantitative real-time PCR analysis of MdMEX expression patterns
Total RNA was extracted from apple tissues according to
the modified hot borate method of Wan and Wilkins (1994).
Residual DNA was digested with RQ1 DNase (Promega, Madison,
WI, USA), and RNA clean-up was performed on RNeasy spin
columns (Qiagen Inc., Valencia, CA, USA). RNA integrity was
confirmed by agarose gel electrophoresis. RNA purity and
concentration were measured on a NanoDrop ND-1000
Spectrophotometer. A 500 mg aliquot of RNA was reverse transcribed with the iScript cDNA synthesis kit (Bio-Rad Laboratories,
Hercules, CA, USA).
Quantitative real-time PCR was performed on the ABI Prism
7000 Sequence Detection System (Applied Biosystems, Foster City,
CA, USA) with the SYBR GreenER qPCR SuperMix (Invitrogen,
Carlsbad, CA, USA) according to the manufacturers’ instructions.
On the basis of RNA quantity used in the cDNA synthesis
reaction, 10 mg of cDNA was used as template, along with 300 nM
of each primer in a final volume of 20 ml. MdMEX was amplified
with the primers MdMEX 604–630 (50 -GAT GCG ATG CCT CTA
CCT TAC TTT GTC-30 ) and MdMEX 701–675 (50 -CCA GCA
TTA AGC CAA CCA AAG TAA TTC-30 ). Data were normalized
to an EST UniGene (Mdo.1683) for glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) with primers GAPDH-f (50 -AGG CTG
GAA TTG CAT TGA ACC A-30 ) and GAPDH-r (50 -TGG ATT
TAG AGG GTG GAT GCT ACG-30 ). Primers for GAPDH were
chosen over primers for 18S RNA, ubiquitin and eukaryotic
elongation factor 1A, because GAPDH expression was most stable
across the range of tissue types.
Three subsamples of each cDNA pool and duplicate nontemplate controls from proportionately diluted, DNased RNA
were run for each primer pair. Primer specificity was evaluated by
melting curve analysis, and high-resolution agarose gel electrophoresis was used to confirm expected amplicon sizes. PCR
efficiencies were calculated from the delta Rn values according to
Ramakers et al. (2003). These efficiencies and the threshold cycle
values for each primer–template combination were used in Pfaffl’s
(2001) equation to calculate expression levels relative to mature
leaves.
Engineering a GFP fusion protein and plant transformation
MdMEX was PCR amplified from first-strand cDNA with
the PfuUltra DNA polymerase according to the manufacturer’s
instructions (Stratagene, La Jolla, CA, USA). The forward primer
(50 -TAG GTA CCG GAT CCT ATG GCC GAG AGT CTA-30 )
incorporated a KpnI recognition site. The reverse primer (50 -CAA
TTG AGC TCA AGC CCG GGA CAA TCC AGA AAC CA-30 )
replaced the native stop codon with an XmaI recognition site and
facilitated an in-frame fusion with a codon-optimized GFP gene
(Chiu et al. 1996) via a eight alanine residue linker (Ayre and
Turgeon 2004). The PCR product was restriction-digested and
cloned into a pUC19 intermediate between the cauliflower mosaic
virus (CaMV) 35S promoter and sGFP. The resulting fusion was
named MdMEX:GFP. The expression cassette was cut from the
pUC vector with XbaI and SacI, subcloned into the binary vector
pGPTV-Bar (Becker et al. 1992) and transformed into
Agrobacterium strain GV3101:pMP90 (Koncz and Schell 1986).
Mutant mex1-1 plants (Niittylä et al. 2004) were transformed by
the floral dip procedure (Clough and Bent 1998). Bar geneexpressing T1 transformants were selected by spraying seedlings on
soil with a 0.1% solution of the glufosinate ammonium-containing
herbicide, Finale (Farnam Companies Inc., Phoenix, AZ, USA).
Transformation was confirmed by PCR using genomic DNA as
template. All further experiments were performed on herbicideresistant T2 plants.
Laser scanning confocal microscopy
Expression of the mature GFP fusion protein in planta was
localized via laser scanning confocal microscopy (LSCM). Whole
seedlings of transformed, wild-type and mex1-1 plants were
mounted in water. Microscopy was performed with a Leica TCSSP2 confocal scanning head mounted on a Leica DMRE-7 (SDK)
upright microscope with a HCPLAPO 20 NA 0.7 objective
(Leica Microsystems, Wetzlar, Germany). The 488 nm line of a
four-line argon laser was used to excite both GFP and chlorophyll.
GFP fluorescence was collected between 500 and 600 nm, and
chlorophyll fluorescence was collected between 660 and 700 nm.
Carbohydrate extraction and analysis
Whole leaves from wild-type and MdMEX:GFP plants were
collected after 5 weeks of growth. Leaves of mex1-1 plants were
collected after 6 weeks when these slower growing plants had
developed to a similar stage (Niittylä et al. 2004). Five individual
plants from three independently transformed lines were evaluated.
Leaf samples were taken 12 h apart, at the end of the light and dark
periods. Fresh leaf tissues (65–75 mg) were transferred to 80%
ethanol, and soluble carbohydrates were extracted three times with
80% ethanol at 808C, with xylitol included as an internal standard.
The extract was then treated as described previously (Cheng and
Fuchigami 2002). Briefly, charged solutes were removed by passing
the extract through columns consisting of Amberlite IR-68 (acetate
form; Sigma) layered on Dowex 50W (hydrogen form; SigmaAldrich, St Louis, MO, USA). After vacuum concentration, the
dried extract was dissolved in ddH20 and analyzed on a Dionex
DX-500 series ion chromatography system with a CarboPac PA-1
Column (Dionex Corp., Sunnyvale, CA, USA).
For starch determination, the residual material after soluble
sugar extraction was dried in an 808C water bath. Next, the starch
was gelatinized by adding 3 ml of 100 mM acetate buffer, pH 4.5
and incubating in a boiling water bath for 1 h. Starch was digested
by adding 50 U of amyloglucosidase (Sigma A9228) in 1 ml of
acetate buffer and incubating at 558C for 4 h. Starch was then
quantified as glucose enzymatically in the hexokinase reaction,
following the chemical manufacturer’s instructions (Sigma
GAHK20).
Measurement of chlorophyll fluorescence and leaf chlorophyll
concentration
Maximum quantum efficiency of PSII was measured with a
pulse-modulated fluorometer, FMS2 (Hansatech, Norfolk, UK)
on overnight dark-adapted leaves of intact plants. Pigments were
extracted from leaf discs in 80% aqueous acetone, and total
chlorophyll was measured according to Porra (1989).
Apple maltose transporter
Data analyses
Statistical analyses were performed using SAS software
(SAS Institute, Cary, NC, USA). Measured parameters were
compared among genotypes using Process GLM. Separations of
means were performed using Dunnett’s tailed t-tests for comparisons of all treatments against a control, specified as wild-type
Arbabidopsis Col-0.
Acknowledgments
We thank Dr. William Laing at HortResearch, New Zealand
for EST sequencing information; Dr. Alison Smith at John Innes
Center, UK for sharing seeds of mex1-1 plants; Drs. Susan Liou,
Hye-Ji Kim, and William Miller for assistance with ion chromatography; Monica Vencato Moll and Dr. Denise Duclos for
assistance with real-time PCR; and Dr. Brian Ayre at the
University of North Texas for consultations on cloning and
plant transformation strategies.
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(Received June 14, 2008; Accepted September 2, 2008)