Planta (2009) 229:659–666
DOI 10.1007/s00425-008-0863-5ORIGINAL ARTICLE
Subcellular localization and enzymatic properties of diVerentially
expressed transketolase genes isolated from the desiccation
tolerant resurrection plant Craterostigma plantagineum
Björn C. Willige · Michael Kutzer · Felix Tebartz ·
Dorothea Bartels
Received: 6 August 2008 / Accepted: 13 November 2008 / Published online: 4 December 2008
© Springer-Verlag 2008
Abstract The desiccation tolerant resurrection plant
Craterostigma plantagineum encodes three classes of trans-
ketolase transcripts, which are distinguished by their gene
structures and their expression patterns. One class, repre-
sented by tkt3, is constitutively expressed and two classes,
represented by tkt7 and tkt10, are upregulated upon rehy-
dration of desiccated C. plantagineum plants. The objective
of this work was to characterize the diVerentially expressed
transketolase isoforms with respect to subcellular localiza-
tion and enzymatic activity. Using GFP fusion constructs
and enzymatic activity assays, we demonstrate that C.
plantagineum has novel forms of transketolase which local-
ize not to the chloroplast, but mainly to the cytoplasm and
which are distinct in the enzymatic properties from the
transketolase enzymes active in the Calvin cycle or oxida-
tive pentose phosphate pathway. A transketolase prepara-
tion from rehydrated leaves was able to synthesize the
unusual C8 carbon sugar octulose when glucose-6-phos-
phate and hydroxy-pyruvate were used as acceptor and
donor molecules in in vitro assays. This suggests that a
transketolase catalyzed reaction is likely to be involved in
the octulose biosynthesis in C. plantagineum.
Keywords Carbohydrate synthesis ·
Chloroplast targeting · Octulose · Resurrection plant ·
Transketolase
Introduction
Transketolases (EC 2.2.1.1) are ubiquitously occurring
enzymes which catalyse the reversible transfer of a glycol-
aldehyde group from an activated ketose as donor to the
acceptor, an activated aldose (Volkamer et al. 1998). The
enzyme functions as homodimer and requires thiamine
pyrophosphate as co-substrate. In non-photosynthetic
organisms, transketolase connects the pentose phosphate
pathway to glycolysis to generate reducing power in form
of NADPH. In plants, transketolases are essential enzymes
of the Calvin cycle. Transketolase catalyses the transfer of
a two-carbon ketol group from either fructose-6-phosphate
or sedoheptulose-7-phosphate to glyceraldehyde-3-phos-
phate, to form xylulose-5-phosphate and either erythrose-
4-phosphate or ribose-5-phosphate. The reactions take
place in the opposite directions in the pentose phosphate
cycle.
When the transketolase enzyme was repressed in
tobacco, photosynthesis and phenylpropanoid metabolism
were negatively aVected by limiting the Xux of erythrose-4-
phosphate into the shikimate pathway (Henkes et al. 2001).
This demonstrates the central role of transketolases in the
primary metabolism. The Calvin cycle is localized in plas-
tids. There are some uncertainties about the localization of
the oxidative pentose phosphate pathway, which takes
place in the cytoplasm in animal cells and fungi, whereas
the complete pathway in plants is only localized to chloro-
plasts according to Schnarrenberger et al. (1995), Debnam
and Emes (1999), and Henkes et al. (2001). All plant
B. C. Willige · M. Kutzer · F. Tebartz · D. Bartels (&)
Institute of Molecular Physiology and Biotechnology
of Plants (IMBIO), University of Bonn, Kirschallee 1,
53115 Bonn, Germany
e-mail: dbartels@uni-bonn.de
Present Address:
B. C. Willige
Department of Plant Sciences, Plant Systems Biology,
Technical University Munich, Am Hochanger 4,
85354 Freising-Weihenstephan, Germany123

660 Planta (2009) 229:659–666transketolase enzymes of the Calvin cycle and the oxidative
pentose phosphate pathway reported so far are nuclear
encoded, and transported into the chloroplasts (Flechner
et al. 1996; Teige et al. 1998; Henkes et al. 2001; Gerhardt
et al. 2003). Besides the transketolases of the Calvin cycle
and the oxidative pentose phosphate cycle, transketolases
involved in the isoprenoid biosynthesis have been isolated
from peppermint and pepper; these transketolases deWne a
unique family conserved between bacteria and plants, but
are absent in animals (Bouvier et al.1998; Lange et al.
1998). These enzymes are also localized in plastids as
shown for pepper and indicated by the presence of a con-
served plastidic targeting sequence for the peppermint
enzyme.
Three diVerent cDNA clones (tkt3, tkt7 and tkt10)
encoding transketolases have been isolated from the desic-
cation tolerant resurrection plant Craterostigma plantagi-
neum (Bernacchia et al. 1995). The transketolases diVer in
their coding sequences and in their expression patterns. The
tkt3 transketolase class is constitutively expressed in leaves
and roots, and the transketolases, tkt7 and tkt10 are prefer-
entially associated with the rehydration process of the des-
iccated plant (Bernacchia et al. 1995).
The tkt3 transcript contains a 180 bp long sequence at
the 5
end which has been assumed to contain a plastid
targeting sequence. This is supported by the observation
that the mature tkt3 protein does not contain this sequence
(Schwall 1995). The tkt3 transketolase has all the con-
served features of the transketolase enzyme involved in
the Calvin cycle and the oxidative pentose phosphate
cycle.
A data base search showed that no transketolase genes
are known which do not encode a plastid target sequence,
except for the cDNA clones tkt10 and tkt7 from C. plantag-
ineum (Bernacchia et al. 1995). It is important in this con-
text to note that C. plantagineum has an unusual sugar
metabolism, in hydrated leaves it contains high amounts of
the C8 sugar octulose which is converted to sucrose during
dehydration (Bianchi et al. 1991). Based on the expression
patterns of tkt7 and tkt10, it has been speculated that trans-
ketolases are involved in the sugar conversion from sucrose
to octulose.
The objective of the work reported here was to compare
the diVerent C. plantagineum isoforms with respect to their
subcellular localization and enzymatic activities. Evidence
is presented that tkt3 transketolase is localized in chloro-
plasts, but tkt7 and tkt10 transketolases are localized in the
cytosol and diVer in their substrate requirements from tkt3.
In vitro experiments showed that a transketolase fraction
from rehydrated leaves is able to synthesize octulose. Thus,
we conclude that tkt7 and tkt10 represent novel transketo-
lases.
Materials and methods
Plant material
Craterostigma plantagineum Hochst plants, originally col-
lected by Prof. Volk (University of Würzburg, Germany),
were grown in an artiWcial clay substrate in a controlled
environment as described by Bartels et al. (1990). Plants
were allowed to dry in their pots by withholding water for
at least 7 days. The growth substrate was then carefully
removed and the plants were rehydrated by soaking them in
water for the indicated periods of time. Tissues were col-
lected and rapidly frozen in liquid nitrogen.
Arabidopsis thaliana (ecotype Col-O; seeds obtained
from the collection of the Max Planck Institute of Plant
Breeding, Cologne, Germany) were grown according to
standard protocols.
Green Xuorescent protein (GFP) constructs and plant
transformation
The GFP expression vector pGJ280 was constructed by Dr.
G. Jach (Max Planck Institute of Plant Breeding, Cologne,
Germany) and contains in the following order: the cauli-
Xower mosaic virus (CaMV) 35S promoter with a dupli-
cated transcriptional enhancer, the tobacco etch virus
translational enhancer, the GFP open reading frame (Tsien
1998) and the CaMV 35S polyadenylation site (Reichel
et al. 1996).
Transketolase cDNA sequences were fused to the GFP
open reading frame via an engineered NcoI site to generate
transketolase–GFP expression constructs which were used
in transient protoplast assays as described by Damm et al.
(1989). Biolistic transient transformation of C. plantagi-
neum was performed according to Ditzer and Bartels
(2006). The constructs were also subcloned into pBin19
plasmids (Bevan 1984) which were integrated into the A.
thaliana genome by using Agrobacterium tumefaciens
GV3103 following the Xoral dip inWltration method
(Clough and Bent 1998). Transgenic seeds (T1) were
selected on 50
g mL¡1 kanamycin and transferred to soil
for subsequent propagation.
Microscopy
Green Xuorescent protein Xuorescence was determined
using a confocal laser scanning microscope (e-C1 confocal
microscope system, Nikon GmbH, Düsseldorf, Germany,
or Leica Microsystems AG, Wetzlar, Germany). Excitation
of GFP was performed with standard Xuorescein isothiocy-
anate Wlters. Images were captured using a digital camera
(DXM 1200; Nikon).123