THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 17, pp. 11815–11818, April 28, 2006
© 2006 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
Similar Protein Phosphatases Control Starch Metabolism in
Plants and Glycogen Metabolism in Mammals*□
S
Received for publication, January 18, 2006, and in revised form, February 27, 2006 Published, JBC Papers in Press, March 2, 2006, DOI 10.1074/jbc.M600519200
Totte Niittylä‡1,2, Sylviane Comparot-Moss‡1, Wei-Ling Lue§, Gaëlle Messerli¶, Martine Trevisan储3,
Michael D. J. Seymour**, John A. Gatehouse**, Dorthe Villadsen‡‡, Steven M. Smith§§, Jychian Chen§,
Samuel C. Zeeman¶4, and Alison M. Smith‡
From the ‡Department of Metabolic Biology, John Innes Centre, Norwich NR4 7UH, United Kingdom, §Institute of Molecular
Biology, Academia Sinica, Taipei 115, Taiwan, ¶Institute of Plant Sciences, ETH Zurich, CH-8092 Zurich, Switzerland, 储Institute of
Plant Sciences, University of Bern, Altenbergrain 21, CH-3013 Bern, Switzerland, **School of Biological and Biomedical Sciences,
Durham University, Durham DH1 3LE, United Kingdom, ‡‡Institute of Molecular Plant Sciences, University of Edinburgh,
Edinburgh EH9 3JH, United Kingdom, and §§Australian Research Council Centre of Excellence in Plant Energy Biology,
University of Western Australia, Crawley WA 6009, Australia
We report that protein phosphorylation is involved in the control of
starch metabolism in Arabidopsis leaves at night. sex4 (starch excess 4)
mutants, which have strongly reduced rates of starch metabolism, lack
a protein predicted to be a dual specificity protein phosphatase. We
have shown that this protein is chloroplastic and can bind to glucans
and have presented evidence that it acts to regulate the initial steps of
starch degradation at the granule surface. Remarkably, the most closely
related protein to SEX4 outside the plant kingdom is laforin, a glucanbinding protein phosphatase required for the metabolism of the mammalian storage carbohydrate glycogen and implicated in a severe form
of epilepsy (Lafora disease) in humans.
Starch, the main storage carbohydrate of plants, accumulates as a
product of photosynthesis in leaves during the day and is converted to
sucrose for export from the leaves at night. This conversion of starch to
sucrose is one of the largest daily carbon fluxes on the planet, but nothing is known about how the process is initiated and controlled. The
amounts of enzymes on the pathway change very little through the
diurnal cycle in leaves of the model plant Arabidopsis thaliana, hence
flux must be controlled by modulation of their activities (1).
Much progress in understanding the pathway has been made through
the selection of Arabidopsis mutants impaired in starch degradation at
night. All such mutations identified thus far are in genes encoding enzymes
of the pathway, rather than proteins likely to be involved in modulation of
the activities of these enzymes (2–11). However, a mutation at a locus not
yet identified, the starch excess 4 (or SEX4) locus, gives rise to a phenotype
indicative of a regulatory defect rather than a defect in a structural enzyme.
Mature sex4 leaves contain three to four times more starch than those of
wild-type plants, apparently because a reduced capacity for starch degrada-
* This work was supported by funding from the Biotechnology and Biological Sciences
Research Council of the United Kingdom (to A. M. S. and J. A. G.), from the Swiss
National Science Foundation (National Centre of Competence in Research-Plant Survival) and the Roche Research Foundation (to S. C. Z.), and from the National Science
Council, Taiwan (to J. C.). The costs of publication of this article were defrayed in part
by the payment of page charges. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
□
S
The on-line version of this article (available at http://www.jbc.org) contains supplemental material.
1
These authors contributed equally to this work.
2
Present address: Carnegie Institution, Stanford, CA 94305-1297.
3
Present address: Ctr. for Integrative Genomics, University of Lausanne, CH-1015, Lausanne, Switzerland.
4
To whom correspondence should be addressed: Institute of Plant Sciences, ETH Zurich,
CH-8092 Zurich, Switzerland. Tel.: 41-44-632-8275; Fax: 41-44-632-1044; E-mail
[email protected].
APRIL 28, 2006 • VOLUME 281 • NUMBER 17
tion at night leads to progressive accumulation of starch over the life of the
leaf (12, 13). Starch granules in leaves of the sex4 mutant are much larger
and more rounded than those of wild-type plants (14). Measurements of
activity and protein of enzymes known to be involved in starch degradation
revealed only one significant reduction in the sex4 mutant in the chloroplastic ␣-amylase AMY3 (12, 15). However, although both the activity and
amount of protein of AMY3 are strongly reduced, this is not the cause of the
deficiency in starch degradation in the sex4 mutant. T-DNA insertion lines
lacking AMY3 protein have normal rates of starch degradation (15). The
aim of the work described in this paper was to discover the nature of the
gene at the SEX4 locus and thus shed light on the regulation of starch
degradation.
EXPERIMENTAL PROCEDURES
Positional Identification of the SEX4 Locus—F2 plants from a cross
between sex4-2 (Col-0 background) and Landsberg erecta showing the
mutant phenotype were used for mapping. The mapping population
(562 plants) was genotyped using SSLP and SNP markers available on
the Arabidopsis Information Resource data base. This shows that the
SEX4 gene was located within an 800-kb region between markers
ATEM1 and SGCSNP42 on chromosome 3.
Plant Growth and Transformation—Plants were grown in 12-h light/
12-h dark conditions (20 °C, 60–70% relative humidity, 175 ␮mol of photons m⫺2 s⫺1), unless otherwise stated. The SEX4 cDNA (U14967 from the
Arabidopsis Stock Center) was cloned into the binary vector 53AS with a 35
S cauliflower mosaic virus promoter and introduced into the sex4-1 and
sex4-2 mutants via Agrobacterium-mediated transformation (by floral infiltration). Transgenic plants were selected by glufosinate resistance and confirmed by PCR and immunoblot analyses. Additionally, a C-terminal fusion
construct of SEX4 cDNA and enhanced yellow fluorescent protein (ClontechTM) was cloned into a vector with a double 35 S cauliflower mosaic
virus promoter and introduced into Arabidopsis via Agrobacterium as
described previously (4).
Gels, Antisera, and Immunoblotting—For the renaturation of amylolytic activity, extracts were subjected to electrophoresis on SDS-polyacrylamide gels containing starch. After washing and incubation in
SDS-free medium, the gels were stained with iodine solution (15). For
preparation of an antiserum to SEX4, a construct encoding a fusion
between the full-length SEX4 protein and glutathione S-transferase
(GST)5 in the pGEX-4T-2 vector (Amersham Biosciences) was
5
The abbreviations used are: GST, glutathione S-transferase; BSA, bovine serum albumin; YFP, yellow fluorescent protein; GWD, glucan water dikinase; CBM, carbohydrate
binding module; KIS, kinase interaction sequence.
JOURNAL OF BIOLOGICAL CHEMISTRY
11815
Control of Starch Metabolism in Plants
FIGURE 1. Structure of the SEX4 gene and predicted protein product. Gray boxes
represent exons. DSPc, dual specificity phosphatase catalytic domain. CBM_20, carbohydrate binding module. The alterations in the five mutant alleles are indicated. sex4-3 and
sex4-5 are T-DNA insertion lines from the Salk Institute Genomic Analysis Laboratory and
are lines SALK_102567 and SALK_126784, respectively.
expressed in Escherichia coli (BL21DE3) (15). The fusion protein was
purified from inclusion bodies and used to immunize rabbits. Antiserum for AMY3 was prepared and used as described previously (15)
Starch Analysis—Iodine staining of leaves and quantitative analyses
of starch contents were performed as described previously (15).
Preparation of Chloroplasts—Chloroplasts were isolated from protoplasts and purified on a Percoll gradient and by treatment with protease
(15, 16). The purity of the chloroplast preparation was confirmed by the
absence of activity of cytosolic marker enzymes. Chloroplast extracts
from wild-type plants and leaf extracts from wild-type and mutant
plants were loaded on a 10% SDS-polyacrylamide gel for the immunoblot analysis. Loading was adjusted so that each lane contained the same
activity of chloroplastic phosphoglucose isomerase. A 1:1000 dilution of
crude antiserum was used to detect the SEX4 protein.
Production of GST Fusion Protein—A fusion construct of the putative
carbohydrate binding module of the SEX4 protein and GST was prepared and expressed in E. coli as described previously (17; the carbohydrate binding module is referred to as the kinase interaction sequence
(KIS) domain in this reference).
Glycogen Binding Assays—Protein-free glycogen (5 mg ml⫺1) in 50
mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.1% (v/v) 2-mercaptoethanol,
0.02% (w/v) Brij-35, 0.1 mg ml⫺1 bovine serum albumin (BSA) was
mixed with GST fusion protein. Samples were incubated at 0 °C for 30
min and then centrifuged 90 min at 100,000 ⫻ g at 4 °C to sediment the
glycogen. Pellets were washed in 50 mM Tris-HCl (pH 7.5), 150 mM
NaCl, resuspended in 4⫻ SDS sample buffer, and run on 12.5% SDS gels.
The gels were stained with Coomassie Brilliant Blue R.
Measurement of Maltose—Plants were grown in 8-h light/16-h dark
conditions. Relative levels of maltose were determined by gas chromatography linked to mass spectrometry using methods described previously (18).
RESULTS
The SEX4 locus was mapped to a region of 800 kb on chromosome 3.
Gene discovery was facilitated by the observation that one gene in this
region (At3g52180) displays the same distinctive pattern of diurnal
change in transcript abundance in the leaf as genes encoding enzymes
known to be involved in starch degradation (1). Sequencing revealed
mutations likely to prevent or impair function in this gene in plants
carrying three independent sex4 alleles (Fig. 1). The sex4-1 allele contains a deletion that overlaps the open reading frames of both
At3g52180 and At3g52190. The sex4-2 allele contains a point mutation
in the seventh exon. This is predicted to change the arginine residue of
the signature motif of a protein phosphatase (see last paragraph under
“Results”) to a lysine; hence this change is highly likely to affect protein
function. The sex4-4 allele contains a point mutation that gives rise to a
stop codon and results in a truncated protein (Fig. 1 and data not
shown).
11816 JOURNAL OF BIOLOGICAL CHEMISTRY
FIGURE 2. Starch excess phenotype of sex4 and complemented lines. A, leaves were
decolorized with hot ethanol and stained with iodine at the end of the dark period.
Wild-type leaves (ecotype Columbia (Col)) contain little starch and do not stain; sex4
leaves have high starch contents and hence stain darkly. B, starch contents at the end of
the night (black bars) and the end of the day (white bars) of leaves of wild-type plants
(Col), plants carrying four different mutant alleles of sex4, and for comparison, sex1
mutant plants. Plants were grown in a 12-h light, 12-h dark diurnal regime. Values are
means ⫾ S.E. of measurements made on five samples. Each sample was a rosette of a
non-flowering plant, approximately four weeks old. C, iodine-stained leaf of a sex4 plant
and of a plant of the same line transformed with a construct containing the wild-type
SEX4 cDNA. Immunoblot analysis confirmed the presence of levels of SEX4 protein in this
line comparable with those in wild-type plants (not shown). Expression of this construct
eliminates the starch excess phenotype. D, starch contents at the end of the night (black
bars) and the end of the day (white bars) of leaves of wild-type plants (Col), sex1 and
sex4 mutant plants, and a double mutant sex4/sex1. Experimental details are as
described for B.
To provide further evidence about the identity of the SEX4 gene, we
isolated two T-DNA insertion mutants in At3g52180 (Fig. 1, sex4-3 and
sex4-5) and showed that they have starch excess phenotypes (Fig. 2A).
Levels of starch are similar to those in plants carrying the previously
characterized mutant alleles (Fig. 2B). We also transformed the sex4-1
and sex4-2 mutants with a cDNA encoding the wild-type SEX4 protein.
Transformants no longer displayed a starch excess phenotype (Fig. 2C).
All of the new sex4 mutant alleles had reduced levels of the chloroplastic
␣-amylase AMY3 (see supplemental Fig. S1), as is the case for sex4-1 and
sex4-2 (12, 15).
The SEX4 protein has a predicted N-terminal chloroplast transit peptide. To discover whether the protein is in fact chloroplastic, the sex4-1
mutant was transformed with a construct encoding the wild-type SEX4
protein fused at the C terminus to yellow fluorescent protein (YFP). The
resulting transgenic plants no longer displayed a starch excess phenotype and exhibited YFP fluorescence specifically in the chloroplasts (Fig.
3A). Furthermore, protein gel blots probed with an antiserum raised
against the SEX4 protein revealed that chloroplasts isolated from the
leaves of wild-type plants contained a protein with a similar apparent
mass to that of the predicted SEX4 protein. This protein was missing
from leaves of sex4-1 mutant plants (Fig. 3B).
Previously, we have shown that sex4 mutants have lower levels of
sugars (sucrose, glucose, and fructose) in their leaves at night (13). To
investigate this further, we measured maltose, the major product of
starch breakdown (4 – 6, 19), 1 h prior to the end of the dark period. In
sex4-1, the relative maltose content was statistically significantly
reduced (55% that of the wild-type plants). This suggests that the
reduced availability of starch catabolites limits sucrose synthesis at
night. Second, we crossed sex4-5 (a T-DNA insertion mutant) with a
sex1 mutant. SEX1 encodes a glucan water dikinase (GWD1), which
phosphorylates glucosyl residues within the amylopectin moiety of
VOLUME 281 • NUMBER 17 • APRIL 28, 2006
Control of Starch Metabolism in Plants
FIGURE 3. The SEX4 protein is chloroplastic. A, protein localization by YFP fluorescence. Upper panels, wild-type (untransformed). Lower panels, sex4-1 plant transformed
with a construct encoding a SEX4-YFP fusion protein. Left, leaves harvested at the end of
the dark period and stained with iodine. Micrographs show confocal fluorescence
microscopy of fresh leaf tissue. Left image, YFP fluorescence; middle image, native chlorophyll fluorescence. The red objects are individual chloroplasts. Right image, merged
image showing coincidence of YFP and chlorophyll fluorescence. In the transformed
line, the expression of the SEX4-YFP fusion protein complements the sex phenotype.
Several independently transformed lines with these characteristics were obtained. B,
immunoblot of extracts of leaves of wild-type (Col) and sex4 mutant plants, and of chloroplasts (Chl) isolated from wild-type plants probed with an antiserum to SEX4. Masses of
molecular markers are shown in kDa; SEX4 is marked with an arrow. The SEX4 protein is
present in purified chloroplasts. It is absent from sex4-1 leaves as expected. SEX4 protein
is present in sex4-2 leaves but is expected to be inactive because of the substitution of an
amino acid that is strictly conserved in the active sites of all dual specificity protein
phosphatases (see Fig. 1).
starch (3, 20). Its action is necessary for normal rates of starch degradation; in its absence, starch accumulates to levels approximately twice
those observed in sex4 mutants (3, 13). The phosphate groups are
believed to facilitate access to the starch granule surface by the enzymes
that catalyze the initial attack on the granule (20); hence GWD1 can be
regarded as an initial step on the pathway of starch degradation. The
starch content of leaves of the double mutant sex4/sex1 closely resembled that of sex1 and was different from that of sex4 (Fig. 2D). At the end
of the light period, the starch content of sex4/sex1 was 1.7-fold higher
than that of sex4 and not statistically different from that of sex1. At the
end of the dark period, the starch content of sex4/sex1 was almost 2-fold
higher than that of sex4 and 80% of that of sex1. The simplest explanation for these data is that SEX4 affects GWD or a step immediately
downstream of it but upstream of maltose production.
SEX4 encodes a putative dual specificity protein phosphatase, PTPKIS1 (17). Genes encoding highly similar proteins are found in other
species of plants, including tomato, rice, and maize. The N-terminal
part of the protein contains the phosphatase domain, and the 63% identical tomato orthologue has been shown to have phosphatase activity
both on a generic phosphatase substrate and on the phosphotyrosine
residues of synthetic peptides (17). In addition, PTPKIS1 possesses a
C-terminal domain containing motifs characteristic of a carbohydrate
binding module (CBM_20; Refs. 21 and 22) (see supplemental Fig. S2).
To test whether the Arabidopsis protein can bind to carbohydrate, the
heterologously expressed C-terminal domain was incubated with glycogen in vitro. The protein bound to glycogen in a saturating manner,
and binding was inhibited by increasing concentrations of ␤-cyclodextrin. The protein also bound to amylose and to starch (Fig. 4 and data
not shown).
APRIL 28, 2006 • VOLUME 281 • NUMBER 17
FIGURE 4. The putative carbohydrate binding module of SEX4 binds to glycogen.
The CBM of SEX4 was expressed as a fusion protein with glutathione S-transferase (GST),
incubated with or without glycogen in the presence of BSA, and then subjected to ultracentrifugation. Control incubations contained GST or GST fused with the kinase interaction (KIS) domain of the protein kinase ZmAKIN ␤␥, which bears sequence similarities to
the CBM of SEX4 (17). The SDS-polyacrylamide gel shows proteins in the pellets. Incubations contained GST and BSA (lanes 1 and 2), CBM-GST and BSA (lanes 3 and 4), or KIS-GST
and BSA (lanes 5 and 6). Incubations shown in lanes 1, 3, and 5 contained glycogen; those
shown in lanes 2, 4, and 6 did not. The putative CBM domain of SEX4 binds glycogen (lane
3), whereas the KIS domain of ZmAKIN ␤␥ does not (lane 5). This experiment was
repeated three times with the same result. Further experiments (not shown) revealed
that CBM-GST fusion protein binds glycogen in a saturating manner and that binding is
inhibited by increasing concentrations of ␤-cyclodextrin, as is the case for starch-binding
proteins (37). The CBM-GST fusion protein also binds amylose and soluble starch (not
shown).
DISCUSSION
Taken together, the localization of SEX4 protein in chloroplasts, its
affinity for glucans, the phenotype of the sex4 mutant, and the diurnal
regulation of SEX4 transcript levels (1) suggest that SEX4 interacts with
starch in vivo and is directly necessary for its metabolism. SEX4 may
dephosphorylate and thus modulate the activity of an enzyme or
enzymes that directly exercises control over flux through the pathway of
starch degradation. Alternatively, SEX4 may act indirectly on starch
metabolism. In general, dual specificity protein phosphatases act on
protein kinases (23). SEX4 may thus modulate the activity of a protein
kinase, which in turn modulates the activity of enzyme(s) of starch
degradation.
The enzymes involved in starch degradation are not fully understood,
and there is little evidence thus far that they have regulatory properties
of importance in the control of flux through the pathway (1, 24). The
extent and importance of phosphorylation in modulating their activities
has not been investigated. However, phosphorylation has recently been
shown to be important in modulating the activity of enzymes of starch
synthesis; isoforms of starch-branching enzyme are activated by phosphorylation in chloroplasts and endosperm amyloplasts of wheat (25).
Our fractionation experiments and genetic analyses indicate that targets for modulation via SEX4 lie within the chloroplast and upstream of
maltose production in the pathway of starch degradation. Thus, possible targets include one or more of the following: glucan water dikinase
(SEX1 or GWD1) or phosphoglucan water dikinase (GWD3 or PWD,
thought to act after GWD) (7, 8), isoamylase 3 (10, 11), chloroplastic
␤-amylases (9), and possibly disproportionating enzyme (2). Mutant
plants lacking any one of these proteins have starch excess phenotypes,
and several of these proteins have been shown to be necessary for normal rates of starch granule degradation at night. The reason why the
chloroplastic ␣-amylase AMY3 is reduced in abundance in the absence
of SEX4 remains to be investigated.
Remarkably, the proteins most closely related to the SEX4-like proteins in plants are mammalian laforins (17, 26). These are also dual
specificity protein phosphatases with CBM_20 domains, although the
CBM is N-terminal in laforins (21, 27). Human and mouse laforins have
affinity for both glycogen and starch (27–30). Similar to SEX4, laforins
are necessary for normal metabolism of storage glucans. Humans and
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Control of Starch Metabolism in Plants
mice carrying mutations that affect laforin function accumulate polyglucosan inclusions, putatively arising from abnormal glycogen metabolism. These are composed of glucan polymers with branching patterns
thought to be more similar to those of the amylopectin component of
plant starch than those of glycogen (31). Polyglucosan inclusions are
implicated in neuronal death and consequent progressive myoclonus
epilepsy in humans and mice (32–34). Recently, laforin was shown to
dephosphorylate (and thereby activate) glycogen synthase kinase 3 at an
amino-terminal serine residue (Ser-9) (35). Active glycogen synthase
kinase 3 phosphorylates and inactivates glycogen synthase. Thus, loss of
laforin may allow glycogen synthase activity to proceed unchecked. Arabidopsis has 10 glycogen synthase kinase 3 homologues (36), one of
which (AtK-1/ASK␬, At1g09840) is predicted to be localized within the
chloroplast. This may represent a target for SEX4, although it is worth
noting, first, that the Ser-9 residue is not conserved in any known plant
glycogen synthase kinase 3 homologues (36) and, second, that the existing biochemical data in this and previous studies (12, 13) point toward a
deficiency in starch breakdown rather than activation of the starch biosynthetic pathway.
In conclusion, despite the appreciable differences in the enzymes
directly involved in starch metabolism in plants and glycogen metabolism in mammals, the striking similarities between SEX4 and laforins
indicate a previously unsuspected degree of convergence or conservation in the regulation of glucan metabolism.
Acknowledgments—We thank Dr. Alisdair Fernie and Nicolas Schauer for
assistance with the gas chromatography-mass spectrometry analysis and
Therese Mandel for access to ethane methyl sulfonate-mutagenized Arabidopsis population.
REFERENCES
1. Smith, S. M., Fulton, D. C., Chia, T., Thorneycroft, D., Chapple, A., Dunstan, H.,
Hylton, C., Zeeman, S. C., and Smith, A. M. (2004) Plant Physiol. 136, 2687–2699
2. Critchley, J. H., Zeeman, S. C., Takaha, T., Smith, A. M., and Smith, S. M. (2001) Plant
J. 26, 89 –100
3. Yu, T.-S., Kofler, H., Häusler, R., Hille, D., Flügge, U.-I., Zeeman, S. C., Smith, A. M.,
Kossmann, J., Lloyd, J., Ritte, G., Steup, M., Lue, W.-L., Chen, J., and Weber, A. (2001)
Plant Cell 13, 1907–1918
4. Niittylä, T., Messerli, G., Trevisan, M., Chen, J., Smith, A. M., and Zeeman, S. C.
(2004) Science 303, 87– 89
5. Chia, T., Thorneycroft, D., Chapple, A., Messerli, G., Chen, J., Zeeman, S. C., Smith,
S. M., and Smith, A. M. (2004) Plant J. 37, 853– 863
6. Lu, Y., and Sharkey, T. D. (2004) Planta 218, 468 – 473
7. Kötting, O., Pusch, P., Tiessen, A., Geigenberger, P., Steup, M., and Ritte, G. (2005)
Plant Physiol. 137, 242–252
8. Baunsgaard, L., Lütken, H., Mikkelsen, R., Glaring, M. A., Pham, T. T., and Blennow,
A. (2005) Plant J. 41, 595– 605
11818 JOURNAL OF BIOLOGICAL CHEMISTRY
9. Kaplan, F., and Guy, C. L. (2005) Plant J. 44, 730 –743
10. Wattebled, F., Dong, Y., Dumez, S., Delvallé, D., Planchot, V., Berbezy, P., Vyas, D.,
Colonna, P., Chatterjee, M., Ball, S., and D’Hulst, C. (2005) Plant Physiol. 138,
184 –195
11. Delatte, T., Umhang, M., Trevisan, M., Eicke, S., Thorneycroft, D., Smith, S. M., and
Zeeman, S. C. (February 22, 2006) J. Biol. Chem. 10.1074/jbc.M513661200
12. Zeeman, S. C., Northrop, F., Smith, A. M., and ap Rees, T. (1998) Plant J. 15, 357–365
13. Zeeman, S. C., and ap Rees, T. (1999) Plant Cell Environ. 22, 1445–1453
14. Zeeman, S. C., Tiessen, A., Pilling, E., Kato, L., Donald, A. M., and Smith, A. M. (2002)
Plant Physiol. 129, 516 –529
15. Yu, T.-S., Zeeman, S. C., Thorneycroft, D., Fulton, D. C., Dunstan, H., Lue, W.-L.,
Hegemann, B., Tung, S.-Y., Umemoto, U., Chapple, A., Tsai, D.-L., Wang, S.-M.,
Smith, A. M., Chen, J., and Smith, S. M. (2005) J. Biol. Chem. 280, 9773–9779
16. Fitzpatrick, L. M., and Keegstra, K. (2001) Plant J. 27, 59 – 65
17. Fordham-Skelton, A. P., Chilley, P., Lumbreras, V., Reignoux, S., Fenton, T. R., Dahm,
C. C., Pages, M., and Gatehouse, J. A. (2002) Plant J. 29, 705–715
18. Roessner, U., Luedemann, A., Brust, D., Fiehn, O., Linke, T., Willmitzer, L., and
Fernie, A. R. (2001) Plant Cell 13, 11–29
19. Weise, S. E., Weber, A. P. M., and Sharkey, T. D. (2004) Planta 218, 474 – 482
20. Ritte, G., Lloyd, J. R., Eckermann, N., Rottmann, A., Kossmann, J., and Steup, M.
(2002) Proc. Natl. Acad. Sci. U. S. A. 99, 7166 –7171
21. Machovič, M., Svensson, B., MacGregor, E. A., and Janeček, Š. (2005) FEBS J. 272,
5497–5513
22. Coutinho, P. M., and Henrissat, B. (1999) Recent Advances in Carbohydrate Bioengineering (Gilbert, H. J., Davies, G., Henrissat, B., and Svensson, B., eds) pp. 3–12, The
Royal Society of Chemistry, Cambridge, UK
23. Luan, S. (2003) Annu. Rev. Plant Biol. 54, 63–92
24. Smith, A. M., Zeeman, S.C, and Smith, S. M. (2005) Annu. Rev. Plant Biol. 56, 73–97
25. Tetlow, I. J., Wait, R., Lu, Z., Akkasaeng, R., Bowsher, C. G., Esposito, S., KosarHashemi, B., Morell, M. K., and Emes, M. J. (2004) Plant Cell 16, 694 –708
26. Kerk, D., Bulgrien, J., Smith, D. W., Barsam, B., Veretnik, S., and Gribskov, M. (2002)
Plant Physiol. 129, 908 –925
27. Wang, J., Stuckey, J. A., Wishart, M. J., and Dixon, J. E. (2002) J. Biol. Chem. 277,
2377–2380
28. Wang, W., and Roach, P. J. (2004) Biochem. Biophys. Res. Commun. 325, 726 –730
29. Ganesh, S., Tsurutani, N., Suzuki, T., Hoshii, Y., Ishihara, T., Delgado-Escueta, A. V.,
and Yamakawa, K. (2004) Biochem. Biophys. Res. Commun. 313, 1101–1109
30. Chan, E. M., Ackerley, C. A., Lohi, H., Ianzano, L., Cortez, M. A., Shannon, P., Scherer,
S. W., and Minassian, B. A. (2004) Hum. Mol. Genet. 13, 1117–1129
31. Minassian, B. A. (2001) Pediatr. Neurol. 25, 21–29
32. Minassian, B. A., Lee, J. R., Herbrick, J.-A., Huizenga, J., Soder, S., Mungall, A. J.,
Dunham, I., Gardner, R., Fong, C.-Y. G., Carpenter, S., Jardim, L., Satishchandra, P.,
Andermann, E., Snead, O. C., Lopes-Cendes, I., Tsui, L.-C., Delgado-Escueta, A. V.,
Rouleau, G. A., and Scherer, S. W. (1998) Nat. Genet. 20, 171–174
33. Serratosa, J. M., Gomez-Garre, P., Gallardo, M. E., Anta, B., de Bernabé, D. B., Lindhout, D., Augustijn, P. B., Tassinari, C. A., Malafosse, R. M., Topcu, M., Gris, D.,
Dravet, C., Berkovic, S. F., and de Cordoba, S. R. (1999) Hum. Mol. Genet. 2, 345–352
34. Ganesh, S., Delgado-Escueta, A. V., Sakamoto, T., Avila, M. R., Machado-Salas, J.,
Hoshii, Y., Akagi, T., Gomi, H., Suzuki, T., Amano, K., Agarwala, K. L., Hasegawa, Y.,
Bai, D.-S., Ishihara, T., Hashikawa, T., Itohara, S., Cornford, E. M., Niki, H., and
Yamakawa, K. (2002) Hum. Mol. Genet. 11, 1251–1262
35. Lohi, H., Ianzano, L., Zhao, X.-C., Chan, E. M., Turnbull, J., Scherer, S. W., Ackerley,
C. A., and Minassian, B. A. (2005) Hum. Mol. Genet. 14, 2727–2736
36. Jonak, C., and Hirt, H. (2002) Trends Plant Sci. 7, 457– 461
37. Belshaw, N. J., and Williamson, G. (1991) Biochim. Biophys. Acta 1078, 117–120
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