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Similar Protein Phosphatases Control Starch Metabolism in Plants and Glycogen Metabolism in Mammals *

Similar Protein Phosphatases Control Starch Metabolism in Plants and Glycogen Metabolism in... 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 □ S Plants and Glycogen Metabolism in Mammals 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 ‡1,2 ‡1 § ¶ 3 Totte Niittyla¨ , Sylviane Comparot-Moss , Wei-Ling Lue , Gae¨lle Messerli , Martine Trevisan , ‡‡ §§ § Michael D. J. Seymour**, John A. Gatehouse**, Dorthe Villadsen , Steven M. Smith , Jychian Chen , ¶4 ‡ Samuel C. Zeeman , 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, 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 tion at night leads to progressive accumulation of starch over the life of the starch metabolism in Arabidopsis leaves at night. sex4 (starch excess 4) leaf (12, 13). Starch granules in leaves of the sex4 mutant are much larger mutants, which have strongly reduced rates of starch metabolism, lack and more rounded than those of wild-type plants (14). Measurements of a protein predicted to be a dual specificity protein phosphatase. We activity and protein of enzymes known to be involved in starch degradation revealed only one significant reduction in the sex4 mutant in the chloro- have shown that this protein is chloroplastic and can bind to glucans plastic -amylase AMY3 (12, 15). However, although both the activity and and have presented evidence that it acts to regulate the initial steps of starchdegradationatthegranulesurface.Remarkably,themostclosely amount of protein of AMY3 are strongly reduced, this is not the cause of the related protein to SEX4 outside the plant kingdom is laforin, a glucan- deficiency in starch degradation in the sex4 mutant. T-DNA insertion lines lacking AMY3 protein have normal rates of starch degradation (15). The binding protein phosphatase required for the metabolism of the mam- aim of the work described in this paper was to discover the nature of the malian storage carbohydrate glycogen and implicated in a severe form gene at the SEX4 locus and thus shed light on the regulation of starch of epilepsy (Lafora disease) in humans. degradation. EXPERIMENTAL PROCEDURES Starch, the main storage carbohydrate of plants, accumulates as a Positional Identification of the SEX4 Locus—F2 plants from a cross product of photosynthesis in leaves during the day and is converted to between sex4-2 (Col-0 background) and Landsberg erecta showing the sucrose for export from the leaves at night. This conversion of starch to mutant phenotype were used for mapping. The mapping population sucrose is one of the largest daily carbon fluxes on the planet, but noth- (562 plants) was genotyped using SSLP and SNP markers available on ing is known about how the process is initiated and controlled. The the Arabidopsis Information Resource data base. This shows that the amounts of enzymes on the pathway change very little through the SEX4 gene was located within an 800-kb region between markers diurnal cycle in leaves of the model plant Arabidopsis thaliana, hence ATEM1 and SGCSNP42 on chromosome 3. flux must be controlled by modulation of their activities (1). Plant Growth and Transformation—Plants were grown in 12-h light/ Much progress in understanding the pathway has been made through 12-h dark conditions (20 °C, 60–70% relative humidity, 175 mol of pho- the selection of Arabidopsis mutants impaired in starch degradation at 2 1 tons m s ), unless otherwise stated. The SEX4 cDNA (U14967 from the night. All such mutations identified thus far are in genes encoding enzymes Arabidopsis Stock Center) was cloned into the binary vector 53AS with a 35 of the pathway, rather than proteins likely to be involved in modulation of S cauliflower mosaic virus promoter and introduced into the sex4-1 and the activities of these enzymes (2–11). However, a mutation at a locus not sex4-2 mutants via Agrobacterium-mediated transformation (by floral infil- yet identified, the starch excess 4 (or SEX4) locus, gives rise to a phenotype tration). Transgenic plants were selected by glufosinate resistance and con- indicative of a regulatory defect rather than a defect in a structural enzyme. firmed by PCR and immunoblot analyses. Additionally, a C-terminal fusion Mature sex4 leaves contain three to four times more starch than those of construct of SEX4 cDNA and enhanced yellow fluorescent protein (Clon- wild-type plants, apparently because a reduced capacity for starch degrada- TM tech ) was cloned into a vector with a double 35 S cauliflower mosaic virus promoter and introduced into Arabidopsis via Agrobacterium as * This work was supported by funding from the Biotechnology and Biological Sciences described previously (4). Research Council of the United Kingdom (to A. M. S. and J. A. G.), from the Swiss Gels, Antisera, and Immunoblotting—For the renaturation of amylo- National Science Foundation (National Centre of Competence in Research-Plant Sur- lytic activity, extracts were subjected to electrophoresis on SDS-poly- vival) 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 acrylamide gels containing starch. After washing and incubation in by the payment of page charges. This article must therefore be hereby marked SDS-free medium, the gels were stained with iodine solution (15). For “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 supple- preparation of an antiserum to SEX4, a construct encoding a fusion mental material. between the full-length SEX4 protein and glutathione S-transferase These authors contributed equally to this work. (GST) in the pGEX-4T-2 vector (Amersham Biosciences) was Present address: Carnegie Institution, Stanford, CA 94305-1297. Present address: Ctr. for Integrative Genomics, University of Lausanne, CH-1015, Lau- sanne, Switzerland. 4 5 To whom correspondence should be addressed: Institute of Plant Sciences, ETH Zurich, The abbreviations used are: GST, glutathione S-transferase; BSA, bovine serum albu- CH-8092 Zurich, Switzerland. Tel.: 41-44-632-8275; Fax: 41-44-632-1044; E-mail min; YFP, yellow fluorescent protein; GWD, glucan water dikinase; CBM, carbohydrate [email protected]. binding module; KIS, kinase interaction sequence. APRIL 28, 2006• VOLUME 281 • NUMBER 17 JOURNAL OF BIOLOGICAL CHEMISTRY 11815 This is an Open Access article under the CC BY license. 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, carbohy- drate 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. Anti- serum 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). 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. Preparation of Chloroplasts—Chloroplasts were isolated from proto- Wild-type leaves (ecotype Columbia (Col)) contain little starch and do not stain; sex4 plasts and purified on a Percoll gradient and by treatment with protease 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 (15, 16). The purity of the chloroplast preparation was confirmed by the (Col), plants carrying four different mutant alleles of sex4, and for comparison, sex1 absence of activity of cytosolic marker enzymes. Chloroplast extracts mutant plants. Plants were grown in a 12-h light, 12-h dark diurnal regime. Values are from wild-type plants and leaf extracts from wild-type and mutant 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 plants were loaded on a 10% SDS-polyacrylamide gel for the immuno- and of a plant of the same line transformed with a construct containing the wild-type blot analysis. Loading was adjusted so that each lane contained the same 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 activity of chloroplastic phosphoglucose isomerase. A 1:1000 dilution of eliminates the starch excess phenotype. D, starch contents at the end of the night (black crude antiserum was used to detect the SEX4 protein. bars) and the end of the day (white bars) of leaves of wild-type plants (Col), sex1 and Production of GST Fusion Protein—A fusion construct of the putative sex4 mutant plants, and a double mutant sex4/sex1. Experimental details are as described for B. carbohydrate binding module of the SEX4 protein and GST was pre- pared and expressed in E. coli as described previously (17; the carbohy- drate binding module is referred to as the kinase interaction sequence To provide further evidence about the identity of the SEX4 gene, we (KIS) domain in this reference). isolated two T-DNA insertion mutants in At3g52180 (Fig. 1, sex4-3 and Glycogen Binding Assays—Protein-free glycogen (5 mg ml )in50 sex4-5) and showed that they have starch excess phenotypes (Fig. 2A). mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.1% (v/v) 2-mercaptoethanol, Levels of starch are similar to those in plants carrying the previously 0.02% (w/v) Brij-35, 0.1 mg ml bovine serum albumin (BSA) was characterized mutant alleles (Fig. 2B). We also transformed the sex4-1 mixed with GST fusion protein. Samples were incubated at 0 °C for 30 and sex4-2 mutants with a cDNA encoding the wild-type SEX4 protein. min and then centrifuged 90 min at 100,000  g at 4 °C to sediment the Transformants no longer displayed a starch excess phenotype (Fig. 2C). glycogen. Pellets were washed in 50 mM Tris-HCl (pH 7.5), 150 mM All of the new sex4 mutant alleles had reduced levels of the chloroplastic NaCl, resuspended in 4 SDS sample buffer, and run on 12.5% SDS gels. -amylase AMY3 (see supplemental Fig. S1), as is the case for sex4-1 and The gels were stained with Coomassie Brilliant Blue R. sex4-2 (12, 15). Measurement of Maltose—Plants were grown in 8-h light/16-h dark The SEX4 protein has a predicted N-terminal chloroplast transit pep- conditions. Relative levels of maltose were determined by gas chroma- tide. To discover whether the protein is in fact chloroplastic, the sex4-1 tography linked to mass spectrometry using methods described previ- mutant was transformed with a construct encoding the wild-type SEX4 ously (18). protein fused at the C terminus to yellow fluorescent protein (YFP). The resulting transgenic plants no longer displayed a starch excess pheno- RESULTS type and exhibited YFP fluorescence specifically in the chloroplasts (Fig. The SEX4 locus was mapped to a region of 800 kb on chromosome 3. 3A). Furthermore, protein gel blots probed with an antiserum raised Gene discovery was facilitated by the observation that one gene in this against the SEX4 protein revealed that chloroplasts isolated from the region (At3g52180) displays the same distinctive pattern of diurnal leaves of wild-type plants contained a protein with a similar apparent change in transcript abundance in the leaf as genes encoding enzymes mass to that of the predicted SEX4 protein. This protein was missing known to be involved in starch degradation (1). Sequencing revealed from leaves of sex4-1 mutant plants (Fig. 3B). mutations likely to prevent or impair function in this gene in plants Previously, we have shown that sex4 mutants have lower levels of carrying three independent sex4 alleles (Fig. 1). The sex4-1 allele con- sugars (sucrose, glucose, and fructose) in their leaves at night (13). To tains a deletion that overlaps the open reading frames of both investigate this further, we measured maltose, the major product of At3g52180 and At3g52190. The sex4-2 allele contains a point mutation starch breakdown (4–6, 19), 1 h prior to the end of the dark period. In in the seventh exon. This is predicted to change the arginine residue of sex4-1, the relative maltose content was statistically significantly the signature motif of a protein phosphatase (see last paragraph under reduced (55% that of the wild-type plants). This suggests that the “Results”) to a lysine; hence this change is highly likely to affect protein reduced availability of starch catabolites limits sucrose synthesis at function. The sex4-4 allele contains a point mutation that gives rise to a night. Second, we crossed sex4-5 (a T-DNA insertion mutant) with a stop codon and results in a truncated protein (Fig. 1 and data not sex1 mutant. SEX1 encodes a glucan water dikinase (GWD1), which shown). phosphorylates glucosyl residues within the amylopectin moiety of 11816 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 17 •APRIL 28, 2006 Control of Starch Metabolism in Plants 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 ultra- centrifugation. Control incubations contained GST or GST fused with the kinase interac- tion (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. Incuba- tions 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 FIGURE 3. The SEX4 protein is chloroplastic. A, protein localization by YFP fluores- 3), whereas the KIS domain of ZmAKIN  does not (lane 5). This experiment was cence. Upper panels, wild-type (untransformed). Lower panels, sex4-1 plant transformed repeated three times with the same result. Further experiments (not shown) revealed with a construct encoding a SEX4-YFP fusion protein. Left, leaves harvested at the end of that CBM-GST fusion protein binds glycogen in a saturating manner and that binding is the dark period and stained with iodine. Micrographs show confocal fluorescence inhibited by increasing concentrations of-cyclodextrin, as is the case for starch-binding microscopy of fresh leaf tissue. Left image, YFP fluorescence; middle image, native chlo- proteins (37). The CBM-GST fusion protein also binds amylose and soluble starch (not rophyll fluorescence. The red objects are individual chloroplasts. Right image, merged shown). image showing coincidence of YFP and chlorophyll fluorescence. In the transformed line, the expression of the SEX4-YFP fusion protein complements the sex phenotype. DISCUSSION 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 chlo- Taken together, the localization of SEX4 protein in chloroplasts, its roplasts (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 affinity for glucans, the phenotype of the sex4 mutant, and the diurnal present in purified chloroplasts. It is absent from sex4-1 leaves as expected. SEX4 protein regulation of SEX4 transcript levels (1) suggest that SEX4 interacts with is present in sex4-2 leaves but is expected to be inactive because of the substitution of an starch in vivo and is directly necessary for its metabolism. SEX4 may amino acid that is strictly conserved in the active sites of all dual specificity protein phosphatases (see Fig. 1). 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 starch (3, 20). Its action is necessary for normal rates of starch degrada- metabolism. In general, dual specificity protein phosphatases act on tion; in its absence, starch accumulates to levels approximately twice protein kinases (23). SEX4 may thus modulate the activity of a protein those observed in sex4 mutants (3, 13). The phosphate groups are kinase, which in turn modulates the activity of enzyme(s) of starch believed to facilitate access to the starch granule surface by the enzymes degradation. that catalyze the initial attack on the granule (20); hence GWD1 can be The enzymes involved in starch degradation are not fully understood, regarded as an initial step on the pathway of starch degradation. The and there is little evidence thus far that they have regulatory properties starch content of leaves of the double mutant sex4/sex1 closely resem- of importance in the control of flux through the pathway (1, 24). The bled that of sex1 and was different from that of sex4 (Fig. 2D). At the end extent and importance of phosphorylation in modulating their activities of the light period, the starch content of sex4/sex1 was 1.7-fold higher has not been investigated. However, phosphorylation has recently been than that of sex4 and not statistically different from that of sex1.Atthe shown to be important in modulating the activity of enzymes of starch end of the dark period, the starch content of sex4/sex1 was almost 2-fold synthesis; isoforms of starch-branching enzyme are activated by phos- higher than that of sex4 and 80% of that of sex1. The simplest explana- phorylation in chloroplasts and endosperm amyloplasts of wheat (25). tion for these data is that SEX4 affects GWD or a step immediately Our fractionation experiments and genetic analyses indicate that tar- downstream of it but upstream of maltose production. gets for modulation via SEX4 lie within the chloroplast and upstream of SEX4 encodes a putative dual specificity protein phosphatase, PTP- maltose production in the pathway of starch degradation. Thus, possi- ble targets include one or more of the following: glucan water dikinase KIS1 (17). Genes encoding highly similar proteins are found in other (SEX1 or GWD1) or phosphoglucan water dikinase (GWD3 or PWD, species of plants, including tomato, rice, and maize. The N-terminal thought to act after GWD) (7, 8), isoamylase 3 (10, 11), chloroplastic part of the protein contains the phosphatase domain, and the 63% iden- -amylases (9), and possibly disproportionating enzyme (2). Mutant tical tomato orthologue has been shown to have phosphatase activity plants lacking any one of these proteins have starch excess phenotypes, both on a generic phosphatase substrate and on the phosphotyrosine and several of these proteins have been shown to be necessary for nor- residues of synthetic peptides (17). In addition, PTPKIS1 possesses a mal rates of starch granule degradation at night. The reason why the C-terminal domain containing motifs characteristic of a carbohydrate chloroplastic -amylase AMY3 is reduced in abundance in the absence binding module (CBM_20; Refs. 21 and 22) (see supplemental Fig. S2). of SEX4 remains to be investigated. To test whether the Arabidopsis protein can bind to carbohydrate, the Remarkably, the proteins most closely related to the SEX4-like pro- heterologously expressed C-terminal domain was incubated with gly- teins in plants are mammalian laforins (17, 26). These are also dual cogen in vitro. The protein bound to glycogen in a saturating manner, specificity protein phosphatases with CBM_20 domains, although the and binding was inhibited by increasing concentrations of -cyclodex- CBM is N-terminal in laforins (21, 27). Human and mouse laforins have trin. The protein also bound to amylose and to starch (Fig. 4 and data affinity for both glycogen and starch (27–30). Similar to SEX4, laforins not shown). are necessary for normal metabolism of storage glucans. Humans and APRIL 28, 2006• VOLUME 281 • NUMBER 17 JOURNAL OF BIOLOGICAL CHEMISTRY 11817 Control of Starch Metabolism in Plants 9. Kaplan, F., and Guy, C. L. (2005) Plant J. 44, 730–743 mice carrying mutations that affect laforin function accumulate poly- 10. Wattebled, F., Dong, Y., Dumez, S., Delvalle´, D., Planchot, V., Berbezy, P., Vyas, D., glucosan inclusions, putatively arising from abnormal glycogen metab- Colonna, P., Chatterjee, M., Ball, S., and D’Hulst, C. (2005) Plant Physiol. 138, olism. These are composed of glucan polymers with branching patterns 184–195 thought to be more similar to those of the amylopectin component of 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 plant starch than those of glycogen (31). Polyglucosan inclusions are 12. Zeeman, S. 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Similar Protein Phosphatases Control Starch Metabolism in Plants and Glycogen Metabolism in Mammals *

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American Society for Biochemistry and Molecular Biology
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Copyright © 2006 Elsevier Inc.
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0021-9258
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DOI
10.1074/jbc.m600519200
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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 □ S Plants and Glycogen Metabolism in Mammals 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 ‡1,2 ‡1 § ¶ 3 Totte Niittyla¨ , Sylviane Comparot-Moss , Wei-Ling Lue , Gae¨lle Messerli , Martine Trevisan , ‡‡ §§ § Michael D. J. Seymour**, John A. Gatehouse**, Dorthe Villadsen , Steven M. Smith , Jychian Chen , ¶4 ‡ Samuel C. Zeeman , 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, 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 tion at night leads to progressive accumulation of starch over the life of the starch metabolism in Arabidopsis leaves at night. sex4 (starch excess 4) leaf (12, 13). Starch granules in leaves of the sex4 mutant are much larger mutants, which have strongly reduced rates of starch metabolism, lack and more rounded than those of wild-type plants (14). Measurements of a protein predicted to be a dual specificity protein phosphatase. We activity and protein of enzymes known to be involved in starch degradation revealed only one significant reduction in the sex4 mutant in the chloro- have shown that this protein is chloroplastic and can bind to glucans plastic -amylase AMY3 (12, 15). However, although both the activity and and have presented evidence that it acts to regulate the initial steps of starchdegradationatthegranulesurface.Remarkably,themostclosely amount of protein of AMY3 are strongly reduced, this is not the cause of the related protein to SEX4 outside the plant kingdom is laforin, a glucan- deficiency in starch degradation in the sex4 mutant. T-DNA insertion lines lacking AMY3 protein have normal rates of starch degradation (15). The binding protein phosphatase required for the metabolism of the mam- aim of the work described in this paper was to discover the nature of the malian storage carbohydrate glycogen and implicated in a severe form gene at the SEX4 locus and thus shed light on the regulation of starch of epilepsy (Lafora disease) in humans. degradation. EXPERIMENTAL PROCEDURES Starch, the main storage carbohydrate of plants, accumulates as a Positional Identification of the SEX4 Locus—F2 plants from a cross product of photosynthesis in leaves during the day and is converted to between sex4-2 (Col-0 background) and Landsberg erecta showing the sucrose for export from the leaves at night. This conversion of starch to mutant phenotype were used for mapping. The mapping population sucrose is one of the largest daily carbon fluxes on the planet, but noth- (562 plants) was genotyped using SSLP and SNP markers available on ing is known about how the process is initiated and controlled. The the Arabidopsis Information Resource data base. This shows that the amounts of enzymes on the pathway change very little through the SEX4 gene was located within an 800-kb region between markers diurnal cycle in leaves of the model plant Arabidopsis thaliana, hence ATEM1 and SGCSNP42 on chromosome 3. flux must be controlled by modulation of their activities (1). Plant Growth and Transformation—Plants were grown in 12-h light/ Much progress in understanding the pathway has been made through 12-h dark conditions (20 °C, 60–70% relative humidity, 175 mol of pho- the selection of Arabidopsis mutants impaired in starch degradation at 2 1 tons m s ), unless otherwise stated. The SEX4 cDNA (U14967 from the night. All such mutations identified thus far are in genes encoding enzymes Arabidopsis Stock Center) was cloned into the binary vector 53AS with a 35 of the pathway, rather than proteins likely to be involved in modulation of S cauliflower mosaic virus promoter and introduced into the sex4-1 and the activities of these enzymes (2–11). However, a mutation at a locus not sex4-2 mutants via Agrobacterium-mediated transformation (by floral infil- yet identified, the starch excess 4 (or SEX4) locus, gives rise to a phenotype tration). Transgenic plants were selected by glufosinate resistance and con- indicative of a regulatory defect rather than a defect in a structural enzyme. firmed by PCR and immunoblot analyses. Additionally, a C-terminal fusion Mature sex4 leaves contain three to four times more starch than those of construct of SEX4 cDNA and enhanced yellow fluorescent protein (Clon- wild-type plants, apparently because a reduced capacity for starch degrada- TM tech ) was cloned into a vector with a double 35 S cauliflower mosaic virus promoter and introduced into Arabidopsis via Agrobacterium as * This work was supported by funding from the Biotechnology and Biological Sciences described previously (4). Research Council of the United Kingdom (to A. M. S. and J. A. G.), from the Swiss Gels, Antisera, and Immunoblotting—For the renaturation of amylo- National Science Foundation (National Centre of Competence in Research-Plant Sur- lytic activity, extracts were subjected to electrophoresis on SDS-poly- vival) 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 acrylamide gels containing starch. After washing and incubation in by the payment of page charges. This article must therefore be hereby marked SDS-free medium, the gels were stained with iodine solution (15). For “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 supple- preparation of an antiserum to SEX4, a construct encoding a fusion mental material. between the full-length SEX4 protein and glutathione S-transferase These authors contributed equally to this work. (GST) in the pGEX-4T-2 vector (Amersham Biosciences) was Present address: Carnegie Institution, Stanford, CA 94305-1297. Present address: Ctr. for Integrative Genomics, University of Lausanne, CH-1015, Lau- sanne, Switzerland. 4 5 To whom correspondence should be addressed: Institute of Plant Sciences, ETH Zurich, The abbreviations used are: GST, glutathione S-transferase; BSA, bovine serum albu- CH-8092 Zurich, Switzerland. Tel.: 41-44-632-8275; Fax: 41-44-632-1044; E-mail min; YFP, yellow fluorescent protein; GWD, glucan water dikinase; CBM, carbohydrate [email protected]. binding module; KIS, kinase interaction sequence. APRIL 28, 2006• VOLUME 281 • NUMBER 17 JOURNAL OF BIOLOGICAL CHEMISTRY 11815 This is an Open Access article under the CC BY license. 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, carbohy- drate 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. Anti- serum 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). 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. Preparation of Chloroplasts—Chloroplasts were isolated from proto- Wild-type leaves (ecotype Columbia (Col)) contain little starch and do not stain; sex4 plasts and purified on a Percoll gradient and by treatment with protease 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 (15, 16). The purity of the chloroplast preparation was confirmed by the (Col), plants carrying four different mutant alleles of sex4, and for comparison, sex1 absence of activity of cytosolic marker enzymes. Chloroplast extracts mutant plants. Plants were grown in a 12-h light, 12-h dark diurnal regime. Values are from wild-type plants and leaf extracts from wild-type and mutant 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 plants were loaded on a 10% SDS-polyacrylamide gel for the immuno- and of a plant of the same line transformed with a construct containing the wild-type blot analysis. Loading was adjusted so that each lane contained the same 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 activity of chloroplastic phosphoglucose isomerase. A 1:1000 dilution of eliminates the starch excess phenotype. D, starch contents at the end of the night (black crude antiserum was used to detect the SEX4 protein. bars) and the end of the day (white bars) of leaves of wild-type plants (Col), sex1 and Production of GST Fusion Protein—A fusion construct of the putative sex4 mutant plants, and a double mutant sex4/sex1. Experimental details are as described for B. carbohydrate binding module of the SEX4 protein and GST was pre- pared and expressed in E. coli as described previously (17; the carbohy- drate binding module is referred to as the kinase interaction sequence To provide further evidence about the identity of the SEX4 gene, we (KIS) domain in this reference). isolated two T-DNA insertion mutants in At3g52180 (Fig. 1, sex4-3 and Glycogen Binding Assays—Protein-free glycogen (5 mg ml )in50 sex4-5) and showed that they have starch excess phenotypes (Fig. 2A). mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.1% (v/v) 2-mercaptoethanol, Levels of starch are similar to those in plants carrying the previously 0.02% (w/v) Brij-35, 0.1 mg ml bovine serum albumin (BSA) was characterized mutant alleles (Fig. 2B). We also transformed the sex4-1 mixed with GST fusion protein. Samples were incubated at 0 °C for 30 and sex4-2 mutants with a cDNA encoding the wild-type SEX4 protein. min and then centrifuged 90 min at 100,000  g at 4 °C to sediment the Transformants no longer displayed a starch excess phenotype (Fig. 2C). glycogen. Pellets were washed in 50 mM Tris-HCl (pH 7.5), 150 mM All of the new sex4 mutant alleles had reduced levels of the chloroplastic NaCl, resuspended in 4 SDS sample buffer, and run on 12.5% SDS gels. -amylase AMY3 (see supplemental Fig. S1), as is the case for sex4-1 and The gels were stained with Coomassie Brilliant Blue R. sex4-2 (12, 15). Measurement of Maltose—Plants were grown in 8-h light/16-h dark The SEX4 protein has a predicted N-terminal chloroplast transit pep- conditions. Relative levels of maltose were determined by gas chroma- tide. To discover whether the protein is in fact chloroplastic, the sex4-1 tography linked to mass spectrometry using methods described previ- mutant was transformed with a construct encoding the wild-type SEX4 ously (18). protein fused at the C terminus to yellow fluorescent protein (YFP). The resulting transgenic plants no longer displayed a starch excess pheno- RESULTS type and exhibited YFP fluorescence specifically in the chloroplasts (Fig. The SEX4 locus was mapped to a region of 800 kb on chromosome 3. 3A). Furthermore, protein gel blots probed with an antiserum raised Gene discovery was facilitated by the observation that one gene in this against the SEX4 protein revealed that chloroplasts isolated from the region (At3g52180) displays the same distinctive pattern of diurnal leaves of wild-type plants contained a protein with a similar apparent change in transcript abundance in the leaf as genes encoding enzymes mass to that of the predicted SEX4 protein. This protein was missing known to be involved in starch degradation (1). Sequencing revealed from leaves of sex4-1 mutant plants (Fig. 3B). mutations likely to prevent or impair function in this gene in plants Previously, we have shown that sex4 mutants have lower levels of carrying three independent sex4 alleles (Fig. 1). The sex4-1 allele con- sugars (sucrose, glucose, and fructose) in their leaves at night (13). To tains a deletion that overlaps the open reading frames of both investigate this further, we measured maltose, the major product of At3g52180 and At3g52190. The sex4-2 allele contains a point mutation starch breakdown (4–6, 19), 1 h prior to the end of the dark period. In in the seventh exon. This is predicted to change the arginine residue of sex4-1, the relative maltose content was statistically significantly the signature motif of a protein phosphatase (see last paragraph under reduced (55% that of the wild-type plants). This suggests that the “Results”) to a lysine; hence this change is highly likely to affect protein reduced availability of starch catabolites limits sucrose synthesis at function. The sex4-4 allele contains a point mutation that gives rise to a night. Second, we crossed sex4-5 (a T-DNA insertion mutant) with a stop codon and results in a truncated protein (Fig. 1 and data not sex1 mutant. SEX1 encodes a glucan water dikinase (GWD1), which shown). phosphorylates glucosyl residues within the amylopectin moiety of 11816 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 17 •APRIL 28, 2006 Control of Starch Metabolism in Plants 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 ultra- centrifugation. Control incubations contained GST or GST fused with the kinase interac- tion (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. Incuba- tions 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 FIGURE 3. The SEX4 protein is chloroplastic. A, protein localization by YFP fluores- 3), whereas the KIS domain of ZmAKIN  does not (lane 5). This experiment was cence. Upper panels, wild-type (untransformed). Lower panels, sex4-1 plant transformed repeated three times with the same result. Further experiments (not shown) revealed with a construct encoding a SEX4-YFP fusion protein. Left, leaves harvested at the end of that CBM-GST fusion protein binds glycogen in a saturating manner and that binding is the dark period and stained with iodine. Micrographs show confocal fluorescence inhibited by increasing concentrations of-cyclodextrin, as is the case for starch-binding microscopy of fresh leaf tissue. Left image, YFP fluorescence; middle image, native chlo- proteins (37). The CBM-GST fusion protein also binds amylose and soluble starch (not rophyll fluorescence. The red objects are individual chloroplasts. Right image, merged shown). image showing coincidence of YFP and chlorophyll fluorescence. In the transformed line, the expression of the SEX4-YFP fusion protein complements the sex phenotype. DISCUSSION 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 chlo- Taken together, the localization of SEX4 protein in chloroplasts, its roplasts (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 affinity for glucans, the phenotype of the sex4 mutant, and the diurnal present in purified chloroplasts. It is absent from sex4-1 leaves as expected. SEX4 protein regulation of SEX4 transcript levels (1) suggest that SEX4 interacts with is present in sex4-2 leaves but is expected to be inactive because of the substitution of an starch in vivo and is directly necessary for its metabolism. SEX4 may amino acid that is strictly conserved in the active sites of all dual specificity protein phosphatases (see Fig. 1). 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 starch (3, 20). Its action is necessary for normal rates of starch degrada- metabolism. In general, dual specificity protein phosphatases act on tion; in its absence, starch accumulates to levels approximately twice protein kinases (23). SEX4 may thus modulate the activity of a protein those observed in sex4 mutants (3, 13). The phosphate groups are kinase, which in turn modulates the activity of enzyme(s) of starch believed to facilitate access to the starch granule surface by the enzymes degradation. that catalyze the initial attack on the granule (20); hence GWD1 can be The enzymes involved in starch degradation are not fully understood, regarded as an initial step on the pathway of starch degradation. The and there is little evidence thus far that they have regulatory properties starch content of leaves of the double mutant sex4/sex1 closely resem- of importance in the control of flux through the pathway (1, 24). The bled that of sex1 and was different from that of sex4 (Fig. 2D). At the end extent and importance of phosphorylation in modulating their activities of the light period, the starch content of sex4/sex1 was 1.7-fold higher has not been investigated. However, phosphorylation has recently been than that of sex4 and not statistically different from that of sex1.Atthe shown to be important in modulating the activity of enzymes of starch end of the dark period, the starch content of sex4/sex1 was almost 2-fold synthesis; isoforms of starch-branching enzyme are activated by phos- higher than that of sex4 and 80% of that of sex1. The simplest explana- phorylation in chloroplasts and endosperm amyloplasts of wheat (25). tion for these data is that SEX4 affects GWD or a step immediately Our fractionation experiments and genetic analyses indicate that tar- downstream of it but upstream of maltose production. gets for modulation via SEX4 lie within the chloroplast and upstream of SEX4 encodes a putative dual specificity protein phosphatase, PTP- maltose production in the pathway of starch degradation. Thus, possi- ble targets include one or more of the following: glucan water dikinase KIS1 (17). Genes encoding highly similar proteins are found in other (SEX1 or GWD1) or phosphoglucan water dikinase (GWD3 or PWD, species of plants, including tomato, rice, and maize. The N-terminal thought to act after GWD) (7, 8), isoamylase 3 (10, 11), chloroplastic part of the protein contains the phosphatase domain, and the 63% iden- -amylases (9), and possibly disproportionating enzyme (2). Mutant tical tomato orthologue has been shown to have phosphatase activity plants lacking any one of these proteins have starch excess phenotypes, both on a generic phosphatase substrate and on the phosphotyrosine and several of these proteins have been shown to be necessary for nor- residues of synthetic peptides (17). In addition, PTPKIS1 possesses a mal rates of starch granule degradation at night. The reason why the C-terminal domain containing motifs characteristic of a carbohydrate chloroplastic -amylase AMY3 is reduced in abundance in the absence binding module (CBM_20; Refs. 21 and 22) (see supplemental Fig. S2). of SEX4 remains to be investigated. To test whether the Arabidopsis protein can bind to carbohydrate, the Remarkably, the proteins most closely related to the SEX4-like pro- heterologously expressed C-terminal domain was incubated with gly- teins in plants are mammalian laforins (17, 26). These are also dual cogen in vitro. The protein bound to glycogen in a saturating manner, specificity protein phosphatases with CBM_20 domains, although the and binding was inhibited by increasing concentrations of -cyclodex- CBM is N-terminal in laforins (21, 27). Human and mouse laforins have trin. The protein also bound to amylose and to starch (Fig. 4 and data affinity for both glycogen and starch (27–30). Similar to SEX4, laforins not shown). are necessary for normal metabolism of storage glucans. Humans and APRIL 28, 2006• VOLUME 281 • NUMBER 17 JOURNAL OF BIOLOGICAL CHEMISTRY 11817 Control of Starch Metabolism in Plants 9. Kaplan, F., and Guy, C. L. (2005) Plant J. 44, 730–743 mice carrying mutations that affect laforin function accumulate poly- 10. Wattebled, F., Dong, Y., Dumez, S., Delvalle´, D., Planchot, V., Berbezy, P., Vyas, D., glucosan inclusions, putatively arising from abnormal glycogen metab- Colonna, P., Chatterjee, M., Ball, S., and D’Hulst, C. (2005) Plant Physiol. 138, olism. These are composed of glucan polymers with branching patterns 184–195 thought to be more similar to those of the amylopectin component of 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 plant starch than those of glycogen (31). Polyglucosan inclusions are 12. Zeeman, S. 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Journal

Journal of Biological Chemistry – American Society for Biochemistry and Molecular Biology

Published: Apr 28, 2006

References