Journal of Plant Biochemistry and Biotechnology

, Volume 22, Issue 4, pp 441–452 | Cite as

Molecular cloning and sequence comparison of a cDNA encoding α-glucan, water dikinase (GWD) from potato (Solanum tuberosum L.), and analysis of gene expression



Starch phosphorylation is an important biochemical aspect of plant starch metabolism as it influences the overall structure of the starch granule, and a prerequisite for its degradation. There is a growing interest on the isolation and characterization of α-glucan/glucan-like, water dikinases (GWDs) from plants, particularly agriculturally important crops, because GWD is known to catalyze starch phosphorylation both in leaves and different plant storage organs. In the present study, a 4,789-bp full-length cDNA encoding a GWD isoform was isolated from a commercially important Indian potato cultivar, Kufri Chipsona-1 by RT-PCR approach using tuber RNA. The predicted protein consisted of 1,463 amino acids having N-terminal 77-amino acid transit peptide, and 1,386-amino acid mature protein shorter by one amino acid as compared to the other mature GWDs from potato and tomato. The mature GWD showed 98 % sequence identity with the GWD isolated earlier from the potato cv. Desiree. Variations were found at 25 locations representing mostly non-conservative substitutions. The GWD represents a distinct isoform from potato, as revealed by sequence and phylogenetic analyses. Amino acid composition, segment-wise hydrophobic characters, predicted secondary structures were also analyzed and documented in this report. Broadly, the level of GWD expression as analyzed by semi-quantitative RT-PCR approach was found to be nearly uniform both in the mature tubers and leaves from most of the potato cultivars. By immunodetection technique, a band corresponding to ~155 kDa protein was detected only in the tuber protein extracts. The tuber starch-bound phosphorus content data showed minor variations between the potato cultivars.


GWD cDNA cloning GWD expression Indian potato cultivars RT-PCR Sequence analysis Tuber starch-bound phosphorus 



Carbohydrate binding module


Complementary deoxyribonucleic acid


European Molecular Biology Laboratory


α-glucan water dikinase


Molecular Evolutionary Genetics Analysis


National Center for Biotechnology Information


Phosphoglucan water dikinase


Reverse transcription-polymerase chain reaction


Starch binding domain


Starch excess


  1. Baunsgaard L, Lutken H, Mikkelsen R, Glaring MA, Pham TT, Blennow A (2005) A novel isoform of Glucan, water dikinase phosphorylates pre-phosphorylated α-glucans and is involved in starch degradation in Arabidopsis. Plant J 41:595–605PubMedCrossRefGoogle Scholar
  2. Bay-Smidt A, Wischmann B, Olsen CE, Nielsen TH (1994) Starch bound phosphate in potato as studied by a simple method for determination of organic phosphate and 31P-NMR. Starch-Starke 46:167–172Google Scholar
  3. Blennow A, Bay-Smidt AM, Olsen CE, Møller BL (2000) The distribution of covalently bound phosphate in the starch granule in relation to starch crystallinity. Int J Biol Macromol 27:211–218PubMedCrossRefGoogle Scholar
  4. Blenow A, Engelsen SB, Nielson TH, Baunsgaard L, Mikkelsen R (2002) Starch Phosphorylation: a new front line in starch research. Trends Plant Sci 7:445–450CrossRefGoogle Scholar
  5. Chou PY, Fasman GD (1974a) Prediction of protein conformation. Biochem 13:222–245CrossRefGoogle Scholar
  6. Chou PY, Fasman GD (1974b) Conformational parameters for amino acids in helical, β-sheet, and random coil regions calculated from proteins. Biochem 13:211–222CrossRefGoogle Scholar
  7. Corpet F (1988) Multiple sequence alignment with hierarchical clustering. Nucleic Acids Res 16:10881–10890PubMedCrossRefGoogle Scholar
  8. Deleage G, Roux B (1987) Amino acid scale: conformational parameter for coil. Protein Eng 1:289–294PubMedCrossRefGoogle Scholar
  9. Doolittle RF (1989) Redundancies in protein sequences. In: Fasman GD (ed) Prediction of protein structure and the principles of protein conformation. Plenum Press, New York, pp 599–623CrossRefGoogle Scholar
  10. Gilman M (1987) Phenol/SDS method for plant RNA preparation. In: Ausubel FM et al (eds) Current protocols in molecular biology. John Wiley and Sons, New York, pp 431–434Google Scholar
  11. Glaring MA, Baumann MJ, Hachem MA, Nakai H, Nakai N, Santelia D, Sigurskjold BW, Zeeman SC, Blennow A, Svensson B (2011) Starch-binding domains in the CBM45 family-low-affinity domains from glucan, water dikinase and α-amylase involved in plastidial starch metabolism. FEBS J 278:1175–1185PubMedCrossRefGoogle Scholar
  12. Hejazi M, Fettke J, Haebel S, Edner C, Paris O, Frohberg C, Steup M, Ritte G (2008) Glucan, water dikinase phosphorylates crystalline Maltodextrins and thereby initiates solubilization. Plant J 55:323–334PubMedCrossRefGoogle Scholar
  13. Hizukuri S, Tabata S, Nikuni Z (1970) Studies on starch phosphate. Part 1. Estimation of glucose-6-phosphate residues in starch and the presence of other bound phosphate(s). Starch-Starke 22:338–343CrossRefGoogle Scholar
  14. Jobling S (2004) Improving starch for food and industrial applications. Curr Opin Plant Biol 7:210–218PubMedCrossRefGoogle Scholar
  15. Kolaskar AS, Tongaonkar PC (1990) A semi-empirical method for prediction of antigenic determinants on protein antigens. FEBS Lett 276:172–174PubMedCrossRefGoogle Scholar
  16. Kötting O, Pusch K, Tiessen A, Geigenberger P, Steup M, Ritte G (2005) Identification of a novel enzyme required for starch metabolism in Arabidopsis leaves. The Phosphoglucan, Water Dikinase. Plant Physiol 137:242–252PubMedCrossRefGoogle Scholar
  17. Kötting O, Kossman J, Zeeman SC, Lloyd J (2010) Regulation of starch metabolism: the age of enlightenment? Curr Opin Plant Biol 13:321–329PubMedCrossRefGoogle Scholar
  18. Kyte J, Doolittle RF (1982) A simple method for displaying the hydropathic character of a protein. J Mol Biol 157:105–132PubMedCrossRefGoogle Scholar
  19. Li CY, Weiss D, Goldschmidt EE (2003) Effects of carbohydrate starvation on gene expression in citrus root. Planta 217:11–20PubMedGoogle Scholar
  20. Lorberth R, Ritte G, Willmitzer L, Kossmann J (1998) Inhibition of a starch-granule-bound protein leads to modified starch and repression of cold sweetening. Nat Biotechnol 16:473–477PubMedCrossRefGoogle Scholar
  21. Mikkelsen R, Blennow A (2005) Functional domain organization of the potato α-glucan, water dikinase (GWD): evidence for separate site catalysis as revealed by limited proteolysis and deletion mutants. Biochem J 385:355–361PubMedCrossRefGoogle Scholar
  22. Mikkelsen R, Baunsgaard L, Blennow A (2004) Functional characterization of α-glucan, water dikinase, the starch phosphorylating enzyme. Biochem J 377:525–532PubMedCrossRefGoogle Scholar
  23. Mikkelsen R, Suszkiewicz K, Blennow A (2006) A novel type carbohydrate-binding module identified in alpha-glucan, water dikinases is specific for regulated plastidial starch metabolism. Biochem 45:4674–4682CrossRefGoogle Scholar
  24. Morrison WR (1964) A fast, simple and reliable method for the microdetermination of phosphorus in biological materials. Anal Biochem 7:218–224PubMedCrossRefGoogle Scholar
  25. Nashilevitz S, Bessudo CM, Aharoni A, Kossmann J, Wolf S, Levy AA (2009) The legwd mutant uncovers the role of starch Phosphorylation in pollen development and germination in tomato. Plant J 57:1–13PubMedCrossRefGoogle Scholar
  26. Nielsen TH, Wischmann B, Enevoldsen K, Moller BL (1994) Starch phosphorylation in potato tubers proceeds concurrently with de novo biosynthesis of starch. Plant Physiol 105:111–117PubMedGoogle Scholar
  27. Ritte G, Eckermann N, Haebel S, Lorberth R, Steup M (2000a) Compartmentation of the starch-related R1 protein in higher plants. Starch-Starke 52:145–149CrossRefGoogle Scholar
  28. Ritte G, Lorberth R, Steup M (2000b) Reversible binding of the starch-related R1 protein to the surface of transitory starch granules. Plant J 21:387–391PubMedCrossRefGoogle Scholar
  29. Ritte G, Lloyd JR, Ekermann N, Rottmann A, Kossmann J, Steup M (2002) The starch-related R1 protein is an α-glucan, water dikinase. Proc Natl Acad Sci USA 99:7166–7171PubMedCrossRefGoogle Scholar
  30. Ritte G, Heydenreich M, Mahlow S, Haebel S, Kötting O, Steup M (2006) Phosphorylation of C6 and C3-positions of Glucosyl residues in starch is catalysed by distinct dikinases. FEBS Lett 580:4872–4876PubMedCrossRefGoogle Scholar
  31. Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425PubMedGoogle Scholar
  32. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar
  33. Santelia D, Zeeman SC (2011) Progress in Arabidopsis starch research and potential biotechnological applications. Curr Opin Biotechnol 22:271–280PubMedCrossRefGoogle Scholar
  34. Smith AM (2008) Prospects for increasing starch and sucrose yields for bioethanol production. Plant J 54:546–558PubMedCrossRefGoogle Scholar
  35. Smith AM, Zeeman SC, Smith SM (2005) Starch degradation. Annu Rev Plant Biol 56:73–97PubMedCrossRefGoogle Scholar
  36. Tabata S, Nagata K, Hizukuri S (1975) Studies on starch phosphates. Part 3. On the esterified phosphates in some cereal starches. Staerke 27:333–335CrossRefGoogle Scholar
  37. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739PubMedCrossRefGoogle Scholar
  38. Vriet C, Welham T, Brachmann A, Pike M, Pike J, Perry J, Parniske M, Sato S, Tabata S, Smith AM, Wang TL (2010) A suite of Lotus japonicus starch mutants reveals both conserved and novel features of starch metabolism. Plant Physiol 154:643–655PubMedCrossRefGoogle Scholar
  39. Yu TS, Kofler H, Häusler RE, Hille D, Flügge U, Zeeman SC, Smith AM, Kossmann J, Lloyd J, Ritte G, Steup M, Lue W, Chen J, Weber A (2001) The Arabidopsis sex1 mutant is defective in the R1 protein, a general regulator of starch degradation in plants, and not in the chloroplast hexose transporter. Plant Cell 13:1907–1918PubMedGoogle Scholar
  40. Zeeman SC, Kossmann J, Smith AM (2010) Starch: its metabolism, evolution, and biotechnological modification in plants. Annu Rev Plant Biol 61:209–234PubMedCrossRefGoogle Scholar

Copyright information

© Society for Plant Biochemistry and Biotechnology 2012

Authors and Affiliations

  1. 1.Department of Biotechnology and Environmental SciencesThapar UniversityPatialaIndia

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