Plant Molecular Biology Reporter

, Volume 30, Issue 4, pp 904–914 | Cite as

Genomic Structure, Sub-Cellular Localization, and Promoter Analysis of the Gene Encoding Sorbitol-6-Phosphate Dehydrogenase from Apple

Original Paper


Sorbitol is the primary photosynthetic product and the translocatable and storage carbohydrate in apple (Malus domestica) and other fruit tree species within the Rosaceae family. Sorbitol-6-phosphate dehydrogenase (S6PDH, EC is the key enzyme in the biosynthesis of sorbitol. In this study, we isolated two full-length genomic sequences for S6PDH from “Gala” apple. The two sequences have same six exons and first two introns, but the sizes of their last three introns are different. The two sequences were mapped to the same loci on chromosome 10. Immunogold electron microscopy analysis demonstrates that the S6PDH is localized mainly in leaf cytosol and chloroplasts. We also isolated and analyzed the promoter region of S6PDH and constructed a series of promoter deletion derivatives with β-glucuronidase (GUS) gene to identify the upstream region of the S6PDH gene required for promoter activity. The GUS activity in Agrobacterium-mediated transient transformation of tobacco leaves reveals that the -1719 region is more important for gene expression contrasting with other regions in the S6PDH promoter. The promoter region can be induced by cold, dark, and abscisic acid treatment.


Apple Sorbitol-6-phosphate dehydrogenase (S6PDH) Genomic structure Sub-cellular localization Promoter analysis 



This work was supported by the earmarked fund for China Agriculture Research System. The authors are grateful to Priscilla Licht for her help in revising our English composition.


  1. An C, Ichinose Y, Yamada T, Tanaka Y, Shiraishi T, Oku H (1993) Organization of the genes encoding chalcone synthase in Pisum sativum. Plant Mol Biol 21(5):789–803PubMedCrossRefGoogle Scholar
  2. Bieleski RL (1982) Sugar alcohols. In: Loewus F, Tanner W (eds) Encyclopedia of plant physiology new series, Vol. 13A. Springer, Berlin, pp 158–192Google Scholar
  3. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254PubMedCrossRefGoogle Scholar
  4. Breathnach R, Chambon P (1981) Organization and expression of eukaryotic split genes coding for proteins. Annu Rev Biochem 50:349–383PubMedCrossRefGoogle Scholar
  5. Brown PH, Hu H (1996) Phloem mobility of boron is species dependent: evidence for phloem mobility in sorbitol-rich species. Ann Bot 77:497–505CrossRefGoogle Scholar
  6. Brown PH, Bellaloui N, Hu H, Dandekar AM (1999) Transgenically enhanced sorbitol synthesis facilitates phloem boron transport and increases tolerance of tobacco to boron deficiency. Plant Physiol 19:17–20CrossRefGoogle Scholar
  7. Chang S, Puryear J, Cairney J (1993) Simple and efficient method for isolating RNA from pine trees. Plant Mol Biol Report 11:113–116CrossRefGoogle Scholar
  8. Cheng LL, Zhou R, Reidel EJ, Sharkey TD, Dandekar AM (2005) Antisense inhibition of sorbitol synthesis leads to up-regulation of starch synthesis without altering CO2 assimilation in apple leaves. Planta 220:767–776PubMedCrossRefGoogle Scholar
  9. Cui SM, Sadayoshi K, Ogawa Y, Nii N (2004) Effects of water stress on sorbitol content in leaves and roots, anatomical changes in cell nuclei, and starch accumulation in leaves of young peach trees. J Japan Soc Hortic Sci 73(1):25–30CrossRefGoogle Scholar
  10. Dellaporta SL, Wood J, Hicks JB (1983) A plant DNA mini-preparation, version II. Plant Mol Biol Report 1:19–21CrossRefGoogle Scholar
  11. Diaz de Leon F, Klotz KL, Lagrimini M (1993) Nucleotide sequence of the tobacco (Nicotiana tabacum) anionic peroxidase gene. Plant Physiol 101(3):1117–1118PubMedCrossRefGoogle Scholar
  12. Escobar-Gutiérrez AJ, Zipperlin B, Carbonne F, Moing A, Gaudillère JP (1998) Photosynthesis, carbon partitioning and metabolite content during drought stress in peach seedlings. Aust J Plant Physiol 25:197–205CrossRefGoogle Scholar
  13. Fan RC, Peng CC, Xu YH, Wang XF, Li Y, Shang Y, Du SY, Zhao R, Zhang XY, Zhang LY, Zhang DP (2009) Apple sucrose transporter SUT1 and sorbitol transporter SOT6 interact with cytochrome b5 to regulate their affinity for substrate sugars. Plant Physiol 150:1880–1901PubMedCrossRefGoogle Scholar
  14. Feldbrugge M, Sprenger M, Dinkelbach M, Yazaki K, Harter K, Weisshaar B (1994) Functional analysis of a light-responsive plant bZIP transcriptional regulator. Plant Cell 6(11):1607–1621PubMedGoogle Scholar
  15. Feldbrugge M, Sprenger M, Hahlbrock K, Weisshaar B (1997) PcMYB1, a novel plant protein containing a DNA-binding domain with one MYB repeat, interacts in vivo with a light-regulatory promoter unit. Plant J 11(5):1079–1093PubMedCrossRefGoogle Scholar
  16. Gao M, Tao R, Miura K, Dandekar AM, Sugiura A (2001) Transformation of Japanese persimmon (Diospyros kaki Thunb.) with apple cDNA encoding NADP-dependent sorbitol-6-phosphate dehydrogenase. Plant Sci 160(5):837–845PubMedCrossRefGoogle Scholar
  17. Gao Y, Zhao Y, Li TT, Ren CX, Liu Y, Wang ML (2010) Cloning and characterization of a G protein β subunit gene responsive to plant hormones and abiotic stresses in Brassica napus. Plant Mol Biol Report 28:450–459CrossRefGoogle Scholar
  18. Hennig J, Dewey RE, Cutt JR, Klessig DF (1993) Pathogen, salicylic acid and developmental dependent expression of a RT beta-1,3-glucanase/GUS gene fusion in transgenic tobacco plants. Plant J 4(3):481–493PubMedCrossRefGoogle Scholar
  19. Higo K, Ugawa Y, Iwamoto M, Korenaga T (1999) Plant cis-acting regulatory DNA elements (PLACE) database. Nucl Acid Res 27:297–300CrossRefGoogle Scholar
  20. Hirai M (1981) Purification and characteristics of sorbitol-6-phosphate dehydrogenase from loquat leaves. Plant Physiol 67:221–224PubMedCrossRefGoogle Scholar
  21. Jefferson R (1987) Assaying chimeric genes in plants: the GUS gene fusion system. Plant Mol Biol Report 5:387–405CrossRefGoogle Scholar
  22. Kanamarua N, Itob Y, Komorib S, Saitob M, Katob H, Takahashib S, Omurac M, Soejimab J, Shiratakea K, Yamadaa K, Yamaki S (2004) Transgenic apple transformed by sorbitol-6-phosphate dehydrogenase cDNA: switch between sorbitol and sucrose supply due to its gene expression. Plant Sci 167(1):55–61CrossRefGoogle Scholar
  23. Kanayama Y, Mori H, Imaseki H, Yamaki S (1992) Nucleotide sequence of a cDNA encoding sorbitol-6-phosphate dehydrogenase from apple. Plant Physiol 100:1607–1608PubMedCrossRefGoogle Scholar
  24. Kanayama Y, Watanabe M, Moriguchi R, Deguchi M, Kanahama K, Yamaki S (2006) Effects of low temperature and abscisic acid on the expression of the sorbitol-6-phosphate dehydrogenase gene in apple leaves. J Jpn Soc Hortic Sci 75(1):20–25CrossRefGoogle Scholar
  25. Kim SY, Wu R (1990) Multiple protein factors bind to a rice glutelin promoter region. Nucl Acid Res 18(23):6845–6852CrossRefGoogle Scholar
  26. Kim JK, Wu R (1992) Nucleotide sequence of a high-pI rice (Oryza sativa)-amylase gene. Plant Mol Biol 18(2):399–402PubMedCrossRefGoogle Scholar
  27. Kobashi K, Gemma H, Iwahori S (2000) Abscisic acid content and sugar metabolism of peaches grown under water stress. J Am Soc Hortic Sci 125:425–428Google Scholar
  28. Lescot M, Dehais P, Thijs G, Marchal K, Moreau Y, van de Peer Y, Rouze P, Rombauts S (2002) PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucl Acid Res 30:325–327CrossRefGoogle Scholar
  29. Li F, Lei HJ, Zhao XJ, Tian RR, Li TH (2011) Characterization of three sorbitol transporter genes in micropropagated apple plants grown under drought stress. Plant Mol Biol Report. doi: 10.1007/s11105-011-0323-4
  30. Liaud MF, Zhang DX, Cerff R (1990) Differential intron loss and endosymbiotic transfer of chloroplast glyceraldehyde-3-phosphate dehydrogenase genes to the nucleus. Proc Natl Acad Sci 87(22):8918–8922PubMedCrossRefGoogle Scholar
  31. Lo Biancoa R, Riegera M, Sung SS (2000) Effect of drought on sorbitol and sucrose metabolism in sinks and sources of peach. Physiol Plant 108:71–78CrossRefGoogle Scholar
  32. Loescher WH (1987) Physiology and metabolism of sugar alcohols in higher plants. Physiol Plant 70(3):553–557CrossRefGoogle Scholar
  33. Loescher WH, Marlow GC, Kennedy RA (1982) Sorbitol metabolism and sink–source interconversions in developing apple leaves. Plant Physiol 70:335–339PubMedCrossRefGoogle Scholar
  34. Nadwodnik J, Lohaus G (2008) Subcellular concentrations of sugar alcohols and sugars in relation to phloem translocation in Plantago major, Plantago maritima, Prunus persica, and Apium graveolens. Planta 227:1079–1089PubMedCrossRefGoogle Scholar
  35. Oura Y, Yamada K, Shiratake K, Yamaki S (2000) Purification and characterization of a NAD+-dependent sorbitol dehydrogenase from Japanese pear fruit. Phytochemistry 54(6):567–572PubMedCrossRefGoogle Scholar
  36. Pastuglia M, Roby D, Dumas C, Cock JM (1997) Rapid induction by wounding and bacterial infection of an S gene family receptor-like kinase in Brassica oleracea. Plant Cell 9:1–13Google Scholar
  37. Pichersky E, Bernatzky R, Tanksley SD, Breidenbach RB, Kausch AP, Cashmore AR (1985) Molecular characterization and genetic mapping of two clusters of genes encoding chlorophyll a/b-binding proteins in Lycopersicon esculentum (tomato). Gene 40(2–3):247–258PubMedCrossRefGoogle Scholar
  38. Rouster J, Leah R, Mundy J, Cameron-Mills V (1997) Identification of a methyl jasmonate-responsive region in the promoter of a lipoxygenase 1 gene expressed in barley grain. Plant J 11(3):513–523PubMedCrossRefGoogle Scholar
  39. Sakanishi K, Kanayama Y, Mori H, Yamada K, Yamaki S (1998) Expression of the gene for NADH-dependent sorbitol-6-phosphate dehydrogenase in peach leaves of various developmental stages. Plant Cell Physiol 39:1372–1374CrossRefGoogle Scholar
  40. Sheveleva EV, Marquez S, Chmara W, Zegeer A, Jensen RG, Bohnert HJ (1998) Sorbitol-6-phosphate dehydrogenase expression in transgenic tobacco. High amounts of sorbitol lead to necrotic lesions. Plant Physiol 117:831–839PubMedCrossRefGoogle Scholar
  41. Soria-Guerra RE, Rosales-Mendoza S, Gasic K, Wisniewski ME, Band M, Korban SS (2011) Gene expression is highly regulated in early developing fruit of apple. Plant Mol Biol Report. doi: 10.1007/s11105-011-0300-y
  42. Sparkes IA, Runions J, Kearns A, Hawes C (2006) Rapid, transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants. Nat Protoc 1(4):2019–2025PubMedCrossRefGoogle Scholar
  43. Stolf-Moreira R, Lemos EGM, Carareto-Alves L (2011) Transcriptional profiles of roots of different soybean genotypes subjected to drought stress. Plant Mol Biol Report 29:19–34CrossRefGoogle Scholar
  44. Suleman P, Steiner PW (1994) Relationship between sorbitol and solute potential in apple shoots relative to fire blight symptom development after infection by Erwinia amylovora. Phytopathology 84(10):1244–1250CrossRefGoogle Scholar
  45. Takaiwa F, Oono K, Wing D, Kato A (1991) Sequence of three members and expression of a new major subfamily of glutelin genes from rice. Plant Mol Biol 17(4):875–885PubMedCrossRefGoogle Scholar
  46. Tao R, Uratsu SL, Dandekar AM (1995) Sorbitol synthesis in transgenic tobacco with apple cDNA encoding NADP-dependent sorbitol-6-phosphate dehydrogenase. Plant Cell Physiol 36(3):525–532PubMedGoogle Scholar
  47. Teo G, Suzuki Y, Uratsu SL, Lampinen B, Ormonde N, Hu WK, DeJong TM, Dandekar AM (2006) Silencing leaf sorbitol synthesis alters long-distance partitioning and apple fruit quality. Proc Natl Acad Sci 103(49):18842–18847PubMedCrossRefGoogle Scholar
  48. Velasco R, Zharkikh A, Affourtit J, Dhingra A, Cestaro A, Kalyanaraman A, Fontana P, Bhatnagar SK, Troggio M, Pruss D, Salvi S, Pindo M, Baldi P, Castelletti S, Cavaiuolo M, Coppola G, Costa F, Cova V, Dal Ri A, Goremykin V, Komjanc M, Longhi S, Magnago P, Malacarne G, Malnoy M, Micheletti D, Moretto M, Perazzolli M, Si-Ammour A, Vezzulli S, Zini E, Eldredge G, Fitzgerald LM, Gutin N, Lanchbury J, Macalma T, Mitchell JT, Reid J, Wardell B, Kodira C, Chen Z, Desany B, Niazi F, Palmer M, Koepke T, Jiwan D, Schaeffer S, Krishnan V, Wu C, Chu VT, King ST, Vick J, Tao Q, Mraz A, Stormo A, Stormo K, Bogden R, Ederle D, Stella A, Vecchietti A, Kater MM, Masiero S, Lasserre P, Lespinasse Y, Allan AC, Bus V, Chagne D, Crowhurst RN, Gleave AP, Lavezzo E, Fawcett JA, Proost S, Rouze P, Sterck L, Toppo S, Lazzari B, Hellens RP, Durel CE, Gutin A, Bumgarner RE, Gardiner SE, Skolnick M, Egholm M, Van de Peer Y, Salamini F, Viola R (2010) The genome of the domesticated apple (Malus x domestica Borkh.). Nat Genet 42:833–839PubMedCrossRefGoogle Scholar
  49. Wang XL, Xu YH, Peng CC, Fan RC, Gao XQ (2009) Ubiquitous distribution and different subcellular localization of sorbitol dehydrogenase in fruit and leaf of apple. J Exp Bot 60(3):1025–1034PubMedCrossRefGoogle Scholar
  50. White AJ, Dunn MA, Brown K, Hughes MA (1994) Comparative analysis of genomic sequence and expression of a lipid transfer protein gene family in winter barley. J Exp Bot 45:1885–1892CrossRefGoogle Scholar
  51. Xu WR, Yu YH, Ding JH, Hua ZY, Wang YJ (2010) Characterization of a novel stilbene synthase promoter involved in pathogen- and stress-inducible expression from Chinese wild Vitis pseudoreticulata. Planta 231:475–487PubMedCrossRefGoogle Scholar
  52. Yamaguchi SK, Shinozaki K (1993) Arabidopsis DNA encoding two desiccation-responsive rd29 genes. Plant Physiol 101(3):1119–1120CrossRefGoogle Scholar
  53. Yamaki S (1980) Property of sorbitol-6-phosphate dehydrogenase and its connection with sorbitol accumulation in apple. Hortic Sci 15:268–270Google Scholar
  54. Yamaki S (1981) Subcellular localization of NADP-dependent sorbitol-6-phosphate dehydrogenase in protoplast from apple cotyledons. Plant Cell Physiol 22:359–367Google Scholar
  55. Yamaki S (1982) Distribution of sorbitol, neutral sugars, free amino acids, malic acid and some hydrolytic enzymes in vacuoles of apple cotyledons. Plant Cell Physiol 23:881–889Google Scholar
  56. Zhang LY, Peng YB, Pelleschi ST, Fan Y, Lu YF, Lu YM, Gao XP, Shen YY, Delrot S, Zhang DP (2004) Evidence for apoplasmic phloem unloading in developing apple fruit. Plant Physiol 135:574–586PubMedCrossRefGoogle Scholar
  57. Zhang J, Yao Y, Streeter JG, Ferree DC (2010) Influence of soil drought stress on photosynthesis, carbohydrates and the nitrogen and phosphorus absorb in different section of leaves and stem of Fugi/M.9EML, a young apple seedling. Afric. J Biotechnol 9(33):5320–5325Google Scholar
  58. Zhang X, Zhen JB, Li ZH, Kang DM, Yang YM, Kong J, Hua JP (2011) Expression profile of early responsive genes under salt stress in upland cotton (Gossypium hirsutum L.). Plant Mol Biol Report. doi: 10.1007/s11105-010-0269-y
  59. Zhou R, Cheng LL, Wayne R (2003) Purification and characterization of sorbitol-6- phosphate phosphatase from apple leaves. Plant Sci 165:227–232CrossRefGoogle Scholar
  60. Zhou R, Cheng LL, Dandekar AM (2006) Down-regulation of sorbitol dehydrogenase and up-regulation of sucrose synthase in shoot tips of the transgenic apple trees with decreased sorbitol synthesis. J Exp Bot 57(14):3647–3657PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  1. 1.State Key Laboratory of Crop Stress Biology in Arid Areas, College of HorticultureNorthwest A&F UniversityYanglingChina

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