Plant Molecular Biology

, Volume 51, Issue 2, pp 249–262 | Cite as

Identification of water-deficit responsive genes in maritime pine (Pinus pinaster Ait.) roots



Root adaptation to soil environmental factors is very important to maritime pine, the main conifer species used for reforestation in France. The range of climates in the sites where this species is established varies from flooded in winter to drought-prone in summer. No studies have yet focused on the morphological, physiological or molecular variability of the root system to adapt its growth to such an environment. We developed a strategy to isolate drought-responsive genes in the root tissue in order to identify the molecular mechanisms that trees have evolved to cope with drought (the main problem affecting wood productivity), and to exploit this information to improve drought stress tolerance. In order to provide easy access to the root system, seedlings were raised in hydroponic solution. Polyethylene glycol was used as an osmoticum to induce water deficit. Using the cDNA-AFLP technique, we screened more than 2500 transcript derived fragments, of which 33 (1.2%) showed clear variation in presence/absence between non stressed and stressed medium. The relative abundance of these transcripts was then analysed by reverse northern. Only two out of these 33 genes showed significant opposite behaviour between both techniques. The identification and characterization of water-deficit responsive genes in roots provide the emergence of physiological understanding of the patterns of gene expression and regulation involved in the drought stress response of maritime pine.

cDNA-AFLP drought stress gene expression Pinus pinaster reverse northern root 


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  1. Arabidopsis Genome Initiative. 2000. Analysis of the genome se-quence of the flowering plant Arabidopsis thaliana. Nature 408: 796-815.Google Scholar
  2. Aziz, A. and Larher, F. 1998. Osmotic stress induced changes in lipid composition and peroxidation in leaf discs of Brassica napus L. J. Plant. Physiol. 153: 754-762.Google Scholar
  3. Bachem, C.W.B., van der Hoeven, R.S., de Bruijn, S.M., Vreug-denhil, D., Zabeau, M. and Visser, R.G.F. 1996. Visualisation of differential gene expression using a novel method of RNA fin-gerprinting based on AFLP: analysis of gene expression during potato tuber development. Plant J. 9: 745-753.Google Scholar
  4. Bassam, B.J., Caetano-Anolles, G. and Gresshoff, P.M. 1991. Fast and sensitive silver staining of DNA in polyacrylamide gels. Anal. Biochem. 196: 80-83.Google Scholar
  5. Brendel, O., Pot, D., Plomion, C., Rozenberg, P. and Guehl, J.M. 2002. Genetic parameters and QTL analysis of ä 13 C and ring width in maritime pine. Plant Cell Env. (in press).Google Scholar
  6. Büssis, D., Kauder, F. and Heineke, D. 1998. Acclimation of potato plants to polyethylene glycol-induced water deficit. I. Photosynthesis and metabolism. J. Exp. Bot. 49: 1349-1360.Google Scholar
  7. Chalhoub, B.A., Thibault, S., Laucou, V., Rameau, C., Höfte, H. and Cousin, R. 1997. Silver staining and recovery of AFLP amplification products on large denaturing polyacrylamide gel. BioTechniques 22: 216-220.Google Scholar
  8. Champoux, M.C., Wang, G., Sarkarung, S., Mackill, D.J., O'Toole, C.O., Huang, N. and McCouch, S.R. 1995. Locating genes as-sociated with root morphology and drought avoidance in rice via linkage to molecular markers. Theor. Appl. Genet. 90: 969-981.Google Scholar
  9. Chang, S., Puryear, J. and Cairney, J. 1993. A simple and efficient method for isolating RNA from pine trees. Plant Mol. Biol. Rep. 11: 113-116.Google Scholar
  10. Chang, S., Puryear, J.D., Dias, M., Funkhouser, E.A., Newton, R.J. and Cairney, J. 1995. Gene expression under water deficit in loblolly pine (Pinus taeda L.): isolation and characterization of cDNA clones. Physiol. Plant. 97: 139-148.Google Scholar
  11. Costa, P. 1999. Réponse moléculaire, physiologique et génétique du pin maritime à une contrainte hydrique. PhD thesis, Université Henry Poincaré, Nancy, France, 116 pp.Google Scholar
  12. Cushman, J.C. and Bohnert, H.J. 2000. Genomic approaches to plant stress tolerance. Curr. Opin. Plant Biol. 3: 117-124.Google Scholar
  13. Dellagi, A., Birch, P.R.J., Heilbronn, J., Lyon, G.D. and Toth, I.K. 2000. cDNA-AFLP analysis of differential gene expression in the prokaryotic plant pathogen Erwinia carotovora. Microbiology 146: 165-171.Google Scholar
  14. Desprez, T., Amselem, J., Caboche, M. and Höfte, H. 1998. Dif-ferential gene expression in Arabidopsis monitored using cDNA arrays. Plant J. 14: 643-652.Google Scholar
  15. Diatchenko, L.D., Lau, Y., Campbell, A.P., Chenchik, A., Mo-qadam, F., Huang, B., Lukyanov, S., Gurskaya, N., Sverdlov, E.D. and Siebert, P.D. 1996. Suppression subtractive hybridiza-tion: a method for generating differentially regulated or tissue-specific cDNA probes and libraries. Proc. Natl. Acad. Sci. USA 93: 6025-6030.Google Scholar
  16. Durrant, W.E., Rowland, O., Piedras, P., Hammond-Kosack, K.E. and Jones, J.D.G. 2000. cDNA-AFLP reveals a striking overlap in race-specific resistance and wound response gene expression profiles. Plant Cell 12: 963-977.Google Scholar
  17. Guyon, J.P. and Kremer, A. 1982. Stabilité phenotypique de la crois-sance en hauteur et cinétique journalière de pression de sève et de la transpiration chez le pin maritime (Pinus pinaster Ait.). Can. J. For. Res. 12: 936-946.Google Scholar
  18. Habu, Y., Fukada-Tanaka, S., Hisatomi, Y. and Iida, S. 1997. Am-plified restriction fragment length polymorphism-based mRNA fingerprinting using a single restriction enzyme that recognizes a 4-bp sequence. Biochem. Biophys. Res. Com. 234: 516-521.Google Scholar
  19. Hubank, M. and Schatz, D.G. 1994. Identifying differences in mRNA expression by representational difference analysis of cDNA. Nucl. Acids. Res. 22: 5640-5648.Google Scholar
  20. Ishitani, M., Nakamura, T., Han, S.Y. and Takabe, T. 1995. Ex-pression of the betaine aldehyde dehydrogenase gene in barley in response to osmotic stress and abscisic acid. Plant Mol. Biol. 27: 307-315.Google Scholar
  21. Jones, J.T. and Harrower, B.E. 1998. A comparison of the efficiency of differential display and cDNA-AFLPs as tools for the isolation of differentially expressed parasite genes. Fund. Appl. Nematol. 21: 81-88.Google Scholar
  22. Jones, C.S., Davies, H.V. and Taylor, M.A. 2000. Profiling of changes in gene expression during raspberry (Rubus ideaus) fruit ripening by application of RNA fingerprinting techniques. Planta 211: 708-714.Google Scholar
  23. Kawalleck, P., Plesch, G., Hahlbrock, K. and Somssich, I.E. 1992. Induction by fungal elicitor of S-adenosyl-L-methionine synthase and S-adenosyl-L-homocysteine hydrolase mRNAsin cultured cells and leaves of Petroselinum crispum. Proc. Natl. Acad. Sci. USA 89: 4713-4717.Google Scholar
  24. Kinlaw, C.S. and Neale, D.B. 1997. Complex gene families in pine genomes. Trends Plant Sci. 2: 356-359.Google Scholar
  25. Leone, A., Costa, A., Tucci, M. and Grillo, S. 1994. Compara-tive analysis of short-and long-term changes in gene expression caused by low water potential in potato (Solanum tuberosum) cell-suspension cultures. Plant. Physiol. 106: 703-712.Google Scholar
  26. Liang, P. and Pardee, A.B. 1992. Differential display of eukary-otic messenger RNA by means of the polymerase chain reaction. Science 257: 967-971.Google Scholar
  27. Liotenberg, S., North, H. and Marion-Poll, A. 1999. Molecular biol-ogy and regulation of abscisic acid biosynthesis in plants. Plant Physiol. Biochem. 37: 341-350.Google Scholar
  28. McCully, M.E. 1999. Roots in soil: unearthing the complexities of roots and their rhizospheres. Annu. Rev. Plant. Mol. Biol. 50: 695-718.Google Scholar
  29. Mittler, R. and Zilinskas, B.A. 1994. Regulation of pea cytoso-lic ascorbate peroxidase and other antioxidant enzymes during the progression of drought stress and following recovery from drought. Plant. J. 5: 397-405.Google Scholar
  30. Montero, E., Cabot, C., Poschenrieder, C.H. and Barcelo, J. 1998. Relative importance of osmotic-stress and ion-specific ef-fects on ABA-mediated inhibitation of leaf expansion growth in Phaseolus vulgaris. Plant Cell Env. 21: 54-62.Google Scholar
  31. N'Guyen, A. and Lamant, A. 1988. Pinitol and myo-inositol ac-cumulation in water-stressed seedlings of maritime pine. Phyto-chemistry 27: 3423-3427.Google Scholar
  32. N'Guyen, A. and Lamant, A. 1989. Variation in growth and os-motic regulation of roots of water-stressed maritime pine (Pinus pinaster Ait.) provenances. Tree Physiol. 5: 123-133.Google Scholar
  33. N'Guyen-Queyrens, A., Ferhi, A., Loustau, D. and Guehl, J.M. 1998. Within ä 13 C spatial variability and interannual varia-tions in wood cellulose of two contrasting provenances of Pinus pinaster. Can. J. For. Res. 28: 766-773.Google Scholar
  34. N'Guyen-Queyrens, A., Costa, P., Loustau, D. and Plomion, C. 2002. Osmotic adjustment in Pinus pinaster cuttings in response to a soil drying cycle. Ann. For. Sci. (in press).Google Scholar
  35. Parry, M.L. 2000. Assessment of potential effects and adaptations for climate change in Europe: summary and conclusions. Jackson Environment Institute, University of East Anglia, Norwich, UK, 24 pp.Google Scholar
  36. Peng, Z., Lu, Q. and Verma, D..P..S. 1996. Reciprocal regulation of 1-pyrroline-5-carboxylate synthetase and proline dehydroge-nase genes controls proline levels during and after osmotic stress in plants. Mol. Gen. Genet. 253: 334-341.Google Scholar
  37. Piétu, G., Mariage-Samson, R., Fayein, N., Matigou, C., Eveno, E., Houlgatte, R., Decraene, C., Vandenbrouck, Y., Tahi, F., Devignes, M., Wirkner, U., Ansorge, W., Cox, D., Nagase, T., Nomura, N. and Auffray, C. 1999. The Genexpress IMAGE knowledge base of the human brain transcriptome: a prototype integrated resource for functional and computational genomics. Genome Res. 9: 195-209.Google Scholar
  38. Plomion, C., Hurme, P., Frigerio, J.-M., Ridolphi, M., Pot, D., Pionneau, C., Avila, C., Gallardo, F., David, H., Neutlings, G., Campbell, M., Canovas, F.M., Savolainen, O., Bodénès, C. and Kremer, A. 1999. Developing SSCP markers in two Pinus species. Mol. Breed. 5: 21-31.Google Scholar
  39. Price, A.H., Steele, K.A., Moore, B.J., Barraclough, P.B. and Clark, L.J. 2000. A combined RFLP and AFLP linkage map of upland rice (Oryza sativa L.) used to identify QTLs for root-penetration ability. Theor. Appl. Genet. 100: 49-56.Google Scholar
  40. Riccardi, F., Gazeau, P., de Vienne, D. and Zivy, M. 1998. Protein changes in response to progressive water deficit in maize. Plant Physiol. 117: 1253-1263.Google Scholar
  41. Rychlik, W. 1995. Priming efficiency in PCR. BioTechniques 18: 84-89.Google Scholar
  42. Sambrook, J., Fritsch, E.F. and Maniatis, T. 1989. Small-scale preparation of plasmid DNA. In: Molecular Ccloning: A Lab-oratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Plainview, NY, pp. 1: 25-28.Google Scholar
  43. Schena, M., Shalon, D., Davis, R.W. and Brown, P.O. 1995. Quantitative monitoring of gene expression patterns with a com-plementary DNA microarray. Science 270: 467-470.Google Scholar
  44. Seillac, P. 1960. Contribution à l'étude de la nutrition du pin mar-itime: variation saisonnière de la teneur des pseudophylles en azote, potassium et acide phosphorique. PhD thesis, Université de Bordeaux, France.Google Scholar
  45. Seki, M., Narusaka, M., Abe, H., Kasuga, M., Yamaguchi-Shinozaki, K., Carninci, P., Hayashizaki, Y. and Shinozaki, K. 2001. Monitoring the expression pattern of 1300 Arabidopsis genes under drought and cold stresses by using a full-length cDNA microarray. Plant Cell 13: 61-72.Google Scholar
  46. Shoko, K., Tadashi, O., Hiroko, K., Osamu, C. and Kousaku, O. 1999. Expression profiling by iAFLP: a PCR-based method for genome-wide gene expression profiling. Genome Res. 9: 1305-1312.Google Scholar
  47. Tudela, D. and Primo-Millo, E. 1992. 1-aminocyclopropane-1-carboxylic acid transported from roots to shoots promotes leaf abscission in Cleopatra mandarin (Citrus reshni Hort. ex Tan.) seedlings rehydrated after water stress. Plant Physiol. 100: 131-137.Google Scholar
  48. Velculescu, V.E., Zhang, L., Vogelstein, B. and Kinzler, K.W. 1995. Serial analysis of gene expression. Science 270: 484-487.Google Scholar
  49. Voiblet, C., Duplessis, S., Encelot, N. and Martin, F. 2000. Iden-tification of symbiosis-regulated genes in Eucalyptus globulus -Pisolithus tinctorius ectomycorrhiza by differential hybridiza-tion of arrayed cDNAs. Plant J. 25: 181-191.Google Scholar
  50. Wang, S.X., Hunter, W. and Plant, A. 2000. Isolation and purifica-tion of functional total RNA from woody branches and needles of sitka and white spruce. BioTechniques 28: 292-296.Google Scholar
  51. Xu, N., Johns, B., Pullman, G.S. and Cairney, J. 1997. Rapid and reliable differential display from minute amounts of tissue: mass cloning and characterization of differentially expressed genes from loblolly pine embryos. Plant Mol. Biol. Rep. 15: 377-391.Google Scholar
  52. Zegzouti, H., Marty, C., Jones, B., Bouquin, T., Latché, A., Pech, J. and Bouzayen, M. 1997. Improved screening of cDNA gener-ated by mRNA differential display enables the selection of true positives and the isolation of weakly expressed messages. Plant Mol. Biol. Rep. 15: 238-245.Google Scholar
  53. Zhou, L.J. and Ye, C.L. 1999. Effect of hight temperature stress on metabolism of nitrogen and carbohydrates in seedlings of cucumber. J. Fujian Agric. Univ. 28: 289-293.Google Scholar

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© Kluwer Academic Publishers 2003

Authors and Affiliations

  1. 1.Equipe de Génétique et Amélioration des Arbres Forestiers, INRACestasFrance

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