Transcriptional profiling approaches to understanding how plants regulate growth and defence: A case study illustrated by analysis of the role of vitamin C

  • Christine H. Foyer
  • Guy Kiddle
  • Paul Verrier
Part of the Experientia Supplementum book series (EXS, volume 97)


In this chapter, basic technical aspects concerning the design of DNA microarray experiments are discussed including sample preparation, hybridisation conditions and statistical significance of the acquired data are detailed. Given that microarrays are perhaps the most used tool in plant systems biology there is much experience in the pitfalls in using them. Herein important considerations are presented for both the experimental biologists and data analyst in order to maximise the utility of these resources. Finally a case study using the analysis of vitamin C deficient plants is presented to illustrate the power of this approach in enhancing comprehension of important and complex biological functions.


Gibberellic Acid Late Embryogenesis Abundant Robust Multichip Average Abi4 Mutant Plant Cell Cycle 
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  1. 1.
    Foyer CH, Noctor G (2005) Redox homeostasis and antioxidant signalling: a metabolic interface between stress perception and physiological responses. Plant Cell 17: 1866–1875PubMedCrossRefGoogle Scholar
  2. 2.
    Foyer CH, Noctor G (2005) Oxidant and antioxidant signalling in plants: a re-evaluation of the concept of oxidative stress in a physiological context. Plant Cell Environ 28: 1056–1071CrossRefGoogle Scholar
  3. 3.
    Fry SC (1998) Oxidative scission of plant cell wall polysaccharides by ascorbate-induced hydroxyl radicals. Biochem J 332: 507–515PubMedGoogle Scholar
  4. 4.
    Potters G, Horemans N, Caubergs RJ, Asard H (2000) Ascorbate and dehydroascorbate influence cell cycle progression in tobacco cell suspension. Plant Physiol 124: 17–20PubMedCrossRefGoogle Scholar
  5. 5.
    Barth C, Moeder W, Klessig DF, Conklin PL (2004) The timing of senescence and response to pathogens is altered in the ascorbate-deficient mutant vitamin C-1. Plant Physiol 134: 178–192CrossRefGoogle Scholar
  6. 6.
    Pavet V, Olmos E, Kiddle G, Mowla, S, Kumar S, Antoniw J, Alvarez ME, Foyer CH (2005) Ascorbic acid deficiency activates cell death and disease resistance responses in Arabidopsis thaliana. Plant Physiol 139: 1291–1303PubMedCrossRefGoogle Scholar
  7. 7.
    Thomas H, Ougham HJ, Wagstaff C, Stead AD (2003) Defining senescence and death. J Expt Bot 54: 1127–1132CrossRefGoogle Scholar
  8. 8.
    Finkel T, Holbrook NJ (2003) Oxidants, oxidative stress and the biology of ageing. Nature 408: 239–247CrossRefGoogle Scholar
  9. 9.
    Partridge L, Gems D (2002) Mechanism of ageing: public or private. Nature Rev Genetics 3: 165–175CrossRefGoogle Scholar
  10. 10.
    Kurzweil R, Grossman T (2005) Fantastic voyage: Live long enough to live forever. Emmaus, Pennsylvania, Rodale Press, 1–452Google Scholar
  11. 11.
    Wheeler GL, Jones MA, Smirnoff N (1998) The biosynthetic pathway of vitamin C in higher plants. Nature 393: 365–369PubMedCrossRefGoogle Scholar
  12. 12.
    Smirnoff N, Running JA, Gatzek S (2004) Ascorbate biosynthesis: a diversity of pathways. In: H Asard, JM May, N Smirnoff (eds.) Vitamin C. Functions and Biochemistry in Animals and Plants. Bios Scientific Publishers, Oxon, UK, Chapter 1: 7–29Google Scholar
  13. 13.
    Agius F, González-Lamothe R, Caballero JL, Munoz-Blanco J, Botella MA, Valpuesta V (2003) Engineering increased vitamin C levels in plants by overexpression of a D-galacturonic acid reductase. Nature Biotechnol 21: 177–181CrossRefGoogle Scholar
  14. 14.
    Conklin PL, Norris SR, Wheeler GL, Williams EH, Smirnoff N, Last RL (1999) Genetic evidence for the role of GDP-mannose in plant ascorbic acid (vitamin C) biosynthesis. Proc Natl Acad Sci USA 30: 4198–4203CrossRefGoogle Scholar
  15. 15.
    Keller R, Springer F, Renz A, Kossmann J (1999) Antisense inhibition of the GDP mannose pyrophosphorylase reduces the ascorbate content in transgenic plants leading to developmental changes during senescence. Plant J 19: 131–141PubMedCrossRefGoogle Scholar
  16. 16.
    Gatzek S, Wheeler GL Smirnoff N (2002) Antisense suppression of L-galactose dehydrogenase in Arabidopsis thaliana provides evidence for its role in ascorbate synthesis and reveals light-modulated L-galactose synthesis. Plant J 30: 541–553PubMedCrossRefGoogle Scholar
  17. 17.
    Tabata K, Ôba K, Suzuki K, Esaka M (2001) Generation and properties of ascorbic acid-deficient transgenic tobacco cells expressing antisense RNA for L-galactono-1,4-lactone dehydrogenase. Plant J 27: 139–148PubMedCrossRefGoogle Scholar
  18. 18.
    Bartoli CG, Guiamet JJ, Kiddle G, Pastori G, Di Cagno R, Theodoulou FL, Foyer CH (2005) The relationship between L-galactono-1, 4-lactone dehydrogenase (GalLDH) and ascorbate content in leaves under optimal and stress conditions. Plant Cell and Environment 28: 1073–1081CrossRefGoogle Scholar
  19. 19.
    Bartoli CG, Yu J, Gómez F, Fernández L, Yu J, McIntosh L, Foyer CH (2006) Inter-relationships between light and respiration in the control of ascorbic acid synthesis and accumulation in Arabidopsis thaliana leaves. J Exp Bot 57: 1621–1631PubMedCrossRefGoogle Scholar
  20. 20.
    Bartoli CG, Pastori GM, Foyer CH (2000) Ascorbate biosynthesis in mitochondria is linked to the electron transport chain between complexes III and IV. Plant Physiol 123: 335–343PubMedCrossRefGoogle Scholar
  21. 21.
    Tabata K, Takaoka T, Esaka M (2002) Gene expression of ascorbic acid-related enzymes in tobacco. Phytochemistry 61: 631–635PubMedCrossRefGoogle Scholar
  22. 22.
    Tamaoki M, Mukai F, Asai N, Nakajima N, Kubo A, Aono M, Saji H (2003) Light-controlled expression of a gene encoding L-galactono-γ-lactone dehydrogenase which affects ascorbate pool size in Arabidopsis thaliana. Plant Sci 1164: 1111–1117CrossRefGoogle Scholar
  23. 23.
    Pignocchi C, Fletcher JM, Wilkinson JE, Barnes JD, Foyer CH (2003) The function of ascorbate oxidase in tobacco. Plant Physiol 132: 1631–1641PubMedCrossRefGoogle Scholar
  24. 24.
    Chen Z, Young TE, Ling J, Chang SCh, Gallie DR (2003) Increasing vitamin C content of plants through enhanced ascorbate recycling. Proc Natl Acad Sci USA 100: 3525–3530PubMedCrossRefGoogle Scholar
  25. 25.
    Conklin PL, Saracco SA, Norris SR, Last RL (2000) Identification of ascorbic acid deficient Arabidopsis thaliana mutants. Genetics 154: 847–856PubMedGoogle Scholar
  26. 26.
    Conklin PL, Williams EH, Last RL (1996) Environmental stress sensitivity of an ascorbic acid-deficient Arabidopsis mutant. Proc Natl Acad Sci USA 3: 9970–9974CrossRefGoogle Scholar
  27. 27.
    Veljovic-Jovanovic SD, Pignocchi, C, Noctor G, and Foyer CH (2001) Low ascorbic acid in the vtc-1 mutant of Arabidopsis is associated with decreased growth and intracellular redistribution of the antioxidant system. Plant Physiol 127: 426–435PubMedCrossRefGoogle Scholar
  28. 28.
    Mulle-Moule P, Conklin PL, Niyogi KK (2002) Ascorbate deficiency can limit violaxanthin de-epoxidase activity in vivo. Plant Physiol 128: 970–977CrossRefGoogle Scholar
  29. 29.
    Radzio A, Lorence A, Chevone BI, Nessler CL (2003) L-Gulono-1, 4-lactone oxidase expression rescues vitamin-C deficient Arabidopsis (vtc) mutants. Plant Mol Biol 53: 837–844PubMedCrossRefGoogle Scholar
  30. 30.
    Dijkwel PP, Huijser C, Weisbeek P, Chua N-M, Smeekens SCM (1997) Sucrose control of phytochrome A signaling in Arabidopsis. Plant Cell 9: 583–595PubMedCrossRefGoogle Scholar
  31. 31.
    Martin T, Hellmann H, Schmidt R, Willmitzer L, Frommer WB (1997) Identification of mutants in metabolically regulated gene expression. Plant J 11: 53–62PubMedCrossRefGoogle Scholar
  32. 32.
    Arenas-Huertero F, Arroyo, A, Zhou L, Sheen J, Leon P (2000) Analysis of Arabidopsis glucose insensitive mutants, gin5 and gin6, reveals a central role of the plant hormone ABA in the regulation of plant vegetative development by sugar. Genes Dev 14: 2085–2096PubMedGoogle Scholar
  33. 33.
    Sheen J, Zhou L, Jang JC (1999) Sugars as signaling molecules. Curr Opin Plant Biol 2: 410–418PubMedCrossRefGoogle Scholar
  34. 34.
    Smeekens S, Rook F (1997) Sugar sensing and sugar-mediated signal transduction in plants. Plant Physiol 115: 7–13PubMedGoogle Scholar
  35. 35.
    Zhou L, Jang JC, Jones TL, Sheen J (1998) Glucose and ethylene signal transduction crosstalk revealed by an Arabidopsis glucose-insensitive mutant. Proc Natl Acad Sci USA 95: 10294–10299PubMedCrossRefGoogle Scholar
  36. 36.
    Huijser C, Kortstee A, Pego J, Weisbeek P, Wisman E, Smeekens S (2000) The Arabidopsis SUCROSE UNCOUPLED-6 gene is identical to ABSCISIC ACID INSENSITIVE-4: involvement of abscisic acid in sugar responses. Plant J 23: 577–585PubMedCrossRefGoogle Scholar
  37. 37.
    Signora L, De Smet I, Foyer CH, Zhang H (2001) ABA plays a central role in mediating the regulatory effects of nitrate on root branching in Arabidopsis. Plant J 28: 655–662PubMedCrossRefGoogle Scholar
  38. 38.
    De Smet I, Signora L, Beeckman T, Inze D, Foyer CH, Zhang H (2003) An ABA-sensitive lateral root developmental checkpoint in Arabidopsis. Plant J 33: 543–555PubMedCrossRefGoogle Scholar
  39. 39.
    Kiddle G, Pastori GM, Bernard B, Pignocchi C, Antoniw J, Verrier PJ, Foyer CH (2003) Effects of leaf ascorbate content on defense and photosynthesis gene expression in Arabidopsis thaliana. Antioxidants and Redox Signalling 5: 23–32CrossRefGoogle Scholar
  40. 40.
    Yang YH, Dudoit S, Luu P, Speed T (2001) Normalization for cDNA microarry data. Berkley Technical report. Scholar
  41. 41.
    Allissul DA, Cui X, Page GP, Sabripour M (2005) Micoarray data analysis from disarry to consolidation and consensus. Nature Rev Genetics 7: 55–65Google Scholar
  42. 42.
    Bolstad BM, Irizarry RA, Astrand M, Speed TP (2003) A comparison of normalization methods for high density oligonucleotide array data based on bias and variance. Bioinformatics 19: 185–193PubMedCrossRefGoogle Scholar
  43. 43.
    Ernst J, Nau GJ, Bar-Jospeh Z (2005) Clustering short time series gene expression data. Bioinformatics 21(supp1): i159–i168PubMedCrossRefGoogle Scholar
  44. 44.
    Wit E, McClure J (2004) Statistics for microarrays, design, analysis and inference. John Wiley & Sons, Chichester, UKGoogle Scholar
  45. 45.
    Pastori GM, Kiddle G, Antoniw J, Bernard S, Veljovic-Jovanovic S, Verrier PJ, Noctor G, Foyer CH (2003) Leaf vitamin C contents modulate plant defense transcripts and regulate genes controlling development through hormone signaling. Plant Cell 15: 939–951PubMedCrossRefGoogle Scholar
  46. 46.
    Pignocchi C, Kiddle G, Hernández I, Foster SJ, Asensi A, Taybi T, Barnes J, Foyer CH (2006) Ascorbate-oxidase-dependent changes in the redox state of the apoplast modulate gene transcription leading to modified hormone signaling and defense in tobacco. Plant Physiol 141: 423–435PubMedCrossRefGoogle Scholar
  47. 47.
    Arrigoni O, de Tullio MC (2000) The role of ascorbic acid in cell metabolism: between gene-directed functions and unpredictable chemical reactions. J Plant Physiol 157: 481–488Google Scholar
  48. 48.
    Pignocchi C, Foyer CH (2003) Apoplastic ascorbate metabolism and its role in the regulation of cell signalling. Curr Opin Plant Biol 6: 379–389PubMedCrossRefGoogle Scholar
  49. 49.
    Potters G, Horemans N, Bellone S, Caubergs R J, Trost P, Guisez Y, Asard H (2004) Dehydroascrobate influences the plant cell cycle through a glutathione-independent reduction mechanism. Plant Physiol 134: 1479–1487PubMedCrossRefGoogle Scholar
  50. 50.
    Olmos E, Kiddle G, Pellny T, Kumar S, Foyer CH (2006) Modulation of plant morphology, root architecture and cell structure by low vitamin C in Arabidopsis thaliana. J Exp Bot 57: 1645–1655PubMedCrossRefGoogle Scholar
  51. 51.
    Fath A, Bethke PC, Jones RL (2001) Enzymes that scavenge reactive oxygen species are down-regulated prior to gibberellic acid-induced programmed cell death in barley aleurone. Plant Physiol 126: 156–166PubMedCrossRefGoogle Scholar
  52. 52.
    Fath A, Bethke P, Beligni V, Jones R (2002) Active oxygen and cell death in cereal aleurone cells. J Exp Botany 53: 1273–1282CrossRefGoogle Scholar
  53. 53.
    Dong JG, Fernandez-Maculet JC, Yang SF (1992) Purification and characterization of 1-aminocyclopropane-1-carboxylate oxidase from apple fruit. Proc Natl Acad Sci USA 89(20): 9789–9793PubMedCrossRefGoogle Scholar
  54. 54.
    Hedden P (1992) 2-Oxoglutarate-dependent dioxygenases in plants: mechanism and function. Biochem Soc Trans 20(2): 373–377PubMedGoogle Scholar
  55. 55.
    Hedden P, Kamiya Y (1997) Gibberellin biosynthesis: Enzymes, genes and their regulation. Ann Rev Plant Physiol Plant Mol Biol 48: 431–460CrossRefGoogle Scholar
  56. 56.
    Quaedvlieg N, Dockx J, Rook F, Weisbeek P, Smeekens S (1995) The homeobox gene ATH1 of Arabidopsis is de-repressed in the hotomorphogenicmutants cop1 and det1. Plant Cell 7(1): 117–129PubMedCrossRefGoogle Scholar
  57. 57.
    Bellaoui M, Pidkowich MS, Samach A, Kushalappa K, Kohalmi SE, Modrusan Z, Crosby WL, Haughn GW (2001) The Arabidopsis BELL1 and KNOX TALE homeodomain proteins interact through a domain conserved between plants and animals. Plant Cell 13(11): 2455–2470PubMedCrossRefGoogle Scholar
  58. 58.
    Phillips AL, Ward DA, Uknes S, Appleford NE, Lange T, Huttly AK, Gaskin P, Graebe JE, Hedden P (1995) Isolation and expression of three gibberellin 20-oxidase cDNA clones from Arabidopsis. Plant Physiol 108(3): 1049–1057PubMedCrossRefGoogle Scholar
  59. 59.
    Xu YL, Gage DA, Zeevaart JA (1997) Gibberellins and stem growth in Arabidopsis thaliana. Effects of photoperiod on expression of the GA4 and GA5 loci. Plant Physiol 114(4): 1471–1476PubMedCrossRefGoogle Scholar
  60. 60.
    Chiang HH, Hwang I, Goodman HM (1995) Isolation of the Arabidopsis GA4 locus. Plant Cell 7(2): 195–201PubMedCrossRefGoogle Scholar
  61. 61.
    Coles JP, Phillips AL, Croker SJ, Garcia-Lepe R, Lewis MJ, Hedden P (1999) Modification of gibberellin production and plant development in Arabidopsis by sense and antisense expression of gibberellin 20-oxidase genes. Plant J 17(5): 547–556PubMedCrossRefGoogle Scholar
  62. 62.
    Kyriakis JM, Avruch J (1996) Sounding the alarm: protein kinase cascades activated by stress and inflammation. J Biol Chem 271: 24313–24316PubMedCrossRefGoogle Scholar
  63. 63.
    Lu C, Man MH, Guevara-Garcia A, Fedoroff NV (2002) Mitogen-activated protein kinase signaling in postgermination arrest of development by abscisic acid. Proc Natl Acad Sci USA 99: 15812–15817PubMedCrossRefGoogle Scholar
  64. 64.
    Dewitte W, Riou-Khamlichi C, Scofield S, Healy JM, Jacqmard A, Kilby NJ, Murray JA (2003) Altered cell cycle distribution, hyperplasia, and inhibited differentiation in Arabidopsis caused by the D-type cyclin CYCD3. Plant Cell 15: 79–92PubMedCrossRefGoogle Scholar
  65. 65.
    de Jager SM, Maughan S, Dewitte W, Scofield S, Murray JA (2005) The developmental context of cell-cycle control in plants. Semin Cell Dev Biol 16: 385–396PubMedCrossRefGoogle Scholar
  66. 66.
    Reicheld J-P, Venoux T, Lardon F, Van Montagu M, Inze D (1999) Specific checkpoints regulate plant cell cycle progression in response to oxidative stress Plant J 17: 647–656CrossRefGoogle Scholar
  67. 67.
    Roudier F, Fedorova E, Györgyey J, Feher A, Brown S, Kondorosi A, Kondorosi E (2000) Cell cycle function of a Medicago sativa A2-type cyclin interacting with a PSTAIRE-type cyclin-dependent kinase and a retinoblastoma protein. Plant J 23(1): 73–83PubMedCrossRefGoogle Scholar
  68. 68.
    Dewitte W, Murray JAH (2003) The plant cell cycle. Annu Rev Plant Biol 54: 235–264.PubMedCrossRefGoogle Scholar
  69. 69.
    Vanacker H, Lu H, Rate DN, Greenberg JT (2001) A role for salicylic acid and NPR1 in regulating cell growth in Arabidopsis. Plant J 28: 209–216PubMedCrossRefGoogle Scholar
  70. 70.
    Peterman TK, Ohol YM, McReynolds LJ, Luna EJ (2004) Patellin1, a novel sec14-like protein, localizes to the cell plate and binds phosphoinositides. Plant Physiol 136: 3080–3094PubMedCrossRefGoogle Scholar
  71. 71.
    Lee YR, Liu B (2004) Cytoskeletal motors in Arabidopsis. Sixty-one kinesins and seventeen myosins. Plant Physiol 136: 3877–3883PubMedCrossRefGoogle Scholar

Copyright information

© Birkhäuser Verlag/Switzerland 2007

Authors and Affiliations

  • Christine H. Foyer
    • 1
  • Guy Kiddle
    • 1
  • Paul Verrier
    • 2
  1. 1.Crop Performance and Improvement DivisionRothamsted ResearchHarpenden, HertfordshireUK
  2. 2.Biomathematics and Bioinformatics DivisionRothamsted ResearchHarpenden, HertfordshireUK

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