Impact of carbon and phosphate starvation on growth and programmed cell death of maritime pine suspension cells

  • Herlânder Azevedo
  • Pedro Humberto Castro
  • Joana Ferreira Gonçalves
  • Teresa Lino-Neto
  • Rui Manuel Tavares


Programmed cell death is a fundamental aspect of plant development and defense. In suspension cultures of maritime pine (Pinus pinaster Ait.), cell death was associated with the simultaneous depletion of sugar and phosphate. This present work suggests that sugar rather than phosphate deprivation induced programmed cell death events, including degradation of nuclear DNA and remobilization of phosphate. However, phosphate starvation may have a synergistic effect on programmed cell death mediated by the lack of carbon source. Sugar and phosphate analogs were used to evaluate the nature of signaling events, and results suggested that programmed cell death induction by sugar starvation occurs downstream of hexokinase-based sugar sensing mechanisms, and that the synergistic effect of lack of phosphate is independent of phosphate sensing.


Pinus pinaster Programmed cell death PCD Phosphate Sugar 



H. A. was supported by the project “Genomics and Evolutionary Biology” co-financed by North Portugal Regional Operational Programme 2007/2013 (ON.2–O Novo Norte), under the National Strategic Reference Framework (NSRF), through the European Regional Development Fund (ERDF). P.H.C. was supported by Fundação para a Ciência e Tecnologia, grant reference SFRH/BD/44484/2008.


  1. Adams VD (1991) Water and wastewater examination manual. Lewis Publishers, Boca Raton, Florida, USAGoogle Scholar
  2. Adams WW III, Watson AM, Mueh KE, Amiard V, Turgeon R, Ebbert V, Logan BA, Combs AF, Demmig-Adams B (2007) Photosynthetic acclimation in the context of structural constraints to carbon export from leaves. Photosynth Res 94:455–466PubMedCrossRefGoogle Scholar
  3. Aubert S, Gout E, Bligny R, Marty-Mazars D, Barrieu F, Alabouvette J, Marty F, Douce R (1996) Ultrastructural and biochemical characterization of autophagy in higher plant cells subjected to carbon deprivation: control by the supply of mitochondria with respiratory substrates. J Cell Biol 133:1251–1263PubMedCrossRefGoogle Scholar
  4. Azevedo H, Conde C, Gerós H, Tavares RM (2006) The non-host pathogen Botrytis cinerea enhances glucose transport in Pinus pinaster suspension-cultured cells. Plant Cell Physiol 47:290–298Google Scholar
  5. Azevedo H, Dias ACP, Tavares RM (2008a) Establishment and characterization of Pinus pinaster suspension cell cultures. Plant Cell Tiss Org Cult 93:115–121Google Scholar
  6. Azevedo H, Lino-Neto T, Tavares RM (2008b) The necrotroph Botrytis cinerea induces a non-host Type II resistance mechanism in Pinus pinaster suspension-cultured cells. Plant Cell Physiol 49:386–395Google Scholar
  7. Azevedo H, Amorim-Silva V, Tavares RM (2009) Effect of salt on ROS homeostasis, lipid peroxidation and antioxidant mechanisms in Pinus pinaster suspension cells. Ann Forest Sci 66:211Google Scholar
  8. Azevedo H, Dias A, Tavares RM (2010) Analysis on the role of phenylpropanoid metabolism in the Pinus pinaster-Botrytis cinerea interaction. J Phytopathol 158:641–646Google Scholar
  9. Baena-Gonzalez E (2010) Energy signaling in the regulation of gene expression during stress. Mol Plant 3:300–313PubMedCrossRefGoogle Scholar
  10. Baena-González E, Sheen J (2008) Convergent energy and stress signalling. Trends Plant Sci 13:474–482PubMedCentralPubMedCrossRefGoogle Scholar
  11. Bassham DC (2007) Plant autophagy—more than a starvation response. Curr Opin Plant Biol 10:587–593PubMedCrossRefGoogle Scholar
  12. Bolduc N, Brisson LF (2002) Antisense down regulation of NtBI-1 in tobacco BY-2 cells induces accelerated cell death upon carbon starvation. FEBS Lett 532:111–114PubMedCrossRefGoogle Scholar
  13. Chen M-H, Liu L-F, Chen Y-R, Wu H-K, Yu S-M (1994) Expression of α-amylases, carbohydrate metabolism, and autophagy in cultured rice cells is coordinately regulated by sugar nutrient. Plant J 6:625–636PubMedCrossRefGoogle Scholar
  14. Contento AL, Kim S-J, Bassham DC (2004) Transcriptome profiling of the response of Arabidopsis suspension culture cells to Suc starvation. Plant Physiol 135:2330–2347Google Scholar
  15. Cortès S, Gromova M, Evrard A, Roby C, Heyraud A, Rolin DB, Raymond P, Brouquisse RM (2003) In plants, 3-O-methylglucose is phosphorylated by hexokinase but not perceived as a sugar. Plant Physiol 131:824–837PubMedCentralPubMedGoogle Scholar
  16. Coruzzi G, Bush DR (2001) Nitrogen and carbon nutrient and metabolite signaling in plants. Plant Physiol 125:61–64PubMedCentralPubMedCrossRefGoogle Scholar
  17. Csaikl UM, Bastian H, Brettschneider R, Gauch S, Meir A, Schauerte M, Scholz F, Sperisen C, Vrnam B, Ziegenhagen B (1998) Comparative analysis of different DNA extraction protocols: a fast, universal maxi-preparation of high quality plant DNA for genetic evaluation and phylogenetic studies. Plant Mol Biol Rep 16:69–86CrossRefGoogle Scholar
  18. del Pozo JC, Allona I, Rubio V, Leyva A, de la Peña A, Aragoncillo C, Paz-Ares J (1999) A type 5 acid phosphatase gene from Arabidopsis thaliana is induced by phosphate starvation and by some other types of phosphate mobilising/oxidative stress conditions. Plant J 19:579–589PubMedCrossRefGoogle Scholar
  19. Dominguez F, Moreno J, Cejudo FJ (2012) The scutellum of germinated wheat grains undergoes programmed cell death: identification of an acidic nuclease involved in nucleus dismantling. J Exp Bot 63:5475–5485PubMedCentralPubMedCrossRefGoogle Scholar
  20. Doyle JJ, Doyle JL (1990) Isolation of plant DNA from fresh tissue. Focus 12:13–15Google Scholar
  21. Duff S, Plaxton W, Lefebvre D (1991) Phosphate-starvation response in plant cells: de novo synthesis and degradation of acid phosphatases. Proc Natl Acad Sci U S A 88:9538–9542PubMedCentralPubMedCrossRefGoogle Scholar
  22. Escobar-Gutiérrez AJ, Daudet F-A, Gaudillère J-P, Maillard P, Frossard J-S (1998) Modelling of allocation and balance of carbon in walnut (Juglans regia L.) seedlings during heterotrophy-autotrophy transition. J Theor Biol 194:29–47PubMedCrossRefGoogle Scholar
  23. Eveland AL, Jackson DP (2012) Sugars, signalling, and plant development. J Exp Bot 63:3367–3377PubMedCrossRefGoogle Scholar
  24. Gibson SI (2000) Plant sugar-response pathways. Part of a complex regulatory web. Plant Physiol 124:1532–1539PubMedCentralPubMedCrossRefGoogle Scholar
  25. Hammond JP, White PJ (2008) Sucrose transport in the phloem: integrating root responses to phosphorus starvation. J Exp Bot 59:93–109PubMedCrossRefGoogle Scholar
  26. Inoue Y, Moriyasu Y (2006) Autophagy is not a main contributor to the degradation of phospholipids in tobacco cells cultured under sucrose starvation conditions. Plant Cell Physiol 47:471–480PubMedCrossRefGoogle Scholar
  27. Jain A, Poling MD, Karthikeyan AS, Blakeslee JJ, Peer WA, Titapiwatanakun B, Murphy AS, Raghothama KG (2007) Differential effects of sucrose and auxin on localized phosphate deficiency-induced modulation of different traits of root system architecture in Arabidopsis. Plant Physiol 144:232–247PubMedCentralPubMedCrossRefGoogle Scholar
  28. Jain A, Nagarajan VK, Raghothama KG (2012) Transcriptional regulation of phosphate acquisition by higher plants. Cell Mol Life Sci 69:3207–3224PubMedCrossRefGoogle Scholar
  29. Karthikeyan AS, Varadarajan DK, Jain A, Held MA, Carpita NC, Raghothama KG (2007) Phosphate starvation responses are mediated by sugar signaling in Arabidopsis. Planta 225:907–918PubMedCrossRefGoogle Scholar
  30. Klionsky DJ, Abeliovich H, Agostinis P, Agrawal DK (2008) Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes. Autophagy 4:151–175PubMedCentralPubMedCrossRefGoogle Scholar
  31. Kroemer G, Levine B (2008) Autophagic cell death: the story of a misnomer. Nat Rev Mol Cell Biol 9:1004–1010PubMedCentralPubMedCrossRefGoogle Scholar
  32. Lei M, Liu Y, Zhang B, Zhao Y, Wang X, Zhou Y, Raghothama KG, Liu D (2011) Genetic and genomic evidence that sucrose is a global regulator of plant responses to phosphate starvation in Arabidopsis. Plant Physiol 156:1116–1130PubMedCentralPubMedCrossRefGoogle Scholar
  33. Lin W-Y, Lin S-I, Chiou T-J (2009) Molecular regulators of phosphate homeostasis in plants. J Exp Bot 60:1427–1438PubMedCrossRefGoogle Scholar
  34. Lopez-Fernandez MP, Maldonado S (2013) Programmed cell death during quinoa perisperm development. J Exp Bot 64:3313–3325PubMedCentralPubMedCrossRefGoogle Scholar
  35. Love AJ, Milner JJ, Sadanandom A (2008) Timing is everything: regulatory overlap in plant cell death. Trends Plant Sci 13:589–595PubMedCrossRefGoogle Scholar
  36. Mittler R, Shulaev V (2004) Programmed cell death in plants: future perspectives, applications, and methods. In: Gray J (ed) Programmed cell death in plants. Blackwell, Ohio, USA, pp 251–264Google Scholar
  37. Moriyasu Y, Inoue Y (2008) Use of protease inhibitors for detecting autophagy in plants. Method Enzymol 451:557–580CrossRefGoogle Scholar
  38. Moriyasu Y, Ohsumi Y (1996) Autophagy in tobacco suspension-cultured cells in response to sucrose starvation. Plant Physiol 111:1233–1241PubMedCentralPubMedGoogle Scholar
  39. Müller R, Nilsson L, Nielsen LK, Nielsen TH (2005) Interaction between phosphate starvation signalling and hexokinase-independent sugar sensing in Arabidopsis leaves. Physiol Plantarum 124:81–90CrossRefGoogle Scholar
  40. Müller R, Morant M, Jarmer H, Nilsson L, Nielsen TH (2007) Genome-wide analysis of the Arabidopsis leaf transcriptome reveals interaction of phosphate and sugar metabolism. Plant Physiol 143:156–171Google Scholar
  41. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plantarum 15:473–475CrossRefGoogle Scholar
  42. Oliveira J, Tavares RM, Gerós H (2002) Utilization and transport of glucose in Olea europaea cell suspensions. Plant Cell Physiol 43:1510–1517PubMedCrossRefGoogle Scholar
  43. Phillips HJ (1973) Dye exclusion tests for cell viability. In: Kruse P Jr, Patterson M Jr (eds) Tissue culture: methods and applications. Academic, New York, USA, pp 406–408CrossRefGoogle Scholar
  44. Raghothama KG (1999) Phosphate acquisition. Annu Rev Plant Physiol Plant Mol Biol 50:665–693PubMedCrossRefGoogle Scholar
  45. Reape TJ, McCabe PF (2008) Apoptotic-like programmed cell death in plants. New Phytol 180:13–26PubMedCrossRefGoogle Scholar
  46. Reape TJ, Molony EM, McCabe PF (2008) Programmed cell death in plants: distinguishing between different modes. J Exp Bot 59:435–444PubMedCrossRefGoogle Scholar
  47. Rolland F, Baena-Gonzalez E, Sheen J (2006) Sugar sensing and signaling in plants: conserved and novel mechanisms. Annu Rev Plant Biol 57:675–709PubMedCrossRefGoogle Scholar
  48. Rose TL, Bonneau L, Der C, Marty-Mazars D, Marty F (2006) Starvation-induced expression of autophagy-related genes in Arabidopsis. Biol Cell 98:53–67PubMedCrossRefGoogle Scholar
  49. Sedmak JJ, Grossberg SE (1977) A rapid, sensitive, and versatile assay for protein using Coomassie Brilliant Blue G250. Anal Biochem 79:544–552PubMedCrossRefGoogle Scholar
  50. Singh VK, Wood SM, Knowles VL, Plaxton WC (2003) Phosphite accelerates programmed cell death in phosphate-starved oilseed rape (Brassica napus) suspension cell cultures. Planta 218:233–239PubMedCrossRefGoogle Scholar
  51. Takatsuka C, Inoue Y, Higuchi T, Hillmer S, Robinson DG, Moriyasu Y (2011) Autophagy in tobacco BY-2 cells cultured under sucrose starvation conditions: isolation of the autolysosome and its characterization. Plant Cell Physiol 52:2074–2087PubMedCrossRefGoogle Scholar
  52. Thompson AR, Vierstra RD (2005) Autophagic recycling: lessons from yeast help define the process in plants. Curr Opin Plant Biol 8:165–173PubMedCrossRefGoogle Scholar
  53. Ticconi CA, Delatorre CA, Abel S (2001) Attenuation of phosphate starvation responses by phosphite in Arabidopsis. Plant Physiol 127:963–972PubMedCentralPubMedCrossRefGoogle Scholar
  54. van Doorn WG (2011) Classes of programmed cell death in plants, compared to those in animals. J Exp Bot 62:4749–4761PubMedCrossRefGoogle Scholar
  55. van Doorn WG, Woltering EJ (2005) Many ways to exit? Cell death categories in plants. Trends Plant Sci 10:117–122PubMedCrossRefGoogle Scholar
  56. van Doorn WG, Beers EP, Dangl JL, Franklin-Tong VE, Gallois P, Hara-Nishimura I, Jones AM, Kawai-Yamada M, Lam E, Mundy J, Mur LA, Petersen M, Smertenko A, Taliansky M, Van Breusegem F, Wolpert T, Woltering E, Zhivotovsky B, Bozhkov PV (2011) Morphological classification of plant cell deaths. Cell Death Differ 18:1241–1246PubMedCentralPubMedCrossRefGoogle Scholar
  57. Vance CP, Uhde-Stone C, Allan DL (2003) Phosphorus acquisition and use: critical adaptations by plants for securing a nonrenewable resource. New Phytol 157:423–447CrossRefGoogle Scholar
  58. Wang H-J, Wan A-R, Hsu C-M, Lee K-W, Yu S-M, Jauh G-Y (2007) Transcriptomic adaptations in rice suspension cells under sucrose starvation. Plant Mol Biol 63:441–463PubMedCrossRefGoogle Scholar
  59. Yen C-H, Yang C-H (1998) Evidence for programmed cell death during leaf senescence in plants. Plant Cell Physiol 39:922–927CrossRefGoogle Scholar

Copyright information

© The Society for In Vitro Biology 2014

Authors and Affiliations

  • Herlânder Azevedo
    • 1
  • Pedro Humberto Castro
    • 2
  • Joana Ferreira Gonçalves
    • 2
  • Teresa Lino-Neto
    • 2
  • Rui Manuel Tavares
    • 2
  1. 1.CIBIO, Centro de Investigação em Biodiversidade e Recursos GenéticosUniversidade do PortoVairãoPortugal
  2. 2.Center for Biodiversity, Functional & Integrative Genomics (BioFIG), Plant Functional Biology CenterUniversity of MinhoBragaPortugal

Personalised recommendations