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. 1996 Jun 2;133(6):1251–1263. doi: 10.1083/jcb.133.6.1251

Ultrastructural and biochemical characterization of autophagy in higher plant cells subjected to carbon deprivation: control by the supply of mitochondria with respiratory substrates

PMCID: PMC2120909  PMID: 8682862

Abstract

Autophagy triggered by carbohydrate starvation was characterized at both biochemical and structural levels, with the aim to identify reliable and easily detectable marker(s) and to investigate the factors controlling this process. Incubation of suspension cells in sucrose- free culture medium triggered a marked degradation of the membrane polar lipids, including phospholipids and galactolipids. In contrast, the total amounts of sterols, which are mainly associated with plasmalemma and tonoplast membranes, remained constant. In particular, phosphatidylcholine decreased, whereas phosphodiesters including glycerylphosphorylcholine transiently increased, and phosphorylcholine (P-Cho) steadily accumulated. P-Cho exhibits a remarkable metabolic inertness and therefore can be used as a reliable biochemical marker reflecting the extent of plant cell autophagy. Indeed, whenever P-Cho accumulated, a massive regression of cytoplasm was noticed using EM. Double membrane-bounded vacuoles were formed in the peripheral cytoplasm during sucrose starvation and were eventually expelled into the central vacuole, which increased in volume and squeezed the thin layer of cytoplasm spared by autophagy. The biochemical marker P-Cho was used to investigate the factors controlling autophagy. P-Cho did not accumulate when sucrose was replaced by glycerol or by pyruvate as carbon sources. Both compounds entered the cells and sustained normal rates of respiration. No recycling back to the hexose phosphates was observed, and cells were rapidly depleted in sugars and hexose phosphates, without any sign of autophagy. On the contrary, when pyruvate (or glycerol) was removed from the culture medium, P-Cho accumulated without a lag phase, in correlation with the formation of autophagic vacuoles. These results strongly suggest that the supply of mitochondria with respiratory substrates, and not the decrease of sucrose and hexose phosphates, controls the induction of autophagy in plant cells starved in carbohydrates.

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Selected References

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  1. Ashworth D. J., Lee R. Y., Adams D. O. Characterization of Acetate and Pyruvate Metabolism in Suspension Cultures of Zea mays by C NMR Spectroscopy. Plant Physiol. 1987 Oct;85(2):463–468. doi: 10.1104/pp.85.2.463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Aubert S., Gout E., Bligny R., Douce R. Multiple effects of glycerol on plant cell metabolism. Phosphorus-31 nuclear magnetic resonance studies. J Biol Chem. 1994 Aug 26;269(34):21420–21427. [PubMed] [Google Scholar]
  3. Baba M., Takeshige K., Baba N., Ohsumi Y. Ultrastructural analysis of the autophagic process in yeast: detection of autophagosomes and their characterization. J Cell Biol. 1994 Mar;124(6):903–913. doi: 10.1083/jcb.124.6.903. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bligny R., Foray M. F., Roby C., Douce R. Transport and phosphorylation of choline in higher plant cells. Phosphorus-31 nuclear magnetic resonance studies. J Biol Chem. 1989 Mar 25;264(9):4888–4895. [PubMed] [Google Scholar]
  5. Chan M. T., Chao Y. C., Yu S. M. Novel gene expression system for plant cells based on induction of alpha-amylase promoter by carbohydrate starvation. J Biol Chem. 1994 Jul 1;269(26):17635–17641. [PubMed] [Google Scholar]
  6. Chen M. H., Liu L. F., Chen Y. R., Wu H. K., Yu S. M. Expression of alpha-amylases, carbohydrate metabolism, and autophagy in cultured rice cells is coordinately regulated by sugar nutrient. Plant J. 1994 Nov;6(5):625–636. doi: 10.1046/j.1365-313x.1994.6050625.x. [DOI] [PubMed] [Google Scholar]
  7. Couée I., Jan M., Carde J. P., Brouquisse R., Raymond P., Pradet A. Effects of glucose starvation on mitochondrial subpopulations in the meristematic and submeristematic regions of maize root. Plant Physiol. 1992 Dec;100(4):1891–1900. doi: 10.1104/pp.100.4.1891. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dieuaide M., Couée I., Pradet A., Raymond P. Effects of glucose starvation on the oxidation of fatty acids by maize root tip mitochondria and peroxisomes: evidence for mitochondrial fatty acid beta-oxidation and acyl-CoA dehydrogenase activity in a higher plant. Biochem J. 1993 Nov 15;296(Pt 1):199–207. doi: 10.1042/bj2960199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Douce R., Joyard J. Biochemistry and function of the plastid envelope. Annu Rev Cell Biol. 1990;6:173–216. doi: 10.1146/annurev.cb.06.110190.001133. [DOI] [PubMed] [Google Scholar]
  10. Dunn W. A., Jr Studies on the mechanisms of autophagy: formation of the autophagic vacuole. J Cell Biol. 1990 Jun;110(6):1923–1933. doi: 10.1083/jcb.110.6.1923. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dunn W. A., Jr Studies on the mechanisms of autophagy: maturation of the autophagic vacuole. J Cell Biol. 1990 Jun;110(6):1935–1945. doi: 10.1083/jcb.110.6.1935. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gancedo J. M. Carbon catabolite repression in yeast. Eur J Biochem. 1992 Jun 1;206(2):297–313. doi: 10.1111/j.1432-1033.1992.tb16928.x. [DOI] [PubMed] [Google Scholar]
  13. Genix P., Bligny R., Martin J. B., Douce R. Transient accumulation of asparagine in sycamore cells after a long period of sucrose starvation. Plant Physiol. 1990 Oct;94(2):717–722. doi: 10.1104/pp.94.2.717. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Graham I. A., Denby K. J., Leaver C. J. Carbon Catabolite Repression Regulates Glyoxylate Cycle Gene Expression in Cucumber. Plant Cell. 1994 May;6(5):761–772. doi: 10.1105/tpc.6.5.761. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Grinde B. Autophagy and lysosomal proteolysis in the liver. Experientia. 1985 Sep 15;41(9):1089–1095. doi: 10.1007/BF01951685. [DOI] [PubMed] [Google Scholar]
  16. Herman E. M., Baumgartner B., Chrispeels M. J. Uptake and apparent digestion of cytoplasmic organelles by protein bodies (protein storage vacuoles) in mung bean cotyledons. Eur J Cell Biol. 1981 Jun;24(2):226–235. [PubMed] [Google Scholar]
  17. Hurkman W. J., Tanaka C. K. Solubilization of plant membrane proteins for analysis by two-dimensional gel electrophoresis. Plant Physiol. 1986 Jul;81(3):802–806. doi: 10.1104/pp.81.3.802. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Journet E. P., Bligny R., Douce R. Biochemical changes during sucrose deprivation in higher plant cells. J Biol Chem. 1986 Mar 5;261(7):3193–3199. [PubMed] [Google Scholar]
  19. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  20. Martin B. A., Tolbert N. E. Factors which affect the amount of inorganic phosphate, phosphorylcholine, and phosphorylethanolamine in xylem exudate of tomato plants. Plant Physiol. 1983 Oct;73(2):464–470. doi: 10.1104/pp.73.2.464. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Marty F. Cytochemical studies on GERL, provacuoles, and vacuoles in root meristematic cells of Euphorbia. Proc Natl Acad Sci U S A. 1978 Feb;75(2):852–856. doi: 10.1073/pnas.75.2.852. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Roberts J. K., Jardetzky O. Monitoring of cellular metabolism by NMR. Biochim Biophys Acta. 1981 Nov 9;639(1):53–76. doi: 10.1016/0304-4173(81)90005-7. [DOI] [PubMed] [Google Scholar]
  23. Roby C., Martin J. B., Bligny R., Douce R. Biochemical changes during sucrose deprivation in higher plant cells. Phosphorus-31 nuclear magnetic resonance studies. J Biol Chem. 1987 Apr 15;262(11):5000–5007. [PubMed] [Google Scholar]
  24. Rose M., Albig W., Entian K. D. Glucose repression in Saccharomyces cerevisiae is directly associated with hexose phosphorylation by hexokinases PI and PII. Eur J Biochem. 1991 Aug 1;199(3):511–518. doi: 10.1111/j.1432-1033.1991.tb16149.x. [DOI] [PubMed] [Google Scholar]
  25. Rébeillé F., Bligny R., Martin J. B., Douce R. Effect of sucrose starvation on sycamore (Acer pseudoplatanus) cell carbohydrate and Pi status. Biochem J. 1985 Mar 15;226(3):679–684. doi: 10.1042/bj2260679. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Schellens J. P., Vreeling-Sindelárová H., Plomp P. J., Meijer A. J. Hepatic autophagy and intracellular ATP. A morphometric study. Exp Cell Res. 1988 Jul;177(1):103–108. doi: 10.1016/0014-4827(88)90028-6. [DOI] [PubMed] [Google Scholar]
  27. Sheen J. Metabolic repression of transcription in higher plants. Plant Cell. 1990 Oct;2(10):1027–1038. doi: 10.1105/tpc.2.10.1027. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Takeshige K., Baba M., Tsuboi S., Noda T., Ohsumi Y. Autophagy in yeast demonstrated with proteinase-deficient mutants and conditions for its induction. J Cell Biol. 1992 Oct;119(2):301–311. doi: 10.1083/jcb.119.2.301. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Tsukada M., Ohsumi Y. Isolation and characterization of autophagy-defective mutants of Saccharomyces cerevisiae. FEBS Lett. 1993 Oct 25;333(1-2):169–174. doi: 10.1016/0014-5793(93)80398-e. [DOI] [PubMed] [Google Scholar]
  30. Ullmann A. Catabolite repression 1985. Biochimie. 1985 Jan;67(1):29–34. doi: 10.1016/s0300-9084(85)80227-3. [DOI] [PubMed] [Google Scholar]
  31. Van der Wilden W., Herman E. M., Chrispeels M. J. Protein bodies of mung bean cotyledons as autophagic organelles. Proc Natl Acad Sci U S A. 1980 Jan;77(1):428–432. doi: 10.1073/pnas.77.1.428. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Wittenbach V. A., Lin W., Hebert R. R. Vacuolar localization of proteases and degradation of chloroplasts in mesophyll protoplasts from senescing primary wheat leaves. Plant Physiol. 1982 Jan;69(1):98–102. doi: 10.1104/pp.69.1.98. [DOI] [PMC free article] [PubMed] [Google Scholar]

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