Autophagy of Chloroplasts During Leaf Senescence

  • Shinya Wada
  • Hiroyuki IshidaEmail author
Part of the Advances in Photosynthesis and Respiration book series (AIPH, volume 36)


During leaf senescence, chloroplasts undergo the programmed breakdown of both stromal and thylakoid components of the photosynthetic apparatus. This strategy has evolved to remobilize nutrients from old leaves into newly developing tissues and sustain maximal growth rates. After the remobilization of chloroplast components, some shrunken chloroplasts called gerontoplasts, which are plastid structures formed by the loss of the thylakoid membrane network, remain in the cytoplasm. Concomitantly, the population of chloroplasts is decreased in mesophyll cells. The morphological traits of senescing cells, including the capture of whole chloroplasts in the vacuole has been observed by electron microscopy since the early 1980s. Chloroplast degradation in the vacuole has been observed.

Recent genome-wide analysis has shed light on autophagy, a bulk protein degradation system well-conserved in eukaryotes ranging from yeast to mammals, in plants. The improvement of techniques for imaging living cells has enabled researchers to describe the characteristic substrate trafficking across membranes from the cytoplasm into the vacuole. By applying our understanding of the degradation mechanism of autophagy characterized in yeasts to plants, chloroplasts were shown to be degraded by autophagy during leaf senescence.

Chloroplast autophagy occurs by two different pathways. The first is partial degradation via vesicle trafficking. Chloroplasts produce vesicles, named Rubisco-containing bodies (RCBs), which contain only the stromal fraction. RCB formation is affected by the carbon status of the cell, and is specifically linked to photosynthesis inside chloroplasts. RCBs are finally transported and degraded in the vacuole by autophagy. The second pathway is the autophagy of whole chloroplasts, and their degradation inside the vacuole. These pathways of chloroplast autophagy exist as one of the degradation mechanisms of chloroplast components during leaf senescence, causing a decrease in chloroplast size and number.


Leaf Senescence Autophagosome Membrane Selective Autophagy Stromal Protein Nitrogen Remobilization 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


ATG genes;

Autophagy related genes;


Green fluorescent protein;


Individually darkened leaves;


Rubisco-containing bodies



We dedicate this review to Prof. Dr. Tadahiko Mae who originated this work in our laboratory, and thank Prof. Dr. Amane Makino and Dr. Yuji Suzuki for helpful advice to guide our investigations. We thank Dr. Louis Irving for critical reading of the manuscript. We also thank all lab members and collaborators, especially Dr. Masanori Izumi, Dr. Kohki Yoshimoto, Prof. Dr. Yoshinori Ohsumi, and Prof. Dr. Maureen R. Hanson. The authors’ work was supported by KAKENHI (grant nos. 18780042, 19039004, 20200061, 20780044, and 22780054).


  1. Caspar T, Huber SC, Somerville C (1985) Alterations in growth, photosynthesis, and respiration in a starchless mutant of Arabidopsisthaliana (L) deficient in chloroplast phosphoglumutase activity. Plant Physiol 79:11–17PubMedCrossRefGoogle Scholar
  2. Caspar T, Lin TP, Kakefuda G, Benbow L, Preiss J, Somerville C (1991) Mutants of Arabidopsis with altered regulation of starch degradation. Plant Physiol 95:1181–1188PubMedCrossRefGoogle Scholar
  3. Chiba A, Ishida H, Nishizawa NK, Makino A, Mae T (2003) Exclusion of ribulose-1,5-bisphosphate carboxylase/oxygenase from chloroplasts by specific bodies in naturally senescing leaves of wheat. Plant Cell Physiol 44:914–921PubMedCrossRefGoogle Scholar
  4. Chung T, Suttangkakul A, Vierstra RD (2009) The ATG autophagic conjugation system in maize: ATG transcripts and abundance of the ATG8-lipid adduct are regulated by development and nutrient availability. Plant Physiol 149:220–234PubMedCrossRefGoogle Scholar
  5. Chung T, Phillips AR, Vierstra RD (2010) ATG8 lipidation and ATG8-mediated autophagy in Arabidopsis require ATG12 expressed from the differentially controlled ATG12A AND ATG12B loci. Plant J 62:483–493PubMedCrossRefGoogle Scholar
  6. Deduve C, Wattiaux R (1966) Function of lysosomes. Annu Rev Physiol 28:435–492CrossRefGoogle Scholar
  7. Farré JC, Manjithaya R, Mathewson RD, Subramani S (2008) PpAtg30 tags peroxisomes for turnover by selective autophagy. Dev Cell 14:365–376PubMedCrossRefGoogle Scholar
  8. Feller U, Anders I, Mae T (2008) Rubiscolytics: fate of Rubisco after its enzymatic function in a cell is terminated. J Exp Bot 59:1615–1624PubMedCrossRefGoogle Scholar
  9. Fujioka Y, Noda NN, Fujii K, Yoshimoto K, Ohsumi Y, Inagaki F (2008) In vitro reconstitution of plant ATG8 and ATG12 conjugation systems essential for autophagy. J Biol Chem 283:1921–1928PubMedCrossRefGoogle Scholar
  10. Guiamét JJ, Pichersky E, Noodén LD (1999) Mass exodus from senescing soybean chloroplasts. Plant Cell Physiol 40:986–992CrossRefGoogle Scholar
  11. Gunning BES (2005) Plastid stromules: video microscopy of their outgrowth, retraction, tensioning, anchoring, branching, bridging, and tip-shedding. Protoplasma 225:33–42PubMedCrossRefGoogle Scholar
  12. Guo YF, Gan SS (2005) Leaf senescence: signals, execution, and regulation. Curr Top Dev Biol 71:83–112PubMedCrossRefGoogle Scholar
  13. Hörtensteiner S, Feller U (2002) Nitrogen metabolism and remobilization during senescence. J Exp Bot 53:927–937PubMedCrossRefGoogle Scholar
  14. Hosokawa N, Sasaki T, Iemura S, Natsume T, Hara T, Mizushima N (2009) Atg101, a novel mammalian autophagy protein interacting with Atg13. Autophagy 5:973–979PubMedCrossRefGoogle Scholar
  15. Inada N, Sakai A, Kuroiwa H, Kuroiwa T (1998) Three-dimensional analysis of the senescence program in rice (Oryza sativa L.) coleoptiles – Investigations of tissues and cells by fluorescence microscopy. Planta 205:153–164PubMedCrossRefGoogle Scholar
  16. Ishida H, Yoshimoto K, Izumi M, Reisen D, Yano Y, Makino A, Ohsumi Y, Hanson MR, Mae T (2008) Mobilization of rubisco and stroma-localized fluorescent proteins of chloroplasts to the vacuole by an ATG gene-dependent autophagic process. Plant Physiol 148:142–155PubMedCrossRefGoogle Scholar
  17. Izumi M, Wada S, Makino A, Ishida H (2010) The autophagic degradation of chloroplasts via Rubisco-containing bodies is specifically linked to leaf carbon status but not nitrogen status in Arabidopsis. Plant Physiol 154:1196–1209PubMedCrossRefGoogle Scholar
  18. Kato Y, Murakami S, Yamamoto Y, Chatani H, Kondo Y, Nakano T, Yokota A, Sato F (2004) The DNA-binding protease, CND41, and the degradation of ribulose-1,5-bisphosphate carboxylase/oxygenase in senescent leaves of tobacco. Planta 220:97–104PubMedCrossRefGoogle Scholar
  19. Kaul S, Koo HL, Jenkins J, Rizzo M, Rooney T, Tallon LJ, Feldblyum T, Nierman W, Benito MI, Lin XY, Town CD, Venter JC, Fraser CM, Tabata S, Nakamura Y, Kaneko T, Sato S, Asamizu E, Kato T, Kotani H, Sasamoto S, Ecker JR, Theologis A, Federspiel NA, Palm CJ, Osborne BI, Shinn P, Conway AB, Vysotskaia VS, Dewar K, Conn L, Lenz CA, Kim CJ, Hansen NF, Liu SX, Buehler E, Altafi H, Sakano H, Dunn P, Lam B, Pham PK, Chao Q, Nguyen M, Yu GX, Chen HM, Southwick A, Lee JM, Miranda M, Toriumi MJ, Davis RW, Wambutt R, Murphy G, Düsterhöft A, Stiekema W, Pohl T, Entian KD, Terryn N, Volckaert G, Salanoubat M, Choisne N, Rieger M, Ansorge W, Unseld M, Fartmann B, Valle G, Artiguenave F, Weissenbach J, Quetier F, Wilson RK, de la Bastide M, Sekhon M, Huang E, Spiegel L, Gnoj L, Pepin K, Murray J, Johnson D, Habermann K, Dedhia N, Parnell L, Preston R, Hillier L, Chen E, Marra M, Martienssen R, McCombie WR, Mayer K, White O, Bevan M, Lemcke K, Creasy TH, Bielke C, Haas B, Haase D, Maiti R, Rudd S, Peterson J, Schoof H, Frishman D, Morgenstern B, Zaccaria P, Ermolaeva M, Pertea M, Quackenbush J, Volfovsky N, Wu DY, Lowe TM, Salzberg SL, Mewes HW, Rounsley S, Bush D, Subramaniam S, Levin I, Norris S, Schmidt R, Acarkan A, Bancroft I, Brennicke A, Eisen JA, Bureau T, Legault BA, Le QH, Agrawal N, Yu Z, Copenhaver GP, Luo S, Pikaard CS, Preuss D, Paulsen IT, Sussman M, Britt AB, Selinger DA, Pandey R, Mount DW, Chandler VL, Jorgensen RA, Pikaard C, Juergens G, Meyerowitz EM, Dangl J, Jones JDG, Chen M, Chory J, Somerville MC, Ar Gen I (2000) Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 408:796–815CrossRefGoogle Scholar
  20. Keech O, Pesquet E, Ahad A, Askne A, Nordvall D, Vodnala SM, Tuominen H, Hurry V, Dizengremel P, Gardeström P (2007) The different fates of mitochondria and chloroplasts during dark-induced senescence in Arabidopsis leaves. Plant Cell Environ 30:1523–1534PubMedCrossRefGoogle Scholar
  21. Köhler RH, Hanson MR (2000) Plastid tubules of higher plants are tissue-specific and developmentally regulated. J Cell Sci 113:81–89PubMedGoogle Scholar
  22. Krupinska K (2007) Fate and activities of plastids during leaf senescence. In: Wise RR, Hoober JK (eds) The structure and function of plastids. Springer, Dordrecht, pp 433–449CrossRefGoogle Scholar
  23. Kura-Hotta M, Hashimoto H, Satoh K, Katoh S (1990) Quantitative-determination of changes in the number and size of chloroplasts in naturally senescing leaves of rice seedlings. Plant Cell Physiol 31:33–38Google Scholar
  24. Kwok EY, Hanson MR (2004) Stromules and the dynamic nature of plastid morphology. J Microsc 214:124–137PubMedCrossRefGoogle Scholar
  25. Lemasters JJ (2005) Perspective – selective mitochondrial autophagy, or mitophagy, as a targeted defense against oxidative stress, mitochondrial dysfunction, and aging. Rejuvenation Res 8:3–5PubMedCrossRefGoogle Scholar
  26. Levine B, Klionsky DJ (2004) Development by self-digestion: molecular mechanisms and biological functions of autophagy. Dev Cell 6:463–477PubMedCrossRefGoogle Scholar
  27. Lin TP, Caspar T, Somerville C, Preiss J (1988) Isolation and characterization of a starchless mutant of Arabidopsisthaliana (L) heynh lacking ADPglucose pyrophosphorylase activity. Plant Physiol 86:1131–1135PubMedCrossRefGoogle Scholar
  28. Mae T, Kai N, Makino A, Ohira K (1984) Relation between ribulose bisphosphate carboxylase content and chloroplast number in naturally senescing primary leaves of wheat. Plant Cell Physiol 25:333–336Google Scholar
  29. Makino A, Osmond B (1991) Effects of nitrogen nutrition on nitrogen partitioning between chloroplasts and mitochondria in pea and wheat. Plant Physiol 96:355–362PubMedCrossRefGoogle Scholar
  30. Martínez DE, Costa ML, Gomez FM, Otegui MS, Guiamet JJ (2008) ‘Senescence-associated vacuoles’ are involved in the degradation of chloroplast proteins in tobacco leaves. Plant J 56:196–206PubMedCrossRefGoogle Scholar
  31. Martinoia E, Heck U, Dalling MJ, Matile P (1983) Changes in chloroplast number and chloroplast constitutents in senescing barley leaves. Biochem Physiol Pflanzen 178:147–155CrossRefGoogle Scholar
  32. Michigan State University Gardieria Database (2004) The Galdieria sulphuraria genome project. Accessed Nov 2010
  33. Minamikawa T, Toyooka K, Okamoto T, Hara-Nishimura I, Nishimura M (2001) Degradation of ribulose-bisphosphate carboxylase by vacuolar enzymes of senescing French bean leaves: immunocytochemical and ultrastructural observations. Protoplasma 218:144–153PubMedCrossRefGoogle Scholar
  34. Mukaiyama H, Baba M, Osumi M, Aoyagi S, Kato N, Ohsumi Y, Sakai Y (2004) Modification of a ubiquitin-like protein Paz2 conducted micropexophagy through formation of a novel membrane structure. Mol Biol Cell 15:58–70PubMedCrossRefGoogle Scholar
  35. Nakatogawa H, Suzuki K, Kamada Y, Ohsumi Y (2009) Dynamics and diversity in autophagy mechanisms: lessons from yeast. Nat Rev Mol Cell Biol 10:458–467PubMedCrossRefGoogle Scholar
  36. Narendra D, Tanaka A, Suen DF, Youle RJ (2008) Parkin is recruited selectively to impaired mitochondria and promotes their autophagy. J Cell Biol 183:795–803PubMedCrossRefGoogle Scholar
  37. Natesan SKA, Sullivan JA, Gray JC (2005) Stromules: a characteristic cell-specific feature of plastid morphology. J Exp Bot 56:787–797PubMedCrossRefGoogle Scholar
  38. National Institute of Agricultural Science (2008) SALAD database. Accessed Nov 2010
  39. Niittylä T, Messerli G, Trevisan M, Chen J, Smith AM, Zeeman SC (2004) A previously unknown maltose transporter essential for starch degradation in leaves. Science 303:87–89PubMedCrossRefGoogle Scholar
  40. Niwa Y, Kato T, Tabata S, Seki M, Kobayashi M, Shinozaki K, Moriyasu Y (2004) Disposal of chloroplasts with abnormal function into the vacuole in Arabidopsis thaliana cotyledon cells. Protoplasma 223:229–232PubMedCrossRefGoogle Scholar
  41. Ohsumi Y (2001) Molecular dissection of autophagy: two ubiquitin-like systems. Nat Rev Mol Cell Biol 2:211–216PubMedCrossRefGoogle Scholar
  42. Okamoto K, Kondo-Okamoto N, Ohsumi Y (2009) Mitochondria-anchored receptor Atg32 mediates degradation of mitochondria via selective autophagy. Dev Cell 17:87–97PubMedCrossRefGoogle Scholar
  43. Ono K, Hashimoto H, Katoh S (1995) Changes in the number and size of chloroplasts during senescence of primary leaves of wheat grown under different conditions. Plant Cell Physiol 36:9–17Google Scholar
  44. Osteryoung KW, Pyke KA (1998) Plastid division: evidence for a prokaryotically derived mechanism. Curr Opin Plant Biol 1:475–479PubMedCrossRefGoogle Scholar
  45. Otegui MS, Noh YS, Martínez DE, Vila Petroff MG, Andrew Staehelin L, Amasino RM, Guiamet JJ (2005) Senescence-associated vacuoles with intense proteolytic activity develop in leaves of Arabidopsis and soybean. Plant J 41:831–844PubMedCrossRefGoogle Scholar
  46. Park H, Eggink LL, Roberson RW, Hoober JK (1999) Transfer of proteins from the chloroplast to vacuoles in Chlamydomonas reinhardtii (Chlorophyta): a pathway for degradation. J Phycol 35:528–538CrossRefGoogle Scholar
  47. Pérez-Pérez ME, Florencio FJ, Crespo JL (2010) Inhibition of target of rapamycin signaling and stress activate autophagy in Chlamydomonas reinhardtii. Plant Physiol 152:1874–1888PubMedCrossRefGoogle Scholar
  48. Prins A, Van Heerden PDR, Olmos E, Kunert KJ, Foyer CH (2008) Cysteine proteinases regulate chloroplast protein content and composition in tobacco leaves: a model for dynamic interactions with ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) vesicular bodies. J Exp Bot 59:1935–1950PubMedCrossRefGoogle Scholar
  49. Pyke KA (1999) Plastid division and development. Plant Cell 11:549–556PubMedGoogle Scholar
  50. Saito C, Ueda T, Abe H, Wada Y, Kuroiwa T, Hisada A, Furuya M, Nakano A (2002) A complex and mobile structure forms a distinct subregion within the continuous vacuolar membrane in young cotyledons of Arabidopsis. Plant J 29:245–255PubMedCrossRefGoogle Scholar
  51. Sakamoto W (2006) Protein degradation machineries in plastids. Annu Rev Plant Biol 57:599–621PubMedCrossRefGoogle Scholar
  52. Shintani T, Huang WP, Stromhaug PE, Klionsky DJ (2002) Mechanism of cargo selection in the cytoplasm to vacuole targeting pathway. Dev Cell 3:825–837PubMedCrossRefGoogle Scholar
  53. Sláviková S, Shy G, Yao YL, Giozman R, Levanony H, Pietrokovski S, Elazar Z, Galili G (2005) The autophagy-associated Atg8 gene family operates both under favourable growth conditions and under starvation stresses in Arabidopsis plants. J Exp Bot 56:2839–2849PubMedCrossRefGoogle Scholar
  54. Stettler M, Eicke S, Mettler T, Messerli G, Hörtensteiner S, Zeeman SC (2009) Blocking the metabolism of starch breakdown products in Arabidopsis leaves triggers chloroplast degradation. Mol Plant 2:1233–1246PubMedCrossRefGoogle Scholar
  55. Su W, Ma HJ, Liu C, Wu JX, Yang JS (2006) Identification and characterization of two rice autophagy associated genes, OsAtg8 and OsAtg4. Mol Biol Rep 33:273–278PubMedCrossRefGoogle Scholar
  56. Suzuki NN, Yoshimoto K, Fujioka Y, Ohsumi Y, Inagaki F (2005) The crystal structure of plant ATG12 and its biological implication in autophagy. Autophagy 1:119–126PubMedCrossRefGoogle Scholar
  57. Takeshige K, Baba M, Tsuboi S, Noda T, Ohsumi Y (1992) Autophagy in yeast demonstrated with proteinase-deficient mutants and conditions for its induction. J Cell Biol 119:301–311PubMedCrossRefGoogle Scholar
  58. Thayer SS, Huffaker RC (1984) Vacuolar localization of endoproteinase-EP1 and endoproteinase-EP2 in barley mesophyll-cells. Plant Physiol 75:70–73PubMedCrossRefGoogle Scholar
  59. Thompson AR, Vierstra RD (2005) Autophagic recycling: lessons from yeast help define the process in plants. Curr Opin Plant Biol 8:165–173PubMedCrossRefGoogle Scholar
  60. Tsukada M, Ohsumi Y (1993) Isolation and characterization of autophagy-defective mutants of Saccharomyces cerevisiae. FEBS Lett 333:169–174PubMedCrossRefGoogle Scholar
  61. Tuttle DL, Dunn WA (1995) Divergent modes of autophagy in the methylotrophic yeast Pichia pastoris. J Cell Sci 108:25–35PubMedGoogle Scholar
  62. Van der Graaff E, Schwacke R, Schneider A, Desimone M, Flügge UI, Kunze R (2006) Trans­­cription analysis of Arabidopsis membrane transporters and hormone pathways during developmental and induced leaf senescence. Plant Physiol 141:776–792PubMedCrossRefGoogle Scholar
  63. Wada S, Ishida H, Izumi M, Yoshimoto K, Ohsumi Y, Mae T, Makino A (2009) Autophagy plays a role in chloroplast degradation during senescence in individually darkened leaves. Plant Physiol 149:885–893PubMedCrossRefGoogle Scholar
  64. Waters MT, Fray RG, Pyke KA (2004) Stromule formation is dependent upon plastid size, plastid differentiation status and the density of plastids within the cell. Plant J 39:655–667PubMedCrossRefGoogle Scholar
  65. Weaver LM, Amasino RM (2001) Senescence is induced in individually darkened Arabidopsis leaves but inhibited in whole darkened plants. Plant Physiol 127:876–886PubMedCrossRefGoogle Scholar
  66. Wittenbach VA, Lin W, Hebert RR (1982) Vacuolar localization of proteases and degradation of chloroplasts in mesophyll protoplasts from senescing primary wheat leaves. Plant Physiol 69:98–102PubMedCrossRefGoogle Scholar
  67. Xiong Y, Contento AL, Bassham DC (2005) AtATG18a is required for the formation of autophagosomes during nutrient stress and senescence in Arabidopsis thaliana. Plant J 42:535–546PubMedCrossRefGoogle Scholar
  68. Yoshimoto K, Hanaoka H, Sato S, Kato T, Tabata S, Noda T, Ohsumi Y (2004) Processing of ATG8s, ubiquitin-like proteins, and their deconjugation by ATG4s are essential for plant autophagy. Plant Cell 16:2967–2983PubMedCrossRefGoogle Scholar
  69. Yoshimoto K, Takano Y, Sakai Y (2010) Autophagy in plants and phytopathogens. FEBS Lett 584:1350–1358PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2013

Authors and Affiliations

  1. 1.Graduate School of Agricultural ScienceTohoku UniversitySendaiJapan

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