Downregulation of ClpR2 leads to reduced accumulation of the ClpPRS protease complex and defects in chloroplast biogenesis in Arabidopsis.
Plastids contain tetradecameric Clp protease core complexes, with five ClpP Ser-type proteases, four nonproteolytic ClpR, and two associated ClpS proteins. Accumulation of total ClpPRS complex decreased twofold to threefold in an Arabidopsis thaliana T-DNA insertion mutant in CLPR2 designated clpr2-1. Differential stable isotope labeling of the ClpPRS complex with iTRAQ revealed a fivefold reduction in assembled ClpR2 accumulation and twofold to fivefold reductions in the other subunits. A ClpR2:(his)(6) fusion protein that incorporated into the chloroplast ClpPRS complex fully complemented clpr2-1. The reduced accumulation of the ClpPRS protease complex led to a pale-green phenotype with delayed shoot development, smaller chloroplasts, decreased thylakoid accumulation, and increased plastoglobule accumulation. Stromal ClpC1 and 2 were both recruited to the thylakoid surface in clpr2-1. The thylakoid membrane of clpr2-1 showed increased carotenoid content, partial inactivation of photosystem II, and upregulated thylakoid proteases and stromal chaperones, suggesting an imbalance in chloroplast protein homeostasis and a well-coordinated network of proteolysis and chaperone activities. Interestingly, a subpopulation of PsaF and several light-harvesting complex II proteins accumulated in the thylakoid with unprocessed chloroplast transit peptides. We conclude that ClpR2 cannot be functionally replaced by other ClpP/R homologues and that the ClpPRS complex is central to chloroplast biogenesis, thylakoid protein homeostasis, and plant development.
Rudella
Andrea
A
Department of Plant Biology, Cornell University, Ithaca, New York 14853, USA.
Friso
Giulia
G
Alonso
Jose M
JM
Ecker
Joseph R
JR
van Wijk
Klaas J
KJ
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000166:cytology / Q000201:enzymology / Q000502:physiology; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D002736:Chloroplasts / Q000378:metabolism* / Q000648:ultrastructure; D015536:Down-Regulation; D045222:Electron Transport Chain Complex Proteins / Q000235:genetics / Q000378:metabolism; D049071:Endopeptidase Clp / Q000235:genetics / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D006706:Homeostasis; D018832:Molecular Chaperones / Q000378:metabolism; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes; D010641:Phenotype; D017440:Photoperiod; D045332:Photosystem II Protein Complex / Q000502:physiology; D010860:Pigments, Biological / Q000378:metabolism; D018515:Plant Leaves / Q000378:metabolism / Q000648:ultrastructure; D030821:Plants, Genetically Modified; D018087:Plastids / Q000201:enzymology; D016415:Sequence Alignment; D013213:Starch / Q000378:metabolism; D020524:Thylakoids / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure
D029681:Arabidopsis Proteins; D045222:Electron Transport Chain Complex Proteins; D018832:Molecular Chaperones; D009097:Multienzyme Complexes; D045332:Photosystem II Protein Complex; D010860:Pigments, Biological; D013213:Starch; D049071:Endopeptidase Clp