Annis, M. Y.; Routray, P.; Bhuiyan, N. H.; Yuan, B.; van wijk, k. J.

Expression of the chloroplast AAA+ chaperone CLPD gene increases during senescence and drought, but its functional role in chloroplast proteostasis is poorly understood. This study provides a comprehensive analysis of Arabidopsis CLPD protein accumulation across development from early seedlings to senescence, and compares results to its homologs CLPC1,2, as well as CLPB3 and cpHSP90. The developmental consequences of complete loss of CLPD expression (clpd-1), as well as overexpression of functional CLPD or CLPD impaired in ATP hydrolysis (CLPD-TRAP), were determined in Arabidopsis. clpd-1 has accelerated seedling development while functional CLPD overexpression lines, but not CLPD-TRAP, have delayed development. To determine if CLPD is a bona fide CLP chaperone associating with the CLPPRT protease and to identify in vivo candidate substrates, we employed the CLPD-TRAP line during the vegetative and flowering (senescent) growth stages. Affinity purification of CLPD-TRAP followed by mass spectrometry showed high enrichment of the CLP protease complex, thus providing direct support for the role of CLPD in substrate delivery to the CLP protease. CLPC1,2 were also highly enriched in the CLPD-TRAP interactome, suggesting hetero-oligomerization and cooperation between the three chaperones is likely. Nine chloroplast candidate substrates were identified in the CLPD-interactomes, including: FHY2 involved in riboflavin synthesis, THI1 and THIC involved in thiamin metabolism, and four proteins of unknown function. Several of these have been previously identified as potential CLPC1 substrates; however, others appear to be specific to CLPD. CLPD acts in substrate selection within a heteromeric CLPC-CLPD hexamer, likely to make unique contributions through its divergent N-terminus.