FtsH metalloproteases and their pseudo-proteases in the chloroplast envelope of Arabidopsis thaliana

Abstract: By cleaving peptide bonds, proteases either activate or degrade proteins and maintain protein quality control in response to various developmental stimuli and environmental factors. My work has focused on elucidating the role of the filamentation temperature sensitive protein H (FtsH) proteases. FtsHs belong to a membrane-embedded class of proteases found in eubacteria, animals and plants, which are located in the organelles of endosymbiosis (mitochondria and chloroplasts). They possess an AAA+ (ATPase associated with various cellular activities) and a peptidase M41 domain containing the HEXXH consensus sequence in the Zn2+ metalloprotease domain. FtsH proteases are known to form ring-like homo- or hetero-hexameric complexes. Arabidopsis thaliana, the model plant used in this study, contains seventeen AtFtsH proteases, of which twelve are presumably proteolytically active and five presumably proteolytic inactive members, known as AtFtsHi (i for inactive). In AtFtsHi members, the HEXXH motif is either deleted (AtFtsHi3) or mutated (AtFtsHi1, 2, 4, 5). Twelve AtFtsHs (AtFtsH 1, 2, 5–9, 11, 12 and AtFtsHi 1-5) are targeted to the chloroplast, whereas the remaining three (AtFtsH 3, 4 and 10) are mitochondrial. In Paper I, we demonstrate that AtFtsH12 interacts with AtFtsHi1, 2, 4, 5 to form a heteromeric complex. Abundance of these AtFtsH12-AtFtsHi complexes alters the accumulation of TIC (translocon on the inner chloroplast membrane) complexes. Transgenic mi12 (miRNA) knockdown plants that express lower amounts of AtFtsH12 displayed a pale-seedling and an aberrant chloroplast phenotype. mi12 plants displayed lowered total chlorophyll (Chla+Chlb) amount compared to wild type (WT), complementation lines and native AtFtsH12 promoter overexpressor (ox12) lines. Our biochemical studies identified drastic modifications in the total proteome of mi12 seedlings. N-terminome analyses of mi12 seedlings showed undisturbed plastidic protein maturation. In Paper II, we have shown that single mutants depleted in AtFTSHI1, 2, 4 or 5 are embryo-lethal, suggesting the pseudo-proteases to have an indispensable role in seed germination. This study further identified “weak” Atftshi1, Atftshi4, Atftshi3-1(kd) and Atftshi3-2 homozygous mutants, which develop into plants with altered photosynthetic efficiency. Field experiments were performed to determine the Darwinian fitness of these homozygous as well as heterozygous AtFtsHi mutants. The results suggested AtFtsHi enzymes to be critical during early developmental stages. A complete Atftshi3 knockdown mutant (Atftshi3-1(kd)) was identified (described in Paper III), which is not embryo-lethal and tolerates drought better than WT plants. Atftshi3-1(kd) leaves were smaller with fewer and smaller stomatal aperture. Above ground, Atftshi3-1(kd) leaves displayed lowered stomatal conductance and increased WUEi (intrinsic water-use efficiency), while below ground, the root-associated bacterial community showed a typical drought stress response. Upregulated transcripts of the ABA-responsive genes in leaves of Atftshi3-1(kd) compared to WT indicate the drought tolerance to be controlled independently of ABA. To conclude, AtFtsHi pseudo-proteases affect various stages of plant development and abiotic stress management, especially drought.