Abundance of metalloprotease FtsH12 modulates chloroplast development in Arabidopsis thaliana

Abstract The ATP-dependent metalloprotease FtsH12 (filamentation temperature sensitive protein H 12) has been suggested to participate in a heteromeric motor complex, driving protein translocation into the chloroplast. FtsH12 was immuno-detected in proplastids, seedlings, leaves, and roots. Expression of Myc-tagged FtsH12 under its native promotor allowed identification of FtsHi1, 2, 4, and 5, and plastidic NAD-malate dehydrogenase, five of the six interaction partners in the suggested import motor complex. Arabidopsis thaliana mutant seedlings with reduced FTSH12 abundance exhibited pale cotyledons and small, deformed chloroplasts with altered thylakoid structure. Mature plants retained these chloroplast defects, resulting in slightly variegated leaves and lower chlorophyll content. Label-free proteomics revealed strong changes in the proteome composition of FTSH12 knock-down seedlings, reflecting impaired plastid development. The composition of the translocon on the inner chloroplast membrane (TIC) protein import complex was altered, with coordinated reduction of the FtsH12-FtsHi complex subunits and accumulation of the 1 MDa TIC complex subunits TIC56, TIC214 and TIC22-III. FTSH12 overexpressor lines showed no obvious phenotype, but still displayed distinct differences in their proteome. N-terminome analyses further demonstrated normal proteolytic maturation of plastid-imported proteins irrespective of FTSH12 abundance. Together, our data suggest that FtsH12 has highest impact during seedling development; its abundance alters the plastid import machinery and impairs chloroplast development.

left panel) as well as genes encoding the thylakoid located FTSH2 and the import complex subunit YCF1.1 (ATCG0100) (right level) were extracted from the Arabidopsis eFP browser (http://bar.utoronto.ca/). Data were normalized by the GCOS method, TGT value of 100 (Winter et. al., 2007). (B) FTSH12 expression in transgenic Arabidopsis T3-lines containing the native FTSH12 promoter fused to the β-glucuronidase (GUS) gene. GUS activity was made visible by histochemical staining in leaves (Ba) and roots (Bb) of 12-day old plants, flowers (Bc), and at embryo stage (Bd). thaliana cell culture (B). A: S-seedlings, L-leaves, R-roots, d-days, w-weeks. Seedlings and two weeks old plants were grown on MS-medium growth plates, while four and eight weeks old plants were grown on soil. B: Cells cultivated in darkness contain proplastids (0 d). On illumination chloroplasts start to develop (day 1), thylakoid structures are established at day 5 and are able to perform photosynthesis at day 7. Cells being illuminated for 14 days contain mature chloroplasts 4 (Dubreuil et al., 2018). 15 µg of protein were loaded onto the SDS-PAGE, three biological replicates were tested.    Significant differences between wt and transgenic plants are indicated by asterisk (Student's t-test, p < 0.05).
(C) Chlorophyll amount (mg g -1 fresh weight) in developing leaves of 10 weeks-old wt and FTSH12 transgenic plants. Values are averages (± SE) of three independent experimental replicates, each with three plants per experiment (Student's t-test, p < 0.05).
(E) Hypocotyl length of wt and transgenic FTSH12 seedlings during de-etiolation. Values represent the mean ± se of measurements conducted on n = 75, n = 71 and n = 73 seedlings for wt, overexpressor and knock-down lines, respectively. Significant differences between wt and transgenic plants are indicated by asterisk (Student's t-test, p < 0.05).   KEGG functional term enrichment, protein interactions and protein coexpression were analysed using the STRING database. Blue edges indicates proteins assocations annotated in databases, pink from experimental evidence, black indicates coexpression. Enriched KEGG pathways are indicated: red, ath01200, carbon metabolism; blue, ath03050, proteasome; green, at03010, ribosome; yellow, ath03040, spliceosome; pink, ath04141, protein processing in the endoplasmic reticulum.

Fig. S11. Graphical depiction of protein interactions in Cluster 2 of the proteome analysis.
KEGG functional term enrichment, protein interactions and protein coexpression were analysed using the STRING database. Blue edges indicates proteins associations annotated in databases, pink from experimental evidence, black indicates coexpression. Enriched KEGG pathways are indicated: red, ath01200, carbon metabolism; blue, ath03050, proteasome; green, ath04145, phagosome.

Fig. S12. Graphical depiction of protein interactions in Cluster 3 of the proteome analysis.
Functional GO term enrichment was analysed using the STRING database. Blue edges indicates proteins associations annotated in databases, pink from experimental evidence, black indicates coexpression. Red, GO:0004004, ATP-dependent RNA helicase activity.

Fig. S13. Graphical depiction of protein interactions in Cluster 4 of the proteome analysis.
KEGG functional term enrichment, protein interactions and protein coexpression were analysed using the STRING database. Blue edges indicates proteins associations annotated in databases, pink from experimental evidence, black indicates coexpression. Enriched KEGG pathways are indicated: red, at03010, ribosome; blue, ath04141, protein processing in the endoplasmic reticulum.

Fig. S14. Graphical depiction of protein interactions in Cluster 5 of the proteome analysis.
KEGG functional term enrichment, protein interactions and protein coexpression were analysed using the STRING database. Blue edges indicates proteins associations annotated in databases, pink from experimental evidence, black indicates coexpression. Enriched KEGG pathways are indicated: red, at03010, ribosome; green, ath03013, RNA transport. KEGG functional term enrichment, protein interactions and protein coexpression were analysed using the STRING database. Blue edges indicates proteins associations annotated in databases, pink from experimental evidence, black indicates coexpression. Enriched KEGG pathways are 20 indicated: red, at03010, ribosome; blue, ath01200, carbon metabolism; green, at00195, photosynthesis; yellow, ath00860, porphyrin and chlorophyll metabolism.