*5.1. FtsHi1*

*Arabidopsis* ARC1 (accumulation and replication of chloroplasts 1) was isolated by map-based cloning and was found to encode FtsHi1 (At4g23940) [72]. Homozygous *ftshi1-2* knock out mutants are embryo lethal, while the missense mutant *ftshi1-1/arc1* displays a pale-seedling phenotype [72]. Albino seeds in developing siliques of *ftshi1-1/arc1* showed arrest at three different stages of embryo development, namely late globular, early heart, and late heart stage [72]. Seeds harvested from field-grown *ftshi1-1/arc* plants displayed a significant delay in germination compared to their respective WT [26]. The *ftshi1-1/arc1* mutant shows an increased number of chloroplasts, and the plants have smaller

rosette sizes throughout their life span [72]. The chloroplast ultrastructure of *ftshi1-1/arc1* showed wavy, swollen, and less organized thylakoids with starch grains accumulating, indicating the chloroplasts still being metabolic active. While chloroplasts of the *ftshi1- 1/arc1* mutant contain assembled Ycf2/FtsH12/FtsHi complexes, these plants are impaired in in vitro protein import, which is most likely caused by miss-folding of the AAA-ATPase domain [58]. Mutated FtsHi1 or FtsH12 might partially substitute FtsHi1 in the complex. Gene expression of *FTSH12* and the other *FTSHis* was significantly lower in the *ftshi1-1/arc1* mutant than in WT [26]. Vice versa, the expression of *FTSHi1* was down-regulated in single *FTSHi* mutants [26]. The overexpression of *FTSHi1 (35S: FTSHi1–YFP)* generated few recovered transgenic plants, which were mildly variegated in appearance [72]. These overexpression plants further showed a substantial increase in chloroplast size, but fewer chloroplasts than WT. *FTSHi1* transcript levels were similar in the white and green sectors of the variegated leaves, but in variegated tissue, the level of FtsHi1–YFP was low, while green tissue accumulated FtsHi1–YFP similar to WT [72].

The FtsHi enzymes of the chloroplast envelope have been suggested to respond to photo-oxidative stress [72]. During de-etiolation in white light at intensities of 15, 100, or 300 μmol m<sup>−</sup><sup>2</sup> s<sup>−</sup>1, *ftshi1-1/arc1* mutant seedlings accumulated only half of the chlorophyll amount compared to WT controls. Low light (1 μmol m<sup>−</sup><sup>2</sup> s<sup>−</sup>1) irradiance with 40% blue or 60% red (BR) light led to a significant increase in the mutant's chlorophyll accumulation. The authors concluded that *ftshi1-1/arc1* at low irradiance still can attenuate chloroplast biogenesis without causing photo-oxidative stress. At normal growth conditions, *ftshi1- 1/arc1* displayed lower non-photochemical quenching (NPQ) values than WT. However, during exposure to various stresses (continuous light, long day/high light, short day/4 ◦C, and long day/30 ◦C), the mutant sustained similar to WT [26], protection mechanisms might have been triggered [76,77]
