Chloroplast SRP54 and FtsH protease coordinate thylakoid membrane-associated proteostasis in Arabidopsis.

Thylakoid membrane protein quality control (PQC), which requires the coordination of membrane protein translocation and degradation of unassembled proteins, determines chloroplast development during de-etiolation. Despite numerous efforts, the regulation of this process in land plants is largely unknown. Here, we report the isolation and characterization of pale green Arabidopsis4 (pga4) mutants in Arabidopsis (Arabidopsis thaliana) with defects in chloroplast development during de-etiolation. Map-based cloning and complementation assays confirmed that PGA4 encodes the chloroplast Signal Recognition Particle 54 kDa (cpSRP54) protein. A heterogeneous Light-Harvesting Chlorophyll a/b Binding-Green Fluorescent Protein (LhcB2-GFP) fusion protein was generated as an indicative reporter for cpSRP54-mediated thylakoid translocation. LhcB2-GFP was dysfunctional and degraded to a short-form dLhcB2-GFP during de-etiolation through an N-terminal degradation initiated on thylakoid membranes. Further biochemical and genetic evidence demonstrated that the degradation of LhcB2-GFP to dLhcB2-GFP was disrupted in pga4 and yellow variegated2 (var2) mutants caused by mutations in the Filamentous Temperature-Sensitive H2 (VAR2/AtFtsH2) subunit of thylakoid FtsH. The yeast two-hybrid assay showed that the N-terminus of LhcB2-GFP interacts with the protease domain of VAR2/AtFtsH2. Moreover, the over-accumulated LhcB2-GFP in pga4 and var2 formed protein aggregates, which were insoluble in mild nonionic detergents. Genetically, cpSRP54 is a suppressor locus for the leaf variegation phenotype of var2. Together, these results demonstrate the coordination of cpSRP54 and thylakoid FtsH in maintaining thylakoid membrane PQC during the assembly of photosynthetic complexes and provide a trackable substrate and product for monitoring cpSRP54-dependent protein translocation and FtsH-dependent protein degradation.

Chloroplast envelope ATPase PGA1/AtFtsH12 is required for chloroplast protein accumulation and cytosol-chloroplast protein homeostasis in Arabidopsis.

The establishment of photosynthetic protein complexes during chloroplast development requires the influx of a large number of chloroplast proteins that are encoded by the nuclear genome, which is critical for cytosol and chloroplast protein homeostasis and chloroplast development. However, the mechanisms regulating this process are still not well understood in higher plants. Here, we report the isolation and characterization of the pale green Arabidopsis pga1-1 mutant, which is defective in chloroplast development and chloroplast protein accumulation. Using genetic and biochemical evidence, we reveal that PGA1 encodes AtFtsH12, a chloroplast envelope-localized protein of the FtsH family proteins. We determined a G703R mutation in the GAD motif of the conserved ATPase domain renders the pga1-1 a viable hypomorphic allele of the essential gene AtFtsH12. In de-etiolation assays, we showed that the accumulation of photosynthetic proteins and the expression of photosynthetic genes were impaired in pga1-1. Using the FNRctp-GFP and pTAC2-GFP reporters, we demonstrated that AtFtsH12 was required for the accumulation of chloroplast proteins in vivo. Interestingly, we identified an increase in expression of the mutant AtFtsH12 gene in pga1-1, suggesting a feedback regulation. Moreover, we found that cytosolic and chloroplast proteostasis responses were triggered in pga1-1. Together, taking advantage of the novel pga1-1 mutant, we demonstrate the function of AtFtsH12 in chloroplast protein homeostasis and chloroplast development.

Interaction of PALE CRESS with PAP2/pTAC2 and PAP3/pTAC10 affects the accumulation of plastid-encoded RNA polymerase complexes in Arabidopsis.

The transcription of photosynthesis genes in chloroplasts is largely mediated by the plastid-encoded RNA polymerase (PEP), which resembles prokaryotic-type RNA polymerases, but with plant-specific accessory subunits known as plastid transcriptionally active chromosome proteins (pTACs) or PEP-associated proteins (PAPs). However, whether additional factors are involved in the biogenesis of PEP complexes remains unknown. Here, we investigated the function of an essential gene, PALE CRESS (PAC), in the accumulation of PEP complexes in chloroplasts. We established that an Arabidopsis leaf variegation mutant, variegated 6-1 (var6-1), is a hypomorphic allele of PAC. Unexpectedly, we revealed that a fraction of VAR6/PAC is associated with thylakoid membranes, where it interacts with PEP complexes. The accumulation of PEP complexes is defective in both var6-1 and the null allele var6-2. Further protein interaction assays confirmed that VAR6/PAC interacts directly with the PAP2/pTAC2 and PAP3/pTAC10 subunits of PEP complexes. Moreover, we generated viable hypomorphic alleles of the essential gene PAP2/pTAC2, and revealed a genetic interaction between PAC and PAP2/pTAC2 in photosynthesis gene expression and PEP complex accumulation. Our findings establish that VAR6/PAC affects PEP complex accumulation through interactions with PAP2/pTAC2 and PAP3/pTAC10, and provide new insights into the accumulation of PEP and chloroplast development.