The FtsH protease heterocomplex in Arabidopsis: dispensability of type-B protease activity for proper chloroplast development

FtsH is an ATP-dependent metalloprotease present as a hexameric heterocomplex in thylakoid membranes. Encoded in the Arabidopsis thaliana YELLOW VARIEGATED2 (VAR2) locus, FtsH2 is one isoform among major Type A (FtsH1/5) and Type B (FtsH2/8) isomers. Mutants lacking FtsH2 (var2) and FtsH5 (var1) are characterized by a typical leaf-variegated phenotype. The functional importance of the catalytic center (comprised by the zinc binding domain) in FtsH2 was assessed in this study by generating transgenic plants that ectopically expressed FtsH2(488), a proteolytically inactive version of FtsH2. The resulting amino acid substitution inhibited FtsH protease activity in vivo when introduced into Escherichia coli FtsH. By contrast, expression of FtsH2(488) rescued not only leaf variegation in var2 but also seedling lethality in var2 ftsh8, suggesting that the protease activity of Type B isomers is completely dispensable, which implies that the chloroplastic FtsH complex has protease sites in excess and that they act redundantly rather than coordinately. However, expression of FtsH2(488) did not fully rescue leaf variegation in var1 var2 because the overall FtsH levels were reduced under this background. Applying an inducible promoter to our complementation analysis revealed that rescue of leaf variegation indeed depends on the overall amount of FtsH. Our results elucidate protein activity and its amount as important factors for the function of FtsH heterocomplexes that are composed of multiple isoforms in the thylakoid membrane.     

EMB1211 is required for normal embryo development and influences chloroplast biogenesis in Arabidopsis

Chloroplast biogenesis is tightly linked with embryogenesis and seedling development. A growing body of work has been done on the molecular mechanisms underlying chloroplast development; however, the molecular components involved in chloroplast biogenesis during embryogenesis remain largely uncharacterized. In this paper, we show that an Arabidopsis mutant carrying a T‐DNA insertion in a gene encoding a multiple membrane occupation and recognition nexus (MORN)‐containing protein exhibits severe defects during embryogenesis, producing abnormal embryos and thereby leading to a lethality of young seedlings. Genetic and microscopic studies reveal that the mutation is allelic to a previously designated Arabidopsis embryo‐defective 1211 mutant (emb1211). The emb1211 +/? mutant plants produce approximately 25% of white‐colored ovules with abnormal embryos since late globular stage when primary chloroplast biogenesis takes place, while the wild‐type plants produce all green ovules. Transmission electron microscopic analysis reveals the absence of normal chloroplast development, both in the mutant embryos and in the mutant seedlings, that contributes to the albinism. The EMB1211 gene is preferentially expressed in developing embryos as revealed in the EMB1211::GUS transgenic plants. Taken together, the data indicate that EMB1211 has an important role during embryogenesis and chloroplast biogenesis in Arabidopsis.     

Anionic lipids are required for chloroplast structure and function in Arabidopsis

Photosynthetic membranes of plants primarily contain non-phosphorous glycolipids. The exception is phosphatidylglycerol (PG), which is an acidic/anionic phospholipid. A second major anionic lipid in chloroplasts is the sulfolipid sulfoquinovosyldiacylglycerol (SQDG). It is hypothesized that under severe phosphate limitation, SQDG substitutes for PG, ensuring a constant proportion of anionic lipids even under adverse conditions. A newly constructed SQDG and PG-deficient double mutant supports this hypothesis. This mutant, sqd2 pgp1-1, carries a T-DNA insertion in the structural gene for SQDG synthase (SQD2) and a point mutation in the structural gene for phosphatidylglycerolphosphate synthase (PGP1). In the sqd2 pgp1-1 double mutant, the fraction of total anionic lipids is reduced by approximately one-third, resulting in pale yellow cotyledons and leaves with reduced chlorophyll content. Photoautotrophic growth of the double mutant is severely compromised, and its photosynthetic capacity is impaired. In particular, photosynthetic electron transfer at the level of photosystem II (PSII) is affected. Besides these physiological changes, the mutant shows altered leaf structure, a reduced number of mesophyll cells, and ultrastructural changes of the chloroplasts. All observations on the sqd2 pgp1-1 mutant lead to the conclusion that the total content of anionic thylakoid lipids is limiting for chloroplast structure and function, and is critical for overall photoautotrophic growth and plant development.     

Interplay between N-terminal methionine excision and FtsH protease is essential for normal chloroplast development and function in Arabidopsis

N-terminal methionine excision (NME) is the earliest modification affecting most proteins. All compartments in which protein synthesis occurs contain dedicated NME machinery. Developmental defects induced in Arabidopsis thaliana by NME inhibition are accompanied by increased proteolysis. Although increasing evidence supports a connection between NME and protein degradation, the identity of the proteases involved remains unknown. Here we report that chloroplastic NME (cNME) acts upstream of the FtsH protease complex. Developmental defects and higher sensitivity to photoinhibition associated with the ftsh2 mutation were abolished when cNME was inhibited. Moreover, the accumulation of D1 and D2 proteins of the photosystem II reaction center was always dependent on the prior action of cNME. Under standard light conditions, inhibition of chloroplast translation induced accumulation of correctly NME-processed D1 and D2 in a ftsh2 background, implying that the latter is involved in protein quality control, and that correctly NME-processed D1 and D2 are turned over primarily by the thylakoid FtsH protease complex. By contrast, inhibition of cNME compromises the specific N-terminal recognition of D1 and D2 by the FtsH complex, whereas the unprocessed forms are recognized by other proteases. Our results highlight the tight functional interplay between NME and the FtsH protease complex in the chloroplast.     

Two electron donation sites for exogenous reductants in chloroplast Photosystem II

Two sites are distinguished for the oxidation of exogenous donors by Photosystem II in non-oxygen evolving chloroplasts. In the presence of lipophilic donors (e.g. phenylenediamine, benzidine, diphenylcarbazide), the rate for Signal IIf rereduction following a flash increases as the concentration of exogenous reductant increases. There is a decrease (20–40%) in Signal IIf magnitude accompanying donor addition at low (< 10^?5M) concentrations, but the extent of the decrease does not change further with increasing donor concentration. Complementary polarographic experiments monitoring donor (phenylenediamine) oxidation show an increase in oxidation rate with increasing donor concentration.In the presence of the hydrophilic donor, Mn²⁺, the Signal IIf decay halftime remains constant with increasing Mn²⁺ concentration. However, the flash-induced Signal IIf magnitude progressively decreases with increasing Mn²⁺ concentration.These results are interpreted in terms of two competing paths for the reduction of P680⁺. In one path P680⁺ reduction is accompanied by the appearance of Signal IIf, and lipophilic donors subsequently rereduce the Signal IIf species in a series reaction. This reduction follows pseudo-first order kinetics as a function of donor concentration. In the second path Mn²⁺ reduces P680⁺ in a parallel reaction that competes with the formation of the Signal IIf species. This results in a decrease in the magnitude of Signal IIf, but no change in its decay time.     

Plastid signals remodel light signaling networks and are essential for efficient chloroplast biogenesis in Arabidopsis

Plastid signals are among the most potent regulators of genes that encode proteins active in photosynthesis. Plastid signals help coordinate the expression of the nuclear and chloroplast genomes and the expression of genes with the functional state of the chloroplast. Here, we report the isolation of new cryptochrome1 (cry1) alleles from a screen for Arabidopsis thaliana genomes uncoupled mutants, which have defects in plastid-to-nucleus signaling. We also report genetic experiments showing that a previously unidentified plastid signal converts multiple light signaling pathways that perceive distinct qualities of light from positive to negative regulators of some but not all photosynthesis-associated nuclear genes (PhANGs) and change the fluence rate response of PhANGs. At least part of this remodeling of light signaling networks involves converting HY5, a positive regulator of PhANGs, into a negative regulator of PhANGs. We also observed that mutants with defects in both plastid-to-nucleus and cry1 signaling exhibited severe chlorophyll deficiencies. These data show that the remodeling of light signaling networks by plastid signals is a mechanism that plants use to integrate signals describing the functional and developmental state of plastids with signals describing particular light environments when regulating PhANG expression and performing chloroplast biogenesis.     

Multiple cellulose synthase catalytic subunits are required for cellulose synthesis in Arabidopsis

The irregular xylem 1 (irx1) mutant of Arabidopsis has a severe deficiency in the deposition of cellulose in secondary cell walls, which results in collapsed xylem cells. This mutation has been mapped to a 140-kb region of chromosome 4. A cellulose synthase catalytic subunit was found to be located in this region, and genomic clones containing this gene complemented the irx1 mutation. IRX1 shows homology to a previously described cellulose synthase (IRX3). Analysis of the irx1 and irx3 mutant phenotypes demonstrates that both IRX1 and IRX3 are essential for the production of cellulose in the same cell. Thus, IRX1 and IRX3 define distinct classes of catalytic subunits that are both essential for cellulose synthesis in plants. This finding is supported by coprecipitation of IRX1 with IRX3, suggesting that IRX1 and IRX3 are part of the same complex.     

Two distinct arginine methyltransferases are required for biogenesis of Sm-class ribonucleoproteins

Small nuclear ribonucleoproteins (snRNPs) are core components of the spliceosome. The U1, U2, U4, and U5 snRNPs each contain a common set of seven Sm proteins. Three of these Sm proteins are posttranslationally modified to contain symmetric dimethylarginine (sDMA) residues within their C-terminal tails. However, the precise function of this modification in the snRNP biogenesis pathway is unclear. Several lines of evidence suggest that the methyltransferase protein arginine methyltransferase 5 (PRMT5) is responsible for sDMA modification of Sm proteins. We found that in human cells, PRMT5 and a newly discovered type II methyltransferase, PRMT7, are each required for Sm protein sDMA modification. Furthermore, we show that the two enzymes function nonredundantly in Sm protein methylation. Lastly, we provide in vivo evidence demonstrating that Sm protein sDMA modification is required for snRNP biogenesis in human cells.     

Photosystem II assembly and repair are differentially localized in Chlamydomonas

Many proteins of the photosynthesis complexes are encoded by the genome of the chloroplast and synthesized by bacterium-like ribosomes within this organelle. To determine where proteins are synthesized for the de novo assembly and repair of photosystem II (PSII) in the chloroplast of Chlamydomonas reinhardtii, we used fluorescence in situ hybridization, immunofluorescence staining, and confocal microscopy. These locations were defined as having colocalized chloroplast mRNAs encoding PSII subunits and proteins of the chloroplast translation machinery specifically under conditions of PSII subunit synthesis. The results revealed that the synthesis of the D1 subunit for the repair of photodamaged PSII complexes occurs in regions of the chloroplast with thylakoids, consistent with the current model. However, for de novo PSII assembly, PSII subunit synthesis was detected in discrete regions near the pyrenoid, termed T zones (for translation zones). In two PSII assembly mutants, unassembled D1 subunits and incompletely assembled PSII complexes localized around the pyrenoid, where we propose that they mark an intermediate compartment of PSII assembly. These results reveal a novel chloroplast compartment that houses de novo PSII biogenesis and the regulated transport of newly assembled PSII complexes to thylakoid membranes throughout the chloroplast.     

Mutations in the Arabidopsis VAR2 locus cause leaf variegation due to the loss of a chloroplast FtsH protease

Variegated plants have green- and white-sectored leaves. Cells in the green sectors contain morphologically normal chloroplasts, whereas cells in the white sectors contain non-pigmented plastids that lack organized lamellar structures. Many variegations are caused by mutations in nuclear genes that affect plastid function, yet in only a few cases have the responsible genes been cloned. We show that mutations in the nuclear VAR2 locus of Arabidopsis cause variegation due to loss of a chloroplast thylakoid membrane protein that bears similarity to the FtsH family of AAA proteins (ATPases associated with diverse cellular activities). Escherichia coli FtsH is a chaperone metalloprotease that functions in a number of diverse membrane-associated events. Although FtsH homologs have been identified in multicellular organisms, their functions and activities are largely unknown; we provide genetic in vivo evidence that VAR2 functions in thylakoid membrane biogenesis. We have isolated four var2 alleles and they have allowed us to define domains of the protein that are required for activity. These include two putative ATP-binding sites. VAR2 protein amounts generally correlate with the severity of the var2 mutant phenotype. One allele lacks detectable VAR2 protein, suggesting that the mechanism of var2 variegation involves the action of a redundant activity in the green sectors. We conclude that redundant activities may be a general mechanism to explain nuclear gene-induced plant variegations.     

Deletion of FtsH11 protease has impact on chloroplast structure and function in Arabidopsis thaliana when grown under continuous light

The membrane‐integrated metalloprotease FtsH11 of Arabidopsis thaliana is proposed to be dual‐targeted to mitochondria and chloroplasts. A bleached phenotype was observed in ftsh11 grown at long days or continuous light, pointing to disturbances in the chloroplast. Within the chloroplast, FtsH11 was found to be located exclusively in the envelope. Two chloroplast‐located proteins of unknown function (Tic22‐like protein and YGGT‐A) showed significantly higher abundance in envelope membranes and intact chloroplasts of ftsh11 and therefore qualify as potential substrates for the FtsH11 protease. No proteomic changes were observed in the mitochondria of 6‐week‐old ftsh11 compared with wild type, and FtsH11 was not immunodetected in these organelles. The abundance of plastidic proteins, especially of photosynthetic proteins, was altered even during standard growth conditions in total leaves of ftsh11. At continuous light, the amount of photosystem I decreased relative to photosystem II, accompanied by a drastic change of the chloroplast morphology and a drop of non‐photochemical quenching. FtsH11 is crucial for chloroplast structure and function during growth in prolonged photoperiod.     

An Arabidopsis pentatricopeptide repeat protein, SUPPRESSOR OF VARIEGATION7, is required for FtsH-mediated chloroplast biogenesis

The Arabidopsis (Arabidopsis thaliana) yellow variegated2 (var2) mutant has green- and white-sectored leaves due to loss of VAR2, a subunit of the chloroplast FtsH protease/chaperone complex. Suppressor screens are a valuable tool to gain insight into VAR2 function and the mechanism of var2 variegation. Here, we report the molecular characterization of 004-003, a line in which var2 variegation is suppressed. We found that the suppression phenotype in this line is caused by lack of a chloroplast pentatricopeptide repeat (PPR) protein that we named SUPPRESSOR OF VARIEGATION7 (SVR7). PPR proteins contain tandemly repeated PPR motifs that bind specific RNAs, and they are thought to be central regulators of chloroplast and mitochondrial nucleic acid metabolism in plants. The svr7 mutant has defects in chloroplast ribosomal RNA (rRNA) processing that are different from those in other svr mutants, and these defects are correlated with reductions in the accumulation of some chloroplast proteins, directly or indirectly. We also found that whereas var2 displays a leaf variegation phenotype at 22°C, it has a pronounced chlorosis phenotype at 8°C that is correlated with defects in chloroplast rRNA processing and a drastic reduction in chloroplast protein accumulation. Surprisingly, the cold-induced phenotype of var2 cannot be suppressed by svr7. Our results strengthen the previously established linkage between var2 variegation and chloroplast rRNA processing/chloroplast translation, and they also point toward the possibility that VAR2 mediates different activities in chloroplast biogenesis at normal and chilling temperatures.     

Photosystem II protein clearance and FtsH function in the diatom Thalassiosira pseudonana

All oxygenic photoautotrophs suffer photoinactivation of their Photosystem II complexes, at a rate driven by the instantaneous light level. To maintain photosynthesis, PsbA subunits are proteolytically removed from photoinactivated Photosystem II complexes, primarily by a membrane-bound FtsH protease. Diatoms thrive in environments with fluctuating light, such as coastal regions, in part because they enjoy a low susceptibility to photoinactivation of Photosystem II. In a coastal strain of the diatom Thalassiosira pseudonana growing across a range of light levels, active Photosystem II represents only about 42 % of the total Photosystem II protein, with the remainder attributable to photoinactivated Photosystem II awaiting recycling. The rate constant for removal of PsbA protein increases with growth light, in parallel with an increasing content of the FtsH protease relative to the substrate PsbA. An offshore strain of Thalassiosira pseudonana, originating from a more stable light environment, had a lower content of FtsH and slower rate constants for removal of PsbA. We used this data to generate the first estimates for in vivo proteolytic degradation of photoinactivated PsbA per FtsH₆ protease, at ~3.9 × 10^?2 s^?1, which proved consistent across growth lights and across the onshore and offshore strains.     

The C-terminal end of LpxC is required for degradation by the FtsH protease

Lipopolysaccharide (LPS) biosynthesis is essential in Gram negative bacteria. LpxC, the key enzyme in LPS formation, catalyses the limiting reaction and controls the ratio between LPS and phospholipids. As overproduction of LPS is toxic, the cellular amount of LpxC must be regulated carefully. The membrane-bound protease FtsH controls the level of LpxC via proteolysis making FtsH the only essential protease of Escherichia coli. We found that the chaperones DnaK and DnaJ co-purified with LpxC. However, degradation of LpxC was DnaK/J-independent in contrast to turnover of the heat shock sigma factor sigma32 (RpoH). The stability of LpxC in a bacterial one-hybrid system suggested that a terminus of LpxC might be important for degradation. Different LpxC truncations and extensions were constructed. Removal of at least five amino acids from the C-terminus abolished degradation by FtsH in vivo. While addition of two aspartic acids to LpxC did not alter its half-life, the exchange of the last two residues against aspartic acids resulted in stabilization. All stable LpxC enzymes were active in vivo as assayed by their high toxicity. Our data demonstrate that the C-terminus of LpxC contains a signal sequence necessary for FtsH-dependent degradation.