The extreme Albino3 (Alb3) C terminus is required for Alb3 stability and function in Arabidopsis thaliana.

MAIN CONCLUSION: The extreme Alb3 C terminus is important for Alb3 stability in a light dependent manner, but is dispensable for LHCP insertion or D1 synthesis. YidC/Oxa1/Alb3 dependent insertion of membrane proteins is evolutionary conserved among bacteria, mitochondria and chloroplasts. Chloroplasts are challenged by the need to coordinate membrane integration of nuclear encoded, post-translationally targeted proteins into the thylakoids as well as of proteins translated on plastid ribosomes. The pathway facilitating post-translational targeting of the light-harvesting chlorophyll a/b binding proteins involves the chloroplast signal recognition particle, cpSRP54 and cpSRP43, as well as its membrane receptor FtsY and the translocase Alb3. Interaction of cpSRP43 with Alb3 is mediated by the positively charged, stromal exposed C terminus of Alb3. In this study, we utilized an Alb3 T-DNA insertion mutant in Arabidopsis thaliana lacking the last 75 amino acids to elucidate the function of this domain (alb3∆C). However, the truncated Alb3 protein (Alb3∆C) proved to be unstable under standard growth conditions, resulting in a reduction of Alb3∆C to 20 % of wild-type levels. In contrast, accumulation of Alb3∆C was comparable to wild type under low light growth conditions. Alb3∆C mutants grown under low light conditions were only slightly paler than wild type, accumulated almost wild-type levels of light harvesting proteins and were not affected in D1 synthesis, therefore showing that the extreme Alb3 C terminus is dispensable for both, co- and post-translational, protein insertion into the thylakoid membrane. However, reduction of Alb3∆C levels as observed under standard growth conditions resulted not only in a severely diminished accumulation of all thylakoid complexes but also in a strong defect in D1 synthesis and membrane insertion.

A tobacco homolog of DCN1 is involved in pollen development and embryogenesis.

KEY MESSAGE: We show that DCN1 binds ubiquitin and RUB/NEDD8, associates with cullin, and is functionally conserved. DCN1 activity is required for pollen development transitions and embryogenesis, and for pollen tube growth. Plant proteomes show remarkable plasticity in reaction to environmental challenges and during developmental transitions. Some of this adaptability comes from ubiquitin-mediated protein degradation regulated by cullin-RING E3 ubiquitin ligases (CRLs). CRLs are activated through modification of the cullin subunit with the ubiquitin-like protein RUB/NEDD8 by an E3 ligase called defective in cullin neddylation 1 (DCN1). Here we show that tobacco DCN1 binds ubiquitin and RUB/NEDD8 and associates with cullin. When knocked down by RNAi, tobacco pollen formation was affected and zygotic embryogenesis was blocked around the globular stage. Additionally, we found that RNAi of DCN1 inhibited the stress-triggered reprogramming of cultured microspores from their intrinsic gametophytic mode of development to an embryogenic state. This stress-induced developmental switch is a known feature in many important crops and leads ultimately to the formation of haploid embryos and plants. Compensating the RNAi effect by re-transformation with a promoter-silencing construct restored pollen development and zygotic embryogenesis, as well as the ability for stress-induced formation of embryogenic microspores. Overexpression of DCN1 accelerated pollen tube growth and increased the potential for microspore reprogramming. These results demonstrate that the biochemical function of DCN1 is conserved in plants and that its activity is involved in transitions during pollen development and embryogenesis, and for pollen tube growth.

Arabidopsis thaliana Oxa proteins locate to mitochondria and fulfill essential roles during embryo development.

Members of the Alb3/Oxa1/YidC protein family function as insertases in chloroplasts, mitochondria, and bacteria. Due to independent gene duplications, all organisms possess two isoforms, Oxa1 and Oxa2 except gram-negative bacteria, which encode only for one YidC-like protein. The genome of Arabidopsis thaliana however, encodes for eight different isoforms. The localization of three of these isoforms has been identified earlier: Alb3 and Alb4 located in thylakoid membranes of chloroplasts while AtOxa1 was found in the inner membrane of mitochondria. Here, we show that the second Oxa1 protein, Oxa1b as well as two Oxa2 proteins are also localized in mitochondria. The last two isoforms most likely encode truncated versions of Oxa-like proteins, which might be inoperable pseudogenes. Homozygous mutant lines were only obtained for Oxa1b, which did not reveal any significant phenotypes, while T-DNA insertion lines of Oxa1a, Oxa2a and Oxa2b resulted only in heterozygous plants indicating that these genes are indispensable for plant development. Phenotyping heterozygous lines showed that embryos are either retarded in growth, display an albino phenotype or embryo formation was entirely abolished suggesting that Oxa1a and both Oxa2 proteins function in embryo formation although at different developmental stages as indicated by the various phenotypes observed.

Nucleus-encoded light-harvesting chlorophyll a/b proteins are imported normally into chlorophyll b-free chloroplasts of Arabidopsis.

Chloroplast-located proteins which are encoded by the nuclear genome have to be imported from the cytosol into the organelle in a posttranslational manner. Among these nuclear-encoded chloroplast proteins are the light-harvesting chlorophyll a/b-binding proteins (LHCPs). After translation in the cytosol, precursor proteins of LHCPs are imported via the TOC/TIC translocase, processed to their mature size to insert into thylakoid membranes where they recruit chlorophylls a and b to form pigment-protein complexes. The translocation of proteins is a highly regulated process which employs several regulators. To analyze whether CAO (chlorophyll a oxigenase) which converts chlorophyll a to chlorophyll b at the inner chloroplast membrane, is one of these regulators, we performed import reactions utilizing a homozygous loss-of-function mutant (cao-1). We imported in vitro translated and (35)S-labeled precursor proteins of light-harvesting proteins of photosystem II LHCB1, LHCB4, and LHCB5 into chloroplasts isolated from cao-1 and show that import of precursor proteins and their processing to mature forms are not impaired in the mutant. Therefore, regulation of the import machinery cannot be responsible for the decreased steady-state levels of light-harvesting complex (LHC) proteins. Regulation does not take place at the transcriptional level either, because Lhcb mRNAs are not down-regulated. Additionally, reduced steady-state levels of LHCPs also do not occur due to posttranslational turnover of non-functional LHCPs in chloroplasts. Taken together, our data show that plants in the absence of CAO and therefore devoid of chlorophyll b are not influenced in their import behavior of LHC proteins.

Biogenesis of the cyanobacterial thylakoid membrane system--an update.

Current molecular analyses suggest that initial steps of the biogenesis of cyanobacterial photosystems progress in a membrane subfraction representing a biosynthetic center with contact to both plasma and thylakoid membranes. This special membrane fraction is defined by the presence of the photosystem II assembly factor PratA. The proposed model suggests that both biogenesis of protein complexes and insertion of chlorophyll molecules into the photosystems occur in this intermediate membrane system.

Alb4 of Arabidopsis promotes assembly and stabilization of a non chlorophyll-binding photosynthetic complex, the CF1CF0-ATP synthase.

All members of the YidC/Oxa1/Alb3 protein family are evolutionarily conserved and appear to function in membrane protein integration and protein complex stabilization. Here, we report on a second thylakoidal isoform of Alb3, named Alb4. Analysis of Arabidopsis knockout mutant lines shows that Alb4 is required in assembly and/or stability of the CF1CF0-ATP synthase (ATPase). alb4 mutant lines not only have reduced steady-state levels of ATPase subunits, but also their assembly into high-molecular-mass complexes is altered, leading to a reduction of ATP synthesis in the mutants. Moreover, we show that Alb4 but not Alb3 physically interacts with the subunits CF1beta and CF0II. Summarizing, the data indicate that Alb4 functions to stabilize or promote assembly of CF1 during its attachment to the membrane-embedded CF0 part.

Interaction of the periplasmic PratA factor and the PsbA (D1) protein during biogenesis of photosystem II in Synechocystis sp. PCC 6803.

The biogenesis of photosynthetic complexes is assisted by a growing number of trans-acting factors in both chloroplasts and cyanobacteria. We have previously shown that the periplasmic PratA factor from Synechocystis sp. PCC 6803 (Synechocystis 6803) is required for adequate C-terminal processing of the PsbA (D1) subunit of photosystem II (PSII) supporting the idea that the early steps of PSII assembly occur at the plasma membrane. Here we report on the molecular analysis of the interaction between PratA and the D1 protein. Both yeast two-hybrid and glutathione S-transferase pulldown assays revealed that PratA binds to the soluble forms of both mature and precursor D1 C-terminal regions. In agreement with that finding, the binding region was mapped to amino acid positions 314-328 of D1 by applying a peptide-scanning approach. Approximately 10-20% of the soluble PratA factor was found to be associated with membranes in a D1-dependent manner. Sucrose density gradient centrifugations allowed the identification of a specific membrane subfraction that contains both PratA and D1 and which might represent a transfer and/or connecting region between plasma and thylakoid membrane. Imaging data obtained with enhanced cyan fluorescent protein-labeled D1 protein in wild-type and pratA mutant backgrounds further supported this notion.

In vivo visualization of Mg-protoporphyrin IX, a coordinator of photosynthetic gene expression in the nucleus and the chloroplast.

The photosynthetic apparatus is composed of proteins encoded by genes from both the nucleus and the chloroplast. To ensure that the photosynthetic complexes are assembled stoichiometrically and to enable their rapid reorganization in response to a changing environment, the plastids emit signals that regulate nuclear gene expression to match the status of the plastids. One of the plastid signals, the chlorophyll intermediate Mg-ProtoporphyrinIX (Mg-ProtoIX) accumulates under stress conditions and acts as a negative regulator of photosynthetic gene expression. By taking advantage of the photoreactive property of tetrapyrroles, Mg-ProtoIX could be visualized in the cells using confocal laser scanning spectroscopy. Our results demonstrate that Mg-ProtoIX accumulated both in the chloroplast and in the cytosol during stress conditions. Thus, the signaling metabolite is exported from the chloroplast, transmitting the plastid signal to the cytosol. Our results from the Mg-ProtoIX over- and underaccumulating mutants copper response defect and genome uncoupled5, respectively, demonstrate that the expression of both nuclear- and plastid-encoded photosynthesis genes is regulated by the accumulation of Mg-ProtoIX. Thus, stress-induced accumulation of the signaling metabolite Mg-ProtoIX coordinates nuclear and plastidic photosynthetic gene expression.