A Sec14 domain protein is required for photoautotrophic growth and chloroplast vesicle formation in Arabidopsis thaliana.

In eukaryotic photosynthetic organisms, the conversion of solar into chemical energy occurs in thylakoid membranes in the chloroplast. How thylakoid membranes are formed and maintained is poorly understood. However, previous observations of vesicles adjacent to the stromal side of the inner envelope membrane of the chloroplast suggest a possible role of membrane transport via vesicle trafficking from the inner envelope to the thylakoids. Here we show that the model plant Arabidopsis thaliana has a chloroplast-localized Sec14-like protein (CPSFL1) that is necessary for photoautotrophic growth and vesicle formation at the inner envelope membrane of the chloroplast. The cpsfl1 mutants are seedling lethal, show a defect in thylakoid structure, and lack chloroplast vesicles. Sec14 domain proteins are found only in eukaryotes and have been well characterized in yeast, where they regulate vesicle budding at the trans-Golgi network. Like the yeast Sec14p, CPSFL1 binds phosphatidylinositol phosphates (PIPs) and phosphatidic acid (PA) and acts as a phosphatidylinositol transfer protein in vitro, and expression of Arabidopsis CPSFL1 can complement the yeast sec14 mutation. CPSFL1 can transfer PIP into PA-rich membrane bilayers in vitro, suggesting that CPSFL1 potentially facilitates vesicle formation by trafficking PA and/or PIP, known regulators of membrane trafficking between organellar subcompartments. These results underscore the role of vesicles in thylakoid biogenesis and/or maintenance. CPSFL1 appears to be an example of a eukaryotic cytosolic protein that has been coopted for a function in the chloroplast, an organelle derived from endosymbiosis of a cyanobacterium

Arabidopsis CSP41 proteins form multimeric complexes that bind and stabilize distinct plastid transcripts

The spinach CSP41 protein has been shown to bind and cleave chloroplast RNA in vitro. Arabidopsis thaliana, like other photosynthetic eukaryotes, encodes two copies of this protein. Several functions have been described for CSP41 proteins in Arabidopsis, including roles in chloroplast rRNA metabolism and transcription. CSP41a and CSP41b interact physically, but it is not clear whether they have distinct functions. It is shown here that CSP41b, but not CSP41a, is an essential and major component of a specific subset of RNA-binding complexes that form in the dark and disassemble in the light. RNA immunoprecipitation and hybridization to gene chips (RIP-chip) experiments indicated that CSP41 complexes can contain chloroplast mRNAs coding for photosynthetic proteins and rRNAs (16S and 23S), but no tRNAs or mRNAs for ribosomal proteins. Leaves of plants lacking CSP41b showed decreased steady-state levels of CSP41 target RNAs, as well as decreased plastid transcription and translation rates. Representative target RNAs were less stable when incubated with broken chloroplasts devoid of CSP41 complexes, indicating that CSP41 proteins can stabilize target RNAs. Therefore, it is proposed that (i) CSP41 complexes may serve to stabilize non-translated target mRNAs and precursor rRNAs during the night when the translational machinery is less active in a manner responsive to the redox state of the chloroplast, and (ii) that the defects in translation and transcription in CSP41 protein-less mutants are secondary effects of the decreased transcript stability

Ion antiport accelerates photosynthetic acclimation in fluctuating light environments

Many photosynthetic organisms globally, including crops, forests and algae, must grow in environments where the availability of light energy fluctuates dramatically. How photosynthesis maintains high efficiency despite such fluctuations in its energy source remains poorly understood. Here we show that Arabidopsis thaliana ​K+ efflux antiporter (​KEA3) is critical for high photosynthetic efficiency under fluctuating light. On a shift from dark to low light, or high to low light, ​kea3 mutants show prolonged dissipation of absorbed light energy as heat. ​KEA3 localizes to the thylakoid membrane, and allows proton efflux from the thylakoid lumen by proton/potassium antiport. ​KEA3’s activity accelerates the downregulation of pH-dependent energy dissipation after transitions to low light, leading to faster recovery of high photosystem II quantum efficiency and increased ​CO2 assimilation. Our results reveal a mechanism that increases the efficiency of photosynthesis under fluctuating light. [EN]This project was funded by the Carnegie Institution for Science, by ERDF-cofinanced grants from the Ministry of Economy and Competitiveness (BIO2012-33655) and Junta de Andalucia (CVI-7558) to K.V., the Natural Sciences and Engineering Research Council of Canada (NSERC) PGS-D3 scholarship to L.P. and Deutsche Forschungsgemeinschaft grants (JA 665/10-1 and GRK 1525 to P.J.; AR 808/1-1 to U.A.).Peer reviewe

Chloroplast Proteins without Cleavable Transit Peptides

Most chloroplast proteins (cp proteins) are nucleus-encoded, synthesized on cytosolic ribosomes as precursor proteins containing a presequence (cTP), and post-translationally imported via the Tic/Toc complex into the organelle, where the cTP is removed. Only a few unambiguous instances of cp proteins that do not require cTPs (non-canonical cp proteins) have been reported so far. However, the survey of data from large-scale proteomic studies presented here suggests that the fraction of such proteins in the total cp proteome might be as large as approximately 30%. To explore this discrepancy, we chose a representative set of 28 putative non-canonical cp proteins, and used in vitro import and Red Fluorescent Protein (RFP)-fusion assays to determine their sub-cellular destinations. Four proteins, including embryo defective 1211, glycolate oxidase 2, protein disulfide isomerase-like protein (PDII), and a putative glutathione S-transferase, could be unambiguously assigned to the chloroplast. Several others ('potential cp proteins') were found to be imported into chloroplasts in vitro, but failed to localize to the organelle when RFP was fused to their C-terminal ends. Extrapolations suggest that the fraction of cp proteins that enter the inner compartments of the organelle, although they lack a cTP, might be as large as 11.4% of the total cp proteome. Our data also support the idea that cytosolic proteins that associate with the cp outer membrane might account for false positive cp proteins obtained in earlier studies

Envelope K +

It is well established that thylakoid membranes of chloroplasts convert light energy into chemical energy, yet the development of chloroplast and thylakoid membranes is poorly understood. Loss of function of the two envelope K(+)/H(+) antiporters AtKEA1 and AtKEA2 was shown previously to have negative effects on the efficiency of photosynthesis and plant growth; however, the molecular basis remained unclear. Here, we tested whether the previously described phenotypes of double mutant kea1kea2 plants are due in part to defects during early chloroplast development in Arabidopsis (Arabidopsis thaliana). We show that impaired growth and pigmentation is particularly evident in young expanding leaves of kea1kea2 mutants. In proliferating leaf zones, chloroplasts contain much lower amounts of photosynthetic complexes and chlorophyll. Strikingly, AtKEA1 and AtKEA2 proteins accumulate to high amounts in small and dividing plastids, where they are specifically localized to the two caps of the organelle separated by the fission plane. The unusually long amino-terminal domain of 550 residues that precedes the antiport domain appears to tether the full-length AtKEA2 protein to the two caps. Finally, we show that the double mutant contains 30% fewer chloroplasts per cell. Together, these results show that AtKEA1 and AtKEA2 transporters in specific microdomains of the inner envelope link local osmotic, ionic, and pH homeostasis to plastid division and thylakoid membrane formation

Growth under fluctuating light reveals large trait variation in a panel of arabidopsis accessions

The capacity of photoautotrophs to fix carbon depends on the efficiency of the conversion of light energy into chemical potential by photosynthesis. In nature, light input into photosynthesis can change very rapidly and dramatically. To analyze how genetic variation in Arabidopsis thaliana affects photosynthesis and growth under dynamic light conditions, 36 randomly chosen natural accessions were grown under uniform and fluctuating light intensities. After 14 days of growth under uniform or fluctuating light regimes, maximum photosystem II quantum efficiency (Fv/Fm) was determined, photosystem II operating efficiency (ΦPSII) and non‐photochemical quenching (NPQ) were measured in low light, and projected leaf area (PLA) as well as the number of visible leaves were estimated. Our data show that ΦPSII and PLA were decreased and NPQ was increased, while Fv/Fm and number of visible leaves were unaffected, in most accessions grown under fluctuating compared to uniform light. There were large changes between accessions for most of these parameters, which, however, were not correlated with genomic variation. Fast growing accessions under uniform light showed the largest growth reductions under fluctuating light, which correlated strongly with a reduction in ΦPSII, suggesting that, under fluctuating light, photosynthesis controls growth and not vice versa.</p

Chloroplast ion homeostasis – what do we know and where should we go?

Plant yields heavily depend on proper macro- and micronutrient supply from the soil. In the leaf cells, nutrient ions fulfill specific roles in biochemical reactions, especially photosynthesis housed in the chloroplast. Here, a well-balanced ion homeostasis is maintained by a number of ion transport proteins embedded in the envelope and thylakoid membranes. Ten years ago, the first alkali metal transporters from the K+ EFFLUX ANTIPORTER family were discovered in the model plant Arabidopsis. Since then, our knowledge about the physiological importance of these carriers and their substrates has greatly expanded. New insights into the role of alkali ions in plastid gene expression and photoprotective mechanisms, both prerequisites for plant productivity in natural environments, were gained. The discovery of a Cl− channel in the thylakoid and several additional plastid alkali and alkali metal transport proteins have advanced the field further. Nevertheless, scientists still have long ways to go before a complete systemic understanding of the chloroplast's ion transportome will emerge. In this Tansley review, we highlight and discuss the achievements of the last decade. More importantly, we make recommendations on what areas to prioritize, so the field can reach the next milestones. One area, laid bare by our similarity-based comparisons among phototrophs is our lack of knowledge what ion transporters are used by cyanobacteria to buffer photosynthesis fluctuations

Data from: High-throughput genotyping of green algal mutants reveals random distribution of mutagenic insertion sites and endonucleolytic cleavage of transforming DNA

A high-throughput genetic screening platform in a single-celled photosynthetic eukaryote would be a transformative addition to the plant biology toolbox. Here, we present ChlaMmeSeq (Chlamydomonas MmeI-based insertion site Sequencing), a tool for simultaneous mapping of tens of thousands of mutagenic insertion sites in the eukaryotic unicellular green alga Chlamydomonas reinhardtii. We first validated ChlaMmeSeq by in-depth characterization of individual insertion sites. We then applied ChlaMmeSeq to a mutant pool and mapped 11,478 insertions, covering 39% of annotated protein coding genes. We observe that insertions are distributed in a manner largely indistinguishable from random, indicating that mutants in nearly all genes can be obtained efficiently. The data reveal that sequence-specific endonucleolytic activities cleave the transforming DNA and allow us to propose a simple model to explain the origin of the poorly understood exogenous sequences that sometimes surround insertion sites. ChlaMmeSeq is quantitatively reproducible, enabling its use for pooled enrichment screens and for the generation of indexed mutant libraries. Additionally, ChlaMmeSeq allows genotyping of hits from Chlamydomonas screens on an unprecedented scale, opening the door to comprehensive identification of genes with roles in photosynthesis, algal lipid metabolism, the algal carbon-concentrating mechanism, phototaxis, the biogenesis and function of cilia, and other processes for which C. reinhardtii is a leading model system