Role of Sulfate Transporters in Chromium Tolerance in Scenedesmus acutus M. (Sphaeropleales)

Sulfur (S) is essential for the synthesis of important defense compounds and in the scavenging potential of oxidative stress, conferring increased capacity to cope with biotic and abiotic stresses. Chromate can induce a sort of S-starvation by competing for uptake with SO42− and causing a depletion of cellular reduced compounds, thus emphasizing the role of S-transporters in heavy-metal tolerance. In this work we analyzed the sulfate transporter system in the freshwater green algae Scenedesmus acutus, that proved to possess both H+/SO42− (SULTRs) and Na+/SO42− (SLTs) plasma membrane sulfate transporters and a chloroplast-envelope localized ABC-type holocomplex. We discuss the sulfate uptake system of S. acutus in comparison with other taxa, enlightening differences among the clade Sphaeropleales and Volvocales/Chlamydomonadales. To define the role of S transporters in chromium tolerance, we analyzed the expression of SULTRs and SULPs components of the chloroplast ABC transporter in two strains of S. acutus with different Cr(VI) sensitivity. Their differential expression in response to Cr(VI) exposure and S availability seems directly linked to Cr(VI) tolerance, confirming the role of sulfate uptake/assimilation pathways in the metal stress response. The SULTRs up-regulation, observed in both strains after S-starvation, may directly contribute to enhancing Cr-tolerance by limiting Cr(VI) uptake and increasing sulfur availability for the synthesis of sulfur-containing defense molecules.

Chloroplast-localized translation for protein targeting in Chlamydomonas reinhardtii

Translation is localized within cells to target proteins to their proper locations. We asked whether translation occurs on the chloroplast surface in Chlamydomonas and, if so, whether it is involved in co-translational protein targeting, aligned spatially with localized translation by the bacterial-type ribosomes within this organelle, or both. Our results reveal a domain of the chloroplast envelope which is bound by translating ribosomes. Purified chloroplasts retained ribosomes and mRNAs encoding two chloroplast proteins specifically on this translation domain, but not a mRNA encoding a cytoplasmic protein. Ribosomes clusters were seen on this domain by electron tomography. Activity of the chloroplast-bound ribosomes is supported by results of the ribopuromycylation and puromycin-release assays. Co-translational chloroplast protein import is supported by nascent polypeptide dependency of the ribosome-chloroplast associations. This cytoplasmic translation domain aligns localized translation by organellar bacterial-type ribosomes in the chloroplast. This juxtaposition the dual translation systems facilitates the targeting and assembly of the polypeptide products.

Residue 49 of AtMinD1 Plays a Key Role in the Guidance of Chloroplast Division by Regulating the ARC6-AtMinD1 Interaction

Chloroplasts evolved from a free-living cyanobacterium through endosymbiosis. Similar to bacterial cell division, chloroplasts replicate by binary fission, which is controlled by the Minicell (Min) system through confining FtsZ ring formation at the mid-chloroplast division site. MinD, one of the most important members of the Min system, regulates the placement of the division site in plants and works cooperatively with MinE, ARC3, and MCD1. The loss of MinD function results in the asymmetric division of chloroplasts. In this study, we isolated one large dumbbell-shaped and asymmetric division chloroplast Arabidopsis mutant Chloroplast Division Mutant 75 (cdm75) that contains a missense mutation, changing the arginine at residue 49 to a histidine (R49H), and this mutant point is located in the N-terminal Conserved Terrestrial Sequence (NCTS) motif of AtMinD1, which is only typically found in terrestrial plants. This study provides sufficient evidence to prove that residues 1–49 of AtMinD1 are transferred into the chloroplast, and that the R49H mutation does not affect the function of the AtMinD1 chloroplast transit peptide. Subsequently, we showed that the point mutation of R49H could remove the punctate structure caused by residues 1–62 of the AtMinD1 sequence in the chloroplast, suggesting that the arginine in residue 49 (Arg49) is essential for localizing the punctate structure of AtMinD11–62 on the chloroplast envelope. Unexpectedly, we found that AtMinD1 could interact directly with ARC6, and that the R49H mutation could prevent not only the previously observed interaction between AtMinD1 and MCD1 but also the interaction between AtMinD1 and ARC6. Thus, we believe that these results show that the AtMinD1 NCTS motif is required for their protein interaction. Collectively, our results show that AtMinD1 can guide the placement of the division site to the mid chloroplast through its direct interaction with ARC6 and reveal the important role of AtMinD1 in regulating the AtMinD1-ARC6 interaction.

Dynamic Cell Imaging: application to the diatom Phaeodactylum tricornutum under environmental stresses

The dynamic movement of cell organelles is an important and poorly understood component of cellular organisation and metabolism. In this work we present a non-invasive non-destructive method (Dynamic Cell Imaging, DCI) based on light scattering and interferometry to monitor dynamic events within photosynthetic cells using the diatom Phaeodactylum tricornutum as a model system. For this monitoring we acquire few seconds movies of the signals that are related to the motion of dynamic structures within the cell (denoted scatterers), followed by a statistical analysis of each pixel time series. Illuminating P.tricornutum with LEDs of different wavelengths associated to short pulsed or continuous-wave modes of illumination revealed that dynamic movements depend on chloroplast activity, in agreement with the reduction in the number of pixels with dynamic behaviour after addition of photosystemII inhibitors. We studied P. tricornutum under two environmentally relevant stresses, iron and phosphate deficiency. The major dynamic sites were located within lipid droplets and chloroplast envelope membranes. By comparing standard deviation and cumulative sum analysis of the time series, we showed that within the droplets two types of scatterer movement could be observed: random motions (Brownian type) but also anomalous movements corresponding to a drift which may relate to molecular fluxes within a cell. The method appears valuable for studying the effects of various environments on a large variety of microalgae in the laboratory as well as in natural aquatic environments.

Comparative Proteomic Analysis on Chloroplast Proteins Provides New Insights Into the Effects of Low Temperature in Sugar Beet

Background: Low temperature, which is one of the main environmental factors that limits geographical distribution and sucrose yield, is a common abiotic stress during the growth and development of sugar beet. As a regulatory hub of plant response to abiotic stress, activity in the chloroplasts is related to many molecular and physiological processes, particularly in response to low temperature stress. Results: The contents of chlorophyll (Chl) and malondialdehyde (MDA), relative electrical conductivity (REL), and superoxide dismutase (SOD) activity were measured. The results showed that sugar beet could manage low temperature stress by regulating the levels of Chl, REL and MDA, and the activity of SOD. The physiological responses indicated that sugar beets respond positively to low temperature treatments and are not significantly damaged. Moreover, to determine the precise time to response low temperature in sugar beet, well-known abiotic stresses-responsive transcript factor family, namely DEHYDRATION RESPONSIVE ELEMENT BINDING PROTEIN (DREB), was selected as the marker gene. The results of phylogenetic analyses showed that BvDREBA1 and BvDREBA4 were in the same branch as the cold- and drought-responsive AtDREB gene. In addition, the expression of BvDREBs reached its maximum level at 24 h after low temperature by RNA-Seq and qRT-PCR analysis. Furthermore, the changes in chloroplast proteome after low temperature at 24 h were detected using a label-free technique. A total of 416 differentially expressed proteins were identified. GO enrichment analysis showed that 16 GO terms were significantly enriched, particularly chloroplast stroma, chloroplast envelope, and chloroplast thylakoid membrane. It is notable that the transport of photosynthetic proteins (BvLTD, BvTOC100, and Toc-Tic complex), the formation of starch granules (BvPU1, BvISA3, and BvGWD3) and the scavenging of reactive oxygen species (BvCu/Zn-SOD, BvCAT, BvPrx, and BvTrx) were the pathways used by sugar beets to respond to low temperatures at an early stage.Conclusions: These results provide a preliminarily analysis of how chloroplasts of sugar beet respond to low temperature stress at the translational level and provide a theoretical basis for breeding low temperature resistant varieties of sugar beet.

Two plastid POLLUX ion channel-like proteins are required for stress-triggered stromal Ca2+ release

Two decades ago, large cation currents were discovered in the envelope membranes of Pisum sativum L. (pea) chloroplasts. The deduced K+-permeable channel was coined fast-activating chloroplast cation (FACC) channel but its molecular identity remained elusive. To reveal candidates, we mined proteomic datasets of isolated pea envelopes. Our search uncovered distant members of the nuclear POLLUX ion channel family. Since pea is not amenable to molecular genetics, we used Arabidopsis thaliana to characterize the two gene homologs. Using several independent approaches, we show that both candidates localize to the chloroplast envelope membrane. The proteins, designated PLASTID ENVELOPE ION CHANNELS (PEC1/2), form oligomers with regulator of K+ conductance (RCK) domains protruding into the intermembrane space. Heterologous expression of PEC1/2 rescues yeast mutants deficient in K+ uptake. Nuclear POLLUX ion channels cofunction with Ca2+ channels to generate Ca2+ signals, critical for establishing mycorrhizal symbiosis and root development. Chloroplasts also exhibit Ca2+ transients in the stroma, probably to relay abiotic and biotic cues between plastids and the nucleus via the cytosol. Our results show that pec1pec2 loss-of-function double mutants fail to trigger the characteristic stromal Ca2+ release observed in wild-type plants exposed to external stress stimuli. Besides this molecular abnormality, pec1pec2 double mutants do not show obvious phenotypes. Future studies of PEC proteins will help to decipher the plant’s stress-related Ca2+ signaling network and the role of plastids. More importantly, the discovery of PECs in the envelope membrane is another critical step towards completing the chloroplast ion transport protein inventory.

Supraoptimal Iron Nutrition of Brassica napus Plants Suppresses the Iron Uptake of Chloroplasts by Down-Regulating Chloroplast Ferric Chelate Reductase

Iron (Fe) is an essential micronutrient for plants. Due to the requirement for Fe of the photosynthetic apparatus, the majority of shoot Fe content is localised in the chloroplasts of mesophyll cells. The reduction-based mechanism has prime importance in the Fe uptake of chloroplasts operated by Ferric Reductase Oxidase 7 (FRO7) in the inner chloroplast envelope membrane. Orthologue of Arabidopsis thaliana FRO7 was identified in the Brassica napus genome. GFP-tagged construct of BnFRO7 showed integration to the chloroplast. The time-scale expression pattern of BnFRO7 was studied under three different conditions: deficient, optimal, and supraoptimal Fe nutrition in both leaves developed before and during the treatments. Although Fe deficiency has not increased BnFRO7 expression, the slight overload in the Fe nutrition of the plants induced significant alterations in both the pattern and extent of its expression leading to the transcript level suppression. The Fe uptake of isolated chloroplasts decreased under both Fe deficiency and supraoptimal Fe nutrition. Since the enzymatic characteristics of the ferric chelate reductase (FCR) activity of purified chloroplast inner envelope membranes showed a significant loss for the substrate affinity with an unchanged saturation rate, protein level regulation mechanisms are suggested to be also involved in the suppression of the reduction-based Fe uptake of chloroplasts together with the saturation of the requirement for Fe.

Visualizing the Integrity of Chloroplast Envelope by Rhodamine and Nile Red Staining

Chloroplasts are essential organelles in plant cells with many important functions. Chloroplasts isolated by Percoll density gradient centrifugation are widely used in the study of chloroplasts. The intactness of isolated chloroplasts is necessary for many of the experiments. In the past, those isolated chloroplasts were either simply believed to be intact or had to be analyzed by indirect biochemical methods. Here we show a new method to check the intactness of isolated chloroplasts by staining their envelope with fluorescent dyes, Rhodamine or Nile red, and then observing them with a fluorescence microscope. With this method, broken chloroplasts and intact chloroplasts can be distinguished easily and their integrity can be checked in a few minutes. Results of this method agreed well with those of biochemical methods. Moreover, we have also found that sometimes the middle layer chloroplasts from the Percoll gradient centrifugation could be mostly broken, which could cause mistakes in the experiment. With our method, this problem can be easily found. This chloroplast envelope staining method can be used in the preparation of isolated chloroplasts to ensure the intactness.

A Lipid Bodies-Associated Galactosyl Hydrolase Is Involved in Triacylglycerol Biosynthesis and Galactolipid Turnover in the Unicellular Green Alga Chlamydomonas reinhardtii

Monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) are the main constituent lipids of thylakoid and chloroplast envelop membranes. Many microalgae can accumulate large amounts of triacylglycerols (TAGs) under adverse environmental conditions, which is accompanied by degradation of the photosynthetic membrane lipids. However, the process mediating the conversion from galactolipids to TAG remains largely unknown. In this study, we performed genetic and biochemical analyses of galactosyl hydrolases (CrGH) identified in the proteome of lipid bodies of the green microalga Chlamydomonas reinhardtii. The recombinant CrGH was confirmed to possess galactosyl hydrolase activity by using o-nitrophenyl-β-D-galactoside as the substrate, and the Michaelis constant (Km) and Kcat of CrGH were 13.98 μM and 3.62 s−1, respectively. Comparative lipidomic analyses showed that the content of MGDG and DGDG increased by 14.42% and 24.88%, respectively, in the CrGH-deficient mutant as compared with that of the wild type cc4533 grown under high light stress conditions, and meanwhile, the TAG content decreased by 32.20%. Up-regulation of CrGH at both a gene expression and protein level was observed under high light stress (HL) conditions. In addition, CrGH was detected in multiple subcellular localizations, including the chloroplast envelope, mitochondria, and endoplasmic reticulum membranes. This study uncovered a new paradigm mediated by the multi-localized CrGH for the conversion of the photosynthetic membranes to TAGs.

Two chloroplastic PLGG1 isoforms function together to transport photorespiratory glycolate and glycerate in rice

The photorespiratory pathway is highly compartmentalized. As such, metabolite shuttles between organelles are critical to ensure efficient photorespiratory carbon flux. Arabidopsis PLGG1 has been reported as a key chloroplastic glycolate/glycerate transporter. Two homologous genes OsPLGG1a and OsPLGG1b have been identified in the rice genome, although their distinct functions and relationships remain unknown. Herein, our analysis of exogenous expression in oocytes and yeast shows that both OsPLGG1a and OsPLGG1b have the ability to transport glycolate and glycerate. Furthermore, we demonstrate in planta, that the perturbation of OsPLGG1a or OsPLGG1b expression leads to extensive accumulation of photorespiratory metabolites, especially glycolate and glycerate. Under ambient CO2 conditions, loss-of-function osplgg1a or osplgg1b mutant plants exhibited significant decreases in photosynthesis efficiency, starch accumulation, plant height, and crop productivity. These morphological defects were almost entirely recovered when the mutant plants were grown under elevated CO2 conditions instead. In contrast to osplgg1a, osplgg1b mutant alleles produced a mild photorespiratory phenotype and had reduced accumulation of photorespiratory metabolites. Subcellular localization analysis showed that OsPLGG1a and OsPLGG1b are located in the inner and outer membranes of the chloroplast envelope, respectively. In vitro and in vivo experiments revealed that OsPLGG1a and OsPLGG1b have a direct interaction. Our results indicate that both OsPLGG1a and OsPLGG1b are chloroplastic glycolate/glycerate transporters required for photorespiratory metabolism and plant growth, and that they may function as a singular complex.