CV-containing vesicle budding from chloroplast inner envelope membrane is mediated by clathrin but inhibited by GAPC

Clathrin-mediated vesicular formation and trafficking are highly conserved in eukaryotic cells and are responsible for molecular cargo transport and signal transduction among organelles. It remains largely unknown whether clathrin-coated vesicles can be generated from chloroplasts. CHLOROPLAST VESICULATION (CV)-containing vesicles (CVVs) generate from chloroplasts and mediate chloroplast degradation under abiotic stress. In this study, we showed that CV interacted with the clathrin heavy chain (CHC) and induced vesicle budding from the chloroplast inner envelope membrane. Defects on CHC2 and the dynamin-encoding DRP1A gene affected CVV budding and releasing from chloroplast. CHC2 is also required for CV-induced chloroplast degradation and hypersensitivity to water stress. Moreover, GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE (GAPC) interacts with CV and impairs the CV-CHC2 interaction. GAPC1 overexpression inhibited CV-mediated chloroplast degradation and hypersensitivity to water stress. CV silencing alleviated the hypersensitivity of gapc1gapc2 plant to water stress. Together, our work revealed a pathway of clathrin-assisted CVV budding from the chloroplast inner envelope membrane, which mediated the stress-induced chloroplast degradation and stress response.

Arabidopsis ORANGE protein regulates plastid pre-protein import through interacting with Tic proteins

Chloroplast-targeted proteins are actively imported into chloroplasts via the machinery spanning the double-layered membranes of chloroplasts. While the key translocons at the outer (TOC) and inner (TIC) membranes of chloroplasts are defined, proteins that interact with the core components to facilitate pre-protein import are continuously being discovered. A DnaJ-like chaperone ORANGE (OR) protein is known to regulate carotenoid biosynthesis as well as plastid biogenesis and development. In this study, we found that OR physically interacts with several Tic proteins including Tic20, Tic40, and Tic110 in the classic TIC core complex of the chloroplast import machinery. Knocking out or and its homolog or-like greatly affects the import efficiency of some photosynthetic and non-photosynthetic pre-proteins. Consistent with the direct interactions of OR with Tic proteins, the binding efficiency assay revealed that the effect of OR occurs at translocation at the inner envelope membrane (i.e. at the TIC complex). OR is able to reduce the Tic40 protein turnover rate through its chaperone activity. Moreover, OR was found to interfere with the interaction between Tic40 and Tic110, and reduces the binding of pre-proteins to Tic110 in aiding their release for translocation and processing. Our findings suggest that OR plays a new and regulatory role in stabilizing key translocons and in facilitating the late stage of plastid pre-protein translocation to regulate plastid pre-protein import.

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.

Electron Tomography Analysis of Thylakoid Assembly and Fission in Chloroplasts of a Single-Cell C4 plant, Bienertia sinuspersici

Bienertia sinuspersici is a single-cell C4 plant species of which chlorenchyma cells have two distinct groups of chloroplasts spatially segregated in the cytoplasm. The central vacuole encloses most chloroplasts at the cell center and confines the rest of the chloroplasts near the plasma membrane. Young chlorenchyma cells, however, do not have large vacuoles and their chloroplasts are homogenous. Therefore, maturing Bienertia chlorenchyma cells provide a unique opportunity to investigate chloroplast proliferation in the central cluster and the remodeling of chloroplasts that have been displaced by the vacuole to the cell periphery. Chloroplast numbers and sizes increased, more notably, during later stages of maturation than the early stages. Electron tomography analyses indicated that chloroplast enlargement is sustained by thylakoid growth and that invaginations from the inner envelope membrane contributed to thylakoid assembly. Grana stacks acquired more layers, differentiating them from stroma thylakoids as central chloroplasts matured. In peripheral chloroplasts, however, grana stacks stretched out to a degree that the distinction between grana stacks and stroma thylakoids was obscured. In central chloroplasts undergoing division, thylakoids inside the cleavage furrow were kinked and severed. Grana stacks in the division zone were disrupted, and large complexes in their membranes were dislocated, suggesting the existence of a thylakoid fission machinery.

Search for high-mass protostars with ALMA revealed up to kilo-parsec scales (SPARKS)

Context. Classical hot cores are rich in molecular emission, and they show a high abundance of complex organic molecules (COMs). The emergence of molecular complexity that is represented by COMs, in particular, is poorly constrained in the early evolution of hot cores. Aims. We put observational constraints on the physical location of COMs in a resolved high-mass protostellar envelope associated with the G328.2551−0.5321 clump. The protostar is single down to ~400 au scales and we resolved the envelope structure down to this scale. Methods. High angular resolution observations using the Atacama Large Millimeter Array allowed us to resolve the structure of the inner envelope and pin down the emission region of COMs. We use local thermodynamic equilibrium modelling of the available 7.5 GHz bandwidth around ~345 GHz to identify the COMs towards two accretion shocks and a selected position representing the bulk emission of the inner envelope. We quantitatively discuss the derived molecular column densities and abundances towards these positions, and use our line identification to qualitatively compare this to the emission of COMs seen towards the central position, corresponding to the protostar and its accretion disk. Results. We detect emission from 10 COMs, and identify a line of deuterated water (HDO). In addition to methanol (CH3OH), methyl formate (CH3OCHO) and formamide (HC(O)NH2) have the most extended emission. Together with HDO, these molecules are found to be associated with both the accretion shocks and the inner envelope, which has a moderate temperature of Tkin ~ 110 K. We find a significant difference in the distribution of COMs. O-bearing COMs, such as ethanol, acetone, and ethylene glycol are almost exclusively found and show a higher abundance towards the accretion shocks with Tkin ~ 180 K. Whereas N-bearing COMs with a CN group, such as vinyl and ethyl cyanide peak on the central position, thus the protostar and the accretion disk. The molecular composition is similar towards the two shock positions, while it is significantly different towards the inner envelope, suggesting an increase in abundance of O-bearing COMs towards the accretion shocks. Conclusions. We present the first observational evidence for a large column density of COMs seen towards accretion shocks at the centrifugal barrier at the inner envelope. The overall molecular emission shows increased molecular abundances of COMs towards the accretion shocks compared to the inner envelope. The bulk of the gas from the inner envelope is still at a moderate temperature of Tkin ~ 110 K, and we find that the radiatively heated inner region is very compact (<1000 au). Since the molecular composition is dominated by that of the accretion shocks and the radiatively heated hot inner region is very compact, we propose this source to be a precursor to a classical, radiatively heated hot core. By imaging the physical location of HDO, we find that it is consistent with an origin within the moderately heated inner envelope, suggesting that it originates from sublimation of ice from the grain surface and its destruction in the vicinity of the heating source has not been efficient yet.

Low dust emissivities and radial variations in the envelopes of Class 0 protostars: possible signature of early grain growth

Context. Analyzing the properties of dust and its evolution in the early phases of star formation is crucial to put constraints on the collapse and accretion processes as well as on the pristine properties of planet-forming seeds. Aims. In this paper, we aim to investigate the variations of the dust grain size in the envelopes of the youngest protostars. Methods. We analyzed Plateau de Bure interferometric observations at 1.3 and 3.2 mm for 12 Class 0 protostars obtained as part of the CALYPSO survey. We performed our analysis in the visibility domain and derived dust emissivity index (β1−3mm) profiles as a function of the envelope radius at 200–2000 au scales. Results. Most of the protostellar envelopes show low dust emissivity indices decreasing toward the central regions. The decreasing trend remains after correction of the (potentially optically thick) central region emission, with surprisingly low β1−3mm < 1 values across most of the envelope radii of NGC 1333-IRAS 4A, NGC 1333-IRAS 4B, SVS13B, and Serpens-SMM4. Conclusions. We discuss the various processes that could explain such low and varying dust emissivity indices at envelope radii 200–2000 au. Our observations of extremely low dust emissivity indices could trace the presence of large (millimeter-size) grains in Class 0 envelopes, in which case our results would point to a radial increase of the dust grain size toward the inner envelope regions. While it is expected that large grains in young protostellar envelopes could be built via grain growth and coagulation, we stress that the typical timescales required to build millimeter grains in current coagulation models are at odds with the youth of our Class 0 protostars. Additional variations in the dust composition could also partly contribute to the low β1−3mm we observe. We find that the steepness of the β1−3mm radial gradient depends strongly on the envelope mass, which might favor a scenario in which large grains are built in high-density protostellar disks and transported to the intermediate envelope radii, for example with the help of outflows and winds.