Chloroplast Aggregation Research Articles

Acclimation Strategies for the Black Sea Diatom Algae Ditylum brightwellii to High Intensity of Light

In cells of a culture of the large diatom Ditylum brightwellii (T. West) Grunow acclimated to weak light (17 μmol photons/(m2 × s)), numerous chloroplasts were evenly distributed throughout the cell cytoplasm. After 10 min of exposure of algae to extremely high illumination (1100 μmol photons/(m2 × s)), their aggregates gradually formed in the center of the cell, which continued until the end of the two-hour exposure period. At light intensities of 510–935 µmol/(photons/(m2 × s) during short-term photoacclimation, chloroplast aggregation was noted for 20–60 min, after which their reverse movement and uniform distribution in the cytoplasm were revealed by the end of the second hour. Under conditions of a longer culture stay at a light intensity of 1100 μmol photons/(m2 × s), the algae retained their viability for only six hours. Long-term photoacclimation of this species, which ended by the end of the second day, was detected when the light weakened by about 2 times. It was expressed as an increase in cell volume and C/Chl a ratio, increased aggregation of chloroplasts in the center of the cell, and a decrease in a number of fluorescent parameters reflecting the efficiency of photosystem II and culture viability.

A kinesin-like protein, KAC, is required for light-induced and actin-based chloroplast movement in Marchantia polymorpha.

Chloroplasts accumulate on the cell surface under weak light conditions to efficiently capture light but avoid strong light to minimize photodamage. The blue light receptor phototropin regulates the chloroplast movement in various plant species. In Arabidopsis thaliana, phototropin mediates the light-induced chloroplast movement and positioning via specialized actin filaments on the chloroplasts, chloroplast-actin filaments. KINESIN-LIKE PROTEIN FOR ACTIN-BASED CHLOROPLAST MOVEMENT (KAC) and CHLOROPLAST UNUSUAL POSITIONING 1 (CHUP1) are pivotal for chloroplast-actin-based chloroplast movement and positioning in land plants. However, the mechanisms by which KAC and CHUP1 regulate chloroplast movement and positioning remain unclear. In this study, we characterized KAC and CHUP1 orthologs in the liverwort Marchantia polymorpha, MpKAC and MpCHUP1, respectively. Their knockout mutants, Mpkack° and Mpchup1k°, impaired the light-induced chloroplast movement. Although Mpchup1k° showed mild chloroplast aggregation, Mpkack° displayed severe chloroplast aggregation, suggesting the greater contribution of MpKAC to the chloroplast anchorage to the plasma membrane. Analysis of the subcellular localization of the functional MpKAC-Citrine indicated that MpKAC-Citrine formed a punctate structure on the plasma membrane. Structure-function analysis of MpKAC revealed that a deletion of the conserved C-terminal domain abrogates the targeting to the plasma membrane and its function. A deletion of the N-terminal motor domain retained the plasma membrane targeting but abrogates the formation of punctate structure and showed severe defect in the light-induced chloroplast movement. Our findings suggest that the formation of the punctate structure on the plasma membrane of MpKAC is essential for chloroplast movement.

Singlet oxygen triggers chloroplast rupture and cell death in the zeaxanthin epoxidase defective mutant aba1 of Arabidopsis thaliana under high light stress

The two Arabidopsis thaliana mutants, aba1 and max4, were previously identified as sharing a number of co-regulated genes with both the flu mutant and Arabidopsis cell suspension cultures exposed to high light (HL). On this basis, we investigated whether aba1 and max4 were generating high amounts of singlet oxygen (1O2) and activating 1O2-mediated cell death. Thylakoids of aba1 produced twice as much 1O2 as thylakoids of max4 and wild type (WT) plants when illuminated with strong red light. 1O2 was measured using the spin probe 2,2,6,6-tetramethyl-4-piperidone hydrochloride. 77-K chlorophyll fluorescence emission spectra of thylakoids revealed lower aggregation of the light harvesting complex II in aba1. This was rationalized as a loss of connectivity between photosystem II (PSII) units and as the main cause for the high yield of 1O2 generation in aba1. Up-regulation of the 1O2 responsive gene AAA-ATPase was only observed with statistical significant in aba1 under HL. Two early jasmonate (JA)-responsive genes, JAZ1 and JAZ5, encoding for two repressor proteins involved in the negative feedback regulation of JA signalling, were not up-regulated to the WT plant levels. Chloroplast aggregation followed by chloroplast rupture and eventual cell death was observed by confocal imaging of the fluorescence emission of leaf cells of transgenic aba1 plants expressing the chimeric fusion protein SSU-GFP. Cell death was not associated with direct 1O2 cytotoxicity in aba1, but rather with a delayed stress response. In contrast, max4 did not show evidence of 1O2-mediated cell death. In conclusion, aba1 may serve as an alternative model to other 1O2-overproducing mutants of Arabidopsis for investigating 1O2-mediated cell death.

Chloroplast aggregation during the cold-positioning response in the liverwort Marchantia polymorpha.

Under low-light conditions, chloroplasts localize along periclinal cell walls at temperatures near 20 °C, but they localize along anticlinal cell walls near 5 °C. This phenomenon is known as the cold-positioning response. We previously showed that chloroplasts move as aggregates rather than individually during the cold-positioning response in the fern Adiantum capillus-veneris. This observation suggested that chloroplasts physically interact with each other during the cold-positioning response. However, the physiological processes underlying chloroplast aggregation are unclear. In this report, we characterized chloroplast aggregation during the cold-positioning response in the liverwort Marchantia polymorpha. Confocal laser microscopy observations of transgenic liverwort plants expressing a fluorescent fusion protein that localizes to the chloroplast outer envelope membrane (OEP7-Citrine) showed that neighboring chloroplast membranes did not fuse during the cold-positioning response. Transmission electron microscopy analysis revealed that a distance of at least 10nm was maintained between neighboring chloroplasts during aggregation. These results indicate that aggregated chloroplasts do not fuse, but maintain a distance of at least 10nm from each other during the cold-positioning response.

The SNARE Protein Syp71 Is Essential for Turnip Mosaic Virus Infection by Mediating Fusion of Virus-Induced Vesicles with Chloroplasts

All positive-strand RNA viruses induce the biogenesis of cytoplasmic membrane-bound virus factories for viral genome multiplication. We have previously demonstrated that upon plant potyvirus infection, the potyviral 6K2 integral membrane protein induces the formation of ER-derived replication vesicles that subsequently target chloroplasts for robust genome replication. Here, we report that following the trafficking of the Turnip mosaic potyvirus (TuMV) 6K2 vesicles to chloroplasts, 6K2 vesicles accumulate at the chloroplasts to form chloroplast-bound elongated tubular structures followed by chloroplast aggregation. A functional actomyosin motility system is required for this process. As vesicle trafficking and fusion in planta are facilitated by a superfamily of proteins known as SNAREs (soluble N-ethylmaleimide-sensitive-factor attachment protein receptors), we screened ER-localized SNARES or SNARE-like proteins for their possible involvement in TuMV infection. We identified Syp71 and Vap27-1 that colocalize with the chloroplast-bound 6K2 complex. Knockdown of their expression using a Tobacco rattle virus (TRV)-based virus-induced gene silencing vector showed that Syp71 but not Vap27-1 is essential for TuMV infection. In Syp71-downregulated plant cells, the formation of 6K2-induced chloroplast-bound elongated tubular structures and chloroplast aggregates is inhibited and virus accumulation is significantly reduced, but the trafficking of the 6K2 vesicles from the ER to chloroplast is not affected. Taken together, these data suggest that Syp71 is a host factor essential for successful virus infection by mediating the fusion of the virus-induced vesicles with chloroplasts during TuMV infection.

Heterologous expression of a chloroplast outer envelope protein fromSuaeda salsaconfers oxidative stress tolerance and induces chloroplast aggregation in transgenic Arabidopsis plants

Suaeda salsa is a euhalophytic plant that is tolerant to coastal seawater salinity. In this study, we cloned a cDNA encoding an 8.4 kDa chloroplast outer envelope protein (designated as SsOEP8) from S. salsa and characterized its cellular function. Steady-state transcript levels of SsOEP8 in S. salsa were up-regulated in response to oxidative stress. Consistently, ectopic expression of SsOEP8 conferred enhanced oxidative stress tolerance in transgenic Bright Yellow 2 (BY-2) cells and Arabidopsis, in which H(2) O(2) content was reduced significantly in leaf cells. Further studies revealed that chloroplasts aggregated to the sides of mesophyll cells in transgenic Arabidopsis leaves, and this event was accompanied by inhibited expression of genes encoding proteins for chloroplast movements such as AtCHUP1, a protein involved in actin-based chloroplast positioning and movement. Moreover, organization of actin cytoskeleton was found to be altered in transgenic BY-2 cells. Together, these results suggest that SsOEP8 may play a critical role in oxidative stress tolerance by changing actin cytoskeleton-dependent chloroplast distribution, which may consequently lead to the suppressed production of reactive oxygen species (ROS) in chloroplasts. One significantly novel aspect of this study is the finding that the small chloroplast envelope protein is involved in oxidative stress tolerance.

Chloroplast Outer Envelope Protein CHUP1 Is Essential for Chloroplast Anchorage to the Plasma Membrane and Chloroplast Movement

Chloroplasts change their intracellular distribution in response to light intensity. Previously, we isolated the chloroplast unusual positioning1 (chup1) mutant of Arabidopsis (Arabidopsis thaliana). This mutant is defective in normal chloroplast relocation movement and shows aggregation of chloroplasts at the bottom of palisade mesophyll cells. The isolated gene encodes a protein with an actin-binding motif. Here, we used biochemical analyses to determine the subcellular localization of full-length CHUP1 on the chloroplast outer envelope. A CHUP1-green fluorescent protein (GFP) fusion, which was detected at the outermost part of mesophyll cell chloroplasts, complemented the chup1 phenotype, but GFP-CHUP1, which was localized mainly in the cytosol, did not. Overexpression of the N-terminal hydrophobic region (NtHR) of CHUP1 fused with GFP (NtHR-GFP) induced a chup1-like phenotype, indicating a dominant-negative effect on chloroplast relocation movement. A similar pattern was found in chloroplast OUTER ENVELOPE PROTEIN7 (OEP7)-GFP transformants, and a protein containing OEP7 in place of NtHR complemented the mutant phenotype. Physiological analyses of transgenic Arabidopsis plants expressing truncated CHUP1 in a chup1 mutant background and cytoskeletal inhibitor experiments showed that the coiled-coil region of CHUP1 anchors chloroplasts firmly on the plasma membrane, consistent with the localization of coiled-coil GFP on the plasma membrane. Thus, CHUP1 localization on chloroplasts, with the N terminus inserted into the chloroplast outer envelope and the C terminus facing the cytosol, is essential for CHUP1 function, and the coiled-coil region of CHUP1 prevents chloroplast aggregation and participates in chloroplast relocation movement.

The action spectrum for chloroplast movements and evidence for blue-light-photoreceptor cycling in the alga Vaucheria

Local stimulation of the coenocytic alga Vaucheria sessilis D.C. by blue light resulted in accumulation of chloroplasts and other organelles. The photoresponse followed a well-defined, wavelength-and fluence-rate-dependent latency period (≧10 s), and could lead to a tenfold decrease in relative cellular transmittance to 675-nm light within 5 min. Light-induced aggregation of chloroplasts was examined at eight wavelengths of light between 385 and 528 nm. A fiber-optic microphotometer was employed and the response was quantitated on the basis of the rate of 675-nm transmittance change after correcting for changes in light scattering. Chloroplast aggregation exhibited a nearly identical quantum-flux-density dependence at all eight wavelenths tested; it showed an action spectrum with a sharp maximum near 470 nm, a trough at 430 nm, and action in the near-ultraviolet spectral region. Light at 454 nm was six times less effective than 473-nm light in stimulating aggregation, a difference which could not be accounted for by chlorophyll screening alone. Beyond the latency period reciprocity did not hold for chloroplast aggregation. Instead, aggregation could be fitted to a kinetic model involving steady-state photoreceptor cycling during continuous irradiation. Chloroplast aggregation in the light was compared with three growth-associated photoresponses in Vaucheria - phototropic bending, branching and apical expansion. Time course and kinetic similarities, and the presence of a cytoplasmic fiber network in growing tips of Vaucheria, indicate that these photoresponses may be related mechanistically.

QUANTITATIVE MICROPHOTOMETRY AT THE CELLULAR LEVEL: A SIMPLE TECHNIQUE FOR MEASURING CHLOROPLAST MOVEMENTS IN VIVO*

Abstract— A recording microphotometer was developed to permit quantitative analysis of light‐induced chloroplast aggregation in single filaments of the alga Vaucheria. Optical fibers were used both to irradiate the cells and to collect the transmitted light. Light transmittance was examined as a function of time at a fixed point along an algal filament. The photoresponse was stimulated by combining an additional actinic beam with the measuring beams such that both actinic and measuring beams followed the same optical path. The quasi‐opalescent properties of the optical fibers and dual‐wavelength measurements compensated for light scattering and variations in sample geometry.Chloroplast aggregation, marked by a passive accumulation of organelles in the irradiated region of the cell, resulted in a decline of cell transmittance to 675 and 743 nm light. During stimulation with moderate intensities of actinic, 473 nm light the relative transmittance to 675 nm light (corrected for scattering, measured at 743 nm) fell below 0.1 within 5 min. The exponential form of this transmittance change as a function of time was consistent with changes in optical geometry of the aggregate during the photoresponse. It follows that the decay in algal transmittance can provide a measure of the rate of chloroplast accumulation in the light path.

A light-dependent current associated with chloroplast aggregation in the alga Vaucheria sessilis

Local irradiation of the alga Vaucheria sessilis (Vauch.) D.C. with blue light, which stimulates cortical fiber reticulation and chloroplast aggregation (M.R. Blatt and W.R. Briggs, 1980, Planta 147, 355-362), also induces an outward-directed current from the irradiated region of the cell. This current appears in conjunction with cortical fiber reticulation and precedes chloroplast aggregation. The current is not photosynthetic in origin, as indicated by experiments with 3(3,4-dichlorophenyl)-1,1-dimethyl urea and carbonyl-cyanide-m-chlorophenylhydrazone (CCCP). It shows a wavelength-dependence similar to that of chloroplast aggregation and reaches a maximum of 500 nA cm(-2) with saturating light intensities. The current is not dependent upon the presence of Na(+), K(+), or Cl(-) in a test medium containing only Na(+), K(+), Ca(2+), and Cl(-), but is inhibited, apparently nonspecifically, in the absence of external calcium. Both the light-induced current and chloroplast aggregation are stimulated by increases in the external KCl concentration and are inhibited by sub-micromolar concentrations of CCCP or by external pHs below approximately 5.5. We suggest that blue light stimulates the local extrusion of cations, possibly of protons, at the plasma membrane, an event which may act to destabilize the cortical fibers in Vaucheria, disrupt cytoplasmic streaming, and eventually lead to organelle aggregation in the light.

66] Preparation and use of protoplasts for studies of lipid metabolism

Abstract Spinach protoplasts are useful in lipid metabolism studies because they provide a homogeneous, differentiated plant tissue that can take up precursors directly from the medium. they are the material of choice for subcellular localization because they can be easily lysed and the intact organelles separated on density gradients. Crude fungal enzyme preparations are used to degrade the cell wall of the spinach leaves, which are stripped of their epidermal cells by brushing and sliced finely to increase the surface for enzyme action. Protoplasts are harvested from the digested leaf slices by filtration and washed and purified by repeated centrifugation. If protoplasts are to be used in metabolism studies, they are stabilized with a salt solution containing divalent cations and incubated in strong light with lipid or fatty acid precursors. For subcellular localization studies, salts are not present in the wash medium to reduce chloroplast aggregation in the gradients and protoplasts are lysed by shearing in a microemulsifying needle or passage through 20- and 10-μm mesh before sucrose density gradient centrifugation. Comparison with marker enzymes for organelles provides a method for the complete subcellular localization of an enzyme activity.

Blue-light-induced cortical fiber reticulation concomitant with chloroplast aggregation in the alga Vaucheria sessilis

Point illumination with low-intensity blue light induces the chloroplasts and other organelles, which normally stream in the cytoplasm of Vaucheria sessilis (Vauch.) D.C. (Xanthophyceae), to aggregate in the illuminated region of the cell. Aggregation is passive and results from the "trapping" of the organelles as they stream into the blue light. Prior to illumination, longitudinal fibers along which the organelles appear to move, can readily be seen through a light microscope fitted with differential interference contrast optics. Upon actinic irradiation, these fibers appear to become destabilized, branching and forming a cortical fiber reticulum in the light. The reticulation process always precedes chloroplast aggregation. Aggregation itself occurs after a lag period which is inversely related to fluence rate. The lag period at high fluence rates (>400 mW m(-2)) may be as short as 20 s. Studies of wavelength dependence show that wavelengths near 480 nm are maximally effective while those longer than 530 nm are inactive.