Chloroplast avoidance movement is not functional in plants grown under strong sunlight

Chloroplast movement in nine climbing plant species was investigated. It is thought that chloroplasts generally escape from strong light to avoid photodamage but accumulate towards weak light to perform photosynthesis effectively. Unexpectedly, however, the leaves of climbing plants grown under strong sunlight showed very low or no chloroplast photorelocation responses to either weak or strong blue light when detected by red light transmittance through leaves. Direct observations of Cayratia japonica leaves, for example, revealed that the average number of chloroplasts in upper periclinal walls of palisade tissue cells was only 1.2 after weak blue‐light irradiation and almost all of the chloroplasts remained at the anticlinal wall, the state of chloroplast avoidance response. The leaves grown under strong light have thin and columnar palisade tissue cells comparing with the leaves grown under low light. Depending on our analyses and our schematic model, the thinner cells in a unit leaf area have a wider total plasma membrane area, such that more chloroplasts can exist on the plasma membrane in the thinner cells than in the thicker cells in a unit leaf‐area basis. The same strategy might be used in other plant leaves grown under direct sunlight.     

Actin-based mechanisms for light-dependent intracellular positioning of nuclei and chloroplasts in Arabidopsis

The plant organelles, chloroplast and nucleus, change their position in response to light. In Arabidopsis thaliana leaf cells, chloroplasts and nuclei are distributed along the inner periclinal wall in darkness. In strong blue light, they become positioned along the anticlinal wall, while in weak blue light, only chloroplasts are accumulated along the inner and outer periclinal walls. Blue-light dependent positioning of both organelles is mediated by the blue-light receptor phototropin and controlled by the actin cytoskeleton. Interestingly, however, it seems that chloroplast movement requires short, fine actin filaments organized at the chloroplast edge, whereas nuclear movement does cytoplasmic, thick actin bundles intimately associated with the nucleus. Although there are many similarities between photo-relocation movements of chloroplasts and nuclei, plant cells appear to have evolved distinct mechanisms to regulate actin organization required for driving the movements of these organelles.Key words: actin, Arabidopsis, blue light, chloroplast positioning, phototropin, nuclear positioning     

Plastid movement impaired 2, a new gene involved in normal blue-light-induced chloroplast movements in Arabidopsis

Chloroplasts move in a light-dependent manner that can modulate the photosynthetic potential of plant cells. Identification of genes required for light-induced chloroplast movement is beginning to define the molecular machinery that controls these movements. In this work, we describe plastid movement impaired 2 (pmi2), a mutant in Arabidopsis (Arabidopsis thaliana) that displays attenuated chloroplast movements under intermediate and high light intensities while maintaining a normal movement response under low light intensities. In wild-type plants, fluence rates below 20 micromol m(-2) s(-1) of blue light lead to chloroplast accumulation on the periclinal cell walls, whereas light intensities over 20 micromol m(-2) s(-1) caused chloroplasts to move toward the anticlinal cell walls (avoidance response). However, at light intensities below 75 micromol m(-2) s(-1), chloroplasts in pmi2 leaves move to the periclinal walls; 100 micromol m(-2) s(-1) of blue light is required for chloroplasts in pmi2 to move to the anticlinal cell walls, indicating a shift in the light threshold for the avoidance response in the mutant. The pmi2 mutation has been mapped to a gene that encodes a protein of unknown function with a large coiled-coil domain in the N terminus and a putative P loop. PMI2 shares sequence and structural similarity with PMI15, another unknown protein in Arabidopsis that, when mutated, causes a defect in chloroplast avoidance under high-light intensities.     

Phototropins and neochrome1 mediate nuclear movement in the fern Adiantum capillus-veneris

In gametophytic cells (prothalli) of the fern Adiantum capillus-veneris, nuclei as well as chloroplasts change their position according to light conditions. Nuclei reside on anticlinal walls in darkness and move to periclinal or anticlinal walls under weak or strong light conditions, respectively. Here we reveal that red light-induced nuclear movement is mediated by neochrome1 (neo1), blue light-induced movement is redundantly mediated by neo1, phototropin2 (phot2) and possibly phot1, and dark positioning of both nuclei and chloroplasts is mediated by phot2. Thus, both the nuclear and chloroplast photorelocation movements share common photoreceptor systems.     

Photoacoustic analysis indicates that chloroplast movement does not alter liquid-phase CO2 diffusion in leaves of Alocasia brisbanensis

Light-mediated chloroplast movements are common in plants. When leaves of Alocasia brisbanensis (F.M. Bailey) Domin are exposed to dim light, mesophyll chloroplasts spread along the periclinal walls normal to the light, maximizing absorbance. Under high light, the chloroplasts move to anticlinal walls. It has been proposed that movement to the high-light position shortens the diffusion path for CO(2) from the intercellular air spaces to the chloroplasts, thus reducing CO(2) limitation of photosynthesis. To test this hypothesis, we used pulsed photoacoustics to measure oxygen diffusion times as a proxy for CO(2) diffusion in leaf cells. We found no evidence that chloroplast movement to the high-light position enhanced gas diffusion. Times for oxygen diffusion were not shorter in leaves pretreated with white light, which induced chloroplast movement to the high-light position, compared with leaves pretreated with 500 to 700 nm light, which did not induce movement. From the oxygen diffusion time and the diffusion distance from chloroplasts to the intercellular gas space, we calculated an oxygen permeability of 2.25 x 10(-)(6) cm(2) s(-)(1) for leaf cells at 20 degrees C. When leaf temperature was varied from 5 degrees C to 40 degrees C, the permeability for oxygen increased between 5 degrees C and 20 degrees C but changed little between 20 degrees C and 40 degrees C, indicating changes in viscosity or other physical parameters of leaf cells above 20 degrees C. Resistance for CO(2) estimated from oxygen permeability was in good agreement with published values, validating photoacoustics as another way of assessing internal resistances to CO(2) diffusion.     

Phototactic Chloroplast Displacement in the Photosynthetic Mutant, Lemna paucicostata Strain 1073

The photosynthetic mutant, strain 1073, of Lemna paucicostataTorr. (L. perpusilla Hegelm.) which has a block in the electrontransport chain between plastoquinone and cytochrome f is capableof light-induced chloroplast displacement movements. At 8000–14000 lx, chloroplasts of the mutant move from their positionadjacent to the inner periclinal wall of the mesophyll cellsto the anticlinal walls, i.e. along those walls parallel tothe direction of the light. Light does not appear to enhancerespiration of the photosynthetic mutant or of the wild typestrain (6746). These and other results support the idea thatchloroplast displacement in light is not solely the result oflight effects on photosynthesis and respiration. Lemna paucicostata Torr., photosynthetic mutant, phototaxis, chloroplast displacement     

Cold‐induced organelle relocation in the liverwort Marchantia polymorpha L.

Organelles change their subcellular positions in response to various environmental conditions. Recently, we reported that cold treatments alter the intracellular position of chloroplasts and nuclei (cold positioning) in the fern Adiantum capillus‐veneris; chloroplasts and nuclei localized to the periclinal cell wall relocated to anticlinal cell wall after cold treatments. To further understand organelle positioning under cold conditions, we studied cold‐induced organelle relocation in the liverwort Marchantia polymorpha L. When sporelings and gemmmalings were treated under low temperature (5 °C), chloroplast cold positioning response was successfully induced both in the sporelings and the gemmmalings of M. polymorpha. Using a genetic transformation, nuclei, mitochondria or peroxisomes were visualized with a fluorescent protein, and the transgenic gemmmalings were incubated under the cold condition. Nuclei and peroxisomes, but not mitochondria, clearly relocated from the periclinal cell wall to the anticlinal cell wall after cold treatments. Our findings suggest that several organelles concurrently change their positions in the liverwort cell to cope with cold temperature.     

Chloroplasts move towards the nearest anticlinal walls under dark condition

Chloroplasts change their intracellular positions in response to their light environment. Under darkness, chloroplasts assume special positions that are different from those under light conditions. Here, we analyzed chloroplast dark positioning using Adiantum capillus-veneris gametophyte cells. When chloroplasts were transferred into darkness, during the first 1–5 h, they moved towards the anticlinal cell walls bordering the adjacent cells rather rapidly. Then, they slowed down and accumulated at the anticlinal walls gradually over the following 24–36 h. The chloroplast movements could be roughly classified into two different categories: initial rapid straight movement and later, slow staggering movement. When the chloroplast accumulation response was induced in dark-adapted cells by partial cell irradiation with a microbeam targeted to the center of the cells, chloroplasts moved towards the beam spot from the anticlinal walls. However, when the microbeam was switched off, they moved to the nearest anticlinal walls and not to their original positions if they were not the closest, indicating that they know the direction of the nearest anticlinal wall and do not have particular areas that they migrate to during dark positioning.     

SPAD chlorophyll meter reading can be pronouncedly affected by chloroplast movement

Non-destructive assessment of chlorophyll content has recently been widely done by chlorophyll meters based on measurement of leaf transmittance (e.g. the SPAD-502 chlorophyll meter measures the leaf transmittance at 650 and 940 nm). However, the leaf transmittance depends not only on the content of chlorophylls but also on their distribution in leaves. The chlorophyll distribution within leaves is co-determined by chloroplast arrangement in cells that depends on light conditions. When tobacco leaves were exposed to a strong blue light (about 340 μmol of photons m⁻² s⁻¹), a very pronounced increase in the leaf transmittance was observed as chloroplasts migrated from face position (along cell walls perpendicular to the incident light) to side position (along cell walls parallel to the incoming light) and the SPAD reading decreased markedly. This effect was more pronounced in the leaves of young tobacco plants compared with old ones; the difference between SPAD values in face and side position reached even about 35%. It is shown how the chloroplast movement changes a relationship between the SPAD readings and real chlorophyll content. For an elimination of the chloroplast movement effect, it can be recommended to measure the SPAD values in leaves with a defined chloroplasts arrangement.     

Low temperature-induced chloroplast relocation mediated by a blue light receptor,phototropin 2, in fern gametophytes

Chloroplast movement in response to light has been known more than 100 years. Chloroplasts move towards weak light and move away from strong light. Dark-induced relocation, called dark positioning, has also been shown. However, the effects of other stimuli on chloroplast movement have not been well characterized. Here we studied low temperature-induced chloroplast relocation (termed cold positioning) in prothallial cells of the gametophytes of the fern Adiantum capillus-veneris. Under weak light chloroplasts in prothallial cells accumulated along the periclinal wall at 25 degrees C, but they moved towards anticlinal walls when the prothalli were subsequently transferred to 4 degrees C. A temperature shift from 25 degrees to 10 degrees C or lower was enough to induce cold positioning, and high-intensity light enhanced the response. Nuclei also relocated from the periclinal position (a position along periclinal walls) to the anticlinal position (a position along anticlinal walls) under cold temperature, whereas mitochondria did not. Cold positioning was not observed in mutant fern gametophytes defective of the blue light photoreceptor, phototropin 2.     

Light-dependent phosphorylation of THRUMIN1 regulates its association with actin filaments and 14-3-3 proteins

Light-dependent chloroplast movements in leaf cells contribute to the optimization of photosynthesis. Low-light conditions induce chloroplast accumulation along periclinal cell surfaces, providing greater access to available light, whereas high light induces movement of chloroplasts to anticlinal cell surfaces, providing photodamage protection and allowing more light to reach underlying cell layers. The THRUMIN1 protein is required for normal chloroplast movements in Arabidopsis (Arabidopsis thaliana) and has been shown to localize at the plasma membrane and to undergo rapid light-dependent interactions with actin filaments through the N-terminal intrinsically disordered region (IDR). A predicted WASP-Homology 2 domain was found in the IDR but mutations in this domain did not disrupt localization of THRUMIN1:YFP to actin filaments. A series of other protein truncations and site-directed mutations of known and putative phosphorylation sites indicated that a phosphomimetic mutation (serine to aspartic acid) at position 170 disrupted localization of THRUMIN1 to actin filaments. However, the phosphomimetic mutant rescued the thrumin1-2 mutant phenotype for chloroplast movement and raises questions about the role of THRUMIN1’s interaction with actin. Mutation of serine 146 to aspartic acid also resulted in cytoplasmic localization of THRUMIN1:YFP in Nicotiana benthamiana. Mutations to a group of putative zinc-binding cysteine clusters implicate the C-terminus of THRUMIN1 in chloroplast movement. Phosphorylation-dependent association of THRUMIN1 with 14-3-3 KAPPA and OMEGA were also identified. Together, these studies provide insights into the mechanistic role of THRUMIN1 in light-dependent chloroplast movements.

Site-directed mutagenesis of THRUMIN1 revealed sites critical to its blue-light-dependent localization to actin filaments, to 14-3-3 proteins, and for its regulation of chloroplast movement.     

Brief irradiation with red or blue light induces orientational movement of chloroplasts in dark-adapted prothallial cells of the fernAdiantum

Orientational movement of chloroplasts was induced by a brief irradiation with red light (R) or blue light (B) in dark-adapted prothallial cells ofAdiantum, whose chloroplasts had gathered along the cell dividing wall (i.e., the anticlinal wall). When the whole dark-adapted prothallia were irradiated from a horizontal direction (i.e., from their lobes) with horizontally vibrating polarized R (H pol. R) for 10 or 3 min, the chloroplast left the anticlinal walls and spread over the prothallial surface (i.e., the periclinal walls) within 1–2 hr after the onset of irradiation, returning to the anticlinal wall (dark-position) within 10 hr. However, vertically vibrating polarized R (V pol. R) for 10 min did not induce the movement towards periclinal walls. The R effect was cancelled by non-polarized far-red light (FR) irradiation just after the R irradiation. Irradiation with H pol. B for 10 or 3 min but not with V pol. B could also induce a similar movement of chloroplasts, although the chloroplasts returned within 4 hr. The effect of H pol. B, however, was not cancelled by the subsequent FR irradiation. When a part of the dark-adapted cell at the prothallial surface was irradiated from above with a microbeam of R or B for 1 min, chloroplasts of the cell in the dark-position moved towards the irradiated locus in subsequent darkness. However, in the neighboring cells, orientational movement was not induced by either R or B microbeams. These results show that in dark-adapted prothallial cells, both brief irradiation with R and B can induce chloroplast photo-orientation and that the photoreceptors are phytochrome and blue light-absorbing pigment, respectively. It is also clear that effects of both R and B irradiation do not transfer to neighboring cells.     

Clumping and dispersal of chloroplasts in succulent plants

Plants have evolved various photoprotective mechanisms to mitigate photodamage. Here we report the diurnal movement of chloroplasts in the leaves of succulent crassulacean acid metabolism (CAM) plants under combined light and water stress. In leaves of water-stressed plants, the chloroplasts became densely clumped in one or sometimes two areas in the cytoplasm under light and dispersed during darkness. The chloroplast clumping resulted in leaf optical changes, with a decrease in absorptance and an increase in transmittance. The plant stress hormone abscisic acid induced chloroplast clumping in the leaf cells under light. We suggest that the marked chloroplast movement in these CAM plants is a photoprotective strategy used by the plants subjected to severe water stress.Abbreviations ABA Abscisic acid - CAM Crassulacean acid metabolism     

Light perception and the role of the xanthophyll cycle in blue-light-dependent chloroplast movements in lemna trisulca L

In most higher plants, chloroplasts move towards the periclinal cell walls in weak blue light (WBL) to increase light harvesting for photosynthesis, and towards the anticlinal walls as an escape reaction, thus avoiding photo-damage in strong blue light (SBL). The photo- receptor(s) triggering these responses have not yet been identified. In this study, the role of zeaxanthin as a blue-light photoreceptor in chloroplast movements was investigated. Time-lapse 3D confocal imaging in Lemna trisulca showed that individual chloroplasts responded to local illumination when one half of the cell was treated with light of different intensity or spectral quality to that received by the other half, or was maintained in darkness. Thus the complete signal perception, transduction and effector system has a high degree of spatial resolution and is consistent with localization of part of the transduction chain in the chloroplasts. Turnover of xanthophylls was determined using HPLC, and a parallel increase was observed between zeaxanthin and chloroplast movements in SBL. Ascorbate stimulated both a transient increase in zeaxanthin levels and chloroplast movement to profile in physiological darkness. Conversely, dithiothreitol blocked zeaxanthin production and responses to SBL and, to a lesser extent, WBL. Norflurazon preferentially inhibited SBL-dependent chloroplast movements. Increases in zeaxanthin were also observed in strong red light (SRL) when no directional chloroplast movements occurred. Thus it appears that a combination of zeaxanthin and blue light is required to trigger responses. Blue light can cause cis-trans isomerization of xanthophylls, thus photo-isomerization may be a critical link in the signal transduction pathway.     

Dynamic changes in the organization of microfilaments associated with the photocontrolled motility of chloroplasts in epidermal cells ofVattisneria

Summary Using time-lapse video microscopy, we performed a semiquantitative investigation of the movement of chloroplasts on the cytoplasmic layer that faces the outer periclinal wall (P side) of epidermal cells of leaves of the aquatic angiospermVallisneria gigantea Graebner. Under continuous irradiation with red light (650 nm, 0.41 W/m²), the movement of chloroplasts on the P side was transiently accelerated within 5 min. The increased movement began to decrease at around 20 min and fell below the original level after 40 to 60 min of irradiation with red light. The acceleration and deceleration of movement of chloroplasts on the P side seemed to lead directly to the increase and the subsequent decrease in the rate of migration of chloroplasts from the P side to the anticlinal layers of cytoplasm, which are responsible for the accumulation of chloroplasts on the P side, as we demonstrated previously. In the presence of inhibitors of photosynthesis, the accelerated movement of chloroplasts was maintained for as long as the chloroplasts were irradiated with red light. The rapid acceleration and deceleration of the movement of chloroplasts could be observed repeatedly with sequential irradiation with red and then far-red light (746 nm, 0.14 W/m²). Concomitantly with the loss of motility of chloroplasts on the P side, a dynamic change in the configuration of microfilaments, from a network to a honeycomb, occurred on the P side.Abbreviations APW artificial pond water - A side cytoplasmic layer that faces the anticlinal wall - ATP adenosine triphosphate - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - F-actin fibrous actin - FITC fluorescein isothiocyanate - PBS phosphate-buffered saline - Pfr farred-light-absorbing form of phytochrome - Pr red-light-absorbing form of phytochrome - P side cytoplasmic layer that faces the outer periclinal wall Dedicated to Professor Eldon H. Newcomb in recognition of his contributions to cell biology     

Decay of membrane potential under phosphorylating conditions in chloroplasts with in vivo activated ATPase

(1) The relation between the membrane potential and phosphorylation was studied in chloroplasts rapidly prepared from illuminated spinach leaves (light chloroplasts) and from dark-adapted leaves (dark chloroplasts). Light chloroplasts had a higher ATP hydrolysis activity than dark chloroplasts. (2) In the presence of ADP or ATP, a rapidly decaying phase of the field-indicating 518 nm absorbance change with a half-time of 15 ms became apparent in addition to the slow phase with a half-time of more than 300 ms in either type of chloroplast. Under these conditions, light chloroplasts showed a larger rapid phase than dark chloroplasts. (3) The rapid phase was suppressed by dicyclohexylcarbodiimide and was assumed to reflect the dissipation of membrane potential due to proton movements inside the CF₁-CF₀ ATP synthetase. (4) A model for the proton movement in ATP synthetase is proposed.     

Photosynthesis-dependent but neochrome1-independent light positioning of chloroplasts and nuclei in the fern Adiantum capillus-veneris

Chloroplasts change their positions in the cell depending on the light conditions. In the dark, chloroplasts in fern prothallia locate along the anticlinal wall (dark position). However, chloroplasts become relocated to the periclinal wall (light position) when the light shines perpendicularly to the prothallia. Red light is effective in inducing this relocation in Adiantum capillus-veneris, and neochrome1 (neo1) has been identified as the red light receptor regulating this movement. Nevertheless, we found here that chloroplasts in neo1 mutants still become relocated from the dark position to the light position under red light. We tested four neo1 mutant alleles (neo1-1, neo1-2, neo1-3, and neo1-4), and all of them showed the red-light-induced chloroplast relocation. Furthermore, chloroplast light positioning under red light occurred also in Pteris vittata, another fern species naturally lacking the neo1-dependent phenomenon. The light positioning of chloroplasts occurred independently of the direction of red light, a response different to that of the neo1-dependent movement. Photosynthesis inhibitors 3-(3,4 dichlorophenyl)-1,1-dimethylurea or 2,5-dibromo-3-isopropyl-6-methyl-p-benzoquinone blocked this movement. Addition of sucrose (Suc) or glucose to the culture medium induced migration of the chloroplasts to the periclinal wall in darkness. Furthermore, Suc could override the effects of 3-(3,4 dichlorophenyl)-1,1-dimethylurea. Interestingly, the same light positioning was evident for nuclei under red light in the neo1 mutant. The nuclear light positioning was also induced in darkness with the addition of Suc or glucose. These results indicate that photosynthesis-dependent nondirectional movement contributes to the light positioning of these organelles in addition to the neo1-dependent directional movement toward light.     

Chloroplast movement provides photoprotection to plants by redistributing PSII damage within leaves

Plants use light to fix carbon through the process of photosynthesis but light also causes photoinhibition, by damaging photosystem II (PSII). Plants can usually adjust their rate of PSII repair to equal the rate of damage, but under stress conditions or supersaturating light-intensities damage may exceed the rate of repair. Light-induced chloroplast movements are one of the many mechanisms plants have evolved to minimize photoinhibition. We found that chloroplast movements achieve a measure of photoprotection to PSII by altering the distribution of photoinhibition through depth in leaves. When chloroplasts are in the low-light accumulation arrangement a greater proportion of PSII damage occurs near the illuminated surface than for leaves where the chloroplasts are in the high-light avoidance arrangement. According to our findings chloroplast movements can increase the overall efficiency of leaf photosynthesis in at least two ways. The movements alter light profiles within leaves to maximize photosynthetic output and at the same time redistribute PSII damage throughout the leaf to reduce the amount of inhibition received by individual chloroplasts and prevent a decrease in photosynthetic potential.     

Chloroplast movements in fern leaves: correlation of movement dynamics and environmental flexibility of the species

The movements of chloroplasts in response to varying levels and wavelengths of incident light were investigated in leaves of four fern species: Adiantum capillus-veneris, Adiantum caudatum, Adiantum diaphanum and Pteris cretica. In all of the species studied blue light induced chloroplast redistribution resulting in face and profile patterns that were typical of low and high fluence rates, respectively. Fluence rate response characteristics and the kinetics of transmission changes accompanying these blue-light-induced movements were similar to those observed in the leaves of higher plants. Only in A. capillus-veneris was the distribution of chloroplasts affected by red light. The response was of the weak-light type, irrespective of the light intensity. The most effective fluence rate for red light was found to be below 7·2 μmol m^–2 s^–1 (1 W m^–2). The effect of red light was far-red reversible, indicating phytochrome involvement. Chloroplast responses were more dynamic in A. capillus-veneris and P. cretica, the two species that exhibited higher environmental flexibility.     

日光温室光温因子对黄瓜叶绿体超微结构及其功能的影响

在日光温室内,研究了光温因子对黄瓜叶绿体超微结构及其功能的影响．结果表明,因季节之间光、温条件不同,日光温室黄瓜叶片显微结构和叶绿体超微结构有一定差异,1月份光照弱叶肉细胞较大,而5月份光照强叶绿体数较多．在该试验条件下,未发现叶片光合速率与叶绿体超微结构之间有直接或密切的相关性．在各生长季节其光合速率均为第4叶>初展叶>基部叶,与叶龄及各叶位的受光量有关．如果将不同叶位叶放在相同的光照下,则差异明显减少．黄瓜叶片的叶肉细胞、叶绿体和淀粉粒的大小以及叶绿体数、基粒数、基粒厚度、基粒片层数都随叶位的下降而呈增加趋势。不同品种、同品种不同生长时期的叶片显微结构和叶绿体超微结构及其功能也有一定的差异．限制日光温室冬季黄瓜光合作用的主要因素是光照弱、有效光照时数少,而在晴天温度的限制作用相对较小。阴天因光照弱而导致的室内低温则是限制黄瓜生长的关键因素．