Second positive phototropism results from coordinated co-action of the phototropins and cryptochromes

Phototropism and hypocotyl growth inhibition are modulated by the coaction of different blue-light photoreceptors and their signaling pathways. How seedlings integrate the activities of the different blue-light photoreceptors to coordinate these hypocotyl growth responses is still unclear. We have used time-lapse imaging and a nontraditional mathematical approach to conduct a detailed examination of phototropism in wild-type Arabidopsis and various blue-light photoreceptor mutants. Our results indicate that high fluence rates of blue light (100 micro mol m(-)(2) s(-)(1)) attenuate phototropism through the coaction of the phototropin and cryptochrome blue-light photoreceptors. In contrast, we also demonstrate that phototropins and cryptochromes function together to enhance phototropism under low fluence rates (<1.0 micro mol m(-)(2) s(-)(1)) of blue light. Based on our results, we hypothesize that phototropins and cryptochromes regulate phototropism by coordinating the balance between stimulation and inhibition of growth of the hypocotyl depending on the fluence rate of blue light.     

Intracellular chloroplast photorelocation in the moss Physcomitrella patens is mediated by phytochrome as well as by a blue-light receptor

 The light-induced intracellular relocation of chloroplasts was examined in red-light-grown protonemal cells of the moss Physcomitrella patens. When irradiated with polarized red or blue light, chloroplast distribution in the cell depended upon the direction of the electrical vector (E-vector) in both light qualities. When the E-vector was parallel to the cross-wall (i.e. perpendicular to the protonemal axis), chloroplasts accumulated along the cross-wall; however, no accumulation along the cross-wall was observed when the E-vector was perpendicular to it (i.e. parallel to the protonemal axis). When a part of the cell was irradiated with a microbeam of red or blue light, chloroplasts accumulated at or avoided the illumination point depending on the fluence rate used. Red light of 0.1–18 W m⁻² and blue light of 0.01–85.5 W m⁻² induced an accumulation response (low-fluence-rate response; LFR), while an avoidance response (high-fluence-rate response; HFR) was induced by red light of 60 W m⁻² or higher and by blue light of 285 W m⁻². The red-light-induced LFR and HFR were nullified by a simultaneous background irradiation of far-red light, whereas the blue-light-induced LFR and HFR were not affected at all by this treatment. These results show, for the first time, that dichroic phytochrome, as well as the dichroic blue-light receptor, is involved in the chloroplast relocation movement in these bryophyte cells. Further, the phytochrome-mediated responses but not the blue-light responses were revealed to be lost when red-light-grown cells were cultured under white light for 2 d. Received: 7 September 1999 / Accepted: 15 October 1999     

Plant blue-light receptors

Plants have several blue-light receptors, which regulate different aspects of growth and development. Recent studies have identified three such receptors: cryptochrome 1, cryptochrome 2 and phototropin. Cryptochromes 1 and 2 are photolyase-like receptors that regulate hypocotyl growth and flowering time; phototropin mediates phototropism in response to blue light. In addition, phytochrome A has also been found to mediate various blue-light responses. Although the signal-transduction mechanisms of blue-light receptors remain largely unclear, phototropin is probably a protein kinase that regulates cytoplasmic calcium concentrations, whereas the cryptochromes might regulate anion-channel activity and changes in gene expression.     

Cryptochromes and phytochromes synergistically regulate Arabidopsis root greening under blue light

To increase their fitness, plants sense ambient light conditions and modulate their developmental processes by utilizing multiple photoreceptors such as phytochrome, cryptochrome and phototropin. Even roots, which are normally not exposed to light, express photoreceptors and can respond to light by developing chloroplasts. In the present study, root greening was observed in Arabidopsis thaliana. Seedlings were grown under monochromatic light and chlorophyll levels in the roots were determined. It was found that blue light was far more effective at inducing chloroplast development in Arabidopsis roots than was red light, and this response was under the control of a strong synergistic interaction between phytochromes and cryptochromes. As expected, the cry1 mutant was deficient in this response. Interestingly, the phyAphyB double mutant failed to respond to blue light under these conditions. This strongly suggests that either phytochrome A or phytochrome B, in addition to cryptochrome, was required for this blue light response. It was further demonstrated that the expression of photosynthetic genes was regulated in the same way. Dichromatic irradiation experiments indicated that this interaction depends on the level of phyB P(FR). Analysis of the cop1, det1 and hy5 mutants indicated that the corresponding factors were involved in the response.     

Structural and functional properties of the coleoptile chloroplast: Photosynthesis and photosensory transduction

Recent studies have shown that guard cell and coleoptile chloroplasts appear to be involved in blue light photoreception during blue light-dependent stomatal opening and phototropic bending. The guard cell chloroplast has been studied in detail but the coleoptile chloroplast is poorly understood. The present study was aimed at the characterization of the corn coleoptile chloroplast, and its comparison with mesophyll and guard cell chloroplasts. Coleoptile chloroplasts operated the xanthophyll cycle, and their zeaxanthin content tracked incident rates of solar radiation throughout the day. Zeaxanthin formation was very sensitive to low incident fluence rates, and saturated at around 800–1000 mol m^–2 s^–1. Zeaxanthin formation in corn mesophyll chloroplasts was insensitive to low fluence rates and saturated at around 1800 mol m^–2 s^–1. Quenching rates of chlorophyll a fluorescence transients from coleoptile chloroplasts induced by saturating fluence rates of actinic red light increased as a function of zeaxanthin content. This implies that zeaxanthin plays a photoprotective role in the coleoptile chloroplast. Addition of low fluence rates of blue light to saturating red light also increased quenching rates in a zeaxanthin-dependent fashion. This blue light response of the coleoptile chloroplast is analogous to that of the guard cell chloroplast, and implicates these organelles in the sensory transduction of blue light. On a chlorophyll basis, coleoptile chloroplasts had high rates of photosynthetic oxygen evolution and low rates of photosynthetic carbon fixation, as compared with mesophyll chloroplasts. In contrast with the uniform chloroplast distribution in the leaf, coleoptile chloroplasts were predominately found in the outer cell layers of the coleoptile cortex, and had large starch grains and a moderate amount of stacked grana and stroma lamellae. Several key properties of the coleoptile chloroplast were different from those of mesophyll chloroplasts and resembled those of guard cell chloroplasts. We propose that the common properties of guard cell and coleoptile chloroplasts define a functional pattern characteristic of chloroplasts specialized in photosensory transduction.Abbreviations Ant or A antheraxanthin - dv/dt fluorescence quenching rate - Fm maximum yield of fluorescence with all PS II reaction centers closed - Fo yield of instantaneous fluorescence with all PS II reaction centers open - Vio or V violaxanthin - Zea or Z zeaxanthin     

Distinct leaf developmental and gene expression responses to light quantity depend on blue-photoreceptor or plastid-derived signals,and can occur in the absence of phototropins

Leaf palisade cell development and the composition of chloroplasts respond to the fluence rate of light to maximise photosynthetic light capture while minimising photodamage. The underlying light sensory mechanisms are probably multiple and remain only partially understood. Phototropins (PHOT1 and PHOT2) are blue light receptors regulating responses which are light quantity-dependent and which include the control of leaf expansion. Here we show that genes for proteins in the reaction centres show long-term responses in wild type plants, and single blue photoreceptor mutants, to light fluence rate consistent with regulation by photosynthetic redox signals. Using contrasting intensities of white or broad-band red or blue light, we observe that increased fluence rate results in thicker leaves and greater number of palisade cells, but the anticlinal elongation of those cells is specifically responsive to the fluence rate of blue light. This palisade cell elongation response is still quantitatively normal in fully light-exposed regions of phot1 phot2 double mutants under increased fluence rate of white light. Plants grown at high light display elevated expression of RBCS (for the Rubisco small subunit) which, together with expected down-regulation of LHCB1 (for the photosynthetic antenna primarily of photosystem II), is also observed in phot double mutants. We conclude that an unknown blue light photoreceptor, or combination thereof, controls the development of a typical palisade cell morphology, but phototropins are not essential for either this response or acclimation-related gene expression changes. Together with previous evidence, our data further demonstrate that photosynthetic (chloroplast-derived) signals play a central role in the majority of acclimation responses.     

Velocity of chloroplast avoidance movement is fluence rate dependent.

In Arabidopsis leaves, chloroplast movement is fluence rate dependent. At optimal, lower light fluences, chloroplasts accumulate at the cell surface to maximize photosynthetic potential. Under high fluence rates, chloroplasts avoid incident light to escape photodamage. In this paper, we examine the phenomenon of chloroplast avoidance movement in greater detail and demonstrate a proportional relationship between fluence rate and the velocity of chloroplast avoidance. In addition we show that the amount of light-activated phototropin2, the photoreceptor for the avoidance response, likely plays a role in this phenomenon, as heterozygous mutant plants show a reduced avoidance velocity compared to that of homozygous wild type plants.     

Structural Adaptation of the Leaf Mesophyll to Shading

Structural characteristics of the mesophyll were studied in five boreal grass species experiencing a wide range of light and water supply conditions. Quantitative indices of the palisade and spongy mesophyll tissues (cell and chloroplast sizes, the number of chloroplasts per cell, the total cell and chloroplast surface area per unit leaf surface area) were determined in leaves of each of the species. The cell surface area and the cell volume in spongy mesophyll were determined with a novel method based on stereological analysis of cell projections. An important role of spongy parenchyma in the photosynthetic apparatus was demonstrated. In leaves of the species studied, the spongy parenchyma constituted about 50% of the total volume and 40% of the total surface area of mesophyll cells. The proportion of the palisade to spongy mesophyll tissues varied with plant species and growth conditions. In a xerophyte Genista tinctoria, the total cell volume, cell abundance, and the total surface area of cells and chloroplasts were 30–40% larger in the palisade than in the spongy mesophyll. In contrast, in a shade-loving species Veronica chamaedris, the spongy mesophyll was 1.5–2 times more developed than the palisade mesophyll. In mesophyte species grown under high light conditions, the cell abundance and the total cell surface area were 10–20% greater in the palisade mesophyll than in the spongy parenchyma. In shaded habitats, these indices were similar in the palisade and spongy mesophyll or were 10–20% lower in the palisade mesophyll. In mesophytes, CO₂ conductance of the spongy mesophyll accounted for about 50% of the total mesophyll conductance, as calculated from the structural characteristics, with the mesophyll CO₂ conductance increasing with leaf shading.     

Non‐invasive,whole‐plant imaging of chloroplast movement and chlorophyll fluorescence reveals photosynthetic phenotypes independent of chloroplast photorelocation defects in chloroplast division mutants

Leaf chloroplast movement is thought to optimize light capture and to minimize photodamage. To better understand the impact of chloroplast movement on photosynthesis, we developed a technique based on the imaging of reflectance from leaf surfaces that enables continuous, high‐sensitivity, non‐invasive measurements of chloroplast movement in multiple intact plants under white actinic light. We validated the method by measuring photorelocation responses in Arabidopsis chloroplast division mutants with drastically enlarged chloroplasts, and in phototropin mutants with impaired photorelocation but normal chloroplast morphology, under different light regimes. Additionally, we expanded our platform to permit simultaneous image‐based measurements of chlorophyll fluorescence and chloroplast movement. We show that chloroplast division mutants with enlarged, less‐mobile chloroplasts exhibit greater photosystem II photodamage than is observed in the wild type, particularly under fluctuating high levels of light. Comparison between division mutants and the severe photorelocation mutant phot1‐5 phot2‐1 showed that these effects are not entirely attributable to diminished photorelocation responses, as previously hypothesized, implying that altered chloroplast morphology affects other photosynthetic processes. Our dual‐imaging platform also allowed us to develop a straightforward approach to correct non‐photochemical quenching (NPQ) calculations for interference from chloroplast movement. This correction method should be generally useful when fluorescence and reflectance are measured in the same experiments. The corrected data indicate that the energy‐dependent (qE) and photoinhibitory (qI) components of NPQ contribute differentially to the NPQ phenotypes of the chloroplast division and photorelocation mutants. This imaging technology thus provides a platform for analyzing the contributions of chloroplast movement, chloroplast morphology and other phenotypic attributes to the overall photosynthetic performance of higher plants.     

Actin cytoskeleton in Arabidopsis thaliana under blue and red light

BACKGROUND INFORMATION: Actin cytoskeleton is the basis of chloroplast-orientation movements. These movements are activated by blue light in the leaves of terrestrial angiosperms. Red light has been shown to affect the spatial reorganization of F-actin in water plants, where chloroplast movements are closely connected with cytoplasmic streaming. The aim of the present study was to determine whether blue light, which triggers characteristic responses of chloroplasts, i.e. avoidance and accumulation, also influences F-actin organization in the mesophyll cells of Arabidopsis thaliana. Actin filaments in fixed mesophyll tissue were labelled with Alexa Fluor 488-conjugated phalloidin. The configuration of actin filaments, expressed as a form factor (4 pi x area/perimeter(2)), was determined for all actin formations which were measured in fluorescence confocal images. RESULTS: In the present study, we compare form-factor distributions and the median form factors for strong and weak, blue- and red-irradiated tissues. Spatial organization of the F-actin network did not undergo any changes which could be attributed specifically to blue light. Actin patterns were similar in blue-irradiated wild-type plants and phot2 (phototropin 2) mutants which lack the avoidance response of chloroplasts. However, significant differences in the shape and distribution of F-actin formations were observed between mesophyll cells of phot2 mutants irradiated with strong and weak red light. These differences were absent in wild-type leaves. CONCLUSIONS: Actin does not appear to be the main target for the blue-light chloroplast-orientation signal. The modes of actin involvement in chloroplast translocations are different in water and terrestrial angiosperms. The results suggest that co-operation occurs between blue- and red-light photoreceptors in the control of the actin cytoskeleton architecture in Arabidopsis.     

Phototropin Mediated Relocation of Myosins in Arabidopsis thaliana

Background

The mechanism of the light-dependent movements of chloroplasts is based on actin and myosin but its details are largely unknown. The movements are activated by blue light in terrestrial angiosperms. The aim of the present study was to determine the role of myosin associated with the chloroplast surface in the light-induced chloroplast responses in Arabidopsis thaliana. The localization of myosins was investigated under blue light intensities generating avoidance and accumulation responses of chloroplasts. The localization was compared in wild type plants and in phot2 mutant lacking the avoidance response.

Results

Wild type and phot2 mutant plants were irradiated with strong (36 µEm⁻²s⁻¹) and/or weak (0.8 µEm⁻²s⁻¹) blue light. The leaf tissue was immunolabeled with antimyosin antibodies. Different arrangements of myosins were observed in the mesophyll depending on the fluence rate in wild type plants. In tissue irradiated with weak blue light myosins were associated with chloroplast envelopes. In contrast, in tissue irradiated with strong blue light chloroplasts were almost myosin-free. The effect did not occur in red light and in the phot2 mutant.

Conclusions

Myosin displacement is blue light specific, i.e., it is associated with the activation of a specific blue-light photoreceptor. We suggest that the reorganization of myosins is essential for chloroplast movement. Myosins appear to be the final step of the signal transduction pathway starting with phototropin2 and leading to chloroplast movements.Key Words: Arabidopsis, blue light, chloroplast movements, myosins, phototropins     

Phototropin‐ and photosynthesis‐dependent mitochondrial positioning in Arabidopsis thaliana mesophyll cells

Mitochondria are frequently observed in the vicinity of chloroplasts in photosynthesizing cells, and this association is considered necessary for their metabolic interactions. We previously reported that, in leaf palisade cells of Arabidopsis thaliana, mitochondria exhibit blue‐light‐dependent redistribution together with chloroplasts, which conduct accumulation and avoidance responses under the control of blue‐light receptor phototropins. In this study, precise motility analyses by fluorescent microscopy revealed that the individual mitochondria in palisade cells, labeled with green fluorescent protein, exhibit typical stop‐and‐go movement. When exposed to blue light, the velocity of moving mitochondria increased in 30 min, whereas after 4 h, the frequency of stoppage of mitochondrial movement markedly increased. Using different mutant plants, we concluded that the presence of both phototropin1 and phototropin2 is necessary for the early acceleration of mitochondrial movement. On the contrary, the late enhancement of stoppage of mitochondrial movement occurs only in the presence of phototropin2 and only when intact photosynthesis takes place. A plasma‐membrane ghost assay suggested that the stopped mitochondria are firmly adhered to chloroplasts. These results indicate that the physical interaction between mitochondria and chloroplasts is cooperatively mediated by phototropin2‐ and photosynthesis‐dependent signals. The present study might add novel regulatory mechanism for light‐dependent plant organelle interactions.     

Chloroplasts can move in any direction to avoid strong light

Chloroplasts migrate in response to different light intensities. Under weak light, chloroplasts gather at an illuminated area to maximize light absorption and photosynthesis rates (the accumulation response). In contrast, chloroplasts escape from strong light to avoid photodamage (the avoidance response). Photoreceptors involved in these phenomena have been identified in Arabidopsis thaliana and Adiantum capillus-veneris. Chloroplast behavior has been studied in detail during the accumulation response, but not for the avoidance response. Hence, we analyzed the chloroplast avoidance response in detail using dark-adapted Adiantum capillus-veneris gametophyte cells and partial cell irradiation with a microbeam of blue light. Chloroplasts escaped from an irradiated spot. Both duration of this response and the distance of the migrated chloroplasts were proportional to the total fluence irradiated. The speed of movement during the avoidance response was dependent on the fluence rate, but the speed of the accumulation response towards the microbeam from cell periphery was constant irrespective of fluence rate. When a chloroplast was only partially irradiated with a strong microbeam, it moved away towards the non-irradiated region within a few minutes. During this avoidance response two additional microbeam irradiations were applied to different locus of the same chloroplast. Under these conditions the chloroplast changed the moving direction after a lag time of a few minutes without rolling. Taken together, these findings indicate that chloroplasts can move in any direction and never have an intrinsic polarity. Similar phenomenon was observed in chloroplasts of Arabidopsis thaliana palisade cells.     

Acclimation of mesophyll and bundle sheath chloroplasts of maize to different irradiances during growth

The regulation by light of the photosynthetic apparatus, and composition of light-harvesting complexes in mesophyll and bundle sheath chloroplasts was investigated in maize. Leaf chlorophyll content, level of plastoquinone, PSI and PSII activities and Lhc polypeptide compositions were determined in plants grown under high, moderate and low irradiances. Photochemical efficiency of PSII, photochemical fluorescence quenching and non-photochemical fluorescence quenching over a range of actinic irradiances were also determined, using chlorophyll a fluorescence analysis. Acclimation of plants to different light conditions caused marked changes in light-harvesting complexes, LHCI and LHCII, and antenna complexes were also reorganized in these types of chloroplasts. The level of LHCII increased in plants grown in low light, even in agranal bundle sheath chloroplasts where the amount of PSII was strongly reduced. Irradiance also affected LHCI complex and the number of structural polypeptides, in this complex, generally decreased in chloroplasts from plants grown under lower light. Surprisingly moderate and low irradiances during growth do not affect the light reaction and fluorescence parameters of plants but generated differences in composition of light-harvesting complexes in chloroplasts. On the other hand, the changes in photosynthetic apparatus in plants acclimated to high light, resulted in a higher efficiency of photosynthesis. Based on these observations we propose that light acclimation to high light in maize is tightly coordinated adjustment of light reaction components/activity in both mesophyll and bundle sheath chloroplasts. Acclimation is concerned with balancing light utilization and level of the content of LHC complexes differently in both types of chloroplasts.     

Fluorescence and Delayed Light Emission from Mesophyll and Bundle Sheath Cells in Leaves of Normal and Salt-Treated Panicum miliaceum

A modified fluorescence microscope system was used to measure chlorophyll fluorescence and delayed light emission from mesophyll and bundle sheath cells in situ in fresh-cut sections from leaves of Panicum miliaceum L. The fluorescence rise in 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (DCMU)-treated leaves and the slow fluorescence kinetics in untreated leaves show that mesophyll chloroplasts have larger photosystem II unit sizes than do bundle sheath chloroplasts. The larger photosystem II units imply more efficient noncyclic electron transport in mesophyll chloroplasts. Quenching of slow fluorescence also differs between the cell types with mesophyll chloroplasts showing complex kinetics and bundle sheath chloroplasts showing a relatively simple decline. Properties of the photosynthetic system were also investigated in leaves from plants grown in soil containing elevated NaCl levels. As judged by changes in both fluorescence kinetics in DCMU-treated leaves and delayed light emission in leaves not exposed to DCMU, salinity altered photosystem II in bundle sheath cells but not in mesophyll cells. This result may indicate different ionic distributions in the two cell types or, alternatively, different responses of the two chloroplast types to environmental change.     

Acclimation of mesophyll and bundle sheath chloroplasts of maize to different irradiances during growth

The regulation by light of the photosynthetic apparatus, and composition of light-harvesting complexes in mesophyll and bundle sheath chloroplasts was investigated in maize. Leaf chlorophyll content, level of plastoquinone, PSI and PSII activities and Lhc polypeptide compositions were determined in plants grown under high, moderate and low irradiances. Photochemical efficiency of PSII, photochemical fluorescence quenching and non-photochemical fluorescence quenching over a range of actinic irradiances were also determined, using chlorophyll a fluorescence analysis. Acclimation of plants to different light conditions caused marked changes in light-harvesting complexes, LHCI and LHCII, and antenna complexes were also reorganized in these types of chloroplasts. The level of LHCII increased in plants grown in low light, even in agranal bundle sheath chloroplasts where the amount of PSII was strongly reduced. Irradiance also affected LHCI complex and the number of structural polypeptides, in this complex, generally decreased in chloroplasts from plants grown under lower light. Surprisingly moderate and low irradiances during growth do not affect the light reaction and fluorescence parameters of plants but generated differences in composition of light-harvesting complexes in chloroplasts. On the other hand, the changes in photosynthetic apparatus in plants acclimated to high light, resulted in a higher efficiency of photosynthesis. Based on these observations we propose that light acclimation to high light in maize is tightly coordinated adjustment of light reaction components/activity in both mesophyll and bundle sheath chloroplasts. Acclimation is concerned with balancing light utilization and level of the content of LHC complexes differently in both types of chloroplasts.