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     

Cooperative regulation of cytoplasmic streaming and ca fluxes by pfr and photosynthesis in vallisneria mesophyll cells

In mesophyll cells of Vallisneria gigantea Graebner, Ca²⁺ regulates the induction and cessation of cytoplasmic streaming. Streaming is induced when the level of calcium in the cytoplasm is lowered through light-accelerated release of Ca²⁺ from the cells (S Takagi, R Nagai [1988] Plant Physiol 88: 228-232). We have now initiated an investigation on the nature of the photoreceptor(s) that are involved in the regulation of Ca²⁺ movements across the cell membrane and of streaming. Streaming is induced only when phytochrome exists in the phytochrome—far redabsorbing form (Pfr)—and photosynthesis is allowed to take place for at least 4 minutes. The former effect is typically photoreversible by red and far-red light, and phytochrome is spectro-photometrically detectable in the crude extract from the leaves. The latter effect is assessed in terms of the wavelength dependency and the effects of diuron and atrazine, two inhibitors of photosynthesis. A similar requirement for Pfr and photosynthesis is found to be associated with the acceleration of Ca²⁺ efflux in the protoplasts. The results suggest that phytochrome and photosynthetic pigment(s) cooperatively regulate cytoplasmic streaming via modulation of the Ca²⁺ transport in the cell membrane.     

Photomorphogenic responses to UV radiation: Involvement of phytochrome and UV photoreceptors in the control of hypocotyl elongation in Lycopersicon esculentum

The photo-inhibition of Lycopersicon esculentum Mill, hypocotyl growth induced by UV radiation may be mediated by both phytochrome and UV-absorbing receptors. The inhibition of growth induced by continuous irradiation with high fluence rate UV radiation is similar in the au mutant, which is severely deficient in spectrophoto metrically and immunochemically detectable phytochrome, and in the isogenic wild type. Parallel irradiation with 692 nm light, which is equivalent to UV radiation for the phytochrome system in our experimental conditions, induced at high photon fluence rates a significant increase in hypocotyl growth in the au mutant. The same light treatments inhibited the hypocotyl growth of the wild type. The responses of water-grown seedlings and chlorophyll-free seedlings (streptomycin and norflurazon treated seedlings) were compared. Water-grown and chlorophyll-free seedlings responded similarly to UV radiation. The presence of chlorophyll correlates with a significant increase in hypocotyl growth of au mutants irradiated with 692 nm light. These results support the conclusion that UV-induced inhibition of growth in the au mutant is independent of phytochrome.     

Comparison on the genotoxic effects of nuclear vs cytoplasmic irradiation from the alteration of CD59 gene locus

Using microbeam to irradiate human-hamster hybrid AL cells withdefined number of a particles in a highly localized spatial region, this paper showed that cytoplasmic irradiation induced very little toxicity. For example, the cell killing by 4 a particle traversal through the cytoplasm was about 10%, and about 70% cells survived after their cytoplasm was irradiated with 32 a particles. In contrast, the survival fractions for nuclear irradiation at the same doses were 35% and less than 1% respectively. Mutation induction showed that while nuclear irradiation induced 3-4-fold more CD59- mutants than cytoplasmic irradiation at equivalent particle traversal, at an equitoxic dose level of 90% survival, the latter exposure mode induced 3.3-fold more mutants than nuclear irradia-tion. Moreover, using multiplex PCR to analyze five marker genes on chromosome 11 (WT, CAT, PTH, APO-A1 and RAS), the results showed that the majority of mutants induced by cytoplasmic irradiation had retained all of the marker genes analyzed. By comparison, the proportion of mutants suffering loss of additional chromosomal markers increased with increasing number of particle traversal through nuclei.     

Photoregulation of asymmetric cell division followed by rhizoid development in the fern Ceratopteris prothalli

Strap-shaped prothalli of CERATOPTERIS: richardii grown in the dark have an apical meristem, a subapical elongation zone and a basal growth cessation zone [Murata et al. (1997) Plant Cell Physiol. 38: 201]. When the dark-grown prothalli were irradiated with continuous white light, marginal cells of the elongation zone divided asymmetrically, and the resulting smaller cells developed into rhizoids. The asymmetric division was also induced by brief irradiation of red light. The effect of red light was cancelled by subsequent irradiation of far-red light, indicating that the asymmetric division was regulated by phytochrome. Since the response to red light was not observed at 10(1) J m(-2) and saturated at 10(2) J m(-2) and the response is photoreversible by far-red light, the photoresponse was classified as a low-fluence response of phytochrome. Although the asymmetric division was induced by brief irradiation of red light, continuous irradiation of white, blue or red light was necessary to induce rhizoid growth. These results indicate that asymmetric division and subsequent cell growth are independently regulated by light in CERATOPTERIS: prothalli.     

Positive and negative tropic curvature induced by microbeam irradiation of protonemal tip cells of the moss Ceratodon purpureus

The photoreceptor phytochrome mediates tropic responses in protonemata of the moss Ceratodon purpureus. Under standard conditions the tip cells grow towards unilateral red light, or perpendicular to the electrical vector of polarized light. In this study the response of tip cells to partial irradiation of the apical region was analysed using a microbeam apparatus. The fluence response curve gave an unexpected pattern: whereas a 15-min microbeam with light intensities around 3 micro mol m (-2) s (-1) induced a growth curvature towards the irradiated side, higher light intensities around 100 micro mol m (-2) s (-1) caused a negative response, the cells grew away from the irradiated side. This avoidance response is explained by two effects: the light intensity is high enough to induce photoconversion into the active Pfr form of phytochrome, not only on the irradiated but also on the non-irradiated side by stray light. At the same time, the strong light on the irradiated side acts antagonistically to Pfr. As a result of this inhibition, the growth direction is moved to the light-avoiding side. Such a Pfr-independent mechanism might be important for the phototropic response to distinguish between the light-directed and light-avoiding side under unilateral light.     

Comparison on the genotoxic effects of nuclear vs cytoplasmic irradiation from the alteration ofCD59 gene locus

Using microbeam to irradiate human-hamster hybrid A_L cells with defined number of α particles in a highly localized spatial region, this paper showed that cytoplasmic irradiation induced very little toxicity. For example, the cell killing by 4 α particle traversal through the cytoplasm was about 10%, and about 70% cells survived after their cytoplasm was irradiated with 32 a particles. In contrast, the survival fractions for nuclear irradiation at the same doses were 35% and less than 1% respectively. Mutation induction showed that while nuclear irradiation induced 3—4-fold more CD59^- mutants than cytoplasmic irradiation at equivalent particle traversal, at an equitoxic dose level of 90% survival, the latter exposure mode induced 3.3-fold more mutants than nuclear irradiation. Moreover, using multiplex PCR to analyze five marker genes on chromosome 11 (WT, CAT, PTH, APO-A1 and RAS), the results showed that the majority of mutants induced by cytoplasmic irradiation had retained all of the marker genes analyzed. By comparison, the proportion of mutants suffering loss of additional chromosomal markers increased with increasing number of particle traversal through nuclei.     

Phytochrome- and blue-light-absorbing pigment-mediated directional movement of chloroplasts in dark-adapted prothallial cells of fernAdiantum as analyzed by microbeam irradiation

When prothalli ofAdiantum capillus-veneris L. were kept for 2 d in the dark, chloroplasts gathered along the anticlinal walls (Kagawa and Wada, 1994, J Plant Res 107: 389–398). In these dark-adapted prothallial cells, irradiation with a microbeam (10 gm in diameter) of red (R) or blue light (B) for 60 s moved the chloroplasts towards the irradiated locus during a subsequent dark period. Chloroplasts located less than 20 gm from the center of the R microbeam (18 J·m^–2) moved towards the irradiated locus. The higher the fluence of the light, the greater the distance from which chloroplasts could be attracted. The B microbeam was less effective than the R microbeam. Chloroplasts started to move anytime up to 20 min after the R stimulus, but with the B microbeam the effect of the stimulus was usually apparent within 10 min after irradiation. The velocity of chloroplast migration was independent of light-fluence in both R and B and was about - 0.3 m·min^–1 between 15 min and 30 min after irradiation. Whole-cell irradiation with far-red light immediately after R- and B-microbeam irradiations demonstrated that these responses were mediated by phytochrome and a blue-light-absorbing pigment, respectively. Sequential treatment with R and B microbeams, whose fluence rates were less than the threshold values when applied separately, resulted in an additive effect and induced chloroplast movement, strongly suggesting that signals from phytochrome and the blue-light-absorbing pigment could interact at some point before the induction of chloroplast movement.Abbreviations B blue light - FR far-red light - IR infrared light - R red light     

Visualization of a rapid,red/far-red light-dependent reaction by centrifuge microscopy

Summary Using a centrifuge microscope with stroboscopic illumination, we examined the effects of light irradiation on the passive movement of chloroplasts in dark-adapted mesophyll cells ofVallisneria gigantea. While irradiation with red light accelerates the passive gliding of chloroplasts produced by centrifugal force, irradiation with far-red light negates this effect. Irradiation with blue light does not accelerate the passive gliding, while red light is completely effective even in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea, an inhibitor of photosynthesis. An apparently active movement of chloroplasts can be induced by irradiation with red or blue light only in the presence of the far-red light-absorbing form of phytochrome. The significance of the reaction in the light with respect to the regulation of cytoplasmic streaming is discussed.Abbreviations APW artificial pond water - CMS centrifuge microscope of the stroboscopic type - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - Pfr phytochrome, far-red light-absorbing form - Pr phytochrome, red light-absorbing form     

A study of beating frequency of a single myocardial cell : II. Ultraviolet micro-irradiation of the nucleus and of the cytoplasm

Single rat myocardial cells were irradiated with the UV micro-irradiation technique over a nuclear or cytoplasmic area of 5 μm of diameter. The contractile response was studied immediately after the irradiation. After 10³ ergs mm⁻² of UV light (254 nm), 4% and 21% of the cells irradiated in the nucleus and the cytoplasm, respectively, showed a temporary increase of the beating rhythm. Moreover, cytoplasmic regions rich in mitochondria were more excitable than other cytoplasmic regions. The ultrastructure and the survival of these cells 24 h after the irradiation did not differ from the control cells. The change of the contractile response according to the localization of the irradiation indicates that the main target organelles are mitochondria; the role of the membrane is not excluded when higher doses of irradiation are considered.     

Control of expression of a gene encoding an extensin by phytochrome and a blue light receptor in spores of Adiantum capillus-veneris L.

In the present study, using a newly developed fluorescent differential display technique, we have carried out large-scale screening for genes whose expression was regulated by phytochrome and antagonistically by a blue light receptor in the spores of the fern Adiantum capillus-veneris L. Spores after imbibition were briefly irradiated with red, red/blue or blue light and collected 8 h after the irradiation. Total RNA was isolated from each sample and used to make cDNA with an oligo-dT primer. The cDNA was then used as a template for PCR with the oligo-dT primer and 80 arbitrary primers. The resulting PCR products were analyzed by an automated fluorescent DNA sequencer. Among 8000 displayed bands, we identified 15 upregulated and four down-regulated bands by red light, and this red light effect was irreversibly reversed by blue light. We cloned one of the up-regulated cDNA fragments and used it to screen a cDNA library prepared from the spores. The isolated insert is predicted to encode Ser-(Pro) _n repeats and showed homology with cell wall-associated extensins. The expression of this cDNA was induced 8 h after a red light treatment and the red light induction was photoreversibly prevented by far-red light and photo-irreversibly by blue light. The mRNA of this gene was detectable 4 h after red light irradiation and gradually increased in germinating spores.     

Metabolic Adaptations of Plant Respiration to Nutritional Phosphate Deprivation

Shoots of the lazy-2 (lz-2) gravitropic mutant of tomato (Lycopersicon esculentum Mill.) have a normal gravitropic response when grown in the dark, but grow downward in response to gravity when grown in the light. Experiments were undertaken to investigate the nature of the light induction of the downward growth of lz-2 shoots. Red light was effective at causing downward growth of hypocotyls of lz-2 seedlings, whereas treatment with blue light did not alter the dark-grown (wild-type) gravity response. Downward growth of lz-2 seedlings is greatest 16 h after a 1-h red light irradiation, after which the seedlings begin to revert to the dark-grown phenotype. lz-2 seedlings irradiated with a far-red light pulse immediately after a red light pulse exhibited no downward growth. However, continuous red or far-red light both resulted in downward growth of lz-2 seedlings. Thus, the light induction of downward growth of lz-2 appears to involve the photoreceptor phytochrome. Fluence-response experiments indicate that the induction of downward growth of lz-2 by red light is a low-fluence phytochrome response, with a possible high-irradiance response component.     

Fluence and wavelength requirements for Arabidopsis CAB gene induction by different phytochromes.

The roles of different phytochromes have been investigated in the photoinduction of several chlorophyll a/b-binding protein genes (CAB) of Arabidopsis thaliana. Etiolated seedlings of the wild type, a phytochrome A (PhyA) null mutant (phyA), a phytochrome B (PhyB) null mutant (phyB), and phyA/phyB double mutant were exposed to monochromatic light to address the questions of the fluence and wavelength requirements for CAB induction by different phytochromes. In the wild type and the phyB mutant, PhyA photoirreversibly induced CAB expression upon irradiation with very-low-fluence light of 350 to 750 nm. In contrast, using the phyA mutant, PhyB photoreversibly induced CAB expression with low-fluence red light. The threshold fluences of red light for PhyA- and PhyB-specific induction were about 10 nmol m-2 and 10 mumol m-2, respectively. In addition, CAB expression was photoreversibly induced with low-fluence red light in the phyA/phyB double mutant, revealing that another phytochrome(s) (PhyX) regulated CAB expression in a manner similar to PhyB. These data suggest that plants utilize different phytochromes to perceive light of varying wave-lengths and fluence, and begin to explain how plants respond so exquisitely to changing light in their environment.     

Phytochrome-mediated branch formation in protonemata of the mossCeratodon purpureus

We have analyzed light induction of side-branch formation and chloroplast re-arrangement in protonemata of the mossCeratodon purpureus. After 12 hr of dark adaptation, the rate of branch formation was as low as 5%. A red light treatment induced formation of side branches up to 75% of the dark-adapted protonema. The frequency of light induced branch formation differed between cells of different ages, the highest frequency being found in the 5th cell, the most distal cell studied from the apex. We examined the effect of polarized light given parallel to the direction of filament growth. The position of branching within the cell depended on the vibration plane of polarized red light. Branch formation was highest when the electric vector of polarized light vibrates parallel to the cell surface and is fluence rate dependent. The positional effect of polarized red light could be nullified to some extent by simultaneous irradiation with polarized far-red light. An aphototropic mutant,ptr116, shows characteristics of deficiency in biosynthesis of the phytochrome chromophore and exhibits no red-light induced branch formation. Biliverdin, a precursor of the phytochrome chromophore, rescued the red-light induced branching when added to the medium, supporting the conclusion that phytochrome acts as photoreceptor for red light induced branch formation. The light effect on chloroplast re-arrangement was also analyzed in this study. We found that polarized blue light induced chloroplast re-arrangement in wild-type cells, whereas polarized red light was inactive. This result suggests that chloroplast re-arrangement is only controlled by a blue light photoreceptor, not by phytochrome inCeratodon.     

Leaf angle in Chenopodium album is determined by two processes: induction and cessation of petiole curvature

Petiole curvature is important in regulating light interception by the leaf. To dissect the determination processes of leaf angle, we irradiated the lamina or petiole of Chenopodium album L. with either one or two spots of actinic light, after dark adaptation. When the abaxial side of the petiole was irradiated with blue light, the petiole curvature increased, and under continuous irradiation, the curvature continued for up to 6 h. The rate of curvature increased with increasing blue light intensity. The curvature induced by irradiation of the abaxial side with blue light ceased when the adaxial side of the petiole was simultaneously irradiated with either blue or red light. When an inhibitor for photosynthesis, 3-(3,4-dichlorophenyl)-1,1-dimethylurea, was applied to the adaxial side of the petiole, the cessation of curvature caused by blue light was only weakly inhibited, while the cessation caused by red light was markedly inhibited. When the adaxial side of the petiole was irradiated alternately with red and far-red light, the far-red light antagonized the cessation of curvature caused by the red light. These results clearly show that the petiole curvature is controlled by two processes, the induction and the cessation of curvature. At least three photoreceptor systems, blue-light receptor, photosynthesis and phytochrome, are involved in the reactions.     

The effect of the laser microirradiation of the cell center on neutrophil motility

The cell center of human neutrophils spread on polylysine-coated coverslips was irradiated with an argon laser microbeam. After the cells were pretreated with acridine orange, the irradiation of the cell center in a dose of over 0.1 J completely and irreversibly suppressed the motility of neutrophils (both random migration and chemotaxis), even though the cells retained their polarization. The same dose, applied to the cell nucleus and the forward and backward edges of the cytoplasm, resulted in little, if any, effect on cell motility, and did not inhibit their movement toward the target. Electron microscopy of the cells with the irradiated center showed the microtubules to persist for no less than 30 minutes; no visible destruction was caused in the cell center structure. Consequently, the cell center directly controls (not through polymerization of microtubules) the motility of neutrophils.     

Effects of Temperature on the Pattern of Anthocyanin Accumulation in Seedlings of Polygonum cuspidatum

Polygonum cuspidatum seedling. Anthocyanin accumulated first in the lower part of hypocotyls and then the site of accumulation gradually extended toward the upper part of hypocotyls when seedlings were irradiated with white light (WL) at 25 C. Etiolated seedlings accumulated anthocyanin only in the upper parts (hook and cotyledons) when the seedlings were irradiated with WL at 5 C. De-etiolated seedlings that had been pre-irradiated with WL for 1 day at 25 C accumulated anthocyanin both in upper and lower parts of the seedlings when the seedlings were irradiated with WL at 5 C. Spectral sensitivity was dependent on the temperature during irradiation. Red light (R), blue light (B), and near ultra-violet light (NUV) induced the accumulation of anthocyanin at 5 C but only NUV was effective in inducing the accumulation of anthocyanin at 25 C. Dichlorophenyl dimethylurea (DCMU) inhibited WL-induced anthocyanin accumulation but did not NUV-induced anthocyanin accumulation at 25 C. However, sucrose promoted NUV action at 25 C, indicating that photosynthesis can promote NUV-induced anthocyanin accumulation. Distribution of phytochrome in etiolated seedlings, that was examined by spectrophotometry, was similar to the distribution of anthocyanin at 5 C. Furthermore, phytochrome remained after 48 hr irradiation with WL at 5 C although phytochrome was rapidly degraded at 25 C. Received 12 July 1999/ Accepted in revised form 24 December 1999