Blue Light Responses Research Articles

Calcium-calmodulin signalling is involved in light-induced acidification by epidermal leaf cells of pea,Pisum sativumL.

Pathways of signal transduction of red and blue lightdependent acidification by leaf epidermal cells were studied using epidermal strips of the Argenteum mutant of Pisum sativum. In these preparations the contribution of guard cells to the acidification is minimal. The hydroxypyridine nifedipine, a Ca2 +-channel blocker, partly inhibited the response to both blue and red light, while the phenylalkylamine, verapamil, a Ca2 +-channel blocker that has been shown in plant cells also to block K+-channels, caused nearly complete inhibition. The Ca2 + -channel activator S(-)Bay K 8644 induced acidification when added in the dark and diminished the light-induced lowering of the extracellular pH. The Ca2 + -ionophores, ionomycin and A23187, also reduced the light response. Furthermore, the light-induced acidification was inhibited by the calmodulin antagonists W-7 and trifluoperazine, but not by W-5. These calmodulin inhibitors completely inhibited the red light-induced acidification, but inhibited the response to blue light by only 60-70%. In general, inhibition by compounds affecting Ca-calmodulin signalling was always stronger on the red light response than that on the blue light response (with the exception of verapamil that blocked both the red and blue light responses equally well). This differential effect on red and blue light-induced responses indicates a role for Ca2 + CaM signalling in both the red and blue light responses, while a second process, independent of Ca2+ is activated by blue light.

Involvement of Calyculin A- and Okadaic Acid-sensitive Protein Phosphatase in the Blue Light Response of Stomatal Guard Cells

Calyculin A (CA) and okadaic acid (OA), inhibitors of protein phosphatases, inhibited blue light (BL)-dependent H + pumping in Vicia guard cell protoplasts at half-inhibitory concentrations of 4.5 nM and 400 nM, respectively. Light-induced stomatal opening in Vicia epidermis was completely suppressed by CA at 100 nM and by OA at 1 fiM. These results suggest that CA- and OA-sensitive protein phosphatase is involved in the BL response of stomatal guard cells.

Inhibition of the stomatal blue light response by verapamil at high concentration.

Blue light-dependent proton pumping in guard cell protoplasts and light-induced stomatal opening in the epidermis were inhibited by 1 mM verapamil, a Ca2+ channel blocker. Proton pumping and stomatal opening induced by fusicoccin, an activator of plasma membrane proton pump, were not inhibited by verapamil. These results suggest that verapamil inhibits blue light signaling in guard cells without inhibiting the pump.

Blue light induced ADP ribosylation of 38 and 56 kDa proteins in the soluble fraction of mycelia of Neurospora crassa

Soluble fractions prepared from the mycelia of wild type (74-OR23-1A) and band ( bd) exhibited an increase in the rate of the ADP ribosylation of a 38 kDa protein from nicotinamide adenine [ 32P]dinucleotide ([ 32P]NAD) in the presence of 10 −7 M riboflavin caused by blue light irradiation in vitro. The soluble fraction was mixed with a reaction mixture containing 5 μCi [ 32P]NAD at 0 °C for 20 s and then it was irradiated with blue light (420 nm, 42 μmol m −2 s −1) for 12.5, 25, 50, 100, 200 or 400 s at 0 °C or for 100 s with photon irradiance of 0.42, 4.2, 6.4 or 42 μmol m −2 s −1. Immediately after irradiation, the reaction was stopped and analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. An increase in the ADP ribosylation of the 38 kDa protein could be detected within 100 s of irradiation, and the enhancement in the rate of ADP ribosylation of the 38 kDa protein was proportional to the increase in the photon irradiance. By the irradiation with blue light for 200 or 400 s, the ADP ribosylation of a 56 kDa protein could also be detected. Analysis by two-dimensional gel electrophoresis of proteins after ADP ribosylation of them revealed that the 38 kDa proteins displayed at least four radioactive protein spots and the 56 kDa protein a single radioactive protein spot. Soluble fractions of mycelia prepared from blind mutants wc-1, wc-2, Δps15-1, lis-1, lis-2 and lis-3 exhibited also the enhancement of the ADP ribosylation of the 38 kDa protein by blue light irradiation, and at least wc-1, Δps15-1, lis-1 and lis-2 displayed a similar blue light response in the 56 kDa protein.

White collar-1, a central regulator of blue light responses in Neurospora, is a zinc finger protein.

The Neurospora crassa blind mutant white collar-1 (wc-1) is pleiotropically defective in all blue light-induced phenomena, establishing a role for the wc-1 gene product in the signal transduction pathway. We report the cloning of the wc-1 gene isolated by chromosome walking and mutant complementation. The elucidation of the wc-1 gene product provides a key piece of the blue light signal transduction puzzle. The wc-1 gene encodes a 125 kDa protein whose encoded motifs include a single class four, zinc finger DNA binding domain and a glutamine-rich putative transcription activation domain. We demonstrate that the wc-1 zinc finger domain, expressed in Escherichia coli, is able to bind specifically to the promoter of a blue light-regulated gene of Neurospora using an in vitro gel retardation assay. Furthermore, we show that wc-1 gene expression is autoregulated and is transcriptionally induced by blue light irradiation.

Close correspondence between the action spectra for the blue light responses of the guard cell and coleoptile chloroplasts, and the spectra for blue light-dependent stomatal opening and coleoptile phototropism.

Fluorescence spectroscopy was used to characterize blue light responses from chloroplasts of adaxial guard cells from Pima cotton (Gossypium barbadense) and coleoptile tips from corn (Zea mays). The chloroplast response to blue light was quantified by measurements of the blue light-induced enhancement of a red light-stimulated quenching of chlorophyll a fluorescence. In adaxial (upper) guard cells, low fluence rates of blue light applied under saturating fluence rates of red light enhanced the red light-stimulated fluorescence quenching by up to 50%. In contrast, added blue light did not alter the red light-stimulated quenching from abaxial (lower) guard cells. This response pattern paralleled the blue light sensitivity of stomatal opening in the two leaf surfaces. An action spectrum for the blue light-induced enhancement of the red light-stimulated quenching showed a major peak at 450 nm and two minor peaks at 420 and 470 nm. This spectrum matched closely an action spectrum for blue light-stimulated stomatal opening. Coleoptile chloroplasts also showed an enhancement by blue light of red light-stimulated quenching. The action spectrum of this response, showing a major peak at 450 nm, a minor peak at 470 nm, and a shoulder at 430 nm, closely matched an action spectrum for blue light-stimulated coleoptile phototropism. Both action spectra match the absorption spectrum of zeaxanthin, a chloroplastic carotenoid recently implicated in blue light photoreception of both guard cells and coleoptiles. The remarkable similarity between the action spectra for the blue light responses of guard cells and coleoptile chloroplasts and the spectra for blue light-stimulated stomatal opening and phototropism, coupled to the recently reported evidence on a role of zeaxanthin in blue light photoreception, indicates that the guard cell and coleoptile chloroplasts specialize in sensory transduction.

Negative Phototropism in the Rhizoid of Bryopsis plumosa

Negative phototropism in the rhizoid of Bryopsis plumosa was analyzed. The growth zone of the rhizoid was confined to the apical hemisphere, as is typical of tip growth. Upon unilateral illumination, the rhizoid bent away from the light source with a “bulging” manner. The photoreceptive site for phototropism was also restricted to the apical hemisphere. The action spectrum for this negative phototropism was determined from fluence-response curves that were obtained after fixing the duration of illumination at 60 min and varying the fluence rate between 0.1 and 3.0 W m-2. The action spectrum had a large peak at 467 nm and smaller peaks at 378 and 414 nm, resembling the action spectra of certain other blue-light responses.

Expression of an Arabidopsis cryptochrome gene in transgenic tobacco results in hypersensitivity to blue, UV-A, and green light.

The Arabidopsis HY4 gene, required for blue-light-induced inhibition of hypocotyl elongation, encodes a 75-kDa flavoprotein (CRY1) with characteristics of a blue-light photoreceptor. To investigate the mechanism by which this photoreceptor mediates blue-light responses in vivo, we have expressed the Arabidopsis HY4 gene in transgenic tobacco. The transgenic plants exhibited a short-hypocotyl phenotype under blue, UV-A, and green light, whereas they showed no difference from the wild-type plant under red/far-red light or in the dark. This phenotype was found to cosegregate with overexpression of the HY4 transgene and to be fluence dependent. We concluded that the short-hypocotyl phenotype of transgenic tobacco plants was due to hypersensitivity to blue, UV-A, and green light, resulting from over-expression of the photoreceptor. These observations are consistent with the broad action spectrum for responses mediated by this cryptochrome in Arabidopsis and indicate that the machinery for signal, transduction required by the CRY1 protein is conserved among different plant species. Furthermore, the level of these photoresponses is seen to be determined by the cellular concentration of this photoreceptor.

EFFECTS OF SHORT PULSES OF BLUE LIGHT ON THE ALKALINIZATION ASSOCIATED WITH THE UPTAKE OF NO3−AND CL−BY THE GREEN ALGAMONORAPHIDIUM BRAUNIIAND RELATED ACTION SPECTRA

AbstractInMonoraphidium braunii, uptake of NO3−, NO2−and Cl−is associated with proton transport and triggered by blue light (BL). Only 10 s after cells able to reduce NO3−to NH4+were irradiated with continuous, low‐fluence BL in the presence of NO3−, an alkalinization of the medium began and only became interrupted by switching off the BL with a 60–90 s time lag. With 30 s BL pulses, the NO3−‐dependent alkalinization lasted 3–5 min until it stopped. When the cells were exposed to continuous BL in the presence of Cl−, the alkalinization also started within 10 s but lasted only 3 min. After that, the pH remained constant and decreased when the BL was switched off. With 30 s BL pulses, the Cl−‐dependent alkalinization lasted 3 min and then decreased to its initial value. The NO3−‐dependent alkalinization shown by cells unable to reduce NO3−to NH4+was similar to that observed in the presence of Cl−. These alkalinization rates fit the Bunsen‐Roscoe reciprocity law. With 2 s pulses of high‐fluence BL, the delay time of the NO3‐ or Cl−‐dependent alkalinizations was only 2 s, one of the fastest BL responses reported so far. The action spectra for Cl−and NO3−uptakes proved to be very similar and matched the absorption spectra of flavins, including the 267 nm peak.

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 μmol m(-2) s(-1). Zeaxanthin formation in corn mesophyll chloroplasts was insensitive to low fluence rates and saturated at around 1800 μmol 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.

Polarity induction versus phototropism in maize: Auxin cannot replace blue light

In a previous study (Nick and Schafer 1991, Planta 185, 415–424), unilateral blue light had been shown, in maize coleoptiles, to induce phototropism and a stable transverse polarity, which became detectable as stable curvature if counteracting gravitropic stimulation was removed by rotation on a horizontal clinostat. This response was accompanied by a reorientation of cortical microtubules in the outer epidermis (Nick et al. 1990, Planta 181, 162–168). In the present study, this stable transverse polarity is shown to be correlated with stability of microtubule orientation against blue light and changes of auxin content. The role of auxin in this stabilisation was assessed. Although auxin can induce reorientation of microtubules it fails to induce the stabilisation of microtubule orientation induced by blue light. This was even true for gradients of auxin able to induce a bending response similar to that ellicited by phototropic stimulation. Experiments involving partial irradiation demonstrated different perception sites for phototropism and polarity induction. Phototropism starts from the very coleoptile tip and involves transmission of a signal (auxin) towards the subapical elongation zone. In contrast, polarity induction requires local action of blue light in the elongation zone itself. This blue-light response is independent of auxin.

Phytochrome-mediated phototropism inAdiantum cuneatum young leaves

Phototropism of youngAdiantum fern leaves is induced by red light as well as blue light. The red light response is mediated by phytochrome. This is the first evidence of phytochrome action in diploid fern tissue. The blue light response is mainly mediated not by phytochrome, but probably by a blue light-absorbing pigment as in the case of almost all plants and fungi. The red light-induced phototropism becomes detectable within 2 hr after the onset of unilateral light. The highest bending rate is about 10 degrees/hr, which occurs between 3–5 hr after the induction of the tropic response. The bending region is about 6–8 mm from the highest point of the coiled crozier where the growth rate becomes slow.

Blue light response of stomata and plasma membrane proton pump

Blue light response of stomata and plasma membrane proton pump

HY4 gene of A. thaliana encodes a protein with characteristics of a blue-light photoreceptor.

Specific responses to blue light are found throughout the biological kingdom. These responses--which in higher plants include phototropism, inhibition of hypocotyl elongation, and stomatal opening--are in many cases thought to be mediated by flavin-type photoreceptors. But no such blue-light photoreceptor has yet been identified or isolated, although blue-light responses in plants were reported by Darwin over a century ago, long before the discovery of the now relatively well characterized red/far-red light photoreceptor, phytochrome. Here we describe the isolation of a gene corresponding to the HY4 locus of Arabidopsis thaliana. The hy4 mutant is one of several mutants that are selectively insensitive to blue light during the blue-light-dependent inhibition of hypocotyl elongation response, which suggests that they lack an essential component of the cryptochrome-associated light-sensing pathway. The HY4 gene, isolated by gene tagging, was shown to encode a protein with significant homology to microbial DNA photolyases. As photolyases are a rare class of flavoprotein that catalyse blue-light-dependent reactions, the protein encoded by HY4 has a structure consistent with that of a flavin-type blue-light photoreceptor.

Reorientation of microtubules at the outer epidermal wall of maize coleoptiles by phytochrome, blue-light photoreceptor, and auxin

The effects of red and blue light on the orientation of cortical microtubules (MTs) underneath the outer epidermal wall of maize (Zea mays L.) coleoptiles were investigated with immunofluorescent techniques. The epidermal cells of dark-grown coleoptiles demonstrated an irregular pattern of regions of parallel MTs with a random distribution of orientations. This pattern could be changed into a uniformly transverse MT alignment with respect to the long cell axis by 1 h of irradiation with red light. This response was transient as the MTs spontaneously shifted into a longitudinal orientation after 1–2 h of continued irradiation. Induction/reversion experiments with short red and far-red light pulses demonstrated the involvement of phytochrome in this response. In contrast to red light, irradiation with blue light induced a stable longitudinal MT alignment which was established within 10 min. The blue-light response could not be affected by subsequent irradiations with red or far-red light indicating the involvement of a separate blue-light photoreceptor which antagonizes the effect of phytochrome. In mixed light treatments with red and blue light, the blue-light photoreceptor always dominated over phytochrome which exhibited an apparently less stable influence on MT orientation. Long-term irradiations with red or blue light up to 6 h did not reveal any rhythmic changes of MT orientation that could be related to the rhythmicity of helicoidal cell-wall structure. Subapical segments isolated from dark-grown coleoptiles maintained a longitudinal MT arrangement even in red light indicating that the responsiveness to phytochrome was lost upon isolation. Conversely auxin induced a transverse MT arrangement in isolated segments even in blue light, indicating that the responsiveness to blue-light photoreceptor was eliminated by the hormone. These complex interactions are discussed in the context of current hypotheses on the functional significance of MT reorientations for cell development.

Action spectrum for subliminal light control of adaptation in Phycomyces phototropism.

Adaptation processes enable phototropism and other blue light responses of Phycomyces to operate over a 10-decade range of fluence rate. Phototropic latency, used routinely to monitor the kinetics of sensitivity recovery after a step down in fluence rate, can be shortened by application of dim light for 35 min during the early part of the latency period. This light is termed subliminal, because it does not elicit phototropism under these experimental conditions; rather, it exerts its influence on the underlying adaptation kinetics. Fluence rate-response data for this latency reduction, obtained at 17 wavelengths of subliminal light from 347 to 742 nm, showed a variety of shapes that could be fit by zero, one, or two sigmoidal components, plus a constant term. At most wavelengths, the fluence-rate threshold for latency reduction by subliminal light tended to be well below the absolute threshold for phototropism, indicating that this effect is highly sensitive. An action spectrum for the sensitivity of the subliminal light effect, derived from the fluence rate-response curves, shows major peaks around 400 and 500 nm and a broad band from 570 to 670 nm, followed by a steep absorption edge. The sensitivity in the near ultraviolet region is relatively very low. The magnitude of the latency reduction also depends strongly on wavelength with a maximum at about 450 nm. The fluence-rate response data and the action spectrum--which is markedly different from that for phototropism and other blue-light responses of Phycomyces--indicate the participation of multiple pigments, or pigment states, in the photocontrol of adaptation.

Evidence for two different blue-light-receptor systems for the fast responses of stimulation of photosynthetic capacity and acidification of the plant surface in brown algae

Two blue-light responses of Phaeophyta that are expressed within a few seconds of a blue-light stimulus were characterized with respect to their photoreception properties. The first response is the activation of red-light-saturated photosynthesis which can be stimulated to values up to 5 times the rates in red light, depending on the species. The second response is a blue-light-induced acidification measurable at the plant surface. Both responses have similar kinetic characteristics and thus led us initially to hypothesise that they were causally connected in the same transduction mechanism. The two responses have action spectra [measured for Ectocarpus siliculosus (Dillwyn) Lyngb. and Laminaria saccharina (L.) Lamouroux] that are indistinguishable within the relatively large limits of error. However, in all species tested, the threshold sensitivity for blue light of the photosynthetic response is lower than that of the pH-shift by a factor of 2 to 150. Furthermore, stimulation of photosynthesis is sensitive to the flavin inhibitors, KI and phenylacetic acid, but the pH response is not affected by these inhibitors. Thus, the blue-light-induced pH-shift does not cause the stimulation of photosynthesis. In contrast, the different fluence-response relationships of the two responses and particularly the differential effect of the inhibitors are clear evidence for the action of two independent transduction pathways and photoreceptor systems for blue light. At least photoreception for stimulation of photosynthesis involves a flavin-or and a pterin.

Modulation of the blue light response of stomata of Commelina communis by CO2

Effects of CO2 on stomatal movements of Commelina communis L. were studied with plants, epidermal strips and guard cell protoplasts. With plants, the stomatal response induced by a blue light pulse was studied for different ambient CO2 concentration ranging from CO2‐deprived air to 100 Pa in darkness or under red light. It was observed that the blue light response could be obtained not only under a red light background but also in darkness and CO2‐free air, the two responses being quite similar.With epidermal strips, the effect of CO2 on ferricyanide reductase activity at the guard cell plasmalemma was studied by transmission electron microscopy. In the presence of ferric ions, reduced ferricyanide gives an electron dense precipitate of Prussian Blue. In darkness and air, no precipitate was observed. In darkness and CO2‐free air as well as under light and normal air, a precipitate was found along the plasmalemma of the guard cells, indicating a ferricyanide reductase activity. With guard cell protoplasts suspended in a medium either in equilibrium with air or in a CO2‐free medium the H+ extrusion induced by a blue light pulse added to a red light background was measured. A low CO2 content was obtained by adding photosynthetic algae to the suspension of guard cell protoplasts. In a CO2‐free medium the rate of H+ extrusion was enhanced.The results are discussed on the basis of a possible competition for reducing power between CO2 fixation and a putative blue light dependent redox chain located on the plasma membrane.

Modulation of the blue light response of stomata of Commelina communis by CO2

Modulation of the blue light response of stomata of Commelina communis by CO2

Transduction of Blue-Light Signals.

BUT IS IT AN ELEPHANT? In the old tale, several blind men are taken to an elephant. In turn, each is asked to examine and identify the mystery object. One blind man examines a leg and claims it to be a tree, another, examining the trunk, claims it to be a snake, and yet another, feeling a tusk, claims it to be a smooth rock. The pattern continues: each describes a component, none identifies the gestalt. There are a wealth of blue-light responses in higher plants. It is clear that many are biochemically, physiologically, or genetically independent, and others are linked. The mechanism by which a photon of blue light is converted into a biochemical signal and transduced into a biological response is under investigation for several of these responses. In many cases, investigators have successfully defined one or more components of the signaling mechanism. Taken together, the identified components have the potentia1 to define an entire signal transduction mechanism; receptor, G-protein, diacylglycerol, inositol triphosphate, calcium, calmodulin, several kinases, ion channels, and so forth. However, direct demonstration of a11 the components has not been achieved in any one system. Indeed, in some cases specific carriers are known to be absent. Further, the locations of the respective carriers differ in accordance with the location of the specific response (Fig. l). Perhaps the mechanisms responsible for transducing the blue-light signals are best represented by a herd of elephants. Signal transduction paradigms are well established. However, recent data suggest that plants may have several nove1 signal carriers (Deng et al., 1992), and it is not certain that the paradigms will be transferable to plants. The majority of studies of signal carriers for blue-light responses in higher plants make use of standard biochemical tools: direct identification based on biochemical properties or antibody crossreactivity, or indirect identification based on inhibitor/activator studies. These biochemical activities are correlated with the respective blue-light response by the photobiological activity (fluence response and/or time course), physical location, or involvement with the response as demonstrated by activator/inhibitor studies. The biochemical approach has recently been joined by a vigorous genetic approach. There are four blue-light responses in higher plants cur' Supported in part by the National Science Foundation and the