Ca is involved in phytochrome A-dependent regulation of the succinate dehydrogenase gene sdh1-2 in Arabidopsis

The mechanism of transduction of the phytochrome signal regulating the expression of succinate dehydrogenase in Arabidopsis has been investigated. Using the phytochrome mutants of Arabidopsis, it is demonstrated that the inhibition of succinate dehydrogenase in the light may result from the phytochrome A-dependent modulation of Ca²⁺ amount in the nuclear fraction of leaves. This leads to the activation of expression of the gene pif3 encoding the phytochrome-interacting factor PIF3, which binds to the promoter of the gene sdh1-2 encoding the SDHA subunit of succinate dehydrogenase and suppresses its expression. It is concluded that Ca²⁺ ions are involved in the phytochrome A-mediated inhibition of succinate dehydrogenase activity in the light.     

Phytochrome-mediated uptake of calcium in Mougeotia cells

Ca²⁺ is proposed to function as a messenger in such phytochrome-mediated responses as localized cell growth, intracellular movements, and control of plasma membrane properties. To test this hypothesis, the uptake of Ca²⁺ in irradiated and non-irradiated regions of individual threads of the green alga Mougeotia was studied with the aid of ⁴⁵Ca²⁺ and low temperature autoradiography: 10–20 cells within 40–60 cell-long threads were irradiated for up to 1 min, transferred to darkness for 3 to 10 min, submersed in a radioactive medium for 1 min, washed in an unlabelled medium for 30 min, and then autoradiographed at-80° C for several days.The autoradiographs show that those cells which had been pre-irradiated with red light did take up 2–10 times more Ca²⁺ than the adjacent non-irradiated cells of the same thread. Cells pre-irradiated with farred light or red light followed by far-red light showed no enhanced uptake of Ca²⁺. These results might be interpreted to indicate, firstly, that phytochrome-Pfr is involved in the enhanced uptake of Ca²⁺ and secondly, that the accumulation of radioactive Ca²⁺ in red light irradiated cells is an expression of an increased intracellular concentration of Ca²⁺. This interpretation is based on the data that (i) the dark interval between irradiation and labelling precluded the involvement of photosynthesis, (ii) the effect of red light was reversible with far-red light, and (iii) the accumulation of Ca²⁺ persisted during the long wash-out period. We speculate, that the red light-enhanced accumulation of Ca²⁺ in Mougeotia cells is caused by a Pfr-mediated increase of the Ca-permeability of the plasma membrane, and perhaps by a Pfr-impeding of an active Ca²⁺-extrusion.Abbreviations APW artificial pond water - EGTA ethylene glycol-bis-(-amino ethyle ether) N,N-tetraacetic acid - R red irradiation - D darkness - FR far-red irradiation - Pfr physiologicallyactive form of phytochrome - Pr physiologically inactive form of phytochrome This paper is part of a Ph. D. Thesis submitted to the University of Erlangen-Nürnberg by E.M. Dreyer     

The phytochrome photoreceptor in the green alga Mesotaenium caldariorum: implication for a conserved mechanism of phytochrome action

Chloroplast movement in the unicellular green alga Mesotaenium caldariorum is one of the earliest documented photomorphogenetic responses in plants. Photobiological studies have established that this response is under the control of phytochrome, whose rigid association with the plasma membrane and/or cytoskeleton enables the algal cells to orientate the chloroplast in response to the direction and intensity of light from the environment. While many of the key components of the algal phytochrome signalling pathway have been elucidated (i.e. Ca²⁺, calmodulin, actin and myosin), the primary biochemical mechanism of algal phytochrome action is unknown. To begin to address this important question, phytochrome and its corresponding genes have been isolated and characterized in this alga. These studies reveal that Mesotaenium cells contain a single type of phytochrome which is encoded by a small family of highly related genes. On the basis of its biochemical properties, primary structure and ability to interfere with the photoregulatory activity of phytochrome in transgenic plant seedlings, it appears likely that the primary mechanism of phytochrome action has been conserved throughout its evolution.     

The effect of membrane stabilizers on phytochrome-controlled anthocyanin biosynthesis in Brassica oleracea

The promotion of anthocyanin synthesis in red-cabbage seedlings by 5 min exposure to R light is inhibited by subsequent application of CaCl₂. The stimulation of dark synthesis of anthocyanin by n-PrOH and by kinetin is also reduced by Ca²⁺ and by cholesterol, both of which are well known to stabilize cell membranes. By contrast, EDTA, which chelates Ca²⁺, promotes dark synthesis of anthocyanin. Assay of native Ca²⁺ extractable from seedlings immersed in EDTA demonstrates that R light exposure promotes a highly significant increase in extractable Ca²⁺. It is suggested that the molecular configuration of the phytochrome molecule affects the ability of a membrane to bind Ca²⁺ and that this in turn affects the permeability to substrates which are required for anthocyanin biosynthesis.     

Phytochrome and calcium ions are involved in light-induced membrane depolarization in Nitella

Isolated internodes of Nitella (N. opaca, N. flexilis) and Nitellopsis spec. were punctured with single microelectrodes and their membrane potentials were recorded continuously during various light treatments. In red light the initial response was always a depolarization. This depolarization began with a lag-time of 0.4-3.5s and reached a steady state within 1–2 min of continuous illumination. Repolarization began within several seconds after turning off the light. The magnitude of the red-light-induced depolarization increased with the Ca²⁺-concentration of the medium. The largest depolarizations were recorded in 5 m mol l^-1 Ca²⁺. Ca²⁺ could not be replaced in this function by Na⁺, Mg²⁺, La³⁺ or mannitol. Far-red light alone had no effect on the resting membrane potential. Far-red light applied immediately after red light accelerated the repolarization of the membrane potential. Far-red light applied simultaneously with red light reduced the amount of depolarization and increased the rate of repolarization. The results indicate that phytochrome and Ca²⁺ are involved in the light-induced depolarization of the membrane. They are consistent with the hypothesis that phytochrome may act by triggering a Ca²⁺-influx at the plasma membrane.Abbreviations APW artificial pond water - Pfr far-red absorbing form of phytochrome - DCMU 3-(3,4-Dichlorphenyl)-1,1-dimethylurea     

Blue light- and genetically-reversed gravitropic response in protonemata of the moss Ceratodon purpureus

In darkness, protonemal filaments of Ceratodon purpureus (Brid.) grow negatively gravitropically (upwards). Red light induces a positive phototropic response mediated by the photoreceptor phytochrome. A red light treatment also has an inhibitory effect on the gravitropic response, an effect also mediated by phytochrome. In this study the effects of blue light on phototropism and on gravitropism were analysed. Unilateral blue light resulted in only a weak phototropic response, but markedly randomised growth direction. Blue light given together with a gravitropic stimulus reversed the gravitropism, changing it from negative to positive (filaments grow downward). The effect of blue light was also analysed with the mutant ptr116, which is defective in the biosynthesis of the phytochrome chromophore, and in a newly isolated mutant wwr2, which is positively gravitropic in darkness. Blue light induced the same reversal of gravitropism in ptr116 as in the wild type, indicating that phytochrome is not involved in this process. In wwr2 the direction of gravitropism was unaltered by the blue light treatment. Light also affects chlorophyll content and the size of plastids, potential statoliths for gravitropism. Red light induced an increase in plastid size and chlorophyll content in the wild type but not in ptr116. Blue light induced a similar change in wild type plastids. It seems as though light-induced alterations of gravitropism are not simply mediated by alterations in plastid properties, and that red light and blue light evoke fundamentally different responses. Received: 11 July 1997 / Accepted: 30 January 1998     

The role of calcium ions in phytochrome-controlled swelling of etiolated wheat (Triticum aestivum L.) protoplasts

Protoplasts from dark-grown wheat (Triticum aestivum L.) maintained at a constant osmotic potential at 22°C, were found to swell upon red irradiation (R) and the effect was negated by subsequent far-red light (FR), indicating phytochrome involvement. Swelling only occurred when Ca²⁺ ions were present in the surrounding medium, or were added within 10 min after R. Furthermore, Mg²⁺, Ba²⁺ or K⁺ could not replace this requirement for Ca²⁺. The presence of K⁺ did not enhance the Ca²⁺-dependent swelling response. When the Ca²⁺-ionophore A 23187 was added to the medium, protoplasts swelled in the dark to the same extent as after R. Both the Ca²⁺-channelblocker Verapamil and La³⁺ inhibited R-induced swelling. It is proposed that R causes the opening of Ca²⁺-channels in the plasma membrane. Boyle-van't Hoff analyses of protoplast volume after R and FR are consistent with the conclusion that R irradiation causes changes in membrane properties.Abbreviations EDTA ethylenediaminetetraacetic acid - FR far-red light - nov non-osmotic-volume - Pfr FR-absorbing form of phytochrome - Pr R-absorbing form of phytochrome - R red light     

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.     

Critical consideration on the relationship between auxin transport and calcium transients in gravity perception of Arabidopsis seedlings

Plants regulate their growth and morphogenesis in response to gravity field, known as gravitropism. In the early process of gravitropism, changes in the gravity vector (gravistimulation) are transduced into certain intracellular signals, termed gravity perception. The plant hormone auxin is not only a crucial factor to represent gravitropism but also a potential signaling molecule for gravity perception. Another strong candidate for the signaling molecule is calcium ion of which cytoplasmic concentration ([Ca²⁺]_c) is known to increase in response to gravistimulation. However, relationship between these two factors, say which is in the first place, has been controversial. This issue is addressed here mainly based on recent progress including our latest studies. Gravistimulation by turning plants 180° induced a two-peaked [Ca²⁺]_c-increase lasting for several minutes in Arabidopsis seedlings expressing apoaequorin; only the second peak was sensitive to the gravistimulation. Peak amplitudes of the [Ca²⁺]_c-increase were attenuated by the 10 µM auxin transport inhibitor (TIBA) and vesicle trafficking inhibitor (BFA), whereas the onset time and rate of rise of the second peak were not significantly altered. This result indicates that polar auxin transport is not involved in the initial phase of the second [Ca²⁺]_c-increase. It is likely that the gravi-induced [Ca²⁺]_c-increase constitutes an upstream event of the auxin transport, but may positively be modulated by auxin since its peak amplitude is attenuated by the inhibition of auxin transport.Key words: auxin, calcium, gravity perception, gravitropism, pin-formed (PIN) protein, Arabidopsis thaliana     

Evidence for A Ca2+ gradient across the plasma membrane of wheat protoplasts

Fluxes of Ca²⁺ across the plasma membrane of isolated wheat protoplasts have been measured both as net accumulation and as uptake under steady-state conditions. The ATPase inhibitors, orthovanadate and diethylstibesterol, and the divalent cation ionophore, A23187, were all found to enhance net Ca²⁺ accumulation by protoplasts. The uptake of Ca²⁺ under steady-state conditions was also stimulated by A23187 but relatively unaffected by a range of plant hormones or by red or far red light. Light treatments were compared to dark controls with protoplasts isolated from etiolated wheat.The results suggest that plant cells maintain a Ca²⁺ gradient across their plasma membrane but it appears not to be under phytochrome control.     

On the Nature and Origin of the Calcium Asymmetry Arising during Gravitropic Response in Etiolated Pea Epicotyls

Seven day old etiolated pea epicotyls were loaded symmetrically with ³H-indole 3-acetic acid (IAA) or ⁴⁵Ca²⁺, then subjected to 1.5 hours of 1g gravistimulation. Epidermal peels taken from top and bottom surfaces after 90 minutes showed an increase in IAA on the lower side and of Ca²⁺ on the upper side. Inhibitors of IAA movement (TIBA, 9-hydroxyfluorene carboxylic acid) block the development of both IAA and Ca²⁺ asymmetries, but substances known to interfere with normal Ca²⁺ transport (nitrendipine, nisoldipine, Bay K 8644, A 23187) do not significantly alter either IAA or Ca²⁺ asymmetries. These substances, however, are active in modifying both Ca²⁺ uptake and efflux through oat and pea leaf protoplast membranes. We conclude that the ⁴⁵Ca²⁺ fed to pea epicotyls occurs largely in the cell wall, and that auxin movement is primary and Ca²⁺ movement secondary in gravitropism. We hypothesize that apoplastic Ca²⁺ changes during graviresponse because it is displaced by H⁺ secreted through auxin-induced proton release. This proposed mechanism is supported by localized pH experiments, in which filter paper soaked in various buffers was applied to one side of a carborundum-abraded epicotyls. Buffer at pH 3 increases calcium loss from the side to which it is applied, whereas pH 7 buffer decreases it. Moreover, 10 micromolar IAA and 1 micromolar fusicoccin, which promote H⁺ efflux, increase Ca²⁺ release from pea epicotyl segments, whereas cycloheximide, which inhibits H⁺ efflux, has the reverse effect. We suggest that Ca²⁺ does not redistribute actively during gravitropism: the asymmetry arises because of its release from the wall adjacent to the region of high IAA concentration, proton secretion, and growth. Thus, the asymmetric distribution of Ca²⁺ appears to be a consequence of growth stimulation, not a critical step in the early phase of the graviresponse.     

Molecular tuning of calcium dependent processes by neuronal calcium sensor proteins in the retina

Vertebrate photoreceptor cells are exquisite light detectors operating under very dim and bright illumination mediated by phototransduction, which is under control of the two secondary messengers cGMP and Ca²⁺. Feedback mechanisms enable photoreceptor cells to regain their responsiveness after light stimulation and involve neuronal Ca²⁺-sensor proteins, named GCAPs (guanylate cyclase-activating proteins) and recoverins. This review compares the diversity in Ca²⁺-related signaling mediated by GCAP and recoverin variants that exhibit differences in Ca²⁺-sensing, protein conformational changes, myristoyl switch mechanisms, diversity in divalent cation binding and dimer formation. In summary, both subclasses of neuronal Ca²⁺-sensor proteins contribute to a complex signaling network in rod and cone cells, which is perfectly suited to match the requirements for sensitive cell responses and maintaining this responsiveness in the presence of different background light intensities.     

Ca-translocating ATPase of the plant plasma membrane

For Ca²⁺ to function as a second messenger in signal transduction, it is essential that plant cells maintain low cytoplasmic Ca²⁺ levels relative to internal organelles and the apoplast. At the plasma membrane, Ca²⁺ is actively transported out of the cytoplasm and current evidence supports the involvement of a primary Ca²⁺-translocating ATPase in mediating this energy-dependent process. This review examines the preliminary biochemical characterization of this transport enzyme.     

Characterization of a calcium/calmodulin-dependent protein kinase homolog from maize roots showing light-regulated gravitropism

Roots of many species respond to gravity (gravitropism) and grow downward only if illuminated. This light-regulated root gravitropism is phytochrome-dependent, mediated by calcium, and inhibited by KN-93, a specific inhibitor of calcium/calmodulin-dependent protein kinase II (CaMK II). A cDNA encoding MCK1, a maize homolog of mammalian CaMK, has been isolated from roots of maize (Zea mays L.). The MCK1 gene is expressed in root tips, the site of perception for both light and gravity. Using the [³⁵S]CaM gel-overlay assay we showed that calmodulin-binding activity of the MCK1 is abolished by 50 M KN-93, but binding is not affected by 5 M KN-93, paralleling physiological findings that light-regulated root gravitropism is inhibited by 50 M KN-93, but not by 5 M KN-93. KN-93 inhibits light-regulated gravitropism by interrupting transduction of the light signal, not light perception, suggesting that MCK1 may play a role in transducing light. This is the first report suggesting a physiological function for a CaMK homolog in light signal transduction.Abbreviations CaM calmodulin - CaMK (II) Ca²⁺/calmodulin-dependent protein kinase (II) - CBP CaM-binding protein - CDPK Ca²⁺-dependent protein kinase - MCK1 maize homolog of mamalian CaMK This work is supported by the National Aeronautics and Space Administration grant No: NAGW 238.     

Calcium and pH patterning at the apical meristem are specifically altered by photoperiodic flower induction in Chenopodium spp.

The apical meristem of the short‐day plant Chenopodium rubrum responds to photoperiodic flower induction with specific changes of pH and Ca²⁺ patterning immediately after the inductive dark span. The red–far‐red reversibility of the pH and Ca²⁺ patterning in response to night break treatments was measured in order to distinguish between the effect of the prolonged dark span per se and the specific effect of photoperiodic flower induction. In addition, the pH and Ca²⁺ patterning in C. rubrum was compared with the long‐day plant Chenopodium murale. The pH was visualized using the fluorescent probe carboxy SNARF‐1. Calcium ion concentrations were studied using a combination of Ca²⁺‐probes Fluo‐3 and Fura Red. It was observed that the specific changes in pH and Ca²⁺ patterning at the apical meristem of C. rubrum were abolished by the red‐light break. This effect was fully reversed with a subsequent single far‐red treatment. These observations infer the influence of phytochrome on both pH and Ca²⁺ patterning. Changes in pH and Ca²⁺ patterning upon flower induction were observed in both long‐day and short‐day plants. These results support the hypothesis that changes of pH and [Ca²⁺] in cells of the apical meristem are part of the pathway in signal transduction triggering flower initiation.     

Reversible inhibition of photochemistry of photosystem II by Ca2+ removal from intact cells of Anacystis nidulans

Depletion of Ca²⁺ from Anacystis nidulans produces an inhibition of O₂ evolution that is accompanied both at 39°C and 77 K by a loss of chlorophyll fluorescence of variable yield. This indicates that Ca²⁺-depletion causes disruption of normal photosystem II function, manifested by the disappearance of photoreduction of Q. Delayed light emission in the ms time range is also eliminated in Ca²⁺-depleted cells, which confirms that Ca²⁺ removal prevents charge separation and recombination in reaction centers of photosystem II. Readdition of Ca²⁺ to depleted cells restores fully the fluorescence of variable yield and delayed light emission, as well as O₂ evolution. Thus, Ca²⁺ may be a required component for photosystem II in A. nidulans.     

Light-affected ca fluxes in protoplasts from vallisneria mesophyll cells

In Vallisneria gigantea Graebner mesophyll cells, red light irradiation induces cytoplasmic streaming by decreasing the Ca²⁺ concentration in the cytoplasm, while far-red light irradiation inhibits it by increasing the concentration (S Takagi, R Nagai 1985 Plant Cell Physiol 26: 941-951). To examine the effects of light irradiation on Ca²⁺ fluxes across the cell membrane, protoplasts are isolated from the mesophyll cells. Changes in Ca²⁺ concentration in a solution bathing the protoplasts are monitored by spectrophotometry, using the Ca²⁺ -sensitive dye murexide. Red light irradiation induces an increase in Ca²⁺ concentration, which means an efflux of Ca²⁺ from the protoplasts. Subsequent far-red light irradiation produces a rapid decrease in Ca²⁺ concentration down to the dark control level; however, this is not observed in the presence of the Ca²⁺ -channel blocker nifedipine. Vanadate inhibits both the streaming and the Ca²⁺ efflux induced by red light irradiation. The results suggest that red light and far-red light control Ca²⁺ movements across the cell membrane, which in turn regulate the streaming.     

Light-Regulated Gravitropism in Seedling Roots of Maize

Red light-induced changes in the gravitropism of roots of Zea mays variety Merit is a very low fluence response with a threshold of 10⁻⁹ moles per square meter and is not reversible by far red light. Blue light also affects root gravitropism but the sensitivity of roots to blue is 50 to 100 times less than to an equal fluence of red. In Z. mays Merit we conclude that phytochrome is the sole pigment associated with light-induced changes in root gravitropism.     

Genetic analysis of the roles of phytochromes A and B1 in the reversed gravitropic response of the lz-2 tomato mutant

The lz-2 mutation in tomato ( Lycopersicon esculentum ) causes conditional reversal of shoot gravitropism by light. This response is mediated by phytochrome. To further elicit the mechanism by which phytochrome regulates the lz-2 phenotype, phytochrome-deficient lz-2 plants were generated. Introduction of au alleles, which severely block chromophore biosynthesis, eliminated the reversal of hypocotyl gravitropism in continuous red and far-red light. The fri ¹ and tri ¹ alleles were introduced to specifically deplete phytochromes A and B1, respectively. In dark-grown seedlings, phytochrome A was necessary for response to high-irradiance far-red light, a complete response to low fluence red light, and also mediated the effects of blue light in a far-red reversible manner. Loss of phytochrome B1 alone did not significantly affect the behaviour of lz-2 plants under any light treatment tested. However, dark-grown lz-2 plants lacking both phytochrome A and B1 exhibited reduced responses to continuous red and were less responsive to low fluence red light and high fluence blue light than plants that were deficient for phytochrome A alone. In high light, full spectrum greenhouse conditions, lz-2 plants grew downward regardless of the phytochrome deficiency. These results indicate that phytochromes A and B1 play significant roles in mediating the lz-2 phenotype and that at least one additional phytochrome is involved in reversing shoot gravitropism in this mutant.     

Calcium in the Regulation of Gravitropism by Light

The red light requirement for positive gravitropism in roots of corn (Zea mays cv “Merit”) provides an entry for examining the participation of calcium in gravitropism. Applications of calcium chelators inhibit the light response. Calcium channel blockers (verapamil, lanthanum) can also inhibit the light response, and a calcium ionophore, A23187, can substitute for light. One can substitute for red light by treatments which have elsewhere been shown to trigger Ca²⁺ influx into the cytosol, e.g. heat or cold shock. Agents which are known to be agonists of the phosphatidylinositol second messenger system (serotonin, 2,4-dichlorophenoxyacetic acid, deoxycholate) can each partially substitute for the red light, and Li⁺ can inhibit the light effect. These experiments suggest that the induction of positive gravitropism by red light involves a rise in cytoplasmic Ca²⁺ concentration, and that a contribution to this end may be made by the phosphatidylinositol second messenger system.