Chloroplast movement in Mougeotia induced by blue light pulses

The profile-to-face chloroplast movement in the green alga Mougeotia has been induced by strong blue and near-ultraviolet light pulses (6 J m^-2). Simultaneously, strong red or far-red light (10 W m^-2) was applied perpendicularly to the inducing beam. The response was measured photometrically. Against the far-red background the reciprocity law was found to hold for pulse durations varying two orders of magnitude. The action spectrum exhibited a maximum near 450 nm and a distinct increase in near-ultraviolet. The time-course and the spectral dependence of pulse responses of chloroplasts in Mougeotia were similar to those recorded for other plants which are sensitive only to blue. This points to an alternative sensor system active in the short-wavelength region in addition to the phytochrome system.Abbreviations FR far-red light - Pr red absorbing form of phytochrome - Pfr far-red absorbing form of phytochrome - R red light This paper is dedicated to the memory of Professor Jan Zurzycki     

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     

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     

Spectral properties of soluble and pelletable phytochrome from epicotyls of etiolated pea seedlings

The red-light(R)-absorbing form of phytochrome (Pr) was detected spectrophotometrically in a 20,000 g particulate fraction prepared from a 1,000 g supernatant fraction from epicotyl tissue of pea (Pisum sativum L.) seedlings grown in the dark and only briefly exposed to dim green light. The difference spectrum of phytochrome in this fraction was essentially the same as that of soluble phytochrome from the same tissue. When the non-irradiated 20,000 g particulate fraction was incubated in the dark at 25° C, an absorbance change (decrease) of Pr after actinic red irradiation was found only in the far-red (FR) region. When the 20,000 g particulate fraction was irradiated with R and then incubated in the dark, the FR-absorbing form of phytochrome (Pfr) disappeared spectrally at a rate about half that in the soluble fraction, and the difference spectrum of the Pr which became detectable after dark incubation of the 20,000 g particulate fraction was markedly distorted. In contrast, Pfr in a 20,000 g particulate fraction prepared from tissues irradiated with R did not change optically during dark incubation at 25° C for 60 min, while Pfr in the soluble fraction from the same tissue disappeared in the dark. No dissociation of either Pr or Pfr from the 20,000 g particulate fraction was indicated during a 60-min dark incubation at 25° C, but Pfr in a 20,000 g particulate fraction prepared in vitro from R-irradiated 1,000 g supernatant fraction in the presence of CaCl₂ disappeared spectrally and the difference spectrum of Pr in the 20,000 g particulate fraction became quite distorted during the dark incubation.Abbreviations Pr red-light-absorbing form of phytochrome - Pfr far-red-light-absorbing form of phytochrome - FR far-red light - FR1 first actinic far-red light - FR2 second actinic far-red light - R red light - R1 first actinic red light - 1kS 1,000 g supernatant fraction - 20kS 20,000 g supernatant fraction - 20kP 20,000 g particulate fraction     

Calcium and phytochrome control of leaf unrolling in dark-grown barley seedlings

The red light-stimulated component of unrolling in sections from 7-d-old dark-grown barley (Hordeum vulgare L.) leaves is inhibited by ethyleneglycol-bis-(-aminoethyl ether)-N,N,N,N-tetracetic acid (EGTA). A free-Ca²⁺ activity of less than 40 M restores the ability to respond to red light, but only if supplied within 1 h of red light. Magnesium ions are an ineffective substitute. At least two processes in unrolling appear to be Ca²⁺-sensitive.Fluence-response measurements indicate that the levels of the far-red-absorbing from of phytochrome (Pfr) still present 4 h after red-light treatment should be above saturation for the unrolling response; consequently, loss of Pfr does not explain the loss in effectiveness of Ca²⁺ during prolonged EGTA treatment. However, if a further red-light treatment is given simultaneously with Ca²⁺ addition 4 h after the initial light stimulus, then full unrolling occurs in EGTA-treated sections. These data indicate that, under normal circumstances, a functional change in the properties of Pfr must occur, uncoupling it from the transduction chain.Abbreviations EGTA ethyleneglycol-bis-(-aminoethylether)-N,N,N,N,-tetracetic acid - FR far-red light - Mes 2-(N-morpholino)ethanesulphonic, acid - Pfr far-red absorbing form of phytochrome - Pr red-absorbing form of phytochrome - R red light     

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     

Blue-light-induced chloroplast orientation in Mougeotia. Evidence for a separate sensor pigment besides phytochrome

Chloroplast orientation in the green alga Mougeotia has been induced by unidirectional red or blue light, given continuously during one hour. In addition, part of the preparations obtained scattered strong far-red light simultaneously with the orienting light. This far-red light completely abolished the response to red light, consistent with phytochrome as the sensor pigment for orientation in Mougeotia. In blue light, however, the response was completely insensitive to far-red light, thus pointing to a different sensor pigment in the shortwavelength region.Abbreviation P_fr far-red-absorbing form of phytochrome     

Light requirement,phytochrome and photoperiodic induction of flowering of Pharbits nil Chois

The low chlorophyll content of cotyledons of Pharbitis nil grown for 24 h in far-red light (FR) or at 18° C in white light from fluorescent lamps (WL) allows spectrophotometric measurement of phytochrome in these tissues. The (A) measurements utilize measuring beams at 730/802 nm and an actinic irradiation in excess of 90 s. The constancy of the relationship between phytochrome content and sample thickness confirms that, under these conditions of measurement, a true maximum phytochrome signal was obtained. These techniques have been used to follow changes in the form and amount of phytochrome during an inductive dark period for flowering. Following exposure to 24h WL at 18° C with a terminal 10 min red (R), P_fr was lost rapidly in darkness and approached zero in less than 1 h; during this period there was no change in the total phytochrome signal. Following exposure to 24 h FR with a terminal 10 min R, P_fr approached zero in 3 h, and the total phytochrome signal decreased by about half. The relevance of these changes to photoperiodic time measurement is discussed.Abbreviations BCJ irradiation from photographic ruby-red lamps - FR far-red light - P_fr far-red-absorbing form of phytochrome - P_r red-absorbing form of phytochrome - P total phytochrome content - R red light - WL white light from fluorescent lamps     

Phytochrome modulation of calcium fluxes in wheat (Triticum aestivum L.) protoplasts

Employing the metallochromic dye murexide and by monitoring the uptake of radiolabelled calcium, photoreversible calcium fluxes were measured in wheat leaf protoplast suspensions. Results obtained by both methods were identical — red light promoted and subsequent far-red irradiation reversed an influx of Ca⁺⁺ ions into the protoplasts. These findings imply phytochrome regulation of Ca⁺⁺ fluxes across the plasma membrane. The influx of Ca⁺⁺ stimulated by 2 min red irradiation could be maintained in total darkness for the initial 16–18 min after illumination, after which a 6–8 min efflux process was triggered and the basal Ca⁺⁺ level restored. Verapamil, a calcium channel blocker, inhibited the red-promoted influx, whereas the far-red mediated efflux could be checked by the use of the ATPase inhibitor vanadate, and also by the calmodulin antagonist chlorpromazine, thus suggesting a role of ion channels and pumps in phytochrome-controlled Ca⁺⁺ fluxes. The possible involvement of phosphoinositides in phytochrome-modulated calcium fluxes was also investigated.Abbreviations A difference in absorbance - CPZ chlorpromazine - FR far-red (light) - MX murexide - PI phosphatidylinositol - PIP₂ phosphatidylinositol 4, 5-bisphosphate - PIPES piperazine-N,N-bis[2-ethanesulfonic acid] - POPOP 1, 4-bis [2-(5-phenyl-1, 3-oxazolyl)]-benzene - PPO 2, 5-diphenyl-1, 3-oxazole - R red (light) - SOV sodium orthovanadate     

Light effects upon dry lettuce seeds

Germination of certain dry seeds (achenes) of Lactuca sativa L. cv. Grand Rapids was increased to ca. 75% after irradiation with 665 nm red light (R; 1x10³ J m^-2); this response was eliminated by far-red light (FR) following the R. The response of dry seeds required an order of magnitude more light than that of wetted seeds, and was not maximal until 48 h after irradiation. Other seeds, which could not be stimulated by R in dry state, showed a partial response after 10 min hydration. Irradiation of dry seeds (or seeds wetted 1 h) with FR (1x10³ J m^-2) reduced dark germination from 26% to 2%. Seeds dehydrated in an oven (60°C, 90 min) showed a decrease in germination if irradiated with R (1x10⁵ J m^-2) before wetting. The results show that phytochrome is present in dry lettuce seeds (and functional in some seed lots) prior to wetting; and that in other seed lots the molecule becomes functional within minutes after wetting the seeds. Transformation of the FR absorbing from of phytochrome (P_FR) to the inactive from (P_R) occurs at lower seed moisture content than the reverse reaction. It appears that dormancy in seeds ripened in sunlight might be assured during seed drying and maturation by the more effective transformation of P_FR to P_R than vice versa as phytochrome is dehydrated.Abbreviations FR far-red - R red - CAL seeds from California - NC seeds from North Carolina (see text)     

Light effects on in vitro rooting of pear cultivars of different rhizogenic ability

The effect of varying light regimes on in vitro rooting of microcuttings of two pear (Pyrus communis L.) cultivars was investigated. Cultures of the easy to-root Conference and the difficult-to-root Doyenne d'Hiver were incubated for 21 days with or without indole-3-butyric acid (IBA) in the medium in darkness or under continuous far-red (8 µmol m^–2 s^–1), blue, white or red (15 or 36 µmol m^–2 s^–1) light. Conference rooted without IBA when exposed to red, blue or white light while no rooting was observed under far-red light and in darkness. The high rooting efficiency under red and, by contrast, the inhibition under far-red light and darkness suggest the involvement of the phytochrome system in rhizogenesis. The addition of IBA to the culture medium enhanced root production under all light regimes in both cultivars. Red light, especially at the lower photon fluence rate, had a positive effect by increasing root extension (number × length of roots) and stimulating secondary root formation.Abbreviations IBA Indole-3-butyric acid - R red light - B blue light - FR far-red light - W white light - D darkness - P_fr active (far-red light absorbing) form of phytochrome - P_tot total phytochrome - BA benzyl-adenine     

Phytochrome Activation of K+ Channels and Chloroplast Rotation in Mougeotia: the Escape Times

Red light mediates chloroplast movement and increased activityof calcium-activated potassium channels on the plasma membraneof the alga Mougeotia sp. (UTEX LB 734). When activation ismediated by phytochrome, a far-red light irradiation given sometime after the red light irradiation will reverse the effectof the red light, due to phytochrome photoreversibility. Wecharacterized the escape times (time required for loss of photoreversibility)for these two processes to compare the transduction pathwaysinvolved in chloroplast rotation and channel activation. Theescape time for chloroplast rotation was 2.5 min after red lightirradiation (red and far-red light irradiations were 30 s).For channel activation, shorter red and far-red light irradiations(10 s) had to be used to obtain an escape time of 20 s. Thedifference in the escape times suggests that there is relativelyrapid divergence in the transduction pathways leading from phytochromeactivation (only one molecular species of phytochrome is foundin Mougeotia) to each of the two responses in the same cellularsystem. Because channel activation occurs 2–4 min afterirradiation while the escape time is 20 s, it is unlikely thatphytochrome acts directly on the channel. (Received September 26, 1995; Accepted December 28, 1995)     

Photocontrol of stem elongation in light-grown plants of Fuchsia hybrida

Stems of the caulescent long-day plant, Fuchsia hybrida cv Lord Byron, showed 2 types of response to light. In one, internode length was increased by far-red irradiation given at the end of an 8 h photoperiod: the response was no greater with prolonged exposure and was less when the start of far-red was delayed. The effect of far-red was reversible by a subsequent exposure to red light. Internode length was inversely proportional to the P_fr/P ratio established before entry to darkness and there was no evidence for loss of P_fr during a 16 h dark period. The inhibitory effect of P_fr acted at a relatively late stage of internode growth. With the development of successive internodes a second response appeared in which stems lengthened following prolonged daily exposures to red or far-red light, or mixtures of the two, or to brief breaks with red or white light. In these later internodes, a short exposure to far-red near the middle of the night was not reversible by red because red alone promoted elongation at this time. Internode length increased with increase in the daily duration of light and, when light was given throughout an otherwise dark period of 16 h, with increase in illuminance to a saturation value of 200 lx from tungsten lamps. Elongation increased as a linear function of decrease in photostationary state of phytochrome down to P_fr/P0.3; however, internodes were shorter in far-red light than in 25% red/red+far-red. It was concluded that stem length is a net response to two modes of phytochrome action. An inductive effect of P_fr inhibits a late stage in internode expansion, and a phytochrome reaction which operates only in light (and may involve pigment cycling) promotes an early stage of internode development. Stem elongation is thus a function both of the daily duration of light and its red/red+far-red content. The outgrowth of axillary buds was controlled by the first type of phytochrome action only.Abbreviations and symbols FR far red light - R red light - P phytochrome - P_fr phytochrome in the far-red light absorbing form - SD 8 h short days - LDP long-day plant - SDP short-day plant     

Rapid photomodulation of stem extension in light-grownSinapis alba L.

Treatment of the whole of aSinapis alba plant with supplementary far-red light (FR), in back-ground white light (WL), induces a rapid increase in stem extension rate. This rapid increase is regulated by the light environment of the stem itself. Supplementary FR to the stem increases extension rate after a lag period of 10–15 min. A lag period of 3–4 h follows FR irradiation of the leaf, before an increase in extension rate is detectable. When the stem is given supplementary FR, the change in extension rate which is induced increases with increasing FR fluence rate, and with decreasing phytochrome photoequilibrium. There is no difference between the effects of supplementary FR _max 719 nm and supplementary FR _max 739 nm for these relationships. The increase in extension rate induced by supplementary FR is reversed by an increase in the fluence rate of red light (R). These data indicate that the response is controlled by phytochrome photoequilibrium.Abbreviations B blue light - FR far-red light - R red light - WL white light - Pfr far-red absorbing form of phytochrome - Pr red absorbing form of phytochrome - P_tot total phytochrome level (=Pr+Pfr); -Pfr/Ptot, measured - ER difference in stem extension rate, before and after treatment     

Phytochrome-mediated changes in the membrane potential of subepidermal cells of Lemna paucicostata 6746

Light-stimulated transmembrane potential changes have been measured continuously after implantation of microelectrodes into subepidermal cells of the short-day plant Lemna paucicostata 6746. Irradiation for 5 min with white or red light caused a transient hyperpolarization. These potential changes could be suppressed with 10^-6 M DCMU. Irradiation of DCMU-inhibited plants with far-red light for 5 min hyperpolarized the membrane potential, which thereafter was not changed by further far-red application. Consecutive red light irradiation for 5 min depolarized the membrane potential. The red/far-red reversibility of the potential changes (which could be repeated several times with a single plant) suggests the participation of phytochrome.Abbreviations EDTA ethylenediaminetetraacetate - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethyl urea - P_r, (P_fr) red- (far-red-) absorbing form of phytochrome     

Light quantity and quality interactions in the control of elongation growth in light-grown Chenopodium rubrum L. seedlings

The spectral control of hypocotyl elongation in light-grown Chenopodium rubrum L. seedlings has been studied. The results showed that although the seedlings responded to changes in the quantity of combined red and far-red radiation, they were also very sensitive to changes in the quantity of blue radiation reaching the plant. Altering the proportion of red: far-red radiation in broad waveband white light caused marked differences in hypocotyl extension. Comparison of the responses of green and chlorophyll-free seedlings indicated no qualitative difference in the response to any of the light sources used, although photosynthetically incompetent plants were more sensitive to all wavelengths. Blue light was found to act primarily of a photoreceptor which is different from phytochrome. It is concluded that hypocotyl extension rate in vegetation shade is photoregulated by the quantity of blue light and the proportion of red: far-red radiation. In neutral shade, such as that caused by stones or overlying soil, hypocotyl extension appears to be regulated primarily by the quantity of light in the blue waveband and secondarily by the quantity of light in the red and far-red wavebands.Abbreviations B blue - FR far-red - k ₁, k ₂ rate constants for photoconverison of Pr to Pfr and Pfr to Pr, respective - k ₁/k ₁ +k ₂= phytochrome photoequilibrium - k ₁ +k ₂= phytochrome cycling rate - Pr=R absorbing form of phytochrome - Pfr=FR absorbing form of phytochrome - P_tot Pr+Pfr - PAR photosynthetically active radiation = 400–700 nm - R red - WL white light     

Nature of the phytochrome effect on the binding of glycolate oxidase to peroxisomes in vitro

The attachment of glycolate oxidase to the peroxisomal fraction derived from etiolated barley leaves (Hordeum vulgare L. cr. Dvir) is affected by light. The effect of red irradiation is reversed by subsequent far-red irradiation, indicating the involvement of phytochrome. This phytochrome effect is assumed to be related to phytochrome binding. Indeed, prevention by filipin (1.2·10^-6 mol g^-1 f wt) or cholesterol of phytochrome binding to membranes abolishes the effect of light on the interaction between glycolate oxidase and the peroxisomal fraction. Glycolate oxidase binding is affected by addition of quasi-ionophores such as gramicidin and filipin at a concentration of 0.6·10^-3 mol g^-1 f wt. This fact indicates that peroxisome-glycolate oxidase interaction may be affected by membrane potential. Since both ion transport and membrane potential are known to be affected by phytochrome, it is proposed that phytochrome acts in the light-induced modulation of glycolate oxidase attachment as a quasi-ionophore.Abbreviations GO glycolate oxidase - Pr and Pfr phytochrome forms absorbing in red and far-red, respectively - R and F red and far-red irradiation - Cumulative 20 Kp 20,000 g pellet obtained by centrifugation of the crude extract - 1 Kp 1,000 g pellet - 20 Kp 20,000 g pellet, obtained by centrifugation of 1 Kp supernatant - 1 Kp, 20 Kp and cumulative 20 Kp pellets obtained after density centrifugation through a sucrose cushion     

Calcium requirement of phytochrome-mediated fern-spore germination: No direct phytochrome-calcium interaction in the phytochrome-initiated transduction chain

Phytochrome-mediated germination of fern spores of Dryopteris paleacea Sw. was initiated by a saturating red-light (R) irradiation after 20 h of imbibition. For its realization external Ca²⁺ was required, with a threshold at a submicromolar concentration, and an optimum was reached around 10^-4 M. At concentrations 10^-1 M only a reduced response was obtained, based probably on an unspecific osmotic or ionic effect. The germination response was inhibited by La³⁺, an antagonist of Ca²⁺. From these results it is concluded that Ca²⁺ influx from the medium into the spores may be an important event in phytochrome-mediated germination. In the absence of Ca²⁺ the R-stimulated system remained capable of responding to Ca²⁺, added as late as 40 h after R. Moreover, Ca²⁺ was effective even if added after the active form of phytochrome, Pfr, had been abolished by far-red (FR) 24 h after R. Thus, the primary effect of Pfr, that initiates the transduction chain, does not require calcium. Coupling of Pfr to subsequent dark reactions has been investigated by R-FR irradiations with various dark intervals. The resulting escape kinetics were characterized by a lag phase (6 h) and half-maximal escape from FR reversibility (19 h). These kinetics were not significantly changed by the presence or absence of calcium. Thus, direct interaction of Pfr and calcium is not a step in the transduction chain initiated by the active form of photochrome.Abbreviations EGTA ethyleneglycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - FR far-red light - Pr red-light-absorbing form of phytochrome - Pfr far red-light-absorbing form of phytochrome - Pipes piperazine-1,4-bis(2-ethanesulfonic acid) - R red light A preliminary report of this work was presented at the XIV Int. Bot. Congr., Berlin (West), Germany, Book of Abstracts, 2-116a-5 (1987)     

Ionic mechanism and role of phytochrome-mediated membrane depolarisation in caulonemal side branch initial formation in the mossPhyscomitrella patens

In caulonemal filaments of the mossPhyscomitrella patens (Hedw.), red light triggers a phytochrome-mediated transient depolarisation of the plasma membrane and the formation of side branch initials. Three-electrode voltage clamp and ion flux measurements were employed to elucidate the ionic mechanism and physiological relevance of the red-light-induced changes in ion transport. Current-voltage analyses indicated that ion channels permeable to K⁺ and Ca²⁺ are activated at the peak of the depolarisation. Calcium influx evoked by red light coincided with the depolarisation in various conditions, suggesting the involvement of voltage-gated Ca²⁺ channels. Respective K⁺ fluxes showed a small initial influx followed by a dramatic transient efflux. A role of anion channels in the depolarising current is suggested by the finding that Cl^– efflux was also increased after red light irradiation. In the presence of tetraethylammonium (10 mM) or niflumic acid (1 M), which block the red-light-induced membrane depolarisation and ion fluxes, the red-light-promoted formation of side branch initials was also abolished. Lanthanum (100 M), which inhibits K⁺ fluxes and part of the initial Ca²⁺ influx activated by red light, reduced the development of side branch initials in red light by 50%. The results suggest a causal link between the red-light-induced ion fluxes and the physiological response. The sequence of events underlying the red-light-triggered membrane potential transient and the role of ion transport in stimulus-response coupling are discussed in terms of a new model for ion-channel interaction at the plasma membrane during signalling.Abbreviations [Ca²⁺]_c cytosolic free Ca²⁺ - I-V current-voltage - E equilibrium potential - Pr red-light-absorbing phytochrome form - Pr far-red-light-absorbing phytochrome form - SPQ 6-methoxy-l-(3-sulphonatopropyl)quinolinium - TEA tetraethylammonium     

Time courses for phytochrome-induced enzyme levels in phenylpropanoid metabolism (phenylalanine ammonia-lyase,naringenin-chalcone synthase) compared with time courses for phytochrome-mediated end-product accumulation (anthocyanin,quercetin)

Time course for changes in the levels of enzymes characteristic of general phenylpropanoid metabolism (phenylalanine ammonia-lyase, PAL; EC 4.3.1.5) and of the flavonoid-glycoside branch pathway (naringenin-chalcone synthase, CHS; EC 2.3.1.74) were measured in the cotyledons of mustard (Sinapis alba L.) seedlings and compared with the rates of accumulation of related end products (anthocyanin and quercetin). Induction of enzyme levels and of end-product accumulation was carried out with red and far-red (FR) light, operating via phytochrome. The data are compatible with the concept that the phytochrome-mediated appearance of enzymes such as PAL and CHS is indeed a prerequisite for the appearance of anthocyanins and flavonols. However, there is no close correlation between enzyme levels and the rates of synthesis of end products which could justify the identification of specific rate-limiting enzymes. Rather, the data indicate that there is a second phytochrome-dependent step, beyond enzyme induction, where the actual rate of flavonoid accumulation is determined. Anthocyanin and quercetin accumulation respond differently to light. However, the relative action of continuous FR, red light pulses and stored phytochrome signal is the same in both cases. This indicates that the mode of operation of phytochrome is the same in both cases. The two syntheses differ only in the degree of responsiveness towards phytochrome. The time course for changes in CHS levels in continuous FR, i.e. under conditions of phytochrome photosteady state, is similar to the time course for PAL levels whereas the time courses in darkness, following transfer from FR to darkness, are totally different. In the case of CHS, a transient rise is observed whereas, with PAL, an instantaneous drop in enzyme level occurs after transfer from FR to darkness. It is concluded that the stored phytochrome signal operates in darkness in the case of CHS but not in the case of PAL.Abbreviations c continuous - CHS naringenin-chalcone synthase (EC 2.3.1.74) - FR far-red light (3.5 W·m^-2) - PAL phenylalanine ammonia-lyase (EC 4.3.1.5) - Pfr phytochrome (far-red absorbing) - Pr phytochrome (red absorbing) - R red light (6.8 W·m^-2) - RG9-light long-wavelength far-red light obtained with RG9 glass filter - [Pfr]/[Ptot], whereby - Ptot total phytochrome (Pr+Pfr)