Inhibition of oxygen evolution following illumination of Chlorella cells with far-red light.

The effect of brief far-red illumination on Chlorella pyrenoidosa cells has been investigated. An inhibition of oxygen evolution occurs 20 to 30 s after the end of the far-red illumination. This inhibition occurs in a step following the initial charge separation process by the System II centers. It is reversible in the light through a purely photochemical process.     

Changes in enzymatic activities in etiolated bean seedling leaves after a brief illumination

The phytochrome controlled increase in total protein in the primary leaf pair of etiolated bean (Phaseolus vulgaris var. Black Valentine) seedlings, which occurs during growth in the dark subsequent to a brief illumination, was investigated. Enzymes from the chloroplasts, the mitochondria, and the soluble cytoplasm all increase in total activity after the illumination.

The total protein and the ribulose carboxylase increases are not inhibited by FUdR, an inhibitor of DNA synthesis. Cycloheximide, an inhibitor of protein synthesis, applied at a time when the ribulose carboxylase activity increase has already commenced, blocks further increase. It was concluded that the total protein and the enzyme increases in the leaf are the result of increases in the per cell levels.

The initial brief illumination is saturating, but 40 minutes later the seedlings have acquired the ability to respond to a second brief illumination. The rate of increase in ribulose carboxylase activity in seedlings that have been illuminated twice is greater than the rate in seedlings that have been illuminated only once.

Far-red light prevents further increase in enzyme activity 48 hours after the initial illumination. There is a lag period interposed between the time of illumination with far-red light and the time at which the seedlings show the greatest effect of far-red light. It was concluded that the phytochrome influence on protein synthesis is not at the terminal steps.

     

Phytochrome mediated induction of nitrate reductase activity in etiolated maize leaves

The effects of red and far-red light on the enhancement of in vitro nitrate reductase activity and on nitrate accumulation in etiolated excised maize leaves were examined. Illumination for 5 min with red light followed by a 4-h dark period caused a marked increase in nitrate reductase activity, whereas a 5-min illumination with far-red light had no effect on the enzyme activity. The effect of red light was completely reversed by a subsequent illumination with the same period of far-red light. Continuous far-red light also enhanced nitrate reductase activity. Both photoreversibility by red and far-red light and the operation of high intensity reaction under continuous far-red light indicated that the induction of nitrate reductase was mediated by phytochrome. Though nitrate accumulation was slightly enhanced by red and continuous far-red light treatments by 17% and 26% respectively, this is unlikely to account for the entire increase of nitrate reductase activity. The far-red light treatments given in water, to leaves preincubated in nitrate, enhanced nitrate reductase activity considerably over the dark control. The presence of a lag phase and inhibition of increase in enzyme activity under continuous far-red light-by tungstate and inhibitors of RNA synthesis and protein synthesis-rules out the possibility of activation of nitrate reductase and suggests de novo synthesis of the enzyme affected by phytochrome.     

Control of Flowering of Xanthium pensylvanicum by Red and Far-red Light

A study was made of the effects of various durations, intensities and combinations of red and far-red light interruptions on the flowering responses of Xanthium pensylvanicum Wallr. A dual response to treatments of far-red light was observed. In short dark periods, far-red light alone did not greatly affect flowering but was able to overcome the inhibition of flowering caused by red light. In dark periods longer than 15 hours, far-red inhibited flowering and added to rather than overcame the inhibition by red light. The dark period length required for far-red inhibition remained the same whether far-red was given at the start or at the eighth hour of darkness.

In 48-hour dark periods Xanthium showed 3 responses to additions of red and far-red light breaks: A) response to red light; B) response to far-red light; and C) response to red followed by far-red light. Red light given any time in the first 30 hours of darkness overcame the inhibitory effect of far-red light given at either the start or the eighth hour of darkness. Red light given later than the thirtieth hour did not overcome the far-red effect.

Approximately the same energy of red light was required to overcome the inhibitory effect of far-red at the second hour of darkness as was required to produce maximum red light inhibition at the eighth hour. Although far-red light was most inhibitory when given early in a long dark period, approximately the same energy of far-red light was required to saturate the far-red response at the fourth, eighth and sixteenth hours.

The results are discussed in relation to other reports of far-red inhibition of flowering in short-day plants.

     

Photophysiology of Kalanchoë Seed Germination

Germination of Kalanchoé blossfeldiana seeds is absolutely light-requiring and needs repeated daily light periods. With increasing length of the photoperiod there was a gradual escape from the far-red inhibition. This escape depended also upon the duration of the far-red exposure: 10-second far-red caused a strong inhibition after a 10- to 30-minute photoperiod and did not inhibit after a 4-hour day, although the effect of the latter was completely suppressed by 5 minutes far-red. The action of a 12-hour photoperiod was not reversed by 10 minutes far-red but it was by 12 hours far-red. Light intensity and temperature during the photoperiod were two other important factors influencing the escape from far-red inhibition. The common features of this escape displayed in very different photomorphological responses are stressed. In order to explain our results in terms of phytochrome action, we distinguish two effects of white light: 1) on the initial photoconversion of the inactive to the active P_FR form 2) on the much slower transformation of P_FR to a reacted form P*_FR; the latter reaction can also proceed in darkness, but is enhanced by light and is dependent upon light intensity and temperature; this reacted phytochrome is not reversible by a brief far-red illumination.     

Formation of tyrosine radicals in photosystem II under far-red illumination

Photosystem II (PS II) contains two redox-active tyrosine residues on the donor side at symmetrical positions to the primary donor, P₆₈₀. Tyr_Z, part of the water-oxidizing complex, is a preferential fast electron donor while Tyr_D is a slow auxiliary donor to P₆₈₀ ⁺. We used PS II membranes from spinach which were depleted of the water oxidation complex (Mn-depleted PS II) to study electron donation from both tyrosines by time-resolved EPR spectroscopy under visible and far-red continuous light and laser flash illumination. Our results show that under both illumination regimes, oxidation of Tyr_D occurs via equilibrium with Tyr_Z ^? at pH 4.7 and 6.3. At pH 8.5 direct Tyr_D oxidation by P₆₈₀ ⁺ occurs in the majority of the PS II centers. Under continuous far-red light illumination these reactions were less effective but still possible. Different photochemical steps were considered to explain the far-red light-induced electron donation from tyrosines and localization of the primary electron hole (P₆₈₀ ⁺) on the Chl_D1 in Mn-depleted PS II after the far-red light-induced charge separation at room temperature is suggested.     

Appearance of Photochemical Activity in Isolated Chloroplasts from Far-red-illuminated Leaves of Phaseolus vulgaris

Chloroplast development was followed in intact bean leaves illuminatedwith far-red light by extracting chloroplasts at various timesto assay photosynthetic activities. Photochemical activity wasdetected in isolated chloroplasts prior to the times which werepreviously reported for intact leaf discs. Cyclic phosphorylationwas observed in isolated chloroplasts after 8 h of far-red illuminationwhile non-cyclic electron transport and phosphorylation weremeasurable after 12 and 16 h of illumination respectively. TheP/2e ratios were less than 0.5 after 24 h of far-red exposurebut approached a value of 1.0 by 60 h of illumination. Ammoniumchloride (10^–3 M) had little effect on electron transportin isolated chloroplasts until after 24 h of far-red illumination.Chlorophyll a accumulated slowly from the onset of far-red illuminationwhile chlorophyll b was not detected until after 48 h of far-redexposure. Leaf fresh weight increased four-fold over the 60h illumination period. Electron microscopy of isolated chloroplasts from far-red-illuminatedleaves indicated the presence of unfused primary thylakoidsby 12 h of exposure and prolamellar bodies throughout the entire60 h illumination period. Grana were not observed in isolatedchloroplasts nor were they induced by a 2 min exposure of thechloroplasts to 172 000 lx of white light. O₂ evolution in leaf discs of far-red-illuminated plants wasmeasurable after 16 h of illumination, attained a maximum valueby 36 h of far-red exposure, and then declined. Net CO₂ fixationwas observed in leaf discs after 8 h of far-red illuminationand the rates remained constant for an additional 16 h, beforeincreasing at least two-fold.     

Phototropism and polarotropism of primary chloronemata of the moss Physcomitrella patens: responses of the wild-type

Primary chloronemata growing from germinated spores of the moss Physcomitrella patens adopt one of two preferred polarotropic orientations depending on the wavelength and photon fluence rate of monochromatic light. Growth is mainly parallel to the electrical vector of plane polarised light in blue light and higher fluence rates of red light, and perpendicular to the electrical vector in the green and far-red regions of the spectrum and in low fluence rates of red light. The transition between the two polarotropic orientations, at wavelengths where it can be observed, usually occurs over a narrow range of fluence rates, and at this point the filaments do not grow randomly but tend to adopt in approximately equal numbers one of the preferred directions of growth. The primary chloronemata are positively phototropic in far-red light and in red light of low fluence rates, but tend to grow at right angles to the incident light in high fluence rates of red light. Simultaneous illumination with a high fluence rate of red light and a low fluence rate of far-red light causes a marked increase in the percentage of filaments growing towards the red light source at the expense of those growing at right angles to it, supporting the hypothesis that in red and far-red light, at least, the responses are controlled by the photoequilibrium of a phytochrome pool.     

Opposite response groups of short-day plants to the spectral composition of the main light period and to end-of-day red or far-red irradiations

Two response groups were found among short-day plants grownin blue green-houses having a high or low far-red admixture.In one group flowering was promoted by low far-red and delayedby high far-red; the other group reacted in an opposite manner.In plants grown under 8-hr day and 16-hr night regimes, floweringin the low far-red group was promoted by red and inhibited byfar-red illumination preceding the 16-hr nights; in the highfar-red group flowering was promoted by far-red and inhibitedby red illumination preceding the dark periods. In both groupsflowering was inhibited by red light applied in the middle ofthe dark period. (Received March 18, 1974; )     

Phytochrome intermediates and action spectra for light perception by dry seeds

It has previously been demonstrated that far-red irradiation of dry Lactuca sativa L. seeds results in inhibition of subsequent germination. Although red has no effect on dry seeds, a red irradiation following a farred irradiation reverses the effect of far-red. This phenomenon is most noticeable in seeds with artificially raised levels of phytochrome in the far-red absorbing form. Qualitatively similar results have been found for the seeds of Plantago major L., Sinapis arvensis L., and Bromus sterilis L. Action spectra studies on Plantago seeds show that the action peaks for promotion and inhibition of germination of hydrated seeds are at 660 and 730 nanometers, respectively. The action spectrum for inhibition of subsequent germination following irradiation of dry seeds is qualitatively and quantitatively similar to that for hydrated seeds, with an action peak at 730 nanometers, indicating absorption by phytochrome in the far-red absorbing form. However, the action spectrum for the reversal of this far-red effect on dry seeds has a broad peak at 680 nanometers and subsidiary peaks at 650 and 600 nanometers. It is proposed that this effect is due to light absorption by the phytochrome intermediate complex meta-Fa, and that the action spectrum reflects the in vivo absorption properties of this intermediate.     

Phytochrome-mediated germination control of maize caryopses

Maize caryopses sown in water germinate equally well either in darkness or under any light regime. However, when they are imbibed in mannitol solutions, continuous far-red light proves to be strongly inhibitory on the final germination as compared to darkness. Similar but less pronounced inhibition is also exhibited by continuous red or blue light. Intermittent far-red light can partially substitute for continuous far-red light in inhibiting maize caryopsis germination, and its effect is reversed to the intermittent red light level when red light is given immediately after each far-red illumination. These results are interpreted as a proof of existence and involvement of phytochrome in the germination control of maize caryopses, though its manifestation is realized only under osmotic stress.Abbreviations D darkness - FR far-red - R red - B blue - c-FR, c-R, c-B continuous FR, R, B, resp. - i-FR, i-R intermittent FR, R, resp.     

Phytochrome mediated regulation of sucrose phosphate synthase activity in maize

The extractable activity of sucrose phosphate synthase was determined in etiolated seedlings of maize (Zea mays L.), soybean (Glycine max [L.] Merr.), and sugar beet (Beta vulgaris L.) following treatments of changing light quality. A 30-minute illumination of 30 microeinsteins per square meter per second white light produced a three-fold increase in sucrose phosphate synthase activity at 2 hours postillumination when compared to seedlings maintained in total darkness. Etiolated maize seedlings treated with 3.6 microeinsteins per square meter per second of red and far-red light showed a 50% increase and a 50% decrease in sucrose phosphate synthase activity, respectively, when compared to etiolated maize seedlings treated with white light. Maize seedlings exposed for 30 minutes to red followed by 30 minutes to far-red showed an initial increase in sucrose phosphate synthase activity followed by a rapid decrease to control level. Neither soybean or sugar beet sucrose phosphate synthase responded to the 30-minute illumination of white light. Phytochrome is involved in sucrose phosphate synthase regulation in maize, whereas it is not responsible for changes in sucrose phosphate synthase activity in soybean or sugar beet.     

Light-induced turnover of chloroplast cytochrome b-559 in the presence of N-methylphenazonium methosulphate

In the presence of 0.1–5 μM N-methylphenazonium methosulphate approx. 50–70% oxidation of cytochrome b-559 can be induced by far-red light. The oxidation is best observed with long wavelength far-red light (732 nm) of moderate intensities (approx. 10⁴ ergs/cm² per s) and is reversed by subsequent illumination with red light. Concentrations of N-methylphenazonium methosulphate above 5 μM are inhibitory probably due to cyclic electron flow. The far-red oxidation is inhibited by low concentrations of the plastoquinone antagonist 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, while 3-(3,4-dichlorophenyl)-1,1-dimethylurea inhibits red light reduction and increases the amplitude of far-red oxidation. The effect of N-methylphenazonium methosulphate is mimicked by N-methyl-phenazonium ethosulphate, but not by pyocyanine or diaminodurene. Low concentrations (2–3 μM) of N-methylphenazonium methosulphate also stimulate a 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone-inhibitable red light reduction of cytochrome f.     

Spontaneous pigment mutants of Anacystis nidulans selected by growth under far-red light

Under far-red (>650 nm) illumination Anacystis nidulans grows poorly and develops a low chlorophyll content. During continued culture over many generations there are increases in growth rate and in the chlorophyll/phycocyanin ratio, usually occurring in concomitant and stepwise fashion. From such selection cultures six clones have been established which differ from the parent in pigment content and show improved growth rate in far-red light. From the evidence at hand the six clones are presumed to be spontaneous mutants selected under the photosynthetically restrictive condition of far-red illumination.     

Nonmonotonic Redox Conversions of P700 in Leaves Irradiated with Far-Red Light Originate from Variable Contributions of Several Alternative Electron Transport Pathways during the Induction Period

The origin of nonmonotonic changes in the redox state of P700, the primary electron donor of PSI, was investigated on predarkened barley (Hordeum vulgare L.) leaves exposed to far-red light. To accomplish this, the relaxation kinetics of absorbance changes at 830 nm, reflecting the dark reduction of P700⁺, were measured at different stages of the induction curve. The onset of far-red light resulted in rapid oxidation of P700, which was followed by its partial reduction and subsequent slow oxidation of P700 to a steady-state level. This steady-state level was usually attained within 10 s under far-red light. The relative contribution of the slow kinetic component of P700⁺ reduction decreased in parallel with the transient photoreduction of P700⁺ and increased upon a subsequent stage of P700 photooxidation. The contribution of the middle component to the dark reduction of P700⁺ increased monotonically with the length of far-red light irradiation. The relative amplitude of the fast component of P700⁺ reduction increased sharply during the first 3 s of irradiation and decreased upon longer light exposures. The rates of fast and slow components of dark reduction of P700⁺ remained constant upon illumination of dark-adapted leaves with far-red light for 1 s and longer periods. Thus, nonmonotonic changes in the redox state of P700 in barley leaves exposed to far-red light reflect variable contributions of few alternative electron transport pathways characterized by different rates of electron donation to PSI. The results show the principle possibility of switching-over between alternative pathways of PSI-related electron transfer within one complex of this photosystem. Such switching may occur irrespective of active operation or inhibition of ferredoxin-dependent electron transport.     

Photobiology of phytochrome-mediated growth responses in sections of stem tissue from etiolated oats and corn

The far-red reversibility of the phytochrome-controlled stimulation of elongation of coleoptile sections by low fluence red light has been characterized in subapical coleoptile sections from dark-grown Avena sativa L., cv Lodi seedlings. The fluence dependence of the far-red reversal was the same whether or not the very low fluence response is also expressed. The capacity of far-red light to reverse the red light-induced response began to decline if the far-red light was given more than 90 minutes after the red irradiation. Escape was complete if the far red irradiation was given more than 240 minutes after the red irradiation. Sections consisting of both mesocotyl and coleoptile tissue from dark-grown Avena seedlings were found to have physiological regulation of the very low fluence response by indole 3-acetic acid and low external pH similar to that seen for sections consisting entirely of coleoptile tissue. The fluence-dependence of the red light-induced inhibition of mesocotyl elongation was studied in mesocotyl sections from dark grown Zea mays L. hybrid T-929 seedlings. Ten micromolar indole 3-acetic acid stimulates the control elongation of the sections, while at the same time increasing the sensitivity of the tissue for the light-induced inhibition of growth by a factor of 100.     

Phytochrome Regulation of Nitrite Reductase--a Chloroplast Enzyme--in Etiolated Maize Leaves

Nitrite reductase in the excised etiolated leaves of maize showedthe photoreversibility by red and far-red light. Five minutesof red light illumination lead to a 130% increase in the enzymeactivity which was reversed by far-red light. The kinetics ofnitrite reductase activity under continuous far-red light showeda lag phase of 1 hr. (Received January 17, 1981; Accepted February 20, 1981)