Red Irradiation Research Articles

Phytochrome Action during Prechilling Induced Germination of Betula papyrifera Marsh

Seeds of paper birch (Betula papyrifera Marsh.) were induced to germinate by prechilling at 3 C or by red light. The light requirement was mediated by phytochrome and the action of phytochrome during prechilling was investigated. Red irradiation (R) prior to prechilling markedly enhanced the effectiveness of the prechilling treatment in inducing subsequent germination at 18 C. Reversal of this enhancement by far-red irradiation (FR) was more effective when FR was supplied after a 1-week prechill treatment than after a 2-week treatment. The R enhancement effect exhibited a sharp drop as prechilling temperature was increased from 5 to 7 C. This decline is consistent with a membrane phase transition at about 7 C where Pfr action is diminished by a loss in sensitivity of its receptor sites. Although phytochrome action was observed during prechilling treatments, the seeds failed to germinate at prechilling temperatures. Therefore, it was concluded that while potentiation of germination by Pfr occurred during prechilling, some other reaction(s) leading to radicle protrusion requires higher temperatures. In one seed source loss of germination potential was observed with protracted storage at 3 C. This was prevented by R supplied during the prechilling treatment. Taken collectively the data suggest that action of phytochrome during prechilling is accentuated in these seeds by two factors: (a) an increase in the sensitivity (or number) of Pfr receptor sites; and (b) preservation of Pfr by deferment of thermal reversion.

Oak seedlings grown in different light qualities

Oak seedlings (Quercus robur L.) were germinated in darkness for 3 weeks and then given continuous long wavelength far‐red light (LFR; wavelengths longer than 700 nm). A control group of seedlings was kept in darkness. After 2 additional weeks the chlorophyll formation ability in red light was examined in the different seedlings. The stability of the protochlorophyll(ide) and chlorophyll(ide) forms to high intensity red irradiation was also measured.Oak seedlings grown in darkness accumulated protochlorophyll(ide) (6 μg per g fresh matter). Absorption spectra and fluorescence spectra indicated the presence of more protochlorophyll(ide)628–632 than protochlorophyllide650–657. The level of protochlorophyll(ide) was higher in leaves of plants cultivated in LFR light (13 μg per g fresh matter) than in leaves of dark grown plants. 12% of the protochlorophyll(ide) was esterified in both cases.The level of protochlorophyll(ide)628–632 in LFR grown oaks varied with the age of the leaves, being higher in the older (basal) leaves, but also in the very youngest (top‐most) leaves. The ability of the leaves to form photostable chlorophyll in red light showed a similar age dependence, being low in rather young and in older leaves. A low ability to form photostable chlorophyll thus appears to be correlated with a high content of protochlorophyll(ide)628–632.Upon irradiation only the protochlorophyllide650–657 was transformed to chlorophyllide. After this phototransformation the chlorophyllide peak at 684 nm shifted to 671 nm within about 30 min in darkness. This shift took place without any accompanying change in photostability of the chlorophyll(ide). Upon irradiation with strong red light a similar shift took place within one minute. This indicates that the chlorophyllide after phototransformation was rather loosely bound to the photoreducing enzyme. The development towards photostable chlorophyll forms consists of three phases and is discussed.

A partial characterization of the long-wavelength ?Activated? far-red absorbing form of phytochrome

The spectral properties of Pfr in extracts prepared from red-irradiated oat tissue are similar to those for Pfr as measured in vivo, with a spectral peak at about 732 nm. The presence or absence of Mg(2+) or EDTA in the extraction medium is without effect on the spectral properties of the Pfr extracted from red-irradiated tissue. In contrast, the spectral properties of Pfr formed in vitro in extracts of non-irradiated oat tissue cleared of particulate matter are markedly different, the absorption peak being at about 725 nm. However, the red irradiation of the crude extract prior to its being cleared of particulate matter, results in the formation of a Pfr-species with spectral properties similar to Pfr as seen in vivo, suggesting the requirement for some particulate fraction for the formation of the long-wavelength Pfr form. The long-wavelength "activated" form of Pfr once extracted and partially purified is highly stable both at 0° and 25°C. Photoconversion of this long-wavelength form of Pfr to Pr initially results in the formation of a Pr species with a peak at 662 nm. In contrast, the peak location of Pr as isolated from non-irradiated tissue is at 667 nm.The 662 nm form of Pr is unstable and with time is transformed in a temperature-dependent reaction to a form with a peak at 667 nm. At 25°C this reaction is rapid, occurring in minutes, while at 0°-4°C it requires a few hours. At 25°C, immediate photoconversion of the 662 nm form of Pr back to Pfr results in the reformation of the long-wavelength form of Pfr. However, after being incubated for 15 min in the Pr form the peak shifts to 667 nm and the Pfr subsequently formed exhibits spectral characteristics identical to those of Pfr as formed in vitro, i.e., with a peak at 725 nm.

Some Observations Related to Red — Far Red Antagonism in Germination of Spores of the Cyanobacterium Anabaena fertilissima

Effects of intermittent short duration exposures of red (650 nm) and/or far red (725-750 nm) illuminations on spore germination were studied in Anabaena fertilissima . Spore germination was induced by red light while far red irradiation inhibited it. In reversal experiments, induction of germination by red light was annulled when the spores were subsequently exposed to far red light whereas inhibition due to far red illumination was overcome when spores were subjected to consequent red light treatment. Such antagonistic effects of red - far red irradiations provide evidence for the operation of photoinduction-photoreversal phenomenon mediated by a pigment which seems to be functionally similar to phytochrome of higher plants.

Phytochrome Responses to End-of-Day Irradiations in Light-grown Corn Grown in the Presence and Absence of Sandoz 9789

Corn seedlings were grown in white light in the absence and presence of the chlorosis-inducing herbicide San 9789. The resulting green and achlorophyllous seedlings were used to investigate phytochrome-mediated responses to end-of-day far red irradiation and reversal of these responses by subsequent red irradiation. Mesocotyl and coleoptile elongation increased in response to end-of-day far red irradiation, whereas the anthocyanin content of the coleoptiles was decreased. All three responses were reversible by red irradiation following the far red. Dose-response curves for far red induction and red reversal of these responses did not differ significantly for plants grown in the presence or absence of San 9789. Thus, San 9789 appears to affect neither phytochrome itself nor the response system involved. Chlorophyll screening likewise does not affect phytochrome relationships for these responses.

Factors Controlling the Retardation of Chlorophyll Degradation during Senescence of Detached Statice (Limonium sinuatum) Flower Stalks

Summary Detachment of green, flowering Limonium sinuatum flower stalks stimulated chlorophyll degradation. The loss of chlorophyll, which is regarded as a marker process of senescence, was accelerated when flower stalks were kept in the dark. Exposure of sections of the stalk to continuous white light or to brief red irradiations inhibited chlorophyll degradation. The effectiveness of the brief red i rradiations was similar to that of continuous white light, and could be completely reversed if each red irradiation was followed by a brief far-red irradiation. It is concluded that phytochrome is present in the flower stalks and mediates the retardation of flower stem senescence by light. Treatment of excised flower stalks with cytokinin (BAP) or gibberellin (GA3) significantly retarded the destruction of chlorophyll which occurred in the dark.

Growth control by phytochrome of de‐etiolated bean hypocotyls mediated by chlorophyll fluorescence

AbstractThe elongation of hypocotyls excised from de‐etiolated seedlings of beans (Phaseolus vulgaris L. cv. British Wax) is inhibited by light, blue and red irradiations being equally effective. Conditions which decrease chlorophyll fluorescence, such as CO2‐free air, abolish the inhibitory effect of blue irradiation and enhance the inhibition by red light. Conversely, conditions which increase chlorophyll fluorescence, such as a N2 atmosphere or irradiation through a chlorophyll filter, abolish the inhibitory effect of red light and enhance the inhibition by blue irradiation. The inhibitory effect of blue light is reversible by red irradiation under increased fluorescence as well as by far red. We propose that the chlorophyll fluorescence excited by blue and red irradiations in λF > 660 nm and λF > 720 nm, respectively, is responsible for the inhibitory effect of blue light and the reduction of the inhibitory effect of non fluorescing red light. Both red and blue wavelengths seem, therefore, to control hypocotyl elongation through phytochrome.

Phytochrome Modification and Light-enhanced, In Vivo-induced Phytochrome Pelletability

Phytochrome that was induced by red irradiation in vivo to pellet with subcellular material and that was released from the pellet by removal of divalent cations exhibited altered characteristics. Compared to phytochrome extracted in a soluble red-absorbing form from etiolated tissue, pelleted and released phytochrome, which was also assayed in the red-absorbing form even though pelleted in the far-red-absorbing form, showed 50% greater micro complement fixation activity, eluted closer to the void volume of a Sephadex G-200 column, and electrophoresed more slowly on sodium dodecyl sulfate-polyacrylamide gels. Data presented here document that phytochrome pelleted in the far-red-absorbing form differs from soluble phytochrome extracted from nonirradiated tissue. These data, however, do not permit the conclusion that there is a causal relationship between pelletability and phytochrome modification.

Phytochrome Modifies Blue-light-induced Electrical Changes in Corn Coleoptiles

Unilateral blue light administered to corn coleoptile segments produces no alteration of transmembrane potential on the light side, and only a small and slow hyperpolarization on the dark side. Red light causes a 5-15 millivolt depolarization in cells on the light side causes and somewhat smaller effects on the dark side. Blue given after red causes a rapid hyperpolarization on both sides of the coleoptile. The effect of the potentiating red preirradiation is probably due to phytochrome, being largely abolished by far-red given after red, but before the blue light. The effect of prior red irradiation decays in the dark, showing a half-time of about 45 minutes at room temperature. This rapid cooperativity between phytochrome and the phototropic pigment may indicate a common locale, possibly in a membrane.

Studies of the Mechanism of Enhancement of Phytochrome-dependent Lettuce Seed Germination by Prechilling

Temperature and kinetic studies were performed to examine the mechanism by which prechilling stimulates phytochrome-dependent seed germination in lettuce, Lactuca sativa, L. cv. Grand Rapids. Imbibed seeds were given a short far red irradiation and one day of dark incubation at 20 C to establish very low levels of the far red-absorbing form of phytochrome-(Pfr). Germination was greatly stimulated by subsequent prechilling treatments when they were followed by a second short far red irradiation. Prechilling therefore increased germination sensitivity to the low, normally inhibitory Pfr levels established by far red irradiation. This sensitivity increased with lowered prechilling temperature to a maximum near 4 C. It was linearly dependent upon duration of prechilling at 4 C up to a near maximal response at 10 hours, and it decayed in a converse manner when seeds were returned to 20 C after 10 hours at 4 C. Prechilling also increased germination responses to subsequent periods of high levels of Pfr which were initiated by red and terminated by far red irradiations. High Pfr periods adequate to promote the germination of unchilled seeds produced sharp inflections at 18 C in the dependence of germination on prechilling temperature. Rates of phytochrome potentiation of germination were not affected by prechilling. The response to prechilling fit a mechanism involving homeoviscous adaptation of membrane lipids to temperature.

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.

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.

Induction of Secondary Dormancy in Chenopodium bonus-henricus L. Seeds by Osmotic and High Temperature Treatments and Its Prevention by Light and Growth Regulators

Factors controlling the establishment and removal of secondary dormancy in Chenopodium bonus-henricus L. seeds were investigated. Unchilled seeds required light for germination. A moist-chilling treatment at 4 C for 28 to 30 days removed this primary dormancy. Chilled seeds now germinated in the dark. When chilled seeds were held in the dark in -8.6 bars polyethylene glycol 6000 solution at 15 C or in water at 29 C a secondary dormancy was induced which increased progressively with time as determined by subsequent germination. These seeds now failed to germinate under the condition (darkness) which previously allowed their germination. Continuous light or daily brief red light irradiations during prolonged imbibition in polyethylene glycol solution at 15 C or in water at 29 C prevented the establishment of the secondary dormancy and caused an advancement of subsequent germination. Far red irradiations immediately following red irradiation reestablished the secondary dormancy indicating phytochrome participation in "pregerminative" processes. The growth regulator combination, kinetin + ethephon + gibberellin A(4)+A(7) (GA(4+7)), and to a relatively lesser extent GA(4+7), was effective in preventing the establishment of the secondary dormancy and in advancing the germination or emergence time. Following the establishment of the secondary dormancy by osmotic or high temperature treatments the regulator combination was relatively more active than light or GA(4+7) in removing the dormancy. Prolonged dark treatment at 29 C seemed to induce changes that were partially independent of light or GA(4+7) control. The data presented here indicate that changes during germination preventing dark treatment determine whether the seed will germinate, show an advancement effect, or will become secondarily dormant. These changes appear to be modulated by light and hormones.

Relationship between Chloroplast Development and ent-Kaurene Biosynthesis in Peas

Treatment of etiolated pea (Pisum sativum (L. cv. Alaska) seedlings with 2'-isopropyl-4'-(trimethylammonium chloride)-5'-methylphenyl piperidine-1-carboxylate (Amo-1618) prior to irradiation with white light inhibits photomorphogenesis and formation and stacking of thylakoid membranes in the chloroplasts, as well as (-)-kaur-16-ene (ent-kaurene)biosynthesis. Exogenous gibberellic acid also inhibits greening. A crudely determined action spectrum for the photoinduction of ent-kaurene biosynthesis shows two peaks, one in the blue region at 458 to 490 nanometers and another in the red region at 606 to 678 nanometers. The possible participation of phytochrome in the photoinduction of ent-kaurene biosynthesis is indicated by comparative effects of red, far red, and alternating red/far red irradiations on enhancement of enzyme activity. The activity of blue light as well as red shows a similarity of the photoinduction of ent-kaurene synthesis activity to the high irradiance responses, and indicates probable participation of a second photoreceptor. From these observations, it is concluded that photoinduction of ent-kaurene biosynthesis and chloroplast development in shoots are closely linked processes.

Photoreversible Calcium Fluxes Induced by Phytochrome in Oat Coleoptile Cells

The chromometallic dye murexide was used to measure photoreversible Ca fluxes in apical tips of etiolated oat coleoptiles and in suspension cultures of protoplasts derived from the coleoptile segments. Phytochrome presence in the protoplasts was indicated by a repeatably photoreversible DeltaA(725 - 800 nm) of >0.001 A centimeters(-1), recorded on a dual wavelength spectrophotometer. Concentrations of Ca in the solution bathing the cells were observed to change photoreversibly, red irradiation inducing an increase in the medium Ca concentration and subsequent farred irradiation inducing a decrease down to near dark control levels. These changes could be measured in media with or without exogenously added Ca. Protoplasts from green primary leaves of oat, which had no spectro-photometrically detectable phytochrome, showed no photoreversible Ca fluxes when measured by the same method. These data imply that red light induces an efflux of Ca from phytochrome-containing cells and that far red light can reverse this change by promoting a Ca reentry into these cells.

Aspects of Seed Dormancy in Fall Panicum (Panicum dichotomiflorum)

Caryopses (seeds) of fall panicum(Panicum dichotomiflorumMichx.) have a dormancy that is not easily overcome. Although accelerated after-ripening for 4 weeks was partially successful in overcoming dormancy, a dark imbibition at 35 C for about a week was more effective and also interacted strongly with an accelerated after-ripening. In either case, activation of phytochrome by red irradiation was required to induce germination. Similarly, alternating temperatures allowed germination of red-treated seeds but constant temperatures did not. More seeds germinated in sealed flasks than in dishes. The enhancement of germination in sealed flasks appeared to be related to increased CO2levels. The data suggested a multiple dormancy in fall panicum seeds. One dormancy, the most intense, was relieved by high-temperature imbibition. The residual dormancy was relieved by active phytochrome and alternating temperatures.

The Influence of Light on Geotropism in Cress Roots

Light affects the growth and orientation of roots of cress seedlings (Lepidium sativum L. cv. Curled). The effects are manifest either as increased rates of geotropic curvature or, if the roots are horizontal, as distorted and crinkled forms of growth. Blue, red, and far-red irradiation can bring about these effects, but with differences of detail: at equal fluence rates during the period of geostimulus, blue is more effective than red at increasing the rate of geocurvature; however, with irradiation prior to a geostimulus, only the stimulatory effects of red irradiation persist for 2-4 h of darkness. Short periods (5 min) of radiation, if given at the time of geostimulus, enhance geocurvature, again with blue most, and far-red least, effective, but there are no clear indications of red/far-red reversibility. The possibility of there being more than one photosystem responsible for the effects of white light on the geotropic responsiveness of roots is discussed.

ANALYSIS OF STARCH ACCUMULATION AND GERMINATION IN ONOCLEA SPORES

Previous studies have shown that low fluences of light accelerate starch accumulation and enhance germination of Onoclea spores. Fluence response curves for induction of starch accumulation were compared with fluence response curves for enhancement of germination in order to determine if the two photoresponses in Onoclea spores have a common photoreceptor. Fluence response curves indicate that both responses were proportional to the log of the fluence and that the relative fluence efficiencies of the four wavelength regions tested were similar for induction of both germination and starch accumulation. Red (600–720 nm) irradiation was the most efficient, while green (500–600 nm), blue (400–520 nm), and far‐red (720–900 nm) irradiations showed a decreasing order of efficiency for induction of the responses. A correlation coefficient between the amount of starch accumulated as a result of red irradiation and the final percent germination was calculated to be 0.964. These results support the hypothesis that a common photoreceptor mediates both photoinduced germination and starch accumulation. 3‐(3,4‐dichlorophenyl)‐1,1‐dimethylurea (DCMU) inhibits photosynthesis by blocking the flow of electrons from Photosystem II to Photosystem I. At 0.1 mM DCMU failed to inhibit both photoinduced starch acumulation and germination. This result and the greater efficiency of red than blue light, the low fluence functional for induction, and the fluence dependency argue against the participation of photosynthesis in photoinduced starch accumulation. A similar conclusion has been previously drawn for photoenhancement of Onoclea spore germination. Additionally, the effects 0.01–1.0 mm cycloheximide and 100 μl/l ethylene on photoinduced starch accumulation were investigated. Neither agent inhibited starch accumulation, whereas both substances inhibited germination 70–90% when applied at a time coincidental with the period of rapid starch accumulation. These results indicate that the photoinduction of starch accumulation does not have an ethylene sensitive stage nor does it require protein synthesis as does photoenhancement of germination of Onoclea spores.

Analysis of Starch Accumulation and Germination in Onoclea Spores

Previous studies have shown that low fluences of light accelerate starch accumulation and enhance germination of Onoclea spores. Fluence response curves for induction of starch accumulation were compared with fluence response curves for enhancement of germination in order to determine if the two photoresponses in Onoclea spores have a common photoreceptor. Fluence response curves indicate that both responses were proportional to the log of the fluence and that the relative fluence efficiencies of the four wavelength regions tested were similar for induction of both germination and starch accumulation. Red (600–720 nm) irradiation was the most efficient, while green (500–600 nm), blue (400–520 nm), and far-red (720–900 nm) irradiations showed a decreasing order of efficiency for induction of the responses. A correlation coefficient between the amount of starch accumulated as a result of red irradiation and the final percent germination was calculated to be 0.964. These results support the hypothesis that a common photoreceptor mediates both photoinduced germination and starch accumulation. 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) inhibits photosynthesis by blocking the flow of electrons from Photosystem II to Photosystem I. At 0.1 mM DCMU failed to inhibit both photoinduced starch acumulation and germination. This result and the greater efficiency of red than blue light, the low fluence functional for induction, and the fluence dependency argue against the participation of photosynthesis in photoinduced starch accumulation. A similar conclusion has been previously drawn for photoenhancement of Onoclea spore germination. Additionally, the effects 0.01–1.0 mm cycloheximide and 100 μl/l ethylene on photoinduced starch accumulation were investigated. Neither agent inhibited starch accumulation, whereas both substances inhibited germination 70–90% when applied at a time coincidental with the period of rapid starch accumulation. These results indicate that the photoinduction of starch accumulation does not have an ethylene sensitive stage nor does it require protein synthesis as does photoenhancement of germination of Onoclea spores.

Reaction of a peroxidase activity to red and far red light in relation to the floral induction of spinach

A basic peroxidase present in spinach leaves ( Spinach oleracea) exhibited a very rapid fluctuation after red or far red irradiations. This response, measured immediately after a 2 min exposure to R or FR, was dependent on the state of the plant (vegetative or floral). The response characteristic of the floral state appeared after one inductive photoperiod (approx. 14 h light) and was still observed in induced plants returned to short days for one week. This peroxidase reaction may be used to detect the first steps of the photoperiodic induction occurring in the leaves.