LIGHT-GROWN PLANTS OF THE CUCUMBER LONG HYPOCOTYL MUTANT EXHIBIT BOTH LONG-TERM AND RAPID ELONGATION GROWTH RESPONSES TO IRRADIATION WITH SUPPLEMENTARY FAR-RED LIGHT

Seedlings of wild type (WT) and the lh mutant of Cucumis sativus were grown in white light and given supplementary far-red radiation either to the whole plant, or via fiber-optic probes directed at the apical region of the hypocotyl. In both WT and mutant seedlings, reducing the ratio of red to far-red radiation resulted in significant increases in extension growth. Direct measurement of extension rate by transducer techniques revealed that the mutant seedlings responded to additional far-red radiation by an increment in extension rate that was equivalent to that observed with the WT seedlings. Assuming that the lh mutant is deficient in phytochrome B (Kendrick and Nagatani, The Plant Journal 1: 133–139, 1991), these results indicate that although phytochrome B clearly has an important role in the induction of the shade avoidance syndrome, it is unlikely to be the sole responsible receptor.     

EXTENSION GROWTH AND ANTHOCYANIN RESPONSES OF PHOTOMORPHOGENIC TOMATO MUTANTS TO CHANGES IN THE PHYTOCHROME: PHOTOEQUILIBRIUM DURING THE DAILY PHOTOPERIOD

Four genotypes of tomato (Lycopersicon esculentum Mill.) in the genetic background ‘Aha Craig’ were used: an aurea (au) mutant, deficient in the bulk light-labile phytochrome pool; a high pigment (hp) mutant, showing exaggerated phytochrome responses at the time of de-etiolation; the au,hp double mutant and the isogenic wild type (WT). A dramatic increase in plant height resulting from an increase in the length of all internodes for each of the genotypes studied was observed upon reduction of the red light: far-red light photon ratio (R:FR) from 6.90 to 0.13 by addition of FR for the whole photoperiod. A concomitant increase in leaf length was also observed. Since au and au,hp mutants, deficient in the bulk light-labile phytochrome pool, respond to this reduction in the R:FR these data demonstrate that the phytochrome pool that mediates this response is present and fully functional. Anthocyanin was detectable in the comparably developed young growing leaves of the WT and hp mutant under the high R:FR, but not in the au and au,hp mutants, suggesting that the potential for anthocyanin synthesis is correlated with the presence of the bulk light-labile phytochrome pool. The kinetics of anthocyanin decrease in the young growing leaves were investigated in the hp mutant and the results suggest a very rapid cessation of flavonoid biosynthesis upon reduction of the R:FR. The functions of different phytochrome types are discussed.     

RESPONSES OF LIGHT-GROWN WILD-TYPE and LONG-HYPOCOTYL MUTANT CUCUMBER SEEDLINGS TO NATURAL and SIMULATED SHADE LIGHT*

The photomorphogenic control of hypocotyl extension growth was characterized in wild type (WT) and long hypocotyl (Ih) mutant seedlings of cucumber (Cucumis sativus L.) grown under natural radiation in outdoor and glasshouse experiments. Hypocotyl extension growth of WT plants was promoted by supplementing sunlight with far-red light during the photoperiod, by reducing the amount of blue light reaching either the whole shoot or the hypocotyl, and by reducing the amount of UV reaching the whole shoot.The Ih seedlings only responded to a reduction in UV-B levels. Both WT and Ih seedlings showed phototropic responses to the direction of blue light. Increasing degrees of vegetational shade promoted hypocotyl growth of WT plants. The Ih mutant showed no hypocotyl growth promotion by natural shade in glasshouse experiments (no UV-B, low water demand) and a reduced response (10-23% of the WT response, according to pretreatment conditions) in outdoor experiments (UV-B, high water demand).     

ACTION SPECTRA FOR INHIBITION OF HYPOCOTYL GROWTH OF WILD-TYPE PLANTS AND OF THE hy2 LONG-HYPOCOTYL MUTANT OF Arabidopsis thaliana L.

Abstract— An analysis was made by action spectroscopy, using the Okazaki Large Spectrograph, of the inhibition of hypocotyl elongation of wild-type plants and the hy2 mutant of Arabidopsis thaliana. Two day old etiolated seedlings were irradiated for 8 h with monochromatic light and left in the dark for 16 h before measurement of hypocotyl length. Spectrophotometric measurement showed that levels of phytochrome in the etiolated tissue of the hy2 mutant were less than 9% of those in the wild type. The action spectra of the wild type looked like those of high irradiance response and showed peaks at 375, 450, 625 and 725 nm, whereas the action spectra of hy2 showed only the peaks at 375 and 450 nm. Monochromatic light of wavelengths longer than 500 nm had no significant inhibitory effects on hy2 plants. Blue and UV-A light were about five times more effective in the wild type than in hy2 plants. Severe inhibitory effects were observed with UV-B light. It is concluded that inhibition of the growth of the hypocotyl involves combined actions of phytochrome and a putative blue/UV-A photoreceptor(s).     

THE IN VIVO SPECTROPHOTOMETRIC ASSAY OF PHYTOCHROME IN TWO MATURE DICOTYLEDONOUS PLANTS*

Abstract— Mature Sinapis alba L. and Impatiens parviflora DC. were treated with the herbicide norfiuorazon prior to development of the third or second leaf, respectively. This treatment yielded a partially bleached plant capable of normal growth and development. The bleached leaves were used for spectrophotometric phytochrome assay. In mature plants an almost constant level of phytochrome is maintained under continuous white light. The dark kinetics and the response of the phytochrome system to light of various qualities provide further evidence of the stable character of the phytochrome system.     

Two native pools of phytochrome A in monocots: Evidence from fluorescence investigations of phytochrome mutants of rice

Fluorescence investigations of phytochrome (phy) in rice (Oryza sativa L. cv. Nipponbare) mutants deficient in phyA, phyB and phyA plus phyB were performed. Total content of the pigment (P(tot)) and its spectroscopic and photochemical characteristics were determined in different parts of the dark-grown and far-red light (FR)-grown coleoptiles. Spectroscopically, phyA in the phyB mutant was identical to phyA in the wild-type (WT) and the extent of the conversion from Pr to lumi-R at 85 K was the same for phyA in both lines and varied similarly, depending on the part of the coleoptile used. The latter finding proved that phyA in rice is heterogeneous and comprises two phyA populations, phyA' and phyA". Functional properties of phyA were also determined. In the dark the phyB mutant had a higher content of phyA, inactive protochlorophyllide (Pchlide633) and active protochlorophyllide (Pchlide655) than WT and its coleoptile was longer, indicating that phyB may affect the development of WT seedlings in the dark. Constant FR drastically reduced the content of phyA, Pchlide633 and Pchlide655 and brought about coleoptile shortening and appearance of the first leaf, whereas pulsed FR of equal fluence was less effective. This suggested that the reactions were primarily of the high irradiance responses type, which are likely to be mediated by phyA'. The effects on protochlorophyllide biosynthesis and growth responses type were more pronounced in the phyB mutant than in the WT seedlings, which can be connected with the higher phyA' content in the phyB mutant and/or phyB interference with its action in WT seedlings. In the phyA mutant induction of Pchlide633 and Pchlide655 biosynthesis was observed under constant FR, indicating that phyC may be responsible for this effect.     

A METHOD FOR MEASURING PHYTOCHROME IN PLANTS GROWN IN WHITE LIGHT

Abstract— Quantitative spectrophotometric measurement of phytochrome in plants grown for relatively long periods of time in white light is not possible due to the presence of chlorophyll. A method is described that prevents the accumulation of chlorophyll permitting such direct spectrophotometric measurement in light-grown tissue. Oat seedlings grown in the presence of the herbicide San 9789† (Norflurazon) for 6 days in constant light have considerably less chlorophyll than etiolated seedlings exposed to 1 min of light. Phytochrome concentrations measured in vivo and in vitro in these herbicide-treated plants were found to be about 2% of the level in etiolated tissue.     

PHOTOCONTROL OF ANTHOCYANIN SYNTHESIS IN TOMATO SEEDLINGS: A GENETIC APPROACH*

The photocontrol of anthocyanin synthesis in dark-grown seedlings of tomato (Lycopersicon esculentum Mill.) has been studied in an aurea (au) mutant which is deficient in the labile type of phytochrome, a high pigment (hp) mutant which has the wild-type level of phytochrome and the double mutant au/hp , as well as the wild type. The hp mutant demonstrates phytochrome control of anthocyanin synthesis in response to a single red light (RL) pulse, whereas there is no measurable response in the wild type and au mutant. After pretreatment with 12 h blue light (BL) the phytochrome regulation of anthocyanin synthesis is 10-fold higher in the hp mutant than in the wild type, whilst no anthocyanin is detectable in the au mutant, thus suggesting that it is the labile pool of phytochrome which regulates anthocyanin synthesis. The au/hp double mutant exhibits a small (3% of that in the hp mutant) RL/far-red light (FR)-reversible regulation of anthocyanin synthesis following a BL pretreatment. It is proposed that the hp mutant is hypersensitive to the FR-absorbing form of phytochrome (P_fr) and that this (hypersensitivity) establishes response to the low level of P_fl. (below detection limits in phytochrome assays) in the au/hp double mutant.     

PHYTOCHROME CONTROL OF THE DEVELOPMENT OF PHOTOPHOSPHORYLATION

In the cotyledons of the mustard (Sinapis ah L.) seedling the development of the capacity for photophosphorylation is strongly influenced by pretreatment of the seedling with red light pulses. The red light acts through phytochrome. After a red light pretreatment the capacity for photophosphorylation increases linearly with the chlorophyll content, at least up to 30 min after the onset of continuous white light. It is proposed that the reaction chain required for photophosphorylation is completed under the influence of phytochrome even in the absence of chlorophyll. As soon as chlorophyll becomes available photophosphorylation functions instantaneously. Without a red light pretreatment there is a lag of more than 15 min before photophosphorylation becomes detectable after the onset of continuous white light even though chlorophyll a is available. Although phytochrome strongly influences the rate of chlorophyll accumulation as well it is improbable that the control by phytochrome of development of photophosphorylation and of chlorophyll accumulation are causally connected.     

Evidence for yellow light suppression of lettuce growth

Researchers studying plant growth under different lamp types often attribute differences in growth to a blue light response. Lettuce plants were grown in six blue light treatments comprising five blue light fractions (0, 2, 6% from high-pressure sodium [HPS] lamps and 6, 12, 26% from metal halide [MH] lamps). Lettuce chlorophyll concentration, dry mass, leaf area and specific leaf area under the HPS and MH 6% blue were significantly different, suggesting wavelengths other than blue and red affected plant growth. Results were reproducible in two replicate studies at each of two photosynthetic photon fluxes, 200 and 500 mumol m-2 s-1. We graphed the data against absolute blue light, phytochrome photoequilibrium, phototropic blue, UV, red:far red, blue:red, blue: far red and 'yellow' light fraction. Only the 'yellow' wavelength range (580-600 nm) explained the differences between the two lamp types.     

PHYTOCHROME AND EFFECTS OF SHADING ON GROWTH OF WOODLAND PLANTS

qrowth of Circaea lutetiaim plants was studied in various locations in or near a mixed deciduous woodland. Morphological changes resulting from increased shading included increases in leaf area ratio, specific leaf area and specific water content. Parallel measurements with a spectroradi-ometer confirmed that shading involved a reduction in both light fluence rate and light quality (e.g. red/far-red ratio). Phytochrome P_fr/P status was also studied by spectrophotometric measurements on Avena seedling test material and by biological (Lactuca seed germination) assay. Attempts were made to demonstrate phytochrome controlled changes in plant morphology under controlled environment, using both end-of-day far-red treatment and far-red enrichment of the main light period. Effects of natural shading were most clearly simulated by varying light fluence rate while maintaining a constant but high red/far-red ratio     

PHOTORESPONSES OF Arabidopsis SEEDLINGS EXPRESSING AN INTRODUCED OAT phyA cDNA: PERSISTENCE OF ETIOLATED PLANT TYPE RESPONSES IN LIGHT-GROWN PLANTS

The photocontrol of hypocotyl elongation has been studied in etiolated and light-grown wild type (WT) Arabidopsis thaliana (L. Heynh) seedlings, and in two homozygous isogenic lines that have been transformed with the oat phy A gene coding sequence under the control of the cauliflower mosaic virus (CaMV) 35S promoter. For etiolated seedlings the inhibition of hypocotyl elongation by continuous broad band far-red light (FR) is saturated at much lower photon fluence rates in the transgenic seedlings compared with WT seedlings. Furthermore, whereas de-etiolation of WT seedlings leads to loss of responsiveness of the hypocotyls to prolonged FR, de-etiolated transgenic seedlings continue to show a pronounced FR-mediated inhibition of elongation. This may reflect the persistence of a FR-high irradiance response (HIR) mediated by the introduced oat phytochrome A. Although the hypocotyls of light-grown transgenic seedlings display a qualitatively normal end-of-day FR growth promotion, such seedlings display an aberrant shade-avoidance response to reduced red:far-red ratio (R:FR). These results are discussed in relation to the proposal that the constitutive expression of phytochrome A leads to the persistence of photoresponse modes normally restricted to etiolated plants.     

Phytochrome Control of Anthocyanin Biosynthesis in Tomato Seedlings: Analysis Using Photomorphogenic Mutants

Anthocyanin biosynthesis has been studied in hypocotyls and whole seedlings of tomato (Lycoperskon esculentum Mill.) wild types (WTs) and photomorphogenic mutants. In white light (WL)/dark (D) cycles the fri¹ mutant, deficient in phytochrome A (phyA), shows an enhancement of anthocyanin accumulation, whereas the tri¹ mutant, deficient in phytochrome Bl (phyBl) has a WT level of anthocyanin. Under pulses of red light (R) or R followed by far-red light (FR) given every 4 h, phyA is responsible for the non-R/FR reversible response, whereas phyBl is partially responsible for the R/FR reversible response. From R and blue light (B) pretreatment studies, B is most effective in increasing phytochrome responsiveness, whereas under R itself it appears to be dependent on the presence of phyBl. Anthocyanin biosynthesis during a 24 h period of monochromatic irradiation at different flu-ence rates of 4 day-old D-grown seedlings has been studied. At 660 nm the fluence rate-response relationships for induction of anthocyanin in the WT are similar, yet complex, showing a low fluence rate response (LFRR) and a fluence rate-dependent high irradiance response (HIR). The high-pigment-1 (hp-1) mutant exhibits a strong amplification of both the LFRR and HIR. The fri¹ mutant lacks the LFRR while retaining a normal HIR. In contrast, a transgenic tomato line overexpressing the oat PHYA3 gene shows a dramatic amplification of the LFRR. The tri¹ mutant, retains the LFRR but lacks the HIR, whereas the fri¹, tri¹ double mutant lacks both components. Only an LFRR is seen at 729 nm in WT; however, an appreciable HIR is observed at 704 nm, which is retained in the tri¹ mutant and is absent in the fri¹ mutant, indicating the labile phyA pool regulates this response component.     

The Development of Solid Spectral Filters for the Regulation of Plant Growth

Phthalocyanine derivatives have been prepared and incorporated into polymer films for use as spectral filters for the modification of plant growth. The unusual absorption characteristics of phthalocyanines, notably the narrow absorption band in the visible region of the electromagnetic spectrum, allows the selective filtering of wavelengths necessary for excitation of either of the two isomeric phytochrome species. The resulting change in the photostationary state for the phytochrome is shown to have a marked effect on the growth characteristics of both chrysanthemums and antirrhinums. Chrysanthemums (short-day plants) grown under a far-red-absorbing film showed a reduction in height (∼14%) and internodal length compared to those grown under a red-absorbing film or a control with no absorption in the visible part of the spectrum; both leaf number and time to flowering were not affected by the presence of the spectral filter. For antirrhinums (long-day plants) both a reduction in height and a substantial increase in leaf area (70%) of the plants grown under the far-red-absorbing film were observed, although flowering was delayed. The potential of these filters as replacements for chemical plant growth regulators is discussed.     

Kinetics of Stem Elongation in Light-Grown Tomato Plants. Responses to Different Photosynthetically Active Radiation Levels by Wild-Type and aurea Mutant Plants

Abstract— In this research, we measured the short- and long-term, stem elongation responses of wild-type and aurea(au) mutant tomato plants to different photosynthetically active radiation (PAR) levels by using linear voltage transducers. Stem elongation was continuously measured in green tomato plants over 2.75 days, under 12 h light/12 h dark photoperiods or in darkness after a 6 h irradiation period. There is no significant difference in stem elongation between wild-type plants pregrown at either LOO or 400 μmol m^?2 s^?1 and then exposed to 12 h photoperiods. However, in the au mutant there is a very large difference between plants pregrown under 100 or 400 umol m ^?2 s^?1 and then exposed either to 12 h photoperiods or to continuous darkness. Total stem elongation of the wild type appears to be maximal at 100 umol m^?2 s^?1, while that of the au mutant appears to be maximal with PAR 400 umol m^?2 s^?1. Wild-type plants displayed PAR-dependent (in the range 100-800 umol m^?2 s^?1) inhibition of growth both during the day and during the night. In contrast, the au mutant showed a fluence-rate-dependent promotion of growth during the dark periods in the range of 10-400 umol m^?2 s^?1. Large, fast and opposite changes in stem elongation rate at the light/dark and dark/light transitions were present in both genotypes. Internode elongation rate in the first half of the night was always modest in wild-type tomato, whereas it increased rapidly in the au mutant. Stem elongation rate of wild type starts to increase after about 6 h in darkness, showing the typical time course of escape from Pfr-mediated inhibition of elongation by an end-of-day response. The role of phytochrome level and type in sensing light quantity is discussed.     

Effects of short-term irradiation on photoinhibition and accumulation of mycosporine-like amino acids in sun and shade species of the red algal genus Porphyra

The effect of irradiance (40 and 840 micromol photons m(-2) s(-1)) of short-term (48 h) irradiation on photosynthetic activity (estimated as oxygen evolution and as chlorophyll fluorescence), specific absorption and fluorescence excitation spectra, photosynthetic pigment accumulation (chlorophyll a and biliproteins) and UV-absorbing compounds (mycosporine-like amino acids, MAAs) was investigated in sun and shade species of the red algal genus Porphyra collected in Trondheimsfjord (Norway). In the sun type, high irradiance exposure (840 micromol photons m(-2) s(-1)) did not alter the Chl a concentration, however, exposure to a lower irradiance (40 micromol photons m(-2) s(-1)) for 48 h significantly increased the chlorophyll concentration. The content of MAAs was significantly higher in the suntype than in the shade type algae. Porphyra-334 is the main MAA in this species followed by shinorine. The total content of MAAs significantly (P<0.05) increased in the sun type after 48 h exposure to both high and low irradiances. However, in the shade type, porphyra-334 significantly decreased (P<0.05) after both high and low irradiance exposure. Photosynthetic activity (as oxygen evolution) and the optimal quantum yield (F(v)/F(m)), as an indicator of photoinhibition, decreased under low and high irradiance in the shade type algae and no full recovery was observed when the algae were transferred to very low irradiation.The sun type algae presented a higher capacity of acclimation to increased irradiance than the shade type algae. This high acclimation of sun type algae to short term high irradiance exposure (48 h) is explained by the higher thermal dissipation. This was estimated as the ratio of nonphotochemical quenching related to the light dose (q(N):dose) and by the accumulation of MAAs.     

AN OSCILLATING SYSTEM REGULATING DEVELOPMENT OF PLANTS

Phytochrome conversion shifts the developmental pattern of dark-grown bean (Phaseolus vulgaris) seedlings. Red light was found to initiate rapid oscillations in a system that links illumination with subsequent growth. A single 8 s flash of red light increased the average leaf weight measured 24 h later by 8%. When total illumination was kept constant but the interval between two 4 s flashes was varied, the resulting leaf weight increase was not uniform but depended on the length of the interval between flashes and showed a series of deep minima followed by sharp maxima. Weight increase at the maximum was 60% greater than at the minimum. The minimum-maximum transitions recurred every 45 s for at least 22 min. Four s of far red light interposed between the two red flashes abolished the oscillation. Temperature between 15 and 30°C had no significant influence on the period of the oscillation but the period varied directly with the duration of the initial red flash. A 2 s initial flash resulted in a 35 s period, while 40 s of red light caused a 2 min period. Oat (Avena sativa) and radish (Raphanus sativus) seedlings were found to possess similar oscillating mechanisms of growth regulation. The lack of pronounced temperature effects as well as our other findings, suggest that this oscillating system may participate in the time measuring as well as growth regulating mechanisms by which phytochrome controls circadian periodicity and development.     

CONTROL OF CHLOROPHYLL b BIOSYNTHESIS BY PHYTOCHROME

In the mustard seedling cotyledons, chlorophyll b appears from the very beginning in white light provided that a red light pulse pretreatment was given 12 h prior to the onset of white light. The red light pulses act through phytochrome. Without pretreatment no chlorophyll b is detectable at least during the first 60 min after the onset of white light (25°C). Biogenesis of chlorophyll b specifically depends on the action of phytochrome during the pre-steady state period as well as during the steady state period of chlorophyll accumulation. In light pulse experiments, it was found that formation of chlorophyll b takes place stoichiometrically at the cost of chlorophyll(ide) a.     

PHYTOCHROME REGULATION OF STEM GROWTH AND INDOLE-3-ACETIC ACID LEVELS IN THE lv AND Lv GENOTYPES OF Pisum

Phytochrome influences stem elongation and the mechanism for this is not understood. The levels of indole-3-acetic acid (IAA) were analyzed in an leLv genotype of Pisum sativum L. which responded to end-of-day far-red light by doubling growth rate. The IAA levels in epidermal peels increased 40% after far-red light whereas IAA levels of the entire stem tissue changed insignificantly. This increase was reversible by red light. Under light-grown conditions, the lv mutation increases stem elongation rates by 2–3-fold and is thought to block the transduction of a phytochrome signal. Analysis of the short-term stem elongation kinetics of dark- and light-grown Lv and lv seedlings suggests that lv blocks the action of the light-stable form of phytochrome. The higher growth rate of lv plants was found to be associated with abnormally high epidermal IAA levels typical of far-red treated Lv plants. End-of-day far-red treatments did not substantially increase epidermal IAA levels in lv plants. These observations support the view that phytochrome regulation of stem elongation may occur in part through modulation of epidermal IAA levels. The lv mutation may result in increased internode growth in part by blocking the ability of phytochrome to decrease epidermal IAA levels.     

Phytochrome-regulated expression of genes encoding light-harvesting chlorophyll a/b-protein in two long hypocotyl mutants and wild type plants of Arabidopsis thaliana

The cab genes which encode the light-harvesting chlorophyll a/b-protein (LHCP) are expressed normally with respect to phytochrome regulation in the hy-3 and hy-5 long hypocotyl mutants of Arabidopsis thaliana. In etiolated seedlings of these mutants as well as of the wild type, 1 min of red light elevates cab mRNA levels substantially within 2 h; this increase is reversed if far-red light is given immediately after the red light treatment. We conclude that the genetic defects in these mutants do not affect steps in the signal transduction pathway leading to the regulated expression of cab genes. Additionally, the mRNA from one of the three known A. thaliana cab genes, AB140, is similar in quantity to the mRNAs from the other two, AB165 and AB180, in dark-grown seedlings of hy-3 and hy-5 as well as the parent A. thaliana (Landsberg) after a brief red light treatment. This aspect of cab gene expression differs from the strain Columbia of A. thaliana in which AB140 mRNA is the predominant message. In mature white light-grown plants of the strain Columbia, AB140 as well as a combination of AB165 and AB180 mRNAs are expressed at high levels, suggesting that AB165 and/or AB180 may be developmentally regulated.