Blue-Light-Regulated Expression of Genes for Two Early Steps of Chlorophyll Biosynthesis in Chlamydomonas reinhardtii

In light:dark-synchronized cultures of Chlamydomonas reinhardtii, the genes encoding the enzymes for two early steps of chlorophyll biosynthesis, glutamate-1-semialdehyde aminotransferase (gsa) and [delta]-aminolevulinic acid dehydratase (alad), are expressed at high levels early in the light phase, just prior to a rapid burst of chlorophyll synthesis. Induction of gsa mRNA in synchronized cells is totally dependent on light, whereas induction of alad mRNA occurs to approximately one-half the light-induced level even in cells kept in the dark during the light phase and appears to be dependent on the cell cycle or a circadian rhythm. gsa mRNA and alad mRNA accumulation is induced by light that was passed through blue (400-480 nm) or green (490-590 nm) filters but not by light that was passed through orange (>560 nm) or red (>610 nm) filters, indicating the participation of a blue-light photoreceptor system rather than a protochlorophyllide- or rhodopsin-based photoreceptor. Light induction of gsa mRNA accumulation is absent in a carotenoid-deficient mutant, which suggests that a carotenoid-containing blue-light photoreceptor is involved. In contrast, pretreatment of wild-type cells with either of two flavin antagonists, phenylacetic acid and KI, does not prevent the light induction. In the later part of the light phase, the gsa mRNA level decreases more rapidly than that of alad mRNA. Turnover studies indicate that the half-life of alad mRNA is twice that of gsa mRNA. This difference in mRNA stability partially accounts for the more rapid decline in gsa mRNA levels after the peak of light induction is reached. Thus, differential blue-light induction and stability of mRNAs regulates the expression of these two chlorophyll biosynthetic genes.     

Red-light and blue-light photoreceptors controlling chlorophyll a synthesis in the red alga Porphyra umbilicalis and in the green alga Ulva rigida

Chlorophyll synthesis is stimulated by red light in the green alga Ulva rigida C. Ag. and in the red alga Porphyra umbilicalis (L.) Kützing. Because the effect of red light showed some far-red reversibility in successive red and far-red light treatments, the involvement of phytochrome or a phytochrome-like photoreceptor is suggested. The extent of the response is dependent on exposure and photon fluence rate of red-light pulses. In addition to the effect of red light, a strong stimulation of chlorophyll synthesis by blue light was only observed in Ulva rigida. The effect of blue light shows also some far-red reversibility. In the green alga the accumulated chlorophyll is higher after blue light pulses than after red light pulses. In Porphyra umbilicalis , however, the contrary is observed. In Ulva rigida the involvement of a blue light photoreceptor in addition to phytochrome or a phytochrome-like photoreceptor is proposed. The different responses to red and blue light in both algae are explained in terms of their adaptation to the natural light environment.     

Regulation of light-harvesting chlorophyll-binding protein mRNA accumulation in Chlamydomonas reinhardi. Possible involvement of chlorophyll synthesis precursors

Light induction of light-harvesting chlorophyll a/b-binding protein (LHCP) mRNA accumulation was studied in light-dark synchronized cultures of Chlamydomonas reinhardi. LHCP mRNA accumulation was prevented by the chlorophyll-synthesis inhibitor alpha,alpha-dipyridyl which blocks late steps in the chlorophyll biosynthetic pathway and leads to the accumulation of the porphyrin intermediate magnesium protoporphyrin methyl ester. LHCP mRNA accumulated normally, however, when chlorophyll synthesis was blocked by inhibitors such as hemin and levulinic acid which interfere with early steps in the chlorophyll biosynthesis pathway prior to the formation of magnesium protoporphyrin methyl ester. Similar effects were observed in the light induction of LHCP mRNA levels in protoporphyrin IX-accumulating mutants, brc-1 and brs-1. These mutants have low levels of LHCP mRNA when grown under heterotrophic conditions in the dark where they accumulate protoporphyrin IX. However, LHCP mRNA is light-induced in brc-1 which synthesizes chlorophyll in the light and presumably consumes porphyrin intermediates in doing so. These results suggest that the chlorophyll-synthesis intermediates, magnesium protoporphyrin methyl ester and its immediate precursors, inhibit by a feedback mechanism the light induction of LHCP mRNA accumulation. Low magnesium protoporphyrin methyl ester levels permit the light-induced accumulation of LHCP mRNA, whereas high magnesium protoporphyrin methyl ester levels destabilize LHCP mRNA regardless of the illumination conditions. Preliminary experiments show that LHCP mRNA accumulation in C. reinhardi is stimulated by blue light, and not by red light which stimulates LHCP mRNA accumulation in higher plants.     

Inhibitory Action of Blue and Far-Red Light in the Flowering of Lemna paucicostata

In a new strain of short-day duckweed (Lemna paucicostata T-101), blue and far-red light-induced inhibition of flowering was investigated. Flowering of this strain failed to be induced under a short-day photoperiod of blue and far-red light, although it responded as a typical short-day plant in red and white light. When the short-day photoperiod of blue or far-red light was terminated by a 15 min red light pulse, flowering recovered completely. This inducing effect of red light was reversed by subsequent exposure to far-red light. Furthermore, it could be demonstrated that 30 min of blue light completely reversed the flowering inductive effect of 5 min red light and vice versa. Evidence is presented suggesting that the inhibitory action of blue and far red light may be due to the lowering of phytochrome P_fr levels below those required to start the dark reactions which lead to flowering. These results are discussed in relation to the time measurement system of photoperiodism.     

Ginkgo biloba retains functions of both type I and type II flowering plant phytochrome

While the photoreceptor systems of flowering plants have been well studied, the origins of these gene families and their functions are only partially understood. To begin to resolve the evolutionary origins of angiosperm photoreceptor function, we have studied the photomorphogenic responses of the early diverging gymnosperm Ginkgo biloba. Here, we describe the effects of continuous white light, red light, far-red light, and blue light on stem length, chlorophyll accumulation, Lhcb mRNA accumulation, and plastid development. Differences in the efficacy of these light regimes on de-etiolation in Ginkgo suggest separate but complementary roles for red and blue light-sensing systems. Additionally, the unique manner in which developmental regulation occurs in Ginkgo reveals a far-red high irradiance response different from both angiosperm and other gymnosperm species. We conclude from these data that Ginkgo contains a functional complement to both flowering plant type I and type II phytochromes, as well as independent blue light-sensing system(s). The implications of these findings are discussed with respect to the evolution of higher plant photoreceptors.     

A Flavin Binding Cryptochrome Photoreceptor Responds to Both Blue and Red Light in Chlamydomonas reinhardtii

Cryptochromes are flavoproteins that act as sensory blue light receptors in insects, plants, fungi, and bacteria. We have investigated a cryptochrome from the green alga Chlamydomonas reinhardtii with sequence homology to animal cryptochromes and (6-4) photolyases. In response to blue and red light exposure, this animal-like cryptochrome (aCRY) alters the light-dependent expression of various genes encoding proteins involved in chlorophyll and carotenoid biosynthesis, light-harvesting complexes, nitrogen metabolism, cell cycle control, and the circadian clock. Additionally, exposure to yellow but not far-red light leads to comparable increases in the expression of specific genes; this expression is significantly reduced in an acry insertional mutant. These in vivo effects are congruent with in vitro data showing that blue, yellow, and red light, but not far-red light, are absorbed by the neutral radical state of flavin in aCRY. The aCRY neutral radical is formed following blue light absorption of the oxidized flavin. Red illumination leads to conversion to the fully reduced state. Our data suggest that aCRY is a functionally important blue and red light-activated flavoprotein. The broad spectral response implies that the neutral radical state functions as a dark form in aCRY and expands the paradigm of flavoproteins and cryptochromes as blue light sensors to include other light qualities.     

Interactions of green or red light with blue light on the dark closure of Albizzia pinnules

The interactions of green or red light with blue light on the dark closing of Albizzia julibrissin Durazz. pinnules have been investigated. Irradiations at 430, 450 and 470 nm progressively delay dark closing with increasing photon fluence rates. Red or green light alone has no effect. However, when the blue fluence rate is low, both red and green light interact with it and increase the delaying effect of the blue light. When the blue fluence rate is high, green light interacts with it to negate some of the effectiveness of the blue light, while red light has no effect. This is similar to results obtained previously with far-red light. It is suggested that the same unidentified photoreceptor is operating in both the far-red and blue regions. The results also indicate the presence of a blue-only absorbing photoreceptor whose action is increased by phytochrome.     

Blue light-dependent in vivo and in vitro phosphorylation of Arabidopsis cryptochrome 1

Cryptochromes are photolyase-like blue/UV-A light receptors that regulate various light responses in animals and plants. Arabidopsis cryptochrome 1 (cry1) is the major photoreceptor mediating blue light inhibition of hypocotyl elongation. The initial photochemistry underlying cryptochrome function and regulation remain poorly understood. We report here a study of the blue light-dependent phosphorylation of Arabidopsis cry1. Cry1 is detected primarily as unphosphorylated protein in etiolated seedlings, but it is phosphorylated in plants exposed to blue light. Cry1 phosphorylation increases in response to increased fluence of blue light, whereas the phosphorylated cry1 disappears rapidly when plants are transferred from light to dark. Light-dependent cry1 phosphorylation appears specific to blue light, because little cry1 phosphorylation is detected in seedlings treated with red light or far-red light, and it is largely independent from phytochrome actions, because no phytochrome mutants tested significantly affect cry1 phosphorylation. The Arabidopsis cry1 protein expressed and purified from insect cells is phosphorylated in vitro in a blue light-dependent manner, consistent with cry1 undergoing autophosphorylation. To determine whether cry1 phosphorylation is associated with its function or regulation, we isolated and characterized missense cry1 mutants that express full-length CRY1 apoprotein. Mutant residues are found throughout the CRY1 coding sequence, but none of these inactive cry1 mutant proteins shows blue light-induced phosphorylation. These results demonstrate that blue light-dependent cry1 phosphorylation is closely associated with the function or regulation of the photoreceptor and that the overall structure of cry1 is critical to its phosphorylation.     

Evidence from studies with acifluorfen for participation of a flavin-cytochrome complex in blue light photoreception for phototropism of oat coleoptiles

The diphenyl ether acifluorfen enhances the blue light-induced absorbance change in Triton X100-solubilized crude membrane preparations from etiolated oat (Avena sativa L. cv. Lodi) coleoptiles. Enhancement of the spectral change is correlated with a change in rate of dark reoxidation of a b-type cytochrome. Similar, although smaller, enhancement was obtained with oxyfluorfen, nitrofen, and bifenox. Light-minus-dark difference spectra in the presence and absence of acifluorfen, and the dithionite-reduced-minus oxidized difference spectrum indicate that acifluorfen is acting specifically at a blue light-sensitive cytochrome-flavin complex. Sodium azide, a flavin inhibitor, decreases the light-induced absorbance change significantly, but does not affect the dark reoxidation of the cytochrome. Hence, it is acting on the light reaction, suggesting that the photoreceptor itself is a flavin. Acifluorfen sensitizes phototropism in dark-grown oat seedlings such that the first positive response occurs with blue light fluences as little as one-third of those required to elicit the same response in seedlings grown in the absence of the herbicide. Both this increase in sensitivity to light and the enhancement of the light-induced cytochrome reduction vary with the applied acifluorfen concentration in a similar manner. The herbicide is without effect either on elongation or on the geotropic response of dark-grown oat seedlings, indicating that acifluorfen is acting specifically close to, or at the photoreceptor end of, the stimulus-response chain. It seems likely that the flavin-cytochrome complex serves to transduce the light signal into curvature in phototropism in oats, with the flavin moiety itself serving as the photoreceptor.     

Adventitious shoot regeneration from Begonia × erythrophylla petiole sections is developmentally sensitive to light quality

The influence of light quality on organogenesis in vitro was investigated using Begonia  ×  erythrophylla petiole explants. Pre-treatment of in vitro donor plants by growth in the dark or under far-red or blue light reduced their competence for shoot formation when compared with those grown under red or white light. Culture of competent petiole explants under far-red, blue light or in the dark reduced the number of shoots produced per explant compared to those cultured under red or white light. Explants were found to be developmentally sensitive to both far-red and blue light, because meristem, but not primordia development was inhibited. In addition, blue light inhibition of shoot formation is not mediated directly through phytochrome, as few shoots formed on explants cultured under a mixture of red and blue light which resulted in a high P _fr/ P _tot (0.82) and would allow shoot formation in the absence of blue light. Unlike the inhibitory influence of far-red light, which is reversible, exposure to blue light permanently reduces an explant's competence for shoot formation. Our results suggest that phytochrome and an independent blue light photoreceptor, possibly a cryptochrome, can regulate shoot production from B. erythrophylla petiole explants.     

Photocontrol of stem growth

Rapid and measurable growth rate changes that occur in seedling stems upon illumination serve as an excellent means to analyze signal transduction. Growth kinetic studies have shown how red, far-red and blue light signals are transduced via the solitary and/or coordinated action of known plant photoreceptors. These reports are consistent with current findings describing light-induced photoreceptor interaction and compartmentation.     

Missense mutation in the amino terminus of phytochrome A disrupts the nuclear import of the photoreceptor

Phytochromes are the red/far-red photoreceptors in higher plants. Among them, phytochrome A (PHYA) is responsible for the far-red high-irradiance response and for the perception of very low amounts of light, initiating the very-low-fluence response. Here, we report a detailed physiological and molecular characterization of the phyA-5 mutant of Arabidopsis (Arabidopsis thaliana), which displays hyposensitivity to continuous low-intensity far-red light and shows reduced very-low-fluence response and high-irradiance response. Red light-induced degradation of the mutant phyA-5 protein appears to be normal, yet higher residual amounts of phyA-5 are detected in seedlings grown under low-intensity far-red light. We show that (1) the phyA-5 mutant harbors a new missense mutation in the PHYA amino-terminal extension domain and that (2) the complex phenotype of the mutant is caused by reduced nuclear import of phyA-5 under low fluences of far-red light. We also demonstrate that impaired nuclear import of phyA-5 is brought about by weakened binding affinity of the mutant photoreceptor to nuclear import facilitators FHY1 (for FAR-RED ELONGATED HYPOCOTYL1) and FHL (for FHY1-LIKE). Finally, we provide evidence that the signaling and degradation kinetics of constitutively nuclear-localized phyA-5 and phyA are identical. Taken together, our data show that aberrant nucleo/cytoplasmic distribution impairs light-induced degradation of this photoreceptor and that the amino-terminal extension domain mediates the formation of the FHY1/FHL/PHYA far-red-absorbing form complex, whereby it plays a role in regulating the nuclear import of phyA.     

Blue Light-Induced Phototropic Response and the Intracellular Photoreceptive Site in Adiantum Protonemata

Blue light-induced phototropism in Adiantum protonemata wasinvestigated with microbeam irradiation. Brief irradiation withblue light effectively induced a phototropic response when itwas applied to a half-side of the apical 200d µm regionof a protonema. The phototropic response was partly reversedby the subsequent far-red light irradiation but the full reversalof the response was not observed even when the fluence of far-redlight was increased. In the far-red reversible part of the response,blue/far-red photoreversibility was repeatedly observed. Thus,both phytochrome and a blue light-absorbing pigment (other thanphytochrome) seem to be involved in the blue light-induced phototropicresponse in Adiantum protonemata. Furthermore, detailed studiesof the far-red light effect on the fluence-response curve forblue lightinduced phototropism revealed that the concomitantmediation by the two receptors was limited to the response inthe relatively higher fluence range of blue light and that theblue light-absorbing pigment alone was responsible in the lowerfluence range. In the higher fluence range, the response mediatedby the blue light-absorbing pigment became saturated and thephytochrome response became evident, indicating a differencein the sensitivities of the two receptor pigments to blue light. When various regions of half-sides of protonemata were irradiatedwith a blue microbeam of 10 µm width, irradiation at theapical 5–25 µm region was most effective both forphytochrome- and blue light-absorbing pigment-mediated response,indicating that the site of blue light perception is almostidentical for each response. (Received July 14, 1986; Accepted September 26, 1986)     

Dark reduction of QA in intact barley leaves as an effect of lowered CO2 concentration monitored by chlorophyll a luminescence and chlorophyll a F0 dark fluorescence

When the CO₂ concentration in the atmosphere above an intact barley leaf was lowered in the dark after illumination, chlorophyll a luminescence and chlorophyll a dark fluorescence were stimulated. The stimulation was induced by lowered levels of CO₂ in a wide concentration range including concentrations well above that saturating photosynthesis. The stimulation of luminescence by lowered CO₂ concentrations was more pronounced after far-red excitation than after white light excitation. The difference in response to lowered CO₂ concentrations after white/far-red excitation was less pronounced for fluorescence than for luminescence. Stimulation of luminescence was more pronounced when the CO₂ concentration was lowered in an O₂-containing atmosphere than under anaerobic conditions. It is concluded that lowering of the CO₂ concentration in the dark after illumination causes a partial reduction of the primary Photosystem II acceptor Q_A.     

AN INTERPRETATION OF DOSE-RESPONSE CURVES FOR LIGHT-INDUCED CELL ELONGATION IN FERN PROTONEMATA

Protonemata of Onoclea sensibilis and Diyopteris filix-mas elongate in response to both red and far-red light. The promotion caused by far-red is larger than that caused by red light. This phenomenon differs from a typical response to phytochrome, the photoreceptor pigment immediately suggested by the activity of red and far-red light. The phenomenon has been explained by two different hypotheses, one of which holds that phytochrome is solely responsible for the response, whereas the other postulates an interaction between phytochrome and P₅₈₀, a yellow-green light absorbing pigment, to account for the response. The hypothesis that phytochrome is the sole photoreceptor leads to some specific predictions concerning the shapes of the dose-response curves for light-induced protonema elongation. These predictions were tested with both continuous and short-term irradiation. In all instances saturating far-red light caused greater elongation than did saturating red light, and no dose of red light duplicated the activity of saturating far-red light. Other experiments tested the interactions of red and far-red light and the effects of different doses of yellow-green light on protonema elongation. The results of many of the experiments were not in agreement with the hypothesis that phytochrome is the sole photoreceptor, whereas they were in agreement with the assumption that filament elongation is controlled by both phytochrome and P₅₈₀.