Down-regulation of phytochrome mRNA abundance by red light and benzyladenine in etiolated cucumber cotyledons

Northern blot analysis revealed that a single 4.2 kb phytochrome mRNA species was detectable in cotyledons excised from five-day-old etiolated cucumber seedlings. Intact etiolated five-day-old cucumber seedlings were given a red light or benzyladenine treatment, and cotyledons were harvested at various times following treatment. The abundance of phytochrome mRNA in the cotyledons was quantitated using ³²P-labeled RNA probes and slot blot analysis. By 2 h after irradiation the phytochrome mRNA level was reduced to 40% of the initial abundance and reaccumulation began by 3 h after irradiation. Reaccumulation of phytochrome mRNA to the time-zero dark control level was achieved by 10 h after treatment. A decrease in phytochrome mRNA abundance was evident by 2 h after benzyladenine treatment, and a maximal reduction to 45% of the time-zero dark control was attained by 4 h after treatment. No recovery of the phytochrome mRNA level was evident by 8 h after benzyladenine treatment. The abundance of actin mRNA was unaffected by benzyladenine treatment.     

Regulation of phytochrome mRNA abundance in green oat leaves

Abstract. Avena sativa L. (oat) seedings were grown 4 d in continuous white light followed by 3 d in darkness. Probes derived from an oat phytochrome cDNA clone (pAP 3.2) were used in slot blot analyses to measure the abundance of phytochrome mRNA in the distinct etiolated and green portions of the leaves produced by these seedlings. Both the green and etiolated portions accumulated phytochrome mRNA to a level of about 85% of the etiolated seedling level. Subsequent experiments with similar seedlings showed that both the green and etiolated portions were capable of inducing a dramatic decline in phytochrome mRNA abundance in response to a saturating red light pulse. Despite the ability of green portions of oat leaves to accumulate phytochrome mRNA and to down-regulate phytochrome mRNA abundance in response to light, no substantial variation in phytochrome mRNA abundance was observed in green oat seedlings maintained on a 12-h day/12-h night cycle. In the same oat seedlings, the abundance of chlorophyll a/b binding protein mRNA fluctuated dramatically during the day/night cycle.     

Phytochrome A, phytochrome B and other phytochrome(s) regulate ATHB-2 gene expression in etiolated and green Arabidopsis plants

The expression of the Arabidopsis ATHB-2 gene is light-regulated both in seedlings and in adult plants. The gene is expressed at high levels in rapidly elongating etiolated seedlings and is down-regulated by a pulse of red light (R) through the action of a phytochrome other than phytochrome A or B, or by a pulse of far-red light (FR) through the action of phytochrome A. In green plants, the expression of the ATHB-2 gene is rapidly and strongly enhanced by lowering the R:FR ratio perceived by a phytochrome other than A or B. Returning the plant to a high R:FR ratio results in an equally rapid decrease of the ATHB-2 mRNA. Consistently, plants overproducing ATHB-2 show developmental phenotypes characteristic of plants grown in low R:FR: elongated petioles, reduced leaf area, early flowering, and reduced number of rosette leaves. Taken together, the data strongly suggest a direct involvement of ATHB-2 in light-regulated growth phenomena throughout Arabidopsis development.     

Establishment of far‐red high irradiance responses in wheat through transgenic expression of an oat phytochrome A gene

A transgenic wheat line over‐expressing an oat phytochrome A gene under the control of the constitutive maize ubiquitin promoter was generated using a biolistic particle delivery system from immature wheat embryos. The resulting line showed increased levels of total phytochrome A protein in both dark‐grown and light‐grown plants. When grown under continuous far‐red light, seedlings of this line showed additional inhibition of the coleoptile extension in comparison with wild‐type seedlings. Unlike the response of wild‐type seedlings to continuous far‐red, this additional inhibition was dependent on fluence rate and was not observed under half‐hourly pulses of far‐red delivering the same total fluence as the continuous irradiation treatment. These observations suggest that increase in phytochrome A levels in wheat leads to the establishment of a far‐red high irradiation reaction in this monocotyledonous plant. Exposure to continuous red light caused a similar inhibition of coleoptile extension in both the wild types and the transgenic seedlings. When wild‐type seedlings were grown under continuous far‐red, their coleoptiles remained completely colourless and first leaves remained tightly rolled. In contrast, transgenic seedlings grown in the same conditions produced significant levels of anthocyanins in their coleoptiles and their first leaves became unrolled. Taken together, our data suggest that the increased levels of phytochrome A in wheat can change the type of response of some developmental processes to light signals, leading to the generation of a high irradiance reaction which is otherwise absent in the wild types under the conditions used.     

Action spectrum for the effect of day-extensions on flowering and apex elongation in green,light-grown wheat (Triticum aestivum L.)

Fluence-rate response curves for wavelengths from 640 nm to 730 nm were constructed for the day-extension promotion of flowering in green, light-grown, wheat (Triticum aestivum L., cv. Alexandria), a long-day plant. The resultant action spectrum had action maxima at 660 nm and 716 nm and resembles spectra for the high-irradiance reaction (HIR) seen in etiolated plants. Because, the HIR is thought to be controlled by type I pytochrome (that which is most abundant in etiolated tissue) our results indicate the involvement of type I phytochrome in the photomorphogenesis of a light-grown, green plant.Abbreviations Pr red-light-absorbing form of phytochrome - Pfr far-red-light-absorbing form of phytochrome - Ptot total phytochrome level (Pr+Pfr) - HIR high-irradiance reaction - SDP short-day plant(s) - LDP long-day plant(s)     

Phytochrome research in whole plant physiology. Wild-type seedlings versus mutants and transgenic plants

In this report I comment on a small selection of the many interesting papers presented at the European Symposium on Photomorphogenesis in Plants. My major concern arises from problems relating to classical phytochrome treatments versus mutants and transgenic plants as tools in photomorphogenic research on whole plant physiology. Plant responses to visible light are especially evident in etiolated seedlings, although they occur throughout the life cycle of the plant. These characteristics of plant photomorphogenesis in nature can be viewed as representing a continuous developmental series of graded responses to subsequent light exposures. Where the individual, photoautotrophic seedling is moving through this developmental gradient depends on its particular light environment, that is, on both the quality and the quantity of light to which the seedling is exposed. On the contrary, phytochrome treatment of mutants and transgenic seedlings having either deficiency or overexpression of photoreceptors will result in an aberrant kind of physiology.     

Dynamic properties of endogenous phytochrome A in Arabidopsis seedlings.

The dynamic behavior of phytochrome A (phyA) in seedlings of the model plant Arabidopsis was examined by in vivo spectroscopy and by western and northern blotting. Rapid accumulation of phyA was observed, reaching a steady state after 3 d. Both red and far-red light initiated a rapid destruction of the far-red-light-absorbing form of phytochrome (Pfr); the apparent half-life was only 4-fold longer in far-red than in red light. Furthermore, the Pfr-induced destruction of the red-light-absorbing form of phytochrome (Pr) of phyA occurred in darkness with a rate identical to that of Pfr destruction. A 2-fold decrease in mRNA abundance was observed after irradiation, irrespective of the applied light quality. However, reaccumulation occurred rapidly after far-red but slowly after red irradiation, indicating different modes of regulation of phytochrome expression after light-dark transitions depending on the light quality of the preceding irradiation. The wavelength dependency of the destruction rates was distinct from that of mustard, a close relative of Arabidopsis, and was explained on the basis of Pfr-induced Pr destruction and a simple kinetic two-step model. No dark reversion was detectable in the destruction kinetics after a red pulse. From these data we conclude that Arabidopsis phyA differs significantly in several aspects from other dicot phytochromes.     

Isolation and characterization of a cDNA-clone coding for potato type A phytochrome

We have isolated and sequenced a cDNA clone encoding the apoprotein of a potato phytochrome. Based on the deduced amino acid sequence, which shows 78% amino acid identity to the Arabidopsis phyA and 50% identity to the Arabidopsis phyB open reading frame, we have classified this cDNA clone as potato phyA phytochrome. The amino acid immediately preceding cysteine 323, which is the homologue of oat cystein 321, to which the chromophore has been shown to be attached, is a tyrosine residue. This contrasts with six other type A phytochrome sequences from both monocots and dicots that encode serine in this position. As already observed in three other cDNAs isolated from dicot species, the potato phyA clone encodes a short open reading frame (13 amino acids) preceding the phyA open reading frame (1123 amino acids), supporting the idea that this type of leader sequence might be involved in the regulated expression of the phytochrome apoprotein. Southern blot analysis revealed a single phyA gene as well as other related phytochrome sequences in the potato genome. phyA mRNA levels varied in different organs and were modulated by white light; in seedlings and sprouts, highest levels of mRNA were detected in the etiolated stage. Upon illumination with white light, mRNA levels decreased to the amount found in leaves of re-etiolated plants. Lowest expression was observed in leaves of plants grown in the light, in tubers irrespective of light treatment, and in roots of plants grown in the dark. In roots of plants grown in the light, elevated levels of phyA mRNA were detected. Using a monoclonal antibody generated against pea phytochrome as an immunochemical probe, the protein was only detectable in protein extracts from etiolated seedlings and sprouts.     

Partial Characterization of Phytochrome I and II in Etiolated and De-Etiolated Tissues of a Photomorphogenetic Mutant (lh) of Cucumber (Cucumis sativus L.) and Its Isogenic Wild Type

A photomorphogenetic mutant (lh) of cucumber has been suggestedto lack light-stable phytochrome function [Adamse et al. (1987)J. Plant Physiol. 127: 481-491]. The present work reports biochemicaland immunochemical characteristics of phytochrome in this cucumbermutant. Spectrophotometric measurement of phytochrome extractedfrom the etiolated seedlings indicated that the mutant containeda similar amount of phytochrome to that of the wild type. Nosignificant differences in apparent molecular mass and reactivityagainst an anti-pea phytochrome monoclonal antibody were observed.Phytochrome in de-etiolated seedlings was partially purifiedto enable spectrophotometric measurement. The phytochrome contentand difference spectrum for photoconversion was very similarin extracts of the mutant and the wild type. Furthermore, thephytochrome extracted from de-etiolated tissues of the mutantand the wild type appear to contain similar amounts of phytochromeI and II, since in both about one quarter of the phytochromein the fraction could be immunoprecipitated by an antibody whichrecognizes phytochrome I. Two possibilities to explain the Ilphenotype are: (i), the mutation changes the function of phytochromeI and/or II without changing its stability and spectrophotometricalcharacteristics; (ii), the mutation results in modificationof transduction chains between the photo-receptor and physiologicalresponses. (Received February 1, 1989; Accepted April 14, 1989)     

The mechanism of rhythmic ethylene production in sorghum. The role of phytochrome B and simulated shading

Mutant sorghum (Sorghum bicolor [L.] Moench) deficient in functional phytochrome B exhibits reduced photoperiodic sensitivity and constitutively expresses a shade-avoidance phenotype. Under relatively bright, high red:far-red light, ethylene production by seedlings of wild-type and phytochrome B-mutant cultivars progresses through cycles in a circadian rhythm; however, the phytochrome B mutant produces ethylene peaks with approximately 10 times the amplitude of the wild type. Time-course northern blots show that the mutant's abundance of the 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase mRNA SbACO2 is cyclic and is commensurate with ethylene production, and that ACC oxidase activity follows the same pattern. Both SbACO2 abundance and ACC oxidase activity in the wild-type plant are very low under this regimen. ACC levels in the two cultivars did not demonstrate fluctuations coincident with the ethylene produced. Simulated shading caused the wild-type plant to mimic the phenotype of the mutant and to produce high amplitude rhythms of ethylene evolution. The circadian feature of the ethylene cycle is conditionally present in the mutant and absent in the wild-type plant under simulated shading. SbACO2 abundance in both cultivars demonstrates a high-amplitude diurnal cycle under these conditions; however, ACC oxidase activity, although elevated, does not exhibit a clear rhythm correlated with ethylene production. ACC levels in both cultivars show fluctuations corresponding to the ethylene rhythm previously observed. It appears that at least two separate mechanisms may be involved in generating high-amplitude ethylene rhythms in sorghum, one in response to the loss of phytochrome B function and another in response to shading.     

A light-regulated pool of phytochrome and rudimentary high-irradiance responses under far-red light in Pinus elliottii and Pseudotsuga menziesii

The presence of a phytochrome pool down-regulated by light and the occurrence of high-irradiance responses to far-red light are well documented in angiosperms but not in gymnosperms. A pool of phytochrome was identified in Pinus elliottii and Pseudotsuga menziesii seedlings grown in darkness with a monoclonal antibody developed against oat phytochrome A. This pool was barely detectable in light-grown tissues. Dark-grown conifer seedlings transferred to continuous red light showed a gradual decrease of the levels of immunodetectable phytochrome. This decrease was significantly slower in gymnosperms than in angiosperms. Dark-grown seedlings of P. elliottii and P. menziesii showed enhanced growth of the cotyledonary whorl and increased anthocyanin pigmentation of the hypocotyl, but no hypocotyl-growth inhibition in response to continuous far-red light. Hourly pulses were significantly less effective than continuos far-red light. The response to far-red light was not observed in seedlings pretreated with red light to reduce the levels of immunodetectable phytochrome. Rudiments of phytochrome A-like function and kinetics are present in P. elliottii and P. menziesii.     

Transient down-regulation of phytochrome mRNA abundance in etiolated cucumber cotyledons in response to continuous white light

Phytochrome mRNA abundance decreased to 20% of the initial level in etiolated cucumber (Cucumis sativus L.) cotyledons exposed to continuous white light. Unexpectedly, by 12 hours of continuous white light, phytochrome mRNA had reaccumulated to 60% of the control level. High stringency RNA blot analyses suggest that it is the mRNA encoding type I phytochrome that reaccumulates.     

PHYTOCHROME CHANGES IN TISSUES OF DARK-GROWN SEEDLINGS REPRESENTING VARIOUS PHOTOPERIODIC CLASSES

Excised tissues of dark-grown seedlings representing long day, short day and daylength indifferent photoperiodic classes were assayed for nonphotochemical changes in phytochrome. In all tissues tested, these changes were qualitatively the same. A brief irradiation with red light was followed in darkness by a decrease in total phytochrome, the disappearance of P_FR, and an increase in detectable P_R. Within the limits of the tissues tested, the kinetics of phytochrome change can be assigned to three groups on the basis of rates. These groups are represented by coleoptiles, hypocotyls and epicotyls, and mesocotyls. The kinetics could not be distinguished on the basis of the photoperiodic class of the mature plant. The significance of these kinetics with respect to the photochemistry of phytochrome conversion is discussed.     

Phytochrome regulation of phytochrome A mRNA levels in the model short-day-plant Pharbitis nil

The exposure of dark-grown Pharbitis nil seedlings to continuous R induces a rapid decrease in PHYA mRNA abundance with a half-life of about 2 h. A 5 min R pulse also induces this decline, and the effect is partially reversible by subsequent FR irradiation, confirming that the regulation of expression is mediated via the Pfr form of a phytochrome. When de-etiolated seedlings are returned to darkness after a W photoperiod, PHYA mRNA slowly reaccumulates from 20% to 50% of the dark level within 24 h. The rate of reaccumulation is greatly accelerated by the removal of Pfr with a FR pulse, resulting in reaccumulation to 100% within approximately 11 h. Without FR irradiation PHYA mRNA expression remains fully repressed for at least 11 h after the end of the photoperiod, suggesting that the controlling Pfr is highly stable.     

Photomorphogenic responses to UV radiation: Involvement of phytochrome and UV photoreceptors in the control of hypocotyl elongation in Lycopersicon esculentum

The photo-inhibition of Lycopersicon esculentum Mill, hypocotyl growth induced by UV radiation may be mediated by both phytochrome and UV-absorbing receptors. The inhibition of growth induced by continuous irradiation with high fluence rate UV radiation is similar in the au mutant, which is severely deficient in spectrophoto metrically and immunochemically detectable phytochrome, and in the isogenic wild type. Parallel irradiation with 692 nm light, which is equivalent to UV radiation for the phytochrome system in our experimental conditions, induced at high photon fluence rates a significant increase in hypocotyl growth in the au mutant. The same light treatments inhibited the hypocotyl growth of the wild type. The responses of water-grown seedlings and chlorophyll-free seedlings (streptomycin and norflurazon treated seedlings) were compared. Water-grown and chlorophyll-free seedlings responded similarly to UV radiation. The presence of chlorophyll correlates with a significant increase in hypocotyl growth of au mutants irradiated with 692 nm light. These results support the conclusion that UV-induced inhibition of growth in the au mutant is independent of phytochrome.