Levels Of Phytochrome Research Articles

Optical Properties of the Pod Wall of the Pea ( Pisum sativum L.). III. The Effects of Senescence and Pod Desiccation

Summary The transmission, reflection and absorption spectra of pods of Pisum sativum L. variety JI 141 were measured at various stages of senescence. These optical properties were correlated with the percentage water content and levels of chlorophyll a, b and total chlorophyll of the pod wall at each stage. Photomorphogenic effects of pod senescence on the seed environment were deduced from changes in the ratio of 660/730 nm light transmitted through the pod wall. Structural aspects of the final stages of pod desiccation were also investigated using scanning electron microscopy. The significance of these results are discussed in relation to the use of optical properties to monitor pod moisture content and the photoenvironment of the senescing pod as it affects 1) phytochrome levels before final seed desiccation and 2) near UV light transmission to the drying seed.

Differential exhibition of photoreversible phytochrome in callus from two woody species, Actinidia deliciosa and Olea europaea

As part of an investigation of the photomorphogenetic control of organogenesis in woody species, the levels of phytochrome in callus tissues of Actinidia deliciosa (kiwi fruit) and Olea europaea (olive) has been studied by in vivo spectrophotometry. Photoreversible signals were detected in Actinidia leaf callus, irrespective of whether it had been grown in red light, far-red light or darkness. In contrast, phytochrome could not be detected in callus generated from Actinidia pith tissues. Analysis of callus derived from olive leaf tissues showed the presence of phytochrome. The results indicate that callus derived from leaf tissues of these two woody plants contains a light stable (Type II) phytochrome. The absence of detectable phytochrome in Actinidia pith callus indicates that biochemical differences in the original tissues are partially retained in the callus derived from them.

Impaired phytochrome‐mediated shade‐avoidance responses in the aurea mutant of tomato

ABSTRACTInternode extension‐growth responses to neighbouring plants and to red to far‐red ratios (R:FR) were investigated in wild‐type (WT) and aurea (au)‐mutant seedlings of tomato grown under natural radiation. The genomic location of the au mutant is not known, but one of its consequences is the reduced phytochrome level. In WT seedlings, internode growth was promoted by the presence of non‐shading neighbours reflecting far‐red light (FR), the shade of a tall canopy, FR provided as a supplement during the photoperiod, and FR pulses either provided at the end of the day or delayed into the dark period. Supplementary FR during the photoperiod also promoted growth in herbicide‐treated partially bleached WT seedlings. The au mutant showed higher background extension‐growth rates, but only responded to the most severe treatments: deep shade light and very low R:FR at the end of the day, i.e. au‐mutant seedlings were less sensitive than WT seedlings to R:FR signals. Wild‐type seedlings were transferred from the glasshouse to a growth room and exposed to white light with two levels of phytochrome‐absorbable radiation but similar phytochrome photoequilibria and radiation for photosynthesis. The plants exposed to the lowest level showed a transient increase of internode extension growth rate and a simultaneous reduction of response to FR pulses, i.e. reproduced some of the features of au‐mutant seedlings. Phytochrome itself could set the degree of response to Pfr during neighbour detection.

Phytochrome A null mutants of Arabidopsis display a wild-type phenotype in white light.

Phytochrome is a family of photoreceptors that regulates plant photomorphogenesis; the best-characterized member of this family is phytochrome A. Here, we report the identification of novel mutations at three Arabidopsis loci (fhy1, fhy2, and fhy3) that confer an elongated hypocotyl in far-red but not in white light. fhy2 mutants are phytochrome A deficient, have reduced or undetectable levels of PHYA transcripts, and contain structural alterations within the PHYA gene. When grown in white light, fhy2 mutants are morphologically indistinguishable from wild-type plants. Thus, phytochrome A appears to be dispensable in white light-grown Arabidopsis plants. fhy2 alleles confer partially dominant phenotypes in far-red light, suggesting that the relative abundance of phytochrome A can affect the extent of the far-red-mediated hypocotyl growth inhibition response. Plants homozygous for the recessive fhy1 and fhy3 mutations have normal levels of functional phytochrome A. The FHY1 and FHY3 gene products may be responsible for the transduction of the far-red light signal from phytochrome A to downstream processes involved in hypocotyl growth regulation.

Photoresponses of transgenic Arabidopsis seedlings expressing introduced phytochrome B‐encoding cDNAs: evidence that phytochrome A and phytochrome B have distinct photoregulatory functions

The photocontrol of hypocotyl elongation has been studied in two transgenic lines of Arabidopsis thaliana which contain elevated levels of phytochrome B encoded by either an introduced rice‐ or Arabidopsis‐derived cDNA driven by the 35S CaMV promoter. Inhibition of hypocotyl growth in etiolated seedlings of the phyB‐transformed lines was saturated at photon fluence rates of continuous red light (R) which were markedly lower than those required for inhibition of growth in seedlings of the isogenic wild‐type (WT). Inhibition of hypocotyl growth in etiolated seedlings of the phyB‐transgenic lines under continuous far‐red irradiation (FR), however, showed the same relationship with fluence rate as WT. Light‐grown seedlings of the phyB‐transgenic lines responded to end‐of‐day FR by an acceleration of growth, in a manner comparable with WT. This response was unaltered when the end‐of‐day FR was extended from a 15 min pulse to 14 h of continuous irradiation. The response of light‐grown, phyB‐transformed seedlings to decreasing R:FR ratio was also qualitatively similar to WT, i.e. increased elongation growth of the hypocotyl and petioles occurred under low R:FR quantum ratio. However, absolute elongation growth was markedly less in the transgenic seedlings at all R:FR ratios tested than in WT. Together, these data indicate that seedlings over‐expressing phytochrome B are more responsive to R than are WT, but are unaltered in their responsiveness to FR. By contrast, seedlings overexpressing phytochrome A are more responsive than WT to both R and FR; whereas the phytochrome B‐deficient mutant hy3 is unresponsive to R while retaining WT‐like responsiveness to FR. These data indicate that in WT etiolated seedlings phytochrome A mediates the effects of continuous FR, and phytochrome B the effects of continuous R. The evidence thus supports the conclusion that these two molecular species of the photoreceptor have differential regulatory roles in the plant.

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).

Temporal and light regulation of the expression of three phytochromes in germinating seeds and young seedlings of Avena sativa L.

An oat (Avena sativa L.) plant contains at least three phytochromes, which have monomeric masses of 125, 124, and 123 kilodaltons (kDa) (Wang et al., 1991, Planta 184, 96-104). The 124-kDa phytochrome is most abundant in dark-grown seedlings, while the other two phytochromes predominate in light-grown seedlings. Using three monoclonal antibodies, each specific to one of the three phytochromes, we have monitored by immunoblot assay the expression of these three phytochromes in the 5 d following onset of imbibition of seeds. On a per-organism basis, each of these three phytochromes increased in abundance for the first 3 d in the light, or for the first 4 d in darkness, after which they each began to decrease in quantity. When 3-d-old dark-grown seedlings were transferred to the light, the abundance of each of these three phytochromes decreased both in absolute amount and relative to the phytochrome levels in control seedlings kept in darkness. In contrast, when 3-d-old light-grown seedlings were transferred to darkness, the abundance of the 124-kDa and 125-kDa phytochromes increased while that of 123-kDa phytochrome remained unchanged. In each case, the level of phytochrome was greater than that of control seedlings maintained in the light. Thus, in addition to temporal regulation, all three phytochromes exhibit photoregulated expression at the protein level, although the magnitude of this photoregulation varies substantially.

Phytochrome levels in the green alga Mesotaenium caldariorum are light regulated.

Experiments undertaken in this investigation examine the influence of light on the levels of phytochrome in the green alga Mesotaenium caldariorum and also provide partial protein sequence of the algal phytochrome. Immunochemical and spectrophotometric measurements reveal that phytochrome levels increase nearly 4-fold upon transfer of light-grown algal cells to total darkness during a 6- to 8-d adaptation period. Within 24 h after return to continuous illumination, the level of phytochrome in dark-adapted cells has decreased to that found in light-grown cells. Red or far-red light experiments show that both effects of light, phytochrome accumulation during dark adaptation and light-dependent decrease of phytochrome, do not depend on the form of the phytochrome photoreceptor (i.e. far-red absorbing or red absorbing) present in the algal cell. The light-dependent reduction of phytochrome in dark-adapted cells is inhibited by the photosynthetic electron transport inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethyl urea, suggesting that this light effect is mediated by photosynthesis. Microsequence analyses of internal peptides indicate that algal phytochrome purified from dark-adapted cells shares the greatest sequence identity with phytochrome from the fern Selaginella (74%). Compared with higher plant photoreceptors, Mesotaenium phytochrome appears to be more closely related to phyB gene products (i.e. 62 and 63% average sequence identity) than to phyA gene products (i.e. 50 and 53% average sequence identity). Because light regulation and the structure of Mesotaenium phytochrome do not conform with either type I (light-labile) or type II (light-stable) phytochromes from higher plants, these results support the hypothesis that the lower green plant photoreceptors represent a distinct class of phytochrome.

Expression of Oat-phyA-cDNA in a Suspension Cell Culture of Transgenic Tobacco: A Single-Cell System for the Study of Phytochrome Function

A culture of callus cells has been developed from a transgenic line of tobacco which contains an introduced phyA-cDNA encoding phytochrome A. Suspension cultures of the cells were shown to accumulate a significant immunodetectable level of the heterologous phytochrome, but not of the native phyA-gene product. The red-irradiated form (Pfr) of the heterologous phytochrome was specifically degraded in vivo, and the red-irradiated (Pfr) and far-red-irradiated (Pr) forms demonstrated different patterns of in vitro proteolytic cleavage. These results strongly suggested that the phytochrome apoprotein was associated with a chromophore moiety which mediated red/far-red sensitive conformational changes of the molecule. Exogenous application of 4-amino-5-hexynoic acid (AHA) to the transgenic suspension cultures resulted in the accumulation of a population of phytochrome which was stable under red light and gave identical patterns of in vitro digestion in the red and far-red irradiated forms, i.e. the spectral activity of phytochrome was inhibited. Application of exogenous 5-aminolevulinic acid (ALA) or biliverdin overcame the inhibitory effects of AHA to restore spectral sensitivity of the phytochrome pool. These results are consistent with the proposed pathway of phytochrome chromophore biosynthesis in intact plant systems. Thus, the transgenic suspension cultures provided a single-cell system in which spectrally-active phytochrome, apparently indistinguishable from the native phytochrome synthesized in etiolated seedlings, was accumulated. Photoregulation of expression of the genes encoding the small subunit of ribulosc-1,5-tephosphate carboxylase and chlorophyll aib binding proteins demonstrated that the heterologous phytochrome population mediated rapid changes in gene expression in the de-differentiated cells. It is therefore proposed that such a suspension culture of transgenic cells offers a model system for the study of phytochrome function.

Initial characterization of a pea mutant with light-independent photomorphogenesis.

We have identified a mutant of pea cultivar Alaska that has many of the characteristics normally associated with light-grown seedlings even when grown in complete darkness. We have designated this mutant lip1, for light independent photomorphogenesis. Etiolated wild-type pea seedlings are white to slightly yellow in color and have a distinct morphology characterized by elongated epicotyls and buds containing unexpanded leaves with small, undifferentiated cells. In contrast, mutant seedlings grown under the same conditions are yellow in color and have short epicotyls and expanded leaves showing clear cellular differentiation. Transmission electron microscopy revealed partially developed, agranal plastids in the dark-grown mutant, unlike wild-type seedlings that contain etioplasts with prolamellar bodies. The mutant also exhibits a much shorter lag period for chlorophyll accumulation when etiolated seedlings are transferred from darkness to white light. The dark-grown mutant has 10-fold less spectrally detectable phytochrome, which can be attributed to a 10-fold reduction in the level of the PHYA polypeptide. Cab, Fed1, and RbcS transcripts are present in dark-grown mutant seedlings at levels comparable to those produced in light-grown material. The levels of these transcripts show a normal decrease when green plants grown for 15 days in a light/dark cycle are transferred to continuous darkness. However, transcript levels remain high during dark treatment of seedlings grown for 9 days in continuous light, indicating that the dark adaptation response in this mutant is developmentally plastic. The lip1 mutant has several features in common with the deetiolated Arabidopsis mutants det1, det2, and cop1. However, there are also several important differences, including varying effects on phytochrome levels, organ-specific gene expression, plastid development, and response to dark adaptation.

The Association of Type I Phytochrome with Wheat Leaf Plasma Membranes

Summary The association of phytochrome with plasma membranes, purified from etiolated wheat leaves, has been analyzed by enzyme-linked immunosorbent assay (ELISA) and Western blotting using antibodies specific for type I phytochrome. Plasma membranes contain 1.9 ng phytochrome μg -1 protein which represents about 0.1% of the total cellular phytochrome. In samples homogenized at pH 8.2 in the absence of Mg 2+ the amount of phytochrome associated with the membranes was unaffected by light treatments given to the tissues. Data from solubilisation experiments are consistent with phytochrome being located predominantly on the cytoplasmic face of the membrane and held by hydrophobic and electrostatic forces. When tissues were transferred from darkness to red light, the onset of the loss of phytochrome from plasma membranes was delayed for one hour as compared with the loss of total phytochrome. Similarly, the inhibition of synthesis by far-red-light pulses was less for plasma membrane-associated than for total phytochrome. The relationship between total and membrane phytochrome was analyzed for a range of total phytochrome levels and indicated that membrane-binding was saturable. It was concluded that plasma membrane phytochrome represents a pool whose regulation is partially independent of the total type I phytochrome.

Photocontrol of biological processes in monocotyledonous plants after relaxation of phytochrome system

Effects of a short-term pre-irradiation of etiolated wheat seedlings by radiation of various wavelengths and various irradiances on the rates of photophospho-rylation, Hill reaction, and chlorophyll accumulation were studied. The curves, constructed for the dependence of inhibitory effect in different processes on pre-irradiance doses, have a saturation level corresponding to photostationary state of the phytochrome system and a pre-threshold site where the inhibition is absent. For an interpretation of the experimental data we suggest a general model that includes both the threshold concept of Mohr and the presence of an additional control mechanism not sensitive to far-red radiation. According to the model, the phytochrome-controlled biological effect is pro-portional to the time during which the level of active phytochrome is higher than the threshold one and may be analysed according to the inhibition degree. The difference in control by red (RR) and green radiation (GR) pretreatments is attributed to the difference of the phytochrome photoconversion rate under RR and GR.

Light-grown plants of transgenic tobacco expressing an introduced oat phytochrome A gene under the control of a constitutive viral promoter exhibit persistent growth inhibition by far-red light

A comparison of the photoregulation of development has been made for etiolated and light-grown plants of wild-type (WT) tobacco (Nicotiana tabacun L.) and an isogenic transgenic line which expresses an introduced oat phytochrome gene (phyA) under the control of a constitutive viral promoter. Etiolated seedlings of both the WT and transgenic line showed irradiance-dependent inhibition of hypocotyl growth under continuous far-red (FR) light; transgenic seedlings showed a greater level of inhibition under a given fluence rate and this is considered to be the result of the heterologous phytochrome protein (PhyA) functioning in a compatible manner with the native etiolated phytochrome. Deetiolation of WT seedlings resulted in a loss of responsiveness to prolonged FR. Light-grown transgenic seedlings, however, continued to respond in an irradiance-dependent manner to prolonged FR and it is proposed that this is a specific function of the constitutive PhyA. Mature green plants of the WT and transgenic lines showed a qualitatively similar growth promotion to a brief end-of-day FR-treatment but this response was abolished in the transgenic plants under prolonged irradiation by this same FR source. Growth inhibition (McCormac et al. 1991, Planta 185, 162-170) and enhanced levels of nitrate-reductase activity under irradiance of low red:far-red ratio, as achieved by the FR-supplementation of white light, emphasised that the introduced PhyA was eliciting an aberrant mode of photoresponse compared with the normal phytochrome population of light-grown plants. Total levels of the oat-encoded phytochrome in the etiolated transgenic tobacco were shown to be influenced by the wavelength of continuous irradiation in a manner which was qualitatively similar to that seen for the native, etiolated tobacco phytochrome, and distinct from that seen in etiolated oat tissues. These results are discussed in terms of the proposal that the constitutive oat-PhyA pool in the transgenic plants leads to a persistence of a mode of response normally restricted to the situation in etiolated plants.

Phytochrome Levels in Light-Grown Avena Change in Response to End-of-Day Irradiations

The effect of 15-minute end-of-day irradiations on photoreversible phytochrome levels in light-grown oat (Avena sativa L., cv Garry) seedlings was investigated. Oat seedlings were grown in a cycle of 8 hours of natural daylight and 16 hours of complete darkness, from sowing until harvest at day 10. The level of extractable, photoreversible phytochrome per unit fresh weight was 60% higher after end-of-day far-red irradiation than after either end-of-day red irradiation or end-of-day far-red followed by end-of-day red. Seedlings irradiated with end-of-day far-red also exhibited a small but significant increase in shoot height and fresh weight per seedling. Extracts of seedlings given each of these end-of-day treatments were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, electroblotted, and immunostained with monoclonal antibodies specific to different phytochromes. Regardless of end-of-day light treatment, phytochrome that is abundant in etiolated tissue was below the limit of detection, indicating that one or more of the phytochromes predominating in green tissue changes in abundance.

PHYSIOLOGICAL CHARACTERIZATION OF A HIGH‐PIGMENT MUTANT OF TOMATO

Abstract— A high‐pigment (hp) mutant, which shows exaggerated phytochrome responses and three other genotypes of Lycopersicon esculenrum Mill. cv. Ailsa Craig: the aurea (au) mutant deficient in the bulk light‐labile phytochrome (PI) pool, the au, hp double mutant, and their isogenic wild type, were used in this study. Measurements of phytochrome destruction in red light (R) revealed that the exaggerated responses of the hp mutant are not caused by a higher absolute phytochrome level or a reduced rate of phytochrome destruction. Fluence‐response relationships for anthocyanin synthesis after a blue‐light pretreatment were studied to test if the hp mutant conveys hypersensitivity to the far‐red light (FR)‐absorbing form of phytochrome (Pfr), i.e. the threshold of Pfr required to initiate the response is lower. The response range for the hp mutant and wild type was identical, although the former exhibited a 6‐fold larger response. Moreover, the kinetics of anthocyanin accumulation in continuous R were similar in the wild‐type and hp‐mutant seedlings, despite the latter accumulating 9‐fold more anthocyanin. Since the properties of phytochrome are the same, the hp mutation appears to affect the state of responsiveness amplification, i.e. the same amount of Pfr leads to a higher response in the hp mutant. We therefore propose that the hp mutation is associated with an amplification step in the phytochrome transduction chain. Escape experiments showed that the anthocyanin synthesis after different light pretreatments terminated with a R pulse was still 50% FR reversible after 4–6 h darkness, indicating that the Pfr pool regulating this response must be relatively stable. However, fluence‐rate response relationships for anthocyanin synthesis and hypocotyl growth induced by a 24‐h irradiation with 451, 539, 649, 693, 704 and 729 nm light showed no or a severely reduced response in the au and au, hp mutants, suggesting the importance of PI in these responses. We therefore propose that the capacity for anthocyanin synthesis (state of responsiveness amplification) could be established by PI, while the anthocyanin synthesis is actually photoregulated via a stable Pfr pool. The Hp gene product is proposed to be an inhibitor of the state of responsiveness amplification for responses controlled by this relatively stable Pfr species.

Phytochrome requires the 6-kDa N-terminal domain for full biological activity.

Phytochrome is a red/far-red-absorbing photoreceptor that controls many aspects of plant photomorphogenesis. Because proteolytic removal of approximately 6 kDa from the N terminus of 124-kDa oat phytochrome substantially alters many physicochemical properties of the chromoprotein, it has been proposed that the N terminus is required for biological activity. Here we test this hypothesis by comparing tobacco plants expressing full-length oat phytochrome (FL) with plants expressing a 118-kDa oat phytochrome lacking amino acids 7-69 (NA phytochrome). NA phytochrome, like its FL counterpart, exists as a homodimer in solution, is capable of covalently binding chromophore to form a red/far-red-photoreversible product, and is rapidly degraded in vivo after photoconversion to the far-red-absorbing form. However, like proteolytically degraded phytochrome missing the N terminus, the absorption maxima of the red- and far-red-light-absorbing forms of NA phytochrome are blue shifted relative to the maxima of the FL chromoprotein, and the rate of dark reversion of the far-red- to red-light-absorbing form is substantially increased. Tobacco plants producing high levels of NA phytochrome do not exhibit the light-exaggerated phenotype characteristic of FL phytochrome overexpression. By comparison of phytochrome-dose-phenotype-response curves generated by using a series of transgenic lines expressing various levels of FL or NA phytochrome, we demonstrate that NA phytochrome has less than 1/5th the biological activity of FL phytochrome expressed in tobacco. Furthermore, the shape of the dose-response curve for plants expressing FL phytochrome indicates that there is a sharp transition between phenotypically normal and abnormal plants over a relatively narrow range of phytochrome content, demonstrating that precise control of phytochrome levels is critical to photomorphogenesis.

The hy3 Long Hypocotyl Mutant of Arabidopsis Is Deficient in Phytochrome B.

The six long hypocotyl (hy) complementation groups of Arabidopsis (hy1, hy2, hy3, hy4, hy5, and hy6) share the common feature of an elongated hypocotyl when grown in white light. The varied responses of these mutants to irradiations of differing wavelengths have suggested that some of the lines may lack elements of the phytochrome signal transduction pathway. We have performed immunoblot and RNA gel blot analyses of the multiple types of phytochrome present in wild-type and mutant Arabidopsis and provide evidence that mutations at the HY3 locus cause a specific deficiency in phytochrome B. Using an Escherichia coli overexpression system, we have developed and identified monoclonal antibodies that selectively recognize phytochromes A, B, and C from Arabidopsis. In wild-type plants, phytochrome A is highly abundant in etiolated tissue, but rapidly decreases about 200-fold upon illumination. Phytochromes B and C are present at much lower levels in etiolated tissue but are unaffected by up to 24 hr of red light illumination, and together predominate in green seedlings. These data establish that phytochromes B and C are "type 2" or photostable phytochromes. Levels of phytochromes A, B, and C similar to those of the wild type are observed in strains containing mutations at the HY4 and HY5 loci. In contrast, all four hy3 mutant alleles tested here exhibit a modest (twofold to threefold) reduction in phyB transcript and a severe (20- to 50-fold) deficiency in phyB-encoded protein, relative to levels in wild-type plants. The levels of phyA- and phyC-encoded mRNA and protein, however, are indistinguishable from the wild type in these mutants. We conclude that the phenotype conferred by hy3 is due to the reduced levels of the light-stable phytochrome B.

Characterization of Tobacco Expressing Functional Oat Phytochrome

Constitutive expression of a chimeric oat phytochrome gene in tobacco (Nicotiana tabacum) results in the accumulation of a functional 124-kilodalton photoreceptor that markedly alters the phenotype of light-grown tobacco (Keller et al. [1989] EMBO J 8: 1005-1012). Here, we provide a detailed phenotypic and biochemical characterization of homozygous tobacco expressing high levels of oat phytochrome. Phenotypic changes include a substantial inhibition of stem elongation, decreased apical dominance, increased leaf chlorophyll content, and delayed leaf senescence. Oat phytochrome synthesized in tobacco is indistinguishable from that present in etiolated oats, having photoreversible difference spectrum maxima at 665 and 730 nanometers, exhibiting negligible dark reversion of phytochrome-far red-absorbing form (Pfr) to phytochrome-red-absorbing form (Pr), and existing as a dimer with an apparent size of approximately 300 kilodaltons. Heterodimers between the oat and tobacco chromoproteins were detected. Endogenous tobacco phytochrome and transgenically expressed oat phytochrome are rapidly degraded in vivo upon photoconversion of Pr to Pfr. Breakdown of both oat and tobacco Pfr is associated with the accumulation of ubiquitin-phytochrome conjugates, suggesting that degradation occurs via the ubiquitin-dependent proteolytic pathway. This result indicates that the factors responsible for selective recognition of Pfr by the ubiquitin pathway are conserved between monocot and dicot phytochromes. More broadly, it demonstrates that the domain(s) within a plant protein responsible for its selective breakdown can be recognized by the degradation machinery of heterologous species.

Spectrophotometric characterization of phytochromes in dimorphic cocklebur seeds in relation to capacity for germination

Phytochrome was measured spectrophotometrically in different tissues of the upper (positively photoblastic) and lower (negatively photoblastic) seeds of the cocklebur (Xanthium pennsylvanicum Wallr.). Axial parts of the seeds, in particular parts of the radicle, contained high levels of phytochrome, while cotyledonary parts contained only low levels. These results were consistent with the distribution of the light‐sensitive areas of the seeds that were associated with germination. Phytochrome levels in both types of dimorphic seeds increased gradually with increasing duration of dark imbibition for 4–8 h, then the rates of increase in levels of phytochrome accelerated. In both types of seed, some phytochrome was measurable even before imbibition. In the lower seeds, up to 20% of the phytochrome was occasionally observed as Pfr in samples imbibed in darkness for a short time (up to 12 h). A slight blue shift of the peak of PT in the difference spectrum of phytochrome was observed in the case of lower seeds imbibed for 0–2 h. These results suggest that, to some extent, the lower axes contain dehydrated Pfr or intermediate(s) in the photoconversion of phytochrome.The dark reactions of Pfr were also examined in excised axes of both types of dimorphic seed after they had been pre‐imbibed for 16 h in darkness. Dark destruction of Pfr was observed in both types of seed. In addition, net increases in levels of Pr were observed in the dark controls and in the samples irradiated with red light after the level of Pfr diminished. No ‘inverse’ dark reversion from Pr to Pfr was detected. Thus, after 16 h of imbibition, there were no differences in terms of properties of phytochrome between the two types of seed, and the different responses to light of upper and lower seeds might depend mainly on a difference in the physiological state of the two types of seed rather than the properties of phytochrome.

Phytochrome content and hypocotyl growth of longhypocotyl mutant and wild-type cucumber seedlings during de-etiolation

Summary Photo-inhibition of hypocotyl growth has been studied in a cucumber ( Cucumis sativus L.) long-hypocotyl ( lh ) mutant and its near isogenic wild type. Hypocotyl growth rate of the mutant and wild type was the same in darkness (D) and was equally inhibited by continuous far-red light (FR). However, under continuous red light (R) the mutant was dramatically less inhibited, resulting after 120 h R in hypocotyls being almost twice as long as those of wild type. Although smaller in absolute length, similar results were obtained under continuous blue light (B). In vivo spectrophotometric phytochrome measurements with Norflurazon-treated plants showed that D-grown mutant and wild-type hypocotyl hooks contain similar amounts of phytochrome, while after 4 days FR, R and B, significantly less phytochrome was detected in the mutant than the wild type. After 4 days in D, morphologically similar mutant and wild-type plants were exposed to R, resulting in the depletion of the labile-phytochrome pool (PI) by destruction. During the first 2.5 h the half-life for depletion of PI for mutant and wild type was similar (≈ 30 min) at 25 °C. However, after 3 h R a significantly lower phytochrome steady-state level was reached in the mutant than the wild type. When plants were pretreated for 4 days with FR before exposure to R, a similar difference in phytochrome steady-state level was observed. Phytochrome resynthesis was measured in seedlings after pretreatment with R and B. Although the rate of resynthesis was slower after B, there were no significant differences in its rate between mutant and wild type. Despite PI destruction and resynthesis being identical in wild type and mutant, the phytochrome level reached after de-etiolation (the PI steady-state plus the light-stable phytochrome [PII]) is different. We propose that the lh mutant exhibits reduced phytochrome function in light-grown plants due to depletion of a stable phytochrome pool.