Molecular analysis of the phytochrome deficiency in an aurea mutant of tomato

Summary The au ^w mutant allele of the aurea locus in tomato has previously been shown to cause deficiency for the phytochrome polypeptide (Parks et al. 1987). We have begun to characterize the molecular basis and consequences of this deficiency. Genomic Southern blot analysis indicates that there are at least two and probably more phytochrome polypeptide structural genes in tomato. RNA blot analysis shows that the au ^w mutant contains normal levels of phytochrome mRNA and in vitro translation of au ^w poly(A)⁺ RNA yields a phytochrome apoprotein that is quantitatively and qualitatively indistinguishable on SDS-polyacrylamide gels from that synthesized from wild-type RNA. These results indicate that the phytochrome deficiency in aurea is not the result of lack of expression of phytochrome genes but is more likely due to instability of the phytochrome polypeptide in planta. Possible reasons for such instability are discussed. Analysis of the molecular phenotype of aurea indicates that the phytochrome-mediated increase in the abundance of the mRNA encoding chlorophyll a/b binding protein (cab) is severely restricted in the mutant as compared with wild-type tomato. Thus, the au w strain exhibits defective photoregulation of gene expression consistent with its very reduced level of the phytochrome photoreceptor.     

Overexpression of rice phytochrome A partially complements phytochrome B deficiency in Arabidopsis

The red/far-red reversible phytochromes play a central role in regulating the development of plants in relation to their light environment. Studies on the roles of different members of the phytochrome family have mainly focused on light-labile, phytochrome A and light-stable, phytochrome B. Although these two phytochromes often regulate identical responses, they appear to have discrete photosensory functions. Thus, phytochrome A predominantly mediates responses to prolonged far-red light, as well as acting in a non-red/far-red-reversible manner in controlling responses to light pulses. In contrast, phytochrome B mediates responses to prolonged red light and acts photoreversibly under light-pulse conditions. However, it has been reported that rice (Oryza sativa L.) phytochrome A operates in a classical red/far-red reversible fashion following its expression in transgenic tobacco plants. Thus, it was of interest to determine whether transgenic rice phytochrome A could substitute for loss of phytochrome B in phyB mutants of Arabidopsis thaliana (L.) Heynh. We have observed that ectopic expression of rice phytochrome A can correct the reduced sensitivity of phyB hypocotyls to red light and restore their response to end-of-day far-red treatments. The latter is widely regarded as a hallmark of phytochrome B action. However, although transgenic rice phytochrome A can correct other aspects of elongation growth in the phyB mutant it does not restore other responses to end-of-day far-red treatments nor does it restore responses to low red:far-red ratio. Furthermore, transgenic rice phytochrome A does not correct the early-flowering phenotype of phyB seedlings. Received: 12 July 1998 / Accepted: 13 August 1998     

Degradation of phytochrome A and the high irradiance response in Arabidopsis: a kinetic analysis

The ability to respond to far‐red‐rich light is essential for seedlings germinating below dense canopies. Physiological and genetic studies have demonstrated that phytochrome A is the only photoreceptor mediating responses to far‐red light. However, all phytochromes including phytochrome A are believed to be activated by red light and to be inactivated by far‐red light. To address the fundamental question of why phytochrome A has its highest physiological activity at presumably inactivating wavelengths, we analysed light‐induced degradation of phytochrome A in Arabidopsis. Rate constants were obtained for all reaction events in a two‐step model of degradation. Based on biochemical data, the model includes a tagging mechanism preceding degradation. The parameterized model describes Pr accumulation, wavelength dependencies of degradation kinetics and steady‐state levels as well as Pfr‐induced Pr degradation. Subsequently, experimentally derived fluence rate response curves, action spectrum and response curves to dichromatic irradiation were compared to simulations based on the model of degradation. Two kinetically defined phytochrome subspecies, untagged Pfr and tagged Pr, have steady‐state levels closely matching the physiological response curves. Therefore, sensing of far‐red light by phytochrome A can be quantitatively explained based exclusively on regulated protein degradation.     

Blue-light-induced chloroplast orientation in Mougeotia. Evidence for a separate sensor pigment besides phytochrome

Chloroplast orientation in the green alga Mougeotia has been induced by unidirectional red or blue light, given continuously during one hour. In addition, part of the preparations obtained scattered strong far-red light simultaneously with the orienting light. This far-red light completely abolished the response to red light, consistent with phytochrome as the sensor pigment for orientation in Mougeotia. In blue light, however, the response was completely insensitive to far-red light, thus pointing to a different sensor pigment in the shortwavelength region.Abbreviation P_fr far-red-absorbing form of phytochrome     

Effects of filipin and steroids on phytochrome pelletability

Red light given to dark-grown etiolated leaves of Hordeum vulgare L. in vivo or to crude homogenates increases the phytochrome content of the 20,000 g pellet on centrifugation. The steroids cholesterol and stigmasterol inhibit this red light-induced phytochrome pelletability. Filipin (a polyene antibiotic, which is known to combine with steroids) inhibits red light-induced phytochrome pelletability. Filipin and steroids at the appropriate concentration applied together prevent the inhibition caused by either when applied alone. These results suggest that phytochrome may bind to a steroid component of membranes. The phospholipid phosphatidyl choline dipalmitoyl has no effect on red light-induced phytochrome pelletability. Preliminary evidence demonstrates a direct association of soluble phytochrome in its active form and steroids. The physiological significance of red light-induced pelletability and the primary mechanism of phytochrome action are discussed in terms of a hypothetical steroid-binding site.     

The mapping of phytochrome genes and photomorphogenic mutants of tomato

The map positions of five previously described phytochrome genes have been determined in tomato (Lycopersicon esculentum Mill.) The position of the yg-2 gene on chromosome 12 has been confirmed and the classical map revised. The position of the phytochrome A (phy A)-deficient fri mutants has been refined by revising the classical map of chromosome 10. The position of the PhyA gene is indistinguishable from that of the fri locus. The putative phyB1-deficient tri mutants were mapped by classical and RFLP analysis to chromosome 1. The PhyB1 gene, as predicted, was located at the same position. Several mutants with the high pigment (hp) phenotype, which exaggerates phytochrome responses, have been reported. Allelism tests confirmed that the hp-2 mutant is not allelic to other previously described hp (proposed here to be called hp-1) mutants and a second stronger hp-2 allele (hp-2 ^j ) was identified. The hp-2 gene was mapped to the classical, as well as the RFLP, map of chromosome 1. Received: 24 May 1996 / Accepted: 14 June 1996     

Dual effect of phytochrome A on hypocotyl growth under continuous red light

The role of phytochrome A in the control of hypocotyl growth under continuous red light (Rc) was investigated using phyA and phyB mutants of Arabidopsis thaliana, which lack phytochrome A (phyA) or phytochrome B (phyB), respectively, and transgenic seedlings of Nicotiana tabacum overexpressing Avena phyA, compared to the corresponding wild type (WT). In WT seedlings of A. thaliana, hypocotyl growth inhibition showed a biphasic response to the fluence rate of Rc, with a brake at 10^?2μmol m^?2 s^?1. At equal total fluence rate, hourly pulses of red light caused slightly more inhibition than Rc. The response to very low fluences of continuous or pulsed red light was absent in the phyA and phyA phyB mutants and present in the phyB mutant. The second part of the response was steeper in the phyA mutant than in the WT but was absent in the phyB mutant. In WT tobacco the response to Rc was biphasic. Overexpression of Avena phyA enhanced the response only at very low fluence rates of Rc (< 10^?2μmol m^?2 s^?1). In both species, the effect of hourly pulses of far-red light was similar to the maximum inhibition observed in the first phase of the response to Rc. Using reciprocity failure (i.e. higher inhibition under continuous than pulsed light) as the operational criterion, a ‘true’ high-irradiance reaction occurred under continuous far-red light but not under Rc or red plus far-red light mixtures. Native and overexpressed phyA are proposed to mediate very low fluence responses under Rc. In WT A. thaliana, this effect is counteracted by a negative action of phyA on phyB-mediated low-fluence responses.     

fhy1 defines a branch point in phytochrome A signal transduction pathways for gene expression

Physiological analysis of the fhy1 mutant of Arabidopsis has led to the proposal that the mutant is deficient in a downstream component of the phytochrome A signal transduction pathway. To define this lesion at the molecular level, we have examined the expression of a range of phytochrome-regulated genes in fhy1. In far-red light, the regulation of genes such as CHS and CHI is blocked in fhy1, whereas the induction of CAB and NR genes is affected minimally. In contrast, the induction of all genes tested is blocked in a phytochrome A-deficient mutant, confirming that gene expression in far-red light is regulated solely by phytochrome A. Thus, fhy1 defines a branch point in phytochrome A signal transduction pathways for gene expression. Contrary to the general opinion that responses to continuous red light are mediated by phytochrome B and other photostable phytochromes, we have shown also that red light-induction of CHS is mediated almost entirely by phytochrome A. Furthermore, phytochrome A-mediated induction of CHS by red light is blocked in fhy1. The induction of CHS by blue light, however, is normal in fhy1, suggesting that although FHY1 is a component of the phytochrome A signaling pathway, it is not a component of the blue-light signaling pathway for CHS expression.     

Far-red light-insensitive, phytochrome A-deficient mutants of tomato

We have selected two recessive mutants of tomato with slightly longer hypocotyls than the wild type, one under low fluence rate (3 mol/m²/s) red light (R) and the other under low fluence rate blue light. These two mutants were shown to be allelic and further analysis revealed that hypocotyl growth was totally insensitive to far-red light (FR). We propose the gene symbol fri (far-red light insensitive) for this locus and have mapped it on chromosome 10. Immunochemically detectable phytochrome A polypeptide is essentially absent in the fri mutants as is the bulk spectrophotometrically detectable labile phytochrome pool in etiolated seedlings. A phytochrome B-like polypeptide is present in normal amounts and a small stable phytochrome pool can be readily detected by spectrophotometry in the fri mutants. Inhibition of hypocotyl growth by a R pulse given every 4 h is quantitatively similar in the fri mutants and wild type and the effect is to a large extent reversible if R pulses are followed immediately by a FR pulse. After 7 days in darkness, both fri mutants and the wild type become green on transfer to white light, but after 7 days in FR, the wild-type seedlings that have expanded their cotyledons lose their capacity to green in white light, while the fri mutants de-etiolate. Adult plants of the fri mutants show retarded growth and are prone to wilting, but exhibit a normal elongation response to FR given at the end of the daily photoperiod. The inhibition of seed germination by continuous FR exhibited by the wild type is normal in the fri mutants. It is proposed that these fri mutants are putative phytochrome A mutants which have normal pools of other phytochromes.     

Photoprotection of phytochrome

High-fluence-rate white light is shown to retard the degradation of phytochrome in etiolated seedlings of four different species: Amaranthus caudatus, Phaseolus radiatus (mung bean), Pisum sativum (garden pea), and Avena sativa (oat). In Amaranthus, a high photon fluence rate (approx. 1000 mol · m^-2 · s^-1) preserved nearly 50% of the total phytochrome over a period of 5 h; at 3 mol · m^-2 · s^-1, less than 8% remained over the same period. Kinetics of the loss of total phytochrome could be interpreted in terms of two populations, one with rapid, and one with slow, turnover rates. A log-linear relationship between fluence rate and proportion of slowly degrading phytochrome was observed; a similar relationship between fluence rate and the amount of phytochrome remaining after a 5-h light treatment was seen. In mung bean, although two populations of differing degradation rates were not resolvable, a similar log-linear relationship between fluence rate and amount remaining after a standard light treatment was evident. Detailed kinetic analyses were not performed with peas and oats, but comparisons of low and high fluence rates demonstrated that photoprotection was similarly effective in these species. In Amaranthus, transfer from high to low fluence rate was accompanied by a rapid increase in degradation rate, indicating that the retarding effect of high-fluence-rate light is not a consequence of the disablement of the degradative machinery.Immunochemical analyses confirmed the existence of photoprotection in all four species, and allowed the extension of the observations to periods of light treatment during which substantial chlorophyll production occurred. Considerable photoprotection was observed in oat seedlings exposed to summer sunlight. These results are interpreted in terms of the accumulation under high fluence rates of photoconversion intermediates not available to the degradative machinery which is specific for the far-red-absorbing form of phytochrome.Abbreviations Pfr far-red absorbing form of phytochrome - Po amount of phytochrome measured at time zero - Pt amount of phytochrome measured at time t - Ptot total phytochrome - WL white light     

Chloroplast movement in Mougeotia induced by blue light pulses

The profile-to-face chloroplast movement in the green alga Mougeotia has been induced by strong blue and near-ultraviolet light pulses (6 J m^-2). Simultaneously, strong red or far-red light (10 W m^-2) was applied perpendicularly to the inducing beam. The response was measured photometrically. Against the far-red background the reciprocity law was found to hold for pulse durations varying two orders of magnitude. The action spectrum exhibited a maximum near 450 nm and a distinct increase in near-ultraviolet. The time-course and the spectral dependence of pulse responses of chloroplasts in Mougeotia were similar to those recorded for other plants which are sensitive only to blue. This points to an alternative sensor system active in the short-wavelength region in addition to the phytochrome system.Abbreviations FR far-red light - Pr red absorbing form of phytochrome - Pfr far-red absorbing form of phytochrome - R red light This paper is dedicated to the memory of Professor Jan Zurzycki     

The aurea mutant of tomato is deficient in spectrophotometrically and immunochemically detectable phytochrome

The aurea locus mutant (au ^w) of tomato contains less than 5% of the level of phytochrome in wild-type tissue as measured by in vivo difference spectroscopy. Immunoblot analysis using antibodies directed against etiolated-oat phytochrome demonstrates that crude extracts of etiolated mutant tissue are deficient in a major immunodetectable protein (116 kDa) normally present in the parent wild type. Analyses of wild-type tissue extracts strongly indicate that the 116-kDa protein is phytochrome by showing that this protein: a) is degraded more rapidly in vitro after a brief far-red irradiation than after a brief red irradiation (Vierstra RD, Quail PH, Planta 156: 158–165, 1982); b) contains a covalently bound chromophore as detected by Zn-chromophore fluorescence on nitrocellulose blots; and c) has an apparent molecular mass comparable to phytochrome from other species on size exclusion chromatography under non-denaturing conditions. The demonstration that the aurea mutant is deficient in this 116-kDa phytochrome indicates that the lack of spectrally detectable phytochrome in this mutant is the result of a lesion which affects the abundance of the phytochrome molecule as opposed to its spectral integrity.     

The active form of phytochrome: a new hypothesis based on phytochrome pelletability studies

Difficulties arising from the current dogma that the far-red absorbing form of phytochrome (P_fr) is the only active form are discussed.A new hypothesis is proposed in which phytochrome is held to be the photoreceptor for both low energy (pulse) and high energy (HIR) responses. There is a common basic mechanism of action involving interaction between phytochrome and a binding site within the cell. The phytochrome involvement in low energy responses exhibits an action spectrum for binding that matches the P_r absorption spectrum and reversibility by far-red irradiation. Upon prolonged irradiation the phytochrome-binding site interaction acquires different characteristics that are reminiscent of those displayed in HIR, e.g. dependence on sustained irradiation for continual binding, dependence of the degree of binding on irradiance and the similarity of the action spectrum with that of HIR action spectra, e.g. that for inhibition of lettuce hypocotyl lengthening.As expected on the basis of the new hypothesis the particulate fraction of phytochrome contains both P_r and P_fr. Arguments are advanced that the presence of P_r in pellets of particulate phytochrome cannot be accounted for by (i) the “induced fit” hypothesis, (ii) the “pigment cycling” hypothesis, and (iii) the “open phytochrome-receptor model”. We conclude that phytochrome molecules, after being sufficiently energized can interact with their intracellular binding sites irrespective of their chromophoric configuration.     

Intracellular redistribution of phytochrome in etiolated soybean (Glycine max L.) seedlings

The intracellular distribution of phytochrome in hypocotyl hooks of etiolated soybean (Glycine max L.) has been examined by immunofluorescence using a newly produced monoclonal antibody (Soy-1) directed to phytochrome purified from etiolated soybean shoots. Cortical cells in the hook region exhibit the strongest phytochrome-associated fluorescence, which is diffusely distributed throughout the cytosol in unirradiated, etiolated seedlings. A redistribution of immunocytochemically detectable hytochrome to discrete areas (sequestering) following irradiation with red light requires a few minutes at room temperature in soybean, whereas this redistribution is reversed rapidly following irradiation with far-red light. In contrast, sequestering in oat (Avena sativa L.) occurs within a few seconds (D. McCurdy and L. Pratt, 1986, Planta 167, 330–336) while its reversal by far-red light requires hours (J. M. Mackenzie Jr. et al., 1975, Proc. Natl. Acad. Sci. USA 72, 799–803). The time courses, however, of red-light-enhanced phytochrome pelletability and sequestering are similar for soybean as they are for oat. Thus, while these observations made with a dicotyledon are consistent with the previous conclusion derived from work with oat, namely that sequestering and enhanced pelletability are different manifestations of the same intracellular event, they are inconsistent with the hypothesis that either is a primary step in the mode of action of phytochrome.Abbreviations DIC differential interference contrast - FR far-red light - Ig immunoglobulin - Pfr, P far-red- and red-absorbing form of phytochrome, respectively - R red light This work was supported by National Science Foundation grant No. DCB-8703057.     

Coupling of phytochrome B to the control of hypocotyl growth in Arabidopsis

Etiolated seedlings of the wild-type (WT) and of the phyB-1 mutant of Arabidopsis thaliana (L.) Heynh. were exposed to red-light (R) and far-red light (FR) treatments to characterize the action of phytochrome B on hypocotyl extension growth. A single R or FR pulse had no detectable effects on hypocotyl growth. After 24-h pre-treatment with continuous FR (FRc) a single R, compared to FR pulse inhibited (more than 70%) subsequent hypocotyl growth in the WT but not in the phyB-1 mutant. This effect of FRc was fluence-rate dependent and more efficient than continuous R (Rc) or hourly FR pulses of equal total fluence. Hypocotyl growth inhibition by Rc was larger in WT than phyB-1 seedlings when chlorophyll screening was reduced either by using broadband Rc (maximum emission 610 nm) or by using narrow-band Rc (658 nm) over short periods (24 h) or with seedlings bleached with Norflurazon. Hourly R or R + FR pulses had similar effects in WT and phyB-1 mutant etiolated seedlings. It is concluded that phytochrome B is not the only photoreceptor of Rc and that the action of phytochrome B is enhanced by a FRc high-irradiance reaction. Complementary experiments with the phyA-201 mutant indicate that this promotion of a phytochrome B-mediated response occurs via co-action with phytochrome A.Abbreviations D darkness - FR far-red light - FRc continuous FR - Pfr FR-absorbing form of phytochrome - HIR high-irradiance reaction - Pfr/P proportion of phytochrome as Pfr - phyA phytochrome A - phyB phytochrome B - R red light - Rc continuous R - WT wild-type I thank Professors R.E. Kendrick and M. Koornneef (Wageningen Agricultural University, The Netherlands) and Professor J. Chory (Salk Institute, Calif., USA) for their kind provision of the original WT and phyB-1 and phyA-201 seed, respectively. This work was financially supported by grants PID and PID-BID from CONICET, AG 040 from Universidad de Buenos Aires and A 12830/1-000019 from Fundación Antorchas.     

The in vivo properties of Amaranthus phytochrome

Summary Phytochrome has been measured in etiolated seedling of Amaranthus caudatus. The phytochrome content increases from the time of germination until 72 hr from sowing, after which it remains constant at 27.5x10^-3 (OD) units per 200 seedlings. After a saturating dose of red light P _fr decays in the dark to a form not detectable photometrically. There is no evidence for the process of dark reversion of P _fr to P _fr found in other dicotyledons. Even in the presence of azide, a selective inhibitor of decay, the process of dark reversion is not observed. The decay of P _fr has been investigated at different temperatures and follows first order decay kinetics throughout. Over the temperature range 15–30° the Q ₁₀ of decay remained constant at 4.3.The photostationary states of phytochrome (P _fr /P _total )maintained by mixed red/far-red light have been measured in both seedlings and partially purified protein extracts, with good agreement. The rate of phytochrome decay can be manipulated by changing the P _fr /P _total ratio. The lag period before a decay curve becomes exponential is characteristic of a particular P _fr /P _total ratio and represents the time for attainment of the photostationary state. The effect of energy on decay has been investigated under red and blue light. The rate of phytochrome decay is dependent on the P _fr /P _total ratio and only becomes energy dependent when the light intensity is so low that the photostationary state is never attained.The process of apparent phytochrome synthesis has been found in Amaranthus. After reducing the phytochrome to a low level by red light treatment a rate of apparent synthesis of 1.35×10^-4 (OD) units per hr per 200 seedlings was observed, levelling off at 29% of the original phytochrome level.Under white tungsten lights of high intensity there is a deviation from the expected first order decay kinetics. The nature of this low rate of decay cannot be explained at the present time.