Temperature- and seed-batch-related variation in the kinetic behaviour of phytochrome under ‘high-irradiance-response’ conditions

The characteristics of the high-irradiance response (HIR) of plant photomorphogenesis are thought to be the result of the interaction of both the light and dark reactions of phytochrome. Thus any variation in the rates of the dark reactions may be expected to lead to variation in the characteristics of the HIR. We report here substantial differences in the rates of the dark reactions between different seed batches of a single species (Sinapis alba L.), and also between different organs of seedlings from each of the batches of seed. Calculations of phytochrome dynamics from the measured dark-reaction rates show that the behaviour of Pfr under HIR conditions will vary considerably according to seed batch and seedling organ. Much larger differences in dark-reaction rates, and the resulting phytochrome dynamics, were found between 25° and 10° C. These lead to the prediction that the HIR will be much reduced at the lower temperature, and may be absent in some cases.Abbreviations and symbols HIR high-irradiance response - Pfr far-red-absorbing form of phytochrome - Pr red-absorbing form of phytochrome - Ptot total phytochrome, Pr+Pfr - _ss Pfr/Ptot ratio which immediately establishes the phytochrome steady state     

Proteolysis alters the spectral properties of 124 kdalton phytochrome from Avena

Native phytochrome from Avena sativa L. is homogeneous with a monomeric molecular weight of 124 kdalton; 6–10 kdalton larger than the heterogeneous 120 kdalton preparations previously considered to be undegraded (Vierstra and Quail, 1982, Proc. Natl. Acad. Sci. USA, 79: 5272–5276). The phototransformation difference spectrum (Pr-Pfr) of 124 kdalton phytochrome measured in crude extracts has a minimum in the farred region at 730 nm, the same as that observed in vivo. These spectral properties contrast with those of 120 kdalton phytochrome purified by column immunoaffinity chromatography where the difference minimum is at 724 nm. When 124 kdalton phytochrome is incubated as Pr in crude extracts, the difference minimum shifts progressively to shorter wavelengths (from 730 to 722 nm) concomitant with the proteolytic degradation of the chromoprotein to the mixture of 118 and 114 kdalton species that comprise 120 kdalton phytochrome preparations. These two effects are inhibited in concert by the serine protease inhibitor, phenylmethylsulfonylfluoride, and or maintenance of the phytochrome in the Pfr form. These results provide further evidence that 124 kdalton phytochrome is the native molecule in Avena and indicate that the peptide segments removed by proteolysis of the Pr form are important to the pigment's spectral integrity. The present data thus resolve the previously unsettled question of why the Pfr form of 120 kdalton phytochrome isolated by various procedures from Avena has been found to absorb at shorter wavelengths than that observed in vivo. Previous spectral studies with 120 kdalton phytochrome preparations are open to reexamination.Abbreviations, symbols PMSF phenylmethylsulfonylfluoride - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - Ig immunoglobulin - A_minimum, A_maximum phototransformation difference spectrum (Pr-Pfr) minimum and maximum - A_r/A_fr ratio spectral change ratio     

Phytochrome-mediated induction of phenylalanine ammonia-lyase in the cotyledons of tomato (Lycopersicon esculentum Mill.) plants

Phenylalanine ammonia-lyase (PAL; EC 4.3.1.5.) induction in cotyledons from 96-h dark-grown Lycopersicon esculentum Mill. was studied in response to continuous light and hourly light pulses (blue, red, far red). The increases of PAL promoted by blue and red pulses are reversed completely by immediately following 758 nm irradiations. The response to continuous red light could be substituted for by hourly 6-min red light pulses. The effect of continuous red treatments is mainly due to a multiple induction effect of phytochrome. In contrast to red light, hourly light pulses with far red and blue, light can only partially substitute for continuous irradiation. The continuous blue response could be due to a combination of a multiple induction response and of a high irradiance response of phytochrome. The continuous far red response, could represent a high irradiance response of phytochrome. Dichromatic irradiations indicate that phytochrome is the photoreceptor controlling the light response (PAL) in tomato seedlings.Abbreviations Norflurazon NF-4-chloro-5-(methylamino)-2-(,,,-trifluoro-m-tolyl)-3 (2H) pyridazinone - PAL phenylalanine ammonia-lyase - phytochrome photoequilibrium P_fr/P_tot - P_fr far-red absorbing form of phytochrome - P_r red absorbing form of phytochrome - P_tot total phytochrome: P_r+P_fr     

The influence of light quality on the phytochrome content of light grown Sinapis alba L. and Phaseolus aureus Roxb.

The effect on the phytochrome system of light regimes establishing a range of photoequilibria was studied in two light grown dicotyledonous plants, both of which were treated with the herbicide SAN 9789 to prevent chlorophyll accumulation. In Sinapis alba L. cotyledons the results are comparable with phytochrome behaviour in etiolated mustard seedlings; the level of Pfr becomes independent of wave-length whereas the total phytochrome level is wave-length dependent. Contrasting properties are exhibited in Phaseolus aureus Roxb. leaves in which total phytochrome is unaffected by light quality; consequently the Pfr level is dependent on wavelength. Nevertheless, the amount of phytochrome in mung leaves increased after transfer to darkness suggesting that light still has a profound influence on the phytochrome system, even though light quality during the light period and prior to darkness does not.Abbreviations FR far-red light - WL white light - PAR photosynthetically active radiation - Pfr far-red light absorbing form of phytochrome - Pr red light absorbing form of phytochrome - Ptot total phytochrome level (=Pr+Pfr) - Pfr/Pfr+Pr - SAN 9789 4-chloro-5-(methylamino) 2(,, trifluoro-m tolyl)-3(2H)-pyridazinone     

Photocontrol of hypocotyl elongation in light-grown Cucumis sativus L.

The control by phytochrome of hypocotyl elongation of light-grown Cucumis sativus L. after a white-light period was examined. The farred-absorbing form of phytochrome inhibits hypocotyl elongation. The response to phytochrome photostationary state () is not linear; all values of from 0.004 to 0.13 promote growth maximally, in the range of values of from 0.13 to 0.22 there is a linear growth response, between values of of 0.22 and 0.35 there is again no differential effect, and for values above 0.35 there is a strong (near linear) effect of on elongation. A kinetic examination of events following the white-light period shows that the major recovery from the photoperiod requires 8.5 h of darkness. End-of-day far-red treatment produces a very different response pattern, with a minor growth stimulation within 28 min of treatment followed by a major effect after 80 to 90 min. Three hours after far-red treatment there is a transient decline in growth rate which persists for about 2 h. Over the whole time course there is a great stimulation of growth rate compared with the controls. A similar growth-rate pattern also occurs if the end-of-day is 0.48, although the magnitude of the growth stimulation is less. Two components are affected by end-of-day , namely the time at which growth recovers and the subsequent growth rate. In the long term, the latter accounts for most of the differences in elongation growth. The dark recovery when only the hypocotyl is irradiated requires 4 h, but end-of-day far-red treatment reduces this to about 1.5 h. The persistence of the far-red-absorbing form of phytochrome for many hours in darkness in these light-grown plants is also demonstrated.Abbreviations and symbols D darkness - FR far-red light - Pfr far-red-absorbing form of phytochrome - R red light - WL white light (from fluorescent lamps) - photostationary state of phytochrome - _c calculated      

A comparative study of the responsivity of Sinapis alba L. seedlings to pulsed and continuous irradiation

Anthocyanin formation in 36h dark grown Sinapis alba L. seedlings and inhibition of hypocotyl elongation in 36h and 54h dark grown and 54h and 7 day light grown seedlings in response to continuous red light could be substituted for by hourly 5 min light pulses where the total fluence over the irradiation period is the same. These pulses are partially (36h) or almost totally (54h and 7 day) reversible by subsequent far-red (RG 9) light pulses. In contrast to 654 nm light, hourly light pulses with 552 nm, 449 nm and 715 nm can at best only partially substitute for continuous irradiation. These data are discussed with respect to the commonly used models for the phytochrome high irradiance response.Abbreviations P_tr tar-red absorbing form of phytochrome - SAN 9789 4-chloro-5-(methyl-amino)-2-(,,-trifluoro-m-tolyl)-3(2H)-pyridazinone=Norflurazon - HIR High irradiance response     

The effect of light pretreatments on phytochrome-mediated induction of anthocyanin and of phenylalanine ammonia-lyase

Induction by light of phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) and of anthocyanin in cotyledons of the mustard (Sinapis alba L.) seedling is strongly affected by a light pretreatment which operates through phytochrome. If PAL or anthocyanin is induced by a light pulse, the effectiveness of phytochrome (P_fr) is strongly increased by a light pretreatment; however, if the increase of the PAL level or synthesis of anthocyanin is elicited by continuous far-red light (operating via phytochrome in the High Irradiance Response), effectiveness of light is strongly reduced by the same light pretreatment. This reduction of effectiveness is correlated with a decrease of total phytochrome (P_tot) caused by the light pretreatment. It is argued that the observations are compatible only with the open phytochrome-receptor model as suggested by Schäfer (J. Mathem. Biol. 2, 41–56, 1975). The peaks of the time courses of the PAL levels under continous far-red light are located at 48 h after sowing and do not depend on the original level of phytochrome. The decrease of the PAL levels beyond 48 h after sowing takes place independently of phytochrome and of the actual level of PAL.Abbreviations P_r red absorbing form of phytochrome - P_fr far-red absorbing form of phytochrome - P_tot total phytochrome (P_r+P_fr) - {ie369-1} [P_fr] /[P_tot], photoequilibrium of phytochrome at wavelength - HIR High Irradiance Response - PAL phenylalanine ammonialyase (EC 4.3.1.5)     

Molecular mapping of genes for resistance to rice blast (Pyricularia grisea Sacc.)

Two dominant genes conferring complete resistance to specific isolates of the rice blast fungus, Pyricularia grisea Sacc., were located on the molecular map of rice in this study. Pi-l(t) is a blast resistance gene derived from the cultivar LAC23. Its map location was determined using a pair of nearly isogenic lines (NILs) and a B₆F₃ segregating population from which the isoline was derived. RFLP analysis showed that Pi-l(t) is located near the end of chromosome 11, linked to RZ536 at a distance of 14.0±4.5 centiMorgans (cM). A second gene, derived from the cultivar Apura, was mapped using a rice doubled-haploid (DH) population. This gene was located on chromosome 12, flanked by RG457 and RG869, at a distance of 13.5+-4.3 cM and 17.7+-4.5 cM, respectively. The newly mapped gene on chromosome 12 may be allelic or closely linked toPi-ta. (=Pi-4(t)), a gene derived from Tetep that was previously reported to be linked to RG869 at a distance of 15.4±4.7 cM. The usefulness of markers linked to blast resistance genes will be discussed in the context of breeding for durable blast resistance.     

Spectrophotometric phytochrome measurements in light-grown Avena sativa L.

Phytochrome was studied spectrophotometrically in Avena sativa L. seedlings that had been grown for 6 d in continous white fluorescent light from lamps. Greening was prevented through the use of the herbicide San 9789. When placed in the light, phytochrome (P_tot) decreased with first order kinetics (_1/2 2 h) but reached a stable low level (2.5% of the dark level) after 36 h. This concentration of phytochrome remained constant in the light and during the initial hours of a subsequent dark period, but increased significantly after a prolonged dark period. Evidence suggests that the constant pool of phytochrome in the light is achieved through an equilibrium between synthesis of the red absorbing (P_r) and destruction of the far-red absorbing form (P_fr) of phytochrome. It is concluded that the phytochrome system in light-grown oat seedlings is qualitatively the same as that known from etiolated monocotyledonous seedlings, but different than that described for cauliflower florets.Abbreviations P_fr the far-red light absorbing form of phytochroma - P_r the red light absorbing form of phytochrome - P_tot P_r+P_fr - k_s rate constant of P_r synthesis - k_d rate constant of P_fr destruction - MOPS N-morpholino-3-propane-sulfonic acid - IRIS Tris (hydroxymethyl) amino methane - San 9789 4-chloro-5-(methyl amino)-2-(,,-trifluoro-m-tolyl)-3(2H)pyridazinone     

An in vitro association of soluble phytochrome with a partially purified organelle fraction from barley leaves

Red light treatment in vitro increases the pelletability of phytochrome in homogenates of etiolated barley (Hordeum vulgare L. cv. Julia) leaves. When mixtures of soluble phytochrome (100,000 x g supernatant) and partially-purified organelles (Sephadex G-50 eluate) are irradiated the amount of pelletable phytochrome increases by a factor of two. Pre-irradiation treatments show that phytochrome in both components of the mixture must be in the Pfr form for increased pelletability to be observed. Once associated, photoreversion of Pfr to Pr does not result in decreased pelletability. The results are consistent with a non-artifactual in vitro association of soluble phytochrome to organelle membranes. One possible explanation is that Pfr molecules associate to form dimers.     

Persistent effects of changes in phytochrome status on internode growth in light-grown mustard: Occurrence,kinetics and locus of perception

Extension growth of the first internode in fully de-etiolated mustard (Sinapis alba L.) seedlings (11–12.5 d old) is under the control of both the current phytochrome photoequilibrium (Pfr/P, ratio of the far-red-absorbing form of phytochrome to total phytochrome) and that established by short (<12 h) pretreatments. Plants were pretreated with either light pulses providing different calculated Pfr/P followed by dark incubations of different durations (a), or with a 12-h period of white light establishing different Pfr/P (b). After the pretreatments, the plants received either light pulses providing different Pfr/P, followed by dark incubations (c), or continuous white light with or without addtional far-red light (d). Thus, four experimental approaches were followed: (a)(c); (a)(d); (b)(c) and (b)(d). Extension growth during the second period (c or d) was not only affected by the current phytochrome status, but also by that established during the pretreatment period (a or b). The results show the existence of a long-term promotion of stem growth which persists after the end of the low Pfr/P pretreatment. This effect is different from the previously reported rapid effect of far-red light added to background white light as follows: (i) the duration of low Pfr/P required to effect a full response is longer (2.5 h); (ii) the duration of the promotion after returning to high Pfr/P is longer (approx. 24 h) and (iii) the locus of perception is mainly in the leaves, rather than the growing internode.Abbreviations FR far-red light - PAR photosynthetically active radiation - Pfr/P ratio between the FR-absorbing form and total phytochrome - R red light - WL white light     

Role of red light in hair-whorl formation induced by blue light in Acetabularia mediterranea

After a pre-treatment with red light, hair formation at the growing tip of the siphonaceous green alga Acetabularia mediterranea Lamour. (= A. acetabulum (L.) Silva) can be induced by a pulse of blue light. Red light is needed again after the inductive blue-light pulse if the new whorl of hairs is to develop within the next 24 h. In order to investigate the role of this red light, the duration of the red irradiation was varied and combined with periods of darkness. The response of hair-whorl formation was dependent on the total amount of red light, regardless of whether the red irradiation followed the blue pulse immediately or was separated from it by a period of darkness. Furthermore, periods of exposure to the photosynthesis inhibitor 3-(3,4-dichlorophenyl)-1-1dimethylurea had a similar effect to darkness. Both observations indicate that this red irradiation acts as a light source for photosynthesis. Whether or not the red light had an additional effect via phytochrome was tested in another type of experiment. The dependence of hair-whorl formation on red-light irradiance in the presence of simultaneous far-red irradiation was determined for the pre-irradiation period as well as for the irradiation period after the blue pulse. In both experiments, far-red light caused a small promotion of hair-whorl formation when low irradiances of red light were used. However, these differences were attributable to a low level of photosynthetic activity (which in fact was measurable) caused by red light reflected in the growth chamber. Furthermore, lowering the proportion of active phytochrome by far-red light would be expected to suppress hair-whorl formation. The influence of far-red light was also tested in a strain of Acetabularia mediterranea that developed hair whorls in about 20% of cells even when kept in complete darkness after the blue-light pulse. Far-red irradiation had no effect. These results strongly indicate that phytochrome is not involved in hair-whorl formation. Rather it is concluded that the effects of red light are caused by photosynthesis.Abbreviation DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea     

Evidence for phytochrome involvement in light-mediated stomatal movement in Phaseolus vulgaris L.

Observations made with primary leaves of Phaseolus vulgaris L. demonstrated that phytochrome modulates light-induced stomatal movement. Removal of the far-red-absorbing form of the pigment (Pfr) with far-red (FR) radiation decreased the time required by the stomata to reach maximal opening following a dark-to-light transition; this effect of FR was fully reversible with red. Removal of Pfr with FR also decreased the time required to reach maximal closure following a light-to-dark transition, and the rate of closure was dependent on the final irradiation treatment before darkness. No evidence was found for phytochrome involvement in determining stomatal aperture under constant conditions of either darkness of light.Abbreviations and symbols Chl chlorophyll - D darkness - FR far-red - phytochrome photostationary state - Pfr, Pr FR- and R-absorbing forms of phytochrome, respectively - R red     

Inheritance of the dwarf plant type in blackgram (Vigna mungo (L.) Hepper)

Summary The inheritance of the dwarf plant type was studied in blackgram (V. mungo (L.) Hepper). Type 9 has erect plant type with normal internode length. The mutant line, EMSD has reduced internode length. The F₁, F₂ and F₃ generations of a cross between Type 9 and EMSD and its reciprocal were studied. The extreme dwarf plant type appeared to be governed by a single recessive gene, dw ₁ dw ₁ with no cytoplasmic effect.Part of Ph.D. Thesis submitted by the first author     

Photoregulation of germination in seed of transgenic lines of tobacco and Arabidopsis which express an introduced cDNA encoding phytochrome A or phytochrome B

Photoinduction and photoinhibition of germination in seed from a homozygous tobacco (Nicotiana tabacum L.) line containing an introduced oat phyA cDNA (encoding phytochrome A) is compared with that of isogenic wild-type (WT) tobacco. Under continuous irradiation by a light source with a low redfar-red (RFR) ratio the transgenic tobacco seed appeared to be less susceptible to photoinhibition of germination compared with WT seed. However, induction of germination following a short pulse by R (666 nm) was not enhanced in the genotype transformed by oat phyA cDNA compared with the WT; neither did germination of the transgenic tobacco seed show an increased sensitivity to saturating pulses of light of longer wavelengths (666–730 nm). In seeds of transgenic Arabidopsis thaliana (L.) Heynh. which contained an introduced phytochrome-B-encoding cDNA, levels of dark germination were enhanced, consistent with mediation of response by phytochrome B-P_fr. The germination behaviour of Arabidopsis genotypes wich contained an introduced cDNA encoding phytochrome A, however, did not significantly differ from that of the WT.Abbreviations ABO seed transformed with Arabidopsis phyB - cDNA; CaMV cauliflower mosaic virus - FR far-red light - P_fr far-red-absorbing form of phytochrome - P_tot total phytochrome - P_fr/P_tot phytochrome photoequilibrium - R red light - RBO seed transformed with rice phyB cDNA - RFR quantum ratio of red and far-red light - WL white light - WL + FR whitelight supplemented with far-red light - WT wild type The authors wish to thank R.D. Vierstra (Department of Horticulture, University of Wisconsin-Madison, USA) for providing the transgenic tobacco line, and M.T. Boylan, D. Wagner and P.H. Quail (U.C. Berkeley/USDA Plant Gene Expression Center, Albany, Calif. USA) for providing the transgenic Arabidopsis lines. The work presented in this paper was funded by grants from the Agricultural and Food Research Council (H.S., A.C.M., G.C.W.).