Factors controlling rates of nonphotochemical transformation of Pisum phytochrome in vitro

Nonphotochemical transformations of the far-red absorbing formof phytochrome, such as its decay or dark reversion to Pr, werestudied with solutions obtained from etiolated pea epicotyltissues at various steps of purification. At pH 7.8, the rateof dark Pfr reversion became significantly faster after thecrude extract was purified by gel filtration, but that of wellpurified solutions was quite low. Decay of Pfr was not seenduring any purification step at an alkaline pH, but it occurredin the acidic range of pH even in the presence of sulfhydrylcompounds. The rate of Pfr reversion was also influenced bypH; it increased with an increasing pH. Dark reversion of Pisum Pfr was confirmed to proceed in a short,rapid initial phase followed by a slow phase. (Received September 9, 1970; )     

Effects of divalent cations and EDTA on spectral properties of phytochrome in particulate fractions from etiolated pea epicotyls

The photoreversible absorbance change of phytochrome in suspensionsof a 20,000xg particulate fraction (20kP) prepared from a 1,000xgsupernatant (1kS) of etiolated pea epicotyl extracts decreasedremarkably in the presence of 5 mM Cu²⁺, Zn²⁺ and Co²⁺, butremained unchanged in 5 mM Ca²⁺, Mg²⁺, Fe²⁺ or Mn²⁺. This spectraldistortion of phytochrome was more evident in soluble preparationsand in suspensions of pellets prepared from red light (R)-irradiatedtissues than it was in suspensions of pellets prepared in thedark from etiolated tissues that received no actinic irradiation. When Cu²⁺ was added to the red-light-absorbing form of phytochrome(Pr) in resuspended pellets prepared from R-irradiated tissues,the distortion of its difference spectrum took place after irradiationwith the first actinic R. In contrast, when Cu²⁺ was added tothe far-red-light-absorbing form of phytochrome (Pfr) in thesame resuspended pellet, no distortion was seen, unless thePfr in the pellet was first photoconverted to Pr and then photoconvertedback to Pfr. Spectral distortion of Pr remained small during dark incubationat 25°C when suspensions of 20kPs were prepared and incubatedwith a buffer containing EDTA, whether the 20kP was preparedfrom nonirradiated tissue or from R-irradiated tissues. But,when EDTA was added to a suspension of 20kP prepared from 1kS,after the 1kS was irradiated with R in the presence of 10 mMCaCl₂, the spectral distortion of Pr in 20kP occurred instantaneously. (Received April 14, 1980; )     

Spectral properties of soluble and pelletable phytochrome from epicotyls of etiolated pea seedlings

The red-light(R)-absorbing form of phytochrome (Pr) was detected spectrophotometrically in a 20,000 g particulate fraction prepared from a 1,000 g supernatant fraction from epicotyl tissue of pea (Pisum sativum L.) seedlings grown in the dark and only briefly exposed to dim green light. The difference spectrum of phytochrome in this fraction was essentially the same as that of soluble phytochrome from the same tissue. When the non-irradiated 20,000 g particulate fraction was incubated in the dark at 25° C, an absorbance change (decrease) of Pr after actinic red irradiation was found only in the far-red (FR) region. When the 20,000 g particulate fraction was irradiated with R and then incubated in the dark, the FR-absorbing form of phytochrome (Pfr) disappeared spectrally at a rate about half that in the soluble fraction, and the difference spectrum of the Pr which became detectable after dark incubation of the 20,000 g particulate fraction was markedly distorted. In contrast, Pfr in a 20,000 g particulate fraction prepared from tissues irradiated with R did not change optically during dark incubation at 25° C for 60 min, while Pfr in the soluble fraction from the same tissue disappeared in the dark. No dissociation of either Pr or Pfr from the 20,000 g particulate fraction was indicated during a 60-min dark incubation at 25° C, but Pfr in a 20,000 g particulate fraction prepared in vitro from R-irradiated 1,000 g supernatant fraction in the presence of CaCl₂ disappeared spectrally and the difference spectrum of Pr in the 20,000 g particulate fraction became quite distorted during the dark incubation.Abbreviations Pr red-light-absorbing form of phytochrome - Pfr far-red-light-absorbing form of phytochrome - FR far-red light - FR1 first actinic far-red light - FR2 second actinic far-red light - R red light - R1 first actinic red light - 1kS 1,000 g supernatant fraction - 20kS 20,000 g supernatant fraction - 20kP 20,000 g particulate fraction     

Apparent Pfr killer reaction and P* denaturation in segments of etiolated pea epicotyl

When totally etiolated pea epicotyls were cut into segments and incubated with potassium phosphate buffer, pH 6.0, in the dark at 25 C, an instantaneous loss of photoreversible absorbance change, Δ (ΔA) between 660 and 730 nm, was observed after the first irradiation with actinic red light in the spectrophotometric measurement of phytochromein vivo. The shorter the epicotyl segments, and the longer the period of dark incubation, the greater was the loss detected in the measurement. A remarkable decline of Δ(ΔA) in the far-red region was seen inin vivo difference spectra for phytochrome, after the epicotyl segments were incubated in the dark at 25 C. As the period of dark incubation was prolonged, the ratio of the maximal change of Δ(ΔA) in the far-red region to that in the red region was reduced. It decreased to ca. one third of the initial value after incubation for 8 hr. The evidence indicates that Pfr killer activity and P^* denaturation, both of which have so far been known onlyin vitro, can also occur in segments of etiolated pea epicotyls.     

Plant Lectins Induce the Production of a Phytoalexin in Pisum sativum

The effects of several plant lectins on the production of apea phytoalexin, pisatin, were examined. Con A, PHA, PNA andPSA each induced the production of pisatin in pea epicotyl tissues,demonstrating that plant lectins can act as elicitors. The productionof pisatin in response to PHA, PNA or PSA was not affected bythe simultaneous presence of the respective hapten sugars, whereashaptens specific for Con A, such as -D-mannose and methyl--D-mannoside,abolished the induction of pisatin by Con A. These results indicatethat the elicitor effect of Con A is attributable to its abilityto bind to specific carbohydrates in pea cells. Induction ofthe production of pisatin by Con A was markedly inhibited bythe suppressor derived from a pea pathogen, Mycosphaerella pinodes,and by several inhibitors related to signal-transduction pathways.It is suggested, therefore, that the Con A-induced productionof pisatin in pea tissues might be associated with activationof a signal-transduction pathway. An additive effect on theaccumulation of pisatin was observed when Con A was presentwith a polysaccharide elicitor from M. pinodes, suggesting thatexogenous Con A does not compete with the recognition site(s)for the fungal elicitor in pea cells. The present data alsoindicate that Con A may be useful for characterization of thesignal-transduction system that leads to the synthesis of phytoalexinin pea epicotyl tissues. (Received November 16, 1994; Accepted April 20, 1995)     

In vivo phytochrome reversion in immature tissue of the alaska pea seedling

Reversion of far red-absorbing phytochrome to red-absorbing phytochrome without phytochrome destruction (that is, without loss of absorbancy and photoreversibility) occurs in the following tissues of etiolated Alaska pea seedlings (Pisum sativum L.): young radicles (24 hours after start of imbibition), young epicotyls (48 hours after start of imbibition), and the juvenile region of the epicotyl immediately subjacent to the plumule in older epicotyls. Reversion occurs rapidly in the dark during the first 30 minutes following initial phototransformation of red-absorbing phytochrome to far red-absorbing phytochrome. If these tissues are illuminated continuously with red light for 30 minutes, the total amount of phytochrome remains unchanged. Beyond 30 minutes after a single phototransformation or after the start of continuous red irradiation, phytochrome destruction commences. In young radicles, sodium azide inhibits this destruction, but does not affect reversion. In older tissues in which far red-absorbing phytochrome destruction begins immediately upon phototransformation, strong evidence for simultaneous far red-absorbing phytochrome reversion is obtained from comparison of far red-absorbing phytochrome loss in the dark following a single phototransformation with far red-absorbing phytochrome loss under continuous red light.     

Effect of malformin on phytochrome reactions in Vigna and Avena

The rate of destruction of the far red absorbing form of phytochrome(Pfr) in green or etiolated cuttings of Vigna radiata was slowerin the presence of malformin than in its absence. Malforminhad no effect on the accumulation of total phytochrome in thedark, or on the reaccumulation of phytochrome after destructionin red light. The amount of photoconversion of the red absorbingform of phytochrome (Pr) to Pfr or Pfr to Pr by given dosesof red or far red radiation was slightly but consistently lessin malformin-treated cuttings of V. radiata than in controls.Malformin had no effect on the rate of destruction or photoconversionof phytochrome in etiolated shoots of Avena sativa. The decreasein destruction rate of Pfr by malformin in V. radiata may contributeto the inhibition of dark abscission by malformin after lighttreatment. (Received October 3, 1979; )     

Phytochrome properties and the molecular environment

In vitro data support a scheme of phytochrome phototransformation involving intermediates in a sequential pathway. The fraction of total phytochrome maintained as intermediate under conditions of pigment cycling as well as the rate of the dark reversion of the far red-absorbing (Pfr) to the red-absorbing form of phytochrome (Pr) has been shown to depend on the molecular environment of the phytochrome molecules. Inverse dark reversion of Pr to Pfr has been observed in vitro. These results contribute toward an understanding of the observed paradoxes between physiological experiments and measurements of the amount and state of phytochrome in vivo. The in vivo spectrophotometric assay measures an average of the properties of phytochrome in different cellular environments, whereas a particular physiological response may be controlled by phytochrome molecules in one particular environment. It is therefore possible that all phytochrome is potentially active and triggers specific responses by virtue of its localization.     

Developmental control of xylem hydraulic resistances and vulnerability to embolism in Fraxinus excelsior L.: impacts on water relations

The freezing tolerance of many plants, such as pea (Pisum sativum),is increased by exposure to low temperature or abscisic acidtreatment, although the physiological basis of this phenomenonis poorly understood. The freezing tolerance of pea shoot tips,root tips, and epicotyl tissue was tested after cold acclimationat 2C, dehydration/rehydration, applications of 10^–4M abscisic acid (ABA), and deacclimation at 25C. Tests wereconducted using the cultivar ‘Alaska’, an ABA-deficientmutant ‘wil’, and its ‘wildtype’. Freezinginjury was determined graphically as the temperature that caused50% injury (T₅₀) from electrical conductivity. Endogenous ABAwas measured using an indirect enzyme-linked immunosorbant assay,and novel proteins were detected using 2-dimensional polyacrylamidegel electrophoresis. The maximum decrease in T₅₀ for root tissuewas 1C for all genotypes, regardless of treatment. For ‘Alaska’shoot tips and epicotyl tissue, exogenous ABA increased thefreezing tolerance by –1.5 to –4.0C, while coldtreatment increased the freezing tolerance by –7.5 to–14.8C. Cold treatment increased the freezing toleranceof shoot tips by –9 and –15C for ‘wil’and ‘wild-type’, respectively. Cold acclimationincreased endogenous ABA concentrations in ‘Alaska’shoot tips and epicotyls 3- to 4-fold. Immunogold labeling increasednoticeably in the nucleus and cytoplasm of the epicotyl after7 d at 2C and was greatest after 30 d at the time of maximumfreezing tolerance and soluble ABA concentration. Cold treatmentinduced the production of seven, three, and two proteins inshoot, epicotyl, and root tissue of ‘Alaska’, respectively.In ‘Alaska’ shoot tissue, five out of seven novelproteins accumulated in response to both ABA and cold treatment.However, only a 24 kDa protein was produced in ‘wil’and ‘wild-type’ shoot and epicotyl tissues aftercold treatment. Abscisic acid and cold treatment additivelyincreased the freezing tolerance of pea epicotyl and shoot tissuesthrough apparently independent mechanisms that both resultedin the production of a 24 kDa protein. Key words: Pisum sativum, cold acclimation, immuno-localization     

Freezing tolerance, protein composition, and abscisic acid localization and content of pea epicotyl, shoot, and root tissue 3

The freezing tolerance of many plants, such as pea (Pisum sativum),is increased by exposure to low temperature or abscisic acidtreatment, although the physiological basis of this phenomenonis poorly understood. The freezing tolerance of pea shoot tips,root tips, and epicotyl tissue was tested after cold acclimationat 2C, dehydration/rehydration, applications of 10^–4M abscisic acid (ABA), and deacclimation at 25C. Tests wereconducted using the cultivar ‘Alaska’, an ABA-deficientmutant ‘wil’, and its ‘wildtype’. Freezinginjury was determined graphically as the temperature that caused50% injury (T₅₀) from electrical conductivity. Endogenous ABAwas measured using an indirect enzyme-linked immunosorbant assay,and novel proteins were detected using 2-dimensional polyacrylamidegel electrophoresis. The maximum decrease in T₅₀ for root tissuewas 1C for all genotypes, regardless of treatment. For ‘Alaska’shoot tips and epicotyl tissue, exogenous ABA increased thefreezing tolerance by –1.5 to –4.0C, while coldtreatment increased the freezing tolerance by –7.5 to–14.8C. Cold treatment increased the freezing toleranceof shoot tips by –9 and –15C for ‘wil’and ‘wild-type’, respectively. Cold acclimationincreased endogenous ABA concentrations in ‘Alaska’shoot tips and epicotyls 3- to 4-fold. Immunogold labeling increasednoticeably in the nucleus and cytoplasm of the epicotyl after7 d at 2C and was greatest after 30 d at the time of maximumfreezing tolerance and soluble ABA concentration. Cold treatmentinduced the production of seven, three, and two proteins inshoot, epicotyl, and root tissue of ‘Alaska’, respectively.In ‘Alaska’ shoot tissue, five out of seven novelproteins accumulated in response to both ABA and cold treatment.However, only a 24 kDa protein was produced in ‘wil’and ‘wild-type’ shoot and epicotyl tissues aftercold treatment. Abscisic acid and cold treatment additivelyincreased the freezing tolerance of pea epicotyl and shoot tissuesthrough apparently independent mechanisms that both resultedin the production of a 24 kDa protein. Key words: Pisum sativum, cold acclimation, immuno-localization     

Effects on Phytochrome Controlled Germination Produced by Far-Red Irradiation of Seeds Before and During Rehydration

Seeds irradiated with red light and then re-dried will respondto this light treatment on subsequent rehydration in the dark.If such high-Pfr seeds are irradiated in the dry state withfar-red light immediately before rehydration the percentagegermination is significantly reduced in the case of Plantagomajor and Sinapis arvensis but increased in Bromus steriliswhere Pfr inhibits germination. This effect of far-red lightcan be reversed by red light despite the fact that red lightalone has no effect on dry seed. This is due to the interconversionof Pfr and the red light absorbing phytochrome intermediatecomplex meta-Fa. If there is a delay between far-red irradiationand rehydration of Sinapis seeds, the inhibitory effect of thefar-red irradiation becomes progressively less the longer thedelay. This reduction in effectiveness of far-red is interpretedin terms of a dark reversal of meta-Fa to Pfr with a half-lifeof about 4–6 h. The reappearance of Pfr is either veryslow or docs not occur in dehydrated Plantago seeds, as far-redtight given 96 h prior to hydration is just as inhibitory asfar-red light given immediately before hydration. Meta-Fa doesappear to revert to Pfr in darkness in Bromus seeds, but onlyvery slowly. The rapid increase in effectiveness of red irradiationduring rehydration of high-Pfr Plantago seeds suggests that,in this species, the pre-treatment used in preparation of high-Pfrseeds may increase the receptivity or amount of the Pfr reactionpartner. Key words: Phytochrome intermediates, Seeds, Germination     

Olfactory detection of a complex versus a simple substance in advanced age

Thresholds for lavandin oil (highly complex substance) and n-butanol(single compound) averaged higher in subjects over 70 yearsthan in controls under 30, by comparable amounts and with aboutthe same variability.     

Changes in Nucleic Acids in Phytochrome-dependent Elongation of the Alaska Pea Epicotyl

Red light, which produces the physiologically active form of phytochrome (Pfr), inhibited epicotyl elongation in intact dark-grown Alaska pea seedlings. This red light response was detectable 3 hours after the light treatment and became pronounced after 5 hours. The growth inhibition was completely reversed by far red light applied immediately after the red or by pretreatment of the seedlings with the plant hormone gibberellin A₃.     

Distribution and nonphotochemical transformation of phytochrome in subcellular fractions from pisum epicotyls

In etiolated pea (Pisum sativum L. cv. Alaska) shoots about 3% of the total extractable phytochrome was found in the mitochondrial fraction and about 4.5% in the microsomal fraction, while over 70% was soluble in the 105,000g supernatant. The value of Δ(ΔA) per milligram of protein was significantly higher in the 105,000g supernatant than in these particulate fractions. The percentage conversion of Pr to Pfr was approximately proportional to the total dose of red light in every subcellular fraction tested, unless the dose approached a saturation level. After a brief irradiation of intact shoots with red light at 26 C, each subcellular fraction showed different patterns of dark transformation in vivo at 26 C; that is, the amount of the particulate-bound phytochrome increased immediately after the irradiation, and a reversion of Pfr to Pr was indicated for the first 2 hr in the 12,000g supernatant, but not at all in the mitochondrial and microsomal fractions. The amounts of Pr in the mitochondrial and microsomal fractions did not change during the dark incubation, while those in the 12,000g supernatant increased with time. Similar results were obtained with apical shoot segments after exposure to red light at 0 C and a subsequent dark incubation on moist filter paper at 26 C.     

Phosphorylation of Phytochrome B Inhibits Light-Induced Signaling via Accelerated Dark Reversion in Arabidopsis

The photoreceptor phytochrome B (phyB) interconverts between the biologically active Pfr (λ_max = 730 nm) and inactive Pr (λ_max = 660 nm) forms in a red/far-red–dependent fashion and regulates, as molecular switch, many aspects of light-dependent development in Arabidopsis thaliana. phyB signaling is launched by the biologically active Pfr conformer and mediated by specific protein–protein interactions between phyB Pfr and its downstream regulatory partners, whereas conversion of Pfr to Pr terminates signaling. Here, we provide evidence that phyB is phosphorylated in planta at Ser-86 located in the N-terminal domain of the photoreceptor. Analysis of phyB-9 transgenic plants expressing phospho-mimic and nonphosphorylatable phyB–yellow fluorescent protein (YFP) fusions demonstrated that phosphorylation of Ser-86 negatively regulates all physiological responses tested. The Ser86Asp and Ser86Ala substitutions do not affect stability, photoconversion, and spectral properties of the photoreceptor, but light-independent relaxation of the phyB^Ser86Asp Pfr into Pr, also termed dark reversion, is strongly enhanced both in vivo and in vitro. Faster dark reversion attenuates red light–induced nuclear import and interaction of phyB^Ser86Asp-YFP Pfr with the negative regulator PHYTOCHROME INTERACTING FACTOR3 compared with phyB–green fluorescent protein. These data suggest that accelerated inactivation of the photoreceptor phyB via phosphorylation of Ser-86 represents a new paradigm for modulating phytochrome-controlled signaling.     

Direct Organogenesis and Plant Regeneration in Preconditioned Tissue Cultures of Lathyrus cicera L., L. ochrus (L.)DC. and L. sativus L.

This study demonstrates the importance of preconditioning ofsource tissue in regeneration of multiple shoot buds from severalspecies of Lathyrus. Preconditioned multiple shoots of Lathyruscicera L., L. ochrus (L.) DC. and L. sativus L. were obtainedby germinating seeds on Murashige and Skoog (MS) medium containing50 µM N⁵-benzylaminopurine (BAP) for 2 to 3 weeks. Multipleshoot bud formation occurred when epicotyl explants of preconditionedshoots were cultured on MS medium containing 5–50 µMBAP. No shoot regeneration was observed from epicotyl explantswhich were obtained from non-preconditioned shoots. Shoot budswere formed directly on explants without an intervening callusphase after 2 to 3 weeks of culture. Regenerated shoot budsformed healthy shoots which developed prolific and strong rootswhen transferred to MS medium supplemented with 2.5 µMnaphthaleneacetic acid (NAA). Lathyrus cicera L., L. ochrus (L.) DC., Ochrus Vetch, L. sativus L., Lathyrus pea, de novo differentiation, epicotyl, preconditioning with BAP     

A fallacy in phytochrome pelletability due to in vitro instability of the phytochrome-particle association

Two fractionation procedures were used to study the phenomenonof phytochrome pelletability or binding to a particulate fractionof maize coleoptiles. Using a revised procedure, we were unableto show an increase of phytochrome pelletability during darkincubation of red irradiated plant tissue, reported by Manabeand Furuya for pea seedlings (6), and the P_fr-enhanced affinityfor P_r in R/FR irradiated tissue as reported by Quail et al.(11). However, we were able to match these reported observationsusing a procedure which we have regarded as standard. In thestandard procedure, the irradiated tissue is homogenized andthe brei permitted to incubate in the dark at 0?C before fractionationby differential centrifugation prior to measurements of phytochromepelletability. In the revised procedure this incubation is eliminatedand fractionation and measurement follow directly on tissuehomogenization. A progressive decrease of particulate phytochromewas observed during dark incubation at 0?C of the brei fromred irradiated tissue, but no substantial decrease was observedduring dark incubation of the red irradiated tissue at 0?C.The decrease was not dependent on in vitro P_frP_r reversion.In the case of R/FR irradiated tissues, phytochrome pelletabilitywas found to decrease during dark incubation of both the irradiatedtissue and its brei at 0?C. With these results and a recognitionof the tendency of phytochrome to dissociate from the particulatefraction in vitro, we have thus rationalized the results ofQuail et al. (11) and Manabe and Furuya (6). (Received August 12, 1976; )     

A Comparison of the Rate of Maintenance Respiration in Some Crop Legumes and Tobacco Determined by Three Methods

Carbon exchange was measured on whole plants of field bean,lucerne, chick pea, kidney bean, pea and tobacco. The maintenance respiration rate was measured in three ways:(i) by allowing the CO₂ efflux to decay in prolonged darknessto an asymptotic value which was then taken to be the maintenancevalue (the dark decay method); (ii) by plotting the dark CO₂efflux as a function of the net CO₂ uptake over a range of irradiancesand taking maintenance as the dark CO₂ efflux when the net CO₂uptake was zero (the dynamic method); and (iii) by plottingthe total CO₂ uptake as a function of the growth rate and takingmaintenance respiration as the CO₂ efflux when the growth ratewas zero (the zero growth rate method). The range of valuesfor the maintenance coefficient over all species was from 1.6to 2.1 per cent of the dry weight per day, 1.8 to 2.1 per centand 2.7 to 2.9 per cent as determined by these three methodsrespectively. There was a linear relationship, common to allspecies, between the maintenance respiration rate (dark decaymethod) and dry weight, total nitrogen and the organic nitrogencontent. The growth coefficient (0.69±0.01) was the samefor field bean, chick pea and lucerne and was unaffected bythe method of estimation. It was concluded that the dark decay method provided the bestestimate of the minimal maintenance requirements in the plantsstudied. Vicia faba L., Medicago sativa L., Cicer arientinum L., Phaseolus vulgaris L., Pisum sativum L., Nicotiana tobacum L., field bean, lucerne, chick pea, kidney bean, pea, tobacco, respiration, maintenance, growth, nitrogen content