Low temperature spectrophotometry of the phototransformation of Pfr to Pr in pelletable pea phytochrome

Manabe, K. 1987. Low temperature spectrophotometry of the phototransformation of P_fr to P_r, in pelletable pea phytochrome.
Low temperature spectrophotometry was used to study the phototransformation of P_fr to P_r in 1000–7000 g pelletable fractions extracted from dark grown pea ( Pisum sativum L. cv. Alaska) epicotyls which had been irradiated with red and then far-red light. At -170°C, far-red irradiation of the pelletable phytochrome which had been pre-irradiated with saturating fluence of red light before freezing caused formation of an intermediate (named I₆₆₀), the difference spectrum of which showed a marked ab-sorbance decrease at 740 nm and a concomitant small increase at about 660 nm. The inermediate I₆₆₀ was converted to another intermediate (I₆₆₀) when it was warmed above -80°C. The difference spectrum of this intermediate showed a positive peak at 670 nm. This intermediate was photoconverted to P_fr by red irradiation and also underwent dark reversion to P_fr at -60°C. I₆₆₀ formed P_r if the temperature was above -10°C. The basic features of the phytochrome intermediates resemble those obtained in vivo and in degraded purified phytochrome.     

Phototransformation of oat phytochrome with millisecond and submillisecond flashes: The level of the photostationary Pfr/Ptot ratio is modified by photoreversible intermediates

Photoconversion of the red-absorbing form of phytochrome (P_r) to the far-red-absorbing form of phytochrome (P_fr) and vice versa has been measured spectrophotometrically at 10°C in immobilized and soluble phytochrome (118 kdalton), prepared from 5-day-old etiolated oat seedlings ( Avena saliva L. cv. Sol II). The photostationary equilibrium φ= P_frP_tot (with P_tot= total amount of phytochrome P_r+ P_fr) for red light depends on whether it is established by repetitive pulses (≥ 5 s) or by repetitive flashes (≥ 4 ms). In the wavelength region around 660 nm, a lower φ is reached with flashes as compared to that with pulses. This difference becomes negligible if the wavelength is shortened to the 600 nm region, and it also disappears if the fluence of each individual flash is reduced. In contrast, in long-wavelength red light and short-wavelength far-red light, a higher φ is reached with flashes than with pulses.
We relate the differences in φ for flash and pulse irradiation to photochromic systems between P_r and photoreversible intermediates in the phototransformation pathway P_r→ P_fr. Thus, light absorption by phytochrome intermediates can be limiting for the quantitative relationship between light signal and P_fr formed.     

Circular dichroism of phytochrome intermediates

Phototransformation P_t to P_fr was investigated with 124-kDa phytochrome from etiolated oat seedlings ( Avena sativa L. cv. Pirol) using circular dichroism spectroscopy at -110°C to +30°C. Using absorption spectra of the intermediates formed at the respective temperatures, circular dichroism spectra (300–800 nm) of pure intermediates were calculated.
The sign of the circular dichroic absorption bands changed upon formation of lumi-R, the primary photoproduct of P_r. This would be compatible with a Z→E isomerization taking place at this reaction step. The subsequent intermediates (meta-R_a and meta-R_c) as well as P_fr showed only small circular dichroism. Their absorption spectra were drastically shifted, but had similar spectral shapes. The results are discussed in terms of conformational changes of the phytochrome chromophore presumably taking place at the early steps of phototransformation P_r to P_fr.     

Chromophore structure in phytochrome intermediates and bleached forms of phytochrome

Phytochrome (120 kdalton or 60 kdalton) was isolated from etiolated seedlings of Avena sativa L. cv. Pirol (Baywa München). Irradiation with red light of the P_r form at −23°C in aqueous medium or at −40°C in 66% glycerol leads to the intermediate meta-Rb. Acidification of the glycerol solution at −40°C leads to the absorption of the 15(E) phytochrome chromophore (= P_fr chromophore). Subsequent irradiation transforms this into the 15(Z) chromophore (= P_r chromophore). The presence of the 15(E) chromophore was demonstrated by the same methods also in phytochrome bleached either as P_fr in the dark by 4 M urea, methanol, acetone, ethylene glycol, 8-anilinonaphthalene-1-sulfonate, or as P_r by irradiation with red light in the presence of the same agents. Phytochrome bleached by sodium dodecylsulfate or by dehydration was also investigated. It was concluded that bleached phytochrome contains the P_fr chromophore without specific interaction with the protein.     

Light-induced linear dichroism in photoreversibly photochromic sensor pigments. – V. Reinterpretation of the experiments on in vivo action dichroism of phytochrome

Experiments by several authors on the effects of polarized light on phytochromemediated responses in fern gametophytes and in the green alga Mougeotia have earlier been interpreted as showing that the transition moment of phytochrome in the P_r form is parallel to the plasmalemma, but perpendicular to the plasmalemma for the P_fr form of phytochrome. It is now shown that the experimental results can be interpreted differently, and that they are also consistent with a chromophore rotation of about 30° (instead of 90°), as found for immobilized phytochrome molecules in vitro. Thus there is no evidence for a rotation of the whole phytochrome protein. For the gametophyte of Adiantum it is calculated that the P_r transition moment is inclined 17° to the plasmalemma, and the P_fr transition moment ca 50°, corresponding to an in vivo chromophore rotation of ca 33°; however, these values are very approximate.     

Binding of 124-kilodalton oat phytochrome to liposomes and chloroplasts

Binding of the radio-iodinated 124-kDa oat ( Avena sativa L. cv. Garry) phytochrome to liposomes and chloroplasts was investigated as a model system in order to understand the molecular affinity of phytochrome toward cellular organelles in plants. The binding of intact (124 kDa) phytochrome to liposomes and chloroplasts is hydrophobic in nature, as in the case of the degraded (118/114 kDa) phytochrome, but electrostatic interactions play a greater role in the intact phytochrome. The physiologically active P_fr form of the intact phytochrome showed a binding preference over the inactive P_r form with neutral liposomes and chloroplasts. However, the P_fr form of intact phytochrome exhibits smaller binding preference than the P_fr form of degraded phytochrome over their respective P_r forms (see Kim, I.-S. and Song, P.-S. 1981, Biochemistry 20: 5482–5489, for degraded phytochrome binding). These results indicate that the 6/10 kDa N-terminus segment, which is lost in the degraded phytochrome, plays an important role in determining the protein surface properties of the intact phytochrome. A competitive binding study on phytochrome also suggested that the P_fr form had a greater binding affinity for chloroplasts than the P_r form. However, the physiological activity of the P_fr form may not be explained simply by the observed difference in binding affinity between the two forms of phytochrome.     

THE COMBINED EFFECTS OF LOW TEMPERATURE AND SO2+NO2 POLLUTION ON THE NEW SEASON'S GROWTH AND WATER RELATIONS OF PICEA SITCHENSIS

Picea sitchensis (Bong.) Carr. seedlings were exposed to SO₂, NO₂ and SO₂+ NO₂ during dormancy in controlled environments, and were taken to night temperatures of 4, 0, −5, −10 and −15 °C in a freezer. Conditions in the freezer were carefully monitored during the low–temperature treatments. In two experiments, different photoenvironments and temperature regimes were imposed prior to the cold treatments, and different effects were observed. In the first, only limited frost hardiness was achieved and night temperatures of −15 °C were lethal. Temperatures of −5 and − 10 °C led to poor survival of lateral buds, particularly in plants exposed to 45 ppb SO₂. The poor bud break in plants exposed to SO₂ and to − 5 °C resulted in a loss of the effectiveness of this temperature as a chill requirement. Pressure-volume analysis showed that the shoots of plants exposed to NO₂ had greater elasticity (lower elastic moduli, e), so that loss of turgor occurred at lower relative water contents. In contrast, a hardening period (2 weeks in night/day temperatures of 3/10 °C and 8 h days at 50 μmol m⁻² s⁻¹ PAR) gave decreased elasticity and lower solute potentials of spruce shoots. In the second experiment, exposure to 30 ppb SO₂ and SO₂+ NO₂ led to slight, but consistent, increases in frost injury to the needles of plants frozen to − 5 and − 10 °C. The results suggest that the main interaction of low temperatures and winter pollutants may be on bud survival rather than on needle damage, but that effects are subtle, only occurring with certain combinations of pollutant dose and cold treatment.     

Chloroplast ultrastructure in two Crassulacean species and an F1 hybrid with differing biomass δ13C values

Abstract. The chloroplasts of two species of the Crassulaceae and their F₁ hybrid were compared by electron microscopy. The two species had contrasting leaf tissue δ¹³C values of −25°/_∞ ( Sedum greggii ) and −13°/_∞ ( Cremnophila linguifolia ), and the F₁ hybrid had a value of − 18°/_∞. S. greggii had a mean of 8.9 thylakoids per granum in contrast to C. linguifolia which had a mean of only 3.8 thylakoids per granum. The F₁ hybrid had a mean of 6.4 thylakoids per granum. Crystaloids were observed in S. greggii and the hybrid but not in C. linguifolia     

The joint action of phytochrome and alternating temperatures in the control of seed germination in Dactylis glomerata

The promoting effect of light and alternating temperatures on the germination of seeds of three contrasting populations of Dactylis glomerata L. was studied. Irradiation treatments using broad band low irradiance light sources resulted in red/far-red reversible effects, demonstrating the involvement of phytochrome in germination control. Reduction of germination by far-red below the level of a dark control indicated the presence of high pre-existing levels of the active form of phytochrome (P_fr) in some individuals. The capacity for dark germination at 21/11°C (12 h/12 h) was shown to be dependent on P_fr. Although some individuals were capable of germination at constant temperatures following red irradiation, in most seeds germination was dependent on the presence of P_fr and alternating temperatures. Some individuals responded to a single red irradiation, although a large proportion of seeds required high levels of P_fr to be maintained for long periods. Previously published dose response curves for alternating temperatures and a measured dark reversion time of 48 h for P_fr established by a single 60 min red irradiation, provided firm evidence of a direct correlation between the requirements for repeated irradiation and number of alternating temperature cycles.     

CO2 enrichment and development of freezing tolerance in Norway spruce

Plant growth and adaptation to cold and freezing temperatures in a CO₂-enriched atmosphere have received little attention despite their predicted effects on plant distribution and productivity. In this study we looked at the interaction between elevated CO₂ and development of freezing tolerance in Norway spruce ( Picea abies (L.) Karst.). First-year seedlings were grown under controlled conditions in an atmosphere enriched in CO₂ (70 Pa) for one simulated growth season. We measured shoot growth, registered the timing of growth cessation and bud set, measured needle net photosynthetic rate, and determined needle carbohydrate concentration (fructose+pinitol, glucose, sucrose, inositol, raffinose and starch). Freezing tolerance (LT₅₀) was determined after exposing whole seedlings to temperatures ranging from −6.5 to −36.0°C and scoring for visual needle browning. Elevated CO₂ did not affect height growth or the timing of growth cessation and bud set. The only statistically significant effects of CO₂ treatment were on seedling dry weight, percent dry matter and starch content. During the three weeks after growth cessation and bud set, freezing tolerance increased from −10 to −35°C, and there was a marked increase in all soluble sugars except inositol. However, neither freezing tolerance nor the concentration of soluble sugars was significantly influenced by elevated CO₂.     

Action spectra for chlorophyll formation during greening of wheat leaves in continuous light

The contents of Chl a and Chl b in dark-grown leaves of wheat ( Triticum aestivum L. cv. Starke II, Weibull) irradiated for 20 h with light of different wavelengths and intensities have been measured. The amount of Chl b formed revealed a higher maximum in the Chl b /Chl a ratio for light most strongly absorbed by the phytochrome system and giving a high [P_fr]/[P_tot] ratio. This indicates a stronger enhancing effect of P_fr on Chl b formation than on Chl a formation. Similarities between action spectra for Chl a and b formation indicate that Chl b originates from Chl a . A comparatively high effectiveness of light within the wavelength range 500–550 nm as compared to the action spectrum for PChl→Chl phototransformation shows that PChl₆₃₆, in addition to PChl₆₅₀, takes part in the greening process.     

Phytochrome in Norflurazon-treated seedlings of Pharbitis nil

The properties of phytochrome have been measured by dual-wavelength spectropho-tometry in the cotyledons of the short-day plant Pharbitis nil Choisy cv. Violet, where it is known to play a role in flower induction. In plants de-etiolated by a single white light period (4 h or longer), destruction of the far-red absorbing form of phytochrome (P_fr) was twice as rapid as after 10 min red light. A small fraction of P_fr was stable. After de-etiolation by a period of white light (6 h or longer) the rapid decrease of P_fr during the first 30 min was accompanied by a rapid increase of the red absorbing form of phytochrome (P_fr). This rapid increase of P_fr is probably due to dark reversion. Long term synthesis of phytochrome was inhibited by the presence of P_fr. Phytochrome synthesised in darkness showed the etiolated-plant type characteristics and underwent rapid destruction upon photoconversion to P_fr. The stable P_fr identified here is possibly that pool of phytochrome associated with the long term promotive process in flower induction, and the rapidly reverting P_fr is that pool associated with the night break inhibition of flowering.     

The effects of temperature and hyperoxia on arterial PO2 and acid-base status in Piaractus mesopotamicus

The effects of hyperoxia and change of temperature (range 20–30° C) on blood gases were studied in the teleost fish Piaractus mesopotamicus , native to several major river systems in Brazil. Large hyperoxia-induced increases of arterial P o₂ ( P _ao₂) indicated that true branchial blood shunts are negligible in relation to total gill perfusion. This implies that blood gases will be influenced by ventilation rather than by shunts. Acute variations of temperature ( t ) were accompanied by changes of arterial blood pH (on the average Δ p H_aΔt⁻¹ of — 0·015 units °C⁻¹), due mainly to alterations of P _aco₂: 2·4 mmHg at 20° C, 5·0 mmHg at 30° C. Concomitantly, P _ao₂ declined from 116 mmHg (20° C) to 89 mmHg (30° C). The data suggest that temperature-induced changes of acid-base status depend mainly on gill ventilation and that the decrease of P _ao₂ with higher temperature is a result of this regulation.     

Enhancement of Pfr-mediated responses by light pretreatments: persistence of the pretreatment effects

Abstract. In germinating seedlings of Sinapis alba , nitrate reductase activity is under phytochrome control and becomes accessible to phytochrome at about 15 h from sowing. The induction of the enzyme with pulses of light is strongly affected by pretreatments given prior to 15 h, also acting through phytochrome. It is shown that the effects of these pretreatments can persist undiminished for a considerable time (>40 h) but do not alter the pattern of the subsequent responsiveness to P_fr. The nitrate reductase response is compared with other data pertaining to a similar response.     

Effects of raised CO2 on potential CH4 production and oxidation in, and CH4 emission from, a boreal mire

1 In a glasshouse experiment we studied the effect of raised CO₂ concentration (720 p.p.m.) on CH₄ emission at natural boreal peat temperatures using intact cores of boreal peat with living vascular plants and Sphagnum mosses. After the end of the growing season half of the cores were kept unnaturally warm (17–20 °C). The potential for CH₄ production and oxidation was measured at the end of the emission experiment.
2 The vascular cores ('Sedge') consisted of a moss layer with sedges, and the moss cores (' Sphagnum ') of Sphagnum mosses (some sedge seedlings were removed by cutting). Methane efflux was 6–12 times higher from the Sedge cores than from the Sphagnum cores. The release of CH ₄ from Sedge cores increased with increasing temperature of the peat and decreased with decreasing temperature. Methane efflux from Sphagnum cores was quite stable independent of the peat temperatures.
3 In both Sedge and Sphagnum samples, CO₂ treatment doubled the potential CH₄ production but had no effect on the potential CH₄ oxidation. A raised concentration of CO₂ increased CH₄ efflux weakly and only at the highest peat temperatures (17–20 °C).
4 The results suggest that in cool regions, such as boreal wetlands, temperature would restrict decomposition of the extra substrates probably derived from enhanced primary production of mire vegetation under raised CO₂ concentrations, and would thus retard any consequent increase in CH₄ emission.     

Gas exchange and carbon isotope composition of Ananas comosus in response to elevated CO2 and temperature

Ananas comosus L. (Merr.) (pineapple) was grown at three day/night temperatures and 350 (ambient) and 700 (elevated) μ mol mol^–1 CO₂ to examine the interactive effects of these factors on leaf gas exchange and stable carbon isotope discrimination ( Δ ,‰). All data were collected on the youngest mature leaf for 24 h every 6 weeks. CO₂ uptake (mmol m^–2 d^–1) at ambient and elevated CO₂, respectively, were 306 and 352 at 30/20 °C, 175 and 346 at 30/25 °C and 187 and 343 at 35/25 °C. CO₂ enrichment enhanced CO₂ uptake substantially in the day in all environments. Uptake at night at elevated CO₂, relative to that at ambient CO₂, was unchanged at 30/20 °C, but was 80% higher at 30/25 °C and 44% higher at 35/25 °C suggesting that phosphoenolpyruvate carboxylase was not CO₂-saturated at ambient CO₂ levels and a 25 °C night temperature. Photosynthetic water use efficiency (WUE) was higher at elevated than at ambient CO₂. Leaf Δ -values were higher at elevated than at ambient CO₂ due to relatively higher assimilation in the light. Leaf Δ was significantly and linearly related to the fraction of total CO₂ assimilated at night. The data suggest that a simultaneous increase in CO₂ level and temperature associated with global warming would enhance carbon assimilation, increase WUE, and reduce the temperature dependence of CO₂ uptake by A. comosus .     

C4 photosynthesis in Cyperus longus L., a species occurring in temperate climates

Abstract. Cyperus longus L. , which has a widespread but disjunct distribution throughout Europe and extends northwards into Britain, was found to be a C₄ species based upon its Kranz leaf anatomy, low CO₂ compensation point and the labelling of malate as an early product of ¹⁴CO₂ fixation. The photosynthetic characteristics of C. longus are similar to many other C₄ species with a high maximum rate of photosynthesis (> 1.5 mg CO₂ m ⁻² s ⁻¹) and a relatively high temperature optimum (30–35°C), but unlike many C₄ species the rate of photosynthesis does not decline rapidly below the optimum temperature and a substantial rate (0.6 mgCO₂ m⁻²s⁻¹)occursat 15°C. Leaf extension is very slow at 15°C and shows a curvilinear response to temperatures between 15 and 25°C. Leaves extend at a rate of almost 4 cm d⁻¹ at 25°C.     

Growth in elevated CO2 enhances temperature response of photosynthesis in wheat

The temperature dependence of C₃ photosynthesis may be altered by the growth environment. The effects of long-term growth in elevated CO₂ on photosynthesis temperature response have been investigated in wheat ( Triticum aestivum L.) grown in controlled chambers with 370 or 700 μmol mol⁻¹ CO₂ from sowing through to anthesis. Gas exchange was measured in flag leaves at ear emergence, and the parameters of a biochemical photosynthesis model were determined along with their temperature responses. Elevated CO₂ slightly decreased the CO₂ compensation point and increased the rate of respiration in the light and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) V_cmax, although the latter effect was reversed at 15°C. With elevated CO₂, J_max decreased in the 15–25°C temperature range and increased at 30 and 35°C. The temperature response (activation energy) of V_cmax and J_max increased with growth in elevated CO₂. CO₂ enrichment decreased the ribulose 1,5-bisphosphate (RuBP)-limited photosynthesis rates at lower temperatures and increased Rubisco- and RuBP-limited rates at higher temperatures. The results show that the photosynthesis temperature response is enhanced by growth in elevated CO₂. We conclude that if temperature acclimation and factors such as nutrients or water availability do not modify or negate this enhancement, the effects of future increases in air CO₂ on photosynthetic electron transport and Rubisco kinetics may improve the photosynthetic response of wheat to global warming.     

Differences in the nature of thermodormancy and far-red dormancy in lettuce seeds

Lettuce seeds cv. Noran germinate at 23°C in light as well as in darkness. However dormancy can be induced either by a long exposure (24 h) to far-red radiation or by an exposure of 48–72 h to a temperature of 37°C. The difference in response of these two types of dormant seeds to conditions inducing germination indicate that in both types P_fr is inactivated, but that a dark process required for immediate action of P_fr does not proceed at 37°C as it does during far-red radiation.     

A Chemical Indicator for the Rapid Measurement of F0 Values

A new chemical indicator for monitoring steam sterilization processes has been calibrated in F ₀ units. The effective temperature range for F ₀ measurements using this device has been shown to lay between 115 and 123°C. The effective F ₀ range of the device has been shown to be 4–23 F ₀ units. Using the device, measurements can be made within 0.5 units of conventionally calculated F ₀ values.