Phytochrome Photoconversion Research Articles

A DIMERIC MECHANISM FOR THE ACTION OF PHYTOCHROME: EVIDENCE FROM PHOTOTHERMAL INTERACTIONS IN LETTUCE SEED GERMINATION*

Abstract— Sensitization of the phytochrome‐mediated germination at 20°C of lettuce seeds (Lactuca sativa L. cv. Grand Rapids) by pretreatment at 4°C, 28°C, or on 1% ethanol, was studied. The 660 nm fiuence‐response characteristics were similarly biphasic for all sensitizing treatments and displayed responses at very low fluences (VLFR) as well as responses characteristic of non‐sensitized seeds at 10000‐fold higher, low fluences (LFR). Maximum VLFR increased with the duration of sensitizing treatments. However, the fluence ranges required for the two types of responses remained relatively constant. These and additonal responses of sensitized seeds to 730 nm fluences were compared to simulations of a mechanism involving a receptor, X, and based on the dimeric structure of phytochrome in which each monomer is independently phototransformed from the inactive (Pr) to the active (Pfr) form. The fluence requirements for phytochrome photoconversion in seeds were determined to be similar to those of purified Avena phytochrome in vitro, on which photochemical parameters for the simulations were based. The analyses suggest that Pr:Pfr‐Xand Pfr:Pfr‐X are responsible, respectively, for the VLFR and the LFR, and that sensitization involves membrane influences on the activity of Pr:Pr‐X. They also suggest the concentration of X to be about 0.001 that of total phytochrome dimer in this system.

Reexamination of the Partial Quantum Yields for the Phytochrome Photoconversion by Adequate Photokinetic Methods

Summary The partial phototransformation quantum yields Φr and Φfr of phytochrome photoconversion are reexamined using adequate photokinetic methods. The principles of these methods based on formally integrated rate equations and difference equations, respectively, are represented. It is shown that the partial quantum yields determined hitherto using the equations derived by Butler et al. (1964) exhibit systematic errors if the initial absorbance at the irradiation wavelength exceeds a value of 0.009. Before applying the kinetic analysis the photoconversion was confirmed to be a reaction of a two component system composed of only Pr and Pfr.

Phytochrome-mediated phototropism in maize mesocotyls. Relation between light and Pfr gradients, light growth response and phototropism

Unilateral irradiation of maize (Zea mays L.) seedlings results in a fluence-rate gradient, and hence below saturation, a gradient of the far-red-absorbing form of phytochrome (Pfr). The Pfr-gradients established by blue, red and far-red light were spectrophotometrically measured in the mesocotyl. Based on these Pfr-gradients and the fluence-response curves of phytochrome photoconversion the fluence-rate gradients were calculated. The fluence-rate gradient in the blue (460 nm) was steeper than that in the red (665 nm), which in turn was steeper than that in the far-red light (725 nm). The fluence-rate ratios front to rear were 1:0.06 (460 nm), 1:0.2 (665 nm), and 1:0.33 (725 nm). The assumption that phytochrome-mediated phototropism of maize mesocotyls is caused by local phytochrome-mediated growth inhibition was tested in the following manner. Firstly, the Pfr response curve for growth inhibition was calculated; these calculations were based on measurements of Pfr-gradients and data from red-light-induced phototropism. Secondly, the Pfr response curve for growth inhibition was used as a basis for calculating fluence-response curves for blue-and far-red-light-induced phototropism. Finally, these calculated results were compared with experimental data. It was concluded that the threshold for phytochrome-mediated phototropism of maize mesocotyls reflects the apparent photoconversion cross section of phytochrome whereas the maximal inducable curvature depends on the steepness of the light (Pfr) gradient across the mesocotyl.

Early Morphogenetic Changes During Phytochrome-induced Fern Spore Germination I. The Existence of a Pre-photoinduction Phase and the Accumulation of Chlorophyll

The relationship between light induced and dark metabolic processes occurring during synchronous germination of unicellular spores of the fern Pteris vittata was investigated. Light was almost totally ineffective as an inducer during a 12 to 16 h preinduction phase. Spores imbibed in the cold before illumination could not be induced to germinate, indicating that metabolic events occurring during the preinduction phase are required for the photomorphogenetic response. Two brief irradiations separated by two hours of darkness during a later, photoinductive phase were more effective in inducing germination than a single one of equal fluence. These short red-light illuminations induced more than 40% synchronous germination and enabled the separation between hydration and light effects. Chlorophyll formation is a phytochrome induced dark process in germinating spores, preceding the first nuclear division and rhizoid emergence. The formation of chlorophyll in ferns in the dark differs from light mediated chlorophyll synthesis in both angiosperms and gymnosperms as it is an enzymatic process, yet is mediated by phytochrome photoconversion.

Far-Red Light-Induced Changes in Intracellular Potentials of Spinach Mesophyll Cells

In green plants, the large bioelectric changes that photosynthetically active light stimulates make it difficult to observe electrical potential changes related to phytochrome photoconversion. As a first step towards distinguishing between photosynthetic and phytochrome effects, we showed that red light enhances far-red stimulated intracellular potential changes in spinach (Spinacia oleracea) leaf mesophyll cells.For a dark-adapted leaf, the response to far-red light increased during the first 10 to 30 exposures of 2.5 minutes, after which it was constant. The intracellular potential depolarized by an average of 0.3 millivolts during each 2.5-minute far-red light period, and returned to the resting value during each subsequent dark period. Continuous supplementary red light (at 1-5% of the fluence rate of the far-red light that stimulated the depolarizations) increased the response to far-red 2- to 3-fold. Supplementary red light did not amplify the response to alternating 702 nanometers light and dark periods. The Emerson enhancement effect thus does not seem to explain amplification of the response to 730 nanometers light by supplementary red light. This does not prove that photosynthetic pigments are not involved in some other way.

CHANGES IN THE RATES OF PHOTOCONVERSION OF PHYTOCHROME DURING ETIOLATION IN MUSTARD SEEDLINGS

Abstract— The relative phytochrome photoconversion rates in cotyledons and hypocotylar hook of etiolating mustard (Sinapis alba L.) seedlings were measured between 16 and 96 h after sowing. It was found that at constant fluence rates photoconversion rate in red light increases in both organs with time whereas the photoconversion rate in far‐red (756 nm) light decreases with time of development. Since the isosbestic point remains constant, it was concluded that the observed changes cannot be attributed to changes of extinction coefficients. It was not possible, however, to decide whether the observed changes are due to changes of light attenuation or quantum yields.

Phytochrome and Circadian Clocks in Samanea

Previous investigations with the electron microprobe reveal that the movements of Samanea leaflets are correlated with massive redistribution of K within the pulvinus. Evidence is now presented that Cl moves with K, whether plants are in white light or darkness, whether or not the amplitude of free running oscillations has damped, and whether or not the rhythm has been rephased by phytochrome photoconversion. The mid-extensor to mid-flexor ratio of K + Cl is correlated with leaflet angle under all conditions. Total Cl in both inner cortex and motor region is approximately 0.6 as high as K. The stoichiometry between Cl and the migratory fraction of K is close to, but not precisely 1:1 in all regions of the pulvinus, suggesting that other ions or systems may also be involved in the balancing of electrical charges.

Chlorophyll interference with phytochrome measurement

Measurements of phytochrome by Δ (ΔA725-815 nm) were completely suppressed at chlorophyll concentrations of the order of 20-40 μg g(-1) f.wt. in vivo and 37 μg cm(-3) in vitro, and the readings were reduced by 50% at only 12 μg cm(-3) in vitro. At these concentrations of chlorophyll in aqueous methanol, the loss of phytochrome signal in vitro appeared to be due to failure of phytochrome photoconversion rather than to interference with ΔA measuremebt by chlorophyll fluorescence in the 125/815 nm measuring beam.

A Kinetic Theory of First Order Cyclical Processes—Phytochrome Controlled Red Light Induced Cereal Leaf Unfolding Compared with Theory

AbstractThe phytochrome controlled unfolding of cereal leaves was studied as a function of irradiation time and light intensity (narrowband red light) over a wide energy range (5 decades). With different intensities, a family of similarly shaped response curves appear with distinct time‐dependent maxima and minima. A theoretical kinetic model based upon a cyclical phytochrome photoconversion scheme has been calculated by us. The theoretical calculations and the experimental findings are in excellent agreement. The same model explains the early photoresponses (first maxima) as an effect of one active phytochrome form, P2, and the delayed photoresponses as an effect of a second active form Pn. The active transitory form, P2 (although it may not be the primary product), is formed upon light absorption from P1. The P2 decays by a first order dark reaction through several inactive intermediates to Pn (active). The effect of the intermediates is mainly to delay the production of the second active product. It is possible to identify the two active products, P2 and Pn, as Pfr and P*fr, respectively.The presented cyclical phytochrome reaction scheme is a special case of a general first order kinetic cycle which includes all possible feed back loops. The latter scheme also has been calculated and programmed since it has a more general application.

Inverse dark reversion of phytochrome: An explanation

Pea epicotyl tissue freeze dried with phytochrome (P) in the red absorbing (Pr) form, on exposure to red light does not form the far-red absorbing form of P (Pfr), but forms the intermediate P698 which reverts to Pr in darkness. Similar tissue containing the pigment as Pfr undergoes a photoreversible reaction on exposure to alternate red and far-red light. This represents the photoreversibility between Pfr and the intermediate P650. The difference spectrum of this reaction is similar to that of phytochrome photoconversion in dry cucumber seeds. P650 is shown to revert slowly to Pfr in darkness and it is proposed that this reaction accounts for the observation of apparent inverse dark reversion in cucumber seeds. Partial rehydration of freeze-dried tissue containing Pr, by means of 80% glycerol: 20% water (v/v), partially restores photoreversibility between Pr and Pfr. In such samples Pfr formation from intermediates, however, is slow and continues for several min in the dark after exposure to red light. This reaction can simulate the apparent inverse dark reversion observed in many seeds during early stages of imbibition. In this case Pfr appears from an intermediate produced by exposure to red light, which has not had time to form Pfr during the normal assay period. These two processes of Pfr production from intermediates in darkness, present under conditionsof partial or extreme dehydration, can explain previous experimental observations interpreted as inverse dark reversion.It is therefore proposed that the process, formerly described as 'inverse dark reversion', is not a transformation of Pr to Pfr and that there is not a separate form of phytochrome responsible for this reaction.

On the kinetics of phytochrome photoconversion in vivo

Samples for spectrophotometric measurement of phytochrome in vivo are not optically thin. For different cross sections of the sample, the rate constant of a photochemical reaction will, therefore, have different values. We have developed a mathematical model, based on the assumption that the rate of phytochrome phototransformation is proportional to the light intensity and that the light intensity gradient in the sample is exponential. Kinetic curves computed with this model conform closely with the measurements. The simplest explanation of the observed kinetics is that there is only one type of phytochrome and that the light intensity gradient in samples that are not too thin, is close to exponential.