INTERMEDIATES IN THE PHOTOCONVERSION OF FUNCTIONAL PHYTOCHROME IN FERN SPORES OF Dryopteris‐I DEMONSTRATION AND QUANTITATIVE CHARACTERIZATION OF THE PHOTOCHROMIC SYSTEM P_r⇔ Iⁱ₇₀₀USING NANOSECOND‐LASER PULSES*^,†

Abstract

Abstract— Spores of Dryopteris paleacea and D. filix‐mas are positively photoblastic with an optimum in the action spectrum around 665 nm. Light is perceived by phytochrome and the relationship between germination and mole fraction of the far‐red‐absorbing form of this pigment, Pfr, was investigated with saturating irradiations between 662 and 747 nm under low‐fluence‐rate conditions. These control irradiations establish a proportion of the total phytochrome, P,tot, as Pfr with Pfr/Ptot–φ at equilibrium. These φ ‐values were calculated according to data for native oat phytochrome (Kelly and Lagarias, 1985, Biochemistry 24, 6003) and the spectral characteristics of the interference filters. With this method a linear relationship could be found between φ and germination from 2 to 70% for D. paleacea and from 2 to 90% for D. filix‐mas, if probit germination was plotted vs probit φ This correlation formed the basis of investigating the phytochrome photoconversion by dye‐laser pulses of 380 ± 30 ns under high‐fluence‐rate conditions, and thus to test quantitatively the impact of the photoreversibility of intermediate reactions of the photoconversion and the red‐absorbing form of phytochrome, Pfr on the final Pfr‐level. Spore germination was initiated by a single‐laser pulse in the range from 592 to 700 nm. The most effective wavelengths were 649 and 660 nm in both species, and at saturation maximal germination (ca. 50%) was obtained from 592 to 665 nm for D. paleacea or ca. 60% germination from 592 to 670 nm for D. filix‐mas. Both saturation levels correspond to a ø‐value between 0.40 and 0.45. This significantly diminished photoconversion is a consequence of the high‐fluence‐rate conditions during the laser pulse which establishes the photochromic system between Pr and a set of very early intermediates, Ii700, (= Pr⇔ Ii700). This system can be described by the extinction coefficients of Pr and the intermediates Ii700, and by the quantum yields, 4,φ for the forward and reverse reactions as φ If φ is calculated, assuming a quantum yield of 1:1 for both reactions and with the extinction coefficients of Pr and Ii7(l() (= lumi‐R) given by Eilfeld and Riidiger (1985, Z. Naturforsch.40c, 109), significantly higher values are calculated for / as compared to φ found in the control experiments. These results can be explained either: (i) with a quantum yield ratio φpr‐φ1700: φ1700φpr=1:1 and an assumed additional dark reaction leading from Ii700 or later intermediates back to Pr: or (ii) with a quantum yield ratio φpr φ 1700: φ1700 φpr=1:2. In this case all Ii700 have to relax to Pfr. In this case all Ii700 have to relax to Pfr.

Haem oxygenase: A functionally diverse enzyme of photosynthetic organisms and its role in phytochrome chromophore biosynthesis, cellular signalling and defence mechanisms

“…The initial photoproduct produced during the changes is composed of two thermally metastable components, I 700 a and I 700 b , which decay within microseconds into an intermediate possessing a lower absorbance (I bl ), which finally generates the far‐red absorbing form, Pfr A (Pfr form of Phy A). At high fluences (730 nm), the I 700 a intermediate photoconvert back to Pr both in vitro as well as in vivo (Seheuerlein & Koller, ). From the above two forms (Pfr and Pr), the Pfr is the active form that initiates biological responses.…”

NITRATE EFFECT ON Pfr‐MEDIATED AND GA3‐INDUCED GERMINATION IN SPORES OF Anemia phyllitidis (L.) Sw*

“…GA,-induced germination was scored 10 days after sowing. Chlorophyll synthesis was used as germination parameter (see Scheuerlein et al, 1988). For each datum point at least 1200 spores were examined in at least two independent experiments with two samples each.…”

“…Spores were sown on medium with NO, (1 mM; 0 ) or without NO; (control, 0). Germination is shown here and in the following figures in probit and Koller, 1988),. several intermittent pulses of saturating R were necessary to obtain a significant response in Anemia phyllitidis [Fig.…”

INTERMEDIATES IN THE PHOTOCONVERSION OF FUNCTIONAL PHYTOCHROME IN FERN SPORES OF Dryopteris–II. In vivo KINETICS OF THE DECAY OF Ii700 AND OF THE FORMATION OF Pfr STUDIED WITH A DOUBLE‐LASER APPARATUS*

“…90% by a R-irradiation of a low photon flow with prolonged duration to saturate the equilibrium Similar results were described previously for D . paleacea (Scheuerlein and Koller, 1988) where only a mole fraction of Pf, between 0.40 and 0.45 (initiating ca. 50% germination) could be formed by a saturating R-laser pulse of 380 ns.…”

“…In the latter case it is possible to record photoconversion of functional phytochrome. Moreover, Scheuerlein and Koller (1988) presented a quantitative description of this photochromic system, based on a linear relationship between the mole fraction of Pf,, obtained by a saturating irradiation and the physiological response; v i z . , the induction of fern-spore germination in Dryopteris.…”