Low temperature spectroscopy of phytochrome: P_r, P_fr and intermediates

Abstract

Phytochrome (120 kdalton) was isolated from etiolated seedlings of Avena sativa L. cv. Pirol (Baywa, München). Low temperature spectra between −17°C and −160°C are recorded for Pr, Pfr, and irradiated phytochrome samples. The temperature‐dependence of the Pr and Pfr absorption spectra is described. Difference spectra of such temperature effects can erroneously be interpreted as difference spectra of intermediates. Probable absorption spectra of intermediates are calculated from the spectra of irradiated Pr or Pfr, respectively. The calculated spectral data are compared with published data on phytochrome intermediates.

Absorption Spectra of Phytochrome Intermediates

“…The spectrum of lum i-R of native phytochrome ( Fig. 1) turned out to be identical with that of lumi-R of partially degraded phyto chrome [8]. Upon transition from Pr to lum i-R, a small but significant shift occurred in the second absorption band (from 380 to 384 nm) whereas the shift in the first absorption band was more pro nounced (from 667 to 693 nm).…”

“…The first intermediate of irradiated PT which can be stabilized below -100 °C is lum i-R [8]. We con firmed that the spectrum of red-irradiated native P T is identical from -120 °C to -165 °C.…”

“…From the amount of intermediates produced at a defined temperature, the absolute spectrum of the inter m ediate^) can be determined. So far, these experi ments were only performed with partially degraded phytochrome (118 kdalton), furtherm ore only a limited spectral region (500-800 nm) was in cluded [8].…”

“…The mutual conversion of both involves several inter mediate steps which h a \e mainly been character ized by their difference spectra [1][2][3][4][5][6][7]. We have recently obtained the first data on absolute spectra of intermediates [8]. This was achieved by deter mination of absolute absorption spectra o f irra diated phytochrome at low tem perature.…”

“…For lumi-R. calculation was based on = 190000 (see [8]). The amount of m eta-Ra and metaRc was determined via the corresponding am ount of P{r produced by rapid thawing.…”