DIRECT MEASUREMENT OF PHYTOCHROME PHOTOCONVERSION INTERMEDIATES AT HIGH PHOTON FLUENCE RATES

Abstract

Abstract—A custom‐built modulated split‐beam spectrophotometer has been used to measure the absorbance of tissue samples and purified phytochrome whilst exposing the sample to actinic 633 nm laser radiation at fluence rates approaching those of daylight. This approach has allowed the direct observation of the accumulation of phytochrome photoconversion intermediates at high fluence rates. At ca 1250 μmol m−2 s−1 upwards of 35% of the total phytochrome was present in the form of photoconversion intermediates in tissues of maize, sunflower and tomato. In other tissues tested (wheat, bean and Amaranthus) and in purified oat phytochrome, rather smaller levels of intermediates accumulated. Upon “lights‐off” only a proportion of the accumulated intermediates decayed to far‐red absorbing phytochrome (Pfr), the remainder appearing as the red‐absorbing form (Pr). Difference spectra suggested that, at high light levels, Pr may be reformed via a photochemical back‐conversion of an intermediate in the Pr—Pfr pathway, although the involvement of intermediates in the Pfr—Pr pathway cannot be excluded. The implications of the results for the ecological function of phytochrome are discussed.