Phytochrome: molecular properties and biogenesis

Abstract

Native Avena phytochrome, recently shown to have a monomeric molecular mass of 124 kDa, has molecular properties that differ significantly from those of the extensively characterized ‘ 120’ kDa or ‘large’ phytochrome preparations now known to contain a mixture of proteolytically degraded 118 and 114 kDa polypeptides. For example, 124 kDa phytochrome has a blocked N-terminus, a P _fr λ _max of 730 nm, a higher photostationary state in red light (86% P _fr ), exhibits no dark reversion and shows no differential reactivity of P _r and P _fr toward a chemical probe of hydrophobic domains. The data indicate that the proteolytically removed 6-10 kDa polypeptide segment (s) is critical to the spectral and structural integrity of the photoreceptor; that at least part of the cleaved domain is located at the N-terminus of the molecule; that this domain influences the chemical reactivity of the chromophore with the external medium; and that a current hypothesis that P _r —P _fr photoconversion results in the exposure of a hydrophobic domain on the molecule is inconsistent with the properties of native phytochrome. Phytochrome has been found to exert rapid negative feedback control over the level of its own translatable mRNA. P _fr formation in etiolated tissue causes a decline in translatable phytochrome mRNA that is detectable within 15—30 min and that results in more than a 95 % reduction within 2 h. Less than 1 % P _fr is sufficient to induce 60 % of the maximum response, which is saturated at 20 % P _fr or less. The rapidity of this autoregulatory control makes phytochrome itself an attractive system for investigating phytochrome-regulated gene expression. A project to clone phytochrome complementary DNA (cDNA) has been initiated. A major obstacle in this work has been the unexpectedly low abundance of phytochrome mRNA, less than 0.005 % of the poly (A) RNA in etiolated tissue. cDNA made from poly (A) RNA enriched ca. 200-fold in phytochrome mRNA has been cloned and bacterial colonies have been screened with a synthetic oligodeoxynucleotide hybridization probe. The sequence of this probe was derived from a known partial amino acid sequence of the phytochrome protein. Difficulties encountered with this approach are discussed.