In addition to proving the existence of a molecule with the predicted photoreversible absorbance properties, the ratiospec method formed the basis for a dual-wavelength spectrophotometry assay for total phytochrome and the proportion of phytochrome present as Pfr in vivo and in vitro. In this technique the difference in absorbance (AA) at two preselected wavelengths in the red and far red parts of the spectrum, usually 660/720 nm or 730/815 nm, are measured before and after irradiation of the sample with alternating R and FR actinic light of sufficient intensity to saturate the photoconversion reaction. The relative amount of phytochrome present can then be calculated from the reversible change in (AA) i.e. (A(AA)) which is a function of the average concentration of phytochrome in the tissue. The absolute signal depends upon the sample thickness, the light-scattering factor and the molar absorption coefficient as well as the phytochrome content. This innovative method was highly effective for dark-grown tissues but had limited usefulness for green tissues where chlorophyll, which strongly absorbs red light, precludes the assay. Alternative methods were needed to study phytochrome in light-grown tissues which are for the most part the relevant ones for photoperiodism. To this end phytochrome proved to be amenable to study by immunochemical methods, being a large protein which is very effective in eliciting antibody production. However, when antibodies were raised against phytochrome isolated from dark-grown seedlings and then tested against phytochrome in extracts of light-grown plants, little or no reaction was obtained (Shimazaki et ai, 1983; Shimazaki and Pratt, 1985; Thomas et al., 1984; Tokuhisa et al., 1985). This led to the conclusion that the phytochrome protein in light-grown plants was not the same as that in dark-grown seedlings, even though spectrophotometrically (i.e. in their R/FR reversibility) they were very similar. This discovery, made independently by several groups, triggered an explosion of research into the question of the differences between the phytochrome in light-grown plants and that in dark-grown seedlings (Jordan et al., 1986; Furuya, 1989). This was much aided by the availability of monoclonal antibodies against phytochrome. Monoclonal antibodies are clones of single antibodies which, therefore, recognise a specific site (epitope) on the protein. Specific monoclonals which recognized different epitopes provided molecular probes for comparing proteins from different sources for similarities and differences. From such studies, it became evident that antigenic differences between phytochromes in dark- and light-grown tissues were found across the entire molecule. It also became evident there were at least three phytochrome proteins and probably more (Furuya, 1989; Wang et al., 1991; Pratt, 1995).
Conclusive confirmation of the diversity of phytochromes came from phytochrome gene studies. With Arabidopsis, Sharrock and Quail (1989) identified five different phytochrome-related sequences in Southern blot analyses and sequenced cDNAs representing three of the genes which they designated phyA, phyB and phyC. Subsequently, two other genes, phyD and phyE, were also isolated and sequenced (Quail, 1994; Clack et al., 1994). Sequences for phyA, phyB and phyC show about 50% similarity at the amino acid level. Phytochrome genes have now been isolated and sequenced from at least 15 plant species, including monocotyledons, dicotyledons, ferns, mosses and algae. The homologues of phyA and phyB have been fully or partially sequenced from rice, potato and tobacco, and immunological evidence points to at least three phytochrome gene products in Avena (Wang et al., 1991). The sequences of phyA or phyB genes are more similar between species, even between monocotyledon and dicotyledon species, than are the sequences of phyA and phyB within a single species (Fig. 3.3). This indicates that the divergence of the gene families pre-dated the evolutionary dichotomy between monocotyledons and dicotyledons and that the organisation of the phytochrome gene family is an evolutionarily conserved feature of higher plants. Arabidopsis has one of the smallest and least complex plant genomes and the identification of five distinct genes in this species suggests that all angiosperms will have a family of phytochrome genes which is at least as big. This immediately raises the question of whether these molecules have different properties and functions which, in turn, markedly influences our approach to physiological questions. We have to consider the possibility that a specific phyto-
Oat phyA3 Ric ephyA Corn phyA 1
Zucchini phyA Pea phyA
Potato phyA Arabidopsis phyA
Rice phyB Arabidopsis phyB Potato phyB
50 60 70 80 90 100 % Amino acid sequence identity
FIG. 3.3. Phylogeny of phytochrome polypeptides as deduced from sequence similarity. After Quail, chrome may function in photoperiodism and that it may have properties different from those of other phytochromes.
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