The Phytochrome Family

Photosynthetic organisms, from bacteria to higher plants, possess light sensing molecules that enable adaptation to fluctuations in intensity, direction, duration, polarization and spectral quality of light from their environment (26). The most well known of these photoreceptors are the phytochromes, which sense the ambient light conditions via their ability to pho-tointerconvert between red (Pr) and far-red (Pfr) light absorbing forms (17,47,51,57). This unique property of phytochromes is conferred by a linear tetrapyrrole (bilin) prosthetic group that is covalently linked to a large polypeptide. First discovered in plants, phytochrome-like molecules also have been identified in lower eukaryotic plant species (i.e. green algae, mosses, and ferns) (27,64) and, more recently, in cya-nobacteria (21,25,66,70). It is well established that higher plants possess multiple phytochromes that are encoded by a small nuclear gene family termed PHYA-F (5, 45). All phytochrome proteins share a highly conserved photosensory domain, in which the bilin prosthetic group is linked

Heme, Chlorophyll, and Bilins: Methods and Protocols Edited by A.G. Smith and M. Witty ©2002 Humana Press, Totowa, NJ

via a thioether to an invariant cysteine residue. Biochemical and molecular cloning studies indicate that the basic architecture of eukaryotic phytochromes has been preserved, while a growing family of phyto-chrome-related genes in cyanobacteria encode polypeptides considerably more divergent in structure. Experimental methods outlined in this chapter discuss 2 major tools, difference spectroscopy and holo-phytochrome assembly, necessary for establishing whether candidate genes encode bonafide phytochromes.

With only 2 known exceptions, eukaryo-tic phytochromes are soluble homodimeric proteins with a subunit roughly 1100 amino acids in length (Figure 1). The bilin prosthetic group is associated with a highly conserved photosensory domain at the protein's N terminus, which is readily cleaved from the C-terminal region by limited proteolysis to yield a photochemically active 60 to 70 kDa monomer (24). The more diverged 500 amino acid C terminus of eukaryotic phytochromes specifies the high affinity subunit—subunit interaction (23) and also possesses 2 regulatory subdomains that genetic studies have established to be critical

Figure 1. Domain structure of eukaryotic and cyanobacterial phytochromes and phytofluors. (A) Phytochromes exist in pho-tointerconvertible red light absorbing (Pr) and far-red light absorbing (Pfr) forms. Phytochromes possess either phytochromobilin or phycocyanobilin prosthetic groups bound to a conserved cysteine residue indicated with an asterisk. (B) Phytofluors are orange fluorescent biliproteins consisting of phycoerythrobilin thioether-linked to the conserved cysteine residue on an apophytochrome. Regulatory domains include the PAS-related domain (PRD), which contain 2 direct repeats shown as dark boxes, and the histi-dine kinase domain (HKD) and the histidine kinase-related domain (HKRD), which are depicted with cross hatching.

Figure 1. Domain structure of eukaryotic and cyanobacterial phytochromes and phytofluors. (A) Phytochromes exist in pho-tointerconvertible red light absorbing (Pr) and far-red light absorbing (Pfr) forms. Phytochromes possess either phytochromobilin or phycocyanobilin prosthetic groups bound to a conserved cysteine residue indicated with an asterisk. (B) Phytofluors are orange fluorescent biliproteins consisting of phycoerythrobilin thioether-linked to the conserved cysteine residue on an apophytochrome. Regulatory domains include the PAS-related domain (PRD), which contain 2 direct repeats shown as dark boxes, and the histi-dine kinase domain (HKD) and the histidine kinase-related domain (HKRD), which are depicted with cross hatching.

for transducing the light signal (reviewed in References 47 and 63). These include the PRD, a domain related to the PAS domain found on eukaryotic regulatory proteins (30,59), and the HKRD, a domain related to histidine kinase transmitter domains of 2 component sensor proteins from bacteria (56). Like eukaryotic phytochromes, the cyanobacterial phytochrome Cphl from Synechocystis sp PCC 6803 (21,70) possesses the conserved N-terminal photosensory domain and histidine kinase domain (HKD), but lacks the PRD regulatory subdomain (Figure 1). Recent investigations support the hypothesis that both Cphl and eukaryotic phytochromes transduce the light signal perceived by the photosensory domain via changes in protein kinase activity of the regulatory domains (69,70).

It was not until the purification of native phytochrome from plants that a chemical examination of the chromophore of phytochrome took place. 1H-Nuclear magnetic resonance (NMR) analysis of chro-mopeptides from oat phytochrome A revealed that its chromophore and linkage to the apoprotein were very similar to those found in phycobiliproteins (34,52). These studies also revealed that oat phytochrome possessed a phytochromobilin (POB) chromophore (Figure 1), confirming earlier investigations of bilins obtained from phy-tochromes by chemical cleavage (50). The precursors of the chromophores of the other phytochrome genes within a given plant species (i.e., PHYB-E) have not been directly determined, but they are assumed to be POB. One exception is phytochrome from the green alga Mesotaenium caldario-rum, which possesses the phycobiliprotein chromophore precursor phycocyanobilin (PCB) (68). The natural chromophore precursor of Cph1 has not yet been determined. PCB has been proposed as a likely candidate owing to its intermediacy in the phycobiliprotein chromophore biosynthetic pathway in Synechocystis sp. PCC 6803 (6).

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