Poster: Photomorphogenesis

Abs # 498: Phytochrome chromophore deficient pcd1 mutant of pea lacks ferrodoxin dependent heme oxygenase 1 (HO1)

Presenter:	Linley, Philip J., linley@bs.aist-nara.ac.jp
Authors	Linley, Philip J. (A)   Landsberger, Martin  (C)   Kohchi, Takayuki  (A)   Cooper, Jon B. (B)   Terry, Matthew J. (B)
Affiliations:	(A): Nara Institute of Science and Technology (B): University of Southampton (C): Universitat Greifswald

The phytochrome chromophore deficient pcd1 mutant of pea exhibits the classical phenotype of phytochrome chromophore-deficient plants. The leaves of pcd1 are pale yellow green in colour and internodes and stems are elongated. In pcd1 normal responses to far-red and red light are absent or strongly reduced while responses to white or blue light are largely unaffected. Previous biochemical analyses using isolated etioplasts indicated that heme oxygenase (HO) activity, the first committed step in the synthesis of the phytochrome chromophore, phytochromobilin, was absent and pcd1 lacks a protein recognized by an antibody raised to Arabidopsis HO1. We have cloned the HO1 gene from pea (PsHO1) and demonstrated the presence of a mutation in PsHO1 resulting in the formation of a premature stop codon. The intron/exon structure of PsHO1 is conserved with relation to other plant HO1s and phylogenetic analyses show a close alignment with other legume HO1s. Northern analysis indicates that PsHO1 is expressed in all tissues examined but PsHO1 mRNA levels are strongly reduced in pcd1. A ~2 fold increase in PsHO1 expression in leaves and stems was seen in response to constant illumination by white light although different induction profiles were observed. Recombinant PsHO1 was overexpressed in E. coli and purified. Purified PsHO1 was shown to convert heme to biliverdin IXƒ¿ in vitro. Kinetic data for the reaction were determined and found to be consistent with those obtained for AtHO1. Maximal activity required the presence of a primary reductant, ferredoxin, a secondary reductant such as ascorbate and an iron chelator. Modeling of PsHO1 by comparison to human HO1 indicated that the heme-binding pocket is structurally conserved even though the sequence identity is not high.