Complementation of phytochrome chromophore-deficient Arabidopsis by expression of phycocyanobilin:ferredoxin oxidoreductase

Abstract

The covalently bound phytochromobilin (P⌽B) prosthetic group is required for the diverse photoregulatory activities of all members of the phytochrome family in vascular plants, whereas by contrast, green algal and cyanobacterial phytochromes use the more reduced linear tetrapyrrole pigment phycocyanobilin (PCB). To assess the functional consequence of the substitution of P⌽B with PCB in plants, the phytochrome chromophore-deficient hy2 mutant of Arabidopsis was transformed with a constitutively expressed pcyA gene that encodes the cyanobacterial enzyme, PCB:ferredoxin oxidoreductase. Spectroscopic analyses of extracts from etiolated seedlings revealed that PcyA expression restored photoactive phytochrome to WT levels, albeit with blue-shifted absorption maxima, while also restoring light lability to phytochrome A. Photobiological measurements indicated that PcyA expression rescued phytochrome-mediated red high-irradiance responses, lowfluence red͞far-red (FR) photoreversible responses, and very-lowfluence responses, thus confirming that PCB can functionally substitute for P⌽B for these photoregulatory activities. Although PcyA expression failed to rescue phytochrome A-mediated FR high-irradiance responsivity to that of WT, our studies indicate that the FR high-irradiance response is fully functional in pcyA-expressing plants but shifted to shorter wavelengths, indicating that PCB can functionally complement this phytochrome-mediated response in vascular plants.

Effects of Modified Phycobilin Biosynthesis in the CyanobacteriumSynechococcussp. Strain PCC 7002

“…Because of the very high expression levels of HY2 expected when driven by the cpcBA promoter, and the structural similarity of the resulting P⌽B chromophore to the native PCB chromophore, it seemed possible that the large proportion of the PBPs in these cells might carry P⌽B and not PCB chromophores. Previously, it was demonstrated that pcyA can largely complement a HY2-deficient strain of A. thaliana; this indicates that the two chromophores are nearly interchangeable in A. thaliana (24). Because this strain carried functional copies of both pcyA and HY2, it was possible that sufficient PCB was available to allow the selective assembly of some PBPs with PCB, as was seen for the strain overexpressing pebA and pebB.…”

Metabolic engineering to produce phytochromes with phytochromobilin, phycocyanobilin, or phycoerythrobilin chromophore in Escherichia coli

“…Fluorescence was detected using FM-BIO II (Hitachi High-Technologies). Absorbance and difference spectra of phytochrome were obtained with a spectrophotometer (HP8453 UV-Visible system, Hewlett Packard GmbH, Waldbronn, Germany) essentially as described previously [18].…”

TheElm1(ZmHy2) Gene of Maize Encodes a Phytochromobilin Synthase

“…Absorption spectra (450-850 nm) were taken in dark then following 60 s R illumination and 60 s FR illumination. For spectrophotometer and filter details, see Kami et al (2004). Difference spectra were obtained by subtraction of R spectra from FR spectra.…”