In Vivo Characterization of Chimeric Phytochromes in Yeast

Klaus Eichenberg,Tim Kunkel,Thomas Kretsch,Volker Speth,Eberhard Schäfer

doi:10.1074/jbc.274.1.354

Abstract

Phytochromes are plant photoreceptors that play a major role in photomorphogenesis. Two members of the phytochrome family have been characterized in some detail. Phytochrome A, which controls very low fluence and high irradiance responses, is rapidly degraded in the light, forms sequestered areas of phytochrome (SAPs), and does not exhibit dark reversion in monocotyledonous seedlings. Phytochrome B mediates red/far-red reversible responses, is stable in the light, and does not form SAPs. We report on the behavior in yeast of the phytochrome apoproteins of rice PHYA, tobacco PHYB, and chimeric PHYAB and PHYBA and on the behavior of the respective holoprotein adducts after assembly with phycocyanobilin chromophore (PHY*). SAP-like formation in yeast was not observed for PHYB, but was detectable for PHYA, PHYAB, and PHYBA. Rice PHYA* did not undergo dark reversion in yeast. Surprisingly, all other tested phytochrome constructs did exhibit dark reversion, including chimeric phytochromes with a short N-terminal part of tobacco PHYB or parsley PHYA fused to rice PHYA. Furthermore, the proportion of phytochrome undergoing dark reversion and the rate of reversion were increased for both the N terminus-swapped constructs and PHYBA*. These results are discussed with respect to structure/function analysis of phytochromes A and B.

Highlights

The peak positions were blue-shifted compared with native rice phyA because PCB was used as the chromophore instead of phytochromobilin [32]
The far-red peaks were identical to those of PHYA* and PHYB*, whereas the red peaks exhibited a blue shift of 4 nm
We investigated which domains of the phytochrome molecule are responsible for dark reversion and SAP formation

Summary

Introduction

In vivo spectroscopic studies of phytochrome have been performed in light-grown bleached seedlings of certain species; the results of these studies may reflect properties of phyB [13, 14] These studies indicate a very slow destruction of the light-stable phytochrome (half-life of Ͼ8 h) and a weak partial dark reversion. The yeast-derived phytochrome-chromophore adducts are functional in planta [16], and tobacco PHYB* is stable, shows a partial rapid dark reversion from Pfr to Pr, and does not form SAPs. In contrast, rice PHYA* expressed in yeast does not undergo dark reversion, but does exhibit SAPs as well as light-independent formation of SAP-like structures [15]. It was concluded that the Nterminal domains determine photosensory specificity and light lability [19, 20]

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