Temperature Effects on Agrobacterium Phytochrome Agp1

Phytochromes are widely distributed biliprotein photoreceptors with a conserved N-terminal chromophore-binding domain. Most phytochromes bear a light-regulated C-terminal His kinase or His kinase-like region. We investigated the effects of light and temperature on the His kinase activity of the phytochrome Agp1 from Agrobacterium tumefaciens. As in earlier studies, the phosphorylation activity of the holoprotein after far-red irradiation (where the red-light absorbing Pr form dominates) was stronger than that of the holoprotein after red irradiation (where the far red-absorbing Pfr form dominates). Phosphorylation activities of the apoprotein, far red-irradiated holoprotein, and red-irradiated holoprotein decreased when the temperature increased from 25 degrees C to 35 degrees C; at 40 degrees C, almost no kinase activity was detected. The activity of a holoprotein sample incubated at 40 degrees C was nearly completely restored when the temperature returned to 25 degrees C. UV/visible spectroscopy indicated that the protein was not denatured up to 45 degrees C. At 50 degrees C, however, Pfr denatured faster than the dark-adapted sample containing the Pr form of Agp1. The Pr visible spectrum was unaffected by temperatures of 20-45 degrees C, whereas irradiated samples exhibited a clear temperature effect in the 30-40 degrees C range in which prolonged irradiation resulted in the photoconversion of Pfr into a new spectral species termed Prx. Pfr to Prx photoconversion was dependent on the His-kinase module of Agp1; normal photoconversion occurred at 40 degrees C in the mutant Agp1-M15, which lacks the C-terminal His-kinase module, and in a domain-swap mutant in which the His-kinase module of Agp1 is replaced by the Hiskinase/response regulator module of the other A. tumefaciens phytochrome, Agp2. The temperature-dependent kinase activity and spectral properties in the physiological temperature range suggest that Agp1 serves as an integrated light and temperature sensor in A. tumefaciens.