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°C to 35°C; at 40°C, almost no kinase activity was detected. The activity of a holoprotein sample incubated at 40°C was nearly completely restored when the temperature returned to 25°C. UV/visible spectroscopy indicated that the protein was not denatured up to 45°C. At 50°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°C, whereas irradiated samples exhibited a clear temperature effect in the 30–40°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°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 His-kinase/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.

Bacteriophytochromes (BphPs) are light-sensing regulatory proteins encoded in photosynthetic and non-photosynthetic bacteria. This protein class incorporate bilin as their chromophore, with majority of them bearing a light- regulated His kinase or His kinase related module in the C-terminal. We studied the His kinase actives in the temperature range of 5°C to 40°C on two BphPs, Agp1 from Agrobacterium tumefaciens and Cph1 from cyanobacterium Synechocystis PCC 6803. As reported, the phosphorylation activities of the far red (FR) irradiated form of the holoprotein is stronger than that of the red (R) irradiated form in both phytochromes. We observed for the apoprotein and FR irradiated holoprotein of Agp1 an increase in the phosphorylation activities from 5°C to 25°C and a decrease from 25°C to 40°C. At 5°C the activities of the apoprotein were significantly lower than those of the FR irradiated holoprotein, which was opposite at 40°C. A similar temperature pattern was observed for Cph1, but the maximum of the apoprotein was at 20°C while the maximum of the FR irradiated holoprotein was at 10°C. At 40°C, prolonged R irradiation leads to an irreversible bleaching of Cph1, an effect which depends on the C-terminal His kinase module. A more prominent and reversible temperature effect on spectral properties of Agp1, mediated by the His kinase, has been reported before. His kinases in phytochromes could therefore share similar temperature characteristics. We also found that phytochrome B mutants of Arabidopsis have reduced hypocotyl growth at 37°C in darkness, suggesting that this phytochrome senses the temperature or mediates signal transduction of temperature effects.

Bacterial phytochromes are dimeric light-regulated histidine kinases that convert red light into signaling events. Light absorption by the N-terminal photosensory core module (PCM) causes the proteins to switch between two spectrally distinct forms, Pr and Pfr, thus resulting in a conformational change that modulates the C-terminal histidine kinase region. To provide further insights into structural details of photoactivation, we investigated the full-length Agp1 bacteriophytochrome from the soil bacterium using a combined spectroscopic and modeling approach. We generated seven mutants suitable for spin labeling to enable application of pulsed EPR techniques. The distances between attached spin labels were measured using pulsed electron-electron double resonance spectroscopy to probe the arrangement of the subunits within the dimer. We found very good agreement of experimental and calculated distances for the histidine-kinase region when both subunits are in a parallel orientation. However, experimental distance distributions surprisingly showed only limited agreement with either parallel- or antiparallel-arranged dimer structures when spin labels were placed into the PCM region. This observation indicates that the arrangements of the PCM subunits in the full-length protein dimer in solution differ significantly from that in the PCM crystals. The pulsed electron-electron double resonance data presented here revealed either no or only minor changes of distance distributions upon Pr-to-Pfr photoconversion.

We set up six light conditions to investigate the changes in the development of Arabidopsis thaliana hypocotyls and roots. Seedlings grown for 96 h under darkness were scored with shorter roots and longer hypocotyls. In shoot-shaded conditions, seedlings were unable to carry out photosynthesis, resulting in insufficient stored nutrients for root development. In the three groups of different light intensities applied to the roots, total light caused stress in the entire seedlings and the length of roots and hypocotyls were shorter than in conditions when roots were growing within light-dark gradients. Importantly, root lengths were higher within light-dark gradients than in total light. Different light treatments did significantly affect root growth and hypocotyl growth. The addition of ATP-competitive mTOR kinase inhibitor (AZD), drastically reduced root, however, this did not occur with hypocotyl length.

Bacteriophytochromes (BphPs) are light-regulated biliprotein photoreceptors widely occurring in bacteria. BphPs exists in two spectral distinct states: the red-light absorbing state (Pr) and the far-red light-absorbing state (Pfr). BphPs are composed of an N-terminal photosensory core module and a light-controlled C-terminal histidine kinase module. The detailed mechanism on how photoconversion, initiated by a light-triggered isomerization of the biliverdin chromophore, is conducted from the N-terminal to the C-terminal domain and how the histidine kinase is modulated is still not clear. In this work Agp1 from Agrobacterium tumefaciens is used as a model phytochrome. The method of labeling full-length Agp1 mutants with single accessible Cys residue using fluorescence probes is described. Different absorption spectra showed that neither mutagenesis nor fluorescence labeling exerts significant effects on the photoconversion behavior of various Agp1 mutants. The histidine kinase activity assay revealed that the labeled holoproteins exhibit wild-types like autophosphorylation activity that depends on the Pr and the Pfr state. Fluorescence spectroscopic data suggested that fluorescence resonance energy transfer occurs from the donor Atto495 to the acceptor Atto565 in double-labeled Agp1-A362C, K514C, H554C, R603C, I653C, and V674C mutants. Red light irradiation gave rise to small changes on fluorescence emission intensity in some mutants but in some no remarkable inter-subunit distance changes occurred during the photo-conversion from Pr to Pfr in full-length Agp1. In contrast to double-labeled samples, significant changes in fluorescence intensity were observed in single-labeled samples.