Red light-regulated interaction of Per-Arnt-Sim histidine kinases with partner histidine-containing phosphotransfer proteins in Physcomitrium patens.

Multi-step phosphorelay (MSP) is a broadly distributed signaling system in organisms. In MSP, histidine kinases (HKs) receive various environmental signals and transmit them by autophosphorylation followed by phosphotransfer to partner histidine-containing phosphotransfer proteins (HPts). Previously, we reported that Per-Arnt-Sim (PAS) domain-containing HK1 (PHK1) and PHK2 of the moss Physcomitrium (Physcomitrella) patens repressed red light-induced protonema branching, a critical step in the moss life cycle. In plants, PHK homolog-encoding genes are conserved only in early-diverging lineages such as bryophytes and lycophytes. PHKs-mediated signaling machineries attract attention especially from an evolutionary viewpoint, but they remain uninvestigated. Here, we studied the P. patens PHKs focusing on their subcellular patterns of localization and interaction with HPts. Yeast two-hybrid analysis, a localization assay with a green fluorescent protein, and a bimolecular fluorescence complementation analysis together showed that PHKs are localized and interact with partner HPts mostly in the nucleus, as unprecedented features for plant HKs. Additionally, red light triggered the interactions between PHKs and HPts in the cytoplasm, and light co-repressed the expression of PHK1 and PHK2 as well as genes encoding their partner HPts. Our results emphasize the uniqueness of PHKs-mediated signaling machineries, and functional implications of this uniqueness are discussed.

PAS-histidine kinases PHK1 and PHK2 exert oxygen-dependent dual and opposite effects on gametophore formation in the moss Physcomitrella patens.

Two-component systems, versatile signaling mechanisms based on phosphate transfer between component proteins, must have played important roles in adaptation and diversification processes in land plant evolution. We previously demonstrated that two Per-Arnt-Sim (PAS)-histidine kinases, PHK1 and PHK2, repress gametophore formation in the moss Physcomitrella patens under aerobic conditions, and that, in eukaryotes, the presence of their homologs is restricted to early-diverging streptophyte linages. We assessed here whether or not PHKs play a role in oxygen signaling. When submerged under water, the double disruption line for PHK1 and PHK2 formed fewer gametophores than the wild-type line (WT) both under light-dark cycles or continuous light, indicating that PHKs promote gametophore formation under an aquatic environment, in contrast to aerobic conditions. Similarly, in an artificial low-oxygen condition, the double disruption line formed fewer gametophores than WT. These results indicate that PHKs exert dual and opposite effects on gametophore formation depending on oxygen status. This study adds important insight into functional versatility and evolutionary significance of two-component systems in land plants.

Light-regulated PAS-containing histidine kinases delay gametophore formation in the moss Physcomitrella patens.

Two-component systems (TCSs) are signal transduction mechanisms for responding to various environmental stimuli. In angiosperms, TCSs involved in phytohormone signaling have been intensively studied, whereas there are only a few reports on TCSs in basal land plants. The moss Physcomitrella patens possesses several histidine kinases (HKs) that are lacking in seed plant genomes. Here, we studied two of these unique HKs, PAS-histidine kinase 1 (PHK1) and its paralog PHK2, both of which have PAS (Per-Arnt-Sim) domains, which are known to show versatile functions such as sensing light or molecular oxygen. We found homologs of PHK1 and PHK2 only in early diverged clades such as bryophytes and lycophytes, but not in seed plants. The PAS sequences of PHK1 and PHK2 are more similar to a subset of bacterial PAS sequences than to any angiosperm PAS sequences. Gene disruption lines that lack either PHK1 or PHK2 or both formed gametophores earlier than the wild-type, and consistently, more caulonema side branches were induced in response to light in the disruption lines. Therefore, PHK1 and PHK2 delay the timing of gametophore development, probably by suppressing light-induced caulonema branching. This study provides new insights into the evolution of TCSs in plants.

Posttranslationally caused bioluminescence burst of the Escherichia coli luciferase reporter strain.

We continuously monitored bioluminescence from a wild-type reporter strain of Escherichia coli (lacp::luc+/WT), which carries the promoter of the lac operon (lacp) fused with the firefly luciferase gene (luc+). This strain showed a bioluminescence burst when shifted into the stationary growth phase. Bioluminescence profiles of other wild-type reporter strains (rpsPp::luc+ and argAp::luc+) and gene-deletion reporter strains (lacp::luc+/crp- and lacp::luc+/lacI-) indicate that transcriptional regulation is not responsible for generation of the burst. Consistently, changes in the luciferase protein levels did not recapitulate the profile of the burst. On the other hand, dissolved oxygen levels increased over the period across the burst, suggesting that the burst is, at least partially, caused by an increase in intracellular oxygen levels. We discuss limits of the firefly luciferase when used as a reporter for gene expression and its potential utility for monitoring metabolic changes in cells.

Diversity of plant circadian clocks: Insights from studies of Chlamydomonas reinhardtii and Physcomitrella patens.

Arabidopsis thaliana has long been the model plant of choice for elucidating the mechanisms of the circadian clock. Recently, relevant results have accumulated in other species of green plant lineages, including green algae. This mini-review describes a comparison of the mechanism of the A. thaliana clock to those of the green alga Chlamydomonas reinhardtii and the moss Physcomitrella patens, focusing on commonalities and divergences of subsystems of the clock. The potential of such an approach from an evolutionary viewpoint is discussed.

Firefly luciferase as the reporter for transcriptional response to the environment in Escherichia coli.

We demonstrate that firefly luciferase is a good reporter in Escherichia coli for transcription dynamics in response to the environment. E. coli strains, carrying a fusion of the promoter of the ycgZ gene and the coding region of the luciferase gene, showed transient bioluminescence on receiving blue light. This response was compromised in mutants lacking known regulators in manners consistent with each regulator's function. We also show that relA, a gene encoding a (p)ppGpp synthetase, affects ycgZ dynamics when nullified. Moreover, two unstable luciferase variants showed improved response dynamics and should be useful to study quick changes of gene expression.