Photochemical properties of the flavin mononucleotide-binding domains of the phototropins from Arabidopsis, rice, and Chlamydomonas reinhardtii

Abstract

Phototropins (phot1 and phot2, formerly designated nph1 and npl1) are blue-light receptors that mediate phototropism, blue light-induced chloroplast relocation, and blue light-induced stomatal opening in Arabidopsis. Phototropins contain two light, oxygen, or voltage (LOV) domains at their N termini (LOV1 and LOV2), each a binding site for the chromophore flavin mononucleotide (FMN). Their C termini contain a serine/threonine protein kinase domain. Here, we examine the kinetic properties of the LOV domains of Arabidopsis phot1 and phot2, rice (Oryza sativa) phot1 and phot2, and Chlamydomonas reinhardtii phot. When expressed in Escherichia coli, purified LOV domains from all phototropins examined bind FMN tightly and undergo a self-contained photocycle, characterized by fluorescence and absorption changes induced by blue light (T. Sakai, T. Kagawa, M. Kasahara, T.E. Swartz, J.M. Christie, W.R. Briggs, M. Wada, K. Okada [2001] Proc Natl Acad Sci USA 98: 6969-6974; M. Salomon, J.M. Christie, E. Knieb, U. Lempert, W.R. Briggs [2000] Biochemistry 39: 9401-9410). The photocycle involves the light-induced formation of a cysteinyl adduct to the C(4a) carbon of the FMN chromophore, which subsequently breaks down in darkness. In each case, the relative quantum efficiencies for the photoreaction and the rate constants for dark recovery of LOV1, LOV2, and peptides containing both LOV domains are presented. Moreover, the data obtained from full-length Arabidopsis phot1 and phot2 expressed in insect cells closely resemble those obtained for the tandem LOV-domain fusion proteins expressed in E. coli. For both Arabidopsis and rice phototropins, the LOV domains of phot1 differ from those of phot2 in their reaction kinetic properties and relative quantum efficiencies. Thus, in addition to differing in amino acid sequence, the phototropins can be distinguished on the basis of the photochemical cycles of their LOV domains. The LOV domains of C. reinhardtii phot also undergo light-activated spectral changes consistent with cysteinyl adduct formation. Thus, the phototropin family extends over a wide evolutionary range from unicellular algae to higher plants.

Figure 1

Absorption and fluorescence spectra of the Arabidopsis phot1 LOV domains purified from E. coli extracts. A through C, Absorption spectra of LOV1 (A), LOV2 (B), and LOV1+2 (C). The concentrations of FMN bound for each fusion protein were 28, 16, and 25 μm, respectively. D, Fluorescence excitation spectrum for Arabidopsis phot1 LOV2. Fluorescence emission was monitored at 520 nm. E, Fluorescence emission spectrum for Arabidopsis phot1 LOV2. Excitation was at 390 nm. At, Arabidopsis.

Figure 2

The changes in the fluorescence emission spectrum induced by saturating white-light irradiation. A, Emission spectrum for the Arabidopsis LOV2 domain before (upper line) and after (lower line) a 10-s irradiation with white light (5,000 μmol m⁻² s⁻¹). B, Emission spectra for the mutant Arabidopsis LOV2 C39A domain from phot1 before (upper line) and after (lower line) the white light irradiation. In both A and B, the excitation wavelength was 450 nm. At, Arabidopsis.

Figure 3

Time course for fluorescence changes for the Arabidopsis phot2 LOV1 domain. Excitation was at 450 nm and emission was monitored at 490 nm. Before the start of the experiment, the sample was incubated in darkness for 10 min. The time scan was started at 0 s in the dark, and the shutter for the excitation beam was opened at 13 s (first ON). The decay curve for fluorescence emission was used to calculate photoproduct formation. When the fluorescence reached steady state (316 s), the shutter was closed (first OFF). After an additional predetermined time in darkness (74 s), the shutter was reopened. The magnitude of the immediate fluorescence increase reflects the amount of recovery of the initial fluorescent state. As soon as the signal reached steady state again (462 s), the shutter was closed. It was then re-opened to make a lights-on measurement after another predetermined period of time (in this case 29 s), etc. In this way, a time course for the regeneration of a fluorescent species could be followed.

Figure 4

Reaction kinetics for photoproduct formation. The kinetics for photoproduct formation for the Arabidopsis phot1 LOV-domain fusion proteins were monitored at 4°C. A₀ represents the amount of unreacted LOV-domain protein as indicated by the fluorescence detected immediately on opening the excitation lamp shutter. A_t represents the corresponding amount of unreacted protein at time t, calculated from the amount of fluorescence at time t. A, LOV-domain proteins from phot1; B, LOV-domain proteins from phot2. At, Arabidopsis.

Figure 5

Reaction kinetics for the dark recovery of the Arabidopsis LOV-domain fusion proteins from both phot1 (A) and phot2 (B). The recovery kinetics were monitored at room temperature. The amount of photoproduct remaining after dark incubation is plotted against the duration of dark incubation. The fluorescence intensity difference between that obtained immediately on opening the excitation lamp shutter after a 10-min dark incubation and that obtained when the fluorescence intensity reached steady state was used to determine the initial amount of photoproduct. The amount of photoproduct remaining after a certain duration of dark incubation was calculated as the initial amount of photoproduct minus the amount of recovery during the dark incubation. Half-lives calculated from the fitted curves are shown in Table III. At, Arabidopsis.

Figure 6

Time course for photoproduct formation for full-length Arabidopsis phot1 and phot2 produced in insect cells from recombinant baculovirus. Measurements were made at room temperature. Data points calculated as for Figure 4. Protein concentrations for the crude soluble extracts were 18.4 mg/mL (phot1) and 23.4 mg/mL (phot2). At, Arabidopsis.

Figure 7

Reaction kinetics for the dark regeneration of full-length Arabidopsis phot1 and phot2 expressed in insect cells transfected with recombinant baculovirus. Remaining photoproduct was calculated as for Figure 5. Data points and solid line represent the dark recovery for the full-length photoreceptors. Dashed lines are from Figure 5 and represent the dark recovery of the various shorter chromopeptides. Protein concentrations of samples are the same as for Figure 6. At, Arabidopsis.