Zeitlupe Senses Blue-Light Fluence To Mediate Circadian Timing in <i>Arabidopsis thaliana</i>

Plants employ a variety of light, oxygen, voltage (LOV) domain photoreceptors to regulate diverse aspects of growth and development. The Zeitlupe (ZTL), Flavin-Kelch-Fbox-1 (FKF1), and LOV-Kelch-Protein-2 (LKP2) proteins dictate measurement of the day length, flowering time, and regulation of the circadian clock by blue-light regulation of protein complex formation. Previous reports indicated that ZTL photochemistry was irreversible, which is inconsistent with its role in marking the day–night transition. A kinetic model of LOV domain function predicts that ZTL has evolved unique photochemical parameters to allow it to function as a sensor of environmental light intensity. Moreover, our model indicates that a photocatalyzed reverse reaction is required for the sensitivity of LOV domains to light fluence. Inclusion of a photocatalyzed rate constant allows the establishment of a photostationary steady state of light-activated proteins, whose relative population is sensitive to daily (circadian) or positional (phototropism) oscillations in light intensity. Photochemical characterization confirms that ZTL undergoes adduct decay on a time scale of hours in contrast to previous reports. The fast photocycle allows detection of the day–night transition facilitating circadian timing. ZTL kinetics reflect an evolutionary adaptation of the ZTL/FKF1/LKP2 family to function in distinct aspects of blue-light signaling

McLOV photocycles and kinetics.

<p>(A-C) McLOV proteins all demonstrate spectra consistent with C4a adduct formation. Dark-state spectra (black) are consistent with oxidized flavin. Illumination with blue light bleaches the 450 nm absorption bands (red). Subtle differences in degrees of photoactivation in McLOVn (A), 337-TF (B) and McLOVr (C) are indicated by residual presence of absorption bands centered around 450 with vibrational bands at 425 and 478 nm. (D-F) McLOVn (D), 337-TF (E) demonstrate first order kinetics as demonstrated by an absorbance trace at 450 nm and their respective residual plots. In contrast McLOVr (F) is best fit with a biexponential function.</p

Arrhenius and Eyring analysis of McLOV proteins.

<p>Arrhenius (A,C,E) and Eyring (B,D,F) of McLOVn (A,B), 337-TF (C,D), and McLOVr (E,F) constructs. All measurements were carried out in triplicate, and error bars are shown as standard deviations relative to the mean. McLOVn and McLOVr both depict weak temperature dependence that indicates low entropies of activation with entropic compensation. In contrast 337-TF, has markedly increased enthalpies of activation (71 kJ/mole vs. ~50 KJ/mole) with a decrease in the entropic penalty (-44 J/mole*K vs. ~ -100 J/mole*K).</p

Model for competitive dimerization.

<p>(A) Sequence alignments of LOV proteins in <i>Methylocystis</i> compared to fungal analogues and the LOV 2 domain of <i>Avena sativa</i> Phototropin (AsLOV2). Comparisons of a LOV-transcription factor in <i>Methylocystis</i> (McLOV-TF) and <i>Methylocystis rosea</i> (McLOVr-TF) demonstrate high homology to sLOV proteins in the respective organisms (McLOVn, McLOVr). Key elements required for signal transduction are conserved in the fungal sLOV proteins <i>Trichoderma reesei</i> ENVOY (ENV1) and <i>Neurospora crassa</i> Vivid (VVD) as well as fungal LOV transcription factors <i>N</i>. <i>crassa</i> White-Collar 1 (WC1) and <i>T</i>. <i>reesei</i> Blue-light receptor 1 (BLR1). These elements are not conserved in AsLOV2 or short LOV domains from <i>P</i>. <i>putida</i> (PpLOV) or <i>R</i>. <i>sphaeroides</i> (RsLOV). Specifically, a key hinge region is conserved in all bacterial and fugal proteins (light green) within the NCap. Two additional residues are absolutely conserved that form key contacts in organization of the NCap hinge region. Core signaling regions of the LOV domain (blue) are conserved in all species. Residues absolutely conserved in all species (*) are noted, residues conserved in fungal and bacterial species, but not AsLOV2 are shown (^) as well as strongly conserved elements (:). (B) Domain architecture in <i>Methylocystis</i> LOV proteins. (C) SEC of McLOVr (black), McLOVn (green) and 337-TF (blue). All elute as dimers with apparent molecular weights (MW) of 35 kDa (McLOVr), 33.5 kDa (McLOVn) and 32 kDa (337-TF). The expected MW of a monomer was 17 kDa. Some preparations of McLOVn contain a significant monomeric fraction. Two distinct peaks with apparent MWs of 34 kDa and 17 kDa are observed.</p

Short LOV Proteins in <i>Methylocystis</i> Reveal Insight into LOV Domain Photocycle Mechanisms

<div><p>Light Oxygen Voltage (LOV) proteins are widely used in optogenetic devices, however universal signal transduction pathways and photocycle mechanisms remain elusive. In particular, short-LOV (sLOV) proteins have been discovered in bacteria and fungi, containing only the photoresponsive LOV element without any obvious signal transduction domains. These sLOV proteins may be ideal models for LOV domain function due to their ease of study as full-length proteins. Unfortunately, characterization of such proteins remains limited to select systems. Herein, we identify a family of bacterial sLOV proteins present in <i>Methylocystis</i>. Sequence analysis of Methylocystis LOV proteins (McLOV) demonstrates conservation with sLOV proteins from fungal systems that employ competitive dimerization as a signaling mechanism. Cloning and characterization of McLOV proteins confirms functional dimer formation and reveal unexpected photocycle mechanisms. Specifically, some McLOV photocycles are insensitive to external bases such as imidazole, in contrast to previously characterized LOV proteins. Mutational analysis identifies a key residue that imparts insensitivity to imidazole in two McLOV homologs and affects adduct decay by two orders of magnitude. The resultant data identifies a new family of LOV proteins that indicate a universal photocycle mechanism may not be present in LOV proteins.</p></div

External bases do not catalyze some McLOV proteins.

<p>(A,B) Both McLOVn (A) and McLOVr (B) show no dependence of adduct decay on imidazole concentration. In contrast, a Thr27Ile demonstrates spectra consistent with LOV chemistry (E) with a 100-fold decrease in the rate of adduct decay (F). The Thr27Ile variant causes an imidazole concentration dependent rate of adduct scission (C). Thus, Thr27 renders McLOV proteins insensitive to imidazole catalysis. (D) In contrast, 337-TF is imidazole sensitive despite containing a Thr at a position equivalent to 27. (G) A LOV structure (modeled from ENVOY PDB: 4WUJ) containing a Thr at a position equivalent to 27. Thr27 can coordinate a hypothetical water molecule between the N5 position on flavin, C61 and Q124 (McLOVr numbering). In all figures, error bars represent the standard deviation of the mean for experiments conducted in triplicate.</p