PAS/LOV proteins

Abstract

The light, oxygen or voltage (LOV) domain belongs to the Per-ARNT-Sim (PAS) superfamily of domains, and functions with the flavin chromophore as a module for sensing blue light in plants and fungi. The Arabidopsis thaliana PAS/LOV proteins (PLPs), of unknown function, possess an N-terminal PAS domain and a C-terminal LOV domain. Our recent analysis using yeast two-hybrid and Escherichia coli protein production systems reveals that the interactions of Arabidopsis PLPs with several proteins diminish under blue light illumination and that the PLP LOV domain may bind to a flavin chromophore. These results suggest that PLP functions as a blue light receptor. Homologs of PLP exist in rice, tomato and moss. The LOV domains of these PLP homologs form a distinct group in phylogenetic analysis. These facts suggest that PLP belongs to a new class of plant blue light receptor.

Phototropin LOV Domain Senses Blue Light

Plants have sensing and signaling mechanisms that help them respond to their environments. Light is one of the most influential environmental factors. Therefore, it is not surprising that plants have various photoreceptors. In Arabidopsis thaliana (L.) Heynh., two phototropins (phot1 and phot2) have been identified as the photoreceptors responsible for blue light induction of tropism, chloroplast movement and stomatal opening.¹ Phototropins possess two light, oxygen or voltage (LOV) domains and one Ser/Thr protein kinase domain. The LOV domain belongs to the Per-ARNT-Sim (PAS) superfamily of domains and is the blue light-sensing module of the phototropins.² The LOV domain binds with a flavin chromophore flavin mononucleotide (FMN). Blue light irradiation causes the formation of a covalent adduct between the FMN molecule and a conserved cysteine residue in the LOV domain, and this adduct dissociates in the dark. This light/dark-dependent structural change regulates the activity of the protein kinase domain of phototropins, which initiates signaling.³

Other LOV Proteins in Arabidopsis

In addition to phot1 and phot2, Arabidopsis has two other groups of LOV proteins. One is the ADO/FKF/LKP/ZTL family of proteins, which consist of LOV, F-box motif and kelch repeat domains.^4–7 Because the LOV domains of these proteins bind with FMN and absorb blue light,⁸ the proteins are believed to be photoreceptors. ZEITLUPE (ZTL) regulates the circadian clock,⁶ and FLAVIN-BINDING, KELCH REPEAT, F-BOX 1 (FKF1) induces floral transition under long-day conditions.^5,8 The other group of LOV proteins contains the PAS/LOV proteins (PLPs), each of which possesses an N-terminal PAS domain and a C-terminal LOV domain⁹ (Fig. 1A). The PAS domains of Arabidopsis PLPs are distinguishable from the LOV domains because they lack the key cysteine residue for light perception (Fig. 1B). There are three splice variants: PLPA, PLPB and PLPC.¹⁰ Compared with the LOV domains of PLPA and the phototropins, the LOV domain of PLPB has two additional amino acids inserted in the middle, whereas that of PLPC lacks 11 amino acids in its C-terminus¹⁰ (Fig. 1A). The physiological functions of the PLPs remain unknown.

Figure 1.

Alignment of deduced amino acid sequences of PLP homologs. The alignment was analyzed with the CLUSTALW program, and the amino acids were shaded black for 100% identity and gray for ≥50% identity by GENETYX software. (A) Full-length protein sequences of PLP homologs in Arabidopsis thaliana (AtPLPA, AtPLPB and AtPLPC), Oryza sativa (OsPLP), Solanum lycopersicum (SlPLP) and Physcomitrella patens (PpPLP) are compared. The PAS domain and the LOV domain are indicated with double and single lines, respectively, above the alignment. (B) PAS and LOV domains of PLP homologs are compared. Amino acids in phy3 LOV2 that interact with flavin are indicated by solid triangles⁹. The position corresponding to the cysteine residue involved in formation of the covalent adduct with flavin in phy3 LOV2 is marked with an asterisk.

Interaction of PLPs with its Interacting Proteins Decreases Under Blue Light Illumination

As a first step toward understanding the molecular function of PLPs, we performed yeast two-hybrid screening using PLPA as bait, and isolated VITAMIN C DEFECTIVE 2 (VTC2), VTC2-LIKE (VTC2L), BEL1-LIKE HOMEODOMAIN 10A (BLH10A) and BLH10B proteins as PLP-interacting proteins.¹⁰ VTC2 was originally isolated as the product of the wild-type gene associated with the vtc2 mutant,¹¹ which is hypersensitive to ozone and low vitamin C levels.¹² VTC2 has been identified as an enzyme in the L-galactose pathway for vitamin C biosynthesis.^13,14 VTC2L is a paralog of VTC2. BLH10 is a member of the 3-aa loop extension (TALE) homeodomain protein family.¹⁵

Because PLPA and PLPB have LOV domains, we examined the effect of blue light on the interactions of each with their interacting proteins using a yeast two-hybrid system.¹⁰ We found that some of the interactions were diminished under blue light illumination, but not in the dark or under illumination by green, red or far-red light. Photochemical analysis of recombinant proteins demonstrated possible flavin binding to PLPA and PLPB.¹⁰ These results imply that PLPs are blue light receptors.

As in phototropins, a change in the conformation of the LOV domain caused by the formation of a covalent adduct between FMN and the conserved cysteine residue of the LOV domain may affect the interaction of PLPs with PLP-interacting proteins. Among proteins so far examined, the conformational change in the LOV domain by light-dependent adduct formation and dissociation leads to the modulation of protein-protein or protein-DNA interactions. LOV domains of Arabidopsis phot1 bind to the C-terminal protein kinase domain in the dark; blue light irradiation causes dissociation of the LOV domain from the kinase domain, and the dissociation results in increased phot1 autophosphorylation and activation.¹⁶ The DNA-binding activity of Vaucheria frigida AUREOCHROME1, which has a basic region/leucine zipper domain and a LOV domain, is increased after blue light irradiation.¹⁷ ZTL and FKF1 bind with GIGANTEA (GI) in the presence of blue light;^18,19 the binding of ZTL with GI increases the stability of ZTL, and the binding of FKF1 with GI induces the interaction of FKF1 with its ubiquitination substrate, CYCLING DOF FACTOR 1 (CDF1). Therefore, it is important to determine whether adduct formation and dissociation occur in PLPs in response to blue light illumination.

PLPs may be a New Class of Blue Light Receptor in Plants

We searched for PLP homologs in other organisms and found them as annotated products of full-length cDNAs or genes in several plant species: rice (Oryza sativa; Os1g09120, TIGR Rice Genome Annotation), tomato (Solanum lycopersicum; SGN-U315842, Sol Genomics Network) and moss (Physcomitrella patens; XM001775942, GenBank) (Fig. 1A). The LOV domains of these homologs are similar to the LOV domain of PLPA. In these species, we did not find any splice variants with amino acid insertions or deletions. The cysteine residue involved in covalent adduct formation with flavin is conserved in the LOV domains of the homologs. The amino acids that are believed to interact with the flavin, as indicated by the crystal structure of Adiantum capillus-veneris phy3 LOV2,⁹ are highly conserved in the LOV domains of the homologs (Fig. 1B). Phylogenetic analysis revealed that the LOV domains of these homologs formed a distinct group from those of other LOV proteins (Fig. 2). These findings suggest that PLP homologs exist in a broad range of species in the plant kingdom and constitute a new class of LOV proteins.

Figure 2.

Phylogenetic tree of PAS and LOV domains. CLUSTALW was used to analyze deduced amino acid sequences of PAS and LOV domains of PLPs, and LOV domains of phototropins (Arabidopsis thaliana PHOT1 and PHOT2; Oryza sativa PHOT1 and PHOT2; Physcomitrella patens PHOTA1, PHOTB1), Adiantum capillus-veneris phy3, members of the ADO/FKF/LKP/ZTL family (A. thaliana FKF1, LKP2, ZTL; O. sativa Os02g05700, Os06g47890, Os11g34460), Vaucheria frigida AUREOCHROME1, and WHITE COLLAR 1 homologs (Neurospora crassa and Phycomyces blakesleeanus). Bootstrap values are indicated at the nodes. The scale bar indicates the evolutionary distance.

The chromophore-interacting amino acids are also highly conserved in PAS domains of PLP homologs in A. thaliana, O. sativa and S. lycopersicum, although the key cysteine residue is not conserved in these domains (Fig. 1B, top 3 sequences). Surprisingly, the N-terminal domain of the Physcomitrella PLP has the conserved cysteine (Fig. 1B). Furthermore, most of the chromophore-interacting amino acids are conserved in the Physcomitrella PLP N-terminal domain. These facts suggest the possible function of this domain as a blue light sensor. Therefore, we designated the N-terminal and C-terminal domains of the Physcomitrella PLP as LOV1 and LOV2, respectively. The photochemical ability of the Physcomitrella PLP LOV domains as blue light sensors is an interesting topic. Judging from the results of protein-protein interaction analysis of Arabidopsis PLPs and the phylogenetic analysis of PLP homologs, PLPs could be new blue light receptors in plants. Future work on PLPs, such as characterization of their gene expression patterns and phenotypic analysis of loss-of-function and gain-of-function mutants, is needed to clarify the biological function of these proposed blue light receptors.

Addendum to: Ogura Y, Komatsu A, Zikihara K, Nanjo T, Tokutomi S, Wada M, Kiyosue T. Blue light diminishes interaction of PAS/LOV proteins, putative blue light receptors in Arabidopsis thaliana, with their interacting partners. J Plant Res. 2008;121:97–105. doi: 10.1007/s10265-007-0118-8.