Abstract
LOV domains function as blue light-sensing modules in various photoreceptors in plants, fungi, algae, and bacteria. A LOV/LOV protein (LLP) has been found from Arabidopsis thaliana (AtLLP) as a two LOV domain-containing protein. However, its function remains unknown. We isolated cDNA clones coding for an LLP homolog from tomato (Solanum lycopersicum) and two homologs from the moss Physcomitrella patens. The tomato LLP (SlLLP) contains two LOV domains (LOV1 and LOV2 domains), as in AtLLP. Most of the amino acids required for association with chromophore are conserved in both LOV domains, except that the amino acid at the position equivalent to the cysteine essential for cysteinyl adduct formation is glycine in the LOV1 domain as in AtLLP. When expressed in Escherichia coli, SlLLP binds FMN and undergoes a self-contained photocycle upon irradiation of blue light. Analyses using mutant SlLLPs revealed that SlLLP binds FMN in both LOV domains, although the LOV1 domain does not show spectral changes on irradiation. However, when Gly(66) in the LOV1 domain, which is located at the position equivalent to the essential cysteine of LOV domains, is replaced by cysteine, the mutated LOV1 domain shows light-induced spectral changes. In addition, all four LOV domains of P. patens LLPs (PpLLP1 and PpLLP2) show the typical features of LOV domains, including the reactive cysteine in each. This study shows that plants have a new LOV domain-containing protein family with the typical biochemical and photochemical properties of other LOV domain-containing proteins such as the phototropins.
Highlights
NOVEMBER 5, 2010 VOLUME 285 NUMBER 45 many other organisms
The molecular interactions between PAS/LOV protein (PLP) and VTC2L, BLH10A, or BLH10B in yeast were dependent on blue light irradiation, suggesting that PLP may function as a photoreceptor [18]
We isolate cDNAs coding for PLP homologs from tomato (Solanum lycopersicum) and the moss Physcomitrella patens, and phylogenetic analysis shows that the PAS domains of the homologs, even the PAS domain of A. thaliana PLP, are closely related to the LOV domain
Summary
Cloning and Sequencing of SlLLP cDNA—To obtain tomato LLP cDNA, RT-PCR was performed using total RNA from flowers of tomato Expression of Recombinant SlLLP in E. coli—For protein expression of SlLLP, cDNA of SlLLP was amplified by PCR using the two primers LLP-F and LLP-R (supplemental Table S1). The PCR product was digested with XhoI and NotI and cloned into the XhoI-NotI site of pThioHisA plasmid (Invitrogen), and the resulting plasmid was named pThio-SlLLP. Purification of Recombinant SlLLP—The transformants, JM109 cells harboring pThio-SlLLP and pRARE2LysS, were grown overnight at 37 °C in Luria-Bertani medium (30 ml) supplemented with ampicillin (100 g mlϪ1) and chloramphenicol (30 g mlϪ1). Expression and Purification of Recombinant PpLLP1 and PpLLP2 and Each LOV Domain in E. coli—For protein expression, cDNAs were amplified by PCR using the two primers listed in supplemental Table S1. FMN associated with SlLLP, purified protein samples were are conserved in both LOV domains of LLPs (asterisks in Fig. denatured by treatment with 1% SDS, and the concentrations of 1B). Are different in the LOV1 domains of A. thaliana (glycine), O
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