Autophosphorylation, electrophoretic mobility and immunoreaction of oat phototropin 1 under UV and blue Light.

Phototropins are UV-A/blue light photoreceptors containing two flavin mononucleotide (FMN)-binding domains, light, oxygen and voltage (LOV)1 and LOV2, of which LOV2 is more sensitive toward light and more important for the physiological response compared with LOV1. Some physiological responses are plant phototropism, chloroplast migration and stomatal opening. Oat phototropin 1 together with light-dependent autophosphorylation shows a reduced electrophoretic mobility and reduced immunoreaction against a heterologous antiserum; both effects were suggested to be caused by phosphorylation at the same sites (M. Salomon, E. Knieb, T. von Zeppelin and W. Rudiger [2003] Biochemistry 42, 4217-4225). In this study, we show that both effects can be separated from each other: at low temperature, reduced immunoreaction preceded the mobility shift, and irradiation with UV-C light led to the mobility shift without the loss of immunoreactivity. We demonstrated that UV-C light at 280 nm, which does not match any absorption maximum of FMN, leads to autophosphorylation of phototropin. It is hypothesized that UV-C light causes differential activation of the LOV domains via energy transfer from aromatic amino acids.

Tissue-specific and subcellular localization of phototropin determined by immuno-blotting.

Phototropin (phot) is a UV/blue- light receptor mediating phototropic reactions of plants as a response to unilateral irradiation. Using an antiserum directed against the N-terminal part of Arabidopsis phot1, we show here cross-reaction with phototropin from Avena sativa, Eruca sativa, Glycine max, Lepidium sativum, Lycopersicon esculentum, Pisum sativum, Sinapis alba, and Zea mays. In all investigated plants, blue light irradiation led to a gel mobility shift of phototropin corresponding to an apparent increase in size of 2-3 kDa. This increase is transient: the apparent size of the phototropin band reverted back to the original size in the dark within 60-90 min. The capacity for in vitro phosphorylation increased to 350% ( A. sativa) and 200% ( L. sativum) at 90 min after a blue light pulse without an increase in the amount of phototropin protein. Starting from coleoptile tips of monocots that contained the highest concentration of phototropin, we found an exponential decrease in basipetal sections of equal size while a linear decrease was determined for dicots in basipetal sections starting from the section below the hypocotyl hook. We confirmed the membrane association of all phototropin in dark-grown seedlings; after a 2-min blue light pulse, however, 20% of phototropin was found in the cytosolic fraction and only 80% in the membrane fraction. Both fractions showed the gel mobility shift indicating light-dependent autophosphorylation. Detergent-free solubilization of phototropin with chaotropic reagents was investigated with etiolated A. sativa seedlings. Up to 95% of phototropin was solubilized with a mixture of sodium bromide and sodium diphosphate, and subsequently subjected to affinity purification using Cibachron Blue 3GA-agarose as a dinucleotide analogue. Immediately after solubilization, soluble phototropin still showed blue-light-dependent autophosphorylation but lost its activity within less than 1 h.

Mapping of low- and high-fluence autophosphorylation sites in phototropin 1.

Phototropins, originally detected by their blue light-dependent autophosphorylation, are plant photoreceptors involved in several blue light responses such as phototropism, chloroplast relocation, leaf expansion, rapid inhibition of hypocotyl growth, and stomatal opening. Three domains have been identified in phototropin sequences, two chromophore binding domains (LOV1 and LOV2) and a kinase domain. We describe here two additional domains, the N-terminus upstream of LOV1 and the hinge region between LOV1 and LOV2, as the regions for autophosphorylation; the phosphorylation sites were identified by site-directed mutagenesis as S27, S30, S274, S300, S317, S325, S332, and S349 of the PHOT1a sequence of Avena sativa. Investigation of the autophosphorylation in vivo revealed that serines close to the LOV1 domain are phosphorylated at lower fluence of blue light than the serines close to the LOV2 domain. Recovery of phosphorylation in vivo during a dark period after saturating irradiation is caused by dephosphorylation rather than by degradation of the phosphorylated form and new synthesis of nonphosphorylated phototropin. The results were obtained by a combination of autophosphorylation of phototropin with phosphorylation of recombinant domains by protein kinase A, which turned out to have the same site specificity as the phototropin kinase, followed by proteolysis and separation of phosphopeptides. With the knowledge of the phosphorylation sites, the physiological and biochemical consequences of autophosphorylation can now be approached by site-directed mutagenesis of phototropins.