Biological activity and dimerization state of modified phytochrome A proteins

Abstract

To assess potential physical interactions of type I phyA with the type II phyB-phyE phytochromes in vivo, transgenes expressing fusion gene forms of phyA were introduced into the Arabidopsis phyA mutant background. When a single c-Myc (myc) epitope is added to either the N- or C-terminus of phyA, the constructs completely complement phyA mutant phenotypes. However, addition of larger tags, such as six consecutive myc epitopes or the yellow fluorescent protein sequence, result in fusion proteins that show reduced activity. All the tagged phyA proteins migrate as dimers on native gels and co-immunoprecipitation reveals no binding interaction of phyA to any of the type II phys in the dark or under continuous far-red light. Dimers of the phyA 1–615 amino acid N-terminal photosensory domain (NphyA), generated in vivo with a yeast GAL4 dimerization domain and attached to a constitutive nuclear localization sequence, are expressed at a low level and, although they cause a cop phenotype in darkness and mediate a very low fluence response to pulses of FR, have no activity under continuous FR. It is concluded that type I phyA in its Pr form is present in plants predominantly or exclusively as a homodimer and does not stably interact with type II phys in a dimer-to-dimer manner. In addition, its activity in mediating response to continuous FR is sensitive to modification of its N- or C-terminus.

Generation of High-Value Genomic Resource in Rice: A “Subgenomic Library” of Low-Light Tolerant Rice Cultivar Swarnaprabha

“…Although there are five types of phytochromes ( phyA , phyB , phyC , phyD , and phyE ) in Arabidopsis , only three predominant types ( phyA , phyB , and phyC ) are functionally annotated in rice [ 15 ]. PhyA mediates irreversible photo-responses in the very-low-fluence and high-fluence ranges (VLFR and HFR), and primarily in the far-red (FR) spectral region [ 16 ], whereas phyB mediates the “classical” R/FR reversible responses in the low-fluence range (LFR) [ 17 ]. The phyA specificity is determined at three levels: (i) intramolecular events; (ii) turnover; (iii) nuclear–cytoplasmic partitioning and interaction with partner proteins [ 18 ].…”