The Biochemical Study Of Arabidopsis Blue Light Receptor CRY2Photoactivation Mechanism And Function

Cryptochromes are photolyase-like flavin proteins, mediating various light responses in plants and animals. The photochemical mechanism of cryptochromes is not well understood. It has been proposed that photoactivation of cryptochromes involves the blue light-dependent photoreduction of flavin adenine dinucleotide (FAD) via the electron transport chain composed of three evolutionarily conserved tryptophan residues known as the "trp triad." Trp Triad is highly conserved in photolyase/cryptochrome family, its function was first identified in photolyase. But it is known that photoreduction is not required for the enzymatic activity of Escherichia coli photolyase in vivo and also physiological activity of animal type I in vivo. It was also reported that site-mutated trp triad of Arabidopsis CRY1abolished the physiological activities of CRY1in transgenic plants, representing the only genetic evidence thus far supporting the trp-triad photoreduction hypothesis. To study the photoactivation mechanism of plant cryptochromes, we mutated each of the three trp-triad residues of Arabidopsis cryptochrome2(CRY2). We investigated this hypothesis by analyzing the photochemical and physiological activities of the wild type and mutated CRY2. And the results of this study were listed as follows:(1) All trp-triad mutations of CRY2tested lost photoreduction activity in vitro, but none of the physiological and biochemical activities tested, including de-etiolation responses in seedlings, floral initiation in adult plants, blue light-specific CRY2degradation, and protein-protein interactions, was eliminated in any of the trp-triad mutations as expected by the photoreduction hypothesis. Our results support the trp-triad-dependent photoreduction is not required for the function of Arabidopsis CRY2in planta;(2) Several trp-triad CRY2mutants are constitutively active (or "more" active) in all physiological responses examined, such as CRY2W374A. The constitutively active CRY2w374A interacts with CRY2-signaling proteins Suppressor of PHYA1(SPA1) and Cryptochrome-Interaction Basic Helix-Loop-Helix (CIB1). The constitutive interaction supports conformational change hypothesis and a biochemical explanation of why a trp-triad CRY2mutant is physiologically active in the absence of light.(3) CRY2protein bound FAD chromophore and maintain blue light receptor activity throught eukaryotic expression system. CRY2protein response to blue light to form dimer or oligomer in vitro. ATP can promotes dimerization or oligomer-ization of CRY2. Dimer of CRY2W374A existed in both darkness and blue light, the dimerization or oligomerization of CRY2W374A also increased by blue light irradiation.(4) The formation of CRY2-CIB1complex depends on blue light and FAD, consistent with the in vivo response, this is the first time a blue light-specific protein-protein interaction is observed in vitro for a plant cryptochrome. CRY2W374A interacts with CIB1constitutively regardless of blue light in vitro. Those results indicate that blue light caused conformational changes of CRY2, mutated W374to A changed CRY2conformation resemble that of the photoactivated CRY2to act constitutively in plant cells.(5) Limited digestion first showed blue light induce CRY2conformational change, the conformation of photoactivated CRY2close to CRY2W374A. The blue light-induced rearrangement sites of CRY2locate in the CCT domain of CRY2, close to STAESSS in DAS motif which is highly conserved in plants. Light induces changes in CRY2supports light-dependent interactions between CRY2-CIB1/SPA1and the CRY2PHR-CCT dissociation hypothesis.(6) CRY2in different tissue seems to have different functions mainly through different mechanisms, such as promote flower initiation in LD, inhibit hypocotyl elongation under blue light. Our constructs:tissue specific promoter drive GFP, mRNA binding protein PABP, Cryptochrome1or Cryptochrome2expressing in transgenic plants provide us more opporunities to study multiplex functions of Cryptochromes and also the regulation mechamisms.