Heterodimerization of type II phytochromes in Arabidopsis

Abstract

Coimmunoprecipitation of members of the phytochrome red͞far-red photoreceptor family from plant extracts has been used to analyze their heteromeric binding interactions. Phytochrome (phy)B or phyD apoproteins with six myc epitopes fused to their N termini are biologically active when expressed in Arabidopsis. Immunoprecipitation of either of these tagged proteins from seedling extracts coprecipitates additional type II phytochromes: six myc (myc6)-phyB coprecipitates phyC-phyE; and myc 6 -phyD coprecipitates phyB and phyE. No interaction of the epitopetagged proteins with type I phyA was detected. Gel filtration chromatography shows that all five of the Arabidopsis phytochromes are present in seedlings as dimers, and that the heteromeric type II phytochrome complexes migrate at molecular masses characteristic of heterodimers. Similar levels of heterodimer formation are observed in extracts of dark-grown seedlings, where the phytochromes are cytosolic, and light-grown seedlings, where they are predominantly nuclear. These findings indicate that Arabidopsis, which until now has been thought to contain five homodimeric forms of phytochrome, in fact contains multiple species of both homodimeric and heterodimeric phytochromes. The conservation of the phytochrome family throughout angiosperms suggests that heterodimeric red͞far-red receptors may be present in many flowering plants. Phytochromes are soluble chromoprotein photoreceptors that mediate plant responses to red (R) and far-red (FR) light. The light-sensing and biological regulatory activities of phytochromes derive from their photochemical interconversion between a R-absorbing conformation (Pr) and a FR-absorbing conformation (Pfr). Absorption of photons of R converts Pr to Pfr, and absorption of photons of FR converts Pfr back to Pr. For most R͞FR plant responses, the Pr form is inactive and the Pfr form is active, so that either the concentration of Pfr or the ratio of Pfr to total phytochrome correlates with the strength of the response. Phytochrome in the Pr conformation is localized to the cytosol, and photoconversion to Pfr results in translocation of the photoreceptor to the nucleus (1, 2). Several candidate phytochrome-interacting proteins have been identified, including the nuclear DNA-binding protein PIF3, the CRY2 blue-light photoreceptor, and the F-box-containing ZTL protein (3, 4). These findings suggest that phytochromes may be directly positioned at light-response gene promoters, and that they may also be directly involved in integrating signaling input from other receptor systems and the circadian clock.Five genes encoding 120-to 130-kDa phytochrome apoproteins are present in the Arabidopsis genome, PHYA-PHYE (5, 6). Mutations in all five of these genes have been identified, and effects of loss of function for each phytochrome form on plant development and light response have been described (7-11). In addition, the levels of each of the phytochrome A-E holoproteins (phyA-phyE) and their light stabilities have been determined (12). Four of the ...

A Constitutively Active Allele of Phytochrome B Maintains Circadian Robustness in the Absence of Light

“…7A) could reflect the influence of residual phyC(P fr ) that had not fully reverted to phyC (P r ) at the onset of darkness. These results illustrate one consequence of the many interactions between phys that underlie the complex regulation of the circadian system by red light (Sharrock and Clack, 2004).…”

“…Type II phys (phyB-E) form homo-and heterodimers that complicate interpretation of phenotypes of loss-offunction phy mutants and of gain-of-function YHB transgenics (Sharrock and Clack, 2004;Clack et al, 2009;Hu et al, 2009). The additive circadian defect of phyABD mutants compared with phyAB has been assumed to indicate an ability of phyD to provide light input into the circadian system in the absence of phyB (Devlin and Kay, 2000).…”

“…The ability of phyB-E to form homo-and heterodimers introduces complexity into the interpretation of individual phy mutant phenotypes (Sharrock and Clack, 2004;Clack et al, 2009;Hu et al, 2013). In addition, period length in phyABCDE mutants is modestly sensitive to different fluence rates of red light (Hu et al, 2013), suggesting that metabolic processes may complicate the assessment of the role of Phys in light input to the circadian clock.…”

Evolutionary Studies Illuminate the Structural-Functional Model of Plant Phytochromes

“…It is interesting to note that phyC and phyE may not, in many cases, be acting alone, at least in Arabidopsis, where under some conditions they occur largely as heterodimers, predominantly with phyB; phyD also heterodimerizes with phyB ( Figure 1; Sharrock and Clack, 2004;Clack et al, 2009). Notably, in the absence of phyB, phyC and phyE are largely destabilized or monomeric, respectively.…”

“…Many of the determinants of functional specificity that distinguish phyA and phyB have been identified from the characterization of monogenic and multiple phytochrome mutants of Arabidopsis, such as those cited above (along with many others), from the dissection of signaling pathways (Castillon et al, 2007;Jiao et al, 2007;Bae and Choi, 2008), and from studies of expression patterns and protein levels (Goosey et al, 1997;Sharrock and Clack, 2002), dimerization (Sharrock and Clack, 2004;Clack et al, 2009), nuclear localization (Kircher et al, 2002), proteinchromophore interactions (Hanzawa et al, 2002), and phyA dark reversion rates (Eichenberg et al, 2000b). Despite these many advances, aspects of the structural-functional models for these photoreceptors remain elusive.…”