Comparative functional analysis of full‐length and N‐terminal fragments of phytochrome C, D and E in red light‐induced signaling

Abstract

SummaryPhytochromes (phy) C, D and E are involved in the regulation of red/far-red light-induced photomorphogenesis of Arabidopsis thaliana, but only limited data are available on the mode of action and biological function of these lesser studied phytochrome species.We fused N-terminal fragments or full-length PHYC, D and E to YELLOW FLUORESCENT PROTEIN (YFP), and analyzed the function, stability and intracellular distribution of these fusion proteins in planta.The activity of the constitutively nuclear-localized homodimers of N-terminal fragments was comparable with that of full-length PHYC, D, E-YFP, and resulted in the regulation of various red light-induced photomorphogenic responses in the studied genetic backgrounds. PHYE-YFP was active in the absence of phyB and phyD, and PHYE-YFP controlled responses, as well as accumulation, of the fusion protein in the nuclei, was saturated at low fluence rates of red light and did not require functional FAR-RED ELONGATED HYPOCOTYL1 (FHY-1) and FHY-1-like proteins.Our data suggest that PHYC-YFP, PHYD-YFP and PHYE-YFP fusion proteins, as well as their truncated N-terminal derivatives, are biologically active in the modulation of red lightregulated photomorphogenesis. We propose that PHYE-YFP can function as a homodimer and that low-fluence red light-induced translocation of phyE and phyA into the nuclei is mediated by different molecular mechanisms.

Bottom-up Assembly of the Phytochrome Network

“…Coupled with the finding that phyE is biologically active in the absence of other phytochromes (Fig 2), this result strongly suggests that phyE forms homodimers. Furthermore, self-interactions were observed for phyA, phyB, and phyD, consistent with these phytochromes forming homodimers [21, 22]. phyA, phyB, phyD and phyE interacted with each other (S10 Fig).…”

“…This low residual activity of phyC is also consistent with the lack of detectable phyC homodimerization [21] (S10 Fig) and the low accumulation of phyC in the absence of phyB (S2 Fig) [20, 27]. However, Triticum aestivum (wheat) phyC homodimerizes in Arabidopsis and elicits photomorphogenic responses [43], and forcing homodimerization of phyC triggers photomorphogenic responses in Arabidopsis [22]. Hence, it is possible that the residual effects of phyC may be due to very low levels of phyC homodimers.…”

“…Phytochrome fusions to fluorescent proteins were instrumental in studies about the dynamics of phytochrome nuclear localization. However, these studies did not distinguish between phytochrome homodimers and heterodimers [22, 33–35]. On the other hand, the interactions among phytochromes evidenced above could be either direct or indirect.…”

“…phyB and phyA have emerged as the most important phytochromes [1, 2, 18, 29], whereas the roles of phyD, phyE, and phyC have been assumed to be minor and redundant with phyB [6, 11, 12, 14, 15, 22, 24, 40, 41]. However, our knowledge about the capacity of individual phytochromes to elicit specific responses and the interactions among them is still incomplete.…”

“…Increased complexity may also be expected, because heterodimers form among phytochrome family members [20, 21]. In studies in which phytochromes were overexpressed, other phytochromes were present in the genetic background, raising questions as to whether the signal was generated by single or multiple phytochromes [22–25]. …”