These PIFs bind to various G-box sequences found in several light-regulated promoters ( Martinez-Garcia et al., 2000 Huq and Quail, 2002) and probably act as negative regulators for the gene expression ( Kim et al., 2003). Consistent with this observation, phytochromes interact with the nuclear-localized basic helix-loop-helix proteins PIF3 and PIF4 in a light-dependent manner ( Ni et al., 1998, 1999 Huq and Quail, 2002). Phytochromes translocate from the cytoplasm to the nucleus and form nuclear speckles upon light activation ( Kircher et al., 1999, 2002 Yamaguchi et al., 1999 Hisada et al., 2000). The functional distinction is particularly striking between phytochrome A (phyA) and phytochrome B (phyB) in the control of seedling deetiolation, for which the effects of continuous far-red (cFR) light are mediated exclusively by phyA, whereas the effects of continuous red light (cR) are mediated predominantly by phyB. Analysis of mutants deficient in different phytochromes has defined differential, as well as overlapping, physiological roles for members of this family ( Quail et al., 1995 Smith and Whitelam, 1997 Franklin et al., 2003 Monte et al., 2003). In Arabidopsis thaliana, the phytochrome family comprises five genes, PHYTOCHROME A ( PHYA) to PHYE ( Mathews and Sharrock, 1997). They are selectively responsible for sensing different light quality. Higher plants contain ensembles of phytochromes, which differ in amino acid sequence by ∼50%. Dark reversion has been proposed to attenuate phytochrome signaling because it reduces the amount of active Pfr ( Mancinelli, 1994). In addition, in the dark, Pfr is converted back to Pr by a thermally driven process called dark reversion. Thus, phytochrome functions as a light-driven biological switch. Conversely, far-red light inactivates phytochrome by converting Pfr back to Pr. Red light activates phytochrome by converting it from the Pr to the Pfr form. Phytochromes undergo photoreversible absorption changes between two spectrally distinct forms: red light absorbing form of phytochrome (Pr) and far-red light absorbing form of phytochrome (Pfr). The phytochrome monomer is a polypeptide of ∼120 kD, with a single, covalently attached open tetrapyrrole chromophore responsible for the absorption of visible light.
A wide range of physiological and developmental processes are under the control of phytochrome ( Neff et al., 2000). Phytochromes are large, soluble proteins that exist as dimers in solution ( Furuya, 1993). Of these photoreceptors, phytochrome is the best characterized. Plants can sense the intensity, wavelength, direction, and timing of illumination using diverse photoreceptors including red-light photoreceptors, phytochromes, blue-light photoreceptors, cryptochromes, and phototropins ( Briggs and Huala, 1999 Neff et al., 2000 Briggs and Christie, 2002). Light acts not only as an energy source for plants but also as an environmental signal that regulates plant development. Taken together, our findings show that the PHY domain is dispensable for phyB signal transduction but is required for stabilizing the Pfr form of phyB. Consistent with this, plants expressing N450 failed to respond to intermittent light applied at long intervals, indicating that N450 Pfr is short-lived in vivo. In vitro spectral analysis of reconstituted chromopeptides further indicated that the stability of the N450 Pfr form, an active form of phytochrome, is markedly reduced in comparison with the Pfr form of full-length phyB. Analysis of these plants revealed that N450 can act as an active photoreceptor when attached to a short nuclear localization signal and β-glucuronidase. Here, we have established transgenic Arabidopsis thaliana lines expressing an N-terminal 450–amino acid fragment of phyB (N450) lacking the PHY domain on a phyB-deficient background. The phytochrome (PHY) domain, which is located at the C-terminal end of the photosensory domain, is required for the spectral integrity of phytochrome however, little is known about the signal transduction activity of this domain. Recently, the 651–amino acid photosensory domain of phytochrome B (phyB) has been shown to act as a functional photoreceptor in the nucleus. Phytochrome, a major photoreceptor in plants, consists of two domains: the N-terminal photosensory domain and the C-terminal domain.
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