In Vivo Characterization of Chimeric Phytochromes in Yeast
1999; Elsevier BV; Volume: 274; Issue: 1 Linguagem: Inglês
10.1074/jbc.274.1.354
ISSN1083-351X
AutoresKlaus Eichenberg, Tim Kunkel, Thomas Kretsch, Volker Speth, Eberhard Schäfer,
Tópico(s)Photosynthetic Processes and Mechanisms
ResumoPhytochromes are plant photoreceptors that play a major role in photomorphogenesis. Two members of the phytochrome family have been characterized in some detail. Phytochrome A, which controls very low fluence and high irradiance responses, is rapidly degraded in the light, forms sequestered areas of phytochrome (SAPs), and does not exhibit dark reversion in monocotyledonous seedlings. Phytochrome B mediates red/far-red reversible responses, is stable in the light, and does not form SAPs. We report on the behavior in yeast of the phytochrome apoproteins of rice PHYA, tobacco PHYB, and chimeric PHYAB and PHYBA and on the behavior of the respective holoprotein adducts after assembly with phycocyanobilin chromophore (PHY*). SAP-like formation in yeast was not observed for PHYB, but was detectable for PHYA, PHYAB, and PHYBA. Rice PHYA* did not undergo dark reversion in yeast. Surprisingly, all other tested phytochrome constructs did exhibit dark reversion, including chimeric phytochromes with a short N-terminal part of tobacco PHYB or parsley PHYA fused to rice PHYA. Furthermore, the proportion of phytochrome undergoing dark reversion and the rate of reversion were increased for both the N terminus-swapped constructs and PHYBA*. These results are discussed with respect to structure/function analysis of phytochromes A and B. Phytochromes are plant photoreceptors that play a major role in photomorphogenesis. Two members of the phytochrome family have been characterized in some detail. Phytochrome A, which controls very low fluence and high irradiance responses, is rapidly degraded in the light, forms sequestered areas of phytochrome (SAPs), and does not exhibit dark reversion in monocotyledonous seedlings. Phytochrome B mediates red/far-red reversible responses, is stable in the light, and does not form SAPs. We report on the behavior in yeast of the phytochrome apoproteins of rice PHYA, tobacco PHYB, and chimeric PHYAB and PHYBA and on the behavior of the respective holoprotein adducts after assembly with phycocyanobilin chromophore (PHY*). SAP-like formation in yeast was not observed for PHYB, but was detectable for PHYA, PHYAB, and PHYBA. Rice PHYA* did not undergo dark reversion in yeast. Surprisingly, all other tested phytochrome constructs did exhibit dark reversion, including chimeric phytochromes with a short N-terminal part of tobacco PHYB or parsley PHYA fused to rice PHYA. Furthermore, the proportion of phytochrome undergoing dark reversion and the rate of reversion were increased for both the N terminus-swapped constructs and PHYBA*. These results are discussed with respect to structure/function analysis of phytochromes A and B. phytochromes A and B, respectively genes or cDNAs of phytochromes A and B, respectively apoproteins of phytochromes A and B, respectively phycocyanobilin adducts of phytochromes A and B, respectively red-light (660 nm)-absorbing form of phytochrome far-red light (730 nm)-absorbing form of phytochrome sequestered area of phytochrome phycocyanobilin total amount of phytochrome. Plants are sessile organisms that have evolved a wide spectrum of mechanisms to adapt to changes in their natural environment. Light is used not only as the energy source for photosynthesis, but also as a major environmental signal. To monitor changes in light quality and quantity and to regulate temporal and spatial patterns in photomorphogenesis, plants have evolved at least three different photoreceptor systems: UVB photoreceptors, blue UVA photoreceptors, and phytochromes (1$$Google Scholar). Of these photoreceptors, the phytochromes are the best characterized. Five genes encoding phytochrome apoproteins have been identified in Arabidopsis (2Sharrock R.A. Quail P.H. Genes Dev. 1989; 3: 1745-1757Crossref PubMed Scopus (693) Google Scholar, 3Clack T. Mathews S. Sharrock R.A. Plant Mol. Biol. 1994; 25: 413-427Crossref PubMed Scopus (555) Google Scholar). Mutants of phyA,1 phyB, and phytochrome D in Arabidopsis have different effects on photomorphogenesis (4Nagatani A. Reed J.W. Chory J. Plant Physiol. (Bethesda). 1993; 102: 269-277Crossref PubMed Scopus (392) Google Scholar, 5Parks B.M. Quail P.H. Plant Cell. 1993; 5: 39-48Crossref PubMed Scopus (293) Google Scholar, 6Whitelam G.C. Johnson E. Peng J. Carol P. Anderson M.L. Cowl J.S. Harberd N.P. Plant Cell. 1993; 5: 757-768Crossref PubMed Scopus (483) Google Scholar, 7Reed J.W. Nagpal P. Poole D.S. Furuya M. Chory J. Plant Cell. 1993; 5: 147-157Crossref PubMed Scopus (761) Google Scholar, 8Aukerman M.J. Hirschfeld M. Wester L. Weaver M. Clack T. Amasino R.M. Sharrock R.A. Plant Cell. 1997; 9: 1317-1326Crossref PubMed Scopus (245) Google Scholar). The dominant phytochrome of etiolated seedlings, the light-labile phyA, controls the very low fluence and the high irradiance responses, whereas the dominant phytochrome of green seedlings and mature plants, the light-stable phyB, controls responses governed by red/far-red reversibility or continuous red light (9Furuya M. Schäfer E. Trends Plant Sci. 1996; 1: 301-307Abstract Full Text PDF Google Scholar).Phytochromes are dimers composed of 120-kDa monomers, each of which contains two domains linked by a highly conserved hinge region. The N-terminal domain bears the chromophore, and the C-terminal domain is involved in dimerization (Fig.1 A) (10Furuya M. Song P.-S. Kendrick R.E. Kronenberg G.H.M. Photomorphogenesis in Plants. 2nd Ed. Kluwer Academic Publishers, Dordrecht, The Netherlands1994: 105-140Crossref Google Scholar). Phytochromes are synthesized in the dark in their physiologically inactive red light-absorbing form (Pr). Upon photon absorption, Pr is converted into the physiologically active far-red light-absorbing form of phytochrome (Pfr). During this reversible process, the absorption maximum of the molecule shifts from 660 nm (red light) to 730 nm (far-red light). Under saturating red light, 80% or more of the phytochrome is in the Pfr form, whereas only 3% Pfr remains following exposure to saturating far-red light (11Mancinelli A.L. Kendrick R.E. Kronenberg G.H.M. Photomorphogenesis in Plants. 2nd Ed. Kluwer Academic Publishers, Dordrecht, The Netherlands1994: 211-269Crossref Google Scholar). In vivo spectroscopy has demonstrated that phyA undergoes dark reversion in most dicotyledonous seedlings, but not in monocotyledonous seedlings (11Mancinelli A.L. Kendrick R.E. Kronenberg G.H.M. Photomorphogenesis in Plants. 2nd Ed. Kluwer Academic Publishers, Dordrecht, The Netherlands1994: 211-269Crossref Google Scholar). Hence, some of the Pfr in dicotyledonous seedlings is converted to the physiologically inactive Pr form in total darkness.Rapid de novo synthesis of phyA in darkness, rapid degradation of the Pfr form (half-life of 30–60 min) in light, rapid dark reversion of 20% of the Pfr molecules to Pr (half-life of 20 min) in most dicotyledonous seedlings, and light-dependent rapid aggregation (half-life of 2 s) of the protein into SAPs have been demonstrated by in vivo spectroscopy and immunological methods (1$$Google Scholar). Ubiquitin may be involved in this degradation process, which is associated with the formation of SAPs (12Vierstra R.D. Kendrick R.E. Kronenberg G.H.M. Photomorphogenesis in Plants. 2nd Ed. Kluwer Academic Publishers, Dordrecht, The Netherlands1994: 141-162Crossref Google Scholar). In vivocharacterization of phytochromes has been possible only for phyA because the less abundant phyB cannot be spectroscopically distinguished from the abundant phyA, and high levels of chlorophyll prevent measurement of phyB even if phyA is destroyed in the light. Even in mutants lacking phyA, phyB is difficult to detect because of its low concentration. In vivo spectroscopic studies of phytochrome have been performed in light-grown bleached seedlings of certain species; the results of these studies may reflect properties of phyB (13Jabben M. Holmes M.G. Shropshire W. Mohr H. Photomorphogenesis. Springer-Verlag, Berlin1983: 704-722Crossref Google Scholar, 14Heim B. Jabben M. Schäfer E. Photochem. Photobiol. 1981; 34: 89-93Crossref Scopus (42) Google Scholar). These studies indicate a very slow destruction of the light-stable phytochrome (half-life of >8 h) and a weak partial dark reversion.A model system for spectroscopic and immunological characterization of phyB has been developed in yeast (Saccharomyces cerevisiae) (15Kunkel T. Speth V. Büche C. Schäfer E. J. Biol. Chem. 1995; 270: 20193-20200Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). The yeast-derived phytochrome-chromophore adducts are functionalin planta (16Kunkel T. Neuhaus G. Batschauer A. Chua N.-H. Schäfer E. Plant J. 1996; 10: 625-636Crossref PubMed Scopus (58) Google Scholar), and tobacco PHYB* is stable, shows a partial rapid dark reversion from Pfr to Pr, and does not form SAPs. In contrast, rice PHYA* expressed in yeast does not undergo dark reversion, but does exhibit SAPs as well as light-independent formation of SAP-like structures (15Kunkel T. Speth V. Büche C. Schäfer E. J. Biol. Chem. 1995; 270: 20193-20200Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Therefore, phytochromes expressed in yeast appear to exhibit most of the properties demonstrated in planta.The effects of modified and overexpressed phytochromes in transgenic plants have helped define the structure and function of phyA and phyB. The 4–6-kDa N terminus of phyA is important for photoreceptor function. Deletion of this region results in a dominant-negative phenotype in overexpressing lines (17Boylan M. Douglas N. Quail P.H. Plant Cell. 1994; 6: 449-460Crossref PubMed Scopus (78) Google Scholar, 18Emmler K. Stockhaus J. Chua N.-H. Schäfer E. Planta (Heidelb.). 1995; 197: 103-110Crossref PubMed Scopus (29) Google Scholar). Wagner et al.(19Wagner D. Fairchild C.D. Kuhn R.M. Quail P.H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4011-4015Crossref PubMed Scopus (68) Google Scholar) constructed PHYAB and PHYBA chimeras similar to ours, but used oatPHYA and rice PHYB and overexpressed the chimeric genes in wild-type Arabidopsis. From these studies, it was concluded that the N-terminal domains determine photosensory specificity and light lability (19Wagner D. Fairchild C.D. Kuhn R.M. Quail P.H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4011-4015Crossref PubMed Scopus (68) Google Scholar, 20Quail P.H. Boylan M.T. Parks B.M. Short T.W. Xu Y. Wagner D. Science. 1995; 268: 675-680Crossref PubMed Scopus (649) Google Scholar).In this study, we describe the expression and assembly of PHYA, PHYB, and various chimeric apoproteins in yeast and the spectroscopic properties of the phytochrome adducts and their capacity to form SAP-like structures. Our aim was to elucidate the molecular substructures of phytochrome important for SAP formation and dark reversion, both of which appear to be essential in regulating signal transduction.DISCUSSIONWe investigated which domains of the phytochrome molecule are responsible for dark reversion and SAP formation. For this investigation, rice PHYA, tobacco PHYB, and various chimeric phytochrome apoproteins were expressed in yeast and reconstitutedin vivo with PCB as the chromophore to enable in vivo spectroscopic examinations. Table II summarizes the results.Rice PHYA* showed no dark reversion, but showed light-independent SAP-like formation. The opposite was true for tobacco PHYB* (see also Ref. 15Kunkel T. Speth V. Büche C. Schäfer E. J. Biol. Chem. 1995; 270: 20193-20200Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Wagner et al. (19Wagner D. Fairchild C.D. Kuhn R.M. Quail P.H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4011-4015Crossref PubMed Scopus (68) Google Scholar) demonstrated, by domain swap experiments in transgenic wild-type Arabidopsis seedlings, that the chromophore-bearing N-terminal domains of phyA and phyB determine the photosensory specificity and different light lability of the phytochromes. These authors used oat PHYAand rice PHYB to clone chimera for their analysis. We used a yeast expression system and rice PHYA and tobaccoPHYB for our constructs. To obtain more information about the role of the N-terminal domain, we constructed chimeric PHYAB and PHYBA by swapping the 60-kDa domains, and two additional chimera (PHYTBRA and PHYPARA) were constructed by fusing either the 10.5-kDa N terminus of tobacco PHYB or the 9-kDa N terminus of parsley PHYA, respectively, to the truncated part of rice PHYA (Fig.1 A).Photoreversible phytochromes were obtained with all constructs, which was a prerequisite for all subsequent analyses. Interestingly, all of the PHYA* chimeras with a replaced N terminus (PHYBA*, PHYTBRA*, and PHYPARA*) showed a slightly hypsochromic shift of the far-red absorption maxima compared with PHYA* (Fig. 2).A characteristic difference between phyA and phyB is the light-dependent SAP formation of phyA in plantaand the light-independent SAP-like formation of PHYA in yeast. Light-induced SAP formation of phyA has been observed in etiolated monocotyledonous and some dicotyledonous seedlings (33Pratt L.H. Kendrick R.E. Kronenberg G.H.M. Photomorphogenesis in Plants. 2nd Ed. Kluwer Academic Publishers, Dordrecht, The Netherlands1994: 163-185Crossref Google Scholar). As SAP formation and pelletability (34Hofmann E. Grimm R. Harter K. Speth V. Schäfer E. Planta (Heidelb.). 1991; 183: 265-273Crossref PubMed Scopus (10) Google Scholar) show parallel kinetics, temperature dependence, and light dependence, it was concluded that these two observations reflect the same process. Therefore, SAP formation is considered as a molecular property of phyA. Although SAP formation of PHYA* and PHYA has also been found in yeast cells, it could not be obtained for PHYB* and PHYB (15Kunkel T. Speth V. Büche C. Schäfer E. J. Biol. Chem. 1995; 270: 20193-20200Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Therefore, we compared SAP-like formation of PHYAB and PHYBA chimeric phytochromes in yeast cells.The chimeric phytochromes PHYAB and PHYBA both showed SAP-like formation. This might be explained by the fact that the N-terminal half and the C-terminal half of PHYA are each sufficient to allow SAP formation when the domains are fused to the corresponding domains of PHYB. It is possible that the surfaces of the chimeric phytochrome molecules became hydrophobic in order to aggregate more simply in a cytosolic environment. phyB is not prone to aggregate because it is possibly more hydrophilic than phyA; the addition of a hydrophobic part causes it to sequester.Wagner et al. (19Wagner D. Fairchild C.D. Kuhn R.M. Quail P.H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4011-4015Crossref PubMed Scopus (68) Google Scholar) demonstrated that phyAB shows a similar destruction as phyA and that phyBA, like phyB, shows almost no destruction. The sequestering of PHYA in yeast is light-independent (15Kunkel T. Speth V. Büche C. Schäfer E. J. Biol. Chem. 1995; 270: 20193-20200Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar), and this was also the case with PHYBA in our experiments, although no destruction occurred in planta (19Wagner D. Fairchild C.D. Kuhn R.M. Quail P.H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4011-4015Crossref PubMed Scopus (68) Google Scholar). Therefore, SAP formation might be necessary, but not sufficient, for destruction (12Vierstra R.D. Kendrick R.E. Kronenberg G.H.M. Photomorphogenesis in Plants. 2nd Ed. Kluwer Academic Publishers, Dordrecht, The Netherlands1994: 141-162Crossref Google Scholar, 35Speth V. Otto V. Schäfer E. Planta (Heidelb.). 1987; 171: 332-338Crossref PubMed Scopus (38) Google Scholar). Because of the fact that PHYBA* is stable in yeast (Fig.4 D), a specific destruction pathway like that in plants does not exist in yeast.Spectroscopic examinations of dark reversion in vivorevealed that 20% of PHYA* decayed (Fig. 4 A, dashed line), whereas Ptot for all other constructs was stable (Fig. 4, B–F, dashed lines). There was no dark reversion of PHYA*. Kunkel et al. (15Kunkel T. Speth V. Büche C. Schäfer E. J. Biol. Chem. 1995; 270: 20193-20200Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar) did not observe this instability of PHYA* because their measurements were performed at 4 °C, whereas we performed our analysis at 20 °C. This difference in temperature is a sufficient explanation of this result. The amount and rate of phytochrome dark reversion in yeast were independent of temperature (compare Fig. 4, A and B, with Ref.15Kunkel T. Speth V. Büche C. Schäfer E. J. Biol. Chem. 1995; 270: 20193-20200Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar; data not shown for Fig. 4, C–F). In contrast to this, phyA dark reversion is strongly temperature-dependentin planta (36Schäfer E. Schmidt W. Planta (Heidelb.). 1974; 116: 257-266Crossref PubMed Scopus (41) Google Scholar), indicating that this process is not a simple thermal instability of Pfr, but a catalyzed process in planta.In most etiolated dicotyledonous seedlings, a small fraction of Pfr (∼20%) undergoes dark reversion into Pr as detected by in vivo spectroscopy (36Schäfer E. Schmidt W. Planta (Heidelb.). 1974; 116: 257-266Crossref PubMed Scopus (41) Google Scholar, 37Marmé D. Marchal B. Schäfer E. Planta (Heidelb.). 1971; 100: 331-336Crossref PubMed Scopus (31) Google Scholar), whereas in etiolated grasses, no dark reversion of Pfr can be detected in vivo (38Schäfer E. Lassig T.-U. Schopfer P. Photochem. Photobiol. 1975; 22: 193-202Crossref PubMed Scopus (77) Google Scholar). phyA was probably measured in these studies because etiolated plant material was used (see the Introduction). Recently, Kunkel et al. (15Kunkel T. Speth V. Büche C. Schäfer E. J. Biol. Chem. 1995; 270: 20193-20200Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar) demonstrated dark reversion for reconstituted tobacco PHYB* in yeast.In vitro dark reversion of extracts of potato PHYA* and PHYB* expressed in yeast was shown by Ruddat et al. (39Ruddat A. Schmidt P. Gatz C. Braslavsky S.E. Gärtner W. Schaffner K. Biochemistry. 1997; 36: 103-111Crossref PubMed Scopus (50) Google Scholar).In vitro dark reversion of oat phyA is detectable only for the so-called large phytochrome, which lacks the 4–6-kDa N-terminal fragment (40Pratt L.H. Photochem. Photobiol. 1978; 27: 81-105Crossref Scopus (107) Google Scholar).Rice PHYA* did not show any dark reversion. The dark reversion rates of PHYA constructs with an N-terminal replacement (PHYBA*, PHYTBRA*, and PHYPARA*) were three times faster and involved a greater proportion of Pfr molecules than the dark reversion rate of PHYB* (Fig. 4,D–F). Consequently, these molecules are less stable in their physiological Pfr form, which is consistent with the previous observation that the N terminus is very important for the stability of the Pfr form of phytochrome (40Pratt L.H. Photochem. Photobiol. 1978; 27: 81-105Crossref Scopus (107) Google Scholar). We conclude that, among other factors, the very N-terminal part of the monocotyledonous phyA is necessary, but not sufficient, for the prevention of dark reversion. The prevention of dark reversion stabilizes the Pfr form of phytochrome and plays an important role in the regulation of signal transduction. This mechanism in signal transduction of phyA in monocotyledonous plants would only be attenuated by destruction of the phyA molecule.The chimeric adducts PHYAB* and PHYBA* both exhibited dark reversion, which indicates that a special domain of phyB does not mediate dark reversion. PHYAB* showed dark reversion comparable to PHYB* and weaker than the three N terminus-swapped phytochromes (PHYBA*, PHYTBRA*, and PHYPARA*). Thus, the C terminus of phyA has a minor, but necessary, role in the prevention of dark reversion, whereas the N terminus has a major, but not sufficient, role in the prevention of dark reversion. Therefore, the very N terminus (9 kDa) and the C-terminal half of phyA are both needed to hinder dark reversion. For most phyA and phyB phytochromes, dark reversion seems to be a crucial down-regulator of signal transduction because the half-life of ∼20 min is much faster than the competing destruction. The attenuation of the signal transduction of phyA in monocotyledonous and some dicotyledonous plants (e.g. Amaranthus) is obviously due to the destruction with a half-life of ∼30 min, rather than due to dark reversion (41Kendrick R.E. Hillman W.S. Am. J. Bot. 1971; 58: 424-428Google Scholar). Destruction probably replaces dark reversion as a switching-off mechanism of signal transduction. However, in all other cases, dark reversion seems to be an intrinsic molecular property of phytochromes per se and was even measured inSynechocystis phytochrome (42Yeh K.-C. Wu S.-H. Murphy J.T. Lagarias J.C. Science. 1997; 277: 1505-1508Crossref PubMed Scopus (443) Google Scholar).We advance the hypothesis that there exists a mechanism in phytochrome that prevents dark reversion, rather than the existence of a mechanism that promotes dark reversion. Consistent with this hypothesis is the observation that changes in the molecular structure of phytochrome result in a partial instability of Pfr, which leads to enhanced and accelerated dark reversion.If this interpretation holds, it would also have to be taken into account for interpretations of phyA and phyB signaling mutants. The missense mutations in both phyA and phyB in Arabidopsis are all clustered in a region in the C-terminal part of phytochrome (43Xu Y. Parks B.M. Short T.W. Quail P.H. Plant Cell. 1995; 7: 1433-1443Crossref PubMed Scopus (69) Google Scholar,44Wagner D. Quail P.H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8596-8600Crossref PubMed Scopus (67) Google Scholar); they lead to a loss of function without, however, changing the expression level of the photoreceptors. Therefore, this domain has been described as a signaling box (Fig. 1 A). However, alterations of the extent and the velocity of dark reversion may be caused by mutations in this region, thus leading to a non-signaling mutation.Recently, Elich and Chory (45Elich T.D. Chory J. Plant Cell. 1997; 9: 2271-2280Crossref PubMed Scopus (68) Google Scholar) analyzed one of these mutations (phyB-101, a Glu-to-Lys change at amino acid 812) and, after expression of this mutated phytochrome in yeast and assembly with the chromophorein vitro, showed a 3-fold faster dark reversion with three to four times more dark-reverting Pfr obtained compared with wild-type phyB. Thus, the interpretation of these “signaling” mutants necessitates a careful in vivo spectroscopic and physiological study.The data presented here and by Elich and Chory (45Elich T.D. Chory J. Plant Cell. 1997; 9: 2271-2280Crossref PubMed Scopus (68) Google Scholar) show that the amount and velocity of dark reversion may be altered by mutations or domain swapping. Based on the observations that the dark reversion of the Pfr/Pr heterodimer is faster than destruction and that only a small percentage of the Pfr molecules undergo dark reversion, Brockmannet al. (46Brockmann J. Rieble S. Kazarinova-Fukshansky N. Seyfried M. Schäfer E. Plant Cell Environ. 1987; 10: 105-111Google Scholar) hypothesized that dark reversion is only possible starting from the Pfr/Pr heterodimer state. After a light pulse shifts a phytochrome system to a 50% photoequilibrium, only 50% of the molecules can convert into the heterodimer state. This is the maximum of heterodimers in the phytochrome system. As only half of these molecules undergo dark reversion, there are only 25% of the Pfr molecules left to show such a reversion. After a saturating light pulse, the system arrives at a photoequilibrium of 80–86%. Hence, 24–32% of heterodimers exist, so 12–18% of the Pfr molecules should show dark reversion. However, as demonstrated in Fig. 4, PHYB* and PHYAB* exhibited a dark reversion of 30%, and PHYBA*, PHYTBRA*, and PHYPARA* showed a dark reversion of 40–50%. Thus, the heterodimer hypothesis is not true in this context, and dark reversion must consequently also be possible from the Pfr/Pfr homodimer state.Our data show that dark reversion is a crucial mechanism to attenuate signal transduction and that this differs for phyA and phyB. Therefore, the dynamics of the phytochrome molecule, especially dark reversion, must be considered in the analysis of the first steps in the signal transduction chain of phytochrome-dependent photomorphogenesis. Plants are sessile organisms that have evolved a wide spectrum of mechanisms to adapt to changes in their natural environment. Light is used not only as the energy source for photosynthesis, but also as a major environmental signal. To monitor changes in light quality and quantity and to regulate temporal and spatial patterns in photomorphogenesis, plants have evolved at least three different photoreceptor systems: UVB photoreceptors, blue UVA photoreceptors, and phytochromes (1$$Google Scholar). Of these photoreceptors, the phytochromes are the best characterized. Five genes encoding phytochrome apoproteins have been identified in Arabidopsis (2Sharrock R.A. Quail P.H. Genes Dev. 1989; 3: 1745-1757Crossref PubMed Scopus (693) Google Scholar, 3Clack T. Mathews S. Sharrock R.A. Plant Mol. Biol. 1994; 25: 413-427Crossref PubMed Scopus (555) Google Scholar). Mutants of phyA,1 phyB, and phytochrome D in Arabidopsis have different effects on photomorphogenesis (4Nagatani A. Reed J.W. Chory J. Plant Physiol. (Bethesda). 1993; 102: 269-277Crossref PubMed Scopus (392) Google Scholar, 5Parks B.M. Quail P.H. Plant Cell. 1993; 5: 39-48Crossref PubMed Scopus (293) Google Scholar, 6Whitelam G.C. Johnson E. Peng J. Carol P. Anderson M.L. Cowl J.S. Harberd N.P. Plant Cell. 1993; 5: 757-768Crossref PubMed Scopus (483) Google Scholar, 7Reed J.W. Nagpal P. Poole D.S. Furuya M. Chory J. Plant Cell. 1993; 5: 147-157Crossref PubMed Scopus (761) Google Scholar, 8Aukerman M.J. Hirschfeld M. Wester L. Weaver M. Clack T. Amasino R.M. Sharrock R.A. Plant Cell. 1997; 9: 1317-1326Crossref PubMed Scopus (245) Google Scholar). The dominant phytochrome of etiolated seedlings, the light-labile phyA, controls the very low fluence and the high irradiance responses, whereas the dominant phytochrome of green seedlings and mature plants, the light-stable phyB, controls responses governed by red/far-red reversibility or continuous red light (9Furuya M. Schäfer E. Trends Plant Sci. 1996; 1: 301-307Abstract Full Text PDF Google Scholar). Phytochromes are dimers composed of 120-kDa monomers, each of which contains two domains linked by a highly conserved hinge region. The N-terminal domain bears the chromophore, and the C-terminal domain is involved in dimerization (Fig.1 A) (10Furuya M. Song P.-S. Kendrick R.E. Kronenberg G.H.M. Photomorphogenesis in Plants. 2nd Ed. Kluwer Academic Publishers, Dordrecht, The Netherlands1994: 105-140Crossref Google Scholar). Phytochromes are synthesized in the dark in their physiologically inactive red light-absorbing form (Pr). Upon photon absorption, Pr is converted into the physiologically active far-red light-absorbing form of phytochrome (Pfr). During this reversible process, the absorption maximum of the molecule shifts from 660 nm (red light) to 730 nm (far-red light). Under saturating red light, 80% or more of the phytochrome is in the Pfr form, whereas only 3% Pfr remains following exposure to saturating far-red light (11Mancinelli A.L. Kendrick R.E. Kronenberg G.H.M. Photomorphogenesis in Plants. 2nd Ed. Kluwer Academic Publishers, Dordrecht, The Netherlands1994: 211-269Crossref Google Scholar). In vivo spectroscopy has demonstrated that phyA undergoes dark reversion in most dicotyledonous seedlings, but not in monocotyledonous seedlings (11Mancinelli A.L. Kendrick R.E. Kronenberg G.H.M. Photomorphogenesis in Plants. 2nd Ed. Kluwer Academic Publishers, Dordrecht, The Netherlands1994: 211-269Crossref Google Scholar). Hence, some of the Pfr in dicotyledonous seedlings is converted to the physiologically inactive Pr form in total darkness. Rapid de novo synthesis of phyA in darkness, rapid degradation of the Pfr form (half-life of 30–60 min) in light, rapid dark reversion of 20% of the Pfr molecules to Pr (half-life of 20 min) in most dicotyledonous seedlings, and light-dependent rapid aggregation (half-life of 2 s) of the protein into SAPs have been demonstrated by in vivo spectroscopy and immunological methods (1$$Google Scholar). Ubiquitin may be involved in this degradation process, which is associated with the formation of SAPs (12Vierstra R.D. Kendrick R.E. Kronenberg G.H.M. Photomorphogenesis in Plants. 2nd Ed. Kluwer Academic Publishers, Dordrecht, The Netherlands1994: 141-162Crossref Google Scholar). In vivocharacterization of phytochromes has been possible only for phyA because the less abundant phyB cannot be spectroscopically distinguished from the abundant phyA, and high levels of chlorophyll prevent measurement of phyB even if phyA is destroyed in the light. Even in mutants lacking phyA, phyB is difficult to detect because of its low concentration. In vivo spectroscopic studies of phytochrome have been performed in light-grown bleached seedlings of certain species; the results of these studies may reflect properties of phyB (13Jabben M. Holmes M.G. Shropshire W. Mohr H. Photomorphogenesis. Springer-Verlag, Berlin1983: 704-722Crossref Google Scholar, 14Heim B. Jabben M. Schäfer E. Photochem. Photobiol. 1981; 34: 89-93Crossref Scopus (42) Google Scholar). These studies indicate a very slow destruction of the light-stable phytochrome (half-life of >8 h) and a weak partial dark reversion. A model system for spectroscopic and immunological characterization of phyB has been developed in yeast (Saccharomyces cerevisiae) (15Kunkel T. Speth V. Büche C. Schäfer E. J
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