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Light Signaling Revisited

neckardt{at}aspb.org

Plants respond to light in a myriad of ways. Between light absorption by photoreceptors and physiological and developmental responses lies a web of interacting factors and interacting pathways, either directly involved in or otherwise impinging upon light signal transduction. The red and far-red absorbing phytochromes are the major photoreceptors controlling photomorphogenesis and the control of flowering time. More than 50 years ago, Harry Borthwick and colleagues performed the now famous lettuce seed germination experiments, demonstrating that a reversible photoreaction controlled this process (Borthwick et al., 1952). Borthwick and colleagues went on to purify the pigment responsible for this effect, which they named phytochrome, and also described its role in seed germination, seedling development, and flowering (Hendricks and Borthwick, 1966). Since these early experiments, our understanding of phytochrome action has increased greatly, particularly in the model plant Arabidopsis.

Arabidopsis has five genes encoding phytochromes, PHYA to PHYE (Clack et al., 1994). Functional phytochromes are dimers of two 125-kD polypeptides, each of which carries a photosensory chromophore at the N terminus and a dimerization domain and a kinase-related domain at the C terminus. All phytochromes undergo reversible photoconversion between a physiologically inactive red light–absorbing form Pr and a physiologically active far red–absorbing form Pfr. The distinction between phyA as the far-red and very-low-fluence response receptor and phyB (and the other phytochromes) as a red and low-fluence response receptor depends in part upon differences in photostability and in light-induced translocation of these molecules into the nucleus. phyA is extremely photolabile and undergoes rapid proteolytic degradation after conversion to the active Pfr form, whereas the other phytochromes are significantly more highly photostabile (Sharrock and Clack, 2002). Although phyB to phyE are not rapidly degraded upon photoconversion, they (at least phyB and to a greater extent phyE) undergo rapid dark reversion to the Pr form (Eichenberg et al., 2000). In darkness, phytochromes are located mainly in the cytosol in the inactive Pr form. Upon illumination with the appropriate wavelength, phytochromes are translocated into the nucleus, where they interact with many nuclear-localized transcription factors. phyB (and possibly also phyC to phyE) is translocated into the nucleus only by red light (Kircher et al., 1999). By contrast, phyA is translocated into the nucleus by far-red as well as red light.

Phytochromes also undergo autophosphorylation and are capable of phosphorylating other proteins. Phosphorylation has long been postulated to be a part of the signal transducing mechanism of phytochromes, but a direct link between kinase activity and phytochrome signaling has not been established (Quail, 2002; Wang and Deng, 2003). Matsushita et al. (2003) have recently shown that the N terminus alone of phyB positively transduces the light signal, whereas the C terminus (including the kinase-like domain) functions to attenuate signaling activity.

Recent work in several laboratories has demonstrated that one of the principal factors with which phytochrome interacts inside the nucleus is PHYTOCHROME INTERACTING FACTOR3 (PIF3), a basic helix-loop-helix protein that also binds in vitro specifically to the G-box regulatory element found in the promoters of several phytochrome-responsive genes. Its precise regulatory role appears to be complex and likely involves interaction with a number of other factors. Different reports have classified it as principally a positive regulator (Halliday et al., 1999; Ni et al., 1998) or a negative regulator (Kim et al., 2003) of phyB-mediated responses as well as a putative master regulator of phyA-mediated responses (Tepperman et al., 2001). In this issue of The Plant Cell, Bauer et al. (pages 1433–1445) provide new insights into the nature of PIF3 function in phytochrome-mediated signal transduction through an analysis of the subcellular distribution of PIF3-fluorescent protein fusions in transgenic plants and an assessment of PIF3 accumulation and degradation in wild-type and various phytochrome mutant seedlings under different light treatments.

Bauer et al. produced several transgenic plant lines that either expressed PIF3 fused to the red-shifted green fluorescent protein or coexpressed PIF3 fused to cyano fluorescent protein and the various phytochromes fused to yellow fluorescent protein. The distribution of PIF3 and the phytochromes between the nucleus and cytoplasm was monitored under various light treatments. The authors also assessed accumulation and degradation of PIF3, using standard protein blots probed with anti-PIF3 antibody, in extracts from wild-type seedlings, various phytochrome mutants, constitutive photomorphogenic1 (cop1) mutants, and the photo current1 (poc1) mutant, which contains a T-DNA insertion in the PIF3 promoter region. The cop1 mutant was used to determine if PIF3 function is regulated by COP1, which has been identified as an E3 ubiquitin ligase that acts as a major repressor of photomorphogenesis by targeting transcriptional activators of photomorphogenic response genes for degradation (Saijo et al., 2003; Seo et al., 2003). Considered along with other recent reports, the results lead to a significant reassessment of the role of PIF3 in the early stages of phytochrome-mediated signaling.

COP1 functions as a repressor of photomorphogenic responses in the nucleus in the dark but is repartitioned out of the nucleus in response to light, thereby alleviating repression of photomorphogenesis (von Arnim et al., 1997). Bauer et al. show that PIF3 is localized in the nucleus in the dark (Figure 1), and it undergoes rapid, light-induced degradation that is mediated by phyA, phyB, and phyD. Dark accumulation of PIF3 in the nucleus required the presence of COP1, but light-induced degradation of PIF3 was found to be COP1 independent. These results are consistent with the observation that functional COP1 is localized in the nucleus only in the dark. Importantly, they also suggest that the action of PIF3 in response to phytochrome activation is rapid and transient and that PIF3 interacts with several phytochrome species. Bauer et al. concluded that PIF3 accumulation in the dark is effected by COP1-mediated degradation of a PIF3 repressor, which may be a repressor of PIF3 gene expression or a factor controlling PIF3 degradation. The latter scenario, that COP1 targets a factor controlling PIF3 degradation, is attractive because it is consistent with the observation that light induces exclusion of COP1 from the nucleus. Under this scenario, the absence of COP1 in the nucleus in the light would lead to COP1-independent degradation of PIF3, whereas COP1 nuclear localization in the dark would lead to COP1-dependent accumulation of PIF3. The answer lies with identifying the COP1 target responsible for permitting the dark accumulation of PIF3.

View larger version (172K): [in this window] [in a new window]   Figure 1. Colocalization of phyB:YFP (Blue-Green) with PIF3:CFP (Red) in the Nucleus of Hypocotyl Cells in Transgenic Arabidopsis (Overlapping of the Two Colors Appears Yellow).

Further insight into phyB signaling was recently provided by Matsushita et al. (2003), who showed that the phyB N terminus contains the signal-transducing domain, whereas the C terminus acts as a regulatory domain that controls translocation of phyB into the nucleus and also functions to attenuate the activity of the N terminus. These results show that phytochrome signaling is not mediated directly by kinase activity because the kinase-like domain of phytochromes is located at the C terminus. Kinase activity instead appears to be related to the regulatory function of the C terminus. PIF3 binds specifically to the Pfr form of phyB, and independent of whether the presence of both N and C termini (Ni et al., 1999) or only the N terminus (Shimizu-Sato et al., 2002) of phyB are required for efficient binding, it may be hypothesized that the function of PIF3 is to modulate the inhibitory effect of the C terminus on phyB signal transduction.

Large-scale DNA microarray analyses of gene expression by Tepperman et al. (2001) provided indirect support for the notion that PIF3 is one of the master regulators of phytochrome-mediated signal transduction. This report showed that a large percentage of genes whose expression is induced early in response to the phyA-mediated light signal are transcriptional regulators that influence the expression of numerous downstream effector genes related to multiple cellular and developmental processes. The promoters of some of these genes, such as CIRCADIAN CLOCK-ASSOCATED PROTEIN 1 and LATE ELONGATED HYPOCOTYL, are known to contain the G-box element that binds PIF3, leading Tepperman et al. (2001) to postulate that PIF3 and other as yet unidentified phytochrome interacting factors are signaling transcriptional regulators that control the phytochrome-mediated regulation of a master set of transcription factor genes that in turn regulate downstream processes. This hypothesis can now be tested directly by further analysis of gene expression in the PIF3 loss-of-function mutants described by Kim et al. (2003) and Bauer et al. (in this issue).

Hendricks and Borthwick (1966) concluded from the limited evidence available at the time that phytochrome "acts on cell permeability as a first or early step in its control of plant growth and development." In the intervening years, this vague notion of phytochrome effect on "cell permeability" has been replaced by detailed knowledge of the structure of phytochrome, its intracellular movements in response to light, interactions with other proteins, and many of the steps involved in signal transduction along the way to activation or repression of light-responsive genes and their roles in plant physiology and development. The work of Bauer et al. adds to our understanding of the intricacies of this fundamental process of plant life.

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Related articles in Plant Cell:

Constitutive Photomorphogenesis 1 and Multiple Photoreceptors Control Degradation of Phytochrome Interacting Factor 3, a Transcription Factor Required for Light Signaling in Arabidopsis

Diana Bauer, András Viczián, Stefan Kircher, Tabea Nobis, Roland Nitschke, Tim Kunkel, Kishore C.S. Panigrahi, Éva Ádám, Erzsébet Fejes, Eberhard Schäfer, and Ferenc Nagy
Plant Cell 2004 16: 1433-1445. [Abstract] [Full Text]  

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