The Red Side of Photomorphogenesis

The importance of light to normal plant growth and development cannot be overstated. As sessile photoautotrophs, plants depend on efficient light capture to compete and reproduce successfully within a relatively restricted geographical realm. For this purpose, these organisms have evolved very sophisticated sensory networks for monitoring the status of several important features of their illuminated surroundings including light intensity, duration, quality, and direction (Kendrick and Kronenberg, 1994). Response to these light signals in the form of altered plant growth and development is termed photomorphogenesis, a process that is distinct from that of photosynthesis, where far greater quantities of light serve as a source of energy for the fixation of carbon. The entire photomorphogenic process rests upon a set of specialized photochromic sensory receptors falling into at least three distinct known classes: phytochromes, cryptochromes, and phototropins. Structural properties of these photoreceptors essentially restrict spectrally important regions for photomorphogenesis to the UV-A and -B, blue, and red portions of the electromagnetic spectrum. Historically, most progress toward understanding the molecular and cellular processes that underlie photomorphogenesis has come from studies of redlight sensing, which spans a relatively broad spectral region (approximately 600–750 nm) to include both red and far-red (approximately 700–750 nm) light. In terms of photomorphogenesis, this region is unique because phytochrome is the only known photoreceptor that absorbs light here exclusively for photosensory purposes. And as a consequence, this characteristic of red-light sensing provides photobiologists with a unique capacity to probe the mechanics of phytochrome-regulated development in isolation from other photosensory systems. However, as an Update on red-light sensing, the intent here is not to provide a comprehensive review of phytochrome because several excellent and quite recent articles are already available to serve this purpose beautifully (Neff et al., 2000; Smith, 2000; Fankhauser, 2001b; Nagy and Schäfer, 2002; Quail, 2002; Wang and Deng, 2003). Rather, this Update is intended to provide new students and educators with a primer to photomorphogenic research in plants. Specifically, it will use both historical and more recent contexts of phytochrome research to describe how our understanding of red-light sensing has evolved over time as well as the challenges that must be successfully addressed for continued progress in the future. It has been just over 50 years since the discovery of phytochrome by Borthwick et al. (1952), the result of their stunning and now classic finding that lightregulated lettuce (Lactuca sativa) seed germination operates through a red/far-red photoreversible process. This unique photosensing feature greatly facilitated all subsequent physical characterization of phytochrome and the photophysiology that it controls. And over this time, phytochrome research has continued to advance through the exploitation of technological achievements in several areas including spectroscopy, biochemical purification, immunochemistry, and molecular genetics (Kendrick and Kronenberg, 1994). What general picture of phytochrome has emerged from these past decades of research, and where is the field currently poised to uncover it further? In plants, where the majority of physical characterization has been performed, phytochrome is a relatively large dimeric chromoprotein, with each monomeric unit being approximately 125 kD (Kendrick and Kronenberg, 1994). Its light-absorbing entity, the chromophore, is a linear tetrapyrrole (phytochromobilin) that is covalently attached autocatalytically via a thioether linkage to a Cys residue of the polypeptide located about one-third the length along the polypeptide sequence from the amino terminus. The photoreceptor can exist in either of two photointerconvertible conformations—a biologically inactive Pr form (λmax ∼ 670 nm) and a biologically active Pfr form (λmax ∼ 730 nm). Phytochrome is synthesized as Pr and is localized initially in the cytosol. Absorption of light by either form induces a conformational change to the other spectral form, thus enabling a flipping between inactive and active states. Analysis of mutant forms of phytochrome has demonstrated that the amino-terminal portion of the polypeptide is critical to the photosensory properties of the holoprotein, whereas regions within the carboxy-terminal two-thirds of the molecule are important to the regulatory function of the photoreceptor. Is there only one type of phytochrome in plant cells? No. Molecular cloning and sequence analysis have shown that phytochrome is encoded by a small multigene family in higher plants, likely evolving from ancient origins as indicated by the recent discovery of phytochrome in certain bacteria (Hughes et al., 1997; Yeh et al., 1997; Bhoo et al., 2001). In Arabidopsis, where the body of recent and current molecular research is proceeding, phytochrome is represented by five members labeled A through E (Clack et al., 1994). Sequence analysis of these plant phytochromes suggests that they arose through four primary duplication events during plant evolution, with the initial split producing the A and B major groups about the time of seed plant appearance (Mathews and Sharrock, 1997). Further separations occurring later in evolution led to the five established family members recognized presently. Most biophysical and functional characteristics for the phytochromes are known for phytochromes A and B (phyA and phyB, respectively; Quail et al., 1994). The relative amounts of these phytochrome types vary according to the ambient light conditions. In this regard, phyA was found to comprise the great majority of photoreceptor type in dark-grown (etiolated) tissue, whereas light-grown green tissue showed this balance shift toward phyB. This dramatic change in photoreceptor proportion results from differences in the expression and stability of the different phytochrome types. PhyA as Pfr is relatively light labile compared with its Pr conform and suppresses its own expression. In contrast, phyB is constitutively expressed and relatively stable as Pfr (Kendrick and Kronenberg, 1994). The molecular confirmation that plants contain distinct and differentially regulated phytochrome types strongly indicated that they might possess shared and/or unique regulatory functions during normal development. This possibility was specifically addressed and validated with the isolation and subsequent analysis of photomorphogenic mutants deficient in one or more phytochrome types (Nagatani et al., 1991, 1993; Somers et al., 1991; Parks and Quail, 1993; Whitelam et al., 1993). Numerous such studies, both older and recent, have now confirmed that a variety of functional relationships exist among the phytochromes, including shared, distinct, and even seemingly antagonistic signaling programs (Whitelam and Devlin, 1997; Devlin et al., 1998, 1999; Franklin et al., 2003b). One major outcome of these works found that phyA controls many processes associated with de-etiolation occurring during seedling emergence and dominates responsiveness to far-red-enriched conditions (termed the far-red high-irradiance response; Smith, 2000; Quail, 2002). PhyB also appears to regulate many of the same processes associated with de-etiolation, such as the inhibition of stem elongation, but it is more important to conditions rich in red light and for sustained developmental progress under longer term natural lighting conditions including adult adaptive responses such as shade avoidance (Smith, 2000; Quail, 2002). That red and far-red light appeared to affect the same processes in a similar manner but through different photoreceptor types, gave the impression that phyA and phyB act in opposition to each other under a single common light regime. But it now appears that this is actually an evolved adaptation to extend the regulatory capacity of the phytochromes over a broader spectral range (Smith et al., 1997). Of the other phytochrome types found in Arabidopsis, more recent mutant studies indicate that, in general, the roles for phyD and phyE appear to be partially redundant to those of phyB in seedling establishment and flowering and may represent an evolved adaptive means to adjust to varying changes in microenvironment (Franklin et al., 2003b). And recent reports describing phyC-deficient mutants have tested ideas concerning possible roles for this photoreceptor that were suggested by earlier studies of phyC-overexpressing transgenic plants and photoreceptor mutants deficient in all other phytochrome types (Qin et al., 1997; Franklin et al., 2003a, 2003b; Monte et al., 2003). A central and persistent goal of phytochrome research has been to identify and arrange the cellular and molecular steps underlying the photoreceptor's ability to regulate photomorphogenesis. Early focus was directed toward determining a primary mechanism for photoreceptor function. Yet a clear answer here is still not available. In general, two fundamental views for phytochrome action have been considered. One hypothesis argues that phytochromeregulated responses are the direct outcome of lightsignaled alterations in gene expression. The other view sees many light responses as a manifestation of biochemical/biophysical changes occurring in the cell through pre-existing signaling components. In fact, the reality is probably a complicated combination of physiological processes and alterations in gene expression. Numerous reports of phytochromeregulated changes in gene expression have supported the former hypothesis for many years (Kuno and Furuya, 2000). And recent evidence demonstrating that phytochrome moves from the cytoplasm into the nucleus upon phototransformation to Pfr clearly implicates a functional role for phytochrome in the regulation of nuclear gene expression (Sakamoto and Nagatani, 1996; Kircher et al., 1999; Yamaguchi et al., 1999; Nagy and Schäfer, 2002). In this regard, a very tantalizing collection of work demonstrating that phyA and phyB can directly associate with a transcriptional complex to alter expression of developmentally important genes indicates that the signaling cascade can be extremely short (Ni et al., 1999; Martinez-Garcia et al., 2000; Zhu et al., 2000). But these observations supporting such a rudimentary signaling cascade do not necessarily invalidate the possibility for other distinct functions of phytochrome in the processing of light information. One large class of proteins important to many aspects of photomorphogenesis is the COP/DET/FUS family (Schwechheimer and Deng, 2000). This group of nuclear-localized factors appears to affect photomorphogenesis through the selected proteolytic degradation of important downstream-signaling elements (Osterlund et al., 2000; Schwechheimer et al., 2001; Schwechheimer and Deng, 2001). Evidence indicates that the phytochromes (and possibly the cryptochromes) alter the activity of this group by controlling their nuclear abundance (Osterlund and Deng, 1998). How this occurs is still not known. There is now also strong evidence to support the longproposed idea that phytochromes can act as protein kinases to regulate development (Yeh and Lagarias, 1998; Fankhauser, 2001a). The identification of PKS1 as a kinase substrate and putative phytochromesignaling partner supports the hypothesis that phytochromes might control photomorphogenesis by regulating phosphorylation status (Fankhauser et al., 1999). That this factor is cytoplasmically localized leaves open the question of whether phytochrome can control photomorphogenesis through means not wholly centered on regulated nuclear gene expression. 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