Chair: Roger Hangarter, Indiana University
Although much is known about the biochemistry and molecular biology of the phytochrome family of photoreceptors, the mechanism of action of phytochromes has remained elusive. Several presentations described interesting approaches that suggest that an understanding of phytochrome action may be within reach. It has generally been assumed that the phytochromes are cytoplasmic proteins but in two posters, Akira Nagatani and Kojo Sakamoto presented compelling evidence from immunochemical and GUS-PHYB fusion protein experiments suggesting that PHYB is localized in the nucleus. These findings raise the intriguing possibility that phytochromes may express their regulatory activities in the nucleus. Several laboratories have been using transgenic approaches to map the functional regions of the phytochrome protein. Experiments presented by Dorris Wagner, R.M. Kuhn and P.H. Quail using overexpressed hybrid phytochrome A and B constructs in Arabidopsis indicate that the N-terminal domains of phytochromes A and B determine the photosensory specificity of the photoreceptors. In addition, mutational analyses of overexpressed phytochromes indicate that a small region of the C-terminal domain is critical for regulatory activity. Results from tobacco plants (presented by Emily Jordan et al) overexpressing a number of small deletion mutants of oat phytochrome A indicate that the N terminus contains two functional domains: one confers conformational stability and the other is involved in attenuating the response of active phytochrome. The Yeast two-hybrid system was used to identify gene products that interact with a C-terminal fragment of phytochrome B (Ted Elich and Joanne Chory). A single clone that specifically interacted with the PHYB bait was isolated and has been designated PIP for Phytochrome-Interacting-Protein. A full-length PIP cDNA was isolated and plants overexpressing an antisense PIP cDNA exhibited several phenotypes that are opposite of those in phyB mutants. Others have been isolating suppressor mutants of hy2 and hy3. The availability of a potential reaction partner of PHYB (PIP) and several interesting suppressor mutants opens new doors to studies of phytochrome signal transduction.
Blue light perception systems are also critical regulators of plant development. Margaret Ahmad and Tony Cashmore presented recent finding on the molecular and biochemical characterization of the recently cloned blue light photoreceptor, HY4. Molecular studies indicate that the HY4 gene evolved from a DNA photolyase gene: HY4 has strong structural similarities and shares the same blue-light absorbing chromophores. Photobiological analysis of a number of allelic mutants show that some mutations affect the action spectrum and absorption spectrum in similar ways, thus, showing that HY4 is a functional blue light photoreceptor. Because HY4 can carry out blue-light-dependent redox reactions, it's mode of action is expected to be unique from that of the phytochromes. Myeon Cho and Edgar Spalding found that an early response to blue light is a transient depolarization of the plasma membrane in Arabidopsis hypocotyl cells. The depolarization begins within a few seconds and precedes the rapid inhibition of elongation caused by blue light. Their results indicate that the depolarization is caused by an anion channel that could be blocked by the channel blocker, NPPB. Moreover, the depolarization response is greatly attenuated in the hy4 mutant. Because the depolarization response is extremely rapid and dependent on HY4, the anion channel may play a crucial role in blue-light-regulation of cell elongation in Arabidopsis hypocotyls. In addition to the results on HY4, Dr. Ahmad presented data suggesting that blue light responses require the action of phytochrome since hy1phyA and phyAphyB double mutants appeared to lack blue-light-dependent inhibition of hypocotyl elongation. Although coaction between phytochrome- and blue-light-dependent responses has been observed in other species, earlier work had suggested that the phytochrome and blue-light sensory systems work largely independently.
In addition to studies on the photoreceptors and early events in the light-dependent-sensory network, downstream components are also being investigated. Mutants that exhibit photomorphogenic-like development in the absence of light (e.g., det and cop mutants) are providing interesting insights into the mechanisms controlling these processes. Many of the loci encode regulatory proteins which act as suppressors of photomorphogenesis in the absence of light signals. Ning Wei and others from Xing Wang Deng's lab presented recent results that indicate that the COP9 gene product is a component of a large protein complex that includes at least the COP8 and COP11 gene products. Immunochemical and GUS-COP9 localization indicate that the complex is located in the nucleus. It appears that COP9, together with COP8 and COP11, constitute part of a novel regulatory complex mediating light-dependent control of plant development. Evidence was presented by von Armin et al suggesting that the COP1 protein moved between the nucleus and cytoplasm in a light-dependent manner but it is not known if COP1 interacts with the nuclear-localized COP9 complex. The DET1 gene also encodes a novel nuclear encoded protein that acts to suppress photomorphogenesis in darkness (D. Poole, R. Cook and J. Chory). DET1 expression is not regulated by light and it is suggested that its activity is regulated post-transcriptionally. All of these gene products appear to be essential downstream components that are involved in coordinating developmental responses to the phytochrome and blue-light sensory systems and, thus, represent key nodes in the environmental sensory network.
In an attempt to get at genes that may be involved in other parts of the environmental sensory network, Nobuyoshi Mochizuki and Joanne Chory have been isolating and characterizing mutants that express CAB3-promoter reporter genes independently of chloroplast development (gun mutants). Five complementation groups have been identified and some of them appear function as part of an intracellular regulatory pathway that is involved in coordinating the expression nuclear and plastid-encoded genes during plastid development. Thus, the GUN genes seem to represent a specific branch of a complex interorganellar regulatory network that is an important part of the overall network regulating light-dependent development. Another important component of the environmental sensory network is its coordination with circadian regulation. Andrew Millar et al showed that circadian period and phase shifting are controlled by phytochrome and blue-light receptor systems. Moreover, the amplitude of light-dependent CAB2 transcription is dependent on the phase of the circadian clock. Hai Hong Zhong et al found that the Arabidopsis CAT3 gene is negatively regulated by phytochrome and that the phytochrome response is mediated indirectly through the circadian clock.
Although this summary is not inclusive (many other interesting findings related to light dependent developmental responses were presented at the conference), it is clear that plants use a complex signal transduction network to fine-tune their responses to inputs received from a multitude of photoreceptor systems. It is also clear that Arabidopsis is playing a central role in elucidating the complex machinery of the light-dependent sensory-response network.