Targeted knockout in Physcomitrella reveals direct actions of phytochrome in the cytoplasm

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102

Phytochromes are red/far-red photochromic photoreceptors central to regulating plant development. Although they are known to enter the nucleus upon light activation and, once there, regulate transcription, this is not the complete picture. Various phytochrome effects are manifested much too rapidly to derive from changes in gene expression, whereas others seem to occur without phytochrome entering the nucleus. Phytochromes also guide directional responses to light, excluding a genetic signaling route and implying instead plasma membrane association and a direct cytoplasmic signal. However, to date, no such association has been demonstrated. Here we report that a phytochrome subpopulation indeed associates physically with another photoreceptor, phototropin, at the plasma membrane. Yeast two-hybrid methods using functional photoreceptor molecules showed that the phytochrome steering growth direction in Physcomitrella protonemata binds several phototropins specifically in the photoactivated Pfr state. Split-YFP studies in planta showed that the interaction occurs exclusively at the plasma membrane. Coimmunoprecipitation experiments provided independent confirmation of in vivo phy-phot binding. Consistent with this interaction being associated with a cellular signal, we found that phytochrome-mediated tropic responses are impaired in Physcomitrella phot − mutants. Split-YFP revealed a similar interaction between Arabidopsis phytochrome A and phototropin 1 at the plasma membrane. These associations additionally provide a functional explanation for the evolution of neochrome photoreceptors. Our results imply that the elusive phytochrome cytoplasmic signal arises through binding and coaction with phototropin at the plasma membrane.

“…The Pp.phy4 knockout line was as described (18). Double and triple phototropin knockout lines were kindly donated by Masahiro Kasahara (22).…”

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

A phytochrome–phototropin light signaling complex at the plasma membrane

Jaedicke

Lichtenthäler

Meyberg

et al. 2012

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102

“…Because such a gene sequence is also absent from the extensive moss EST and nearly complete Physcomitrella genome databases, it is very unlikely that an Adiantum PHY3 homolog is present in mosses. In any case, targeted knockout of PHY and PHOT genes in Physcomitrella has shown that red-light-induced chloroplast movement in Physcomitrella is mediated by canonical phys with phots acting downstream (6,7).…”

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confidence: 99%

A chimeric photoreceptor gene, NEOCHROME, has arisen twice during plant evolution

Suetsugu

Mittmann

Wagner

et al. 2005

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154

149

Although most plant species from algae to flowering plants use blue light for inducing phototropism and chloroplast movement, many ferns, some mosses, and green algae use red as well as blue light for the regulation of these responses, resulting in better sensitivity at low light levels. During their evolution, ferns have created a chimeric photoreceptor (phy3 in Adiantum) between phytochrome (phy) and phototropin (phot) enabling them to use red light effectively. We have identified two genes resembling Adiantum PHY3, NEOCHROME1 and NEOCHROME2 (MsNEO1 and MsNEO2), in the green alga Mougeotia scalaris, a plant famous for its light-regulated chloroplast movement. Like Adiantum PHY3, both MsNEO gene products show phytochrome-typical bilin binding and red͞far-red reversibility, the difference spectra matching the known action spectra of light-induced chloroplast movement in Mougeotia. Furthermore, both genes rescue red-light-induced chloroplast movement in Adiantum phy3 mutants, indicating functional equivalence. However, the fern and algal genes seem to have arisen independently in evolution, thus providing an intriguing example of convergent evolution. convergent evolution ͉ Mougeotia

“…Data from gene-targeted phytochrome knockout mutants in Physcomitrella patens and Ceratodon purpureus further supports the role of these red-light sensors in phototropism (14,15). Relocation of chloroplasts in nonvascular plants is also controlled by phytochromes, neochromes and phototropins (14,16,17). For these photoresponses, cytosolic photosensors are required to sense the light direction.…”

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confidence: 89%

“…The protonema grows toward unilateral red light (positive phototropism) at high fluence rates (5-10 μmol m −2 s −1 ), away from it (negative phototropism) at low fluence rates (below 1 μmol m −2 s −1 ), and spreads randomly at intermediate fluence rates (25). Phytochromes have been implicated as the main photoreceptor sensing light direction in control of protonemal cell growth in Physcomitrella (14). To test the hypothesis that both FDBRs contribute to the red-light-phototropic response, we analyzed the red-light triggered phototropic response in single and double mutants.…”

mentioning

confidence: 99%

Distinct phytochrome actions in nonvascular plants revealed by targeted inactivation of phytobilin biosynthesis

Chen

2012

The red/far-red light photoreceptor phytochrome mediates photomorphological responses in plants. For light sensing and signaling, phytochromes need to associate with open-chain tetrapyrrole molecules as the chromophore. Biosynthesis of tetrapyrrole chromophores requires members of ferredoxin-dependent bilin reductases (FDBRs). It was shown that LONG HYPOCOTYL 2 (HY2) is the only FDBR in flowering plants producing the phytochromobilin (PΦB) for phytochromes. However, in the moss Physcomitrella patens, we found a second FDBR that catalyzes the formation of phycourobilin (PUB), a tetrapyrrole pigment usually found as the protein-bound form in cyanobacteria and red algae. Thus, we named the enzyme PUB synthase (PUBS). Severe photomorphogenic phenotypes, including the defect of phytochrome-mediated phototropism, were observed in Physcomitrella patens when both HY2 and PUBS were disrupted by gene targeting. This indicates HY2 and PUBS function redundantly in phytochrome-mediated responses of nonvascular plants. Our studies also show that functional PUBS orthologs are found in selected lycopod and chlorophyte genomes. Using mRNA sequencing for transcriptome profiling, we demonstrate that expression of the majority of red-light-responsive genes are misregulated in the pubs hy2 double mutant. These studies showed that moss phytochromes rapidly repress expression of genes involved in cell wall organization, transcription, hormone responses, and protein phosphorylation but activate genes involved in photosynthesis and stress signaling during deetiolation. We propose that, in nonvascular plants, HY2 and PUBS produce structurally different but functionally similar chromophore precursors for phytochromes. Holophytochromes regulate biological processes through light signaling to efficiently reprogram gene expression for vegetative growth in the light.