Plant Gene Register PGR96-067

Hughes JE, Lamparter T and Mittmann F. (1996) CERPU;PHY0;2, a "Normal" Phytochrome in Ceratodon (Accession No. U56698) (PGR96-067). Plant Physiol. 112: 446

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CERPU;PHY0;2, a "Normal" Phytochrome in Ceratodon (Accession No. U56698 )

Hughes JE, Lamparter T and Mittmann F

Instituet fuer Pflanzenphysiolgie, Freie Universitaet, D-14195 Berlin, Germany.

Corresponding Author: JE Hughes, Fax: +49 30 838 4357, E-mail: hughes@zedat.fu-berlin.de

Footnote: The sequence previously termed PHYCER ( S51224 and S20160 ) and the new sequence (previously PHYCER2, X89725 )) have been renamed CERPU;PHY0;1 and 2, respectively, following a provisional consensus for naming non- angiosperm phytochromes according to CPGN rules (three fields: the SwissProt genus/species-abbreviation; PHY plus zero to indicate unknown phytochrome subclass; order-of-discovery number for that species). For brevity we use CP1 and 2 here.

Phytochrome, a family of photochromic photoreceptors probably ubiquitous in green plants, exists in vivo as a cytosolic dimer of two ca. 125kD apoproteins each with a covalently-bound chromophore. The mechanism of action is unknown.

Cytosolic localisation (Cope & Pratt, 1992; Hanstein et al., 1992) obviously conflicts with the current hypothesis regarding polarotropic- and phototropic responses in lower plants that phytochrome is membrane- associated (Kraml, 1994; Lamparter et al., 1995). The paradox possibly masks critical information regarding the mechanism of phytochrome action: we are therefore investigating the phytochrome system in protonemata of the leafy moss Ceratodon purpureus, a species well-suited to photobiological studies.

Thuemmler et al. (1992) described a gene, CERPU;PHY0;1 (CP1) from Ceratodon showing 5' homology to phytochromes but with a non-phytochrome- like 3' region; Kolukisaglu et al. (1993) on the other hand described a "normal" B-like phytochrome in the moss Physcomitrella patens (PHYPA;PHY0;1). We have shown that a "normal" phytochrome predominates in Ceratodon and reported the sequence of an N-terminal gene fragment likely to encode this phytochrome (Lamparter et al., 1995). We have now cloned the corresponding complete CDS and flanking regions from genomic- and cDNA: we call this gene CERPU;PHY0;2 (CP2). The predicted apoprotein has a molecular weight of 124.1kD and comprises 1121 residues. A single canonical phytochrome chromophore binding site occurs in the sequence.

Analyses of CP2 and hypothetical apoprotein sequence show strong homologies to higher plant phytochromes, especially to those of the B- group (Table 1). Most phytochrome CDS's include three introns whereas CP2 shows five. #1, #4 and #5 are positioned typically, whereas #2 is close to the additional intron seen in soybean PHYB (L34843). We have not investigated possible introns in the UTR's of the new gene. Conservation at the nucleotide level between Physcomitrella phytochrome and the CP2 CDS is 85% once gaps are inserted for two codon deletions in the N- terminus of the Ceratodon gene: two apparently recent local frame-shifts are apparent (475..489 and 3648..3657 in CP2). All three residues known to be necessary for PHYB action in transgenic Arabidopsis (Wagner & Quail, 1995) are conserved in CP2, as are 10 of the 14 known functional residues in PHYA (Quail et al., 1995). The perfectly-conserved PHYA M680 residue leads to loss of function when mutated to I, whereas this residue is L in B-type phytochromes and all moss and clubmoss sequences.

Both BLITZ and SBASE domain analyses of the new phytochrome reveal extensive C-terminal homology to bacterial sensor / transmitter protein kinases, as originally observed by Schneider-Poetsch et al. (1991) for other phytochromes. Furthermore, the sequences DLEPYLGL and EDGYLEL centred on Y260 and Y951 respectively resemble known and potential binding sites for N-terminal SH2-domains of mammalian phospholipase C gamma-subunits (Lagarias et al. 1995; Songyang et al. 1993). The tyrosine must be phosphorylated for binding and indeed the EPYLGL motif seen in most phytochromes is the target consensus for mammalian tyrosine kinases. There is, on the other hand, no evidence that either signalling pathway is used by phytochrome.

A potentially even more important similarity is to ORF gi1001165 in the cyanobacterium Synechocystis (Kaneko et al. 1995); CP2 is its closest homolog in the protein database (Table 1). BLASTP analyses reveal 34% identity (50% similarity) to residues 291..338 of CP2, the pocket surrounding the chromophore. The invariate CH at the attachment site is conserved. Even stronger homology (58% identity, 75% similarity) is shown to residues 220..296, spanning the EPYLGL motif. The EDGYLEL motif is absent from the prokaryotic sequence, as is the LIPPIF sequence (see below). Whether the Synechocystis gene does indeed encode a phytochrome- like photoreceptor is not yet known, however.

The close homology to Physcomitrella phytochrome is maintained throughout the CP2 polypeptide (including the revised Physcomitrella C- terminus; see Lamparter et al. 1995), whereas the 3' UTR's are dissimilar. Both moss phytochromes show canonical epitopes for and are bound by the P-25 and Z-3B1 "universal" antiphytochrome monoclonal antibodies. These antibodies do not recognise CP1 but rather a protein with a close N-terminal homology to it. Accordingly, the two Ceratodon phytochromes show ca. 85% identity up to the start of the LIPPIF-motif (L753 in CP2); homology breaks down here and is lost completely 13 residues downstream. The divergence occurs in a region almost perfectly conserved in all other phytochromes (and including the P-25 epitope). The splice site for intron #1 of CP1 and 2 lies upstream of this point although the intron sequences are dissimilar. Thus it would seem that the two phytochromes have diverged functionally following duplication and rearrangement at the start of the LIPPIF-region, between introns #1 and #2. The apparent 5' insertion of 361 bases in CP1 relative to CP2 derives from a hitherto unnoticed direct repeat of similar size 170..490 and 560..910 in CP1: no such repeat is apparent in the new gene (Pustell matrix analyses).

Preliminary Northern data implies that CP2 transcripts are expressed but only weakly light-regulated. The gene thus fulfils all our predictions for the dominant phytochrome in Ceratodon except that the calculated molecular weight is somewhat lower than the 130 kD derived from SDS-PAGE mobility. We have no evidence of further phytochrome- related sequences with an appropriately strong 5' homology to the two now known in Ceratodon, and therefore tentatively assign CERPU;PHY0;2 as the dominant phytochrome in this species.

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Acknowledgments:

This work was supported in part by the FNK of the Free University of Berlin. We thank Cornelia Kreschel for expert ABI 377 sequencing, Lee Pratt and Hansjoerg Schneider-Poetsch for providing P-25 and Z-3B1 antibodies, and Annegret Wilde (Humboldt-University, Berlin) for helpful discussions.

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Table 1: Homologies to CpPHY2

Phytochrome homolog	BLASTN	IDE	WSIM	Source
Synechocystis (prokaryote)	60	23	--	gi1001165
Higher plant A-type	57-67	53-55	70-72	various
Higher plant B-type	60-67	59-61	73-76	various
Adiantum (fern)	54-69	66	76	P42496
Mesotaenium (alga)	64-76	70	--	GI1125699
Selaginella (clubmoss)	70-76	80	86	S31280
Physcomitrella (moss)	78-85	90	92	S37206
BLASTN (NCBI): % base identity in homologous regions IDE (PHD, EMBL; PALIGN, PC/GENE): % amino acid identity WSIM (PHD, EMBL): % amino acid weighted similarity

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Literature cited:

Cope M & Pratt LH (1992) Intracellular redistribution of phytochrome in etiolated soybean (Glycine max L.) seedlings. Planta 188:115-122

Hanstein C, Grolig F & Wagner G (1992)Immunolocalistion of cytosolic phytochrome in the green alga Mougeotia. Bot. Acta 105: 55-62

Kaneko T, Tanaka A, Sato S, Kotani H, Sazuka T, Miyajima N, Sugiura M & Tabata S (1995) Sequence analysis of the unicellular cyanobacterium Synechocystis sp. strain PCC6803. I. Sequence features of the 1 Mb region from map positions 64% to 92% of the genome. DNA res. 2: 153-166

Kolukisaglu, HU, Braun B, Martin WF & Schneider-Poetsch HAW (1993) Mosses do express conventional, distantly B-type related phytochromes - phytochrome from Physcomitrella patens (Hedw.) FEBS Lett. 334: 95-100

Kraml M (1994) Light direction and polarisation. In Kendrick RE & Kronenberg GHM (Eds.) "Photomorphogenesis in plants" 2nd Ed. Kluwer Acad. Publ., Netherlands, pp. 417-490

Lagarias DM, Wu S-H & Lagarias JC (1995) Atypical phytochrome gene structure in the green alga Mesotaenium caldariorum. Pl. Mol. Biol. 29: 1127-1142.

Lamparter T, Podlowski S, Mittmann F, Schneider-Poetsch HAW, Hartmann E and Hughes J (1995) Phytochrome form protonemal tissue of the moss Ceratodon purpureus. J. Pl. Physiol. 147: 426-434

Quail PH, Boylan MT, Parks BM, Short TW, Xu Y & Wagner D (1995) Phytochromes: photosensory perception and signal transduction. Science 268: 675-680

Schneider-Poetsch HAW, Braun B Marx S & Schaumburg A (1991) Phytochromes and bacterial sensor proteins are related by structural and functional homologies: hypothesis on phytochrome-mediated signal- transduction. FEBS Lett. 281: 245-249

Songyang Z et al. (1993) SH2 domains recognise specific phosphopeptide sequences. Cell 72: 767-778

Thuemmler FM, Dufner M, Kreisl P & Dittrich P (1992) Molecular cloning of a novel phytochrome gene of the moss Ceratodon purpureus which encodes a putative light-regulated protein kinase. Plant Mol. Biol. 20: 1003-1017

Wagner D & Quail PH (1995) Mutational analysis of phytochrome B identifies a small COOH-terminal domain critical for regulatory activity. Proc. Nat. Acad. Sci. USA 92: 8596-8600

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