Hansjörg A.W. Schneider-Poetsch

Phytochromes and bacterial sensor proteins are related by structural and functional homologies. Hypothesis on phytochrome-mediated signal-transduction.

Phytochrome and bacterial sensor proteins are related by functional and structural homologies. They are both sensors of environmental stimuli and share structural homologies which comprise a domain of about 250 amino acids (about 28 kg mol−1). This domain is C‐terminal in phytochromes and in several bacterial sensor proteins. In both groups of sensors this domain undergoes conformational changes which are caused by the N‐terminal part sensing the stimulus. In the case of the bacterial sensors, the conformational alteration is, regulated by additional proteins, conferred to a corresponding regulator protein which then acts on transcription. The coincidences between the two group of sensors are striking enough to assume phytochrome to transduce signals in a way comparable to the bacterial two‐component systems.

Signal transduction by phytochrome: phytochromes have a module related to the transmitter modules of bacterial sensor proteins.

A C‐terminal section of phytochromes turned out to share sequence homologies with the full length of the transmitter modules (about 250 amino acids) of bacterial sensor proteins. Coinciding hydrophobic clusters within the homologous domains imply that the overall folding of the two different types of peptides is similar. Hence, phytochromes appear to possess the structural prerequisites to transmit signals in a way bacterial sensor proteins do. The bacterial sensor proteins are known to be environmental stimuli‐regulated kinases belonging to two‐component systems. After sensing a stimulus by the N‐terminal part of the sensor protein, conformational alterations confer the signal to its (mostly) C‐terminal transmitter module which in turn is transitionally autophosphorylated at a conserved histidine. From the histidine the phosphate is transferred to the receiver module of a system‐specific regulator protein which eventually acts on transcription or enzyme activity. The histidine is not conserved in phytochromes. Instead, a conserved tyrosine is found spatially very close to the histidine position. This tyrosine might play the role of histidine, and kinase function might be associated with this part of phytochrome. In spite of this divergence, the structural similarities point to a common evolutionary origin of the phytochrome and bacterial modules.

The evolution of gymnosperms redrawn by phytochrome genes: The Gnetatae appear at the base of the gymnosperms

Abstract. Gymnosperms possess two to four phytochrome types which apparently are the result of successive gene duplications in the genomes of their common ancestors. Phytochromes are nuclear-encoded proteins whose genes, contrary to chloroplast, mitochondrion, and rRNA genes, have hitherto rarely been used to examine gymnosperm phylogenies. Since the individual phytochrome gene types implied phylogenies that were not completely congruent to one another, conflicting branching orders were sorted by the number of gene lineages present in a taxon. The Gnetatae (two gene types) branched at the base of all gymnosperms, a position supported by bootstrap sampling (distance and character state trees, maximum likelihood). The Gnetatae were followed by Ginkgo, Cycadatae, and Pinaceae (three gene types) and the remaining conifers (four gene types). Therefore, in phytochrome trees, the most ancient branch of the conifers (Pinatae) seems to be the Pinaceae. The next split appears to have separated Araucariaceae plus Podocarpaceae from the Taxaceae/Taxodiaceae/Cupressaceae group. Structural arrangements in the plastid genomes (Raubeson and Jansen 1992) corroborate the finding that there is no close connection between Pinaceae and Gnetatae as suggested by some publications. The analyses are based on 60 phytochrome genes (579 positions in an alignment of PCR fragments) from 28 species. According to rough divergence time estimates, the last common ancestor of gymnosperms and angiosperms is likely to have existed in the Carboniferous.

Avenacosidase from oat: purification, sequence analysis and biochemical characterization of a new member of the BGA family of β-glucosidases

A protein consisting of 60 kDa subunits (As-P60) was isolated from etiolated oat seedlings (Avena sativa L.) and characterized as avenacosidase, a β-glucosidase that belongs to a preformed defence system of oat against fungal infection. The enzyme is highly aggregated; it consists of 300–350 kDa aggregates and multimers thereof. Dissociation by freezing/thawing leads to complete loss of enzyme activity. The specificity of the enzyme was investigated with para-nitrophenyl derivatives which serve as substrates, in decreasing order β-fucoside, β-glucoside, β-galactoside, β-xyloside. The corresponding orthonitrophenyl glycosides are less well accepted. No hydrolysis was found with α-glycosides and β-thioglucoside. An anti-As-P60 antiserum was prepared and used for isolation of a cDNA clone coding for As-P60. A presequence of 55 amino acid residues was deduced from comparison of the cDNA sequence with the N-terminal sequence determined by Edman degradation of the mature protein. The presequence has the characteristics of a stroma-directing signal peptide; localization of As-P60 in plastids of oat seedlings was confirmed by western blotting. The amino acid sequence revealed significant homology (>39% sequence identity) to β-glucosidases that are constituents of a defence mechanism in dicotyledonous plants. 34% sequence identity was even found with mammalian and bacterial β-glucosidases of the BGA family. Avenacosidase extends the occurrence of this family of β-glucosidases to monocotyledonous plants.

Cross-reactivity of monoclonal antibodies against phytochrome from Zea and Avena : Localization of epitopes, and an epitope common to monocotyledons, dicotyledons, ferns, mosses, and a liverwort.

The cross-reactivity of diverse monoclonal antibodies against phytochrome from Zea and Avena was tested by enzyme-linked immunosorbentassay (ELISA) and by immunoblotting. About 40 antibodies were selected by means of nondenatured phytochrome; all of them reacted with sodium dodecyl sulfate denatured homologous antigen on immunoblots. The epitopes for 14 antibodies (4 raised against Avena and 10 against Zea phytochrome) were localized in 6 regions of the phytochrome molecule by means of Western blot analysis of proteolytic fragments of known localization. Results of studies on the inhibition of antibody binding by other antibodies were largely compatible with these latter findings. Except in a few cases, inhibition occurred when antibodies were located on the same or a closely adjacent region. As demonstrated by 16 species, cross-reactivity with phytochromes from other Poaceae was high. Greater losses in cross-reactivity were observed only with antibodies recognizing an epitope in the vicinity of the carboxyl terminus of 118-kg · mol-1 phytochrome. Cross-reactivity with phytochrome from dicotyledons was restricted to a few antibodies. However, phytochrome(s) from plants illuminated for 24 h or more could be detected. One of the antibodies that recognized phytochrome from dicotyledons was also found to recognize phytochrome or a protein of 120–125 kg·mol-1 from several ferns, a liverwort and mosses. This antibody (Z-3B1), which was localized within a 23.5-kg·mol-1 section of Avena phytochrome (Grimm et al., 1986, Z. Naturforsch. 41c, 993), seems to be the first antibody raised against phytochrome from a monocotyledon with such a wide range of reactivity. Even though epitopes were recognized on different phytochromes, the strength of antibody binding indicated that these epitopes are not necessarily wholly identical.

Mosses do express conventional, distantly B-type-related phytochromes : phytochrome of Physcomitrella patens (Hedw.)

We have screened a cDNA library of the moss Physcomitrella patens (Hedw.) for phytochrome sequences. The isolated sequences turned out to encode a phytochrome dissimilar to the phytochrome type postulated for the moss Ceratodon [(1 992) Plant Mol. Biol. 20, 1003–1017] Physcomitrella phytochrome was completely alignable to fern phytochrome (Selaginella) and phytochromes of higher plants. The frequency of clones encoding this phytochrome indicated that a Ceratodon‐like type should only be expressed, if at all, with lower frequencies than the sequenced phytochrome cDNA. Sequence differences between lower plant phytochromes are small as compared to phytochrome types of higher plants.

PHYTOCHROME EVOLUTION: A PHYLOGENETIC TREE WITH THE FIRST COMPLETE SEQUENCE OF PHYTOCHROME FROM A CRYPTOGAMIC PLANT: (Selaginella martensii SPRING)

We have sequenced cDNA and genomic clones coding for phytochrome of the fern Selaginella. On the amino acid level, this phytochrome shares sequence homologies with phytochromes of higher plants which range between 62 (phytochrome E of Arabidopsis) and 55 (56)% [phytochrome C of Arabidopsis (Avena)]. Introns in the Selaginella gene are short and occupy positions known from phytochrome sequences of higher plants. A rooted phylogenetic tree based on mutation distances puts Seluginella phytochrome closest to the hypothetical ancestor. A similar tree arises if the tree is constructed with partial sequences (about 200 amino acids) around the chromophore attachment site. An extension of this tree by sequences of other cryptogamic plants (Mougeotia, Ceratodon, Psilotum) shows all these sequences including those of the phytochromes B and C of Arabidopsis on a branch, well separated from the branch formed by phytochromes known to accumulate in etiolated plants. The rooted phytochrome phylogenetic tree, however, is difficult to reconcile with the fossil record.

The amino acid sequence previously attributed to a protein kinase or a TCP1-related molecular chaperone and co-purified with phytochrome is a β-glucosidase

A 60 kDa protein (P60) co‐purified with phytochrome was identified as avenacosidase, a β‐glucosidase which is part of the defense system of Avena sativa. An antiserum raised against P60 was used to isolate a cDNA clone coding for the complete amino acid sequence of P60. The cDNA‐derived amino acid sequence contained the partial sequences described before for a protein kinase [(1989) Planta 178, 199–206] and for a TCP1‐related molecular chaperone [(1993) Nature 363, 644–647] co‐purified with phytochrome. We conclude that these activities were related to minor contaminants and that only sequences of avenacosidase had been obtained.

Phytochrome types in Picea and Pinus. Expression patterns of PHYA-related types

Knowledge of the genes in gymnosperms encoding the apoproteins of the plant photoreceptor phytochrome is currently scanty as for gymnosperm nuclear protein coding sequences in general. Here we report two complete cDNA-derived sequences which code for two different types of gymnosperm phytochrome. One sequence stems from Norway spruce (Picea abies) and the other from Scots pine (Pinus sylvestris). More detailed studies have shown that both types of phytochrome gene are present in Norway spruce. From phylogenetic analyses, these types appear to branch off from progenitors that are also the common ancestors of the angiosperm PHYA/PHYC and PHYB/PHYD/PHYE lineages. Partial phytochrome sequences of other gymnosperms cluster with either the one type or the other of the gymnosperm phytochrome genes characterized here. Southern blot analysis of Picea DNA using probes derived from the full-length Picea gene indicated a family of at least five members. Whether they code for new types may be doubted since only two phylogenetic clusters were found. Studies using RNA-PCR of Picea RNA extracted from either light- or dark-grown seedlings indicated that the steady-state levels of the transcripts of two PHYA/C-related genes were hardly affected by light.

PARTIAL NUCLEOTIDE SEQUENCE OF PHYTOCHROME FROM THE ZYGNEMATOPHYCEAN GREEN ALGA Mougeotia

Following polymerase chain reaction, a fragment of about 800 bp was amplified from genomic Mougeotia DNA using oligonucleotides directed to conserved regions of known phytochrome genes. The nucleotide sequence points to a different exon/intron structure in the neighborhood of the chromophore attachment site of this Mougeotia phytochrome gene, as compared to other phytochromes. Alignment of the derived amino acid sequence to phytochromes of higher and lower plants shows highest homology (>60%) to type II (green type) phytochrome, while Northern blot analysis of total Mougeotia RNA indicates down‐regulation of the phytochrome transcription in light. Signal pattern of hybridized genomic DNA after digestion reveals the presence of probably only one phytochrome gene in Mougeotia.