Evolution of seed storage protein genes: Legumin genes of Ginkgo biloba

Legumin-like seed storage proteins have been intensively studied in crop plants. However, little is known about the molecular evolution of these proteins and their genes and it was assumed that they originated from an ancestral gene that already existed at the beginning of angiosperm evolution. We have evidence for the ubiquitous occurrence of homologous proteins in gymnosperms as well. We have characterized the major seed storage globulin from Ginkgo biloba by amino acid sequencing, which reveals clear homology to legumin-like proteins from angiosperms. The Ginkgo legumin is encoded by a gene family; we describe two of its members. The promoter regions contain sequence motifs which are known to function as regulatory elements involved in seed-specific expression of angiosperm legumins, although the tissues concerned are different in gymnosperms and angiosperms. The Ginkgo legumin gene structure is divergent from that of angiosperms and suggests that the evolution of legumin genes implicated loss of introns. From our data and from functional approaches recently described it becomes obvious that the posttranslational processing site of legumin precursors is less conserved than hitherto assumed. Finally, we present a phylogenetic analysis of legumin encoding sequences and discuss their utility as molecular markers for the reconstruction of seed plant evolution.Correspondence to: K.-P. Häger     

Identification of two differentially regulated isoforms of protochlorophyllide oxidoreductase (POR) from tobacco revealed a wide variety of light- and development-dependent regulations of POR gene expression among angiosperms

NADPH-protochlorophyllide oxidoreductase (POR) catalyzes the light-dependent reduction of protochlorophyllide a in the chlorophyll biosynthetic pathway. Here, we identified two distinct POR cDNAs from tobacco. Both POR isoforms are encoded by a respective single copy gene in tobacco genome. The overall deduced amino acid sequences of two tobacco cDNAs, designated here POR1 and POR2, displayed significant identities (∼75%), but showed different patterns of light and developmental regulation. In contrast to the previously isolated POR isoforms of Arabidopsis thaliana and barley, the expression of both tobacco POR isoforms were not negatively regulated by light and persisted in matured green tissues. Furthermore, the expression of both genes appeared to be regulated by a diurnal regulation. These results show a wide variety of light- and development-dependent regulations of POR gene expression among angiosperms. Furthermore, phylogenetic analysis including tobacco revealed that POR gene family is differentially represented by angiosperms, most of which is probably caused by independent gene duplication in individual plant. Present results imply a modification of the previous concept that chlorophyll biosynthesis and chloroplast differentiation in angiosperms are ubiquitously controlled by unique functions of two POR isoforms. This revised version was published online in June 2006 with corrections to the Cover Date.     

Legumin of Vicia faba major: accumulation in developing cotyledons,purification, mRNA characterization and chromosomal location of coding genes

Summary Experiments were carried out on Vicia faba major involving (1) determination of the pattern of legumin accumulation during seed development, (2) protein purification from mature cotyledons, (3) the characterization of legumin mRNA, and (4) the chromosomal localization of the genes coding for legumins. In developing cotyledons the synthesis of legumin begins 28 days after petal desiccation (DAPD), and 4 days after initiation of vicilin synthesis. The two subunits (_A and _A) of legumin A appear 2 days earlier than those (_B and _B) of legumin B. While the accumulation of vicilin peaks on the 30th DAPD, that of legumin continues during further seed development, and the synthesis of legumin mRNA peaks on the 37th DAPD. Northern blot hybridizations using two DNA plasmids containing cDNA inserts with sequence homology to the A- and B-type legumin genes, respectively, indicated that legumin mRNAs extracted from cotyledons 36 DAPD band below the 18S RNA band. In addition, a faint band below that of the 25S RNA band can be observed in legumin mRNAs extracted from cotyledons at an earlier developmental stage (30 DAPD). By means of polyacrylamide gel electrophoresis in the presence or absence of SDS and 2-mercaptoethanol, two fractions could be eluted after zonal isoelectric precipitation of the globulins from mature seeds: one fraction contains mainly vicilin, the other, legumin. In situ hybridization showed that legumin genes are arranged in two clusters: the genes coding for legumin A are located in the longer arm of the one between the two shortest subtelocentric chromosome pairs whose centromere is in a less terminal position; those coding for legumin B are located in the non-satellited arm of the longer submetacentric pair.     

Characterization and evolutionary relationship of methionine-rich legumin-like protein from buckwheat.

We have isolated and characterized a full-length cDNA for legumin-like storage polypeptide from buckwheat seed (Fagopyrum esculentum Moench) and compared its deduced amino acid sequence with those from different representatives of dicots, monocots and gymnosperms. The cDNA sequence was reconstructed from two overlapping clones isolated from a cDNA library made on mRNA of buckwheat seed at the mid-maturation stage of development. Analysis of the deduced amino acid sequence revealed that this specific buckwheat storage polypeptide should be classified in the methionine-rich legumin subfamily present in the lower angiosperm clades, a representative of which was first characterized in Magnolia salicifolia (clone B 14). The fact that a methionine-rich legumin coexists together with methionine-poor legumins in buckwheat should be an important element regarding the evolutionary position of buckwheat. This may also be supporting evidence that the B14 ortholog was not lost in evolution but was protected under pressure of an increased need for sulfur. Using primers designed from characterized cDNA, we also isolated its corresponding gene from buckwheat genomic DNA and analyzed the characteristic exon/intron structure. The firstly identified two-intron structure of buckwheat legumin gene is an important contribution to study of methionine-rich legumins in lower angiosperms.     

Accumulation of seed storage proteins and the taxonomy ofPoaceae

The accumulation of specific seed proteins is a taxonomically valuable feature and can be used to additionally characterize plant taxa. To date, mainly crop proteins have been analysed in thePoaceae. In this investigation seed proteins from 147 species were screened with emphasis on legumin-like proteins and prolamins. The groups resulting from evaluation of the protein profiles correspond with well-known subfamilies and tribes.Panicoideae are clearly separated fromPooideae. WithinPooideae, theBromeae plusTriticeae tribes revealed obvious similarities.Lolium, Festuca andVulpia, generally included in the tribeFestuceae, revealed a protein profile similar to the profile of theBromeae/Triticeae. Legumin-like proteins are accumulated abundantly inBambusoideae andPooideae exceptBromeae/Triticeae, however, only the species included in theAveninae subtribe produce soluble (globulin-type) legumins as already known fromAvena sativa. Dedicated to emer. Univ.-Prof. DrFriedrich Ehrendorfer on the occasion of his 70th birthday     

Heterogeneity of sulphur content in the storage proteins of pea cotyledons

By means of crossed immunoelectrophoresis of the cotyledonary storage proteins of Pisum sativum L. it was shown that reduced accumulation of the legumin fraction, resulting from severe sulphur deficiency during growth, is accompanied by relative suppression of a quantitatively minor storage protein (Peak 3) shown previously by subunit analysis to be related to the vicilin series of holoproteins. The pattern of isotopic labelling of the storage proteins after injection of [³⁵S]methionine into the pedicel during seed development under normal nutritional conditions indicated that Peak-3 protein, like legumin, has a relatively high content of sulphur amino-acids. Like certain of the vicilin molecules carrying the determinants responsible for Peak-4, Peak-3 protein binds selectively to concanavalin A.     

The legumin gene family: structure and evolutionary implications of Vicia faba B-type genes and pseudogenes

We have characterized several Vicia faba genes encoding methionine residue-free group B subunits of the 11S or legumin storage proteins. The respective gene subfamily consists of 10 to 15 members, six of them having been studied by DNA sequence analysis. Four functional genes (LeB2, LeB4, LeB6, LeB7) are highly homologous in their coding region and 0.3 kb of their 3 flanking sequences. On the other hand, two pseudogenes (LeB1, LeB5) have accumulated a large number of mutations including an identical 0.7 kb internal deletion; they are both flanked by a repetitive element. Analysis of sequence changes show that transitions are nearly double as frequent as transversions. CpG is the most infrequent dinucleotide whereas TpA is significantly underrepresented in exon sequences. End points of deletions are correlated with short direct repeats and preferentially found in the two introns. Our studies indicate that the Vicia faba legumin B gene subfamily contains a group of expressed, highly homologous genes as well as more diverged pseudogenes.     

Polymorphism of legumin subunits from field bean (Vicia faba L. var. minor) and its relation to the corresponding multigene family

Legumin, which amounts to approximately 55% of the seed protein in field beans (Vicia faba L. var. minor), is a representative of the 12S storage globulin family. The 12S storage globulins are hexameric holoprotein molecules composed of different types of polymorphic subunits encoded by a multigene family. Type-A legumin subunits contain methionine whereas type-B are methionine-free subunits. Sequencing of two different type A-specific cDNAs, as well as an FPLC/HPLC-based improvement of subunit fractionation and peptide mapping with subsequent partial amino-acid sequencing, permit the assignment of some of the polymorphic legumin subunits to members of the multigene family. Two different type A subunits (A1 and A2) correspond to the two different cDNA clones pVfLa129 (A2) and 165 (A1), but microheterogeneity in the amino-acid sequences indicates that polymorphic variants of both representatives of this type may exist. Two groups of published type B-specific gene sequences (LeB7, and LeB2, LeB4, LeB6, respectively) are represented by two polymorphic subunit fractions (B3I, B3II, and B4I, B4II). A seventh clone, LeB3, encodes one of the large legumin subunits that is only a minor component of the legumin seed protein complex.     

Chemical modification of intracellular cytosine deaminase fromChromobacterium violaceum YK 391

Cytosine deaminase (cytosine aminohydrolase, EC 3.5.4.1) stoichiometrically catalyzes the hydrolytic deamination of cytosine and 5-fluorocytosine to uracil and 5-fluorouracil, respectively. Amino acid residues located in or near the active sites of the intracellular cytosine deaminase fromChromobacterium violaceum YK 391 were identified by chemical modification studies. The enzymic activity was completely inhibited by chemical modifiers, such as 1 mM NBS, chloramine-T, ρ-CMB, ρ-HMB and iodine, and was strongly inhibited by 1 mM PMSF and pyridoxal 5′-phosphate. This chemical deactivation of the enzymic activity was reversed by a high concentration of cytosine. Furthermore, the deactivation of the enzymic activity by ρ-CMB was also reversed by 1 mM cysteine-HCl, DTT and 2-mercaptoethanol. These results suggested that cysteine, tryptophan and methionine residues might be located in or near the active sites of the enzyme, while serine and lysine were indirectly involved in the enzymic activity. The intracellular cytosine deaminase fromC. violaceum YK 391 was assumed to be a thiol enzyme.     

Amino acid sequence of piratoxin-I, a myotoxin fromBothrops pirajai snake venom, and its biological activity after alkylation withp-bromophenacyl bromide

The complete sequence of the 121 amino acid residues of piratoxin-I (PrTX-I), a phospholipase A₂ (PLA₂)-like myotoxin fromBothrops pirajai snake (Bahia jararacussu) venom, is reported. From the sequence, anM _r of 13,825 and an approximatepI of 8.3 were calculated. PrTX-I shows a high sequence homology with Lys-49 myotoxins from other bothropic (∼95%) and nonbothropic (∼80%) venoms, but only 70–75% homology w hen aligned with the catalytically active Asp-49 PLA₂s. When compared with bothropstoxin-I fromBothrops jararacussu, which is morphologically almost identical toB. pirajai, only two changes out of 121 total amino acid residues have been observed. The approximate minimal lethal doseLD ₅₀ (mice, i.p., 24 hr) of PrTX-I was 8 (6.8–9.1) mg/kg, and the minimal edematogenic dose (MED) in a rat paw model was 39.5±1.8 ug. After alkylation of His-48 withp-bromophenacyl bromide, the MED was 40.1±1.9 ug, but up to 4LD ₅₀ were unable to cause death in any of a group of eight mice after 72 hr. Therefore the edematogenic activity was retained and apparently did not involve His-48, suggesting that at least two biologically active sites are present in PrTX-I.     

Entwicklungsgeschichte und Ultrastruktur von Pollenkitt und Exine bei nahe verwandten entomophilen und anemophilen Angiospermensippen:Ranunculaceae,Hamamelidaceae, Platanaceae undFagaceae

Transfer of fresh pollen either by wind or by insects is strongly related to the degree of its stickiness. The sticky substances involved should be understood as pollenkitt. Ultrastructural investigations indicate that the sticky pollen of entomophilous angiosperms (in this publicationHamamelis vernalis andH. virginiana, Corylopsis platypetala) results from the deposition of usually electron-dense, homogeneous pollenkitt mainly on the outside layer of the exine. In contrast, the pollen of anemophilous angiosperms (in this publicationParrotia persica, Platanus orientalis, Thalictrum flavum andQuercus robur), but also of some entomophilous angiosperms with special pollination ecology (e.g.Aquilegia vulgaris) is rather dry and powdery. In these cases the pollenkitt often is electron-transparent and inhomogeneous and is mainly removed into the cavities of the exine. Ultrastructure and deposition of pollen and pollenkitt in ambophilous angiosperms (in this publicationThalictrum minus, Castanea sativa) are ± intermediate.

     

Amino acid sequence of piratoxin-I, a myotoxin fromBothrops pirajai snake venom, and its biological activity after alkylation withp-bromophenacyl bromide

The complete sequence of the 121 amino acid residues of piratoxin-I (PrTX-I), a phospholipase A₂ (PLA₂)-like myotoxin fromBothrops pirajai snake (Bahia jararacussu) venom, is reported. From the sequence, anM _r of 13,825 and an approximatepI of 8.3 were calculated. PrTX-I shows a high sequence homology with Lys-49 myotoxins from other bothropic (∼95%) and nonbothropic (∼80%) venoms, but only 70–75% homology w hen aligned with the catalytically active Asp-49 PLA₂s. When compared with bothropstoxin-I fromBothrops jararacussu, which is morphologically almost identical toB. pirajai, only two changes out of 121 total amino acid residues have been observed. The approximate minimal lethal doseLD ₅₀ (mice, i.p., 24 hr) of PrTX-I was 8 (6.8–9.1) mg/kg, and the minimal edematogenic dose (MED) in a rat paw model was 39.5±1.8 ug. After alkylation of His-48 withp-bromophenacyl bromide, the MED was 40.1±1.9 ug, but up to 4LD ₅₀ were unable to cause death in any of a group of eight mice after 72 hr. Therefore the edematogenic activity was retained and apparently did not involve His-48, suggesting that at least two biologically active sites are present in PrTX-I.     

Molecular cloning of an insertion sequence-like element from <Emphasis Type="Italic">Vibrio anguillarum</Emphasis> and its functional identification in <Emphasis Type="Italic">E. coli</Emphasis>

The tdh (thermostable direct hemolysin) gene occurs in some strains of Vibrio species. All tdh genes are flanked by insertion sequence-like elements (ISV). All previous attempts have failed to detect transposition of these ISVs. In this work, we have built a transposition detection system in E. coli and succeeded in detecting the transposition of an insertion sequence-like element at a frequency of Km^r mutants of 7.2 × 10⁻⁶. A specific flanking sequence (5′-Py-Pu-3′) was found on either side of the target duplication.     

Production of sialyltrisaccharides using β-galactosidase andtrans-sialidase in one pot

Sialyltrisaccharides based on β-galactosyldisaccharides were synthesized using β-galactosidase andtrans-sialidase in one pot. Using β-galactosidase fromBacillus circulans andtrans-sialidase fromTrypanosoma cruzi simultaneously, 6 mM sialyltrisaccharides composed of about 95% NeuAcα(2,3)Galβ(1,4)GlcNAc and 5% NeuAcα(2,3)Galβ(1,6)GlcNAc were produced from a reaction mixture containing 25 mM 0-nitrophenyl-β-D-galactopyranoside, 100 mM N-acety lglucosamine and 10 mM p-nitrophenyl-α-D-N-acetylneuraminic acid. One beauty of this reaction was that a secondary hydrolysis of the disaccharide intermediate occurring between the activated galactopyranoside and N-acetylglucosamine was prevented. Using β-galactosidase fromEscherichia coli and the sametrans-sialidase, 15 mM sialyltrisaccharides composed of about 90% NeuAcα(2,3)Galβ(1,6)GlcNAc and 10% NeuAcα(2,3)Galβ (1,4)GlcNAc were produced from a reaction mixture containing 400 mM galactose, 800 mM N-acetylglucosamine and 20 mMp-nitrophenyl-α-D-N-acetylneuraminic acid. In this study, the reverse-galactosylation reaction between galactose and N-acetylglucosamine was dominant since the disaccharide intermediate mainly resulted in the sialylated product.     

Primitive Defence: The MiAMP1 Antimicrobial Peptide Family

The founder of the MiAMP1 protein family was originally isolated from Macadamia integrifolia and had antimicrobial activity in vitro. MiAMP1 was the first plant protein with a structure containing a βγ-crystallin precursor fold, a structural superfamily associated with antimicrobial proteins in other kingdoms. In recent times, expanding plant genomics information has revealed that genes encoding homologues of MiAMP1 are conserved across the plant kingdom from lycophytes, gymnosperms to early angiosperms (e.g. Amborella, Papaver) and some monocots (e.g. Zantedeschia, Zea, Sorghum). Many studies of plant–pathogen interactions in gymnosperms have demonstrated a potential role for MiAMP1 family members in defence against fungal pathogens. This commentary describes the discovery and diversity of this protein family and considers current evidence supporting, and future opportunities for substantiating, a role in defence in primitive plants, and why this role may have diminished in higher plants.     

Phylogeny and divergence of basal angiosperms inferred from<Emphasis Type="Italic"> APETALA3</Emphasis>- and<Emphasis Type="Italic"> PISTILLATA</Emphasis>-like MADS-box genes

The B-class MADS-box genes composed of APETALA3 (AP3) and PISTILLATA (PI) lineages play an important role in petal and stamen identity in previously studied flowering plants. We investigated the diversification of the AP3-like and PI-like MADS-box genes of eight species in five basal angiosperm families: Amborella trichopoda (Amborellaceae); Brasenia schreberi and Cabomba caroliniana (Cabombaceae); Euryale ferox, Nuphar japonicum, and Nymphaea tetragona (Nymphaeaceae); Illicium anisatum (Illiciaceae); and Kadsura japonica (Schisandraceae). Sequence analysis showed that a four amino acid deletion in the K domain, which was found in all previously reported angiosperm PI genes, exists in a PI homologue of Schisandraceae, but not in six PI homologues of the Amborellaceae, Cabombaceae, and Nymphaeaceae, suggesting that the Amborellaceae, Cabombaceae, and Nymphaeaceae are basalmost lineages in angiosperms. The results of molecular phylogenetic analyses were not inconsistent with this hypothesis. The AP3 and PI homologues from Amborella share a sequence of five amino acids in the 5 region of exon 7. Using the linearized tree and likelihood methods, the divergence time between the AP3 and PI lineages was estimated as somewhere between immediately after to several tens of millions of years after the split between angiosperms and extant gymnosperms. Estimates of the age of the most recent common ancestor of all extant angiosperms range from ~140–210 Ma, depending on the trees used and assumptions made.     

Molecular phylogeny ofSalicaceae and closely relatedFlacourtiaceae: Evidence from 5.8 S, ITS 1 and ITS 2 of the rDNA

A ribosomal DNA region, including the entire 5.8S RNA gene and the internal transcribed spacers ITS 1 and ITS 2, was used for studying the phylogeny ofSalicaceae and the relationship betweenSalicaceae andFlacourtiaceae. The length of the ITS regions withinSalicaceae andFlacourtiaceae was similar to that found in other angiosperms. The GC content of both ITS regions was high, varying 62.7-72.2%. The most parsimonious tree clusters the wind-pollinatedChosenia bracteosa among theSalix species, suggesting that it should be included in the genusSalix. The grouping withinSalix leaves subg.Salix as paraphyletic, for which reason the subgeneric division is questionable.Populus was monophyletic and formed a sister group toSalix. The interspecific variation of the ITS sequences was very small inSalicaceae, which is in contradiction to the age of the group according to the evidence from fossil data.Idesia polycarpa fromFlacourtiaceae shows great sequence similarity withSalicaceae, but the analysis of 5.8S rDNA supports monophyly of the four species ofFlacourtiaceae sampled for this study.     

Purification of cadmium-binding proteins from related species of terrestrial helicidae (gastropoda,mollusca): A comparative study

Summary Three species of terrestrial Helicidae (Helix pomatia, Cepaea hortensis andArianta arbustorum) were fed cadmium-rich diet in the laboratory. The snails accumulated high amounts of the metal in their hepatopancreas. Most cadmium and some zinc were found, after centrifugation, in the soluble fractions from which a cadmium-binding protein was isolated for each species by ion exchange and gel chromatography. The proteins contained different amounts of cadmium, but little or no zinc, and showed high absorption at 254 nm indicating the presence of cadmium-mercaptide bonds. After gel filtration, a molecular weight of 12000 was found for cadmium-binding proteins fromHelix pomatia andArianta arbustorum, whereas a molecular weight of 10 000 was found for a cadmium-binding protein fromCepaea hortensis. SDS-polyacrylamide gel electrophoresis showed one single band for each protein fromHelix pomatia andArianta arbustorum and suggested a molecular weight of 11000 for both species. Amino acid analysis revealed, for each protein, high amounts of cysteine (12–20%), glycine (15–19%), and serine (12–14%), and moderately elevated contents of lysine (9–13%) and alanine (4–8%), but no methionine and only traces, if any, of aromatic amino acids. The ratios of cadmium to cysteine were 1:5, 1:10 and 1:3 in the proteins fromHelix pomatia,Cepaea hortensis andArianta arbustorum, respectively. Some features of the isolated proteins resembled mammalian metallothioneins. Most characteristics, however, differed from true metallothioneins and were similar to cadmium-binding proteins found in some marine molluscs.     

Globin evolution in the genusXenopus: Comparative analysis of cDNAs coding for adult globin polypeptides ofXenopus borealis andXenopus tropicalis

Summary Globin mRNAs ofXenopus borealis andXenopus tropicalis have been cloned and sequenced. The nucleotide and derived amino acid sequences were compared with each other and with already available data fromXenopus laevis. This analysis rendered clear evidence that the common ancestor ofX. laevis andX. borealis, but not ofX. tropicalis, had lost one amino acid of the -globins prior to a genome duplication event that preceded the segregation of the former two species. Replacement-site substitutions were used to calculate a rough time scale of genome duplication and species segregation. The results suggest an ancient separation between theX. laevis and theX. tropicalis groups occurring approximately 110–120 million years ago. Analysis of the amino acid chains demonstrated various alterations. However, some functional domains, like heme-binding sites and₁₂ contact sites, were subject to a high degree of conservation, indicating the existence of functional constraints on them also in the genusXenopus.