Characterization of matteuccin, the 2.2S storage protein of the ostrich fern. Evolutionary relationship to angiosperm seed storage proteins

The 2.2S spore storage protein (matteuccin) of the ostrich fern, Matteuccia struthiopteris, has been isolated and characterized. It is a small basic protein consisting of two disulfide-linked polypeptides with approximate molecular masses of 3.0 kDa and 8.0 kDa. At least four different isoforms exist where two of the forms differ from the other by having a slightly smaller heavy chain. Amino acid analysis reveals that the 2.2S protein is rich in arginine. Almost complete amino acid sequence information was obtained for the light chain and a partial sequence for the heavy chain. Amino acid sequence comparison reveals that this protein shows a high similarity to seed storage proteins in different angiosperm species in spite of the fact that the common ancestor of ferns and angiosperms lived more than 300 million years ago.     

A vicilin-like seed protein of cycads: similarity to sucrose-binding proteins

Seed storage globulins of the 7S and 11S type are synthesized in the seeds of angiosperms and gymnosperms. We have isolated and characterized a vicilin-like gene expressed in the cycad Zamia furfuraceae. Sequence comparisons reveal clear similarities to a sucrose-binding protein isolated from soybean. We suggest the existence of a superfamily of related genes including both vicilin-like and legumin-like seed globulin genes as well as genes coding for spherulins, germins and sucrose-binding-proteins.     

A fern spore storage protein is genetically similar to the 1.7 S seed storage protein ofBrassica napus

The ostrich fern, Matteuccia struthiopteris L., contains two globulin spore storage proteins of 2.2 S and 11.3 S, with physical characteristics similar to those of seed storage proteins of Brassica napus (rapeseed) and Raphanus sativus (radish). By the use of a cloned cDNA that encodes the 1.7 S B. napus storage protein (napin), gene sequences that hybridized with napin were detected in fern nuclear DNA, and a 900-nucleotide homologous mRNA was detected in developing spores. In vitro translation of this fern mRNA produced a 22-kD polypeptide comparable in size to the 21-kD precursor polypeptide identified in Brassica. No hybridizations were observed between the Brassica 12 S clone and either fern DNA or developing spore mRNA.     

Isolation and Characterization of Mustard (Sinapis alba L.) Seed Storage Proteins

A total storage protein fraction was prepared from mustard (Sinapis alba L.) seeds via isolated protein bodies and characterized by sedimentation, immunological, and electrophoretic techniques. Mustard seed storage protein consists of three fractions (1) a “legumin-like” 13-S complex composed of two pairs of disulfide-linked polypeptides (16.5 + 28.5 kDa and 19.5 + 34 kDa, respectively) and two single polypeptides (18 kDa and 26 kDa), (2) a “vicilin-like” 9-S complex composed of two glycoproteins (64 kDa and 77 kDa), and (3) two small polypeptides (10 kDa and 11 kDa) which probably represent the 1.7-S complex found in other Cruciferae. In contrast to related species, no glycosylated polypeptide was found in the 13-S complex. Immunological relationships were found between the paired polypeptides of the 13-S complex but not between polypeptides of the 13-S complex and polypeptides of the 9-S complex. Pulse-chase labeling and in vitro translation of polysomal RNA from young embryos demonstrated that the polypeptides of the 13-S complex originate from high molecular mass precursors, except for the 18 kDa polypeptide which appears to be synthesized in its final size. The amino-acid composition of the major polypeptides of the mustard storage protein is given.     

Tobacco embryogenesis: Storage-protein-accumulating cells of embryo, suspensor, and endosperm are able to undergo cytokinesis

Summary It is widely accepted that seed storage proteins accumulate only in cells which have entered the cell expansion phase and do not continue to divide. Here we present data indicating that the accumulation of storage globulins in tobacco begins already during early embryogenesis in a period of sustained mitotic activity. Western blot analysis revealed that polypeptides of the legumin-like 12S globulins (M_r 60000, 40000, 20000) appear at mid/late globular stage, whereas the vicilin-like 7S globulin (M_r 50000) follows during the transition from heart to torpedo stage. The accumulation of legumin-like polypeptides begins first in the endosperm during the mid globular stage followed in the embryo-suspensor complex during the heart-shaped stage. The vicilin-related fraction appears first in the endosperm and three days later in the embryo. Examination of individual cells from squash preparations revealed that protein bodies are not confined to intermitotic cells, but are also present in cells undergoing mitosis. Protein bodies of dividing cells situated outside the mitotic apparatus are not metabolized during cytokinesis. The only cell type which loses its protein bodies completely prior to the first mitotic division is the primary hypophysis cell. Our finding that storage proteins can occur in dividing cells independent of their origin and developmental capacity indicates that the cell expansion hypothesis of storage protein accumulation has to be revised.     

Production of synthetic seeds by encapsulating gametophytes in Matteuccia struthiopteris,edible ferns

In Vitro Cellular & Developmental Biology - Plant - Matteuccia struthiopteris (L.) Tod. is one of the edible ferns consumed globally. However, efficient M. struthiopteris propagation methods...     

L-O-Caffeoylhomoserine from Matteuccia struthiopteris

A caffeic acid derivative was isolated from Matteuccia struthiopteris (ostrich fern) as a major radical scavenger. The compound consisted of caffeic acid and L-homoserine. NMR and MS analysis revealed the structure as L-O-caffeoylhomoserine.     

Homology between legumin-like polypeptides from cereals and pea.

The presence of legumin-like constituents within the globulin fractions of wheat (Triticum aestivum), rye (Secale cereale) and corn (maize, Zea mays) was demonstrated. Two-dimensional analysis of wheat globulins in the presence and absence of a reducing agent revealed the existence of reducible approximately 60 kDa polypeptides. Western-blot analysis with 125I-labelled antibodies raised against the oat (Avena sativa) 12S globulin holoprotein or its alpha-subunits demonstrated, firstly, the immunological homology between the alpha- and beta-subunits of pea (Pisum sativum) legumin and oat 12S globulin, and secondly, the similar occurrence in wheat of antigenically homologous approximately 20kDa and approximately 40 kDa polypeptides that associate via disulphide linkage to form approximately 60 kDa dimers. Western blotting also showed the presence of disulphide-linked approximately 20 kDa and approximately 40 kDa legumin-like subunits within the globulin fractions of rye and corn.     

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     

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     

山东植物2个新记录属

报道了山东植物2个新记录属:荚果蕨属(Matteuccia Todaro)及其新记录种荚果蕨(Matteuccia struthiopteris(Linnaeus)Todaro),觿茅属(Dimeria Robert Brown)及其新记录种觿茅(Dimeria ornithopoda Trinius)。凭证标本均存放于山东中医药大学药学院标本室(SDCM)。     

Fungal ferritins: The ferritin from mycelia of Absidia spinosa is a bacterioferritin

Two distinct ferritin like iron containing proteins have been identified and isolated from the fungus Absidia spinosa; one from the spores and another from the mycelia. The mycelial protein has been purified and consists of two subunits of approx. 20 kDa. The N-terminal sequences of both subunits have been determined. The holoprotein as isolated contains approx. 750 iron atoms/molecule and exhibits a heme-like UV-Vis spectrum. Based on the heme spectrum and the high degree of sequence homology found, it has been established that the mycelial protein is a bacterioferritin. This is the first example demonstrating the presence of a bacterioferritin in a eukaryotic organism.     

Vicilin-like seed storage proteins in the gymnosperm interior spruce (Picea glauca/engelmanii)

A seed storage protein cDNA was characterized from a library of interior spruce (Picea glauca/engelmanii complex) cotyledonary stage somatic embryos. The deduced amino acid sequence predicts a 448 amino acid (50 kDa) polypeptide with 28–38% identity with angiosperm vicilin-like 7S globulins. XXC/G codon usage is low (47%) relative to monocot angiosperms while pairwise comparisons show that spruce, monocot, and dicot vicilins are approximately equal in amino acid divergence. Although small by comparison, the spruce vicilin contains an N terminal hydrophilic region characteristic of angiosperm large vicilins. Genomic Southern blotting predicts that the cDNA is encoded by a gene family.     

SPORE GERMINATION AND DEVELOPMENT OF THE YOUNG GAMETOPHYTE OF THE OSTRICH FERN (MATTEUCCIA STRUTHIOPTERIS)

Light is required for the germination of spores of Matteuccia struthiopteris. Histochemical studies show that dormant spores contain no starch, but have an abundance of storage protein granules. Starch accumulates in the numerous chloroplasts of the spore on exposure to light and becomes gradually more extensive. Protein granules disappear as germination progresses. Following this, the centrally located nucleus migrates toward the proximal spore face. Concomitant with the nuclear migration, an increase of cytoplasmic RNA surrounding the nucleus occurs. An equal nuclear division and unequal cell division give rise to a 2-celled gametophyte consisting of a large prothallial cell and smaller rhizoidal cell. A new peripheral wall forms around the entire protoplast at the time of nuclear migration, while a transverse wall forms after nuclear division. The rhizoid emerges through the split raphe along the proximal spore face; it is rich in cytoplasmic RNA but contains very few chloroplasts and little starch. Electron microscopy of the 2-celled stage revealed a greater concentration of mitochondria, Golgi bodies, and a more extensive endoplasmic reticulum in the rhizoid than was found in the prothallial cell, which, however, was far richer in chloroplasts and lipid bodies. As the rhizoid elongates and becomes more vacuolated, cytoplasmic RNA decreases as cytoplasmic protein increases. The rhizoid undergoes no cell divisions, while the prothallial cell retains the potential for further cell division. The possible significance of the distribution of storage products, cell organelles, and other cell components were considered in relation to the non-equational cell division and differentiation of the 2 cells.     

Electrophoretic analysis of seed proteins in the dicotyledoneae

SDS-PAGE was used to analyze seed protein extracts from plants representing 58 orders of the Dicotyledoneae. In most seeds, includingMagnolia grandiflora, the most prevalent storage protein class appeared to be the 11 S or legumin-like proteins. These data suggest that the legumin-like proteins are ubiquitous throughout the plant kingdom. Supported by the Mississippi Agricultural and Forestry Experiment Station, Publication Number J-7788.     

Biochemical and immunocytochemical localization of a BAPAase in developing and mature lupin cotyledons

Summary Activity measurements and specific antibodies were used to detect and localize in developing and mature cotyledons ofLupinus albus seeds an endopeptidase, active on BAPA, previously isolated from the same seeds. Total activity and enzyme amount were highest at full seed maturation and then declined during germination. Protein bodies were isolated from mature dry cotyledons under anhydrous conditions with a yield of intact organelles of about 80% as assessed by dot blotting with antibodies to lupin legumin-like storage globulin. Activity assays on the isolated protein bodies indicated that 72% of BAPAase activity was associated with these organelles. Quantitative immunocytolocalization with antibodies to the enzyme on thin sections of mature lupin cotyledons confirmed that 75% of the enzyme was located inside the protein bodies. The possible involvement of the BAPAase in the proteolytic processing of the storage proteins during seed ontogeny is discussed.Abbreviations BAPA N-benzoyl-L-arginine-4-p-nitroanilide - DAF days after flowering - EM electron microscopy - NaPi sodium phosphate buffer - LRW London resin white - SDS sodium dodecylsulphate - PAGE polyacrylamide gel electrophoresis     

The evolution of angiosperm seed proteins: A methionine-rich legumin subfamily present in lower angiosperm Clades

Analysis of legumin-encoding cDNAs fromDioscorea caucasica Lipsky (Dioscoreaceae) and fromAsarum europaeum L. (Aristolochiaceae) shows that there is an especially methionine-rich legumin subfamily present in the lower angiosperm clades including the Monocotyledoneae. It is characterized by a methionine content of 3–4 mol% which is roughly triple the methionine proportion of most other legumins. These “MetR” legumins, if present, still have to be detected in the higher angiosperms including the important seed crops. Evolutionary analysis suggests that the MetR legumins are the result of a gene duplication allowing the differentiation of legumin genes according to their sulfur content. The duplication event must have taken place before the split into mono- and dicotyledonous plants but probably after the separation of angiosperms and gymnosperms. Correspondence to: H. Fischer