Uwe Jensen

Legumin-like proteins in gymnosperms

Abstract Evidence is given for the production of legumin-like proteins in the seeds of Ginkgo biloba . The M r of the heterogeneous protein and of its SS bonded acidic and basic subunits, the occurrence of two subunit dimer types (‘normal’ and ‘small’), and the electrophoretic characteristics are similar to well known angiosperm legumins. This is the first gymnosperm for which the production of legumin-like proteins has been demonstrated.

Systematics and phylogeny of the Ranunculaceae — a revised family concept on the basis of molecular data

Phylogenetic trees for the Ranunculaceae based on four independent molecular data sets are compared and analyzed. This comparison includes chloroplast DNA restriction site variation; atpB (chloroplast), rbcL, nuclear ribosomal DNA sequences (analysed as a combined data set), nuclear adh sequences, and serological detected characters of the major seed protein, legumin. These trees are highly congruent in terminal branching patterns with high support. This congruency suggests a strong correlation between the evolution of the genes and proteins investigated and the taxa involved and further demonstrates the utility of molecular markers in plant phylogeny. The molecular results are often congruent with non-molecular data as well and are used to develop more reliable systematic classification of the Ranunculaceae.

Abies seed protein profile divergent from other Pinaceae

Legumins, i.e. 11/12 S storage proteins, are the major seed proteins in the legumes Vicia faba (Bayley & Boulter, 1970), Pisum sativum (Croy & al., 1979) and in other Vicieae species (Derbyshire & al., 1976). Similar proteins with the significant legumin characters (acidic subunits of Mr = c. 30 40 KD, basic subunits of Mr = c. 20 KD, linked together by S-S-bridges and forming hexameric molecules in the storage tissues) have also been recognized as major storage proteins in many dicot and monocot species (De Klerk & Engelen, 1985; Fischer & Schopfer, 1988; Hasegawa & al., 1978; Jensen, 1984; Jensen & Bittner, 1981; Konishi & al., 1985; Simon & al., 1985). Jensen & Berthold (1989) reported legumin-like proteins even in the primary endosperm of Ginkgo biloba seeds; their structural homology with angiosperm legumin was recently confirmed by sequence analysis of the P-subunit (Hager & Richardson, in preparation). Similar proteins have been identified in conifers (Gifford, 1988; and our own data). They are mostly insoluble in buffers normally used for legumin extractions. Insoluble seed legumins are also reported for Euphorbiaceae (Gifford & Bewley, 1983; Lalonde & al., 1984) and 7Tphaceae (Bergner & Jensen, 1989). They can be extracted by adding up to 10 %o NaCl (w/v) to the buffers (Bergner & Jensen, 1989) or even better by using SDS and urea buffers. In the latter case they are extractable only in form of a,-dimers. Data from many publications plus our own results demonstrate the generalized occurrence of homologous legumin-like proteins in seed plants, both gymnosperms and angiosperms. They are the main storage proteins in a majority of taxa. They are produced in triploid (angiosperm endosperm), diploid (embryo) and/or haploid tissues (gymnosperm endosperm). In this investigation we analysed the occurrence of the insoluble legumin-like seed proteins in Pinaceae. Gifford (1988) had previously reported electrophoretic data for six species of Pinus. In the seeds of 12 Abies species tested no such proteins were detected. This lack can be used as a diagnostic character for the genus Abies.

Secondary compounds of the Ranunculiflorae

Natural products can provide additional arguments for the relationships within families of the Ranunculiflorae, although parallel evolution and loss of chemical characters probably occurred frequently. Especially the accumulation of benzylisoquinoline alkaloids is a characteristic feature, however, in Ranunculaceae restricted to Coptideae and Isopyreae; they are common in the Berberidaceae, but missing in Podophyllum and Diphylleia and probably other Berberideae. Quinolizidine alkaloids are typical products of the Cimicifugeae (Ranunculaceae) and Leonticeae (Berberidaceae). The Delphiniinae (Ranunculaceae) are characterized by diterpene alkaloids and the accumulation of mannitol. Cyanogenic compounds occur in Isopyreae (Ranunculaceae), but they are observed elsewhere in the Ranunculiflorae. Ranunculins are present only in the Ranunculeae and Anemoneae and additionally in Helleborus (all Ranunculaceae). Lignans are accumulated in the closely related Podophyllum and Diphylleia, but also in Epimedium (all Berberidaceae). For the Fumariaceae the free amino acid acetylornithine is suspected to be the main nitrogen transport molecule.

Chloroplast DNA restriction site polymorphism inGenisteae (Leguminosae) suggests a common origin for European and American lupines

Restriction site polymorphism in cpDNA of 35 legumes was studied in order to address natural relationships and geographic distribution within the tribeGenisteae. 386 sites were studied, 277 were polymorphic, 207 were informative. Phylogenetic inferences with distance and parsimony methods suggest that the American and MediterraneanLupinus species belong to a monophyletic group which arose from a single center of diversification. The data furthermore indicate thatLupinus should not be included in the tribeGenisteae since at the level of cpDNA polymorphismAnagyris foetida (tribeThermopsideae) appears more closely related to otherGenisteae thanLupinus does.

The N-terminal amino acid sequence of the β-subunit of the legumin-like protein from seeds of Ginkgo biloba

The sequence of the first 52 amino acids at the N-terminus of the beta-subunit of a legumin-like protein from seeds of the gymnosperm Ginkgo biloba were determined by automated sequencing and DABITC/PITC microsequence analyses of peptides derived from the protein by enzymatic digestions and chemical cleavage with CNBr. The protein from Ginkgo exhibits sequence homologies (32-49% identities) with the 11S globulins and legume-like proteins from seeds of various angiosperm monocotyledons and dicotyledons.

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.

Legumin-like and Vicilin-like Storage Proteins in Nigella damascena (Ranunculaceae) and six other Dicotyledonous Species

A legumin-like protein and a vicilin-like protein are the essential storage proteins in dicotyledonous plants. Similar, but not identical physicochemical characteristics, as well as serological cross-reactivity, demonstrate the homology of each of these proteins in seeds of different plant taxa. Instead of the native 50,000 type subunit of the vicilin-like proteins and the 40,000 and 20,000 type subunits of the legumin-like proteins, smaller polypeptides have been observed in consequence of temperature, storing conditions or purification procedures. Even proteins from freshly harvested seeds appear to have been partly altered by environmental conditions.

Serological legumin data and the phylogeny of the Ranunculaceae

Legumin, the main seed storage protein in Ranunculaceae, was isolated from species of 20 genera (19 belong to Ranunculaceae, one to Papaveraceae) and used to elicit monospecific antisera. The precipitation bands and the presence of spurs were observed in Ouchterlony two-dimensional double diffusion tests. These data were used to calculate similarity indices as well as neighbor joining and UPGMA phylogenetic trees. Seven groups of closely related genera could be detected, i.e., Anemone/Hepatica/Clematis; Ranunculus/ Ficaria; Adonis/Trollius/Trautvetteria; Cimicifuga/Actaea/Caltha; Aconitum/ Delphinium/ Consolida; Aquilegia/Thalictrum; Xanthorhiza/Coptis. They support the classifications based on other molecular data, and correlate with both morphological and chemical data. This strengthens the status as monophyletic groups.

Proteins in Plant Evolution and Systematics

There are scarcely any characters in systematics that have generated more controversy during their long period of application than that of the proteins. On the one hand their high a priori significance tempts an experimentator to extend, often inadmissibly, the a posteriori significance of his results. While, on the other hand, exaggerated criticism may arise when results from comparative protein experiments are at odds with the current concepts of relationships. An example of this comes from amino acid sequencing, where initial reflections (BOULTER et al., 1970) have led to a too optimistic view, some a posteriori considerations (CRONQUIST, 1976), however, to an underestimation of its significance in systematics.