Storage globulins in Gnetopsida. 1. Recognition of legumin-like proteins

Abstract

Salt soluble storage proteins were extracted from seeds of Ephedra distachya, Ephedra foeminea, Gnetum gnemon, Gnetum montanum and Welwitschia mirabilis and separated by chromatographic procedures. The molecular weight of the main storage globulin ranges from 300 to 350 kD. Denaturation by SDS resolved the holoprotein in monomers of M_r 40 to 60 kD. Oligomers up to 120 kD were observed in Ephedra. Reduction of disulphide bridges by DTE resolved the monomers in paris of polypeptides of M_r 10 to 35 kD. The characters above indicate that the main storage globulin of Gnetopsida is a legumin-like protein.     

C-GLYCOSYLFLAVONES IN THE GNETOPSIDA: A PRELIMINARY REPORT

C-Glycosylflavones appear to be the only detectable flavonoids in representative species of Gnetum, Ephedra and Welwitschia. Ephedra antisyphilitica contains mixed 6,8-di-C-glycosides of apigenin and luteolin and Welwitschia mirabilis produces 6-C-glycosylchrysoeriol and 6,8-di-C-glycosylchrysoeriol. Recently it was reported that Gnetum gnemon contains 6-C-, 8-C and 6,8-di-C-glucosyl derivatives of apigenin, luteolin and their 7-0-methyl ethers. Based on the present preliminary survey the apparent absence of other flavonoid types indicates that C-glycosylflavones may be a unifying characteristic of the Gnetopsida.     

Phylogenetic relationships in gnetophyta deduced fromrbcL gene sequences

Part of the large subunit of the ribulose-1,5-bisphosphate carboxylase gene (rbcL) was sequenced (1333 base pairs) from three species of gymnosperms:Ephedra sinica Gnetum parvifolium, Welwitschia mirabilis. Phylogenetic trees inferred from the neighbor joining, Wagner parsimony and maximum likelihood methods showed thatGnetum andWelwitschia were more closely related to each other than either is toEphedra within Gnetophyta, and the result supports previous cladistical analysis of morphological data.     

C-glycosylflavones of the gnetopsida

C-Glycosylflavones have been identified in Ephedra antisyphilitica, Gnetum gnemon and Welwitschia mirabilis. The C-glycosidic moieties of apigenin and luteolin derivatives have been identified as glucose and/or xylose for these species.     

The exine ultrastructure of pollen grains in Gnetum (Gnetaceae) from China and its bearing on the relationship with the ANITA Group

Pollen grains of six species of Gnetum , G. parvifolium , G. hainanense , G. luofuense , G. pendulum , G. cleistostachyum and G. montanum , collected from China were examined using light, scanning and transmission electron microscopy. Pollen grains of Gnetum are subspheroidal or irregular-apolar, inaperturate, 11.21–22.44 µm in long axis and 9.34–20.47 µm in short axis. The exine surface is covered with spinules, 0.50(0.30–0.71) µm long spaced on average 1.12(0.81–1.46) µm apart. The exine is about 0.55 µm thick and comprises ectexine and endexine. The ectexine includes a thin tectum and an infratectal granular layer. The tectum protrudes outwards, forming the spinules. The endexine is composed of discontinuous lamellae, with lacunae between lamellae. The pollen grains of Gnetum are compared with those of Ephedra and Welwitschia , and also those of the ANITA Group of angiosperms, including Amborellaceae, Nymphaeales, Illiciales, Trimeniaceae and Austrobaileyaceae. The exine ultrastructures of Gnetum , Ephedra and Welwitschia are quite similar, consisting of tectum, granular layer and lamellated endexine. The exine ultrastructure of Gnetum is also similar to that of Nymphaea colorata (Nymphaeaceae) in the transitional region between the proximal and distal poles, but differs from that of Amborellaceae, Illicium religiosum (Illiciaceae), Schisandra (Schisandraceae), Trimeniaceae and Austrobaileyaceae. This comparison of exine ultrastructure provides new evidence for consideration of the relationship between Gnetum and the ANITA Group.  © 2004 The Linnean Society of London, Botanical Journal of the Linnean Society , 2004, 146 , 415–425.     

Sequence Peculiarity of Gnetalean Legumin-Like Seed Storage Proteins

The development of seeds as a specialized organ for the nutrition, protection, and dispersal of the next generation was an important step in the evolution of land plants. Seed maturation is accompanied by massive synthesis of storage compounds such as proteins, starch, and lipids. To study the processes of seed storage protein evolution we have partially sequenced storage proteins from maturing seeds of representatives from the gymnosperm genera Gnetum, Ephedra, and Welwitschia—morphologically diverse and unusual taxa that are grouped in most formal systems into the common order Gnetales. Based on partial N-terminal amino acid sequences, oligonucleotide primers were derived and used for PCR amplification and cloning of the corresponding cDNAs. We also describe the structure of the nuclear gene for legumin of Welwitschia mirabilis. This first gnetalean nuclear gene structure contains introns in only two of the four conserved positions previously characterized in other spermatophyte legumin genes. The distinct phylogenetic status of the gnetalean taxa is also reflected in a sequence peculiarity of their legumin genes. A comparative analysis of exon/intron sequences leads to the hypothesis that legumin genes from Gnetales belong to a monophyletic evolutionary branch clearly distinct from that of legumin genes of extant Ginkgoales and Coniferales as well as from all angiosperms. Received: 5 June 1997 / Accepted: 31 March 1998     

A fossil-calibrated relaxed clock for Ephedra indicates an Oligocene age for the divergence of Asian and New World clades and Miocene dispersal into South America

Ephedra comprises approximately 50 species, which are roughly equally distributed between the Old and New World deserts, but not in the intervening regions （amphitropical range）. Great heterogeneity in the substitution rates of Gnetales （Ephedra, Gnetum, and Welwitschia） has made it difficult to infer the ages of the major divergence events in Ephedra, such as the timing of the Beringian disjunction in the genus and the entry into South America. Here, we use data from as many Gnetales species and genes as available from GenBank and from a recent study to investigate the timing of the major divergence events. Because of the tradeoff between the amount of missing data and taxon/gene sampling, we reduced the initial matrix of 265 accessions and 12 loci to 95 accessions and 10 loci, and further to 42 species （and 7736 aligned nucleotides） to achieve stationary distributions in the Bayesian molecular clock runs. Results from a relaxed clock with an uncorrelated rates model and fossil-based calibration reveal that New World species are monophyletic and diverged from their mostly Asian sister clade some 30 mya, fitting with many other Beringian disjunctions. The split between the single North American and the single South American clade occurred approximately 25 mya, well before the closure of the Panamanian Isthmus. Overall, the biogeographic history of Ephedra appears dominated by long-distance dispersal, but finer-scale studies are needed to test this hypothesis.     

Double fertilization in Gnetum gnemon (Gnetaceae): its bearing on the evolution of sexual reproduction within the Gnetales and the anthophyte clade

The fertilization process in Gnetum is critical to our understanding of the evolution of sexual reproduction within the Gnetales, a monophyletic group of nonfiowering seed plants that are the closest living relatives to flowering plants. Although much is known about the fertilization process in Ephedra, which is basal within the Gnetales, little is known about sexual reproduction in the derived sister groups Gnetum and Welwitschia. Ovules of Gnetum gnemon were collected at various stages after hand pollination and processed for light, fluorescence, and electron microscopy. Approximately 5 d after pollination, pollen tubes reach sexually mature female gametophytes, which are coenocytic. At that time, a binucleate sperm cell is found within each pollen tube. Within 7 d of pollination, double fertilization events occur when each of two sperm nuclei released from a pollen tube fuses with a separate, undifferentiated female nucleus within the free nuclear female gametophyte, which lacks differentiated egg cells. The products of double fertilization are two viable zygotes; endosperm is not formed. The lack of differentiated egg cells in Gnetum gnemon is unparalleled among land plants and the documentation of a regularly occurring process of double fertilization is congruent with the hypothesis that a rudimentary process of double fertilization evolved in a common ancestor of angiosperms and Gnetales.     

Pollenkitt is lacking inGnetatae:Ephedra andWelwitschia; further proof for its restriction to the angiosperms

Three members of theGnetatae (Ephedra campylopoda, E. americana, Welwitschia mirabilis) were investigated by TEM and SEM with respect to their anther tapetum and pollen development. In all three species pollenkitt is lacking. The pretended pollen stickiness thus does not depend on pollenkitt. Considering former observations one can now clearly state that pollenkitt is missing in all recent gymnosperm classes (both anemophilous and ± entomophilous). Pollenkitt thus is restricted and ± omnipresent within the angiosperms, where it represents one of the most important components of the entomophily syndrome. This can be regarded as important proof for the hypothesis that the angiosperms are a single coherent phylogenetic group.     

Phylogeny of gymnosperms inferred fromrbcL gene sequences

Partial nucleotide sequences of the large subunit of ribulose-1,5-bisphosphate carboxylase (rubisco) gene (1333 base pairs: about 90% of the gene) from several seed plants were determined. Phylogenetic trees based on amino acid sequences were inferred by using the neighbor joining and maximum likelihood methods. The results indicate (1) monophyly of gnetum group (Ephedra, Gnetum, Welwitschia), (2) monophyly of extant gymnosperms containing gnetum group, which contradicts the results of morphological data.     

Former diversity of Ephedra (Gnetales): evidence from Early Cretaceous seeds from Portugal and North America

BACKGROUND AND AIMS: The extant species of the seed plant group Gnetales (Ephedra, Gnetum and Welwitschia) have been considered a remnant of a much greater, now extinct, diversity due to the pronounced differences in form and ecology among the genera. Until recently, this hypothesis has not been supported by evidence from the fossil record. This paper adds to the expanding information on Gnetales from the Early Cretaceous and describes coalified seeds from Barremian-Albian localities in Portugal and USA. METHODS: The fossils were extracted from sediment samples by sieving in water. Adhering mineral matrix was removed by chemical treatment. Seeds were investigated using light and scanning electron microscopy. Morphology and anatomy of the seeds were documented and compared with those of extant species. KEY RESULTS: The fossils share characters with extant Ephedra, for example papillae on the inner surface of the seed envelope and in situ polyplicate pollen grains that shed the exine during germination. They differ from extant Ephedra seeds in morphological and anatomical details as well as in their smaller size. Two new species of Ephedra are described together with one species assigned to a new genus of Gnetales. Other Ephedra-like seeds, for which pollen and critical morphological details are currently unknown, are also present in the samples. CONCLUSIONS: These Cretaceous seeds document that key reproductive characters and pollen germination processes have remained unchanged within Ephedra for about 120 million years or more. There is sufficient variety in details of morphology to suggest that a diversity of Ephedra and Ephedra-like species were present in the Early Cretaceous flora. Their presence in Portugal and eastern North America indicates that they were widespread on the Laurasian continent. The fossil seeds are similar to seeds of Erdtmanithecales and this supports the previously suggested relationship between Erdtmanithecales and Gnetales.     

Wood and bark anatomy of the African species of Gnetum

Quantitative and qualitative data are given for the two African species of Gnetum ( Gnetum section Gnetum subsection Micrognemones ). These species are lianoid and lack the fibre-tracheids of G. gnemon but have about the same vessel element and tracheid length as in that species. Vessel diameter is related to stem age and organography. Tori are clearly present in tracheary elements of the African Gnetum species, a first report for the genus. In these two species, origin of the lateral meristem, which produces vascular tissue and cambia, can be traced directly and indirecdy to cortical parenchyma. A second kind of meristematic action, newly reported for Gnetum, is produced by proliferation of axial parenchyma, fragmenting secondary xylem. Both presence of tori and site of origin of lateral meristematic activity in Gnetum contrast with corresponding conditions in Welwitschia.     

Environmental control of crassulacean acid metabolism in Welwitschia mirabilis Hook. Fil. in its range of natural distribution in the Namib desert

Summary Within the area of its natural distribution in South West Africa, Welwitschia mirabilis has a less negative ¹³C value than C₃ plants and a more negative ¹³C value than C₄ species. This indicates that Welwitschia m. assimilates CO₂ partially via CAM when growing in its natural habitat. The difference between the ¹³C values of Welwitschia m. and of the C₃ species is significant in the savanna, whereas it is only small and statistically not significant in the grassland zone. The proportion of CO₂ fixed via CAM is largest in the coastal desert zone. There was no correlation between the ¹³C values and the Cl^- or ash content of the tissue. Thus, CAM in Welwitschia m. seems not to be induced by salt stress. There is no change in the ¹³C values along the persistent Welwitschia m. leaf. The present data indicate that on a broad geographical scale in the area of distribution temperature regime, and water stress as a modifying factor, determine CAM in Welwitschia m. The ecological implications are discussed by comparing the behaviour of Welwitschia m. with other CAM, C₃ and C₄ species of the accompanying flora.     

Morphogenesis of the reproductive shoots of Welwitschia mirabilis and Ephedra distachya (Gnetales), and its evolutionary implications

For decades, Gnetales appeared to be closely related to angiosperms, the two groups together forming the anthophyte clade. At present, molecular studies negate such a relationship and give strong support for a systematic position of Gnetales within or near conifers. However, previous interpretations of the male sporangiophores of Gnetales as pinnate with terminal synangia conflict with a close relationship between Gnetales and conifers. Therefore, we investigated the morphogenesis of the male reproductive structures of Welwitschia mirabilis and Ephedra distachya by SEM and light microscopy. The occurrence of reduced apices to both halves of the antherophores of W. mirabilis gives strong support for the assumption that the male ‘flowers’ of W. mirabilis represent reduced compound cones. We assume that each half of the antherophore represents a lateral male cone that has lost its subtending bract. Although both halves of the antherophores of Ephedra distachya lack apical meristems, the histological pattern of the developing antherophores supports interpreting them as reduced lateral male cones as well. Therefore, the male sporangiophores of Gnetales represent simple organs with terminal synangia. Although extant conifers do not exhibit terminal synangia, similar sporangiophores are reported for some Cordaitales, the hypothetical sister group of conifers. Moreover, several Paleozoic conifers exhibit male cones with terminal sporangia or synangia. Therefore, we propose that conifers, Cordaitales and Gnetales originated from a common ancestor that displayed simple sporangiophores with a terminal cluster of sporangia.     

Epidermal Structure and Ontogeny of Stomata in Vegetative and Reproductive Organs of Ephedra and Gnetum

The epidermal structure and development of stomata in vegetativeand reproductive organs of Ephedra foliata and Gnetum ula isdescribed. The epidermal cells are polygonal, isodiametric,or elongated with thick or thin straight, arched, or slightlysinuous anticlinal walls. The cuticle is thin or thick. Papilla-likeunicellular outgrowths are present in Ephedra foliata. The maturestomata are orientated parallel to the longitudinal axes orirregularly. The mature stomata are anomocytic, paracytic, witha single subsidiary cell, cyclocytic, and actinocytic. Arresteddevelopment, contiguous stomata, and stomata with aborted guardcells have been observed. The ontogeny of stomata on differentorgans of these two plants is typically haplocheilic or perigenousbut the stomatal apparatus varies from organ to organ.     

Wood, bark and stem anatomy of New World species of Gnetum

Quantitative and qualitative data are presented for 11 collections of six species. The wide range of character states for the species is presented in the form of a key to emphasize their potential systematic correlations. Distinctive among these are phcllem characteristics. Fibre-tracheids are newly reported for lianoid Gnetum species. Cells previously thought to be like companion cells in secondary phloem are shown to be uniseriate rays, counterparts to uniseriate xylem rays. Laticifers are abundant in most of the species, and are newly described for secondary tissues of Gnetum. Presence of tyloses in laticifers of two species is apparently a new report for vascular plants. Tori are present in two New World Gnetum species, adding to the report in African species. Perforation plates are simple except near or in primary xylem, where they are simple or foraminate. Torus presence and foraminate perforation plate presence are features more reminiscent of Ephedra and other gymnosperms than of angiosperms. The bark of Gnetum is also very similar to that of Ephedra.     

Synthese von Pigmenten und Entwicklung des Photosyntheseapparates in einigen Höheren Pflanzen

Die Synthese von Pigmenten und die Entwicklung des Photosyntheseapparates wurde an Keimlingen von Ephedra distachya und Welwitschia mirabilis untersucht, die im Dunkeln bzw. im Licht angezogen wurden. Zum Vergleich wurden Keimlinge der “typischen” Gymnosperme Pinus silvestris untersucht. Die Chlorophyllbildung bei Keimlingen von Welwitschia gleicht derjenigen der Angiospermen, sie synthetisieren Chlorophyll nur im Licht und entwickeln parallel dazu einen funktionsfähigen Photosyntheseapparat. Ephedra bildet ebenso wie die Gymnosperme Pinus Chlorophyll auch im Dunkeln. Die Dunkelsynthese von Chlorophyllen bei Pinus und Ephedra wird nach Überführung ins Licht (nach einer eintägigen lag-Phase) erhöht fortgesetzt. Dunkelangezogene Keimlinge zeigen keine Photosynthesekapazität. Erst nach 5 bis 10 Minuten Dauerlicht beginnt die Sauerstoffentwicklung. Wird die photosynthetische O₂-Entwicklung während der weiteren Ergrünung auf gleichen Chlorophyllgehalt bezogen, zeigt sich während der ersten beiden Tage im Licht ein steiler Anstieg, gefolgt von einem kontinuierlichen Abfall. Dieser wird hervorgerufen durch bevorzugte Synthese von Antennenchlorophyll, welches für eine bessere Ausnutzung von Licht geringerer Intensität sorgt. In der Chlorophyllbiosynthese und der Entwicklung des Photosyntheseapparates folgt Welwitschia den Charakteristika der Angiospermen, während Ephedra ausgesprochene Gymnospermenmerkmale zeigt. Wir danken Prof. Dr. C. H. Bornman und der Staatsdarre Wolfgang (Hanau) für die Überlassung von Samen von Welwitschia mirabilis bzw. Pinus silvestris und Frau I. Krieger und Herrn H. Becker für wertvolle Hilfe bei der Anfertigung des Manuskriptes.     

Leaf‐morphology and leaf‐anatomy in Ephedra altissima Desf. (Ephedraceae,Gnetales) and their evolutionary relevance

Leaves in most extant Ephedra ‐species represent only rudimentary scales without chlorophyll. Photosynthesis is completely restricted to green shoots. Only some species, e.g. Ephedra altissima, develop folious leaves even when being mature. Morphology and anatomy of cotyledons, primary leaves, and subsequent folious leaves of Ephedra altissima were examined particularly with special focus on their vasculature. The results show that the reduction of the leaves is achieved by an extreme reduction of the adaxial leaf surface. Most parts of the leaf surface are therefore developed by the abaxial side. Apart from a reduced lamina, a thick cuticle and sunken stomata are the only xerothermic adaptations in leaves of Ephedra altissima. A hypodermis and endodermis are also not developed. The anatomical results indicate that ancestors of modern Ephedra‐ species might have evolved in a more humid climate, contrasting to the arid habitats in which extant Ephedra‐ species are native today. (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)