Observations on the fine structure of the root nodule endophyte of Hippophaë rhamnoides L.

Summary Electron microscopy of ultra-thin sections of Hippophaë rhamnoides root nodules has been carried out in order to elucidate the nature of the endophyte. The organism is seen as a branching, septate filament approximately 0.6 microns in diameter bearing on its terminal ends spherical sub-divided vesicles 3–4 microns in diameter. In the mature nodule the vesicles are the most prominent endophyte form and appear to be formed by swelling of the hyphal tips. It is concluded that the endophyte is an actinomycete closely related to but not identical with that of Alnus glutinosa.     

Feedback regulation of nodule formation in Hippophaë rhamnoides

Seedlings of Hippophaë rhamnoides possessing two equally infectible root systems (split roots) were used in conjunction with specific Frankia strains to investigate plant control over nodulation. When a wild-type Frankia strain was inoculated onto both root systems simultaneously or 1, 2, 4, or 8 weeks apart, an inhibitory response occurred which retarded nodulation on the root exposed to the delayed inoculum. Similar suppressive responses were also observed when two different wild-type Frankia inocula were applied onto opposite sides of a split-root system at different times. The depressed response shown by the delayed inoculum was more pronounced as the delay period was increased. The roots exposed to the delayed inoculum displayed a complete lack of nodulation when the delay was 4 or 8 weeks. The nodulation response on the root inoculated first depended on subsequent inoculation of the second root system of the plant, so that maximum nodulation of the first root was observed when the second root was unnodulated. These results provide evidence that sea buckthorn has an active, systemic mechanism for feedback control of nodulation that suppresses further nodule formation and prevents excessive nodulation. The significance of these results to the understanding of nodule ontogeny is discussed.     

Occurrence and infective potential of the endophyte ofHippophaë rhamnoides L. ssp.rhamnoides in coastal sand-dune areas

Summary The infective potential (IP) of nodule homogenates from field-grownHippophaë rhamnoides L. ssp.rhamnoides was determined by counting the number of nodules formed on test plants after inoculation with various dilutions of the homogenates. The IP was almost constant,i.e. 10⁵ to 10⁶ per gram of fresh nodule material. Methods to store nodule material without loss of IP were tested. The IP of air-dried nodule powders stored at 6°C hardly decreased during a period of more than a year.Data are presented on the IP of soil samples from sites representing various stages of dune formation. BeforeH. rhamnoides appeared, the IP was low: 1 to 36 nodules were formed on test plants per kg of soil. This low IP was due to low numbers of infective endophyte particles in these soils. During the succession of theH. rhamnoides scrub, the IP of the soil increased, due to the increase in the number of endophyte particles in the soil. Gradually, however, nodulation was limited by other environmental factors. The nature of these factors is discussed.     

The of gamma-radiation for induction of apomixis in sea-buckthorn (Hippophaë rhamnoides L.)

The results of studying of the induced apomixis in pollen of sea-buckthorn irradiated by the 60Co gamma-Radiation are considered. Was established that the most effective dose for pollination of the experimental plants is 50 k Gy. In total, from 46 seedlings 19 cases of apomictic origin were revealed, 7 individuals were found to be haploid (n = = 12) and 19 ones were diploid (2n = 24) of maternal origin. Was supposed that apomictic plants (19 seedlings) have parthenogenetic origin. The reason for such conclusion is that the irradiated anomalous pollen tubes despite not having spermia, are entering embryo sac and stimulate the development of apomictic embryo from non-fertilized female gametes. Apparently, pollen tubes cause the induction of DNA replication in the ovules and the development of parthenogenetic plants. Consequently, the described method can be used for the regulation of parthenogenesis in sea-buckthorn to change natural ratio (1 : 1) of male to female plants in desirable quantity.     

Nodulation and nitrogen-fixation byHippophaë rhamnoides L. in the field

Summary 1. The extent of nodulation and nitrogen fixation by a population ofHippophaë rhamnoides bushes occurring on a sand dune system on the east coast of England has been studied.2. Nodules were found on all plants (the latter ranged from 3 to 16 years in age) and using the isotope N¹⁵ it was confirmed that these nodules fixed nitrogen. The nodules on the youngest plants were most active in fixation but because of the greater total nodule mass older plants fixed the largest quantities of nitrogen.3. Large increases in total nitrogen (i.e. soil plus plant nitrogen) occurred with increase in age of the plants, such nitrogen increases ranging from 27 kg per hectare per annum in the presence of bushes 0 to 3 years old to 179 kg per hectare per annum in the presence of bushes 13 to 16 years old.4. The N¹⁵-technique indicated that a proportion of the nitrogen accumulated was due to fixation byHippophaë but nitrogen contributions from other sources could not be ruled out.5. The levels of nitrate-nitrogen and ammonium-nitrogen in the soil underHippophaë showed a marked seasonal variation, increasing to a maximum in the winter and to a minimum in the summer. These levels were in general inversely related to the level of average monthly rainfall. Because of the low levels of combined nitrogen in the soil during the summer months it seems unlikely that combined nitrogen should markedly inhibit fixation in this situation.     

Die vordringlichsten Zuchtziele bei Sanddorn (Hippophaë rhamnoides L.)

Ohne ZusammenfassungMit 9 TextabbildungenHans Lembke zum 75. Geburtstag.     

Inositols and methylinositols in sea buckthorn (Hippophaë rhamnoides) berries

Sea buckthorn (Hippophaë rhamnoides L.) berries, especially of ssp. sinensis, contain significant quantities of an unknown, water-soluble compound, evidently a cyclitol derivative. The compound was isolated by HPLC and analyzed by GC–MS [trimethylsilyl (TMS) derivative, selected ion monitoring (SIM) and total ion chromatogram (TIC) analyses], by ¹H and ¹³C NMR and by optical activity measurements. The results together with analyses of reference compound verified the unambiguous structure (?)-2-O-methyl-l-chiro-inositol (l-quebrachitol). In addition, chiro-inositol and myo-inositol existing in trace amounts were identified based on reference compounds, chromatographic data and mass spectra of the TMS derivatives. Methyl-myo-inositol was tentatively identified based on chromatography and mass spectrometry. Inositols and methyl inositols are bioactive compounds essential for regulating physiological processes of plants and humans. To our knowledge, this is the first report on the presence of chiro-inositol and myo-inositol in sea buckthorn and l-quebrachitol in edible berries. The identification of the inositols and l-quebrachitol in sea buckthorn may bring new insights into the sensory properties and also mechanisms behind the health effects of the berry.     

Nematodes (Longidorus sp. andTylenchorhynchus microphasmis loof) in growth and nodulation of sea buckthorn (Hippophaë rhamnoides L.)

Summary Hippophaë rhamnoides seedlings were grown in sterilized and unsterilized soil from a decliningH. rhamnoides scrub, to which different numbers ofLongidorus sp. andTylenchorhynchus microphasmis were added. In sterilized and unsterilized soil, retardation of growth, content of dry matter in the shoots, and incidence of deformed short lateral roots of test plants were positively correlated with counts of both nematode species. Nitrogen content in the shoots, nodulation on the roots of test plants and increase increase in nematodes were negatively correlated with the initial number of both nematode species in sterilized soil. In unsterilized soil, an unknown biotic factor was present that reduces growth ofH. rhamnoides, nodulation and multiplication of the nematodes. This factor seems to interact with the nematodes in reducing growth ofH. rhamnoides.Deceased.     

Changes in Triacylglycerol Composition in Maturing Sea Buckthorn (Hippophaë rhamnoides) Seeds

Sea buckthorn (Hippophaë rhamnoides L.) seeds on the 29th, 53rd, 80th, and 107th day after pollination were used for determining, by lipase hydrolysis, the qualitative and quantitative composition of the triacylglycerol (TAG) positional types, groups, and positional species, as well as the factor of selectivity of incorporation of unsaturated fatty acids, octadecenoic, linoleic, and linolenic, into the sn-2-position of TAGs. Until the 80th day after pollination, there was a predominant formation of triunsaturated TAGs, which included linolenic and linoleic acid residues. After the 80th day, the absolute content of these major components of total TAGs markedly decreased, and an increase in total TAG content was mainly accounted for by the rise in the level of those TAG species, which included saturated fatty acids, palmitic and stearic (monosaturated–diunsaturated and disaturated–monounsaturated), as well as in the level of sn-2-octadecenoyl species belonging to the triunsaturated and palmito–diunsaturated types of TAGs. At each maturation stage, the quantitative dynamics of separate TAG species was determined by the content of fatty acid species available for TAG formation and the factor of selectivity of these species. The decrease in the content of a certain group of triunsaturated TAGs found here seems to be caused by their metabolization during seed maturation.     

Factors affecting growth and nodulation ofHippophaë rhamnoides L. ssp.Rhamnoides in soils from two successional stages of dune formation

Summary To explain the decline of Hippopha? scrub in the vegetation succession in the dunes of The Netherlands, the growth and nodulation of Hippopha? plants grown in pots, using soil from an early stage (site AH) and a post-optimum stage (site HP), were investigated. In HP-soil nodulation, yield, and the nitrogen and phosphorus content of test plants were always lower and the number of necrotic nodules and the dry matter content were always higher than in AH-soil, even after inoculation with crushed nodules and the addition of a nutrient solution. Plants in HP-soil also had darker roots, less root hairs, a higher number of short lateral roots and a higher percentage of dead roots than those in AH-soil. These characteristics of adverse growth conditions disappeared upon ignition or gamma-irradiation of HP-soil. Possible explanations of these results are discussed. The degeneration of Hippopha? scrub cannot be ascribed to the age of the plants, the absence of sufficient infective endophyte particles or to abiotic factors such as unfavourable physical (particle size) or chemical soil conditions but is caused by biotic factors. No indications were obtained that plant-pathogenic fungi and bacteria are involved. HP-soil in contrast to AH-soil, however, contained large numbers of the nematodeLongidorus sp., a species known to cause root deformations. The conclusion was that this nematode is one of the biotic factors involved in the degeneration of the Hippopha? scrub. This degeneration is due to a restriction of the root system resulting in a low phosphate uptake, a low nodulation capacity and, as a consequence, a low nitrogen content. The results demonstrate that biotic soil factors are important in influencing succession in higher plant communities.     

Absence of “void area” in freeze-etched vesicles of the Alnus crispa var. mollis Fern. root nodule endophyte

Nitrogen-fixing root nodules of Alnus crispa var. mollis Fern. were studied by transmission electron microscopy and by freeze-etching technique. Ultrathin sectioning of septate vesicles of the actinomycetal endophyte showed an electron transparent zone, the so-called void area, between the vesicle cell wall and its encapsulation material. This void area was not observed in the freeze-etching replicas of cryoprotected nodular tissue. It is suggested that the void area is the result of the coming-off of the vesicle cell wall from the capsule and that its formation reflects difficulty in fixing the voluminous mature vesicle of the root nodule endophyte.     

Mycorrhizal improvement in non-leguminous nitrogen fixing associations with particular reference toHippophaë rhamnoides L.

Summary The roots ofHippophaë rhamnoides which regularly bear actinomycete induced nodules when growing on Scottish sand dunes have also been found to support an endomycorrhizal association withGlomus fasciculatus. Ultrastructural and cytochemical studies carried out on the indigenous infections of establishedHippophaë mycorrhizal roots would support the postulate that transport is indeed occurring between the fungal symbiont and the host plant and vice versa in respect of phosphate and carbohydrate. Experiments using various inoculation regimes, demonstrated the significant improvement in the mycorrhizal/nodulated plants compared to the nodulated-only and the mycorrhizal-only plants with respect to plant growth, uptake of phosphate and nitrogenase activity, when grown in a medium poor in combined nitrogen and soluble phosphate. Preliminary work onAlnus andMyrica species growing in Central Scotland indicates that the mycorrhizae associated with these nodulated root systems exhibit a different interaction pattern which may be dependent on habitat type and associated angiosperm species.     

Late Quaternary history of Hippophaë rhamnoides L. (Elaeagnaceae) inferred from chalcone synthase intron (Chsi) sequences and chloroplast DNA variation

Fossil pollen records indicate that Hippophaë rhamnoides (Sea Buckthorn) was widespread on late‐ and early postglacial raw soils throughout much of central and northern Europe, but that Early Holocene reforestation restricted populations to northern coastal habitats, or along mountain streams in the Alps, Pyrenees, and Carpathians. We used sequence variation at the nuclear chalcone synthase intron (Chsi), in conjunction with chloroplast DNA–restriction fragment length polymorphism data, to investigate the intraspecific phylogeny, phylogeographic structure, and expansion demographic history of this dioecious and wind‐pollinated shrub at its range‐wide scale in Europe and Asia Minor. Four major Chsi phylogroups of unresolved relationships were identified with estimated divergences ～172 000 years ago. Large‐scale phylogeographic structures of nuclear and cytoplasmic markers were congruent in identifying (i) southeastern Europe as the most likely source of colonization into central Europe and Scandinavia, and (ii) the area just north of the Alps as a contact zone between populations from the Alps and the east/central European‐Scandinavian lineage. Coalescence‐based analyses (i.e. nested clade analysis and mismatch distributions) of Chsi variation were able to detect at least four major episodes of population growth, all within about the last 40 000 years. In particular, these analyses identified a nearly synchronized timing of population expansions in various parts of the species’ range in central‐eastern Europe/Asia Minor, most likely correlating with the Younger Dryas Stadial (～13 000–11 600 years ago). It remains to be established whether the phylogeographic history of H. rhamnoides, and particularly its rapid response to the rapid environmental changes of the Younger Dryas cold snap, is unique to the species, or whether it is shared with other cold‐tolerant shrub (or grassland) species known from late‐glacial raw soils in Europe.     

Genetic structure of natural populations of the grass endophyte Epichloë festucae in semiarid grasslands

Plants of red fescue (Festuca rubra), a commercially important turf grass, are infected by the fungal endophyte Epichloë festucae in semiarid natural grasslands, known as dehesas, in western Spain. We used amplified fragment length polymorphism (AFLP) markers to analyse the genetic polymorphism existing in two natural populations of Epichloë festucae. Linkage disequilibrium and the presence of clonal lineages indicated that nonrecombinant asexual reproduction predominates in both populations. However, most genetic variation detected was found to occur within populations, with only a moderate amount of genetic differentiation between populations (F_ST: 0.197). Overall, the study suggests that dehesa grasslands are useful reservoirs of Epichloë festucae endophytes, and provides information on population structure which is relevant to design sampling strategies.     

Out of the Qinghai-Tibet Plateau: evidence for the origin and dispersal of Eurasian temperate plants from a phylogeographic study of Hippophaë rhamnoides (Elaeagnaceae)

Numerous temperate plants now distributed across Eurasia are hypothesized to have originated and migrated from the Qinghai-Tibet Plateau (QTP) and adjacent regions. However, this hypothesis has never been tested through a phylogeographic analysis of a widely distributed species. Here, we use Hippopha? rhamnoides as a model to test this hypothesis. We collected 635 individuals from 63 populations of the nine subspecies of H. rhamnoides. We sequenced two maternally inherited chloroplast (cp) DNA fragments and also the bi-paternally inherited nuclear ribosomal ITS. We recovered five major clades in phylogenetic trees constructed from cpDNA and internal transcribed spacer (ITS) sequence variation. Most sampled individuals of six subspecies that are distributed in northern China, central Asia and Asia Minor/Europe, respectively, comprised monophyletic clades (or subclades) nested within those found in the QTP. Two subspecies in the QTP were paraphyletic, while the placement of another subspecies from the Mongolian Plateau differed between the ITS and cpDNA phylogenetic trees. Our phylogeographic analyses supported an 'out-of-QTP' hypothesis for H. rhamnoides followed by allopatric divergence, hybridization and introgression. These findings highlight the complexity of intraspecific evolutions and the importance of the QTP as a center of origin for many temperate plants.     

The α-glycerophosphate cycle inDrosophila melanogaster. I. Biochemical and developmental aspects

The two α-glycerophosphate dehydrogenases ofDrosophila melanogaster (mitochondrial αGPO and soluble αGPDH) have been biochemically characterized in a preliminary investigation of the α-glycerophosphate cycle inDrosophila. The soluble enzyme is NAD linked and can be distinguished from the mitochondrial oxidase in terms of locational specificity,pH optimum, salt precipitation, and electrophoretic behavior. The mitochondrial enzyme is NAD independent and exhibits behavior typical of a lipoprotein. Extraction procedures are described for αGPO with nonionic detergents. Isoelectric focusing of αGPO on polyacrylamide gels resolved two molecular forms of αGPO which differ in isoelectric point, ease of extraction, and developmental and spatial distribution. Developmental profiles of both αGPO and αGPDH are presented. The occurrence of multiple forms of both the soluble (Wright and Shaw, 1969) and the mitochondrial forms of the enzymes is discussed in light of a multifunctional role of the α-glycerophosphate cycle inDrosophila. This project was supported by a Genetics Training Grant T1 GM 1035 from the National Institute of General Medical Sciences.