The detection of leghemoglobin-line sequences in legumes and non-legumes

Summary Leghemoglobin is a major component of the nitrogen-fixing nodules formed by legumes in association with bacterial symbionts of the genusRhizobium. It is thought to be involved in regulating the oxygen tension within nodules. In a series of Southern blot experiments using cloned soybean leghemoglobin cDNAs as hybridization probes, cross-hybridizing sequences have been detected in legumes closely related to soybean (members of the Leguminosae subfamily Papilionoideae), as well as in a distantly related legume not reported to be nodulated (subfamily Caesalpinioideae). With the same probes, the presence of cross-hybridizing sequences has also been detected in plants outside the Leguminosae, including two nitrogen-fixing non-legumes and one species which is not nodulated. These results suggest that the genes for oxygen-binding proteins may be more widely dispersed than previously thought.     

Natural abundance of 15N in actinorhizal plants and nodules

Substantial enrichment of some plant parts in ¹⁵N relative to the rest of the plant is unusual, but is found in the nitrogen-fixing nodules of many legumes. A range of actinorhizal plants was surveyed to determine whether the nodules of any of them are also substantially enriched in ¹⁵N. The nonlegume Parasponia, nodulated by a rhizobium, was also included. Four of the actinorhizal genera and Parasponia were grown in N-free culture, and three actinorhizal genera were collected from the field. Nodules of Parasponia, Casuarina and Alnus were¹⁵N enriched relative to other plant parts, but only Parasponia approached the degree of enrichment found in some legume nodules. The nodules of Datisca, Myrica, Elaeagnus, Shepherdia, and Coriaria were depleted in ¹⁵N. Thus many actinorhizal nodules are depleted in ¹⁵N compared to other plant parts and enrichment is modest when it does occur. Whole plant ¹⁵N content (¹⁵N) in four actinorhizal plants and Parasponia showed a relatively narrow range of –1.41 to –1.90. Hence regardless of the degree of nodule enrichment or depletion, whole plant ¹⁵N content appears to vary little in plants grown in N-free culture.     

Molecular phylogeny of the actinorhizal Hamamelidae and relationships with host promiscuity towards Frankia

Several of the most studied actinorhizal symbioses involve associations between host plants in the subclass Hamamelidae of the dicots and actinomycetes of the genus Frankia. These actinorhizal plants comprise eight genera distributed among three families of ‘higher’ Hamamelidae, the Betulaceae, Myricaceae, and Casuarinaceae. Contrasting promiscuity towards Frankia is encountered among the different actinorhizal members of these families, and a better assessment of the evolutionary history of these actinorhizal taxa could help to understand the observed contrasts and their implications for the ecology and evolution of the actinorhizal symbiosis. Complete DNA sequences of the chloroplast gene coding for the large subunit of ribulose 1,5-bisphosphate carboxylase (rbcL) were obtained from taxa representative of these families and the Fagaceae. The phylogenetic relationships among and within these families were estimated using parsimony and distance-matrix approaches. All families appeared monophyletic. The Myricaceae appeared to derive first before the Betulaceae and the Casuarinaceae. In the Casuarinaceae, the genus Gymnostoma derived before the genera Casuarina and Allocasuarina, which were found closely related. The analysis of character-state changes in promiscuity along the consensus tree topology suggested a strong relationship between the evolutionary history of host plants and their promiscuity toward Frankia. Indeed, the actinorhizal taxa that diverged more recently in this group of plants were shown to be susceptible to a narrower spectrum of Frankia strains. The results also suggest that the ancestor of this group of plant was highly promiscuous, and that evolution has proceeded toward narrower promiscuity and greater specialization. These results imply that a tight relationship between the phytogenies of both symbiotic partners should not be expected, and that host promiscuity is likely to be a key determinant in the establishment of an effective symbiosis.     

Nitrogen fixation in legumes and actinorhizal plants in natural ecosystems: values obtained using 15N natural abundance

Background: Nitrogen fixation has been quantified for a range of crop legumes and actinorhizal plants under different agricultural/agroforestry conditions, but much less is known of legume and actinorhizal plant N₂ fixation in natural ecosystems.

Aims: To assess the proportion of total plant N derived from the atmosphere via the process of N₂ fixation (%Ndfa) by actinorhizal and legume plants in natural ecosystems and their N input into these ecosystems as indicated by their ¹⁵N natural abundance.

Methods: A comprehensive collation of published values of %Ndfa for legumes and actinorhizal plants in natural ecosystems and their N input into these ecosystems as estimated by their ¹⁵N natural abundance was carried out by searching the ISI Web of Science database using relevant key words.

Results: The %Ndfa was consistently large for actinorhizal plants but very variable for legumes in natural ecosystems, and the average value for %Ndfa was substantially greater for actinorhizal plants. High soil N, in particular, but also low soil P and water content were correlated with low legume N₂ fixation. N input into ecosystems from N₂ fixation was very variable for actinorhizal and legume plants and greatly dependent on their biomass within the system.

Conclusions: Measurement of ¹⁵N natural abundance has given greater understanding of where legume and actinorhizal plant N₂ fixation is important in natural ecosystems. Across studies, the average value for %Ndfa was substantially greater for actinorhizal plants than for legumes, and the relative abilities of the two groups of plants to utilise mineral N requires further study.     

The occurrence of amoeboid plastids in the actinorhizal root nodules of Alnus glutinosa (L.) Gaertn.

Abstract. The invasion of the actinomycete Frankia into the root cells of Alnus glutinosa with subsequent nodule formation effects a number of ultrastructural changes in the host cell cytoplasm. Among other changes the amyloplasts rapidly lose their starch and acquire an amoeboid or pleomorphic form. Such plastids occur predominantly in the mature vesicle-containing, nitrogen-fixing cells of the nodule. They lack starch, have an electron dense stroma and a complex lamellar system. This last would appear to be associated with a distinct membranous reticulum which can be extensive. The flexible form of these plastids is mirrored in their ability to enclose portions of host cytoplasm together with organelles and even other plastids. Their close association with cristate mitochondria suggests an active metabolic role in the nodule symbiosis.     

Simultaneous parsimony jackknife analysis of 2538rbcL DNA sequences reveals support for major clades of green plants, land plants, seed plants and flowering plants

The ever-larger data matrices resulting from continuing improvements in DNA sequencing techniques require faster and more efficient methods of phylogenetic analysis. Here we explore a promising new method, parsimony jackknifing, by analyzing a matrix comprising 2538 sequences of the chloroplast generbcL. The sequences included cover a broad taxonomic range, from cyanobacteria to flowering plants. Several parsimony jackknife analyses were performed, both with and without branch-swapping and multiple random addition sequences: 1) including all positions; 2) including only first and second codon positions; 3) including only third positions; and 4) using only transversions. The best resolution was obtained using all positions. Removal of third positions or transitions led to massive loss of resolution, although using only transversions somewhat improved basal resolution. While branch-swapping improved both resolution and the support found for several groups, most of the groups could be recovered by faster simple analyses. Designed to eliminate groups poorly supported by the data, parsimony jackknifing recognizes 1400 groups on the basis of allrbcL positions. These include major taxa such as green plants, land plants, flowering plants, monocots and eudicots. We include appendices of supported angiosperm families, as well as larger groups.     

Genome modifications in protoplast-derived tobacco plants: Contents of repetitive DNA sequences

Plasticity of the tobacco genome was studied by testing the DNAs of protoplast-derived regenerants with three different repetitive DNA sequences by the method of quantitative DNA/DNA hybridizations. A large population of 91 regenerants belonging to 35 different protoclones was analysed and a high degree of heterogeneity in the contents of the different DNA repeats was detected. The contents of middle repetitive sequences of two types were more stable or changed in the same direction, while the highly repetitive sequence varied independently and displayed a significant reduction in comparison with the two other sequences. Comparing the variation within the subpopulations of plants of the same clonal origin and the variation among the protoclones led to a conclusion that the pre-existing DNA variability in the starting plant material and/or thein vitro stress during the very early stages of protoclone regeneration played a decisive role in the formation of modified genomes in regenerants.     

Recent developments in the actinorhizal symbioses

Over 200 species of angiosperms in eight different families are capable of forming root nodule symbioses with the actinomycetal genusFrankia as endosymbiont. Several thorough reviews of the biology of these actinorhizal associations have appeared in recent years (Benson and Silvester, 1993; Schwintzer and Tjepkema, 1990; Tjepkema et al., 1986). The purpose of the present discussion is to provide a summary overview of the actinorhizal symbioses, with an emphasis on recent research activities. A few areas of comparative interest with other symbiotic diazotrophs will be highlighted, especially regarding the question of oxygen protection and nitrogen fixation.     

Choosing a reporter for gene expression studies in transgenic actinorhizal plants of the Casuarinaceae family

Transgenic Casuarinaceae and reporter genes provide valuable tools to study gene expression in transgenic actinorhizal nodules. In this paper, we discuss the use of ß-glucuronidase for the histochemical localization and quantification of gene expression in transgenic plants of Allocasuarina verticillata and Casuarina glauca nodulated by the actinomycete Frankia. We also report on the genetic transformation of A. verticillata by the Agrobacterium tumefaciens strain C58C1(pGV2260) containing the 35S-mgfp5-ER construct encoding a modified green fluorescent protein of Aequorea victoria in a binary vector. The evolution of the GFP fluorescence was monitored through all stages of the regeneration process. The data indicate that GFP is not toxic in Casuarinaceae and that this reporter gene can be used for visual screening of transformed calli and transgenic plants. The fluorescence pattern of gfp provides a new tool for monitoring in vivo transgene expression in actinorhizal plants.     

A method to study zhizosphere processes in thin soil layers of different proximity to roots

Frankia is the diverse bacterial genus that fixes nitrogen within root nodules of actinorhizal trees and shrubs. Systematic and ecological studies of Frankia have been hindered by the lack of morphological, biochemical, or other markers to readily distinguish strains. Recently, nucleotide sequence of 16 S RNA from the small ribosomal subunit has been used to classify and identify a variety of microorganisms. We report nucleotide sequences from portions of the 16 S ribosomal RNA from Frankia strains AcnI1 isolated from Alnus viridis ssp. crispa (Ait.) Turrill and PtI1 isolated from Purshia tridentata (Pursh) DC. The number of nucleotide base substitutions and gaps we find more than doubles the previously reported sequence diversity for the same variable regions within other strains of Frankia.     

DNA structures associated with autonomously replicating sequences from plants

DNA fragments capable of conferring autonomous replicating ability to plasmids inSaccharomyces cerevisiae were isolated from four different plant genomes and from the Ti plasmid ofAgrobacterium tumefaciens. The DNA structure of these autonomously replicating sequences (ARSs) as well as two from yeast were studied using retardation during polyacrylamide gel electrophoresis and computer analysis as measures of sequence-dependent DNA structures. Bent DNA was found to be associated with the ARS elements. An 11 bp ARS consensus sequence required for ARS function was also identified in the elements examined and was flanked by unusually straight structures which were rich in A+T content. These results show that the ARS elements from genomes of higher plants have structural and sequence features in common with ARS elements from yeast and higher animals.Supported by Grant 1RO1-GM41708-O1 from the National Institute of Health.     

Molecular ecology ofFrankia: Advantages and disadvantages of the use of DNA probes

Ecological studies on the actinomyceteFrankia are often influenced by the difficulty to isolate and identify this microorganism. The application of molecular biological techniques offers possibilities to detect microbes without isolation and cultivation.Nif genes or whole plasmids can serve as targets for the design of specific probes. Alternatively, ribosomal RNA (rRNA), commonly used in modern phylogenetic studies, can be used as a target molecule in ecological studies. This paper gives an overview of new developments on the use of 16S rRNA as a target molecule for oligonucleotide probes. Group-specific sequences in the 16S rRNA ofFrankia can be used as targets for oligonucleotide probes that a) recognize ineffectiveFrankia strains onAlnus, b) recognize effective strains onAlnus, c) recognize allFrankia strains tested so far. The present paper summarizes the essential steps needed for the use of these probes for the detection ofFrankia strains in soil without isolation and cultivation.     

小麦族植物DNA重复序列研究

近些年来从小麦族植物中分离到了许多DNA重复序列,并对其组织结构特点,物种分布特异性和在染色体上的分布特征做了广泛的研究,其中一些重复序列已被成功地用于检测遐入小麦的外源染色质和小麦族有关种属的进化研究,本文就以上诸方面进行了简要介绍。     

The evolution of actinorhizal symbioses: Evidence for multiple origins of the symbiotic association

According to morphologically based classification systems, actinorhizal plants, engaged in nitrogen-fixing symbioses with Frankia bacteria, are considered to be only distantly related. However, recent phylogenetic analyses of seed plants based on chloroplast rbcL gene sequences have suggested closer relationships among actinorhizal plants. A more thorough sampling of chloroplast rbcL gene sequences from actinorhizal plants and their nonsymbiotic close relatives was conducted in an effort to better understand the phylogenetic relationships of these plants, and ultimately, to assess the homology of the different actinorhizal symbioses. Sequence data from 70 taxa were analyzed using parsimony analysis. Strict consensus trees based on 24 equally parsimonious trees revealed evolutionary divergence between groups of actinorhizal species suggesting that not all symbioses are homologous. The arrangement of actinorhizal species, interspersed with nonactinorhizal taxa, is suggestive of multiple origins of the actinorhizal symbiosis. Morphological and anatomical characteristics of nodules from different actinorhizal hosts were mapped onto the rbclL-based consensus tree to further assess homology among rbcL-based actinorhizal groups. The morphological and anatomical features provide additional support for the rbcL-based groupings, and thus, together, suggest that actinorhizal symbioses have originated more than once in evolutionary history.     

Are there magnet plants in Australian ecosystems: Pollinator visits to neighbouring plants are not affected by proximity to mass flowering plants

Aggregations of resource-rich plants can act as “magnets” drawing pollinators from other plants. Magnets can have positive and negative impacts on co-flowering neighbours: enhanced pollination via a ‘spillover-effect’ or a reduction in pollination via competition. Support for the importance of magnets largely comes from studies conducted in the northern hemisphere. We used a comparative approach to test two hypotheses for three pairs of Australian native plants: (1) putative magnets attract a greater number and more diverse suites of pollinators than co-flowering species; and (2) the quantity, diversity and specificity of pollinators varies with distance from putative magnets. We surveyed pollinator activity on co-flowering plants before and after bagging to experimentally exclude pollinators from putative magnets, dominant flowering species with populations ranging in size from 700 to 4000 m². Selected focal species were found to be pollinator magnets, but did not appear to influence the pollination of neighbours. Prior to bagging, putative magnets received more visits but visitors were predominantly (90–100%) exotic honeybees (Apis mellifera). The number and diversity of pollinators on co-flowering species did not consistently increase when magnets were bagged. Moreover, pollinator visitation, diversity and constancy did not vary with distance from putative magnets before or after bagging. All sampled (n = 388) honeybees had pollen of only one plant species on their bodies and no honeybee sampled on a co-flowering species carried pollen of magnet species (n = 212). We found that interactions between pollinators and co-flowering Australian plants differ substantially from those reported for the northern hemisphere; this is most likely due to the impact of abundant, introduced honeybees.     

The branching pattern of major groups of land plants inferred from parsimony analysis of ribosomal RNA sequences

The parsimony and bootstrap branching pattern of major groups of land plants derived from relevant 5S rRNA sequence trees have been discussed in the light of paleobotanical and morphological evidences. Although 5S rRNA sequence information is not useful for dileneating angiosperm relationships, it does capture the earlier phase of land plant evolution. The consensus branching pattern indicates an ancient split of bryophytes and vascular plants from the charophycean algal stem. Among the bryophytes,Marchantia andLophocolea appear to be phylogenetically close and together withPlagiomnium form a monophyletic group.Lycopodium andPsilotum arose early in vascular land plant evolution, independent of fem-sphenopsid branch. Gymnosperms are polyphyletic; conifers, Gnetales and cycads emerge in that order with ginkgo joiningCycas. Among the conifers,Metasequoia,Juniperus andTaxus emerge as a branch independent ofPinus which joins Gnetales. The phylogeny derived from the available ss-RNA sequences shows that angiosperms are monophyletic with monocots and dicots diverging from a common stem. The nucleotide replacements during angiosperm descent from the gymnosperm ancestor which presumably arose around 370 my ago indicates that monocots and dicots diverged around 180 my ago, which is compatible with the reported divergence estimate of around 200 my ago deduced from chloroplast DNA sequences. Since deceased.     

四种荒漠植物的光合响应

研究了胡杨（Populus euphratica Oliv）、疏叶骆驼刺（Alhagi sparsifolia B.Keller et Shap)、多枝柽柳（Tamarix ramosissima Lbd)和头状沙拐枣（Calligonum caput-medusae Schrenk)等四种塔克拉玛干荒漠植物的光合响应曲线,结果表明：（1）四种植物中,疏叶骆驼刺光呼吸（Rp)和CO2补偿点（Г）最高,净光合速率（An）以及水分利用效率（WUE）最低;而C4植物头状沙拐枣无论Rp还是Г都最低,它的An、光补偿点（LCP）及光饱和点（LSP）都显著高于其它3种植物（P≤0．05）,而且因为非常低的蒸腾速率（E）,沙拐枣的WUE也较高。（2）4种植物都为喜光植物,但仍然表现出一定的差异。根据它们光曲线中LCP以及暗呼吸速率（Rd),4种喜光植物的喜光顺序为：头状沙拐枣,疏叶骆驼枣刺,多枝柽柳,胡杨。（3）柽柳光合响应曲线与日变化中的An,gs等值差异较大,可能是该植物的温度生态位较其它三种植物范围较窄,设置的25℃不在其最适温度范围内,或者是在这个温度下气孔对光强变化的不敏感。（4）综合前人的结果表明,在特定的环境条件,发育阶段以及经过一定的诱导处理,胡杨可以因诱导而表现出一些C4植物特征。     

Spittlebug indicators of nitrogen-fixing plants

Abstract.

• 1 Spittlebugs (Homoptera: Cercopoidea) are preferentially associated with nitrogen-fixing angiosperms. Generally, the Aphrophoridae associate with legumes, the Clastopteridae with actinorhizal plants, and the Cercopidae with associative nitrogen-fixing grasses. This is the first reported insect—host association specific to nitrogen-fixing plants.
• 2 It is probable that spittlebugs are attracted to nitrogen-fixing plants because these hosts provide a relatively rich, reliable source of organic nitrogen compounds for xylem-sucking insects.
• 3 Many spittlebugs live on plants that do not fix nitrogen, and ecological, mechanical or chemical factors may bar some spittlebugs from otherwise suitable nitrogen-fixing plants.
• 4 Among nitrogen-fixing hosts, spittlebugs appear to prefer plants that transport fixed nitrogen as amino acids and amides to plants that transport fixed nitrogen as ureides.