An Alternative Mode of Early Land Plant Colonization by Putative Endomycorrhizal Fungi

Rhizomatous axes of Nothia aphylla, a land plant from the 400-myr-old Rhynie chert, host a fungus that closely resembles Glomites rhyniensis (Glomeromycota), the endomycorrhizal fungus of the Rhynie chert plant Aglaophyton major. However, G. rhyniensis is an intercellular endophyte that becomes intracellular exclusively within a well-defined region of the cortex, while the fungus in N. aphylla initially is intracellular but later becomes intercellular in the cortex. We hypothesize that N. aphylla displays an alternative mode of colonization by endomycorrhizal fungi, perhaps related to the peculiar internal anatomy of the lower portion of the rhizomatous axis, in which the radial arrangement of cells, along with the virtual absence of intercellular spaces, provides no intercellular infection pathway into the cortex.Key Words: Aglaophyton major, endomycorrhiza, Glomeromycota, Nothia aphylla, Early Devonian, Rhynie chertThe Early Devonian (c. 400 Ma) Rhynie chert is an in situ silicified hot springs environment that has become significant in our understanding of the complexity of life in early terrestrial ecosystems because of the extraordinary preservation of plants, animals, and microorganisms.¹ Moreover, various associations and interactions between different organisms can be directly examined,² including the earliest fossil examples of arbuscular endomycorrhizae.^3,4 The Rhynie chert land plant Aglaophyton major is characterized by arching, stomatiferous prostrate axes that grow along the substrate surface, and form rhizoid-bearing bulges, usually around stomata, upon contact with the substrate. Extramatrical hyphae of the mycorrhizal fungal enter the axes through these stomata, and spread out through the intercellular system of the hypodermis and cortex, subsequently penetrating individual cells within a well-defined region of the cortex (i.e., the mycorrhizal arbuscule-zone) to form arbuscules.⁴A recently published study⁵ reports on three fungal endophytes that (co-)occur in the Rhynie chert plant Nothia aphylla. This plant consists of upright aerial axes arising from a system of non-stomatiferous, subterranean rhizomatous axes characterized by a prominent ventral rhizoidal ridge.^6,7 The rhizoidal ridge, which is unique among Rhynie chert land plants, consists of a rhizoid-bearing epidermis, a multi-layered hypodermis, files of parenchyma cells that connect to the stele, and extra-stelar conducting elements (Fig. 1A); intercellular spaces are virtually absent.Open in a separate windowFigure 1Nothia aphylla from the Lower Devonian Rhynie chert. (A) Ventral portion of a rhizomatous axis with rhizoidal ridge (cross section); bar = 250 µm. (B) Fungal hypha [arrows] entering the axis through a rhizoid; bar = 30 µm. (C) Sheathed intracellular hyphae [arrows] in hypodermal cells (transverse section); bar = 20 µm. (D) Intercellular hyphae and vesicles in the cortex (longitudinal section); bar scale = 50 µm. (E) Hyphae, vesicles and a thick-walled spore in the cortex (longitudinal section); bar = 100 µm. All images from the original paper; reproduced with permission.One of the fungal endophytes in N. aphylla closely resembles Glomites rhyniensis (Glomeromycota), the endomycorrhizal fungus of A. major.⁴ In N. aphylla, this fungus occurs as an intracellular endophyte in rhizoids and tissues of the rhizoidal ridge. Moreover, it is abundant in the intercellular system of the cortex of both prostrate and proximal portions of aerial axes. The fungus enters the axes through rhizoids (Fig. 1B). Once in the hypodermis, hyphae become sheathed by cell wall material (Fig. 1C). In the cortex, the fungus produces intercellular vesicles (Fig. 1D) and thick-walled spores (Fig. 1E). Based on the presence of vesicles that are similar to those of G. rhyniensis, and spores like those in extant Glomeromycota, we hypothesize that this fungus is an endomycorrhizal member of the Glomeromycota; however, arbuscules have not been observed to date.If this interpretation is accurate, N. aphylla displays an alternative pattern of colonization by endomycorrhizal fungi. Although the morphology of the fungus and distribution in N. aphylla correspond to that of G. rhyniensis in A. major, the infection pathway is distinctly different. While G. rhyniensis is an intercellular endophyte that penetrates individual cells exclusively within the mycorrhizal arbuscule-zone,⁴ the fungus of N. aphylla enters the plant as an intracellular endophyte, and remains intracellular until it reaches the cortex. The host plant apparently does not respond to the invading fungus because infected rhizoids are not altered morphologically. Once in the hypodermis, however, hyphae become separated from the host cell protoplast. This feature suggests a shift from (i) uncontrolled intracellular occurrence of the fungus in the rhizoids, to (ii) controlled intracellular occurrence in the rhizoidal ridge, to (iii) intercellular occurrence in the cortex.The fact that the rhizomatous axes of N. aphylla are subterranean, along with the peculiar internal anatomy of the rhizoidal ridge, may have provided the selective pressure for an alternative mode of colonization by endomycorrhizal fungi. The fungus probably enters the plant through rhizoids because the axes are non-stomatiferous. Moreover, the morphology and radial arrangement of cells in the rhizoidal ridge, along with the virtual absence of intercellular spaces, perhaps does not provide an intercellular infection pathway into the cortex. We speculate that N. aphylla tolerated intracellular penetration in the rhizoids and within the tissues of the rhizoidal ridge in order to become inoculated. Tolerating (or even facilitating) intracellular penetration within a limited area of the axis may simultaneously have provided the plant with a means of recognizing and subsequently distinguishing the endomycorrhizal fungus from potentially harmful parasites (e.g., by surface features of the hyphae or chemical signals). Once recognized, the endomycorrhizal fungi become sheathed and “guided” through the ridge without being able to extract nutrients from the host, and into the cortex where intracellular penetration is not longer possible. The parasites, once recognized, are confined in the tissues of the rhizoidal ridge by specific or unspecific host responses, e.g., secondarily thickened cell walls.⁵ Conversely, if the endomycorrhizal fungus entered the plant through surface openings, and spread out exclusively through the intercellular system, the mechanisms that might confine simultaneous parasite infections were probably much more limited.Endomycorrhizal relationships are believed to have evolved from parasitic interactions.⁸ It has been postulated that modern enodomycorrhizal fungi in some way control parasites because both compete for the same resources.⁹ It may be that, during the evolution of fungal endophytism, the initial benefits of mycorrhizae included protection of the host from pathogenic fungi.¹⁰ Nothia aphylla from the Lower Devonian Rhynie chert adds support to this hypothesis, and may demonstrate that more than a single pattern of colonization by endomycorrhizal fungi occurred during the early evolution of land plants.     

Inhibiting alternative pathway complement activation by targeting the factor D exosite

By virtue of its amplifying property, the alternative complement pathway has been implicated in a number of inflammatory diseases and constitutes an attractive therapeutic target. An anti-factor D Fab fragment (AFD) was generated to inhibit the alternative complement pathway in advanced dry age-related macular degeneration. AFD potently prevented factor D (FD)-mediated proteolytic activation of its macromolecular substrate C3bB, but not proteolysis of a small synthetic substrate, indicating that AFD did not block access of the substrate to the catalytic site. The crystal structures of AFD in complex with human and cynomolgus FD (at 2.4 and 2.3 Å, respectively) revealed the molecular details of the inhibitory mechanism. The structures show that the AFD-binding site includes surface loops of FD that form part of the FD exosite. Thus, AFD inhibits FD proteolytic function by interfering with macromolecular substrate access rather than by inhibiting FD catalysis, providing the molecular basis of AFD-mediated inhibition of a rate-limiting step in the alternative complement pathway.     

Characterization of antigenic sialoglycoprotein subunits of the placental brush border membranes: Comparison with liver and kidney membrane subunits by two-dimensional electrophoresis

The sialoglycoprotein subunits of human placental brush border membranes were labeled by sequential treatment with periodate and (³H)-sodium borohydride, which trititates sialic acid, and by lactoperoxidase-catalyzed (¹²⁵I) iodination of tyrosine residues. The labeled subunits were characterized with respect to their affinity for antisera raised against Triton X-100 extracts of placental brush border membranes. The immunochemically reactive components were analyzed by two-dimensional electrophoresis according to a modification of the O'Farrell technique [20] enabling the assignment of estimated M_r? and pI. Of the 33 ³H-labeled brush border subunits present in Triton X-100-solubilized membrane preparations, 18 subunits reacted with antiplacental brush border antisera insolubilized on CNBr-activated Sepharose or in immunoprecipitates. Fourteen of these tritiated subunits were also labeled with ¹²⁵I, confirming that these are glycoproteins. The plasma membranes of normal human liver and microsomes from kidney were examined for the placental brush border glycoprotein subunits by reaction with insolubilized antiplacental brush border antisera and two-dimensional electrophoresis of the reacting tritium-labeled subunits. Comparison of the two-dimensional electrophoretic maps of the immunochemically reacting glycoproteins from liver, kidney, and placenta resulted in the identification of seven placental subunits in common with liver and kidney on the basis of antigenic cross-reactivity, M_r?, and pI. Four placental glycoproteins were not found in the other tissues and are potentially specific to the placenta. Three of the placental subunits were only seen in placenta and kidney. Three of the subunits ran at the dye front and could not be assigned molecular weights. One of the subunits was poorly labeled by tritiation of sialic acid and was not considered.     

Adduct formation between the carcinogen N-acetoxy-2-acetylaminofluorene and synthetic polydeoxyribonucleotides.

The chemical carcinogen N-acetoxy-2-acetylaminofluorene (NA-AAF) was reacted with poly(dG-dC) - poly(dG-dC); poly dG - poly dC; poly(dA-dT) - poly (dA-dT); and poly dA - poly dT under a variety of conditions. Poly (dG-homo GC polymer and 10--20 more reactive the A + T polymers. Lowering the ionic strength increased the extent of reaction, while pH change (8.9 vs. 5.5) had only a small effect. If ionic strength was adjusted so that the two guanine-containing polymers showed equal thermal stability (as judged by Tm) then the alternating copolymer was 7 times as reactive as the homopolymer. In aggreement with previous investigators, the major product was found to be 8-(N-2-fluorenylacetamido) deoxyguanosine.     

Early Devonian fungi: a blastocladalean fungus with sexual reproduction

In this paper we describe a fossil fungus–Paleoblastocladia milleri gen. et sp. nov.–from the 400 million-year-old Early Devonian Rhynie chert that shares numerous features with modern zoosporic fungi placed in the order Blastocladiales. The fungus occurs in tufts that arise from stomata or between the cuticle and epidermis of Aglaophyton major axes. Thallus development begins from an irregular bipolar basal cell that produces a system of intramatrical rhizoids and clavate-shaped extramatrical, nonseptate hyphae. These hyphae develop into two types of mature thalli. Sporothalli are characterized by several orders of dichotomous branching and the production of terminal, globose zoosporangia, as well as thick-walled, pitted resting sporangia. On separate dichotomously branched thalli (gametothalli) are terminal chains of two or three gametangia, in which the terminal one is slightly larger. Despite the fact that all of the reproductive organs contain either zoospores or gametes, none show evidence of discharge papillae. The fossil fungus is compared with extant members of the Blastocladiales, and the presence of sexual reproduction is discussed.     

Isolation and partial characterization of monophosphoglycerate mutase from human erythrocytes.

Monophosphoglycerate mutase has been purified to homogeneity from outdated human erythrocytes as indicated by exclusion chromatography, polyacrylamide gel electrophoresis, and equilibrium centrifugation. Occasionally, the recommended purification procedure yields a small amount (3% or less) of a single extraneous protein which can be deleted from the enzyme preparation by employing an additional purification step. The native enzyme has a molecular weight of 54,000 to 56,000 as determined by equilibrium centrifugation and exclusion chromatography. Disc gel electrophoresis in the presence of sodium dodecyl sulfate yields a single protein band with a molecular weight of 28,600, indicating that the native macromolecule is a dimer composed of subunits of similar mass. Homogeneous monophosphoglycerate mutase is free of diphosphoglycerate mutase, enolase, and nonspecific phosphatase activities; however, the enzyme manifests intrinsic 2,3-diphospho-D-glycerate phosphatase activity as shown by thermal denaturation studies. The diphosphatase activity is stimulated by PPi and glycolate-2-P, but is inhibited by Cl-, HSO3-, and Pi. The pH optimum for both the diphosphatase and the mutase is 6.8. The Km for 2,3-diphospho-D-glycerate in the phosphatase reaction is 82 muM at 37 degrees and pH 7.2. The amino acid composition of homogeneous monophosphoglycerate mutase is given.