Uninfected Roots Research Articles

Immunolocalization of Tomato Mosaic Tobamovirus in Roots of Red Spruce Seedlings

AbstractThe objective of this study was to determine the location of tomato mosaic tobamovirus (ToMV) in red spruce (Picea rubens) seedling roots. Roots were collected from uninfected and infected spruce seedlings at biweekly intervals, fixed in formaldehyde‐glacial acetic acid‐50% ethanol (1 : 1 : 18), embedded in paraffin, sectioned, and tested by indirect immunofluorescence (IF). ToMV was detected by IF in infected, but not in uninfected red spruce roots. All seedlings that tested positive by IF had previously tested positive by enzyme‐linked immunosorbent assay. Fluorescence was observed most frequently 2 weeks after inoculation in cortical and epidermal cells, as well as in the lateral root primordia. The ToMV was not observed in vascular tissue, the root cap, or meristematic cells. Virus distribution was uneven, and occurred in localized, concatenated pockets, parallel to the axis of the apical meristem. In older seedlings, fluorescence was associated with irregular‐shaped vesicles in cortical cells.

Changes in actin microfilament arrays in developing pea root nodule cells

Various microorganisms that form symbiotic associations with plant roots alter the cytoskeleton of host cells. The objective of this study was to determine the organization of actin microfilaments in developing Pisum sativum L. (pea) root nodule cells at various stages after infection by Rhizobium leguminosarum bv. viciae. Fluorescently labelled microfilaments in uninfected pea root nodule cells occur in association with the nucleus, along cytoplasmic strands, and as long microfilament bundles randomly organized in the cortex of the cell. These actin arrays are also present in recently infected cells that have been invaded by an infection thread and contain a small number of bacteroids. In addition, the recently infected cells contain diffuse cytoplasmic actin, long actin microfilament bundles near the vacuole, and a nuclear-associated network of microfilament bundles. In older infected cells, the predominant array is a network of cytoplasmic microfilaments that are wavy and extend in multiple directions within the cell; the network is equally abundant in all regions of the cytoplasm and may interact with the bacteroids and organelles. Thus, actin microfilaments reorganize during the pea root nodule infection process to form distinct arrays whose organization depends on the stage of infection.Key words: nodule, actin microfilaments, Rhizobium, pea, symbiosis.

Macronutrient concentrations of soybean infected with soybean cyst nematode

Soybean cultivars (Glycine max(L.) Merr.) infected with soybean cyst nematode (SCN; Heterodera glycinesIchinohe) often show symptoms similar to K deficiency. The objectives of this experiment were to determine if SCN infection affected macronutrient concentrations in soybean seedling vegetative tissues, determine whether increased K fertility can overcome these possible effects, and to determine if these possible effects are localized at the site of infection or expressed systemically throughout the root system. Soybean plants were grown with root systems split into two halves. This allowed differential K (0.2, 2.4 and 6.0 mM K nutrient solutions) and SCN (0 and 15 000 eggs/plant) treatments to be applied to opposite root-halves of the same plant. Thirty days after plants were inoculated with SCN, macronutrient concentrations of shoot and root tissues were determined. Potassium concentration in leaf blades was not affected; but K concentrations in leaf-petiole and stem tissues were increased with SCN infection. Roots infected with SCN contained lower K concentrations than uninfected roots, but only for the 2.4 mM K treatment. Thus, at the medium level of K fertility, SCN reduced K concentration in soybean roots, and increasing K fertility to the high level overcame the effect. Because K concentrations in the shoot tissues were not reduced by SCN infection, above ground portions of the plant may be able to overcome limitations that occur in roots during the first 30 days of infection. Increasing K fertility level in soybean fields may not benefit vegetative growth of soybean infected with SCN.

Peptides isolated from cell walls of Medicago truncatula nodules and uninfected root

The hydroxyproline-rich root nodules of legumes provide a microaerobic niche for symbiotic nitrogen-fixing Rhizobacteria. The contributions of the cell wall and associated structural proteins, particularly the hydroxyproline-rich glycoproteins (HRGPs), are therefore of interest. Our approach involved identification of the protein components by direct chemical analysis of the insoluble wall. Chymotryptic peptide mapping showed a “P3-type” extensin containing the highly arabinosylated Ser-Hyp 4-Ser-Hyp-Ser-Hyp 4-Tyr 3-Lys motif as a major component. Cell wall amino acid analyses and quantitative hydroxyproline arabinoside profiles, predominantly of tri- and tetraarabinosides, confirmed this extensin as the major structural protein in the cell walls of both root nodules and uninfected roots. On the other hand, judging from the Pro, Glu and non-glycosylated Hyp content, the nodule-specific proline-rich glycoproteins, such as the early nodulins (ENOD-PRPs), are present in much lesser amounts. Although we isolated no PRP peptides from nodule cell walls, a single PRP peptide from root cell walls confirmed the presence of a PRP in roots and represented the first direct evidence for a crosslinked PRP in muro. Compared with root cell walls (∼7% protein dry weight) nodule cell walls contained significantly more protein (∼13% dry weight) with an overall amino acid and peptide composition indicating the presence of structural protein unrelated to the HRGPs.

Changes in mRNA abundance within Heterodera schachtii-infected roots of Arabidopsis thaliana.

Gene expression changes in plant roots infected by plant-parasitic cyst nematodes are involved in the formation of nematode feeding sites. We analyzed mRNA abundance changes within roots of Arabidopsis thaliana during the early compatible interaction with Heterodera schachtii, the sugarbeet cyst nematode. Approximately 1,600 root sections, each containing a single parasitic nematode and its feeding site, and 1,600 adjacent, nematode-free root sections were excised from aseptic A. thaliana cultures 3 to 4 days after inoculation with H. schachtii. These tissue samples were termed infected and uninfected, respectively. Preparasitic nematodes were added to the uninfected tissue sample to maintain the nematode to plant tissue proportion. mRNA extracted from these two tissue samples was subjected to differential display analysis. Thirty-six cDNA clones corresponding to mRNA species with different abundance between both tissue samples were isolated. Of these clones, 24 were of A. thaliana origin and 12 were from H. schachtii. Differential display data predicted that the A. thaliana cDNA clones corresponded to 13 transcripts that were more abundant in the infected root sections and 11 transcripts that were more abundant in the uninfected root sections. H. schachtii cDNA clones were predicted to correspond to four transcripts that were more abundant in parasitic nematodes and to eight transcripts that were more abundant in preparasitic nematodes. In situ hybridization experiments confirmed the mRNA abundance changes in A. thaliana roots predicted by the differential display analyses for two A. thaliana clones.

Flavan-containing cells delimit Frankia-infected compartments in Casuarina glauca nodules.

We investigated the involvement of polyphenols in the Casuarina glauca-Frankia symbiosis. Histological analysis revealed a cell-specific accumulation of phenolics in C. glauca nodule lobes, creating a compartmentation in the cortex. Histochemical and biochemical analyses indicated that these phenolic compounds belong to the flavan class of flavonoids. We show that the same compounds were synthesized in nodules and uninfected roots. However, the amount of each flavan was dramatically increased in nodules compared with uninfected roots. The use of in situ hybridization established that chalcone synthase transcripts accumulate in flavan-containing cells at the apex of the nodule lobe. Our findings are discussed in view of the possible role of flavans in plant-microbe interactions.

Transcription of a Gene Encoding a Lectinlike Glycoprotein Is Induced in Root Cells Harboring Arbuscular Mycorrhizal Fungi in Pisum sativum

The gene PsNlec1, which encodes a lectinlike glycoprotein, is strongly expressed in pea nodule tissue. Using gene-specific polymerase chain reaction (PCR) primers, in situ hybridization probes, and specific antisera derived from the PsNlec1 sequence, we investigated gene expression associated with the arbuscular mycorrhizal (AM) symbiosis of pea roots. With the use of reverse transcription (RT)-PCR and cold in situ hybridization, strong expression of the transcript was demonstrated not only in root nodules but also in mycorrhizal roots when the cells were colonized by the endomycorrhizal fungus Glomus versiforme. No transcript was detectable in uninfected pea roots. With an antiserum raised against PsNLEC-1 polypeptide, a single antigenic band (25 kDa) was observed following gel electrophoresis of extracts from mycorrhizal roots. However, the amount of antigen was apparently too low to be detected by immunogold localization in tissue sections of pea mycorrhizal roots.

Apical meristems of tomato roots and their modifications induced by arbuscular mycorrhizal and soilborne pathogenic fungi

Functional morphological patterns in root apices of tomato (Lycopersicon esculentum) dependent on growth, ageing and infection by the arbuscular mycorrhizal (AM) fungus Glomus mosseae and/or by the soilborne pathogenic fungus Phytophthora nicotianae var parasitica (P. parasitica) were studied. Uninfected root apices were characterized by closed, tri‐layered meristems with nonreticulate nuclei; however, some apices of each treatment lost their meristematic nature, stopped growing and differentiated, becoming ‘parenchymatized’. The pathogenic fungus reduced the apex diameter and the number of mitotically active and viable apices inducing plasmolysis, cell and nucleus degeneration, and necrosis. The AM fungus, on the other hand, produced an increase in apex size and reduced the percentage of necrosis both in uninfected roots and in roots infected by P. parasitica. Thus, the AM fungus protected the apices from the pathogenic infection, allowing normal root growth. Furthermore, larger apices, which produce thicker roots, might indirectly contribute to plant protection. Increased volumes of colonizable tissues favour the spreading of the symbiont, and P. parasitica hyphae are always excluded from arbuscule‐containing cells.

Genomic organization and expression properties of the MtSucS1 gene, which encodes a nodule-enhanced sucrose synthase in the model legume Medicago truncatula.

We have isolated and sequenced a sucrose synthase (SucS) cDNA from the model legume Medicago truncatula. This cDNA (MtSucS1) contains an ORF of 2418 bp, coding for a protein of 805 amino acids with a molecular mass of 92.29 kDa. The deduced amino acid sequence shows significant homology to other plant sucrose synthases, in particular to the nodule-enhanced sucrose synthases from pea and broad bean. Northern analysis revealed that the corresponding gene shows a ten-fold higher expression level in root nodules than in uninfected root, stem and leaf tissues. SucS protein was detected in root nodules from a variety of legumes, including M. truncatula. Whereas only one SucS isoform was detectable in root nodules, an additional sucrose synthase of slightly larger molecular weight was present in uninfected root, stem and flower tissues of M. truncatula. From our expression and sequence data we infer that the MtSucS1 gene encodes a nodule-enhanced sucrose synthase in M. truncatula. Southern hybridization data indicate that MtSucS1 is a single-copy gene. An analysis of a genomic MtSucS1 sequence revealed that the gene consists of 14 exons with the start codon being located on exon II. As is common for SucS genes, the MtSucS1 gene contains a large intron of 747 bp in the 5' untranslated region. The transcriptional start of MtSucS1 was mapped and putative regulatory elements in the MtSucS1 promoter were identified.

Expression of a soybean gene encoding the tetrapyrrole-synthesis enzyme glutamyl-tRNA reductase in symbiotic root nodules.

Heme and chlorophyll accumulate to high levels in legume root nodules and in photosynthetic tissues, respectively, and they are both derived from the universal tetrapyrrole precursor delta-aminolevulinic acid (ALA). The first committed step in ALA and tetrapyrrole synthesis is catalyzed by glutamyl-tRNA reductase (GTR) in plants. A soybean (Glycine max) root-nodule cDNA encoding GTR was isolated by complementation of an Escherichia coli GTR-defective mutant for restoration of ALA prototrophy. Gtr mRNA was very low in uninfected roots but accumulated to high levels in root nodules. The induction of Gtr mRNA in developing nodules was subsequent to that of the gene Enod2 (early nodule) and coincided with leghemoglobin mRNA accumulation. Genomic analysis revealed two Gtr genes, Gtr1 and a 3' portion of Gtr2, which were isolated from the soybean genome. RNase-protection analysis using probes specific to Gtr1 and Gtr2 showed that both genes were expressed, but Gtr1 mRNA accumulated to significantly higher levels. In addition, the qualitative patterns of expression of Gtr1 and Gtr2 were similar to each other and to total Gtr mRNA in leaves and nodules of mature plants and etiolated plantlets. The data indicate that Gtr1 is universal for tetrapyrrole synthesis and that a Gtr gene specific for a tissue or tetrapyrrole is unlikely. We suggest that ALA synthesis in specialized root nodules involves an altered spatial expression of genes that are otherwise induced strongly only in photosynthetic tissues of uninfected plants.

Isolation of lipoxygenase cDNA clones from pea nodule mRNA.

The pattern of lipoxygenase (LOX) gene expression was investigated in pea nodule tissues using the technique of in situ hybridization. Five lipoxygenase cDNAs were cloned from nodule mRNA by the RT-PCR and 3' RACE procedures. These clones (loxN1 to loxN5) show a high degree of sequence homology, except in the 3'-untranslated region. Gene-specific riboprobes were therefore generated from subclones carrying the 3'-untranslated regions in order to investigate tissue-specific gene expression. Northern blotting analysis revealed that loxN1 corresponded to a transcript that was expressed exclusively in roots and nodules but not in the aerial parts of the plant. However, none of the LOX genes appeared to be up-regulated in nodule tissue relative to uninfected roots. Starting with the incomplete cDNA clone for loxN1, the full coding sequence termed lox1:P.s:1 was obtained by further rounds of RT-PCR and 5' RACE procedures. In situ hybridization with nodule tissues revealed several different patterns of expression for the various LOX probes. However, none of the corresponding transcripts was expressed exclusively in the invasion zone, as might have been expected if one LOX gene product had been uniquely associated with the invasion process. In conclusion, this study provides no evidence for a direct role for any LOX gene product in plant-microbe interaction or host defence, but the fact that all the transcripts were expressed at the nodule apex suggests that LOX could be involved in the development of this organ.

A bacterial artificial chromosome library for soybean PI 437654 and identification of clones associated with cyst nematode resistance.

We have constructed a soybean bacterial artificial chromosome (BAC) library using the plant introduction (PI) 437654. The library contains 73 728 clones stored in 192 384-well microtiter plates. A random sampling of 230 BACs indicated an average insert size of 136 kb with a range of 20 to 325 kb, and less than 4% of the clones do not contain inserts. Ninety percent of BAC clones in the library have an average insert size greater than 100 kb. Based on a genome size of 1115 Mb, library coverage is 9 haploid genome equivalents. Screening the BAC library colony filters with cpDNA sequences showed that contamination of the genomic library with chloroplast clones was low (1.85%). Library screening with three genomic RFLP probes linked to soybean cyst nematode (SCN) resistance genes resulted in an average of 18 hits per probe (range 7 to 30). Two separate pools of forward and reverse suppression subtractive cDNAs obtained from SCN-infected and uninfected roots of PI437654 were hybridized to the BAC library filters. The 488 BACs identified from positive signals were fingerprinted and analyzed using FPC software (version 4.0) resulting in 85 different contigs. Contigs were grouped and analyzed in three categories: (1) contigs of BAC clones which hybridized to forward subtracted cDNAs, (2) contigs of BAC clones which hybridized to reverse subtracted cDNAs, and (3) contigs of BAC clones which hybridized to both forward and reverse subtracted cDNAs. This protocol provides an estimate of the number of genomic regions involved in early resistance response to a pathogenic attack.

Differential Display Analysis of the Early Compatible Interaction Between Soybean and the Soybean Cyst Nematode

The marked cellular changes during feeding site formation of the soybean cyst nematode (Heterodera glycines) indicate major changes in soybean gene expression. We used differential display of mRNA to detect host gene expression changes during the early compatible interaction between soybean and H. glycines. Fifteen cDNA clones corresponding to mRNAs with different abundances in H. glycines-infected versus uninfected roots were identified. Differential display results indicated that abundances of five mRNAs increased in infected roots, whereas abundances of 10 mRNAs decreased. Transcripts for nine of these 15 cDNAs could be detected on RNA blots, and their hybridization signals confirmed the differential display results for eight of these nine cDNAs. Sequence analyses identified five cDNAs with decreased mRNA levels in infected roots as corresponding to two putative aldolase genes, a transcription-factor TFIIA homologue, the soybean small GTP-binding protein gene sra1, and the soybean auxin down-regulated gene ADR12. RNA blot analyses of other auxin down-regulated genes revealed a decrease in their mRNA abundances in H. glycines-infected roots as well.

Evidence that the plant host synthesizes the heme moiety of leghemoglobin in root nodules

Although it is well established that the plant host encodes and synthesizes the apoprotein for leghemoglobin in root nodules, the source of the heme moiety has been uncertain. We recently found that the transcript for coproporphyrinogen III oxidase, one of the later enzymes of heme synthesis, is highly elevated in soybean (Glycine max L.) nodules compared with roots. In this study we measured enzyme activity and carried out western-blot analysis and in situ hybridization of mRNA to investigate the levels during nodulation of the plant-specific coproporphyrinogen oxidase and four other enzymes of the pathway in both soybean and pea (Pisum sativum L.). We compared them with the activity found in leaves and uninfected roots. Our results demonstrate that all of these enzymes are elevated in the infected cells of nodules. Because these are the same cells that express apoleghemoglobin, the data strongly support a role for the plant in the synthesis of the heme moiety of leghemoglobin.

Patterns of ENOD40 gene expression in stem-borne nodules of Sesbania rostrata.

At the base of adventitious root primordia, located on the stem of the tropical legume Sesbania rostrata, nitrogen-fixing nodules are formed upon inoculation with the microsymbiont Azorhizobium caulinodans. This pattern of nodule development presents features of indeterminate and determinate nodules in early and later stages, respectively. A S. rostrata cDNA clone homologous to early nodulin ENOD40 genes was isolated from a cDNA library of developing stem nodules. SrENOD40-1 contained the conserved regions I and II of other ENOD40 genes. By reverse transcriptase PCR, enhanced SrENOD40-1 expression was observed in the adventitious root primordia between 4 and 8 h after inoculation with A. caulinodans. In situ hybridization showed that SrENOD40-1 transcripts, present around the central vascular bundle of the uninfected root primordia, were strongly enhanced upon induction of nodule development. De novo SrENOD40-1 expression was observed in the initiating and growing nodule primordia and around vascular bundles. When cell type specification sets in, the expression became pronounced in cells derived from the meristematic regions. In other parts of the plant, weak SrENOD40-1 expression was associated with vascular bundles and was observed in leaf and stipule primordia.

Expression of Lupinus luteus cDNA coding for PR10 protein in Escherichia coli: purification of the recombinant protein for structural and functional studies.

The cDNA clones coding for two pathogenesis-related protein homologues of PR10 class, LlPR10.1A and LlPR10.1B, were identified in yellow lupin expression library of uninfected roots. The contribution of PR10 proteins to the overall mechanism of plant defence still remains unknown. In order to elucidate the structure and function of lupin PR10.1A protein, a substantial quantity of the protein was produced in an E. coli expression system using plasmids of pET-series: pET-3a and pET-15b, carrying the T7 promoter. Both plasmids with subcloned Llpr10.1a gene were overexpressed in E. coli, strain BL21(DE3)pLysS. The recombinant LlPR10.1A protein, overproduced in bacterial cells transformed with the pET-3a/Llpr10.1a plasmid, was purified to homogeneity from the insoluble "inclusion bodies" by ammonium sulphate fractionation and two sequential chromatographic steps: ion-exchange chromatography on DE 52 cellulose followed by size exclusion chromatography on Superdex 75 FPLC column. The (His)6 LlPR10.1A protein overproduced in E. coli cells harbouring the pET-15b/Llpr10.1a plasmid was purified by chromatography on Ni2+-charged His. Bind Resin. Western blot analysis with rabbit serum containing anti-LlPR10.1AN antibody revealed identical immunochemical properties of the two recombinant polypeptides and native LlPR10.1A protein. The recombinant protein produced in pET-3a plasmid was renatured from its insoluble form, concentrated up to 22 mg/ml and submitted to crystallisation. However, the LlPR10.1A protein expressed in pET-15b plasmid precipitated from the solution when at a higher concentration (10 mg/ml). This preparation was used at a lower concentration as an antigen for the preparation of polyclonal antibodies for immunochemical studies.

The use of inositol hexaphosphate as a phosphorus source by mycorrhizal and non‐mycorrhizal Scots Pine (Pinus sylvestris)

1. The external mycelia of the ectomycorrhizal fungi Thelephora terrestris and Suillus luteus, associated with Pinus sylvestris roots, exhibited a substantial extracellular acid phosphatase activity. The activity was positively correlated with the ergosterol concentration in the growth substratum and decreased with an increasing P nutrition. 2. The pioneer species T. terrestris grew best at a high Pi nutrition level whereas S. luteus, a ‘late‐stage’ mycobiont, produced more active biomass at a low Pi nutrition level. 3. The phytase activity of the external mycelia could not be detected; at the root surface a phytase activity was observed. Mycorrhizas had significantly higher activities than uninfected roots. 4. The addition of a relatively high concentration of a soluble phytate to the growth substratum resulted in an increased relative growth rate (RGR) in both mycorrhizal and non‐mycorrhizal plants. The influence of the mycorrhizal fungi on the use of the phytate‐P was small, despite the phytase activity of the mycorrhizal feeder roots. 5. The addition of phytate fixed on a HPLC resin did not result in an increase of the RGR and P uptake neither in the non‐mycorrhizal nor in the mycorrhizal Pines. The experiment did not support the hypothesis that phytate, which has a low solubility in soils, is a useful P source for ectomycorrhizal plants.

The asparagine synthetase gene VfAS1 is strongly expressed in the nitrogen-fixing zone of broad bean ( Vicia faba L.) root nodules

A full-length transcript sequence encoding the broad bean ( Vicia faba L.) asparagine synthetase (AS) VfAS1 was isolated from a nodule cDNA library. Sequence homologies indicated that VfAS1 belonged to the glutamine-dependent type of AS enzymes. The corresponding gene was highly expressed in root nodules and at a three-fold lower level in uninfected roots. Additionally, traces of VfAS1 transcripts were detected in epicotyl and stem tissues. Tissue-print hybridizations revealed that the VfAS1 gene was strongly expressed in the nitrogen-fixing zone III of root nodules. VfAS1 transcripts were absent from the meristem, the prefixing zone II as well as from peripheral nodule tissues.

Expression of Cell Cycle Genes During Sesbania rostrata Stem Nodule Development

Upon infection of Sesbania rostrata with Azorhizobium caulinodans, nodules are formed on roots and stems. Stem nodules develop from abundantly distributed dormant root primordia. To acquire more insight into the meristem organization during stem nodule development, the expression patterns of a mitotic B1-type cyclin gene (Sesro; CycB1;1), a cyclin-dependent kinase gene (Cdc-2-1Sr), and a histone H4 gene (H4-1Sr) of S. rostrata were followed by in situ hybridization. Cdc2-1Sr transcripts were found in all cells of uninfected and infected root primordia. In uninfected root primordia, Sesro;CycB1;1 transcripts were detected in a few cells of the apical root meristem whereas H4-1Sr transcripts were abundant in this region. Interestingly, after inoculation with A. caulinodans, H4-1Sr transcripts disappeared in the root meristem and a patchy pattern of Sesro;CycB1;1 and H4-1Sr expression appeared in the cortex of the root primordium, reflecting the formation of globular nodule primordia. When bacterial invasion started, a distal nodule meristem was delimited wherein Sesro;CycB1;1 and H4-1Sr expression was concentrated. Approximately 1 week after inoculation, meristem activity ceased, indicated by the loss of Sesro;CycB1;1 and H4-1Sr expression.

A mycorrhizal fungus changes microtubule orientation in tobacco root cells

Cortical cells of mycorrhizal roots undergo drastic morphological changes, such as vacuole fragmentation, nucleus migration, and deposition of cell wall components at the plant-fungus interface. We hypothesized that the cytoskeleton is involved in these mechanisms leading to cell reorganization. We subjected longitudinal, meristem to basal zone, sections of uninfectedNicotiana tabacum roots to immunofluorescence methods to identify the microtubular (MT) structures associated with root cells. Similar sections were obtained from tobacco roots grown in the presence ofGigaspora margarita, an arbuscular mycorrhizal fungus which penetrates the root via the epidermal cells, but mostly develops in the inner cortical cells. While the usual MT structures were found in uninfected roots (e.g., MTs involved in mitosis in the meristem and cortical hoops in differentiated parenchyma cells), an increase in complexity of MT structures was observed in infected tissues. At least three new systems were identified: (i) MTs running along large intracellular hyphae, (ii) MTs linking hyphae, (iii) MTs binding the hyphae to the host nucleus. The experiments show that mycorrhizal infection causes reorganization of root MTs, suggesting their involvement in the drastic morphological changes shown by the cortical cells.