| 1. |
- Ilina, Elena L., et al.
(författare)
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Composite Cucurbita pepo plants with transgenic roots as a tool to study root development
- 2012
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Ingår i: Annals of Botany. - : Oxford University Press (OUP). - 0305-7364 .- 1095-8290. ; 110:2, s. 479-489
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Tidskriftsartikel (refereegranskat)abstract
- In most plant species, initiation of lateral root primordia occurs above the elongation zone. However, in cucurbits and some other species, lateral root primordia initiation and development takes place in the apical meristem of the parental root. Composite transgenic plants obtained by Agrobacterium rhizogenes-mediated transformation are known as a suitable model to study root development. The aim of the present study was to establish this transformation technique for squash. The auxin-responsive promoter DR5 was cloned into the binary vectors pKGW-RR-MGW and pMDC162-GFP. Incorporation of 5-ethynyl-2-deoxyuridine (EdU) was used to evaluate the presence of DNA-synthesizing cells in the hypocotyl of squash seedlings to find out whether they were suitable for infection. Two A. rhizogenes strains, R1000 and MSU440, were used. Roots containing the respective constructs were selected based on DsRED1 or green fluorescent protein (GFP) fluorescence, and DR5::Egfp-gusA or DR5::gusA insertion, respectively, was verified by PCR. Distribution of the response to auxin was visualized by GFP fluorescence or -glucuronidase (GUS) activity staining and confirmed by immunolocalization of GFP and GUS proteins, respectively. Based on the distribution of EdU-labelled cells, it was determined that 6-day-old squash seedlings were suited for inoculation by A. rhizogenes since their root pericycle and the adjacent layers contain enough proliferating cells. Agrobacterium rhizogenes R1000 proved to be the most virulent strain on squash seedlings. Squash roots containing the respective constructs did not exhibit the hairy root phenotype and were morphologically and structurally similar to wild-type roots. The auxin response pattern in the root apex of squash resembled that in arabidopsis roots. Composite squash plants obtained by A. rhizogenes-mediated transformation are a good tool for the investigation of root apical meristem development and root branching.
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| 2. |
- Ilina, Elena L., et al.
(författare)
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Lateral root initiation and formation within the parental root meristem of Cucurbita pepo : is auxin a key player?
- 2018
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Ingår i: Annals of Botany. - : Oxford University Press (OUP). - 0305-7364 .- 1095-8290. ; 122:5, s. 873-888
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Tidskriftsartikel (refereegranskat)abstract
- Background and Aims In some plant families. including Cucurbitaceae, initiation and development of lateral roots (LRs) occur in the parental root apical meristem. The objective of this study was to identify the general mechanisms underlying LR initiation (LRI). Therefore, the first cellular events leading to LRI as well as the role of auxin in this process were studied in the Cucurbita pepo root apical meristem.Methods Transgenic hairy roots harbouring the auxin-responsive promoter DR5 fused to different reporter genes were used for visualizing of cellular auxin response maxima (ARMs) via confocal laser scanning microscopy and 3-D imaging. The effects of exogenous auxin and auxin transport inhibitors on root branching were analysed.Key Results The earliest LRI event involved a group of symmetric anticlinal divisions in pericycle cell files at a distance of 250-350 pm from the initial cells. The visualization of the ARMs enabled the precise detection of cells involved in determining the site of LR primordium formation. A local ARM appeared in sister cells of the pericycle and endodermis files before the first division. Cortical cells contributed to LR development after the anticlinal divisions in the pericycle via the formation of an ARM. Exogenous auxins did not increase the total number of LRs and did not affect the LRI index. Although exogenous auxin transport inhibitors acted in different ways. they all reduced the number of LRs formed.Conclusions Literature data, as well as results obtained in this study, suggest that the formation of a local ARM before the first anticlinal formative divisions is the common mechanism underlying LRI in flowering plants. We propose that the mechanisms of the regulation of root branching are independent of the position of the LRI site relative to the parental root tip.
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| 3. |
- Demchenko, Kirill N., et al.
(författare)
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Plasmodesmata without callose and calreticulin in higher plants - open channels for fast syrnplastic transport?
- 2014
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Ingår i: Frontiers in Plant Science. - : Frontiers Reseach Foundation. - 1664-462X. ; 5
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Tidskriftsartikel (refereegranskat)abstract
- Plasmodesmata (PD) represent membrane-lined channels that link adjacent plant cells across the cell wall. PD of higher plants contain a central tube of endoplasmic reticulum (ER) called desmotubule. Membrane and lumen proteins seem to be able to move through the desmotubule, but most transport processes through PD occur through the cytoplasmic annulus (Brunkard (Bale 2013). Calreticulin (CRT), a highly conserved Ca2+-binding protein found in all multicellular eukaryotes, predominantly located in the ER, was shown to localize to PD, though not all PD accumulate CRT. In nitrogen-fixing actinorhizal root nodules of the Australian tree Casuarina glauca, the primary walls of infected cells containing the microsymbiont become lignified upon infection. TEM analysis of these nodules showed that during the differentiation of infected cells, PD connecting infected cells, and connecting infected and adjacent uninfected cells, were reduced in number as well as diameter (Schubert eta, 2013). In contrast with PD connecting young infected cells, and most PD connecting mature infected and adjacent uninfected cells, PD connecting mature infected cells did not accumulate CRT. Furthermore, as shown here, these PD were not associated with callose, and based on their diameter, they probably had lost their desmotubules. We speculate that either this is a slow path to PD degradation, or that the loss of callose accumulation and presumably also desmotubules leads to the PD becoming open channels and improves metabolite exchange between cells.
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| 4. |
- Demina, Irina V., et al.
(författare)
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Accumulation of and Response to Auxins in Roots and Nodules of the Actinorhizal Plant Datisca glomerata Compared to the Model Legume Medicago truncatula
- 2019
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Ingår i: Frontiers in Plant Science. - : Frontiers Media SA. - 1664-462X. ; 10
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Tidskriftsartikel (refereegranskat)abstract
- Actinorhizal nodules are structurally different from legume nodules and show a greater similarity to lateral roots. Because of the important role of auxins in lateral root and nodule formation, auxin profiles were examined in roots and nodules of the actinorhizal species Datisca glomerata and the model legume Medicago truncatula. The auxin response in roots and nodules of both species was analyzed in transgenic root systems expressing a beta-glucuronidase gene under control of the synthetic auxin-responsive promoter DR5. The effects of two different auxin on root development were compared for both species. The auxin present in nodules at the highest levels was phenylacetic acid (PAA). No differences were found between the concentrations of active auxins of roots vs. nodules, while levels of the auxin conjugate indole-3-acetic acid-alanine were increased in nodules compared to roots of both species. Because auxins typically act in concert with cytokinins, cytokinins were also quantified. Concentrations of cis-zeatin and some glycosylated cytokinins were dramatically increased in nodules compared to roots of D. glomerata, but not of M. truncatula. The ratio of active auxins to cytokinins remained similar in nodules compared to roots in both species. The auxin response, as shown by the activation of the DR5 promoter, seemed significantly reduced in nodules compared to roots of both species, suggesting the accumulation of auxins in cell types that do not express the signal transduction pathway leading to DR5 activation. Effects on root development were analyzed for the synthetic auxin naphthaleneacetic acid (NAA) and PAA, the dominant auxin in nodules. Both auxins had similar effects, except that the sensitivity of roots to PAA was lower than to NAA. However, while the effects of both auxins on primary root growth were similar for both species, effects on root branching were different: both auxins had the classical positive effect on root branching in M. truncatula, but a negative effect in D. glomerata. Such a negative effect of exogenous auxin on root branching has previously been found for a cucurbit that forms lateral root primordia in the meristem of the parental root; however, root branching in D. glomerata does not follow that pattern.
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| 5. |
- Kiryushkin, Alexey S., et al.
(författare)
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Do DEEPER ROOTING 1 Homologs Regulate the Lateral Root Slope Angle in Cucumber (Cucumis sativus)?
- 2024
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Ingår i: International Journal of Molecular Sciences. - 1661-6596 .- 1422-0067. ; 25:4
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Tidskriftsartikel (refereegranskat)abstract
- The architecture of the root system is fundamental to plant productivity. The rate of root growth, the density of lateral roots, and the spatial structure of lateral and adventitious roots determine the developmental plasticity of the root system in response to changes in environmental conditions. One of the genes involved in the regulation of the slope angle of lateral roots is DEEPER ROOTING 1 (DRO1). Its orthologs and paralogs have been identified in rice, Arabidopsis, and several other species. However, nothing is known about the formation of the slope angle of lateral roots in species with the initiation of lateral root primordia within the parental root meristem. To address this knowledge gap, we identified orthologs and paralogs of the DRO1 gene in cucumber (Cucumis sativus) using a phylogenetic analysis of IGT protein family members. Differences in the transcriptional response of CsDRO1, CsDRO1-LIKE1 (CsDRO1L1), and CsDRO1-LIKE2 (CsDRO1L2) to exogenous auxin were analyzed. The results showed that only CsDRO1L1 is auxin-responsive. An analysis of promoter-reporter fusions demonstrated that the CsDRO1, CsDRO1L1, and CsDRO1L2 genes were expressed in the meristem in cell files of the central cylinder, endodermis, and cortex; the three genes displayed different expression patterns in cucumber roots with only partial overlap. A knockout of individual CsDRO1, CsDRO1L1, and CsDRO1L2 genes was performed via CRISPR/Cas9 gene editing. Our study suggests that the knockout of individual genes does not affect the slope angle formation during lateral root primordia development in the cucumber parental root.
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| 6. |
- Kiryushkin, Alexey S., et al.
(författare)
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Hairy CRISPR : Genome Editing in Plants Using Hairy Root Transformation
- 2022
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Ingår i: PLANTS. - : MDPI AG. - 2223-7747. ; 11:1
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Tidskriftsartikel (refereegranskat)abstract
- CRISPR/Cas-mediated genome editing is a powerful tool of plant functional genomics. Hairy root transformation is a rapid and convenient approach for obtaining transgenic roots. When combined, these techniques represent a fast and effective means of studying gene function. In this review, we outline the current state of the art reached by the combination of these approaches over seven years. Additionally, we discuss the origins of different Agrobacterium rhizogenes strains that are widely used for hairy root transformation; the components of CRISPR/Cas vectors, such as the promoters that drive Cas or gRNA expression, the types of Cas nuclease, and selectable and screenable markers; and the application of CRISPR/Cas genome editing in hairy roots. The modification of the already known vector pKSE401 with the addition of the rice translational enhancer OsMac3 and the gene encoding the fluorescent protein DsRed1 is also described.
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| 7. |
- Kiryushkin, Alexey S., et al.
(författare)
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Lateral Root Initiation in Cucumber (Cucumis sativus) : What Does the Expression Pattern of Rapid Alkalinization Factor 34 (RALF34) Tell Us?
- 2023
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Ingår i: International Journal of Molecular Sciences. - 1661-6596 .- 1422-0067. ; 24:9
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Tidskriftsartikel (refereegranskat)abstract
- In Arabidopsis, the small signaling peptide (peptide hormone) RALF34 is involved in the gene regulatory network of lateral root initiation. In this study, we aimed to understand the nature of the signals induced by RALF34 in the non-model plant cucumber (Cucumis sativus), where lateral root primordia are induced in the apical meristem of the parental root. The RALF family members of cucumber were identified using phylogenetic analysis. The sequence of events involved in the initiation and development of lateral root primordia in cucumber was examined in detail. To elucidate the role of the small signaling peptide CsRALF34 and its receptor CsTHESEUS1 in the initial stages of lateral root formation in the parental root meristem in cucumber, we studied the expression patterns of both genes, as well as the localization and transport of the CsRALF34 peptide. CsRALF34 is expressed in all plant organs. CsRALF34 seems to differ from AtRALF34 in that its expression is not regulated by auxin. The expression of AtRALF34, as well as CsRALF34, is regulated in part by ethylene. CsTHESEUS1 is expressed constitutively in cucumber root tissues. Our data suggest that CsRALF34 acts in a non-cell-autonomous manner and is not involved in lateral root initiation in cucumber.
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| 8. |
- Kiryushkin, Alexey S., et al.
(författare)
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Lateral Root Initiation in the Parental Root Meristem of Cucurbits : Old Players in a New Position
- 2019
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Ingår i: Frontiers in Plant Science. - : Frontiers Media SA. - 1664-462X. ; 10
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Tidskriftsartikel (refereegranskat)abstract
- While in most higher plants, including the model system Arabidopsis thaliana, the formation of lateral root primordia is induced in the elongation zone of the parental root, in seven plant families, including Cucurbitaceae, an alternative root branching mechanism is established such that lateral roots are initiated directly in the apical meristem of the parental root. In Arabidopsis, the transcription factor GATA23 and MEMBRANE-ASSOCIATED KINASE REGULATOR4 (MAKR4) are involved in the gene regulatory network of lateral root initiation. Among all marker genes examined, these are the earliest known marker genes up-regulated by auxin during lateral root initiation. In this study, putative functional orthologs of Arabidopsis GATA23 and MAKR4 were identified in cucumber (Cucumis sativus) and squash (Cucurbita pepo). Both cucurbits contained 26 genes encoding GATA family transcription factors and only one MAKR4 gene. Phylogenetic and transcriptional analysis of up-regulation by auxin led to the identification of GATA23 putative functional orthologs in Cucurbitaceae - CpGATA24 and CsGATA24. In squash, CpMAKR4 was up-regulated by naphthylacetic acid (NM) and, similar to MAKR4 in Arabidopsis, indole-3-butyric acid (IBA). A detailed analysis of the expression pattern of CpGATA24 and CpMAKR4 in squash roots from founder cell specification until emergence of lateral root primordia was carried out using promoter-fluorescent reporter gene fusions and confocal microscopy. Their expression was induced in the protoxylem, and then expanded to founder cells in the pericycle. Thus, while the overall expression pattern of these genes was significantly different from that in Arabidopsis, in founder cells their expression was induced in the same order as in Arabidopsis. Altogether, these findings suggest that in Cucurbitaceae the putative functional orthologs of GATA23 and MAKR4 might play a role in founder cell specification and primordium positioning during lateral root initiation. The role of the protoxylem in auxin transport as a trigger of founder cells specification and lateral root initiation is discussed.
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| 9. |
- Pawlowski, Katharina, et al.
(författare)
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The diversity of actinorhizal symbiosis
- 2012
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Ingår i: Protoplasma. - : Springer Science and Business Media LLC. - 0033-183X .- 1615-6102. ; 249:4, s. 967-979
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Forskningsöversikt (refereegranskat)abstract
- Filamentous aerobic soil actinobacteria of the genus Frankia can induce the formation of nitrogen-fixing nodules on the roots of a diverse group of plants from eight dicotyledonous families, collectively called actinorhizal plants. Within nodules, Frankia can fix nitrogen while being hosted inside plant cells. Like in legume/rhizobia symbioses, bacteria can enter the plant root either intracellularly through an infection thread formed in a curled root hair, or intercellularly without root hair involvement, and the entry mechanism is determined by the host plant species. Nodule primordium formation is induced in the root pericycle as for lateral root primordia. Mature actinorhizal nodules are coralloid structures consisting of multiple lobes, each of which represents a modified lateral root without a root cap, a superficial periderm and with infected cells in the expanded cortex. In this review, an overview of nodule induction mechanisms and nodule structure is presented including comparisons with the corresponding mechanisms in legume symbioses.
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| 10. |
- Schubert, Maria, et al.
(författare)
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Lignification of cell walls of infected cells in Casuarina glauca nodules that depend on symplastic sugar supply is accompanied by reduction of plasmodesmata number and narrowing of plasmodesmata
- 2013
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Ingår i: Physiologia Plantarum. - : Wiley. - 0031-9317 .- 1399-3054. ; 147:4, s. 524-540
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Tidskriftsartikel (refereegranskat)abstract
- The oxygen protection system for the bacterial nitrogen-fixing enzyme complex nitrogenase in actinorhizal nodules of Casuarina glauca resembles that of legume nodules: infected cells contain large amounts of the oxygen-binding protein hemoglobin and are surrounded by an oxygen diffusion barrier. However, while in legume nodules infected cells are located in the central tissue, actinorhizal nodules are composed of modified lateral roots with infected cells in the expanded cortex. Since an oxygen diffusion barrier around the entire cortex would also block oxygen access to the central vascular system where it is required to provide energy for transport processes, here each individual infected cell is surrounded with an oxygen diffusion barrier. In order to assess the effect of these oxygen diffusion barriers on oxygen supply for energy production for transport processes, apoplastic and symplastic sugar transport pathways in C. glauca nodules were examined. The results support the idea that sugar transport to and within the nodule cortex relies to a large extent on the less energy-demanding symplastic mechanism. This is in line with the assumption that oxygen access to the nodule vascular system is substantially restricted. In spite of this dependence on symplastic transport processes to supply sugars to infected cells, plasmodesmal connections between infected cells, and to a lesser degree with uninfected cells, were reduced during the differentiation of infected cells.
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| 11. |
- Shumilina, Julia, et al.
(författare)
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Integrative Proteomics and Metabolomics Analysis Reveals the Role of Small Signaling Peptide Rapid Alkalinization Factor 34 (RALF34) in Cucumber Roots
- 2023
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Ingår i: International Journal of Molecular Sciences. - 1661-6596 .- 1422-0067. ; 24:8
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Tidskriftsartikel (refereegranskat)abstract
- The main role of RALF small signaling peptides was reported to be the alkalization control of the apoplast for improvement of nutrient absorption; however, the exact function of individual RALF peptides such as RALF34 remains unknown. The Arabidopsis RALF34 (AtRALF34) peptide was proposed to be part of the gene regulatory network of lateral root initiation. Cucumber is an excellent model for studying a special form of lateral root initiation taking place in the meristem of the parental root. We attempted to elucidate the role of the regulatory pathway in which RALF34 is a participant using cucumber transgenic hairy roots overexpressing CsRALF34 for comprehensive, integrated metabolomics and proteomics studies, focusing on the analysis of stress response markers. CsRALF34 overexpression resulted in the inhibition of root growth and regulation of cell proliferation, specifically in blocking the G2/M transition in cucumber roots. Based on these results, we propose that CsRALF34 is not part of the gene regulatory networks involved in the early steps of lateral root initiation. Instead, we suggest that CsRALF34 modulates ROS homeostasis and triggers the controlled production of hydroxyl radicals in root cells, possibly associated with intracellular signal transduction. Altogether, our results support the role of RALF peptides as ROS regulators.
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| 12. |
- Voitsekhovskaja, Olga V, et al.
(författare)
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Evidence for functional heterogeneity of sieve element-companion cell complexes in minor vein phloem of Alonsoa meridionalis.
- 2009
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Ingår i: Journal of experimental botany. - : Oxford University Press (OUP). - 1460-2431 .- 0022-0957. ; 60:6, s. 1873-83
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Tidskriftsartikel (refereegranskat)abstract
- Two modes of phloem loading have been proposed, apoplastic and symplastic, depending on the structure of sieve element-companion cell complexes (SE-CCCs) in minor vein phloem. Species are usually classified as either apoplastic or symplastic loaders although the cytology of SE-CCCs in minor veins of the majority of plants indicates that both mechanisms can be simultaneously involved in phloem loading. The functions of structurally different SE-CCCs in minor veins of the stachyose-translocating plant Alonsoa meridionalis were examined. A stachyose synthase gene, AmSTS1, was expressed in intermediary cells but not in the ordinary companion cell of the same vein. In contrast, sucrose transporter AmSUT1 protein was present in ordinary companion cells but not in the neighbouring intermediary cells. These data reveal the principles of phloem sap formation in A. meridionalis and, probably, in many other dicots. The two types of SE-CCCs within one and the same minor vein load different carbohydrates, using contrasting mechanisms for their delivery into the phloem. Lateral sieve pores in the minor vein phloem lead to mixing of the carbohydrates soon after loading. While symplastic and apoplastic pathways can function simultaneously during phloem loading, they are separated at the level of different SE-CCCs combined in phloem endings.
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| 13. |
- Wibberg, Daniel, et al.
(författare)
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Frankia-Enriched Metagenomes from the Earliest Diverging Symbiotic Frankia Cluster : They Come in Teams
- 2019
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Ingår i: Genome Biology and Evolution. - : Oxford University Press (OUP). - 1759-6653. ; 11:8, s. 2273-2291
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Tidskriftsartikel (refereegranskat)abstract
- Frankia strains induce the formation of nitrogen-fixing nodules on roots of actinorhizal plants. Phylogenetically, Frankia strains can be grouped in four clusters. The earliest divergent cluster, cluster-2, has a particularly wide host range. The analysis of cluster-2 strains has been hampered by the fact that with two exceptions, they could never be cultured. In this study, 12 Frankia-enriched metagenomes of Frankia cluster-2 strains or strain assemblages were sequenced based on seven inoculum sources. Sequences obtained via DNA isolated from whole nodules were compared with those of DNA isolated from fractionated preparations enhanced in the Frankia symbiotic structures. The results show that cluster-2 inocula represent groups of strains, and that strains not represented in symbiotic structures, that is, unable to performsymbiotic nitrogen fixation, may still be able to colonize nodules. Transposase gene abundance was compared in the different Frankia-enriched metagenomes with the result that NorthAmerican strains contain more transposase genes than Eurasian strains. An analysis of the evolution and distribution of the host plants indicated that bursts of transposition may have coincided with niche competition with other cluster-2 Frankia strains. The first genome of an inoculum from the Southern Hemisphere, obtained from nodules of Coriaria papuana in Papua NewGuinea, represents a novel species, postulated as Candidatus Frankiameridionalis. All Frankia-enrichedmetagenomes obtained in this study contained homologs of the canonical nod genes nodABC; the North American genomes also contained the sulfotransferase gene nodH, while the genome from the Southern Hemisphere only contained nodC and a truncated copy of nodB.
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| 14. |
- Zdyb, Anna, et al.
(författare)
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Allene oxide synthase, allene oxide cyclase and jasmonic acid levels in Lotus japonicus nodules
- 2018
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 13:1
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Tidskriftsartikel (refereegranskat)abstract
- Jasmonic acid (JA), its derivatives and its precursor cis-12-oxo phytodienoic acid (OPDA) form a group of phytohormones, the jasmonates, representing signal molecules involved in plant stress responses, in the defense against pathogens as well as in development. Elevated levels of JA have been shown to play a role in arbuscular mycorrhiza and in the induction of nitrogen-fixing root nodules. In this study, the gene families of two committed enzymes of the JA biosynthetic pathway, allene oxide synthase (AOS) and allene oxide cyclase (AOC), were characterized in the determinate nodule-forming model legume Lotus japonicus JA levels were to be analysed in the course of nodulation. Since in all L. japonicus organs examined, JA levels increased upon mechanical disturbance and wounding, an aeroponic culture system was established to allow for a quick harvest, followed by the analysis of JA levels in whole root and shoot systems. Nodulated plants were compared with non-nodulated plants grown on nitrate or ammonium as N source, respectively, over a five week-period. JA levels turned out to be more or less stable independently of the growth conditions. However, L. japonicus nodules formed on aeroponically grown plants often showed patches of cells with reduced bacteroid density, presumably a stress symptom. Immunolocalization using a heterologous antibody showed that the vascular systems of these nodules also seemed to contain less AOC protein than those of nodules of plants grown in perlite/vermiculite. Hence, aeroponically grown L. japonicus plants are likely to be habituated to stress which could have affected JA levels.
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