This study identified the phenotypic and genotypic characteristics of the bacteria that nodulate wild Lathyrus and Vicia species natural distribution in the Gaziantep province of Turkey. Principle component analysis of phenotypic features revealed that rhizobial isolates were highly resistant to stress factors such as high salt, pH and temperature. They were found to be highly sensitive to the concentrations (mg/mL) of the antibiotics neomycin 10, kanamycin, and tetracycline 5, as well as the heavy metals Ni 10, and Cu 10, and 5. As a result of REP-PCR analysis, it was determined that the rhizobial isolates were quite diverse, and 5 main groups and many subgroups being found. All of the isolates nodulating wild Vicia species were found to be related to Rhizobium sp., and these isolates were found to be in Clades II, III, IV, and V of the phylogenetic tree based on 16S rRNA. The isolates that nodulated wild Lathyrus species were in Clades I, II, IV, V, VI, VII, and VIII, and they were closely related to Rhizobium leguminasorum, Rhizobium sp., Phyllobacterium sp., Serratia sp., and Pseudomonas sp. According to the genetic analyses, the isolates could not be classified at the species level, the similarity ratio was low, they formed a distinct group that was supported by strong bootstrap values in the phylogenetic tree, and the differences discovered in the network analysis revealed the diversity among the isolates and gave important findings that these isolates may be new species.
- MeSH
- antibakteriální látky farmakologie MeSH
- Bacteria genetika klasifikace izolace a purifikace účinky léků MeSH
- DNA bakterií genetika MeSH
- fenotyp MeSH
- fylogeneze * MeSH
- genetická variace MeSH
- genotyp * MeSH
- kořenové hlízky rostlin * mikrobiologie MeSH
- Lathyrus * mikrobiologie MeSH
- Rhizobium genetika klasifikace izolace a purifikace MeSH
- RNA ribozomální 16S * genetika MeSH
- těžké kovy MeSH
- vikev * mikrobiologie MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Turecko MeSH
Alders (Alnus spp.) often dominate at nutrient-poor sites by symbiotic relations with atmospheric nitrogen-fixing bacteria. However, little is known about quantitative relationships between root nodule as a nitrogen acquisition organ and leaf as a carbon acquisition organ. To examine carbon allocation, nitrogen acquisition and net production in nutrient-poor conditions, we examined allocation patterns among organs of shrub Alnus fruticosa at a young 80-year-old moraine in Kamchatka. Slopes of double-log allometric equations were significantly smaller than 1.0 for the root mass, leaf mass and root nodule mass against stem mass, and for the root nodule mass against root mass, indicating that smaller individuals invested disproportionally more biomass into resource-acquiring leaf and root tissues than to supportive tissues compared to older individuals. The slope of allometric equation of root depth against stem height was 0.542, indicating that smaller/younger individuals allocate disproportionally more biomass into root length growth than stem height growth. On the contrary, the root nodule mass isometrically scaled to leaf mass. The whole-plant nitrogen content also isometrically scaled to root nodule mass, indicating that a certain ratio of nitrogen acquisition depended on root nodules, irrespective of plant size. Although the net production per plant increased with the increase in stem mass, the slope of the double-log regression was smaller than 1.0. On the contrary, the net production per plant isometrically increased with leaf mass, root nodule mass and leaf nitrogen content per plant. Since the leaf mass isometrically scaled to root nodule mass, growth of each individual occurred at the leaves and root nodules in a coordinated manner. It is suggested that their isometric increase contributes to the increase in net production per plant for A. fruticosa in nutrient-poor conditions.
- MeSH
- biomasa MeSH
- dusík metabolismus MeSH
- kořenové hlízky rostlin růst a vývoj fyziologie MeSH
- kořeny rostlin růst a vývoj fyziologie MeSH
- ledový příkrov MeSH
- olše růst a vývoj fyziologie MeSH
- stonky rostlin růst a vývoj fyziologie MeSH
- symbióza MeSH
- uhlík metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Rusko MeSH
Legume mutants have shown the requirement for receptor-mediated cytokinin signaling in symbiotic nodule organogenesis. While the receptors are central regulators, cytokinin also is accumulated during early phases of symbiotic interaction, but the pathways involved have not yet been fully resolved. To identify the source, timing, and effect of this accumulation, we followed transcript levels of the cytokinin biosynthetic pathway genes in a sliding developmental zone ofLotus japonicusroots.LjIpt2andLjLog4were identified as the major contributors to the first cytokinin burst. The genetic dependence and Nod factor responsiveness of these genes confirm that cytokinin biosynthesis is a key target of the common symbiosis pathway. The accumulation ofLjIpt2andLjLog4transcripts occurs independent of theLjLhk1receptor during nodulation. Together with the rapid repression of both genes by cytokinin, this indicates thatLjIpt2andLjLog4contribute to, rather than respond to, the initial cytokinin buildup. Analysis of the cytokinin response using the synthetic cytokinin sensor,TCSn, showed that this response occurs in cortical cells before spreading to the epidermis inL. japonicusWhile mutant analysis identified redundancy in several biosynthesis families, we found that mutation ofLjIpt4limits nodule numbers. Overexpression ofLjIpt3orLjLog4alone was insufficient to produce the robust formation of spontaneous nodules. In contrast, overexpressing a complete cytokinin biosynthesis pathway leads to large, often fused spontaneous nodules. These results show the importance of cytokinin biosynthesis in initiating and balancing the requirement for cortical cell activation without uncontrolled cell proliferation.
- MeSH
- biologické modely MeSH
- cytokininy biosyntéza MeSH
- kořenové hlízky rostlin cytologie genetika růst a vývoj fyziologie MeSH
- kořeny rostlin cytologie genetika růst a vývoj fyziologie MeSH
- Lotus cytologie genetika růst a vývoj fyziologie MeSH
- regulace genové exprese u rostlin MeSH
- regulátory růstu rostlin biosyntéza MeSH
- Rhizobiaceae fyziologie MeSH
- rostlinné proteiny genetika metabolismus MeSH
- signální transdukce * MeSH
- symbióza MeSH
- tvorba kořenových hlízek MeSH
- vývojová regulace genové exprese genetika MeSH
- Publikační typ
- časopisecké články MeSH
Cytokinins are required for symbiotic nodule development in legumes, and cytokinin signaling responses occur locally in nodule primordia and in developing nodules. Here, we show that the Lotus japonicus Ckx3 cytokinin oxidase/dehydrogenase gene is induced by Nod factor during the early phase of nodule initiation. At the cellular level, pCkx3::YFP reporter-gene studies revealed that the Ckx3 promoter is active during the first cortical cell divisions of the nodule primordium and in growing nodules. Cytokinin measurements in ckx3 mutants confirmed that CKX3 activity negatively regulates root cytokinin levels. Particularly, tZ and DHZ type cytokinins in both inoculated and uninoculated roots were elevated in ckx3 mutants, suggesting that these are targets for degradation by the CKX3 cytokinin oxidase/dehydrogenase. The effect of CKX3 on the positive and negative roles of cytokinin in nodule development, infection and regulation was further clarified using ckx3 insertion mutants. Phenotypic analysis indicated that ckx3 mutants have reduced nodulation, infection thread formation and root growth. We also identify a role for cytokinin in regulating nodulation and nitrogen fixation in response to nitrate as ckx3 phenotypes are exaggerated at increased nitrate levels. Together, these findings show that cytokinin accumulation is tightly regulated during nodulation in order to balance the requirement for cell divisions with negative regulatory effects of cytokinin on infection events and root development.
- MeSH
- alely MeSH
- buněčná diferenciace MeSH
- cytokininy metabolismus MeSH
- dusičnany metabolismus MeSH
- fenotyp MeSH
- fixace dusíku genetika MeSH
- fylogeneze MeSH
- homeostáza * MeSH
- kořenové hlízky rostlin genetika růst a vývoj MeSH
- Lotus enzymologie genetika růst a vývoj MeSH
- meristém cytologie růst a vývoj MeSH
- mutace genetika MeSH
- oxidoreduktasy genetika metabolismus MeSH
- promotorové oblasti (genetika) MeSH
- rostlinné geny MeSH
- rostlinné proteiny genetika metabolismus MeSH
- tvorba kořenových hlízek genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Medicago truncatula represents a model plant species for understanding legume-bacteria interactions. M. truncatula roots form a specific root-nodule symbiosis with the nitrogen-fixing bacterium Sinorhizobium meliloti. Symbiotic nitrogen fixation generates high iron (Fe) demands for bacterial nitrogenase holoenzyme and plant leghemoglobin proteins. Leguminous plants acquire Fe via "Strategy I," which includes mechanisms such as rhizosphere acidification and enhanced ferric reductase activity. In the present work, we analyzed the effect of S. meliloti volatile organic compounds (VOCs) on the Fe-uptake mechanisms of M. truncatula seedlings under Fe-deficient and Fe-rich conditions. Axenic cultures showed that both plant and bacterium modified VOC synthesis in the presence of the respective symbiotic partner. Importantly, in both Fe-rich and -deficient experiments, bacterial VOCs increased the generation of plant biomass, rhizosphere acidification, ferric reductase activity, and chlorophyll content in plants. On the basis of our results, we propose that M. truncatula perceives its symbiont through VOC emissions, and in response, increases Fe-uptake mechanisms to facilitate symbiosis.
- MeSH
- biomasa MeSH
- chlorofyl analýza MeSH
- FMN-reduktasa metabolismus MeSH
- koncentrace vodíkových iontů MeSH
- kořenové hlízky rostlin mikrobiologie MeSH
- Medicago truncatula chemie růst a vývoj metabolismus mikrobiologie MeSH
- půda chemie MeSH
- Sinorhizobium meliloti metabolismus MeSH
- těkavé organické sloučeniny metabolismus MeSH
- železo metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: Fluorescent tagging of nodule bacteria forming symbioses with legume host plants represents a tool for vital tracking of bacteria inside the symbiotic root nodules and monitoring changes in gene activity. The constitutive expression of heterologous fluorescent proteins, such as green fluorescent protein (GFP), also allows screening for nodule occupancy by a particular strain. Imaging of the fluorescence signal on a macro-scale is associated with technical problems due to the robustness of nodule tissues and a high level of autofluorescence. SCOPE: These limitations can be reduced by the use of a model species with a fine root system, such as Vicia tetrasperma. Further increases in the sensitivity and specificity of the detection and in image resolution can be attained by the use of a fluorescence scanner. Compared with the standard CCD-type cameras, the availability of a laser source of a specified excitation wavelength decreases non-specific autofluorescence while the photomultiplier tubes in emission detection significantly increase sensitivity. The large scanning area combined with a high resolution allow us to visualize individual nodules during the scan of whole root systems. Using a fluorescence scanner with excitation wavelength of 488 nm, a band-pass specific emission channel of 532 nm and a long-pass background channel of 555 nm, it was possible to distinguish nodules occupied by a rhizobial strain marked with one copy of cycle3 GFP from nodules colonized by the wild-type strain. CONCLUSIONS: The main limitation of the current plant model and GFP with the wild-type emission peak at 409 nm is a sharp increase in root autofluorescence below 550 nm. The selectivity of the technique can be enhanced by the use of red-shifted fluorophores and the contrasting labelling of the variants, provided that the excitation (482 nm) and emission (737 nm) maxima corresponding to root chlorophyll are respected.
In rhizobial symbiosis with legume plant hosts, the symbiotic tissue in the root nodules of indeterminate type is localized to the basal part of the nodule where the symbiotic zones contain infected cells (IC) interspersed with uninfected cells (UC) that are devoid of rhizobia. Although IC are easily distinguished in nodule sections using standard histochemical techniques, their observation in intact nodules is hampered by nodule tissue characteristics. Tagging of Rhizobium leguminosarum bv. viciae strain 128C30 with a constitutively expressed gene for green fluorescent protein (nonshifted mutant form cycle3) in combination with the advantages of the tiny nodules formed by Vicia tetrasperma (L.) SCHREB . allowed for vital observation of symbiotic tissue using fluorescence microscopy. Separation of a red-shifted background channel and digital image stacking along z-axis enabled us to construct a nodule image in a classical fluorescence microscopy of nodules exceeding 1 mm in diameter. In parallel, visualization of nodule bacteria inside the symbiotic tissue by confocal microscopy at the excitation wavelength 488 nm clearly distinguished IC/UC pattern in the nodule virtual sections and revealed red-shifted fluorescence of nonrhizobial origin. This signal was located on the periphery of IC and increased with their degradation, thus suggesting accumulation of secondary metabolites, presumably flavonoids. The simultaneous detection of bacteria and secondary metabolites can be used for monitoring changes to intact nodule physiology in the model legumes. The advantage of V. tetrasperma as a suggested laboratory model for pea cross-inoculation group has been demonstrated.
- MeSH
- fluorescenční mikroskopie MeSH
- kořenové hlízky rostlin fyziologie chemie mikrobiologie MeSH
- půdní mikrobiologie MeSH
- Rhizobium leguminosarum cytologie fyziologie genetika chemie MeSH
- symbióza MeSH
- vikev cytologie fyziologie chemie mikrobiologie MeSH
- zelené fluorescenční proteiny analýza genetika metabolismus MeSH
The root nodules of Phaseolus mungo (a herbaceous leguminous pulse) contained a high amount of 3-indolylacetic acid (IAA). A tryptophan pool present in the nodule might play the role of precursor for IAA production. From the root nodule a Rhizobium sp. was isolated. The symbiont produced a large amount of IAA (142 microg/mL) from L-tryptophan-supplemented basal medium. The production of IAA by the symbiont was much increased over the control when a L-tryptophan (2 mg/mL) supplemented C-free mineral medium was enriched with mannitol (1 %), L-asparagine (0.3 %) and thiamine hydrochloride (1 microg/mL). The possible role of the rhizobial production of IAA on the rhizobia-legume symbiosis is discussed.
