Browsing by Subject "AMT"

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    Interactions between non-symbiotic N2-fixing bacteria and plant roots in plant-microbial associations
    (2009) Calvo Alegre, Olga-Cristina; von Wirén, Nicolaus
    The development of biofertilizers on the basis of plant growth promoting rhizobacteria (PGPR) may be a promising approach to partially substitute costly and energy-consuming mineral fertilizers in agricultural plant production and to support agriculture in developing countries. A successful and competitive rhizosphere colonization of PGPR strains has been identified as a prerequisite for the expression of plant growth promoting effects. Apart from a wide range of external factors with an impact on the colonization process, such as soil properties, temperature, soil moisture and fertilization levels, in particular plant-microbial interactions may play an important role for the successful establishment of compatible associations. In this context, certain plant root exudates may act as signals to mediate bacterial responses with importance for root colonisation (e.g. motility and chemotaxis, production of extracellular polysaccharides). On the other hand, the induction of bacterial plant growth promotion may also depend on ability of the host plant to respond to the presence and the activity of the associated bacteria. It was therefore the aim of this thesis to investigate the contribution of putative PGPR to growth and N uptake in wheat plants and characterize the underlying mechanisms in root-bacterial associations. In the first part of this thesis, the contribution of various non-symbiotic diazotrophic rhizobacteria to plant growth promotion and N nutrition has been studied in a series of greenhouse pot inoculation experiments with wheat (Triticum aestivum L.). Different bacterial inoculants, plant genotypes, soil properties, water regimes and N fertilization levels have been varied as factors with potential impact on plant growth promotion by diazotrophs. The contribution of biological nitrogen fixation was assessed by the 15N dilution method. Plant growth and grain yield were influenced by the different N fertilization levels but no stimulation of growth or N uptake was note upon bacterial inoculation. These observations suggested a high degree of specificity or limiting factors, determining a successful plant-microbial association. The second part describes possible mechanisms that may be involved in the establishment of diazotrophs in the rhizosphere of suitable host plants. As an initial step of the colonization process, a targeted movement of the bacteria to the root surface is required and root exudates may act as attractants. Since dicarboxylic acids are known to exert chemotactic activity on diazotrophic bacteria, seed and root exudates of two graminaceous crops (Triticum aestivum L. and Zea mays L.) and for comparison also of a non-graminaceous plant species (Phaseolus vulgaris L.) were collected in hydroponic culture with and without N supply, and organic acid profiles in these root exudates were analysed. Bacterial motility assays were conducted with the major carboxylates detected in the root exudates of the selected plant species and compared to glucose and water, using Brevibacillus reuszeri as a model bacterium. Pure malate, which was found at high levels in root exudates of bean and wheat, and particularly malonate (bean) and t-aconitate (maize) stimulated the motility of Brevibacillus reuszeri as compared with glucose or water. A particularly intense promotion of bacterial motility was recorded in the presence of crude root exudates of wheat and maize plants grown under N limitation, which was not observed for root exudates of bean. However, this was not related with comparable changes of malate or t-aconitate concentrations in the root exudates. In wheat exudates, malate concentrations even decreased in response to N limitation. These findings suggest the presence of specific factors released in root exudates of N-deficient cereals, promoting the rhizosphere colonisation with B. reuszeri. For an identification of the respective factors, a more comprehensive profiling of the root exudates is necessary. In associations with diazotrophic bacteria, host plants are supplied with ammonium by the bacterial partner. This raised the question whether plant ammonium uptake systems have an impact on the efficiency of the association. To address this problem, an antisense approach was conducted with tomato, with characterised ammonium transporters (LeAMT1;1 and LeAMT1;2). The final goal was the inhibition of the ammonium transporters by production of LeAMT antisense lines to study their putative role in plant associations with diazotrophic bacteria. Northern blot analysis revealed a strong repression of LeAMT1;2 expression in three independent antisense lines associated with a lower ammonium uptake capacity under N-sufficient and N-deficient growth conditions. In contrast, LeAMT1,1 expression was only weakly repressed in antisense lines and there was no impact on N uptake. A faster decline of chlorophyll in older leaves indicates a physiological function of LeAMT1;1 and LeAMT1;2 in ammonium uptake and retrieval in shoot and root cells. The absence of consistent effects on N acquisition of the investigated antisense lines limited the suitability of this approach for studies on associations with diazotrophic bacteria.
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    Transporters mediating ammonium uptake in plants and their regulation by the abiotic stress signaling pathway
    (2023) Porras Murillo, Romano; Ludewig, Uwe
    Nitrogen nutrition refers to the uptake, assimilation, and utilization of ammonium, nitrate, and organic nitrogen sources. Ammonium is energetically a more cost-effective nitrogen source than nitrate but can be toxic for plants, and its use by plants is regulated at different levels. Ammonium transporters (AMTs) take up ammonium and are localized primarily in plant roots, working as trimers in the plasma membrane. Under high external ammonium concentrations, phosphorylation in AMTs C-termini shuts down transport to avoid toxicity. This phosphorylation is performed by CIPK23, a kinase shown to be inhibited by Clade A PP2Cs. This study aimed to characterize AMTs from wheat and analyze their transcriptional response to ammonium. Another aim was to determine the role of clade A PP2Cs and PYR/PYL receptor proteins for abscisic acid in ammonium nutrition. Chapter I describes the physiological responses of winter wheat to different nitrogen sources and ammonium concentrations. The plants mainly used root morphological responses to adapt to differences in the nitrogen source. High external concentrations of ammonium reduced plant growth, while these conditions induced the expression of TaAMT1;1 and TaAMT1;2. In Chapter II, we studied the capacity of TaAMT2s to transport ammonium and their transcriptional responsiveness to ammonium nutrition. From the six TaAMT2s, only TaAMT2;1 could transport ammonium in a yeast complementation line. Besides, its expression in roots is lower under ammonium than under nitrate. The expression pattern among the remaining TaAMT2s (TaAMT2;2-TaAMT2;6) is similar, with higher expression under ammonium, in both roots and leaves, compared to nitrate. Chapter III focused on the role of the PP2C phosphatase ABI1 (ABA-insensitive 1) in ammonium nutrition and the effect of external ammonium concentrations on ABA concentrations. Ammonium increased ABA concentrations in roots by activating ABA-GE, meaning ammonium toxicity could be sensed as abiotic stress through ABA. Without ammonium, ABI1 dephosphorylates AMTs and inhibits CIPK23; with ammonium, ABA-PYR/PYL complex-mediated inhibition of ABI1 releases CIPK23 to phosphorylate AMTs and avoids ammonium toxicity. Finally, in Chapter IV, we studied the role of AIP1 and its ammonium-dependent regulator, PYL8, in nitrogen nutrition. We described the function of AIP1, which was redundant to ABI1 in AMT regulation. Based on ammonium-dependent root architecture changes, and higher auxin accumulation in pyl8-1 root tips compared to the wild type, we suggest that PYL8 is involved in root-phenotype modulation in an ammonium-dependent manner.
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    Understanding the role of the Calcineurin B-like (CBL) proteins and the CBL-Interacting Protein Kinases (CIPK) of wheat (Triticum aestivum) in the regulation of its high affinity ammonium transporters
    (2021) Ijato, Toyosi; Ludewig, Uwe
    Ammonium is an important nitrogen source whose potential for toxicity (in plants) has been a limitation to its use as a fertilizer. In this work, the response of wheat to ammonium nutrition and the regulatory mechanism governing its high affinity ammonium transporters was investigated. Wheat was able to utilize sole ammonium in the 1 mM range just like sole nitrate. An elevated ammonium concentration of 10 mM caused ammonium induced toxicity effects. Unexpectedly, the wheat seedlings failed to downregulate TaAMT1;1 and TaAMT1;2 in response to elevated ammonium concentration. Nevertheless, TaAMT1;1 and TaAMT1;2 complement ammonium transport in the mep yeast strain (lacking endogenous ammonium transporters). The Voltage dependent ammonium induced currents of the transporters saturated in a concentration dependent manner with Km AMT1;1 = 76 μM and Km AMT1;2 = 196 μM. The affinity of the transporters for ammonium was voltage dependent and indicated that ammonium passes around 35 % of the membrane electric field before rate limiting deprotonation in both transporters. Furthermore, phospho-proteomics study showed the differential phosphorylation of TaAMT1;1 and TaAMT1;2 specific proteotypic phosphopetides. Interestingly, a mutation that mimics phosphorylation at the conserved threonine T453 (T453D) was able to inactivate the ammonium transport function of the transporters. Additionally TaCBL1, and TaCBL2 were able to independently regulate the activity of TaAMT1;1, while TaCBL1, TaCBL2 and TaCBL6 were able to independently impair the ammonium induced current of TaAMT1;2. Subsequently, TaCBL1, TaCBL2 and TaCBL6 interacted with TaCIPK9 to further reduce the ammonium induce current caused by the single TaCBLs proteins on TaAMT1s. TaCIPK23 and TaCIPK32 were only able to regulate TaAMT1;2 by interacting with both TaCBL2 and TaCBL6. Thus, the robust response of wheat to ammonium N was shown to include the phospho-regulation of its high affinity ammonium transporters in a manner that recruits the activity of its TaCBL and TaCIPK proteins.