The multiple plant response to high ammonium conditions: The Lotus japonicus AMT1; 3 protein acts as a putative transceptor

Plant evolved a complex profile of responses to cope with changes of nutrient availability in the soil. These are based on a stringent control of expression and/or activity of proteins involved in nutrients transport and assimilation. Furthermore, a sensing and signaling system for scanning the concentration of substrates in the rooted area and for transmitting this information to the plant machinery controlling root development can be extremely useful for an efficient plant response. Ammonium represents for plants either a preferential nitrogen source or the trigger for toxicity symptoms depending by its concentration. We propose a role for the high affinity Lotus japonicus ammonium transporter LjAMT1;3 as an intracellular ammonium sensor to achieve a convenient modulation of the root development in conditions of potentially toxic external ammonium concentration

Characterization of a Developmental Root Response Caused by External Ammonium Supply in Lotus japonicus1[C][W]

Plants respond to changes of nutrient availability in the soil by modulating their root system developmental plan. This response is mediated by systemic changes of the nutritional status and/or by local perception of specific signals. The effect of nitrate on Arabidopsis (Arabidopsis thaliana) root development represents a paradigm of these responses, and nitrate transporters are involved both in local and systemic control. Ammonium (NH4+) represents an important nitrogen (N) source for plants, although toxicity symptoms are often associated with high NH4+ concentration when this is present as the only N source. The reason for these effects is still controversial, and mechanisms associating ammonium supply and plant developmental programs are completely unknown. We determined in Lotus japonicus the range of ammonium concentration that significantly inhibits the elongation of primary and lateral roots without affecting the biomass of the shoot. The comparison of the growth phenotypes in different N conditions indicated the specificity of the ammonium effect, suggesting that this was not mediated by assimilatory negative feedback mechanisms. In the range of inhibitory NH4+ conditions, only the LjAMT1;3 gene, among the members of the LjAMT1 family, showed a strong increased transcription that was reflected by an enlarged topology of expression. Remarkably, the short-root phenotype was phenocopied in transgenic lines by LjAMT1;3 overexpression independently of ammonium supply, and the same phenotype was not induced by another AMT1 member. These data describe a new plant mechanism to cope with environmental changes, giving preliminary information on putative actors involved in this specific ammonium-induced response

Phase contrast and fluorescence microscopy analysis of SF214.

<p>Observation of the same microscopy field by phase contrast (left), autofluorescence (middle) and DAPI staining (right). The same section of each panel is enlarged. The arrows in the enlarged sections point to a doublet of cells, still partially attached and deriving from the same mother cell, in which only one cell (grey arrows) is autofluorescent.</p

Autofluorescence at different growth conditins.

<p>Microscopy fields of SF214 cells grown at different conditions and observed by phase contrast and autofluorescence and compared to assess the proportion of fluorescent vs not fluorescent cells. Left panel: exponential vs. stationary growth phase (in LB medium at 37°C); middle panel: 25°C vs. 37°C as growth temperature (in LB medium for 24 hours); right panel: minimal (S7) vs. rich (LB) growth medium (stationary cells grown at 25°C). For each panel a graph reports the percentage of fluorescent (gray bars) vs. not fluorescent (dark gray bars) cells. For each condition a total of 1.000 cells from five different microscopy fields were counted. Spores and cells containing a prespore were not counted.</p

Cell and spore survival after treatment with H₂O₂.

<p>Cells and spores were treated with 30 mM H₂O₂ for various times. For each time point the CFU of cells (gray symbols) and spores (white symbols) of the wild type (squares) and the mutant (circles) strains was obtained by plating on LB plates and incubation for 24 hours at 37°C.</p

Autofluorescence of sporulating cells.

<p>The same microscopy field observed by phase contrast (left), autofluorescence (middle). The right panel reports the merge of phase contrast and autofluorescence images. Cells were grown in rich (LB) medium for 15 hours.</p

Fluorescence and immunofluorescence microscopy with anti-CotE antibody.

<p>Microscopy analysis of cells from different fields observed by phase contrast, DAPI-staining, immunofluorescence with anti-CotE primary antibody and Texas Red conjugated secondary antibody. Merged panels of DAPI-immunofluorescence and autofluorescence-immunofluorescence are shown.</p

Microscopy analysis of SF214 and of its unpigmented mutant.

<p>Microscopy analysis of SF214 and of its unpigmented mutant (SF214-Mut). For each strain the same microscopy field is shown by phase contrast (left) and autofluorescence (right). The same conditions of exposure were used for the two microscopy fields.</p