Figure 1.
Exogenous phosphate and nitrate inhibit root colonization by Rhizophagus irregularis.
Plants inoculated with Rhizophagus irregularis were watered with the basic nutrient solution, additionally supplemented with the indicated nutrient concentrations. The first column to the left in each graph corresponds to the concentration in the basic nutrient solution, except for KH2PO4 (see Table S2); the other columns represent elevated nutrient levels as indicated. Columns represent the average of four replicate plants with standard deviations.
Figure 2.
Inhibition of AM colonization by exogenous phosphate depends on the supply with other nutrients.
KH2PO4 was applied to inoculated plants at the indicated concentrations either with basic nutrient solution (black columns) or alone (white columns). Additional treatments involved the application of KH2PO4 with only micronutrients (light grey) or only macronutrients (dark grey), respectively. Columns represent the average of four replicate plants with standard deviations. Asterisks indicate significant differences between phosphate alone (white bars) and phosphate with micronutrients (light grey bars), respectively, vs. the treatment with a combination of Pi and basic nutrient solution (black bars).
Figure 3.
Dynamics of fungal root colonization and plant growth under different nutritional conditions.
Plants inoculated or not with R. irregularis were supplied with water (blue), basic nutrient solution (red), 5 mM KH2PO4 (green), or with a combination of 5 mM KH2PO4 and basic nutrient solution (purple). Samples were harvested at the indicated time points to determine root colonization (a), shoot fresh weight (b,c), and mycorrhizal growth response (MGR; (d)). MGR is defined as the ratio of the shoot weight of mycorrhizal versus non-mycorrhizal control plants. Values are the mean of three biological replicates. Error bars represent standard deviations.
Figure 4.
R. irregularis increases nutrient content of plants supplied with water or with nutrient solution.
Nutrient levels in leaves were determined 36 days after inoculation in the plants shown in Figure 3. Values are the mean of three biological replicates. Error bars represent standard deviations. Asterisks indicate significant differences between mycorrhizal roots (black columns) and non-mycorrhizal controls (white columns). (a) Plants were fertilized with basic nutrient solution. Values are expressed relative to the non-mycorrhizal fertilized controls that were set to 100% for each nutrient. (b) As in (a), but without nutrient solution. Values are expressed relative to the non-colonized water-treated controls that were set to 100% for each nutrient.
Figure 5.
Treatment with KH2PO4 alone causes depletion of other nutrients and interferes with nutrient acquisition in mycorrhizal roots.
Nutrient levels in leaves were determined 36 days after inoculation in the plants shown in Figure 3. Values are the mean of three replicates. Error bars represent standard deviations. Asterisks indicate significant differences between Pi and water treatment (a) or between mycorrhizal and non-mycorrhizal roots (b). (a) Treatment of non-inoculated plants with 5 mM KH2PO4 alone (black columns) compared to controls with water alone (white columns). Values are expressed relative to the water-treated controls that were set to 100% for each nutrient. (b) Nutrient content of mycorrhizal plants treated with 5 mM KH2PO4 (black columns) compared to non-inoculated controls treated with 5 mM KH2PO4 (white columns; corresponding to black columns in (a)). Values are expressed relative to the non-colonized Pi-treated controls that were set to 100% for each nutrient.
Figure 6.
Shoot weight, shoot/root ratio and N/P ratio as indicators of nutritional status.
Treatments were as in Figure 3, shown are the values of the final time point (48 days after inoculation). Columns represent the average of three biological replicates, error bars represent standard deviations. Asterisks indicate significant differences between mycorrhizal and non-mycorrhizal plants (white vs. black columns), crosses indicate significant differences between the non-mycorrhizal nutrient treatments vs. the non-mycorrhizal water treatment (i.e. between the different white columns). (a) Shoot weight of plants grown with R. irregularis (black column) or without (white columns) under different nutritional conditions. (b) Shoot/root ratio of plants inoculated with R. irregularis (black columns) or without (white columns) under various nutritional conditions. A ratio of 3.5–4 indicates that plants are well supplied with mineral nutrients, whereas a ratio around 2 indicates that plants are starved and allocate relatively large amounts of resources to the root system to compensate nutritional deficits. (c) N/P ratio of the same plants as in (a),(b). In the absence of exogenous Pi supply, mycorrhizal plants (black columns) exhibited lower N/P ratios than non-mycorrhizal controls, reflecting increased mycorrhizal Pi supply. Administration of 5 mM KH2PO4 reduced N/P ratio even stronger than AM, in particular if only Pi was supplied.
Figure 7.
Phylogenetic analysis of Pi-responsive transporters of Petunia hybrida compared to Arabidopsis transporters for nitrate, nitrite, and peptides.
For the EST sequences listed in Table 1 the full-length predicted cDNA sequences were derived from the petunia genome sequence. Predicted petunia protein sequences were compared with Arabidopsis thaliana transporters for nitrate and nitrite (NRT and Nitr1, respectively), and for peptide transporters (PTR). Note the clear separation of the nitrate transporter subfamilies NRT1 and NRT2. The NRT1 family also comprises the nitrite transporter AtNitr1 and several peptide transporters, of which only two are represented (AtPTR2 and AtPTR5). Petunia has two very closely homologous representatives of the high affinity nitrate transporter family NRT2 (cn8666 and cn7864). In addition, there is a putative nitrite transporter (corresponding to the EST sequences CL1918 and cn5943), and two additional members of the low affinity NRT1 family. Genes boxed in green were analyzed by qPCR (Figure 8). Cn8665, which is almost identical to cn8666, and CL5245, which is predicted to encode a nitrogen limitation adaptation gene (see Table 1), were omitted from phylogenetic analysis.
Figure 8.
Quantitative real time PCR analysis (qPCR) of marker genes for nitrogen and phosphorus acquisition.
qPCR analysis was performed to determine the expression of the N transporters boxed in Figure 7, and of the AM-specific phosphate transporter PhPT4. Treatments were as in Figures 3–6 and Table 1. Expression values were first normalized to GAPDH and then expressed as induction ratios for the indicated treatments relative to the standard treatment (non-mycorrhizal fertilized plants), as in Table 1. Columns represent the mean of five biological replicates. Error bars represent the standard deviations derived from the two standard deviations of the compared treatments (see Materials and Methods). Numbers below the x-axis reflect the percentage of root colonization in the respective sample.
Figure 9.
Withdrawal of individual nutrients interferes with the inhibitory effect of Pi.
Effects of withdrawal of individual nutrients from the basic nutrient solution applied together with 52PO4. Omitted nutrients were replaced by other nutrients to maintain osmotic relationships. The strong inhibitory effect of Pi (P) was reduced particularly by removal of nitrate (P-N), but also to a lesser extent by removal of S, K, Ca, and Fe. The control treatment (c) represents fertilization with low Pi levels (0.03 mM). Columns represent the average of six biological replicates, error bars represent the standard deviations. Asterisks indicate significant differences between the treatments lacking individual nutrients and the treatment with basic nutrient solution and high Pi.
Table 1.
Microarray analysis of N-related transporter genes in relation to the nutritional status.