Nitrogen acquisition in Agave tequilana from degradation of endophytic bacteria

Abstract

Plants form symbiotic associations with endophytic bacteria within tissues of leaves, stems, and roots. It is unclear whether or how plants obtain nitrogen from these endophytic bacteria. Here we present evidence showing nitrogen flow from endophytic bacteria to plants in a process that appears to involve oxidative degradation of bacteria. In our experiments we employed Agave tequilana and its seed-transmitted endophyte Bacillus tequilensis to elucidate organic nitrogen transfer from (15)N-labeled bacteria to plants. Bacillus tequilensis cells grown in a minimal medium with (15)NH4Cl as the nitrogen source were watered onto plants growing in sand. We traced incorporation of (15)N into tryptophan, deoxynucleosides and pheophytin derived from chlorophyll a. Probes for hydrogen peroxide show its presence during degradation of bacteria in plant tissues, supporting involvement of reactive oxygen in the degradation process. In another experiment to assess nitrogen absorbed as a result of endophytic colonization of plants we demonstrated that endophytic bacteria potentially transfer more nitrogen to plants and stimulate greater biomass in plants than heat-killed bacteria that do not colonize plants but instead degrade in the soil. Findings presented here support the hypothesis that some plants under nutrient limitation may degrade and obtain nitrogen from endophytic microbes.

Figure 1. Bacillus tequilensis: an endophyte of Agave tequilana.

Intracellular bacteria in meristematic root cells (A) and oxidation within root cortical cell (B), detail of cell morphology of the isolate grown on TSA medium (C), and bacteria on root surface showing H₂O₂ concentrations (brown) around bacterial cells (D).

Figure 2. Quantification of ¹⁵N-Trp in foliar tissue of A. tequilana using HPLC-MS/MS.

The sample groups analyzed were H₂O treated, unlabeled B. tequilensis (¹⁴N-Bteq) and ¹⁵N-labeled B. tequilensis (¹⁵N-Bteq). Nitrogen content was calculated as the quantity of ¹⁵N-Trp (ng/mg). Data are the mean values ± standard error of the mean from three independent experiments. (*)¹⁵N-labeled B. tequilensis data are significantly different when compared with the H₂O and unlabeled B. tequilensis groups (p<0.05). For multiple comparisons, t-test was applied.

Figure 3. HPLC-MS/MS analysis of ¹⁵N-labeled 2′-deoxynucleosides from A. tequilana supplemented with ¹⁵N-labeled B. tequilensis.

2′-Deoxynucleosides were detected by the loss of the 2-deoxyribose moiety: ¹⁵N₅-dG, m/z 273→157 (A), ¹⁵N₃-dC, m/z 231→115 (B), ¹⁵N₅-dA, m/z 257→141 (C) and ¹⁵N₂-dT, m/z 245→129 (D).

Figure 4. Relative abundance of pheophytin isotopomers from A. tequilensis.

(A) Chemical structure; (B) Percentage increase of pheophytin isotopomers calculated from plants supplemented with unlabeled B. tequilensis (C) and with ¹⁵N-labeled B. tequilensis (D). The data points shown are the mean values ± standard error of the mean from three independent experiments. p<0.08; p<0.02; p<0.05; p<0.01 and p<0.003, respectively for m/z 871.57, 872.57, 873.57, 874.57 and 875.57, comparing ¹⁴N and ¹⁵N-labeled B. tequilensis groups.