Nitrogen acquisition in Agave tequilana from degradation of endophytic bacteria

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 15N-labeled bacteria to plants. Bacillus tequilensis cells grown in a minimal medium with 15NH4Cl as the nitrogen source were watered onto plants growing in sand. We traced incorporation of 15N 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.

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 NH 4 Cl 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. L ike all living things, plants require nitrogen (N) throughout their development. The N incorporated as NO 3 2 and NH 4 1 represents about 2% of total plant dry matter, and is a component of proteins, nucleic acids, cofactors, signalling molecules, storage and numerous plant secondary products. The availability of N to plant roots is often an important limiting factor for plant growth. Plants obtain N from nitrogen fixing bacteria and decomposition of dead tissues of both plants and animals by microorganisms [1][2][3][4][5] . Moreover, a tiny fraction (0.00024%) of planetary N is available to plants in soils, where plants compete with microbes to absorb N 6 . The atmospheric formation of nitrate by photo-oxidation through the effect of lightning also is minimal 9,10 . The limited bioavailability of N, and the need to enhance crop growth, have provoked a continual expansion in use of N fertilizer with from 12 to 104 additional teragrams used each year 7,8 . On average only 30-50% of the applied N is taken up by plants, with the remainder being lost to surface run-off, leaching of nitrates and ammonia volatilization 11 . The excessive use of fertilizers is a highly contaminating process 9 and the energy required to produce N through the Haber-Bosch process requires at least 1% of the world annual energy supply 12 .
Historically, it was thought that plants derived all N nutrition from the inorganic forms of N, NO 3 2 and NH 4 1 . However, it is now known that the principal form of N entering soils that do not receive inorganic fertilizer is organic N derived from microbial breakdown of organic matter, including amino acids, di-and tri-peptides, DNA and proteins [13][14][15] . In addition, some plants can scavenge organic N from insects (carnivorous plants) or by entomopathogenic fungus-mediated N translocation from insects 16,17 . Further, some plants have been shown to consume and degrade bacteria and yeasts through endocytosis 3 . However, it is unclear whether endocytosis of microbes by plants is a purely defensive process or rather is a means to acquire nutrients [3][4][5] . Plants are naturally colonised by endophytic bacteria that inhabit internal spaces without deleterious effects on host plants 1,2 . Endophytic bacteria that spend some time in soil enter roots or shoots and may spread to all plant tissues, often transmitting in seeds, thus ensuring transfer to the next plant generation 2 . However, controversy exists regarding the significance of nitrogen-fixing endophytes in plants 1,2 . It is frequently argued that positive effects of bacteria on plant growth may be the result of auxins and other growth regulators rather than enhanced N acquisition due to microbial N fixation [18][19][20][21] . Also there is no clear evidence as to how N is transferred from endophytic diazotrophs to the host plant. It has been suggested that N transfer to the host is via amino acids, ammonia, small organic molecules or mineralization of dead bacterial cells 22 . Whether N is actually transferred from endophytic bacteria to plant host has proven difficult to resolve. The question of the mechanism of transfer of N from endophyte to host plant is an important gap in our knowledge. One possible mechanism for N transfer to host is that plants may scavenge organic nitrogen by oxidation and degradation of endophytic bacteria or their proteins using reactive oxygen species (ROS) to lyse cells and denature proteins. This mechanism has been termed 'oxidative nitrogen scavenging' 4,23 . The experiments reported here were done to evaluate whether 15 N incorporated into bacterial endophyte biomolecules such as proteins and nucleic acids could be traced into plant molecules; and whether the transfer process involves evidence of oxidative degradation of microbes.

15
N 2 gas assimilation into seedlings. Shoots of seedlings grown in 15 N 2 -enriched air showed higher d 15 N vs air content (54.93 6 5.65 d 15 N vs (%); mean 6 standard error of mean) than shoots from seedlings grown in non-enriched air (3.07 6 1.12 d 15 N vs (%)). This large difference in the 15 N/ 14 N ratios between the two treatments shows that N was fixed in the seedling tissues.
Bacillus tequilensis endophyte. For use in our tests to evaluate 15 N transfer/translocation from bacterial endophyte to plant, we isolated an endophyte from seeds, seedlings and young plantlets (bulbillos) of Agave tequilana Weber, and identified it as endospore-forming Bacillus tequilensis (accession number KF792125) 24 (Fig. 1) using sequence data. Bacillus tequilensis was present in both roots (Fig. 1C) and shoots of the host plant.
Visualization of bacteria in plant tissues. In seedling infection experiments using B. tequilensis we observed intracellular colonization by the bacterium into meristems and root epidermal cells of seedlings ( Fig. 1A and 1B) and young plantlets. We further observed oxidation of bacterial cells within and on the surface of root epidermal cells ( Fig. 1B and 1D) 25 Supplementary Fig. S1). We detected tryptophan by high-performance liquid chromatography coupled to mass spectrometry in tandem (HPLC-MS/MS), although this amino acid was not quantified (Supplementary Fig.  S4) [26][27][28] .
Pheophytin analysis. Analysis of unlabeled pheophytin a ( Fig. 4B) Fig. 4D and inset 4B and 4C). The 15 N uptake to form the isotopomer m/z 876.57 pheophytin was notably greater, than that of unlabeled leaves, attesting to the incorporation of 15 N atoms into the tetrapyrrole ring of the pheophytin molecule.

Experiment 2
Biomass increases in plants. In this experiment we treated plants with live or heat-killed 15 N-labeled Bacillus (121uC, 10 min); and  with MMN solution (see methods), non-endophytic E. coli (not labeled) and water as controls. Biomass measurements after two months of treatment showed that the greatest biomass increase was seen in plants treated with living B. tequilensis, where mean biomass increase was 1.63 6 0.28 g (mean 6 standard deviation). Plants treated with heat-killed B. tequilensis or mineral nutrient solution (MMN) showed biomass increases of 0.83 6 0.16 g for heat-killed B. tequilensis; and 0.52 6 0.34 g for MMN. Plants treated with non-endophytic Escherichia coli showed a biomass increase of 0.71 6 0.14 g. Plants treated only with water showed the least biomass increase (0.15 6 0.05 g).

Discussion
The assimilation of 15 N 2 gas into tissues of Agave seedlings is indicative of the presence of nitrogen-fixing microorganisms within seedling tissues. From seedlings, seeds and bulbillos, we consistently isolated Bacillus tequilensis; however, plants may also have contained other non-cultured bacteria that could have been responsible for N fixation in plant tissues. We do not consider B. tequilensis to be responsible for N 2 fixation in plant tissues; however, because of its ease of culturing and its endophytic nature, we employed B. tequilensis in experiments to evaluate the hypothesis that nitrogenous nutrients may flow from bacterial endophyte populations to plants.
We labeled proteins and nucleic acids of B. tequilensis with 15 N; then inoculated plants with suspensions of bacteria. Using these bacteria we conducted two experiments. In the first experiment we watered plants weekly with a 4 mL suspension of 80.10 6 CFU mL 21 of 15 N-labeled Bacillus for a six-month period. Our results show that plants incubated with the bacterial endophyte came to contain N that was originally contained within the bacterium. Detection of 15 Nlabeled tryptophan and 29-deoxynucleosides suggests that 15 Nlabeled bacterial endophytes enter into or otherwise associate with plant tissues. Because both bacteria and plants possess tryptophan and 29-deoxynucleosides, with this data alone we cannot evaluate transfer of nitrogen from bacterium to plant. However, we were able to detect the 15 N in pheophytin a, a molecule derived from plant chlorophylls. Because chlorophyll is unique to the plant cells, the presence of the 15 N label there is a definitive confirmation of transference of N from bacterium to plant tissues.
A second experiment was conducted to evaluate whether soil absorption from dead bacteria could account for some 15 N movement into plant tissues, we developed an experiment in which we www.nature.com/scientificreports treated plants with live or heat-killed 15 N-labeled bacteria (121uC, 10 min); and with MMN solution, non-endophytic E. coli (not labeled) and water as controls. Plants were watered weekly for a two-month period using 4 mL of bacterial suspension at a concentration of 80.10 6 CFU mL 21 or MMN solution. In this experiment we found significantly more incorporation of 15 N into 29-deoxynucleosides in plants treated with live Bacillus than in plants treated with heat-killed Bacillus. We further evaluated the growth of these plants compared with those treated with water, E. coli or mineral nutrient solution. The amount of total acquired biomass was almost double in plants treated with living Bacillus than in plants treated with heat-killed Bacillus and E. coli; and more than 3.3 times that seen in plants treated with mineral solution. Enhanced growth of plants, and incorporation of the 15  Microscopic observations of plant roots treated with the living Bacillus and stained using a reactive oxygen probe showed that degrading bacterial cells were associated with reactive oxygen and were often internalized into plant roots. Our observations are similar to those of Paungfoo-Lonhienne et al. 3 , where microbes were shown to enter into root cells of tomato and Arabidopsis where they were degraded. The involvement of reactive oxygen in procurement of organic nutrients from bacteria is consistent with 'oxidative nitrogen scavenging', where reactive oxygen and proteases may be involved in nutrient extraction from bacteria 4,33 . We did not visualize bacteria or their degradation in shoot tissues; however, this is likely due to failure of the aqueous stains to penetrate into the shoot, rather than their absence from shoots.
In a recent study that evaluated the contribution of organic N in wheat from direct microorganism consumption against nitrate, L-alanine and L-tetraalanine absorption, it was found that plants absorbed nutrients through endocytosis of soil microbes, but the N obtained through this process was up to two orders of magnitude slower than other forms of organic and inorganic N in the soil 5 . Our study examining nutrient transfer from B. tequilensis to Agave suggests a process where endophytic bacterial degradation may supply N for plant growth; however, this mechanism may not be rapid and it could depend on occurrence of N deficiency or other nutrient depletion in soils. Our initial experiment to evaluate movement of 15 N into plant molecules lasted six months. We do not know the minimum amount of time or precise plant growth conditions needed to see movement of N from bacterial endophytes to host plants. We propose that efficiency of this mode of nutrition depends on the particular host and microbe association. Agave tequilana is a desert plant adapted to growth under low N soil conditions and B. tequilensis is a native endophytic microbe of that plant. Internal colonization of plant tissues may increase the probability of transference of nutrients from endophytes to host plants. In this respect endophytic microbes that fix N may represent a nutritional resource that may be tapped into when soil N is limiting. Equally important to plants could be N derived from soil microbes that grow and obtain nutrients in the soil then colonize growing plants where they may be degraded. Whether N derived from microbes is a significant source of N for plants is a question that requires additional investigation. While our experiments on N transfer from endophyte to host in Agave are not exhaustive, they do provide further evidence that plants may obtain N through degradation of symbiotic microbes 3-5, 33 .  Methods Plant materials. For experiments we used one-yr-old seeds and asexual plantlets (bulbillos) derived from plants of Agave tequilana Weber that were originally collected on an A. tequilana plantation near Atotonilco el Alto, Jalisco, Mexico at coordinates 20u34922.710N, 102u32900.85, 1900masl. 15 N 2 gas assimilation experiment with seedlings. An experiment was conducted to evaluate whether endophytic microbes fix nitrogen within intact plant tissues. In this experiment seeds were surface disinfected in 3% sodium hypochlorite for 20 min with constant agitation to remove external bacteria, then rinsed three times using sterile water. Five seeds were plated onto 0.7% agarose media in each of eight Petri dishes. Four Petri plates were placed in a 1-liter gas chamber in which the air was enriched with 33 mL of 15 N 2 gas. The other four Petri dishes were placed in a chamber in which the air was not enriched with 15 N 2 . Both chambers were placed under fluorescent lighting with alternating light/dark periods (10 hr/14 hr) at laboratory ambient temperature for 21 days. After incubation shoots were excised from roots, washed to remove any superficial bacteria, then dried for 14 hr in an oven at 60uC. All shoots from a plate were combined to ensure sufficient material for analysis. For massspectroscopic 14 N/ 15 N ratio analysis, we sent 0.9-1.0 g of dried shoot material to the Stable Isotope/Soil Biology Laboratory at the Odum School of Ecology at the University of Georgia, Athens, USA.
Isolation of bacteria from A. tequilana seeds. Seeds were surface sterilized to remove epiphytic microbes. In this process seeds were immersed in a 3% hypochlorite solution for twenty minutes with constant agitation. The seeds were then rinsed with sterile distilled water and placed in 85% ethyl alcohol for ten minutes, followed by three rinses with sterile distilled water. To confirm the disinfection process, aliquots of the sterile water used in the final rinse were plated in tryptic soy agar (TSA; Difco, Sparks, MD, USA) and incubated at 28uC for 15 days, after which plates were examined for the presence of microorganism growth. To isolate bacteria, we used two procedures: 1) 30 sterilized seeds were placed onto 15 cm diameter Petri plates containing potato dextrose agar (PDA, Difco, Sparks, MD); and 2) Ten seeds were ground with 5 mL of aqueous solution (0.9% NaCl) using a sterile mortar and pestle and the seed extract was plated on TSA plates with different volumes (50 to 350 microliters per plate). All plates were incubated at 28uC for seven days until creamcolored colonies appeared.
Identification of bacterium by 16S rRNA and sequencing. For bacterial identification, 980 base pairs of the 16S rDNA region were sequenced 24 . Sequences were compared to sequences available in the NCBI GenBank database to identify the closest matches. We submitted a representative sequence to NCBI (GenBank accession number KF792125).

Experiment 1
Plant growth conditions. To reactivate bulbillos of A. tequilana collected from the field, plants were grown in sterile soil in a growth chamber at 27uC with 50% humidity under 14 h light and 10 h dark photoperiods and lamp intensities of 80 Watts/m 2 for three weeks. Then plantlets were rinsed with sterile water to eliminate organic soil on roots and transferred immediately to N-free sand (30 g per glass bottle) for another three weeks maintained under the same conditions.
Visualization of bacterial oxidation in A. tequilana seedlings and bulbillos. To visualize bacterial oxidation in plant tissues, we stained seedlings and bulbillos with DAB/horseradish peroxidase for a 6-12 h period. We then excised seedling roots and shoots and placed them on a slide containing 0.1% aqueous toluidine blue. The DAB/ peroxidase probe was prepared with a 5 mL solution of 100 mM potassium phosphate buffer, pH 6.9, 2.5 mM diaminobenzidine tetrachloride and 5purpurogallin units/mL of horseradish peroxidase (Type VI, Sigma, St. Louis, MO, USA) 25,33 . Slides were examined using bright field microscopy on a Zeiss AxioskopeH with a Spot Insight TM 4 megapixel digital camera.
Bacterial growth and [ 15 N]-labeling. Bacillus tequilensis was grown and maintained in TSA. To evaluate transferral of organic N from bacteria to plants under limited nutrient conditions, we labeled B. tequilensis using 14 NH 4 Cl or 15 NH 4 Cl as the sole N sources in minimal medium M9 and incubated for 18 h at 200 rpm. All bacteria cultured with 15 N contained nucleic acids labeled with isotopic N (Supplementary Fig.  S8). 15 N-Labeling of Bacillus tequilensis was confirmed by reversed phase HPLC-MS analysis of DNA bases (Supplementary Fig. S8). The HPLC-MS results showed complete 15 N-labeling of nitrogen atoms of purine and pyrimidine bases through detection of 15 N-labeled guanine at m/z 157 ( Supplementary Fig. S8B), 15 N-labeled cytosine at m/z 115 ( Supplementary Fig. S8C), 15  Extraction of pheophytin from A. tequilana. To confirm that N transferred from microbes to plant tissue, an analysis in high-resolution mass spectrometry was carried out to evaluate the relative abundance of isotopomers of pheophytin (Fig. 4). Pheophytin is a magnesium-free derivative of chlorophyll, which has advantages over chlorophylls for isotopic analysis because of its stability, simpler mass spectra and better ionization characteristics. Pheophytin was extracted following the method of Perkins and Roberts 26,27 , with some modifications. Briefly, 0.5 g fresh leaves were frozen in liquid N 2 and ground to a fine powder using mortar and pestle. For pheophytin extraction equal volumes of 85% acetone in 1 mL water, and ethyl ether were added and centrifuged at 10, 000 rpm for 10 min. The ether phase was collected and the procedure was repeated two times. Chlorophyll was converted to pheophytin by adding 10 mL diluted 6 M HCl to the ether extract. The excess of HCl was removed by adding 500 mL of water and centrifuging at 10, 000 rpm for 10 min, three times. The ether phase was collected, dried and prepared for HPLC/MS analysis by adding methanol to obtain a final sample concentration of 1 mg/mL.
HPLC/MS analysis of pheophytin. Pheophytin samples were analyzed using a MicroTOFQ-II mass spectrometer (Bruker) coupled to a Shimadzu HPLC system (Tokyo, Japan) with two pumps LC-20AD, automatic injector SIL-20A, column oven CTO-20A, UV detector SPD-20A and controller CBM-10A. A column Phenomenex Luna 5 mm (PFP2 150 3 2 mm, 100A particle size) was used and chromatography was performed with a flux of 200 mL/min using acetonitrile:H 2 O (1 0.1% formic acid) as mobile phase in a gradient of 0 to 5 min 60% of acetonitrile, from 5 to 30 min 60 to 100% of acetonitrile. The column oven was kept in 40uC, UV detector was recorded at 400 and 600 nm. The mass spectrophotometer was operating in electrospray positive mode, with a nebulization and drying gas at 4 Bar and 8 L/min, respectively. Capillar voltage was set to 4500 V and drying temperature in 200uC. Collision cell and quadrupole energy were set to 20 eV and 10 eV, respectively. The molecular formula of pheophytin a is C 55 H 74 N 4 O 5 and the base peak was detected at [M1H] 1 5 872.5731.
Analysis of 15 N 3 -dC and 15 N 5 -dA extracted from DNA of plants treated for two months with live or heat-killed 15 N-labeled bacteria was performed as described above.
HPLC-MS analysis of DNA from bacteria. See Supplementary Note 4.

Experiment 2
Plant mass accumulation study. To evaluate whether soil absorption from dead bacteria could account for some 15 N movement into plant tissues, we developed an experiment in which we treated plants (six replicates per treatment) over a two month period with live or heat-killed 15 N-labeled B. tequilensis (heated to 121uC, 10 min), and with 50% MMN solution, non-endophytic E. coli (not labeled) and water as controls. Bacteria were applied as described in Experiment 1. To assess increase of 15 N in leaves of Agave plants treated with 15 N-labeled bacteria, deoxynucleosides were extracted from leaves and 29-deoxycytidine and 29-deoxyadenosine were measured. Measurements of plant mass with water-washed roots were made before and after two months of treatments. Biomass increase is the change in whole plant wet weight as a result of the treatments.
Statistical analyses. Statistical tests were performed with Origin (version 8.0) and GraphPad PRISM (version 5.0) programs. Significant differences were determined by t-test and one-way ANOVA (applying Dunnett's post-test) with the level of significance set at P , 0.05 for 15 N-Trp quantification, detection of nucleosides ( 15 N 3 -dC and 15 N 5 -dA) and biomass increase, respectively.