Influence of External Nitrogen on Nitrogenase Enzyme Activity and Auxin Production in Herbaspirillum seropedicae (Z78)

Abstract

The production of nitrogenase enzyme and auxins by free living diazotrophs has the potential to influence the growth of host plants. In this study, diazotrophs were grown in the presence of various concentrations of nitogen (N) to determine the optimal concentration of N for microbial growth stimulation, promotion of gaseous N (N₂) fixation, and phytohormone production. Therefore, we investigate whether different levels of N supplied to Herbaspirillum seropedicae (Z78) have significant effects on nitrogenase activity and auxin production. The highest nitrogenase activity and the lowest auxin production of H. seropedicae (Z78) were both recorded at 0 gL⁻¹ of NH₄Cl. Higher levels of external N caused a significant decrease in the nitrogenase activity and an increased production of auxins. In a subsequent test, two different inoculum sizes of Z78 (10⁶ and 10¹² cfu/ml) were used to study the effect of different percentages of acetylene on nitrogenase activity of the inoculum via the acetylene reduction assay (ARA). The results showed that the optimal amount of acetylene required for nitrogenase enzyme activity was 5% for the 10⁶ cfu/ml inoculum, whereas the higher inoculum size (10¹² cfu/ml) required at least 10% of acetylene for optimal nitrogenase activity. These findings provide a clearer understanding of the effects of N levels on diazotrophic nitrogenase activity and auxin production, which are important factors influencing plant growth.

Nitrogen (N) is an essential nutrient for all life, and the vast majority of organisms have adapted to process N through various environmental sources. In plants, N is taken up only in the reduced form of ammonia (NH₃). The reduction takes place via biological N fixation (BNF), a high-energy process requiring hydrolysis of many adenosine triphosphate (ATP) molecules within the cell (Hartmann et al. 1986). BNF involves symbiotic or associative relationships between the diazotroph and the host plant, as the diazotrophic nitrogenase enzyme (encoded by the diazotroph nifHDK genes) catalyses the reduction of gaseous nitrogen (N₂) to NH₃ (de Campos et al. 2006). However, the process is suppressed in many bacterial species when an ample or excess supply of fixed N is available. In the presence of excess fixed N, the diazotrophic nitrogenase enzyme is no longer active, either due to down-regulation of protein synthesis and/or inactivation of the protein (Rudnick et al. 1997). Early findings by Eady et al. (1978) and Postgate (1982) reported that nitrogenase enzyme is not synthesised by Azospirillum brasilense when supplied with high concentrations of ammonium chloride (NH₄Cl). It has also been shown that when diazotrophs are exposed to even low amounts of fixed N (including NH₃, ammonium [NH₄⁺] and nitrate ion [NO₃⁻]), the BNF process can be suppressed by a strict regulatory control mechanism (Eady et al. 1978; Postgate 1982; Merrick & Edwards 1995). The fixation process and its regulation are known to be controlled via transcriptional repression of the nif A gene, which encodes the N fixation activator protein (Yan et al. 2010). The presence of fixed forms of N in the cell prevents N₂ fixation through the action of N regulatory protein C (NtrC), which prevents nitrogenase enzyme synthesis by repression of the nif A gene. The nif A gene encodes a positive regulatory protein that activates transcription of other nif genes leading to nitrogenase enzyme synthesis. Under limited N conditions, NtrC is active and allows transcription of nif A, leading to the synthesis of genes required for N fixation. A similar inactivation mechanism is employed by Rhodospirillum rubrum, in which the presence of NH₄⁺ leads to the suppression of nitrogenous enzyme transcription through covalent modification of an Fe protein (Pope et al. 1985). In this case, an excess of NH₄⁺ causes the addition of an adenosine diphosphate (ADP) molecule to dinitrogenase reductase (Fe protein), which results in loss of enzyme activity. In addition, the amount of fixed N influences the colonisation and growth of diazotrophs on the root surfaces. Excess of NH₄⁺ causes a reduction in the number of diazotrophs and a decrease in BNF activity (Rivera et al. 1991). Decreased nitrogenase enzyme activity of A. diazotrophicus due to high concentrations of NH₄Cl and ammonium nitrate (NH₄NO₃) has long been recorded (Muthukumarasamy et al. 2002). These various findings clarify the reasons why optimal N concentrations are crucial for maximising N₂ fixation activities of diazotrophs. In addition to fixation of N₂, diazotrophs promote rooting and growth of host plants by synthesising different growth-promoting hormones such as gibberellins and auxins (indole-3-acetic acid, IAA) (Asghar et al. 2002; Ozturk et al. 2003). Previous reports show that 80% of bacteria isolated from the rhizosphere are capable of producing phytohormones essential for plant growth (Patten & Glick 1996). For example, IAA production is actively stimulated in plants and increases the cell elongation of roots. Moreover, IAA promotes cell division and differentiation of the vascular tissues of the plant (Tien et al. 1979). The secretion of auxins is influenced by microbial culture conditions, diazotroph growth stage and availability of substrates (Frankenberger & Arshad 1995). Several experiments have involved inoculating free-living diazotrophs such as Azospirillum brasilense onto paddy plants, sugar cane and in vitro oil palm shoots, showing the promotion of host-plant root growth by auxin secretion (Keyeo et al. 2011; Noor Ai’shah et al. 2013). However, it is unclear whether phytohormone production of any diazotroph is also influenced by external N concentrations and, if so, what level of external N constitutes the optimal amount. Thus, the objectives of the present experiment are as follows: 1) to observe the effects of external N sources on N₂ fixation activities and auxin production of Herbaspirillum seropedicae (Z78) and 2) to determine the optimal external N concentration for maximising N₂ fixation and phytohormone production by Z78.

The bacteria H. seropedicae Z78 (ATCC 35893) was cultured in 250 ml Erlenmeyer flasks containing minimal N medium (Okon et al. 1977). The culture was shaken continuously at 160 rpm (28°C) for 48–72 hours on a rotary shaker until the medium became turbid. The optical density for the inoculum was measured at 530 nm, using a spectrophotometer (Lambda Bio + spectrophotometer, Perkin Elmer, USA). A total of 20 μl of broth culture was transferred to semisolid culture media with varied N concentrations and prepared for nitrogenase enzyme analysis. In addition, a total of 1 ml of bacterial culture was inoculated to fresh minimal N medium containing L-tryptophan for auxin production analysis.

The nitrogenase enzyme activity of H. seropedicae (Z78) was determined using the acetylene reduction assay (ARA) (Hardy et al. 1968; Ohyama & Pham 2006). The assay was performed in airtight 30 ml universal bottles containing 10 ml N-free semisolid media (NFb) with different N concentrations (0, 0.25, 0.75 and 1.0 gL⁻¹) of NH₄Cl. A total volume of 5% air was removed from the headspace of each universal bottle and replaced with acetylene gas (C₂H₂) (99.8% purity), followed by 24 hours incubation at 30°C (Elbeltagy et al. 2001). At the end of incubation, a total of 1 ml gas mixture was withdrawn and transferred into a vacuum tube before it was injected into a GC-2014 gas chromatograph (Shimadzu, USA) to assay for the presence of ethylene (C₂H₄) gas. The gas chromatograph was fitted with a Supelco Carboxen 1004 stainless steel micropacked column, 2 m × 0.76 mm ID, and equipped with a flame ionization detector (FID). N was used as the carrier gas at a flow rate of 30 ml min⁻¹, while the column, injection and FID temperatures were maintained at 80°C, 180°C and 180°C, respectively. The actual concentration of ethylene produced was determined based on the prepared standard curve of C₂H₄ gas and the peak area (Somasegaran & Hoben 1985; Elbeltagy et al. 2001). The N fixation activity (μmol C₂H₄ cfu ⁻¹ h⁻¹) was defined based on the C₂H₄ concentration (μmol C₂H₄) and the viable cell number (cfu) of Z78.

The auxin production of Z78 was assayed based on Salkowski’s colourimetric technique (Asghar et al. 2002; Patten & Glick 2002). The inocula were cultured in a 250 ml Erlenmeyer flask containing 100 ml of minimal N medium (Okon et al. 1977) and treated with different concentrations of NH₄Cl (0, 0.25, 0.75 and 1.0 gL⁻¹ of NH₄Cl). All of the media preparations were supplemented with 0.5 gL⁻¹ L-tryptophan as a precursor for auxin biosynthesis (Glickmann & Dessaux 1995; Theunis et al. 2004; Zahir et al. 2010; Noor Ai’shah et al. 2013). The presence of auxins in the broth culture was detected through the appearance of pinkish red colour (Barazani & Friedman 1999). The colour intensity was measured by a spectrophotometer at 530 nm (Dobbelaere et al. 1999).

The results were analysed statistically by analysis of variance (ANOVA) using SPSS 15.0 for data analysis. The ANOVA was performed to test the significance of the treatment effects. One-way analysis of variance for a split-plot factorial design with Duncan’s multiple-range test was used to compare the means.

The ability of Z78 to fix N and to produce auxins when supplied with different levels of NH₄Cl was studied. The highest nitrogenase activity (2.54 × 10⁻⁷ μmol C₂H₄/cfu/h) was recorded at 0 gL⁻¹ of NH₄Cl supply relative to the cultures treated with 0.25, 0.75 and 1.0 gL⁻¹ of NH₄Cl (Fig. 1). This result indicates that higher levels of N sources have an impact on the acetylene reduction activity, while the nitrogenase enzyme activity of the diazotroph decreases with increasing N supply in the growth medium. The results were different for auxin production, where the highest activity was recorded for inoculum supplied with higher amounts of N (0.25–1.0 gL⁻¹ of NH₄Cl) (Fig. 2). A trend of increased production was noted when N was supplied to the growth medium. A concentration of 0 gL⁻¹ of NH₄Cl resulted in the lowest production of auxins (2.43 μg/ml), while 1.0 gL⁻¹ of NH₄Cl resulted in the highest level of auxin production at 11.93 μg/ml.

Figure 1:

Nitrogenase enzyme activity with different NH₄Cl concentrations (gL⁻¹) for Z78 (H. seropedicae).

Notes: Data shown are the means of triplicate tests. Means accompanied by different letters are significantly different (Duncan’s test, p<0.05).

Figure 2:

Production of auxins for Z78 (H. seropedicae) treated with different NH₄Cl concentrations (gL⁻¹).

Notes: Data shown are the means of triplicate tests. Means accompanied by different letters are significantly different (Duncan’s test, p<0.05).

Previous studies have shown that the BNF activity of diazotrophic microorganisms is influenced by fixed N input because NH₄⁺ transport consumes an inducible and energy-dependent system that is repressed by NH₃ (Hartmann & Kleiner 1982; Pedrosa & Yates 1983). For example, Rasmussen et al. (2012) describe the influence of cattle slurry-N fertiliser on suppressed N₂-fixation activity of white and red clover inoculated with selected diazotrophs. Another finding by Naudin et al. (2011) highlighted that extensive N fertilisation reduced the biomass and N₂ fixed by peas in an intercropping system of wheat and pea. In the present study, the process might increase the biomass of wheat but not of pea, and it is likely that the diazotroph population had decreased. Rivera et al. (1991) found that higher mineral N concentration decreased the population size of diazotrophic microorganisms in sugar cane and led to a reduction in nitrogenase enzyme activity. The use of NH₄Cl and NH₄NO₃ is said to reduce both the colonisation and the acetylene-reduction activity of Acetobacter diazotrophicus. Even in small amounts, exogenous NH₄⁺ is known to quickly and reversibly inhibit the nitrogenase activity in the whole cells of H. seropedicae (Fu & Burris 1989). All of these studies contribute to the interpretation of our results, wherein the highest nitrogenase activity was recorded for Z78 grown in medium treated with 0 gL⁻¹ of NH₄Cl and wherein higher levels of N sources (0.25, 0.75 and 1.0 gL⁻¹ of NH₄Cl) influenced the nitrogenase enzyme activity. A similar response to N input on nitrogenase activity was reported by Klassen et al. (2001), in which the addition of 0.2 mmol L⁻¹ NH₄Cl caused almost immediate inhibition of the nitrogenase activity of A. brasilense FP2. However, the activity was fully recovered following exhaustion of ammonium ions from the medium. The N sources (e.g., NH₄Cl and NH₄NO₃) also have an impact on root colonisation and the acetylene-reduction activity of A. diazotrophicus (Muthukumarasamy et al. 2002). These various findings also explain why the nitrogenase activity is the highest when the N supply is 0 gL⁻¹ of NH₄Cl and decreases once the N supply increases. Our experimental results are best explained by the regulatory control mechanism of the nitrogenase enzyme at the transcriptional and post-translational levels (Chubatsu et al. 2012). In addition, culture conditions, which include nutrient availability, pH and bacterial strain, will also influence the “NH₄⁺ switch off” mechanisms (Hartmann et al. 1986). Vose et al. (1981); Rudnick et al. (1997), and Steenhoudt and Vanderleyden (2000) also highlighted the inhibition of nitrogenase activity for A. brasilense and H. seropedicae due to inactivation of NifA under excess N conditions. Upon exhaustion of N sources from the medium, the NifA was activated through a process that involved the signal transduction protein PII. Recent findings by Yan et al. (2010) show that the transcriptional regulation of the nif gene depends on both nif-specific and ntr gene regulatory systems. Suppression of the nitrogenase activity may be due to the inhibition of nif A gene expression (Souza et al. 1999). The nif A gene is important for inducing other nif gene expression including the iron-molybdenum cofactor (FeMo-co) nitrogenase structural genes.

It has been demonstrated that diazotrophs can be beneficial to plants not only for their ability to fix N₂ but also for their ability to produce beneficial phytohormones such as IAA (Rodrigues et al. 2008). Auxin production is important and reportedly plays a vital role in stem and root elongation in higher plants and growth stimulation of microorganisms (Goodwin 1978; Tsavkelova et al. 2006). Auxin production is also reported to affect photosynthesis in inoculated host plants. In addition, the resistance of plants to stress factors is also influenced by auxin biosynthesis (Tsavkelova et al. 2006). IAA is synthesised by microbes including the epiphytic and tissue-colonising bacteria in soil (Patten & Glick 1996). Auxins are also produced by isolates from the rhizosphere as secondary metabolites due to a rich supply of substrates available (Strzelczyk & Pokojska-Burdziej 1984). One of the most prominent diazotrophs and auxin producers is Azospirillum spp., which is reported to form an association with both cereal and non-cereal plants (Bashan et al. 2004). Thus, auxinsynthesising rhizobacteria are better studied than other rhizobacteria (Tsavkelova et al. 2006; Spaepen et al. 2007). Our results have shown that higher levels of auxins were produced by Z78 when the media was supplied with a higher amount of N. An increasing trend is observed, with 0 gL⁻¹ of NH₄Cl resulting in the lowest production of auxins and 1.0 gL⁻¹ of NH₄Cl resulting in the highest. Similar findings by Tharwat et al. (2004) showed that application of various N sources such as NH₄Cl, ammonium sulphate [(NH₄)₂SO₄], ammonium phosphate monobasic (NH₄H₂PO₄) and NH₄NO₃ in low concentrations stimulated indole production in Azospirillum strains. However, application of potassium nitrate (KNO₃), sodium nitrate (NaNO₃) and potassium (KNO₂) inhibited the production of indole compounds for all bacteria tested. Noor Ai’shah et al. (2013) reported that the viable cell numbers of diazotrophs also influenced the production of optimum IAA and can be related to N₂ fixation capacity of diazotrophs. It was reported that higher viable cell numbers of diazotrophs (up to 10¹⁰ cfu ml⁻¹) influenced the production of optimum IAA. The overall IAA productivity of Z78 was recorded at 0.165 μg ml⁻¹ hr⁻¹, while the viable cell count increased gradually (Noor Ai’shah et al. 2013). Any declines in IAA production reflected the decline in viable cell numbers of inoculum tested. Although the diazotroph could produce more IAA, under in vitro conditions, IAA production may show detrimental effects to the host plants. Tharwat et al. (2004) noted that high amounts of indoles produced by Azospirillum strains reduced the length of roots and stems of the host plants. Similarly, Keyeo et al. (2011) found that plants inoculated with A. brasilense (Sp7) and H. seropedicae (Z78) both showed inferior growth due to excess auxin production. Higher concentrations of IAA in the range of 0.001–1.0 μg/ml can also inhibit nitrogenase activity of Azospirillum lipoferum (Silveira & Drozdowicz 1983). These findings clearly support the conclusion that the optimal concentration of N required by Z78 to stimulate N₂ fixation activity was recorded at 0 gL⁻¹ of NH₄Cl. A higher supply of exogenous N would suppress nitrogenase enzyme activity. The optimal concentration of N needed for auxin production is also 0.25 gL⁻¹ of NH₄Cl.

In conclusion, H. seropedicae (Z78) shows the highest nitrogenase activity when supplied with 0 gL⁻¹ of NH₄Cl based on the ARA test. When higher levels of N were supplied, a significant decrease in the nitrogenase activity of these bacteria resulted. The production of auxins requires at least 0.25 gL⁻¹ of NH₄Cl. High auxin production therefore does not correspond to high nitrogenase activity.