Inoculation of sugarcane with Pantoea sp. increases amino acid contents in shoot tissues; serine, alanine, glutamine and asparagine permit concomitantly ammonium excretion and nitrogenase activity of the bacterium

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Summary

Pantoea sp. is an endophytic nitrogen-fixing bacterium isolated from sugarcane tissues. The aim of the present study was to determine the contents of amino acids in sugarcane as a result of inoculation of nodes and nodal roots with Pantoea sp. strain 9C and to evaluate the effects of amino acids on growth, nitrogenase activity and ammonium excretion of the bacterium. Content of almost all amino acids increased in 30-day-old plantlets by root inoculation. The most abundant amino acids in shoot tissues were asparagine and proline, and those in nodal roots were asparagine, proline, aspartic acid, glutamic acid and serine. The bacterium was able to grow on all tested amino acids except histidine, isoleucine and leucine. Nitrogenase Pantoea sp. was partially inhibited by 1, 2 or 5 mmol L−1 and completely inhibited by 10 mmol L−1 of NH4+ in the media. Pantoea sp. showed nitrogenase activity in 5 mmol L−1 of serine, asparagine, threonine, alanine, proline, tyrosine, valine, methionine, lysine, phenylalanine, cysteine, tryptophan, citrulline and ornithine. Pantoea sp. did not excrete ammonium when it grew in vivo conditions favoring nitrogen fixation; however, ammonium was detected in the supernatant when 5 mmol L−1 asparagine, aspartic acid, alanine, serine or glutamine was added to the medium. The highest ammonium concentration in the supernatant was detected, when Pantoea grew on serine. Ammonium in the supernatant and nitrogenase activity were only detectable concomitantly when the medium was supplemented with serine, alanine, glutamine or asparagine. We discuss roles of amino acids on plant–bacteria interaction during the colonization of sugarcane plants.

Introduction

Gramineous plants are worldwide spread and, like legumes, are the primary food sources for human beings and animals. To optimize the crop yield extensive amounts of fertilizers are used in agriculture; however, biological nitrogen fixation (BNF) is known to be a beneficial alternative to reduce the consumption of fertilizers and to minimize the harmful impact of chemicals on the biosphere.

Several N-fixing bacteria are associated with sugarcane. The most common endophytic bacteria isolated from sugarcane tissues are Gluconacetobacter diazotrophicus, Herbaspirillum rubrisubalbicans and Herbaspirillum seropedicae (Baldani et al., 1997; James, 2000; Baldani et al., 2002; Boddey et al., 2003). Other microorganisms such as Enterobacter cloacae, Klebsiella oxytoca, Pantoea, Burkholderia unamae and Burkholderia tropica have also been found inside sugarcane (Mirza et al., 2001; Loiret et al., 2004; Perin et al., 2006). These endophytic microorganisms could have effects on plant yield and growth promotion (Ryan et al., 2008).

It is well known that legumes, in which symbiotic BNF takes place, mainly transport N as ureides or amides. The knowledge about the N compounds in the tissues of sugarcane is still scarcely. Tejera et al. (2004) found that amino acids and NH4+ appear at different concentrations in apoplastic and symplastic sap of sugarcane tissues. Moreover, the amino acid concentrations in sugarcane change in response to the inoculation with G. diazotrophicus (Tejera et al., 2006). N-fixing bacteria living in close association with plants release normally amino acids but it depends on several factors (González-López et al., 2005). The role of amino acids in sugarcane–endophyte interaction needs to be elucidated.

In the present study we determined the contents of amino acids in sugarcane as a result of inoculation with Pantoea sp. and evaluated the effects of amino acids on growth, nitrogenase activity and bacterial excretion of ammonium.

Section snippets

Inoculation

Pantoea sp. strain 9C (Loiret et al., 2004) was grown for 15 h in 3L DYGS medium (Rodrigues-Neto et al., 1986), pelleted by centrifugation, washed and re-suspended in fresh LGI medium (Cavalcante and Döbereiner, 1988). The bacterial suspension (1010 cell mL−1) was used as inoculant in two different ways. For treatment 1 (nodes inoculation), nodes of sugarcane (Saccharum spp. hybrids, var. C1051-73) with approximately 5 cm of internodal tissues on each side were dried for 2 h in an oven at 60 °C,

Results

HPLC analysis enabled detection of 15 different l-amino acids in plantlets. Due to similar retention times, individual quantification of Ala and Arg was not possible. The amino acids with the highest contents in roots of non-inoculated plants were Asn, Pro and Asp, whereas in shoots Asn and Pro were the highest. Among the amino acids Asn gave rise to 80% and 85% of the total amino acid content in shoot and root tissue, respectively. The amino acids Asn, Pro, Asp, Glu, Ser and Gln added up to

Discussion

Asparagine was found to be the most predominant molecule among the 15 detectable amino acids (Figure 1) and accounted for 72–85% of the total amino acid content in sugarcane tissues. Our results differ from previous data about predominant amino acids and their contents in sugarcane tissues. Tejera et al. (2006) reported that Ser, Pro, Ala and Asp were the main amino acids (60% of the entire amino acid pool) in apoplastic or symplastic sap of three different varieties of fertilized sugarcane,

Acknowledgments

This work was supported by the Federal Ministry of Education and Research in Germany (Bundesministerium fur Bildung und Forschung, BMBF) and by the Humboldt University of Berlin. The authors appreciate the support provided by these institutions. We acknowledge the proof reading by Mike Zirkle and Virginia Waddick.

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