Key physiological properties contributing to rhizosphere adaptation and plant growth promotion abilities of Azospirillum brasilense

Authors

  • Sharon Fibach-Paldi,

    1. Department of Plant Pathology and Microbiology and The Otto Warburg Minerva Center for Agricultural Biotechnology, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot, Israel
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  • Saul Burdman,

    1. Department of Plant Pathology and Microbiology and The Otto Warburg Minerva Center for Agricultural Biotechnology, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot, Israel
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  • Yaacov Okon

    Corresponding author
    • Department of Plant Pathology and Microbiology and The Otto Warburg Minerva Center for Agricultural Biotechnology, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot, Israel
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Correspondence: Yaacov Okon, Department of Plant Pathology and Microbiology and The Otto Warburg Minerva Center for Agricultural Biotechnology, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, PO Box 12, Rehovot 76100, Israel. Tel.: +972 8 9489216; fax: +972 8 9466794; e-mail: okon@agri.huji.ac.il

Abstract

Azospirillum brasilense is a plant growth promoting rhizobacterium (PGPR) that is being increasingly used in agriculture in a commercial scale. Recent research has elucidated key properties of A. brasilense that contribute to its ability to adapt to the rhizosphere habitat and to promote plant growth. They include synthesis of the auxin indole-3-acetic acid, nitric oxide, carotenoids, and a range of cell surface components as well as the ability to undergo phenotypic variation. Storage and utilization of polybetahydroxyalkanoate polymers are important for the shelf life of the bacteria in production of inoculants, products containing bacterial cells in a suitable carrier for agricultural use. Azospirillum brasilense is able to fix nitrogen, but despite some controversy, as judging from most systems evaluated so far, contribution of fixed nitrogen by this bacterium does not seem to play a major role in plant growth promotion. In this review, we focus on recent advances in the understanding of physiological properties of Abrasilense that are important for rhizosphere performance and successful interactions with plant roots.

Introduction

The rhizosphere is the area of the soil that is influenced by the plant roots. It is rich in microorganisms, with their composition differing from the rest of the soil owing to the activity of plant roots (the so called rhizosphere effect). Among microorganisms inhabiting the rhizosphere, several bacterial species, known as plant growth promoting rhizobacteria (PGPRs), are able to promote root and plant growth (Hartmann et al., 2008; Spaepen et al., 2009). Some PGPRs promote plant growth directly by producing and secreting plant growth substances, supplying readily available micro- and macroelements, biological nitrogen fixation (diazotrophs), and/or solubilization of phosphorus, among others (Spaepen et al., 2009). Other rhizosphere bacterial species benefit plant growth through indirect effects, which are mainly associated with reduction of damage caused by plant pathogens (Van Loon, 2007; Weller, 2007).

The Azospirillum genus belongs to the alphaproteobacteria class and comprises free-living, nitrogen-fixing, vibrio- or spirillum-shaped rods, which produce polar and peritrichous flagella (Baldani et al., 2005) (Fig. 1). Azospirilla exert beneficial effects on plant growth and yield of many agronomically important crops (Okon, 1985; Spaepen et al., 2009; Helman et al., 2011). Commercial inoculants of azospirilla have been tested and applied in hundreds of thousands of hectares, mainly in Latin America (Fuentes-Ramirez & Caballero-Mellado, 2005; Cassan & Garcia de Salamone, 2008; Hungria et al., 2010; Helman et al., 2011).

Figure 1.

Flagella and aerotaxis in Azospirillum brasilense. (a) Vibrion-shaped cells of Azospirillum brasilenseSp74031 (chsA mutant) containing one polar flagellum with a longer wavelength that is used for swimming, and several peritrichous flagella with shorter wavelength that are used for swarming. Picture modified from Carreño-López et al. (2009) (b) Typical aerotactic band (white arrow) of Azospirillum brasilenseCd moving in a capillary containing 0.1 M phosphate buffer without any other attractant. Bacteria follow optimal oxygen concentration created by cell respiration. Picture modified from Okon et al. (1980).

About 16 Azospirillum species have been described so far; however, Azospirillum brasilense and Azospirillum lipoferum have been studied in more detail than the others (Baldani et al., 2005). Draft genomic sequences of A. brasilense Sp245 and A. lipoferum CRT1 have been obtained, but the full annotations of these genomes have not yet been published (I. Zhulin and F. Wisniewsky-Dye, pers. commun.). Preliminary data from the A. brasilense genome sequencing project are available at http://genomics.ornl.gov/research/azo.

Azospirilla are able to fix nitrogen in association with plants, but apparently, nitrogen fixation does not play a major role in plant growth promotion in most systems evaluated so far (Spaepen et al., 2009; Helman et al., 2011). On the other hand, azospirilla are able to produce and secrete plant growth regulators (phytohormones) such as auxins (indole-3-acetic acid; IAA), cytokinins, and gibberellins, as well as nitric oxide (NO), which likely are key signals and components of plant growth promotion effects (Dobbelaere & Okon, 2007; Spaepen et al., 2007, 2009; Molina-Favero et al., 2008; Bashan & de-Bashan, 2010; Helman et al., 2011).

Basic knowledge about the physiological properties of PGPRs is crucial for understanding diverse aspects related to rhizosphere performance and successful interactions with plant roots. For instance, this knowledge might help understanding the modes of colonization of plant surfaces by PGPRs, their interactions with other microorganisms, and the modes of action by which these microorganisms benefit plants. In addition, this knowledge might stimulate ideas about how to improve the production and application of PGPR inoculants. Here we focus on recent advances on the understanding of A. brasilense physiological properties that are important for rhizosphere performance and successful interactions with plant roots (Table 1). Reviewing the effects of Azospirillum strains in root morphology and physiology of diverse plant species as well as the success of field experimentation with Azospirillum-based inoculants are beyond the scope of the present mini-review. However, for the convenience of the reader, these aspects and corresponding references are summarized in Tables 2 and 3, respectively.

Table 1. Rhizosphere competence-related properties of Azospirillum strains
PropertiesReferences
Biological nitrogen fixation; microaerophilic metabolismOkon (1985)
Assimilatory and dissimilatory nitrate reduction; production of NO by aerobic periplasmic nitrate reductaseSteendhoudt et al. (2001); Molina-Favero et al. (2008)
Motility by polar and lateral flagella; chemotaxis and aerotaxis to low oxygen concentration; energy taxis for optimal proton motive force generationAlexandre et al. (2000); Alexandre (2010)
Synthesis and utilization of PHAs; involvement of glycogen in cell survivalKadouri et al. (2002, 2003, 2005); Lerner et al. (2009a)
Synthesis of carotenoids and their role in protection from oxidative damageNur et al. (1981); Hartmann & Hurek (1988); Mishra et al. (2011)
Synthesis of auxins by the IPA and other pathways; IAA signalingSpaepen et al. (2007); Van Puyvelde et al. (2011)
Variability in cell surface polysaccharides and proteins and their influence on cell aggregation, root adsorption, and cell survivalBurdman et al. (2000ab, 2001); Lerner et al. (2009bc)
Phase/phenotypic variationVial et al. (2006); Wisniewski-Dye & Vial (2008); Lerner et al. (2010)
Table 2. Plant growth promotion effects following inoculation with Azospirillum in the greenhouse and in the field
PropertiesReferences
Root hair proliferation in root tips of grasses, cereals, legumes, and tomatoDobbelaere & Okon (2007); Molina-Favero et al. (2008)
Enhancement of root elongation and branchingDobbelaere & Okon (2007)
Enhancement of water and mineral uptake by roots and improvement of water/mineral status of the plantDobbelaere & Okon (2007); Cassan & Garcia de Salamone (2008)
Increase of leaf surface area and stem diameter; improvement of tillering and yieldDobbelaere & Okon (2007); Helman et al. (2011)
Improvement of respiration rate, enzymatic activity, and hydrolysis of glycosides, and auxin and gibberellin conjugates; changes in root secondary metabolites (e.g. benzoxazine derivatives in maize)Dobbelaere & Okon (2007); Cassan & Garcia de Salamone (2008); Walker et al. (2011)
Improvement of growth by ammonium-secreting strainsVan Dommelen et al. (2009)
Slight increase in biological nitrogen fixation and nitrogen enrichment in roots of grasses and cerealsDobbelaere & Okon (2007)
Enhanced flavonoid signaling, nodulation, and nitrogen fixation in co-inoculation with rhizobiaDardanelli et al. (2008); Helman et al. (2011); Star et al. (2011)
Importance of IAA and NO signaling in the rhizosphere; effect of Pseudomonas fluorescens diacethylphloroglucinol; cadaverine productionCassan et al. (2009); Combes-Meynet et al. (2011); Couillerot et al. (2011); Van Puyvelde et al. (2011)
Table 3. Field inoculation experiments with Azospirillum-based commercial inoculants in Israel and Latin America
CountryCropsNumber of experimentsAverage yield increase over controls (%)
  1. a

    Most experiments were carried out in 1979–2000, with freshly prepared inoculants of Azospirillum brasilense (Dobbelaere et al., 2001).

  2. b

    Experiments were carried during 1999–2000 with commercial peat-based inoculants of A. brasilense, at various locations (Dobbelaere et al., 2001; Fuentes-Ramirez & Caballero-Mellado, 2005).

  3. c

    Experiments performed in various locations during 2002–2007 with commercial liquid inoculants of A. brasilense (Cassan & Garcia de Salamone, 2008; Diaz-Zorita & Fernandez-Canigia, 2009).

  4. d

    Experiments performed during 2005–2010 with commercial liquid inoculants of A. brasilense (reported by ASINAGRO and Lage y Cia.).

  5. e

    Experiments performed during 3 years for maize, 2 years for wheat in two locations, with liquid and peat-based inoculants of six A. brasilense and two Azospirillum lipoferum strains (Hungria et al., 2010; Reis et al., 2011).

IsraelaMaize, wheat, sorghum328–17
MexicobMaize, wheat, sorghum, barley5722–42
ArgentinacMaize, wheat3835–41
UruguaydMaize, sorghum, rice206–23
BrazileMaize, wheat178–30

Production of auxin and NO

Auxins are a major class of phytohormones that are involved in the coordination of plant growth and development. The effects of azospirilla on plant root morphology (e.g. elongation of primary roots and increase of the number and length of lateral roots) have been shown to correlate with exogenous levels of the auxin indole-3-acetate (IAA), evidencing that positive effects on roots upon inoculation with azospirilla are mainly owing to the production and secretion of IAA by these bacteria (Dobbelaere & Okon, 2007; Spaepen et al., 2007, 2009).

In A. brasilense, 90% of IAA is produced by the indole-3-pyruvate (IPA) pathway in the presence of tryptophan (Vande Broek et al., 1999; Spaepen et al., 2007, 2009). In this bacterium, the rate-limiting step in IAA synthesis is catalyzed by the enzyme IPA decarboxylase, which catalyzes the conversion of IPA into IAA, and is encoded by the ipdC gene. Transcription of the ipdC gene is positively regulated by its end product IAA, which constitutes a positive feedback loop regulation (Spaepen et al., 2007).

Dual inoculation of several legumes with rhizobia and azospirilla significantly increases nodulation, nitrogen fixation, accumulation of macro- and microelements, and biomass as compared to inoculation with rhizobia alone (Helman et al., 2011; Table 2). An A. brasilense ipdC mutant was partially defective in nodulation and nitrogen fixation of common bean roots co-inoculated with rhizobia, in comparison with co-inoculation with the parental type Sp245. This indicates that there is a differential response of the plant roots to the auxin produced by bacteria (Remans et al., 2008). In agreement, recent experiments with vetch showed that the ipdC mutant induced less root hair formation and induction of secretion of nod gene inducers by roots, relative to the wild type (Star et al., 2011). Moreover, comparison between the ipdC mutant and the wild type in inoculation experiments with wheat plants demonstrated a direct link between IAA production and effects on root morphology (Spaepen et al., 2008). When the native ipdC promoter was replaced by a constitutive or a plant-inducible promoter in strain Sp245, effects on root morphology were similar as those observed with the wild type, but at lower inoculum concentrations (Spaepen et al., 2008).

The transcriptome of the ipdC mutant and the wild type were recently compared in absence or presence of exogenously added IAA by microarrays (Van Puyvelde et al., 2011). Inactivation of ipdC or addition of IAA resulted in broad transcriptional changes, leading to the conclusion that IAA is a signaling molecule in A. brasilense. Moreover, it was proposed that when exposed to IAA, the bacterium adapts itself to the plant rhizosphere, mainly by changing its arsenal of transport and cell surface proteins. Interestingly, IAA addition upregulates genes encoding a type VI secretion system (T6SS), a kind of secretion system that has been specifically implicated in bacterium–eukaryotic host interactions. Moreover, many transcription factors showed altered expression in the different treatments, indicating that the regulatory machinery of the bacterium is altered in response to IAA (Van Puyvelde et al., 2011).

Increasing evidence indicates that NO is a key signaling molecule that is involved in a wide range of functions in plants (Creus et al., 2005; Molina-Favero et al., 2008). It has been demonstrated that NO plays an important role in auxin-regulated signaling cascades, influencing root growth and development (Pagnussat et al., 2003). NO is produced by A. brasilense Sp245 under aerobic conditions, mainly owing to the activity of periplasmic nitrate reductase (Nap) (Steendhoudt et al., 2001). A nap A. brasilense mutant produces only 5% of the NO produced by the wild type and is not able to promote lateral and adventitious root formation and plant development like the wild type (Molina-Favero et al., 2008). The relationship between NO and IAA production in A. brasilense is still to be elucidated. However, a recent study revealed that a nap mutant of A. brasilense possesses a reduced ability to induce root hair formation and nodulation by rhizobia in vetch roots. Moreover, vetch roots inoculated with this mutant secreted less nod gene inducers than roots inoculated with wild-type A. brasilense, and the indole content of the growth solution of napA-inoculated plants was reduced at a lower rate than those of wild-type-inoculated plants (Star et al., 2011).

Production of polybetahydroxyalkanoates

A wide variety of taxonomically different groups of microorganisms within the Bacteria and Archaea domains produce intracellular homopolymers or copolymers containing different alkyl groups at the β position, described as polybetahydroxyalkanoates (PHAs). These polymers are used as energy and carbon storage compounds (Madison & Huisman, 1999). In A. brasilense, PHAs are major determinants for overcoming periods of carbon and energy starvation (Fig. 2). Increased survival upon starvation in phosphate buffer was observed in A. brasilense Sp7 relative to a phaC (PHA synthase) mutant defective in PHA production (Kadouri et al., 2002, 2003, 2005; Castro-Sowinski et al., 2010) (Fig. 2).

Figure 2.

Production of PHAs in Azospirillum brasilense and its role in cell survival. (a) Transmission electron microscope of an Azospirillum brasilenseSp7 cell filled with PHA granules, after growth in a high C : N ratio medium. (b) Effect of PHA on survival ability of starved bacteria. Wild-type cells (Sp7; triangles) and a Sp7 phaC mutant (circles) were grown in high C : N ratio media and transferred to phosphate buffer where they were maintained for a period of 12 days. Bacterial survival during this period was assessed by dilution plating. Wild-type cells were shown to possess significantly higher survival ability than the mutant. Pictures taken from Kadouri et al. (2002). Similar effects of PHA in cell survival were observed following heat and UV-irradiation stresses (Kadouri et al., 2003).

The abilities of A. brasilense phaC and phaZ (PHA depolymerase) mutants to tolerate and survive to various stresses, including UV-irradiation, heat, osmotic shock, desiccation, and oxidative stress, were significantly impaired as compared with wild-type cells (Kadouri et al., 2003, 2005). In addition, PHA accumulation in A. brasilense was shown to support chemotaxis, motility, and cell multiplication. Therefore, it is well established that production of PHAs in A. brasilense is of critical importance for improving shelf life, efficiency, and reliability of commercial inoculants (Kadouri et al., 2005).

PHA production appears to be an important trait for root colonization and plant growth promotion by azospirilla. Plant growth promotion effects are more consistent with A. brasilense inoculants containing cells with high amounts of PHA. For instance, field experiments carried out in South America with maize and wheat revealed that increased crop yields were consistently obtained using inoculants prepared with PHA-rich Azospirillum cells (Dobbelaere et al., 2001; Helman et al., 2011; Table 3).

Production of carotenoids

Carotenoids are tetraterpenoid organic pigments that occur in plants and in some bacteria and fungi. In bacteria, carotenoids counteract photo-oxidative damage (Krinsky, 1979). They are known to quench singlet oxygen and to have chain-breaking ability in radical-mediated autoxidation reactions (Burton & Ingold, 1984; Ziegelhoffer & Donohue, 2009). Many azospirilla produce carotenoids (Fig. 3), and 30 years ago, Nur et al. (1981) suggested that in this bacterium, carotenoids play an important role in protecting nitrogenase against oxidative damage, thus being critical for nitrogen fixation under nitrogen-deficient conditions. This hypothesis was confirmed by comparative studies using A. brasilense strains producing different levels of carotenoids (Hartmann & Hurek, 1988; Baldani et al., 2005).

Figure 3.

Carotenoid production and phenotypic variation in Azospirillum brasilense. The parental strain Sp7 (a) was exposed to starvation for 12 days, after which a phenotypic variant with a pink-carotenoid pigmentation was isolated (b) Pictures taken from Lerner et al. (2010).

Bacteria that live in the rhizosphere experience variations in temperature, salinity, osmolarity, pH, and availability of nutrients and oxygen (Zahran, 1999). In response to specific stimuli, bacterial sigma factors alter the pattern of gene expression by changing the affinity and specificity of RNA polymerase to different promoters during initiation of transcription (Heimann, 2002). Among the different sigma factors, group 4 s70 sigma factors were initially thought to be involved in responses to changes in the extra-cytoplasmic compartment of the cell and hence were called extracytoplasmic function (ECF) sigma factors (Heimann, 2002). In the case of rhizosphere bacteria, it is assumed that these sigma factors are critical in adaptation, survival, and proliferation in the soil, particularly under stressful conditions.

The involvement of the ECF sigma factor RpoE (also known as σE) in regulation of carotenoid synthesis in A. brasilense as well as in its tolerance to abiotic stresses was recently investigated by Mishra et al. (2011). An in-frame rpoE deletion mutant of A. brasilense Sp7 was carotenoidless and slow-growing, and was more sensitive than the wild type to salt, ethanol, and methylene blue stresses. Expression of rpoE in the rpoE deletion mutant complemented the defects in growth, carotenoid biosynthesis, and sensitivity to the different stresses (Mishra et al., 2011). It was also shown that a mutation in the gene encoding an anti-sigma factor (ChrR) belonging to the Zn2+ anti-sigma family caused overproduction of carotenoids in A. brasilense (Thirunavukkarasu et al., 2008; Mishra et al., 2011).

Chemotaxis

Chemotaxis is the ability bacteria have to sense gradients of compounds and to drive motility toward the most appropriate niche and is an important trait for survival in the rhizosphere and in plant–microbe interactions (Alexandre, 2010). Signal transduction systems enable cells to detect and adapt to these changes by executing appropriate cellular responses, such as regulation of gene expression or modulation of the swimming pattern. The best characterized signal transduction system is the one regulating the run or tumble swimming bias via chemotaxis in Escherichia coli (Wadhams & Armitage, 2004). This signal transduction system consists of a set of conserved proteins, which includes CheA, CheW, CheY, CheB, and CheR and a set of chemoreceptors known as methyl-accepting proteins that perceive environmental cues.

In A. brasilense, energy taxis is dominant (Fig. 1), with responses to most stimuli in this bacterium being triggered by changes in the electron transport system (Alexandre et al., 2000). Greer-Phillips et al. (2004) identified a novel chemoreceptor-like protein, named Tlp1, which serves as an energy taxis transducer. A tlp1 mutant was shown to be deficient in chemotaxis toward several rapidly oxidizable substrates, to taxis to the terminal electron acceptors oxygen and nitrate, and to redox taxis, suggesting that Tlp1 controls energy taxis in A. brasilense. The tlp1 mutant is also impaired in colonization of plant roots (Greer-Phillips et al., 2004).

Stephens et al. (2006) characterized the CheB and CheR components of the chemotaxis-like signal transduction pathway Che1 in A. brasilense. Characterization of cheB, cheR, and cheBR null mutants showed that these genes significantly influence chemotaxis and aerotaxis but are not essential for these behaviors, suggesting that multiple chemotaxis systems are present and contribute to chemotaxis and aerotaxis in A. brasilense. A further study characterized mutants for genes cheA1 and cheY1, also components of the Che1 system. As for the cheB/cheR mutants, these mutants were defective but not null for chemotaxis and aerotaxis, and showed a minor defect in swimming pattern. Detailed characterizations of these mutants lead the authors to propose that the Che1 chemotaxis-like pathway modulates cell length as well as flocculation (Bible et al., 2008).

Recently, Carreño-López et al. (2009) identified gene chsA as an important component of the chemotaxis signaling pathway in A. brasilense. The encoded protein, ChsA, displays characteristic signaling protein architecture, containing a PAS sensory domain and an EAL domain. The authors showed that a chsA null mutant was impaired in surface motility and chemotactic response, although it was not affected in synthesis of polar and lateral flagella, thus strengthening a key role of this gene in chemotaxis.

Cell surface components

Cell surface exopolysaccharides (EPS) and lipopolysaccharides (LPS) as well as outer membrane proteins (OMPs) and flagellin have been shown to be involved in cell aggregation as well as in attachment, adherence, and colonization of root surfaces by A. brasilense (Burdman et al., 2000a; Vanbleu et al., 2004).

The A. brasilense Cd 47.7-kDa major OMP was shown to act as an adhesin involved in root adsorption and cell aggregation (Burdman et al., 2001). Recently, a 67-kDa outer membrane lectin (OML) produced by A. brasilense Sp7 was also proposed to be involved in cell aggregation. This lectin recognizes and binds specifically to the bacterial EPS, and mediates adhesion of Azospirillum cells through EPS bridges (Mora et al., 2008).

Comparative analyses of A. brasilense strains differing in cell aggregation ability indicated a strong and direct correlation between EPS concentration and cell aggregation (Burdman et al., 2000b). In addition, arabinose, one of the monosaccharides found in both EPS and capsular polysaccharide (CPS) of A. brasilense, was suggested to be an important determinant for aggregation ability. The concentration of arabinose in EPS and CPS of A. brasilense positively correlated with the level of cell aggregation and this monosaccharide could not be detected in strains lacking aggregation ability (Burdman et al., 2000b; Bahat-Samet et al., 2004; Jofre et al., 2004).

Azospirillum lipoferum LPS are composed mainly of glucose and rhamnose, while those of A. brasilense contain glucose, galactose, xylose, rhamnose, fucose, and glucosamine (Jofre et al., 2004; Vanbleu et al., 2005). The LPS O-antigenic structures of A. brasilense strains Sp245 were shown to be composed of linear pentasaccharide repeats containing only d-rhamnose residues (Konnova et al., 2008). In A. brasilense Sp245 and Sp7, plasmids p120 and p90, respectively, were found to be involved in the synthesis of LPS, EPS, and polar and lateral flagella, strengthening the importance of these plasmids in Azospirillum–plant root interaction (Vanbleu et al., 2004; Petrova et al., 2005).

Two genes homologous to rhizobial nodulation genes nodPQ are located on plasmid p90. A nodPQ mutant of A. brasilense Sp7 lacks sulfate groups in its LPS (Vanbleu et al., 2005). An A. brasilense Cd mutant disrupted in the dTDP-rhamnose synthesis gene rmlD showed a modified LPS core structure, a significant reduction of LPS rhamnose, a nonmucoid colony morphology, increased EPS production, and was affected in maize root colonization (Bahat-Samet et al., 2004; Jofre et al., 2004).

Three additional genes located in the p90 plasmid of strain Sp7 were recently characterized following mutagenesis. The wzm gene encodes an inner membrane protein of an ABC transporter, which in gram-negative bacteria transports extracellular polysaccharides such as LPS, CPS, and EPS across the two membranes. The noeL gene encodes GDP-mannose 4,6-dehydratase, one of the enzymes involved in conversion of mannose to fucose; and noeJ encodes mannose-6-phosphate isomerase that catalyzes the interconversion of d-fructose-6-phosphate and d-mannose-6-phosphate. Impairment in wzm, noeL, and noeJ leads to defective LPS in strain Sp7. In wzm and noeJ mutants, only low-molecular-weight LPS bands were observed (Lerner et al., 2009bc), while no LPS band was observed for the noeL mutant (Lerner et al., 2009b). In addition, substantial changes in the profile of OMPs were observed for noeJ, noeL, and wzm mutants in comparison with the wild type by SDS-PAGE (Lerner et al., 2009bc). These mutants also showed deficient survival to salt, heat, and osmotic stresses; however, the effect of these mutations in plant–A. brasilense interaction is still to be investigated.

A recent study using atomic force microscopy revealed distinct morphological properties of flocculating A. brasilense Che1 mutants, in comparison with the wild type. Whereas wild-type cells were shown to produce a smooth mucosal extracellular matrix, flocculating Che1 mutants produced distinctive extracellular fibril structures, and likely a different structure and composition of EPS (Edwards et al., 2011).

Nitrogen fixation

Biological nitrogen fixation by azospirilla occurs in pure culture and under optimal oxygen pressure, temperature, and carbon and energy sources (Okon, 1985). Measurable nitrogen fixation activities of azospirilla in association with plants have been demonstrated many times (Okon, 1985; Spaepen et al., 2009; Bashan & de-Bashan, 2010). However, extensive quantitative measurements of nitrogen fixation in greenhouse and field experiments as well as characterization of nitrogen fixation mutants showed that contribution of fixed nitrogen by A. brasilense does not play a major role in plant growth promotion in most systems evaluated so far (Okon, 1985; Spaepen et al., 2009). Nevertheless, nitrogen fixation ability is considered a positive attribute for rhizosphere competence of azospirilla (Okon, 1985).

The two primary environmental modulators of nitrogenase synthesis and activity in A. brasilense are ammonium ions (inline image) and oxygen (O2) (Pedrosa & Elmerich, 2007; Cassan & Garcia de Salamone, 2008). The nitrogenase complex is sensitive to oxygen and, as mentioned, carotenoids are thought to play an important role in protection against oxidative damage in A. brasilense (Hartmann & Hurek, 1988; Baldani et al., 2005).

Transcription of the nitrogen fixation (nif) genes in proteobacterial diazotrophs is generally activated by the NifA protein. In many nitrogen-fixing bacteria, the nifA promoter is under control of the general nitrogen regulation (Ntr) system through the direct action of the transcriptional activator NtrC (Pedrosa & Elmerich, 2007). In contrast, in A. brasilense, NtrC is not involved in direct activation of the nifA promoter (Pedrosa & Elmerich, 2007). The activity of the NifA protein in A. brasilense is controlled by a signal transduction protein of the PII family in response to fluctuations in inline image levels. In this bacterium, the activity of NifA is also affected by molecular oxygen (Cassan & Garcia de Salamone, 2008). Once synthesized, nitrogenase activity of A. brasilense, as well as of other Rhodospirillales, is reversibly inactivated in vivo by inline image or anaerobiosis. This inactivation involves ADP-ribosylation of the Fe-protein (dinitrogenase reductase) catalyzed by dinitrogenase reductase ADP-ribosyltransferase (DraT) and is reversed, upon inline image exhaustion, by dinitrogenase reductase activating glycohydrolase (DraG) (Cassan & Garcia de Salamone, 2008). The activities of both DraT and DraG enzymes are regulated according to the levels of ammonium through direct interactions with the PII proteins GlnB and GlnZ. DraG interacts with GlnZ both in vivo and in vitro, and DraT interacts with GlnB in vivo (Huergo et al., 2009).

Phenotypic variation

Bacteria have developed mechanisms to maintain cell viability during starvation and resume growth when nutrients become available. These include among others phase variation (Kussell et al., 2005). Phase variation has been proposed as an important mechanism by which microorganisms adapt to environmental changes, such as those existing in the soil rhizosphere (Van den Broek et al., 2005). In phase variation, the expression of a given gene is either in an ‘ON’ or an ‘OFF’ mode, with these changes usually being reversible. Phase variation has been defined as a random event that occurs at high frequency, involves changes in the DNA, and leads to a phenotypically heterogeneous population (Van der Woude & Baumler, 2004; Wisniewski-Dye & Vial, 2008).

Several studies with Azospirillum have identified and characterized phenotypic variants. In A. lipoferum 4B, phenotypic variation was associated with loss of a 750-kb plasmid (Vial et al., 2006). In A. brasilense Sp245, a spontaneous variant was shown to lose plasmids p40, p85, and p120; however, it gained a new plasmid of more than 300 MDa (Katsy et al., 2002). Phenotypic variants of A. brasilense Sp7 also showed altered plasmid composition, as well as changes in LPS structure (Petrova et al., 2005). New phenotypic variants of A. brasilense Sp7 were retrieved recently, after exposure of the parental strain mainly to starvation, but also after colonization of maize roots (Lerner et al., 2010). Two variants, Sp7E and Sp7EPS, were found to produce significantly higher EPS concentrations relative to the Sp7 parental strain and were LPS-defective. The variants were also shown to carry alterations in DNA rearrangement, EPS monosaccharide composition, and OMP profile as compared to the parental strain (Lerner et al., 2010). Importantly, the variants differed from the parental strain in cell pigmentation (Fig. 3), susceptibility to stresses, antibiotics, and capability of biofilm formation (Lerner et al., 2010). Future studies may determine how phenotypic variation is associated with survival in bacterial inoculants, root colonization, and plant growth promotion.

Conclusion and perspectives

The annotation of bacterial and plant genomes and the use of high-throughput techniques are contributing significant advances in the elucidation of signal molecule exchanges between pathogenic and beneficial bacteria and their plant hosts, as well as the physiological features contributing to improved performance of a given bacterium in its ecological niche (Spaepen et al., 2007). Similar advances are needed in the area of Azospirillum– and other PGPR–plant interactions (Pothier et al., 2007; Van Puyvelde et al., 2011).

Investigating the traits that contribute to bacterial survival under adverse conditions during inoculant production, storage, inoculation, and colonization of seeds and plants is very important. For example, it is crucial to better understand the roles of cell storage materials like PHAs (Kadouri et al., 2005; Castro-Sowinski et al., 2010), glycogen (Lerner et al., 2009a), polyphosphates, and others, and cell surface components like EPS, LPS, and surface proteins in enhanced resistance of bacteria to diverse stress conditions (e.g. salinity, desiccation, osmotic pressure, suboptimal temperature, and more).

Further investigation using the available mutants as reported in this review could focus on the clarification of the complex interactions between different rhizosphere features, in contributing to a successful ecological performance of A. brasilense. This knowledge could contribute with new ideas as to which traits could be improved for more efficient plant growth promotion inoculants for the benefit of agriculture.

Dedication

This Minireview is dedicated to the memory of Robert H. Burris and Jesus Caballero-Mellado, for their extensive contribution to the research of diazotrophic PGPR.

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