Latest revision as of 13:38, 24 March 2017

Abstract

The symbiotic diazotrophs comprise with a very diverse group of Gram negative soil bacteria, collectively called as rhizobia found in nodule of legume plant. Rhizobia adopt themselves in different environment including soil, rhizosphere and grown within legume roots, where they fix nitrogen. The establishment of symbiosis is a very complicated process involving a coordinated exchange of signal between legume plants and the symbionts. The nodule development requires synthesis of signal molecules such as Nod factors that are important for induction of nodule development. There are different types of surface polysaccharides such as lipopolysaccharides, capsular polysaccharides, neutral and acidic polysaccharides found in rhizobia. The production of symbiotically active polysaccharides may allow rhizobial strains to adapt themselves to changing environmental conditions and interact efficiently with legume plants. Despite extensive research, the actual molecular function of the surface polysaccharides of rhizobia in symbiosis remains unclear. This review emphasized on the structural composition of extracellular polysaccharide of different rhizobia isolated from different legume plants. The compositions of extracellular polysaccharides are different in different rhizobia. The various compositions of extracellular polysaccharides produced by the symbionts are considered as the signaling molecules essential for determining host plant specificity. The present status of the biological functions of the exo-polysaccharide in symbiosis such as host specificity, successful invasion, formation of infection thread and induction of nodule formation in legume plants is also summarized here.

Abbreviations

EPS , extracellular polysaccharide ; CPS , capsular polysaccharide ; LPS , lipopolysaccharides ; CG , cyclic beta glucan ; KPS , K-antigen polysaccharide ; NP , neutral polysaccharide

Keywords

Rhizobium spp. ; Extracellular polysaccharide ; Production ; Composition ; Succinoglycan ; Galactoglucan

Introduction

Polysaccharide is the most abundant organic macromolecules in nature. The biosyntheses of polysaccharide are found in different organisms ranging from bacteria to eukarya, including plants. Gram negative soil bacteria belonging to (α and β-proteobacteria) have the ability to produce root nodule by symbiosis with legume plants (Skorupska et al., 2006 ). This interaction is initiated by the exchange of signal by diverse molecules between the two partners. Among them, plants liberate flavonoids into the rhizospheric region that upregulate rhizobial genes responsible for nodule formation (Spaink, 2000  ;  Schulze et al ., 1998 ). Recognition of the symbiont is made possible through the exchange of nod factor perception and Ca^2 + /calmodulin-dependent protein signaling (Geurts and Bisseling, 2002  ;  Oldroyd and Downie, 2004 ). The establishment of successful symbiosis in legume plant was made by the production of nod factor signal and exposure of the correct surface and/or extracellular polysaccharide.

This signal exchange between the two partners seems to work at a distance in the rhizosphere and before binding of rhizobial symbiont to the host root hairs, to induce morphogenetic changes in plant roots. These signal molecules, the Nod factors, are sufficient for initiation of root hair changes, bacterial cell entry for infection, thread formation and activation of cortical cell division Schulze et al. (1998) to generate the nodule primordium (Nod) (Kijne, 1992 ). Rhizobia colonize with plant root hairs, develops infection, the bacteria multiply to form infection thread. In this thread the bacteria encircled by the peribacteroid membrane of plant origin that differentiate into bacteroids (Fisher and Long, 1992 ).

Bacteroids were found to fix nitrogen by synthesizing the nitrogenase enzyme and other proteins. In return, plant supplies carbohydrates to bacteria as a source of carbon and energy. The establishment of symbiosis is stringently controlled through a complex network of signaling cascades Schulze et al. (1998) . This process is partner specific and signifies that the rhizobial species can only nodulate a limited but defined range of legume plants.

The key factors for the interaction are a number of rhizobial genes which are responsible for production of different types of cell-surface polysaccharides such as capsular polysaccharide (CPS) that form as adherent cohesive layer on the cell surface. However, the term exopolysaccharides (EPS) is used for polysaccharides with little or no cell association (Becker and Pühler, 1998 ). Cyclic beta-(1-2)-glucan is concentrated in periplasmic space of rhizobia, which plays an important role in osmotic adaptation of bacteria Breedveld et al. (1993) . Lipopolysaccharides (LPS) are anchored in outer membrane and consist of lipid A, a core polysaccharide and repeating O-side antigen polysaccharides. Despite extensive research, the precise role of surface polysaccharides in symbiosis remains unclear. So the role of rhizobial polysaccharide has been the goal of many studies.

The present review describes the production and structure of different exopolysaccharides of rhizobia isolated from legume root nodule. Attempts were also made to discuss the possible role of the exopolysaccharide in legume — rhizobia symbiosis and nodule formation.

Structural Features of Rhizobial Exopolysaccharides

Rhizobial cell produces different types of surface polysaccharides into environment or retained at the cell surface. They comprise extracellular polysaccharide (EPS), lipopolysaccharide (LPS), capsular polysaccharide (CPS), cyclic beta glucan (CG), K-antigen polysaccharide (KPS), neutral polysaccharide (NP), gel-forming-polysaccharide (GPS), and cellulose fibrils. They are species as well as strain-specific heteropolymers and consisting of repeating units containing mainly common monosaccharides (d -glucose, d -mannose, d -galactose, l -rhamnose, d -glucuronic and d -galacturonic acids) (Table 1 ). A large diversity in EPS chemical structures can be found among rhizobia, concerning sugar composition, linkage of subunit, repeating unit size and degree of polymerization as well as non-carbohydrate decoration (Table 1 ) (Laus et al ., 2005  ; Skorupska et al ., 2006  ; Downie, 2010  ;  Janczarek, 2011 ). EPS are mainly two types, succinoglycan (EPS I) and galactoglucan (EPS II) produced by several rhizobial strains (Reinhold et al., 1994 ) (Fig. 1 ). EPS-I composed of octasaccharide repeating units containing one galactose and seven glucose residues (in molar ratio 1:7), joined by β-1,3; β-1,4 and β-1,6 glycosidic linkages whereas EPS II is a polymer of disaccharide repeating unit and joined by α-1,3 and β-1,3 glycosidic bonds (Her et al ., 1990  ;  Zevenhuizen, 1997 ).

Fig. 1. Chemical structure of rhizobial exopolysaccharides (EPS) (based on Skorupska et al ., 2006  ; Downie, 2010  ;  Janczarek, 2011 and Janczarek et al., 2014 review works). Abbreviations: Glc = glucose, GlcA = glucuronic acid, Gal = galactose, Succ = succinate and Ac = acetyl.

Single repeating unit is decorated by different non-carbohydrate such as acetyl, pyruvyl and succinyl groups. Both EPS I and II are secreted in two major fractions — High Molecular Weight (HMW) consisting of hundreds to thousands of repeating units and Low Molecular Weight (LMW) that represents monomers, dimers and trimers in a case of EPS I and oligomers (15–20) in the case of EPS II (Gonzalez et al ., 1996  ; Gonzalez et al ., 1998  ;  Wang et al ., 1999 ). The pattern of non-carbohydrate modifications of EPS may be different in various strains of the same species and depend on the phase of bacterial growth and culture medium. Non-carbohydrate modifications located in the side chain of the EPS units proved to be very important for the signaling properties of EPS in the symbiosis (Ivashina and Ksenzenko, 2012  ;  Janczarek et al ., 2014 ).

Production of Exopolysaccharides in Culture by Rhizobia

Rhizobium spp. are able to produce large amount of EPS in culture rather than EPS produced in symbiotic condition ( Table 2 ). The growth environment was very important for maximum exopolysaccharide production (Sutherland, 1972 ). Utilization of different carbon sources for the growth and EPS production by Rhizobium sp. was reported earlier ( Stowers, 1985  ;  Breedveld et al ., 1993 ). However, carbon source in medium was a determining factor for the amount of EPS produced by R. tropici CIAT899 ( Staudt et al., 2012 ). Different carbon sources supplemented at 1% level promoted the bacterial growth and EPS production by a Rhizobium sp. to different extent and mannitol was the most effective promoter. Higher concentrations of polysaccharides were obtained when carbon:nitrogen ratio was higher in medium ( Breedveld et al., 1993 ). Sulfate was less preferred nitrogen source for most of nitrogen sources for some strain of Rhizobium , whereas nitrate, glycine and ammonium conjugate were most preferred as nitrogen source ( Ghosh et al., 2005 ). Mandal et al. (2007) have reported l -asparagine increased both growth and production by Rhizobium sp. VMA301 from V. mungo (L.) Hepper. Various studies have found that EPS production is favored under conditions of nitrogen limitation ( Doherty et al ., 1988  ;  Dudman, 1964 ). Under such nitrogen-limited conditions, any excess sugars remaining can be used specifically for polysaccharide synthesis (Kucuk and Kivanc, 2009 ). However, thiamine hydrochloride and nicotinic acid increased EPS production by different species of Rhizobium were observed Amemura et al ., 1983  ;  Ghosh et al ., 2011 . Varied degrees of promotion of growth by isolates in the presence of vitamins might be due to differential requirement of vitamins as cofactors. Watson et al. (2001) stated that biotin might be involved as metabolic regulator in rhizobial growth.

a. Ethanol-precipitated material from supernatant.

b. Ethanol precipitated material/carbon, ND = not detected.

EPS production also varies with time, as a function of growth phase, for many bacteria. For many rhizobacterial species, growth and exopolysaccharide production occur simultaneously, because EPS biosynthesis being growth-associated (Datta and Basu, 1999  ;  Ghosh et al ., 2005 ). Several studies have indicated that the EPS yields vary with bacterial growth phase, while EPS composition remains constant through the batch cycle of growth (Breedveld et al ., 1993  ;  Ghosh et al ., 2015 ).

In regards to oxygen availability, the agitation rate controls both bacterial growth and EPS production. Bacterial growth (in biomass) tends to be increase with higher agitation rates, under conditions where oxygen limitation is not imposed. However, the impact of agitation on EPS production is unclear. Studies by Dudman (1964) and Kucuk and Kivanc (2009) indicate that EPS production is optimal under conditions of low agitation. However, others have indicated that polysaccharide production is maximized under conditions of high aeration, when oxygen limitation is not imposed (Duta et al., 2006 ).

Both bacterial growth and rates of EPS production are dependent upon the pH of the growth medium (Martinez-Romero et al ., 1991  ;  Staudt et al ., 2012 ). High level of pH resulted dramatic decrease in EPS production, particularly in the acidic range (Kucuk and Kivanc, 2009  ;  Staudt et al ., 2012 ) whereas optimum EPS production was reported at slight alkaline pH value of 8.0 (Sayyed et al., 2011 ). The role of bacterial growth temperature in EPS production is variable. EPS production is often favored by suboptimal temperature Sutherland (1972) . However, other studies found maximum EPS production occurring at optimal temperature conditions by rhizobia (Sayyed et al ., 2011  ;  Ghosh et al ., 2015 ).

Functions of Rhizobial Exopolysaccharide in Symbiosis

Several possible biological functions of rhizobial EPS are suggested by different workers (Fig. 2 ). These includes role in protecting bacteria against environmental stresses, initial attachment of bacteria to the roots, involvement in stage of infection, structural role in the infection thread formation, release of bacteria from the infection threads, bacteroid development, suppression of plant defense responses and protection against plant antimicrobial compounds (Becker and Pühler, 1998  ;  Fraysse et al ., 2003 ).

Fig. 2. Different types of function related to rhizobial EPS production.

The primary attachment is established by recognition of root-hair lectins by the surface carbohydrates of rhizobial cell Dazzo et al. (1984) . EPS may enhance the chance of adhesion of bacteria to the tip of growing root hairs. However, EPS-deficient mutants are not significantly affected in adhesion to the roots of several species such as clover (Rolfe et al., 1996 ), vetch (Van Workum et al., 1998 ) or alfalfa (Cheng and Walker, 1998 ). The surface proteins rhicadhesins could also play an important role in the initial attachment (Smit et al., 1992 ) and involvement of cellulose fibrils is suggested in this process (Laus et al., 2005 ). After initial attachment of R. leguminosarum to root hairs, bacteria aggregate around the root hair surface in a cap or a biofilm formation which requires cellulose fibrils ( Laus et al ., 2005  ;  Williams et al ., 2008 ). Rhizobial strain produce EPS in large amount were characterized by biofilm formation that enhanced bacterial cell to better adaptation inside the host roots (Fujishige et al., 2006 ).

The low molecular mass of EPS in Sinorhizobium meliloti Rm1021 has been studied to know the structural requirement of EPS for an effective nodulation ( Gonzalez et al., 1996 ). Normally, this strain requires the presence of a succinoglycan to efficiently infect Medicago sativa root hairs Reinhold et al. (1994) . The importance of this polysaccharide in the symbiosis was confirmed in several non-EPS producing strains of Sinorhizobium meliloti and Rhizobium leguminosarum bvs. trifolii and viciae , which were symbiotically defective due to induction of empty or almost uninfected nodules on the respective host plants, being a result of aborted infection thread elongation within the peripheral cells of the developing nodule ( Ivashina and Ksenzenko, 2012 ).

The most important among the suggested EPS functions is its role as a signaling molecule. Rhizobial lipochitin oligosaccharides (Nod factors), bacterial cell-surface components and low-molecular weight metabolites are engaged in the signaling. The EPS of Mesorhizobium loti played a signaling role in symbiosis with Lotus plants forming determinate type of nodules Kelly et al. (2013) . Although several studies about rhizobial EPS and its precise function as a signal molecule in the symbiosis have not yet been clearly established (Janczarek et al., 2014 ).

Moreover, EPS is especially important in symbioses with legumes that form indeterminate-type nodules, where long infection threads are formed, as a compound indispensable for initiation and propagation of infection threads, bacterial release from the infection threads and development of bacteroids (Brewin, 2004 ; Skorupska et al ., 2006  ;  Ivashina and Ksenzenko, 2012 ). The acidic EPS essential for the development of an indeterminate type of nodules: Sinorhizobium meliloti /alfalfa , Rhizobium leguminosarum bv. Viciae /Vicia sativa and bv. Trifoli /trifolium ssp. NGR234/Leucaena ( Breedveld et al ., 1993  ;  Djordjevic et al ., 1987 ).

Besides, there are several ways that plant pathogenic and symbiotic bacteria used to avoid plant defense system and to protect themselves (D'Haeze et al., 2004 ). The rhizobial surface polysaccharides and glucan play important roles in protection against the host defense (D'Haeze et al., 2004 ). It is suggested that EPS might be act by suppressing bacterial antigens at the stage of nodule cell infection (Wielbo et al., 2004 ). Recently Ciesla et al. (2016) reported that EPS plays an essential role in electrophoretic mobility of rhizobial cells and their higher amounts depend on mobility as well as its acidic nature of the bacterial cell surface.

Conclusion

Extracellular polysaccharides (EPS) are species-specific complex polymers of different carbohydrate unit secreted by bacterial cell. This subject has been a great interest for a long time because of their importance in successful development of symbiosis with legume hosts. The production of EPS by the rhizobia is considered to have important physiological implications and its critical constituents seemed to be only for the invasion process leading to indeterminate and determinate nodule types. Several putative roles have been considered for EPS synthesis such as specific signaling in the root invasion process, inhibition of plant defense response structural requirement for this process and electrophoretic mobility of rhizobial cells.

On the other hand, new development of molecular methods that advances our understanding of the biosynthesis, regulation and secretion of exopolysaccharides. The important goal for the future research should be the enlightenment of mechanisms of EPSs action as signaling molecules in the initiation and development of symbiosis and mechanisms of control of the plant defense systems which enable rhizobia to invade legume plants and their survival.

Acknowledgements

Authors gratefully acknowledged the financial support provided by the UGC , New Delhi, India, through CAS, Department of Botany, and The University of Burdwan , Burdwan, West Bengal, India. The authors also gratefully acknowledged DST (SERB), New Delhi, India (NPDF/2016/00323 dt. 05.07.2016) for providing National Post-Doctoral Fellowship to the first author.

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