Enzymatic role for soybean ecto-apyrase in nodulation

Abstract

Root nodulation is regulated by a variety of mechanisms. Ecto-apyrase is an enzyme proposed to control the concentration of extracellular nucleotides. Transgenic expression of the soybean GS52 ecto-apyrase was shown to stimulate nodulation. However, mutation of the enzyme to disrupt enzymatic activity prevented this effect. Therefore, the data suggest that the enzymatic activity of the ecto-apyrase is critical for nodulation enhancement, suggesting a direct effect on extracellular nucleotide hydrolysis. In this article, we propose a hypothetical mechanism for plant ecto-apyrase function during nodulation.

Legume plants and bacteria of the family Rhizobiaceae have the ability to establish a symbiosis in which the bacteria fix nitrogen within a newly emerged plant organ, the root nodule. Root nodule development is initiated by signal exchange between the symbiotic bacteria and their plant hosts. Flavonoids produced by the plants trigger the induction of bacterial nodulation genes that encode proteins involved in production and transport of specific lipochitooligosaccharide signal molecules, so-called Nod factor, that initiate nodule organogenesis. The Nod factor is the key rhizobial signal required for nodulation and is essential for the induction of many of the early nodulation responses.^1,2

Ecto-apyrase is an extracellular nucleoside triphosphate diphosphohydrolase that modulates the nucleotide concentration in the extracellular matrix.^3,4 Previous studies suggested an important role for the soybean ecto-apyrase GS52 (Glycine soja 52 kDa protein) in rhizobial root hair infection and root nodule formation. For example, GS52 expression is rapidly induced upon rhizobial inoculation by Bradyrhizobium japonicum.⁵ The GS52 ecto-apyrase falls into a legume specific clade of apyrase enzymes suggesting a legume-specific role (e.g., nodulation).⁶ The GS52 ecto-apyrase was localized to the plasma membrane and treatment of roots with anti-GS52 antibody was shown to block nodulation.⁵ Furthermore, ectopic expression of GS52 ecto-apyrase in Lotus japonicus roots resulted in increased infection by rhizobia and higher levels of nodulation.⁷ RNA interference-mediated gene silencing of GS52 expression in soybean transgenic roots led to a marked reduction in nodulation.⁸

Although these previous studies suggested an important role for the soybean ecto-apyrase in nodulation, there remained the question as to whether the enzymatic function of the protein was essential for nodulation. In our recent publication,⁹ the question was addressed by biochemical and nodulation analysis of mutant forms of the enzyme defective in catalysis. Specifically, the wild-type GS52 protein or inactive mutant forms were expressed in the roots of Medicago truncatula and soybean (Glycine max), and the levels of nodulation were measured. The results showed that roots expressing wild-type GS52 formed significantly more nodules, whereas no increase in nodulation was observed in roots expressing the mutant GS52 proteins. In addition to these results, the recent paper also described the biochemical properties of the soybean GS52 ecto-apyrase. For example, the enzyme showed broad substrate specificity (its activity on pyrimidine nucleotides and diphosphate nucleotides was significantly higher than on ATP), which was corroborated by structural modeling that predicted low adenine-base specificity in the substrate-binding pocket of the enzyme. This characteristic is likely conserved in the legume-specific clade of apyrase enzymes.^6,9 Taken together, these studies provide new information to define the specific function of the ecto-apyrase enzyme in the nodulation process.

Interestingly, it was reported that the activity of ecto-apyrase is directly regulated by the Nod factor. For example, DbLNP, an ecto-apyrase from Dolichos biflorus was reported to bind to the Nod factors derived from various rhizobia.¹⁰ This binding increased the enzymatic activity of the DbLNP apyrase,¹⁰ suggesting a possible signaling role for the ecto-apyrase in nodulation. To assess whether the function of the GS52 enzyme is directly affected by Nod factor addition, we examined the enzymatic activity of the recombinant GS52 protein in the presence of increasing concentrations of the B. japonicum Nod factor. The recombinant protein was isolated after expression in E. coli or after expression in tobacco leaves (for the detailed methods, see ref. 9 and 11). The results showed that the activity of the GS52 enzyme was not significantly affected by Nod factor addition (Fig. 1). These data suggest that the GS52 ecto-apyrase likely has no direct interaction with the Nod factors. However, the Nod factor does affect the soybean ecto-apyrase in an indirect manner, i.e., at the transcriptional level since the expression of the GS52 gene is induced upon B. japonicum inoculation.^5,8,12 Our results appear to be at odds with the results previously published for the DbLNP apyrase.¹⁰ However, in these studies, the DbLNP enzyme was directly isolated from D. biflorus tissues, where in our case recombinant protein was analyzed. It is known that apyrases can be modified by glycosylation.^13,14 This was the major reason why we also sought to analyze the GS52 protein isolated after expression in tobacco. Indeed, the enzymatic properties of the enzyme isolated from E. coli or tobacco were somewhat different. For example, the V_max values of the enzyme from tobacco are >100 times higher than that from E. coli⁹ (ATPase activity: K_m = 300 µM, V_max = 285.7 µmol min⁻¹ mg⁻¹; ADPase activity: K_m = 286 µM, V_max = 555.6 µmol min⁻¹ mg⁻¹). In conclusion, although it is quite possible that the GS52 protein does not respond to Nod factor addition, we cannot rule out the possibility that GS52 protein isolated directly from soybean tissue might behave differently and more in line with the earlier reports using DbLNP.

Figure 1.

Effect of Nod factor on apyrase activity of GS52. The recombinant GS52 proteins purified from E. coli (A) and tobacco (B) were preincubated for 1 h in 40 mM Tris-MES buffer (pH 6.5) containing various concentrations of B. japonicum USDA110 Nod factor. Subsequently, apyrase enzymatic activity (ATPase activity) was measured as described in our previous publication.⁹ Similar results were obtained on the ADPase activity (data not shown).

The catalytic domain of the GS52 ecto-apyrase is predicted to be extracellular and therefore, must act to hydrolyze extracellular nucleotides. Indeed, the addition of Nod factors to root hairs of M. truncatula was shown to trigger extracellular nucleotide release.⁹ Therefore, it is interesting to postulate that this release of ATP (or other nucleotides) is somehow involved in modulating downstream signaling events critical for rhizobial infection and nodulation. For example, exogenous addition of extra-cellular ATP was shown to induce the production of reactive oxygen species (ROS), nitric oxide and elevation of cytosolic calcium,¹⁵ which are all responses known to be triggered by Nod factors.^2,16,17 In addition, Nod factor addition to legume root hair cells causes calcium oscillations, whose frequency and amplitude are apparently a specific signal to initiate downstream intracellular signaling events.¹⁸ Similar, but not identical, calcium oscillations were seen in Arabidopsis roots upon addition of nucleotides.¹⁹

Cardenas et al. demonstrated that the Nod factor induced transient ROS production and suggested that rhizobia are allowed to enter the host plant without triggering a defense response by regulating ROS production because excessive ROS might provoke infection thread abortion.²¹ Since Nod factors induce the release of nucleotides⁹ and extracellular nucleotides cause ROS production in root hairs,^20,22,23 the available data lead to a model (Fig. 2) suggesting a role for the ecto-apyrase in optimizing extracellular nucleotide levels to permit root hair growth, cell wall modification and subsequent formation of the infection thread.²⁴ Optimizing this process would be essential to prevent detrimental effects that might, for example, be caused by excessive production of ROS. Although this model is consistent with current evidence, key features still lack strong experimental support, which will be addressed by ongoing research efforts.

Figure 2.

Schematic showing the hypothesized role of the ecto-apyrases in maintaining an optimal extracellular nucleotide concentration at the root hair surface to allow rhizobial infection. Nod factor increases the extracellular nucleotide concentration ([Nuc]_ext) at the root hair surface.⁹ Concurrently, the Nod factor maximizes the ecto-apyrase (APY) function by increased the gene expression level⁵,⁸,¹²,²⁵ or perhaps, by directly affecting its enzymatic activity.¹⁰ The ecto-apyrase then controls [Nuc]_ext at an optimal level to induce a proper concentration of ROS ([ROS]) production that contributes to changing plant cell wall formation and to regulation of the infection thread rigidity. Optimizing [ROS] levels may also prevent the induction of a plant defense response.

Addendum to: Tanaka K, Nguyen CT, Libault M, Cheng J, Stacey G. Enzymatic activity of the soybean ecto-apyrase GS52 is essential for stimulation of nodulation. Plant Physiol. 2011;155:1988–1998. doi: 10.1104/pp.110.170910.