Specific expression and activity of acid phosphatases in common bean nodules

Abstract

Under phosphorus (P) deficiency, sensitivity of the N₂-fixing legumes increases since the large amount of P-dependent carbon and energy turnover required during N₂ fixation are not satisfied. However, despites the fact that these crops have been widely characterized under P-deficiency and a number of tolerance traits have been identified, abilities of the nodules to cope with this environmental constraint have still to be further investigated. Increases both of activity and gene expression of acid phosphatases (APases) are among mechanisms that lead to increase both of N₂ fixation and nodule respiration under P-deficiency. Our findings have revealed that expression of phosphoenol pyruvate phosphatase (PEPase) and trehalose 6P phosphatase (TPP) genes and activities of the corresponding enzymes were positively correlated with increases both of the rhizobial symbiosis efficiency in use of P for N₂ fixation and nodule O₂ permeability. Under P-deficiency, this positive correlation was more significant for the recombinant inbred line (RIL) of Phaseolus vulgaris RIL115 that is tolerant to P-deficiency than the sensitive RIL147. Overall, the present work suggests that the tissue-specific localized PEPase and TPP transcripts of infected cells and nodule cortex play a role in adaptation to P-deficiency and are likely involved in nodule respiration linked to symbiotic nitrogen fixation (SNF).

Introduction

Low phosphorus (P) availability is a widespread constraint of nutrient deficiency for plant growth especially in acidic and alkaline soils.¹ The limited availability of P reduces not only allocation of P into nodules and P use efficiency by shoot² but also has deleterious effect on N₂ fixation that has been reported to be directly related to declines in biomass of nodules and shoot.^3-7 Although P-deficiency has a large negative impact on plant growth and development, it appears to be especially detrimental for N₂-fixing legumes because of the specific requirement of P for symbiotic N₂ fixation.⁸ In spite of widely documented sensitivity of N₂-fixing legumes to P-deficiency, the ability of nodules to act as strong P sinks is still not fully understood, although it predicts the important role of P in nodule functioning.⁹ Recently, it has been demonstrated that high nodule P content may contribute to the energy metabolism in N₂-fixing nodules under P-deficiency.^3,5,9 This adaptation to P-deficiency seems to be multi-coordinated at the physiological and molecular levels as recently demonstrated by Molina et al.¹⁰ under salinity and Iturbe-Ormaetxe et al.¹¹ under oxidative stress.

Secretion of acid phosphatases (APases) that hydrolyze esterified-P compounds is among the intricate array of adaptations to P-deficiency. These exo-enzymes are involved in P nutrition by increasing P acquisition from extracellular environment and enhancing P utilization by internal mechanisms associated with efficient use of P at the cellular level.^8-13 Induction of intracellular and secreted APases appears to be a universal plant response to nutritional Pi limitation which participates in systemic inorganic P (Pi) mobilization from soil organic matter localized-P, including nucleic acids.¹⁴ In N₂-fixing nodules, recent studies have suggested that stimulations of such APases as phytase,³ phosphoenol pyruvate phosphatase (PEPase)¹⁵ and trehalose 6P phosphatase (TPP)¹⁶ within P. vulgaris nodules, may be an adaptive mechanism to P-deficiency. In this context, Penheiter et al.¹⁷ concluded that APases in the symbiosome membrane of Glycine max was involved in P homeostasis within the nodules.¹⁸

In nodules, expression of large number of Glycine max purple acid phosphatase “GmPAP” genes was mainly detected under P-deficiency.¹⁹ This organ was reported to be strongly enriched in highly tissue-specific genes.^20,21 Likewise, several studies have reported that transcripts abundance of APases^15,16 under P-deficiency and ascorbate peroxidase (APX)^10-22 under salinity and P limitation¹⁶ within the nodule cortex seems to be an adaptive response at least against these adverse conditions. This abundance was associated with an increase in nodule conductance to O₂ diffusion.^5-16 Such a finding opens up a new research question on the relationship between the above specific sub-cellular localization in nodule cortex and the regulation of the nodule respiration since this zone, more particularly the inner cortex, is postulated to be a physical barrier for the regulation of nodule permeability to gas diffusion^23-25 and osmotic conditions.⁹ In this revue, we highlight the differential expression of PEPase and TPP in relation with APX localization within the nodules of P. vulgaris under P-deficiency and address whether this expression might influence the nodule O₂ permeability and the subsequent respiration that supports N₂ fixation in legumes.

Specific Expression and Activity of APases in Nodule Cortex

Differential in situ expression of PEPase and TPP genes was visualized within nodules of common bean. These genes were highly expressed under P-deficiency (Fig. 1B,D,F and H) with high transcription level in the infected zone, nodule cortex and vascular traces for the RIL115 as compared with RIL147 (Figs. 1 and 2C,E and F). Abundance of PEPase and TPP transcripts in infected cells next to inner cortex and outer cortex cells (Figs. 1 and 2) for both RILs is the first observation of these genes expression among APases genes that are overexpressed in legume nodules in response to P-deficiency. This finding highlights a new mechanism by which nodules adapt to P-deficiency constraint. The increase both in transcripts and enzyme activities (Table 1) of PEPase and TPP especially in the P-efficient RIL115 evidences a functional role in supplying large amount of Pi as a necessary requirement for bacteroids metabolism, as well as in the overall tolerance of the rhizobial symbiosis to P-deficiency. This functional role is consistent with the high positive correlation between PEPase and TPP enzyme activities and efficiency of rhizobial symbiosis (EURS), N₂ fixation¹⁵ and nodule respiration (Fig. 3B and C). Coherently, Li et al.¹¹ have claimed that some members of the purple acid phosphatases gene family in soybean are involved in the response of host plant to symbiosis with rhizobia or arbuscular mycorrhizal fungi under P-limited conditions. In addition, the TPP activation under P-deficiency would not only release Pi but also trehalose of which the accumulation in nodules of P. vulgaris,²⁷ Lotus japonicus²⁸ and in Aspergillus nidulans²⁹ correlated with resistance to several abiotic constraints. Nevertheless, although the plant-cell concentration of trehalose 6-P is generally very low and is not much documented for N₂-fixing nodules,³⁰ catalyzing the dephosphorylation of trehalose 6-P may supply some Pi for bacteroids metabolism in relation with nodule function.

Figure 1. In situ RTPCR localization of phosphoenol pyruvate phosphatase (PEPase), trehalose 6P phosphatase (TPP) and ascorbate peroxidase (APX) transcripts (green spots) in common bean nodules of RILs 115 and 147 grown under sufficient P (250 P) vs. deficient P (75P) supply. IC, inner cortex; IZ, infected zone; OC, outer cortex; VT, vascular traces parenchyma. Bar = 500 µm in all the pictures.^15,16

Figure 2. Nodule cortex and infected cells transcript localization of phosphoenol pyruvate phosphatase (PEPase), trehalose 6P phosphatase (TPP) and ascorbate peroxidase (APX) in common bean nodules of RILs 115 and 147 grown under sufficient P (250 P) vs. deficient P (75P) supply. control, nodule section without reverse transcriptase. IC, inner cortex; InC, infected cells; OC, outer cortex; UC, uninfected cells; VT, vascular trace parenchyma. Bars = 500µm in (A andB), 200 µm in (C, E, F, G and H) and 50µm in (D).^15,16

Table 1.

RILs	P-level	APases	PEPase	TPP	Peroxidase	N2 fixation	EURS
115	250P	4.3b ± 0.77	0.68ab ± 0.13	0.03bc ± 0.002	14a ± 2.78	155a ± 41.5	13.9
	75P	5.5b ± 0.10	0.96a ± 0.10	0.05a ± 0.004	16a ± 1.06	109ab ± 10.5	16.2
147	250P	6b ± 0.10	0.53b ± 0.03	0.03c ± 0.001	6b ± 0.99	135a ± 27.5	7.93
	75P	9.75a ± 0.20	0.94a ± 0.20	0.04b ± 0.002	7d ± 1.18	87b ± 12.3	19

Enzyme activities of total acid phosphatase (APases, µmol PNPP min⁻¹ g⁻¹ nodule fresh weight, i.e., nfw), phosphoenol pyruvate phosphatase (PEPase, µmol NADH min⁻¹ g⁻¹ nfw), trehalose 6P phosphatase (TPP, µmol P min⁻¹ g⁻¹ nfw), peroxidase (µmol H₂O₂ min⁻¹ g⁻¹ nfw), N₂ fixation and efficiency in use of rhizobial symbiosis (EURS) defined as biomass’ ratio of shoot/nodule of common bean RILs 115 and 147 grown under P sufficient (250P) vs. deficient P (75P) supply. Data are means ± SE of six replicates harvested at 42 d after transplantation.^15,16

Figure 3. Relationship between O₂ permeability, P concentration (A) and enzyme activity of PEPase (B), TPP (C) and peroxidase (D) in nodules of common bean RILs 115 and 147 inoculated with R. tropici CIAT899 and grown under sufficient (empty circles) vs. deficient (filled circles) P supply. Data are individual values of ten replicates harvested at 42 d after transplanting. Asterisks denote significant difference at p < 0.05.^5,15,16

Most APases may hydrolyze Pi from a broad spectrum of Pi mono-esters. However, they may have different functions. Thus, overexpression of GmPAP3 exhibiting alkaline peroxidase activity increased tolerance to oxidative damage under salinity.^31,32 Therefore, basing on the large specificity of APases,³³ the PEPase activation in cortex of P-deficient nodules would lead to the production of pyruvate which plays an essential role in limiting production of active oxygen species through a non-enzymatic oxidative decarboxylation by hydrogen peroxide (H₂O₂).^33,34 Similarly PEPase and TPP would not only to be involved in intra-nodular Pi remobilization, but also in alleviating oxidative stress in senescent cells as well as the cortical cells, (Fig. 4) that are considered the main target of H₂O₂ accumulation.³⁵ This is in agreement with nodule enzyme peroxidase stimulation and especially the elevated levels of APX transcript in nodule cortex of alfalfa, chickpea and common bean.^10,16,22

Figure 4. Conceptual schema of the relationships between nodule P content, phosphatases expression, nodule respiration and oxidative stress in N₂-fixing nodules under P-deficiency. The filled (green and red) arrows indicate direct relationships of nodule P with either nodule O₂ permeability or nodule oxidative stress. High nodule P stimulates nodulation, N content and nodule respiration.⁵ The latter, with carbon supply regulation and N feedback, contribute to regulating SNF.⁴³ Moreover, oxidative stress is not induced only under P-deficiency,^34,44 but also in response to high P concentration in nodules where respiration is expected to be not associated to SNF.^5-16 This is evidenced by the absence of correlation between the efficiency in use of rhizobial symbiosis and SNF.⁵ The discontinued green arrows indicate plausible speculative relationships between oxidative stress, APases, peroxidase and nodule respiration linked to SNF. P-deficiency increases PEPase activity and thus, liberation of Pi and pyruvate.¹⁵ The latter might act as a non-enzymatic antioxidant to limit production of active oxygen species during oxidative stress. Likewise, peroxidase should play the same role, inducing along with pyruvate, an increased respiration unlinked to SNF.^16,34 Additionally, activation of TPP¹⁶ and phytase³ under P-deficiency contributes to the release of Pi, but satisfaction of the demand in trehalose rather than phytate is likely to play a role in nodule O₂ permeability regulation. This is still to be further verified especially whether expression of TPP in nodule cortex is associated with abundance of trehalose in this zone.

APases Transcripts in Nodule Cortex as Related to Nodule Respiration

The positive correlations of nodule O₂ permeability with PEPase and TPP activities in nodules and the increase of the EURS under P-deficiency (Fig. 3A–C) are new evidences claiming that nodule respiration might be linked to P status and APases activity. Thus, abundance of PEPase and TPP within nodules, especially nodule cortex, are likely be involved to higher nodule demands for ATP generation. This energy couples to nodule O₂ respiration since for each N₂ reduced at least 16 ATP is consumed.²⁶ In addition, transcriptional profiles of PEPase and TPP in nodule cortex may link with the O₂ flux²³ that is postulated to be osmo-regulated through this tissue.⁹ This is consistent with the correlation between the P content of nodules and the nodule permeability to O₂ diffusion (Fig. 4).^4-9 Moreover, both the activation of TPP especially in infected cells and the appearance of trehalose in nodules at the onset of N₂ fixation,³⁶ substantiate a physiological role of TPP in regulating nodule O₂ diffusion. Our results report that bacteroids in infected cells exhibit high TPP and supports the hypothesis that trehalose may be respired within bean nodules in relation with P status and APases activities. In this context, we suggest that trehalose could be involved as an osmoticum as well as the occlusion of intercellular spaces of middle-cortex and inner-cortex cells, nodule zones that were proposed as a physical barrier mechanism for osmo-regulation of the nodule permeability.^9,37-39

Furthermore, we suggest that increase in nodule P content results in an increase in nodulation, N₂ fixation and nodule O₂ permeability (Fig. 4).⁵ This suggestion is confirmed by the existence, under P-sufficiency, of positive correlations between nodule O₂ permeability and both P and peroxidase activity (Fig. 3A–D). Alternative respiratory component would be induced by high H₂O₂ content in N₂-fixing nodules as this is likely consistent with the enhanced respiratory activity since pericycle and companion cells of the vascular traces are rich of mitochondria which generate large amount of H₂O₂.²² these statements are in concordance with the enhanced APX in the parenchyma immediately surrounding vascular traces of alfalfa²² and supports the suggestion of Denison and Kinraide³⁸ that the diffusion barrier contains a respiration component that reflects respiratory activity of nodule parenchyma and vascular traces.

Relation with Oxidative Stress in N₂-Fixing Nodules: Specific Expression of APX

Unlike PEPase and TPP outer cortex-specific localization, APX gene was preferentially expressed in the inner cortex (Fig. 2G and H) of both RILs especially under P-sufficiency (Fig. 1I and K). At the physiological level, this high level of APX transcript in the inner cortex zone emphasized with a positive correlation (r = 0.64*) between peroxidase activity and nodule O₂ permeability (Figs. 3D,  2G and H). However under P-sufficiency, nodule respiration is not directly associated with SNF (Fig. 4). This is substantiated with the absence of any relation with the EURS for plant growth, as compared with that under P-deficiency. It suggests that high P in nodules may produce an additional constraint (Fig. 4), leading to an alternative respiratory pathway unrelated to the N₂ fixation.^5-16 Such respiratory pathway is likely related to a generation of intracellular reactive oxygen species (ROS) which is associated with high leakage of the electron transport chain during activation of the antioxidants reactions such as mitochondrial Cytochrome c oxidase and malate dehydrogenase in nodule cortex.¹¹ Interestingly, in Arabisdopsis thaliana, upregulation of ROS responsive genes has been linked with trehalose concentration.^40,41 Moreover, upregulation of a large number of antioxidant genes such as sAPX (L-ascorbate peroxidase, stromal, AT4G08390), PER22 (Peroxidase 22, AT2G38380) and PRO1 (the mitochondrial proline oxidase stress responsive gene, AT3G30775) was associated with the overexpression of TPP in Arabisdopsis thaliana (AtTPP, putative, AT4G12430).⁴⁰

Furthermore, the elevated level of APX transcript in the inner cortex compared with the outer cortex opens up the possibility of inner cortex’s role in coping with oxidative stress in N₂-fixing nodules as recently reported that the gradual increase of pea-nodule respiration from nodule meristem to the late symbiotic zone is correlated with a localization of succinate dehydrogenase.⁴² We suggest that, the inner cortex-localized APX transcript could protect nodule’s internal tissues under P-deficiency but also under P-sufficiency if high P accumulation in nodules may become toxic or induce an additional oxidative stress. High level of H₂O₂ concentration may, therefore, be induced and stimulate the abundance of APX transcript we observed within the nodule vascular traces. Additionally, the sub-cellular localization of APX transcript in the periphery of the infected cells (Fig. 2G and H) may be likely linked to peroxisomes that were specifically located at the peripheral infected cells of the pea nodules.³⁵ Thus, the peripheral infected cells-localized APX may constitute a systemic response against peroxisome activities since these organelles, besides mitochondria, are the major sites for ROS productions.

Conclusion

Overall, this revue has revealed that N₂-fixing nodules have the potential to activate a large number of APases genes for their tolerance to P-deficiency. The elevated APases transcripts and activities may likely coincide with high nodule P requirement depletion of the vacuolar Pi pool and points to an intracellular mechanism of Pi utilization. The localized-nodule cortex APases transcripts under P-deficiency predicts not only an optimal intracellular Pi scavenging ability but also opens up a challenge to understand whether such sub-cellular localization is related to other biochemical functions during N₂ fixation in bean nodules. Moreover, despites the above discussed roles of APases against P limitation, their involvement in coping with multiples stressful conditions, such as oxidative stress, remains very expected in legume nodules. This seems to be highlighted through the simultaneously differential expression of APases and APX in the nodule cortex. They may alleviate oxidative stress and very likely involved in nodule permeability to O₂ diffusion. However, despites all of this knowledge, additional studies are needed to deeply understand how do these tissues-specific expressions adjust N₂ fixation throughout the regulation of nodule gas diffusion.

Avonce N, Leyman B, Mascorro-Gallardo JO, Van Dijck P, Thevelein JM, Iturriaga G. The Arabidopsis trehalose-6-P synthase AtTPS1 gene is a regulator of glucose, abscisic acid, and stress signaling. Plant Physiol. 2004;136:3649–59. doi: 10.1104/pp.104.052084.

Streeter JG. Carbohydrates in soybean nodules. II. Distribution of compounds in seedlings during the onset of nitrogen fixation. Plant Physiol. 1980;66:471–6. doi: 10.1104/pp.66.3.471.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.