Jean Jacques Drevon

Phosphatase and phytase activities in nodules of common bean genotypes at different levels of phosphorus supply

Plants grown at limited P supply can increase the activity of phosphatases in roots to hydrolyse organic-P compounds in the soil thus improving plant P acquisition, but little information is available about the role of these enzymes for internal plant metabolism at limited-P conditions. This work intended to measure the activities of acid phosphatases and phytases in nodules of common bean (Phaseolus vulgaris) genotypes at different levels of P supply. The experiment was carried out in a 5u2009×u20095 factorial design with four replicates, comprising five bean genotypes and five P levels (20, 40, 80, 160 and 320xa0μmol P plant−1 week−1) in nutrient solution. Root seedlings were inoculated with Rhizobium tropici and plants were grown in 1-l bottles. Nodule samples were detached from 39-day-old plants and enzyme activities were determined in crude extracts. Plants were harvested at the stage of pod setting. Polynomial models fitted to data indicated maximal values at the level of 194xa0μmol P for shoot mass, at 206xa0μmol P for nodule mass and at 221xa0μmol P for shoot N. Whereas shoot mass was 1.7 times lower at 20 than at 160xa0μmol P, nodule mass was 7.5 times lower. Concentration of P in nodules increased from 40 to 320xa0μmol P but remained stable between 20 and 40xa0μmol P, suggesting a minimal threshold concentration of 3xa0mg P g−1 for nodule growth. Activities of phosphatases and phytases in nodules decreased strongly as P supply was raised from 20 to 80xa0μmol P, remaining almost stable at higher P levels. Phosphatase activity ranged from 1,154 to 406xa0nmol min−1 g−1 (nodule fresh mass) from 20 to 80xa0μmol P respectively, while the phytase activity ranged from 55 to 14xa0nmol min−1 g−1 from 20 to 80xa0μmol P. Bean genotypes differed in shoot and nodule mass at the levels of 80 and 160xa0μmol P, whilst they differed in nodule enzyme activities only at the lowest P level, the relationship between nodule enzyme activities and growth of different bean genotypes was not evident. It is concluded that bean plants at P-deficient conditions increase the activities of phosphatases and phytases in nodules. This may constitute an adaptive mechanism for N2-fixing legumes to tolerate P deficiency, by increasing the utilisation of the scarce P within the nodules.

Variation in Nitrate Tolerance of Nitrogen Fixation in Soybean (Glycine max) — Bradyrhizobium Symbiosis

Summary The long-term inhibition of symbiotic nitrogen fixation by nitrate was compared in five cultivars of soybean (Glycine max L.) screened for tolerance to nitrate, with cultivar Kingsoy as non-tolerant control. The plants were inoculated with the same strain of Bradyrhizobium japonicum and grown hydroponically in the greenhouse. Nodule mass, nitrate reductase activity, nitrogenase activity (C2H2 reduction) and its response to oxygen partial pressure were determined in the presence or absence of nitrate. Nodulation and acetylene reduction rates were inhibited by nitrate in all plants but there were significant differences among cultivars. The cultivars maintaining nodule activity in the presence of nitrate had low induction of nodule cytosolic nitrate reductase. Moreover, there was a correlation between cytosolic NR induction and NO3- inhibition, which suggests that nitrate assimilation in nodules is involved in the N2 fixation inhibition. Comparisons among cultivars showed that those having large increases in ARA in response to O2 also had the highest capacity to fix N2 in the presence of nitrate. A superior cultivar for both of these characters was cv. Tielingbaime. These results suggest a decrease in the energetic supply of bacteroids in the presence of nitrate.

Effect of phosphorus deficiency on acid phosphatase and phytase activities in common bean (Phaseolus vulgaris L.) under symbiotic nitrogen fixation

Changes in growth, symbiotic nitrogen fixation (SNF), acid phosphatase (ACP), and phytase activities to phosphorus availability (15 and 60 μmol KH2PO4 plant−1 week−1) were compared in two recombinant lines (115 and 147) of common bean. Plant growth, nodulation and SNF were genotype and P level-dependent. 147 was more affected by P shortage (15 μmol P) than 115. Four ACP types were revealed in the nodules of both lines, ACP1 exhibiting a higher specific activity under P shortage as compared to the 60 μmol P treatment, especially in 115. A single phytase was revealed for the nodules of both lines and was significantly enhanced by P deficiency. Three ACP types were found in roots and leaves, showing increasing activity under P deficiency, especially in 115. Regardless of P supply, leaf ACP specific activity was higher than that of nodules and roots in the both lines. Interestingly, phosphorus use efficiency for N2 fixation significantly correlated to nodule ACP activity under P shortage in the both lines. The relatively better performance of 115 as compared to 147 under P deficiency could be partly ascribed to the ability of 115 to maintain higher ACP activity. This enzyme might be involved in the remobilization of the plant Pi and its utilization for SNF.

Root Proliferation, Proton Efflux, and Acid Phosphatase Activity in Common Bean (Phaseolus vulgaris) Under Phosphorus Shortage

The impact of phosphorus (P) availability on root proliferation, proton efflux, and acid phosphatase activities in roots and leaves was investigated in two lines of common bean (Phaseolus vulgaris): BAT 477 and CocoT. Phosphorus was supplied as KH2PO4 at 0 and 60u2009µmol per plant (0P and 60P, respectively). Under P shortage, the plant growth was more restricted in CocoT than in BAT 477, shoots being more affected than roots. The root area increased significantly at 0P in both lines. Up to 1xa0week following P shortage, the proton efflux increased in both lines despite a higher extent in BAT 477 as compared to CocoT. Root acid phosphatase activity was significantly higher under P limitation in the both lines, this trend being more pronounced in BAT 477 than in CocoT. This was also true for the leaf acid phosphatase. Regardless of the bean line, higher values were recorded for the old leaves as compared to the young ones for this parameter. Interestingly, a significant correlation between Pi content in old leaves and their acid phosphatase activity was found in P-lacking (0P) plants of the both bean lines, suggesting that acid phosphatase may contribute to increase the phosphorus use efficiency in bean through the P remobilization from the old leaves. As a whole, our results highlight the significance of the root H+ extrusion and the acid phosphatase activity rather than the root proliferation in the relative tolerance of BAT 477 to severe P deficiency.

Is genotypic variation of H+ efflux under P deficiency linked with nodulated-root respiration of N2 – Fixing common-bean (Phaseolus vulgaris L.)?

To examine genotypic variation of common bean in growth, phosphorus uptake, nodulated-root proton release, and nodule gas permeability, seven common bean recombinant inbred lines (RIL) from the cross of BAT477×DOR364 were inoculated by Rhizobium tropici CIAT 899 and grown in hydroaeroponic culture under glasshouse conditions. A positive correlation was observed between shoot and nodule biomass for most of the studied RILs. Under P deficiency, the tolerant common bean RILs acidified more of their rhizosphere than the sensitive ones. The proton release of the RILs 147, 124, 104, 75 and RIL34 was positively correlated with nodule O₂ permeability. We conclude that nodulated common bean plants release a substantial amount of H⁺ into the rhizosphere that is linked to the symbiotic N₂ fixation. It depends upon the nodule permeability to O₂ diffusion, and varies with genotype.

The nodule conductance to O2 diffusion increases with phytase activity in N2-fixing Phaseolus vulgaris L

To understand the relationship between phosphorus use efficiency (PUE) and respiration for symbiotic nitrogen fixation (SNF) in legume nodules, six recombinant inbred lines of common bean (RIL Phaseolus vulgaris L.), contrasting in PUE for SNF, were inoculated with Rhizobium tropici CIAT899, and grown under hydroaeroponic culture with sufficient versus deficient P supply (250 versus 75 μmol P plant(-1) week(-1)). At the flowering stage, the biomass of plants and phytase activity in nodules were analyzed after measuring O2 uptake by nodulated roots. Our results show that the P-deficiency significantly increased the phytase activity in nodules of all RILs though with highest extent for RILs 147, 29 and 83 (ca 45%). This increase in phytase activity was associated with an increase in nodule respiration (ca 22%) and in use of the rhizobial symbiosis (ca 21%). A significant correlation was found under P-deficiency between nodule O2 permeability and phytase activity in nodules for RILs 104, 34 and 115. This observation is to our knowledge the first description of a correlation between O2 permeability and phytase activity of a legume nodule. It is concluded that the variation of phytase activity in nodules can increase the internal utilization of P and might be involved in the regulation of nodule permeability for the respiration linked with SNF and the adaptation to P-deficiency.

Discrimination against 15N among recombinant inbred lines of Phaseolus vulgaris L. contrasting in phosphorus use efficiency for nitrogen fixation

Although isotopic discrimination processes during nitrogen (N) transformations influence the outcome of (15)N based quantification of N2 fixation in legumes, little attention has been given to the effects of genotypic variability and environmental constraints such as phosphorus (P) deficiency, on discrimination against (15)N during N2 fixation. In this study, six Phaseolus vulgaris recombinant inbred lines (RILs), i.e. RILs 115, 104, 34 (P deficiency tolerant) and 147, 83, 70 (P deficiency sensitive), were inoculated with Rhizobium tropici CIAT899, and hydroaeroponically grown with P-sufficient (250 μmol P plant(-1) week(-1)) versus P-deficient (75 μmol P plant(-1) week(-1)) supply. Two harvests were done at 15 (before nodule functioning) and 42 (flowering stage) days after transplanting. Nodulation, plant biomass, P and N contents, and the ratios of (15)N over total N content ((15)N/Nt) for shoots, roots and nodules were determined. The results showed lower (15)N/Nt in shoots than in roots, both being much lower than in nodules. P deficiency caused a larger decrease in (15)N/Nt in shoots (-0.18%) than in nodules (-0.11%) for all of the genotypes, and the decrease in shoots was greatest for RILs 34 (-0.33%) and 104 (-0.25%). Nodule (15)N/Nt was significantly related to both the quantity of N2 fixed (R(2)=0.96***) and the P content of nodules (R(2)=0.66*). We conclude that the discrimination against (15)N in the legume N2-fixing symbiosis of common bean with R. tropici CIAT899 is affected by P nutrition and plant genotype, and that the (15)N/Nt in nodules may be used to screen for genotypic variation in P use efficiency for N2 fixation.

Expression of a phosphate-starvation inducible fructose-1,6-bisphosphatase gene in common bean nodules correlates with phosphorus use efficiency

While increased P-hydrolysing acid phosphatases (APase) activity in bean nodules is well documented under phosphorus (P) limitation, gene expression and subcellular localization patterns within the N2-fixing nodule tissues are poorly understood. The aim of this research was to track the enzyme activity along with the intra-nodular localization of fructose-1,6-bisphosphatase (FBPase), and its contribution to P use efficiency (PUE) under symbiotic nitrogen fixation (SNF) in Phaseolus vulgaris. The FBPase transcript were localized in situ using RT-PCR and the protein activity was measured in nodules of two contrasting recombinant inbred lines (RILs) of P. vulgaris, namely RILs 115 (P-efficient) and 147 (P-inefficient), that were grown under sufficient versus deficient P supply. Under P-deficiency, higher FBPase transcript fluorescence was found in the inner cortex as compared to the infected zone of RIL115. In addition, both the specific FBPase and total APase enzyme activities significantly increased in both RILs, but to a more significant extent in RIL115 as compared to RIL147. Furthermore, the increased FBPase activity in nodules of RIL115 positively correlated with higher use efficiency of both the rhizobial symbiosis (23%) and P for SNF (14% calculated as the ratio of N2 fixed per nodule total P content). It is concluded that the abundant tissue-specific localized FBPase transcript along with induced enzymatic activity provides evidence of a specific tolerance mechanism where N2-fixing nodules overexpress under P-deficiency conditions. Such a mechanism would maximise the intra-nodular inorganic P fraction necessary to compensate for large amount of P needed during the SNF process.

Role of acid phosphatase in the tolerance of the rhizobial symbiosis with legumes to phosphorus deficiency

Phosphorus (P) deficiency initiates a myriad of transcriptional, biochemical and physiological responses stimulating either the root’s extracellular abilities to acquire soil P in the rhizosphere or optimize its intracellular use efficiency and allocation through all plant organs. Enhancing activity of acid phosphatase (APase) to acquire and remobilize Pi from organic P compounds is one important strategy for improving plant P nutrition. The release of APase to the rhizosphere is a typical and almost universal P-starvation response in higher plants. However, relatively little is known about the functions of intracellular APase in legume nodules. The aim of this review was to track the enzyme activity along with the intra-nodular localization of APase, and its contribution in the rhizobial symbiosis tolerance to P-deficiency. Our findings have revealed that expression of APase and phytases genes and activities of the corresponding enzymes were positively correlated with increases both of the P use efficiency for N2 fixation and nodule O2 permeability in the rhizobial symbiosis with legumes. The induced enzyme activity and the marked transcripts localization of APase and phytase in nodule cortex would control nodule respiration and contribute to adaptation of nodulated legumes to low-P availability. Thus, the increase of APase and phytase activities in legume nodules supports a physiological role of these enzymes in the regulation of nitrogenase activity in connection with the nodule-P status, and opens up a new scenario for a better understanding of the regulation of N2 fixation in legumes.

Genotypic Variation in Nodule Iron Content of Common Bean (Phaseolus Vulgaris L.) in Response to Phosphorus Deficiency

Common bean (Phaseolus vulgaris L.) can supply all of the iron that humans require for metabolism. Also, it fixes atmospheric nitrogen (N2) in symbiosis with rhizobia. In order to analyze the relation between phosphorus (P) and iron (Fe) elements in nodules and their roles for the plant N2-dependent growth, six common bean recombinant inbred lines (RIL) of the cross of BAT477 and DOR364 were inoculated with Rhizobium tropici CIAT 899 (originating from International Center of Tropical Agriculture, Colombia) and grown with sufficiency versus deficiency P supply in hydroaeroponic culture. Under P deficiency, the Fe content in nodules decreased in all studied genotypes and was significantly the highest for RIL 34. The nodule contents of Fe and P were significantly correlated under P deficiency. It is concluded that the regressions of nodule Fe content as a function of P content in nodules, roots and shoots, depend upon P supply and genotype.