Phaseolus vulgaris (Smith, 1977)

López-Gómez, Miguel, Cobos-Porras, Libertad, Hidalgo-Castellanos, Javier & Lluch, Carmen, 2014, Occurrence of polyamines in root nodules of Phaseolus vulgaris in symbiosis with Rhizobium tropici in response to salt stress, Phytochemistry 107, pp. 32-41 : 33-37

2.1. Occurrence of polyamines in nodules of P. vulgaris

Polyamines (PAs) levels in leaves, roots and nodules of P. vulgaris (inoculated with Rizobium tropici ) were determined by HPLC as described in the material and methods section. In Fig. 1 is shown a typical elution chromatogram corresponding to the three organs mentioned above and as can be observed, the number of PAs detected was higher in nodules compared to leaves and roots. In the chromatogram corresponding to nodules, four peaks between Spd and Spm were detected and three of them could not be assigned to any of the standard PAs used. These peaks were only observed in the nodular tissue while in leaves and roots, only common PAs such as Put, Spd and Spm were found. The peak posterior to Spd corresponded to the Homspd standard and seems to be the most abundant polyamine in nodules, as confirmed by the quantitative assay shown in Table 1 , which has been previously observed in nodules of P. vulgaris ( Smith, 1977) as well as in others legumes such as M. sativa ( López-Gómez et al., 2014) or V. angularis ( Fujihara et al., 1995). On the contrary, common PAs such as Put and Spm were majority in leaves, compared to roots and nodules, while cadaverine (Cad) was minority in all plant organs, with no detectable levels in leaves ( Table 1 ). The major presence of Homspd in the nodular tissue has been attributed to its bacteroidal origin ( Fujihara, 2009). In this regard, the distribution of PAs in different nodular fractions were analyzed and compared with the free-living bacteria in order to determine their plant or bacterial origin in the nodular tissue ( Table 2 ). Homospd was the most abundant polyamine in the free-living bacteria and in bacteroids, confirming the bacteroidal origin of this polyamine in nodules. On the contrary, Cad was only detected at low concentrations in the plant cytosolic fraction ( Table 2 ) which suggest its plant origin.

To identify the standard unavailable compounds ( X, Y, Z), the dansylated nodule extracts were analyzed by UPLC-MS and the molecular formula was established by high accuracy quasi-molecular ion such as [M+H] + within a mass error of 5 ppm. Then the most rational molecular formula was searched in chemical databases (www.chemspider.com). The mass fragmentation pattern of the compound X showed ion at m / z 873 ( Fig. 1 S ) with a mass error of 0.3 ppm which matched with dansylated 4-ABcad. However, no coincidences with PAs were found for the peaks Y and Z. The occurrence of 4-ABcad has only been described in root nodules of V. angularis ( Fujihara et al., 1995) and has been considered to be an unusual polyamine occurring in rhizobial cells under specific environmental conditions. Indeed, the analysis of different nodular fractions ( Table 2 ) reveals a higher relative level of 4-ABcad in bacteroids than in the cytosol, while this polyamine was not detected in the free living bacteria, which suggest that this compound is synthesized by the bacteroids to counteract the hyper-osmotic conditions found into the nodule. It is also possible that within the nodule bacteroids are supplied with Cad, precursor for the synthesis of 4-ABcad, as described by Fujihara et al. (1995) in Bradyrhizobium japonicum bacteroids isolated from V. angularis plants. This possibility is supported by the low levels of Cad detected in the nodular tissue ( Table 1 ) that would indicate its consumption by the bacteroids to produce 4-ABcad.

2.2. Response of nodular polyamines levels to salt stress conditions

In order to study the implication of PAs in the nodular response to salt stress, P. vulgaris plants were subjected to 100 mM NaCl treatments for 3 and 7 days before harvest. The quantification of free PAs shown in Fig. 2 reveals a reduction in response to salinity of about 20% for all of them, except for Cad which augmented 20% under salt stress. This may be due to the fact that Cad, differing from PAs of the Put family (Spd and Spm), is the product of another metabolic pathway starting from aspartate and formed by lysine decarboxylation (Kuznetsov et al., 2007), and would not be in competition with Put derived from glutamate. Therefore, Cad accumulation under salt stress conditions compensates the decrease in the content of Put-family PAs, as previously observed by Kuznetsov et al. (2007). The Homspd reduction observed in nodules is consistent with previous studies where rhizobial strains subjected to salt stress showed a reduction in the cellular Homspd levels ( Fujihara and Yoneyama, 1993).

The nodule specific polyamine 4-ABcad displayed a similar response to salt stress that Cad with an induction in its concentration of about 25% and 70% after 3 and 7 days, respectively ( Fig. 3 ). This result would support the supply of Cad from the plant to the bacteroid for the synthesis of 4-ABcad as previously reported by Fujihara et al. (1995). In addition, the fact that 4-ABcad is considered to be an unusual polyamine occurring in rhizobial cells under specific environments, would justify the occurrence of this compound in the nodular tissue. Moreover, the increment of the concentration of this polyamine in nodules under salt stress would support its participation in the anti stress response induced by the bacteroids in response to hyper-osmotic conditions provoked by salinity.

The analysis of soluble conjugated PAs in nodules reveals an increment of theirs concentration under salt stress conditions ( Fig. 4 ), with triple and double Put and Spd levels, respectively. Therefore, the conversion of free Put and Spd to soluble conjugated forms could be behind the reduction of the free forms under salt stress conditions. On the contrary Cad soluble conjugated form displayed the same pattern that the free form and no conjugated forms of Spm and Homspd were detected. Soluble conjugated PAs has been postulated to be important for the control of intracellular polyamine concentrations ( Bagni and Tassoni, 2001), interaction with compounds of the cell wall ( Lam et al., 1992) and also could serve for polyamine transport or even as substrate for peroxidases ( Havelamge et al., 1996). It is not excluded that conjugates could represent a storage polyamine pool used under extreme conditions ( Kuznetsov and Shevyakova, 2007). Considering the sum of free and soluble conjugated PAs in the nodule, the amount of Put, Spd and Cad augmented under salt stress conditions which would be in agreement with previous reports in which an increase of Put and Spd levels in response to abiotic stresses, including salinity, has been postulated ( Jiménez-Bremont et al., 2007; Hernandez-Lucero et al., 2008; Alcazar et al., 2010).

2.3. Differential expression of polyamines biosynthetic genes in different plant organTranscript levels of Adc, Odc, Spds, Spms and Samdc genes were analysed by RT-PCR in different bean tissues ( Fig. 5 ). Expression level of Adc gene in the nodular tissue was very low compared to leaves and roots. However, Odc gene transcripts were mainly detected in the nodular tissue which suggests that ODC activity is essential for the PAs synthesis in this organ. Adc transcript accumulation has been previously reported to be tissue-specific and developmentally regulated with highly expression levels in young developing tissues such as meristems, young leaflets and flower buds ( Perez-Amador et al., 1995). The nodules used in our study were determined and fully developed without a persistent meristem which would have contributed to the low expression level of the Adc gene. Odc transcription has been associated to cell growth and proliferation in roots compared to leaves or stems were the cell division is limited ( Kwak and Lee, 2001). The expression of Odc in the nodular tissue could be related to the cell proliferation experimented by the root cortex cells to generate the nodule and similarly to a previous work in P. vulgaris ( Jimenez-Bremont et al., 2006) , no transcription were detected in leaves. The Spds transcripts were present at higher level in the nodular tissue with intermediate and low expression level in roots and leaves, respectively. This result would be in agreement with a more active polyamine biosynthetic metabolism in nodules compared with other plant organs and is consistent with the higher polyamine concentration detected in nodules compared to shoot and roots ( Fig. 1 ) ( Fujihara et al., 1994). In contrast, in Arabidopsis thaliana Spds transcripts were expressed in all plant organs at similar level ( Hanzawa et al., 2002). Samdc transcripts were present in leaves and nodules at similar levels while in roots the expression was higher. In this regard, it has been reported that Samdc transcription in pea plants varied among tissues and in response to the developmental conditions ( Marco and Carrasco, 2002). Higher accumulation of Samdc transcripts have been described in young tissues and organs where active cell divisions occur whereas its expression levels were reduced in mature and non-dividing tissues ( Mad Arif et al., 1994). Finally, Spms expression was rather low in all organs tested which is consistent with the low levels of Spm detected in nodules. In general, these results indicate that the expression levels of the genes involved in the biosynthetic pathway of PAs are tissue-specific an do not follow a common pattern in different plant species which is probably the result of different regulation mechanism as stated by Jimenez-Bremont et al. (2006).

2.4. Effect of salt stress on the expression of genes involved in polyamine biosynthesis in root nodules

The effect of salt stress on the expression of the genes involved in the synthesis of PAs in nodules was analyzed as shown in Fig. 6 . Adc and Odc genes, involved in the synthesis of Put, were up-regu-lated by salt stress after 3 days while only Adc was up-regulated after 7 days. Indeed, the induction level of Odc by salinity after 3 days was higher compared to Adc, which together with the detection of the Odc transcripts mainly in the nodule ( Fig. 5 ), suggest that this gene might play an important role in the polyamine adjustment during the early response to salt stress in nodules. On the contrary, in most of the studies concerning salt-stress in different plant species, PAs response has been assumed to rely mainly on Adc activation ( Bouchereau et al., 1999). Similarly to Adc and Odc, Samdc and Spds genes showed 3 and 2-fold up-regulation by 100 mM NaCl after 3 and 7 days, respectively which is consistent with the induction observed in the Spd, level ( Fig. 4 ).

2.5. Enzyme activities ADC and ODC

In order to analyze the existence of post-transcriptional mechanisms involved in the regulation of ADC and ODC enzyme activities, responsible for the synthesis of Put and its derived PAs Spd and Spm, both enzymes were assayed as shown in Fig. 7 . ADC activity did not show response to salinity after 3 days of treatment and a slight inhibition of 10% was detected after 7 days. Interestingly, ODC activity displayed a 10% induction after 3 days and a 17% reduction at 7 days by salinity which together with the higher induction of the expression of the Odc gene 3 days after treatment would support a main role for this metabolic pathway in the early response to salinity in nodules. However, ADC and ODC enzyme activities did not correlate with the free PAs levels after 3 days ( Fig. 2 ) which would support the transformation of free Put and Spd in its respective soluble conjugated forms as the mechanism responsible for the free PAs decline under salinity.

2.6. Nitrogen fixation under salt stress conditions

In order to evaluate the nodule functioning under salt stress conditions, several parameters related to the nitrogenase activity were determined as shown in Fig. 8 . Nitrogenase activity was strongly inhibited by salinity in a time dependent manner with more than 50% reduction of the nitrogen fixation rate ( NFR) after 3 days of salt treatment and 90% reduction after 7 days. Nitrogenase sensitivity to salinity has been previously described in P. vulgaris ( Khadri et al., 2006) as well as in other legumes such as M. sativa ( López-Gómez et al., 2014) and Pisum sativum ( Cordovilla et al., 1999) . This negative effect of salt stress on the nitrogen fixation process has been attributed to its negative effect on the root infection and nodule organogenesis, the efficiency of the fully formed nodules or the differentiation of the bacteroids into active nitrogen-fixing ( Zahran, 1999). In our experimental conditions, the nitrogenase inhibition cannot be associated to the nodule organogenesis since the salt treatment was imposed once the symbiosis was established and the nodules were active. In this regard, our data only showed about 20% inhibition of the nodule biomass ( NFW) by the salinity, which indicate that the nitrogenase activity reduction was due to the negative effect of salt on the nodular metabolism rather than to the nodule formation. The drastic reduction of the nitrogenase activity would be also a limiting factor for the synthesis of free PAs in nodules under salt stress conditions, as previously observed in M. sativa ( López-Gómez et al., 2014) .

2.7. Plant growth pattern was modified by the salinity

Concomitantly to the negative effect of salt stress on the nitrogen fixation, a decline of the plant biomass production was detected ( Fig. 9 ) with 17% reduction of the plant dry weight ( PDW) 7 days after salt treatment initiation. This plant growth inhibition may be related to the nitrogen limitation since, in these plants, nitrogen supply depends entirely on the nitrogenase activity. Depressive effect of NaCl on plant growth has been previously reported in several legumes such as pea, faba-bean, soy-bean ( Delgado et al., 1994) and chick-pea ( Tejera et al., 2006). However, in P. vulgaris the plant growth inhibition did not affect equally to shoot and roots, as shown by the increment of around 55% of the root to shoot ratio ( RSR) at 7 days. Similar results have been detected in previous experiments with P. vulgaris ( Khadri et al., 2006) and in other legumes such as Cicer arietinum ( Soussi et al., 1998) , being considered this behaviour profitable since it could improve plant water status by increasing the root requirements to explore a higher soil volume.

3. Concluding remarks

PAs have been shown to be an important part of plant stress responses based on their ability to act as antioxidants, stabilizers of nucleic acids and biomembranes, and also on their effect as compatible osmolytes. Root nodules of legumes, of great importance for nitrogen supply through symbiotic nitrogen fixation, constitutes a complex organ for the study of the adaptation mechanisms to salt stress by the coexistence of the rhizobial and plant metabolic responses. In this context, P. vulgaris root nodules were found to contain a higher variety of PAs than roots and leaves, most of them more abundant in the bacteroids due to their bacterial origin. Among the bacteroidal PAs detected, 4-ABcad has been identified in this work together to Homspd as the most abundant PAs in nodules. These PAs are considered to be uncommon because of their limited distribution in nature and the absence of the diaminobutyl group, characteristic of the common PAs Put, Spd and Spm. Under salt stress conditions, the concentration of soluble conjugated Put and Spd increased in detriment of the free forms which imply that soluble conjugated PAs could play a role in the response to salt stress in nodules. In addition, the concentration of free Cad and 4-ABcad increased by the salinity which suggest the involvement of the bacteroidal PAs metabolism in the response of P. vulgaris nodules to salinity. This result indicates that the alteration of the bacterial metabolism towards the production of uncommon polyamines such as 4-ABcad would be one of the mechanisms to tolerate salt stress in the rhizobia-legume symbiosis.

Figures

Fig. 1. Elution profile of polyamines in P. vulgaris nodules (A), roots (B) and leaves (C) standard polyamines (D). Polyamines were extracted and derivatized with dansyl chloride before subjecting to HPLC analysis.1,7-diaminoheptane was used as a internal standard (IS). Peaks X, Y and Z in panel A denotes unknown compounds. Peaks in panel D: Dap; diaminopropane, Put; putrescine, Cad; cadaverine, Spd; spermidine, Homspd; homospermidine, Spm; spermine.

Fig. 2. Free putrescine (Put), spermidine (Spd), spermine (Spm), homospermidine (Homspd) and cadaverine (Cad) content in nodules of P. vulgaris inoculated with R. tropici CIAT 899 treated with 0 and 100 mM NaCl for 3 and 7 days. Date are mean ± SE (n = 3). Mean values followed by the same letter do not differ (P 6 0.05) using LSD test.

Fig. 3. Free 4-aminobutylcadaverine (4-ABcad) content expressed in relative values (%) in nodules of P. vulgaris inoculated with R. tropici CIAT 899 treated with 0 and 100 mM NaCl for 3 and 7 days. Relative values expressed as % are mean ± SE (n = 3). Mean values followed by the same letter do not differ (P 6 0.05) using LSD test.

Fig. 4. Soluble conjugated putrescine (Putc), spermidine (Spdc) and cadaverine (Cadc) content in nodules of P. vulgaris inoculated with R. tropici CIAT 899 treated with 0 and 100 mM NaCl for 3 and 7 days. Date are mean ± SE (n = 3). Mean values followed by the same letter do not differ (P 6 0.05) using LSD test.

Fig. 5. Expression analyses of arginine decarboxylase (Adc), ornithine decarboxylase (Odc), spermidine synthase (Spds), spermine synthase (Spms) and S-adenosylmethionine decarboxylase (Samdc) in leaves (L), roots (R) and nodules (N) of P. vulgaris inoculated with R. tropici CIAT899. 10 µl of the RT-PCR product was loaded on each lane and separated by electrophoresis on 1% (w/v) agarose gel. Acting product was used as a loading control.

Fig. 6. Quantitative RT-PCR analysis of arginine decarboxylase (Adc), ornithine decarboxylase (Odc), S-adenosylmethionine decarboxylase (Samdc) and spermidine synthase (Spds) transcripts in nodules of P. vulgaris inoculated with R. tropici CIAT899 treated with 0 and 100 mM NaCl for 3 and 7 days. Expression levels are normalized to the housekeeping gene Actin and are presented as relative to untreated plants. The data are expressed as the means and SE of three biological replications.

Fig. 7. Enzyme activities arginine decarboxylase (ADC) and ornithine decarboxylase (ODC) in nodules of P. vulgaris inoculated with R. tropici CIAT899 treated with 0 and 100 mM NaCl for 3 and 7 days. Date are mean ± SE (n = 3). Mean values followed by the same letter do not differ (P 6 0.05) using LSD test.

Fig. 8. Nitrogen fixation parameters: apparent nitrogenase activity (ANA), total nitrogenase activity (TNA), nitrogen fixation rate (NFR), and nodule fresh weight (NFW) in P. vulgaris inoculated with R.tropici CIAT 899 treated with 0 and 100 mM NaCl for 3 and 7 days. Date are mean ± SE (n = 8). Mean values followed by the same letter do not differ (P 6 0.05) using LSD test.

Fig. 9. Plant dry weight (PDW) and root to shoot rate (RSR) in P. vulgaris inoculated with R. tropici CIAT 899 treated with 0 and 100 mM NaCl for 3 and 7 days. Date are mean ± SE (n = 8). Mean values followed by the same letter do not differ (P 6 0.05) using LSD test.