Nodulation and Delayed Nodule Senescence: Strategies of Two Bradyrhizobium Japonicum Isolates with High Capacity to Fix Nitrogen

The purpose of this work was to study further two Bradyrhizobium japonicum strains with high nitrogen-fixing capacity that were identified within a collection of approximately 200 isolates from the soils of Argentina. Nodulation and nitrogen-fixing capacity and the level of expression of regulatory as well as structural genes of nitrogen fixation and the 1-aminocyclopropane-1-carboxylate (ACC) deaminase gene of the isolates were compared with that of E109-inoculated plants. Both isolates of B. japonicum, 163 and 366, were highly efficient to fix nitrogen compared to commercial strain E109. Isolate 366 developed a higher number and larger biomass of nodules and because of this fixed more nitrogen. Isolate 163 developed the same number and nodule biomass than E109. However, nodules developed by isolate 163 had red interiors for a longer period, had a higher leghemoglobin content, and presented high levels of expression of acdS gene, that codes for an ACC deaminase. In conclusion, naturalized rhizobia of the soils of Argentina hold a diverse population that might be the source of highly active nitrogen-fixing rhizobia, a process that appears to be based on different strategies.


Introduction
Rhizobia-legume interactions lead to the development of nitrogen-fixing nodules where atmospheric N 2 is reduced to ammonia and assimilated by the plant. This process, known as Biological Nitrogen fixation, is one of the most important biotechnological tools in sustainable agriculture management [29,48].
Soybean (Glycine max [L.] Merr) is nowadays the most important legume worldwide. The crop was introduced in Argentina and Brazil, in both countries its expansion started in the 1970 and nowadays conform the most important area of soybean production worldwide. In Argentina, 90% of the soybean-cultivated area is inoculated at seeding with bacteria of the genus Bradyrhizobium, mainly B. japonicum and occasionally with B. elkanii. Because of this, most of the soils, contain naturalized populations of bradyrhizobia [17,28,38,43], which often outcompete selected strains provided by commercial inoculants [2,25,27,30]. Soil bacteria populations are under continuous evolution due to genetic changes that might lead to the appearance of new isolates, which might be highly competitive and proficient saprophytic strains [2,6,25]. In line with this, bradyrhizobia isolated from the soils are often more competitive than the highly efficient nitrogen-fixing bacteria introduced by means of commercial inoculants [1,25]. The genetic changes that occur in soil bacteria are frequently driven by a negative selection against nitrogen fixation, which is frequently impaired within naturalized populations of rhizobia [2,6].
The amount of N fixed by a legume along its life cycle is a function of nodule number as well as nitrogen-fixing efficiency and the length of time nodules remain active, before senescence when a complex set of interactions occur. Nitrogen fixation in nodules increases 15 days after inoculation and remains at high levels within 2-4 weeks, decreasing thereafter due to nodule senescence [47].

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During senescence, nodules' interior shifts from a pink to green color because of the nitration of the heme group of leghemoglobin [36]. Furthermore, Vance et al. [57], Pfeiffer et al. [42], Pladys and Rigaud [44,45], demonstrated that age-related senescence, in alfalfa, soybean, and French beans, respectively, was related to the activity of plant proteolytic enzymes. Pladys and Vance [46] also found that in nitrogen-fixing nodules of alfalfa proteolysis increased slowly within 21 days. The targets of these proteases are nodule cytosolic proteins, especially leghemoglobin, whose degradation affects the O 2 supply to bacteroides. Membranes and bacteroides also are digested by proteolytic enzymes; iron is released into the host cell cytoplasm and because of this nitrogen fixation decreases [10].
Ethylene is a plant hormone that plays a key role in plant and nodule senescence [16,34]. Like other hormones, it plays many roles particularly in symbiosis; it affects nodule number and development as well as the location of nodule primordia, but it is also involved in nodule senescence [16,18,20,39,53]. 1-aminocyclopropane-1-carboxylate (ACC) deaminase is an enzyme that cleaves 1-aminocyclopropane-1-carboxylate (ACC) to ammonia and α-ketobutyrate, reducing in this way ethylene production [15,49,52]. Interestingly, some plant growth-promoting bacteria promote growth due to the synthesis and activity of ACC deaminases.
ACC deaminases coding genes acdS have been described in many different bacteria, fungi, and actinomycetes [35]. Among nitrogen-fixing organisms commonly known as rhizobia, acdS has also been described in Bradyrhizobium. In Mesorhizobium loti, acdS is under the transcriptional control of the nifA promoter, which regulates the transcription of nif genes [33,37,51]. The activity of the ACC deaminase has been associated with nitrogen fixation and to the delay of nodule senescence [53], in such a way that a decrease in fixation might lead to ethylene synthesis and nodule senescence [13,47]. Thus, in this scenario, a reduction in ethylene synthesis by an increase in ACC deaminase activity might delay nodule senescence and increase the period of nitrogen fixation. Consequently, the production of ACC deaminase might be a biological strategy to improve nitrogen fixation [16,53]; however, the mechanisms that govern all the events that lead to nodule senescence remain elusive.
In this work, we characterized the nodulation and nitrogen fixation ability of soybean plants inoculated with two naturalized strains of bradyrhizobia with high nitrogen-fixing capacity that were isolated from soybean fields with different histories of management in Argentina. We evaluated the expression level of genes related to nodulation and nitrogen fixation and the gene coding for the ACC deaminase, in both free-living bacteria and nodule-forming bacteroides, as well as the content of leghemoglobin and ammonia in nodules.

Bradyrhizobium Cultures
Two hundred representatives of Bradyrhizobium japonicum and B. elkanii were isolated from soybean (Glycine max [L]. Merr) plants growing in a field located at Runciman, Santa Fe, Argentina (located at latitude: − 33.900, longitude: − 61.250) [38]. They were all slow-growing bacteria that developed 1-3 cm diameter colonies on yeast extract mannitol (YEM) after an incubation period of 7 days at 28 °C. These organisms were identified by PCR amplification of the internal transcribed spacer (ITS) 23S-16S rDNA [22] and RSα, a specific fragment that corresponds to repetitive DNA of Bradyrhizobium [26] and then were incorporated to the culture collection of the CIDEFI.
Bacteria were cultured on liquid YEM medium [58] at 28 °C in the darkness for 144 h. Stock cultures were prepared by adding 10% glycerol to a 1 × 10 9 cell ml −1 culture and then 1.5 ml aliquots were frozen in liquid nitrogen. Stock cultures were kept at − 70 °C. B. japonicum E109 was included as reference strain in all the studies (Culture Collection of INTA -Argentina); this strain is a derivative of USDA138 that has been used to formulate commercial inoculants in Argentina for a long time.
Based on the outstanding nitrogen-fixing capacity compared to control strain E109, two isolates of B. japonicum were selected 163 and 366. Isolates as well as control strains were grown on YEM agar medium at 28 °C or in liquid YEM medium, at 150 rev min −1 [58]. Tubes containing agar slants grown on YEM agar were kept at 4 °C and were used as starter cultures. Isolates

Plant Assays
Soybean (Glycine max [L] Merr) seeds cv DM4670 were surface sterilized by immersing them in 50% ethanol for 5 min and then 5 additional minutes in 5% v/v sodium hypochlorite, which was followed by ten washings with distilled water until no vapors of hypochlorite were perceived. Then, seeds were germinated in water agar at 28 °C in the darkness. Forty-eight hours later seedlings were transplanted to 2 l plastic pots filled with vermiculite and were inoculated with 10 7 cell ml −1 of B. japonicum strain E109 (positive control), isolate 163 or 366. Plants were grown under controlled conditions in the greenhouse at 24 ± 2 °C, 50% RH, 16 h photoperiod, and were watered with a nitrogen-free nutrient solution [58]. Three different experiments were performed, with twenty-four replicates of each assayed combination. Plants were harvested 6 weeks after seeding. Cultivar DM4670 is produced by the company Don Mario and is a cultivar that belongs to group four (a long one that is why it is a 4.67) and it is a widely used cultivar within Argentina.
Nitrogen-fixing activity was estimated by means of the acetylene reduction assay [5,50]. Detached nodulated roots were enclosed in 250 ml airtight bottles. Roots were supplied through rubber stoppers with C 2 H 2 (10% v/v) at time zero and incubated for 10 min at room temperature (25 ± 2 °C). Samples (0.5 ml) taken from airtight bottles were analyzed in a Gas Chromatograph equipped with a flame ionization detector (FID, KONIX 3000).
Dry weight of nodules and plant shoots were determined. Nodules were detached from roots, counted, placed in paper bags, and desiccated in an oven at 70 °C until constant dry weight is achieved. Similarly, plant shoots also were placed in paper bags and dried at 70 °C in an oven until constant weight is achieved.

Gene Expression in Nodules and Free-Living Bacterial Cultures
RNA was extracted from both liquid cultures and nodules. In order to extract RNA, cultures were grown on Peptone-Salts-Yeast Extract-Arabinose medium at 28 °C in a rotary shaker at 150 rev min −1 [31].
Bradyrhizobium Japonicum cultures were grown to mid-exponential phase, which corresponded to an optical density at 600 nm of 0.4 to 0.5 in microoxic cultures (peptone-salts-yeast extract-arabinose medium). Cultures were rapidly transferred to cold tubes, centrifuged for 5 min (13,000 rev min −1 , 4 °C), the supernatant was decanted, and cells were frozen in liquid nitrogen and stored at − 80 °C.
RNA of nodules was extracted from 1 g of nitrogen-fixing nodules that were harvested and ground in a mortar previously sterilized twice at 120 °C for 45 min and washed with DEPC-treated water. The powder was homogenized in 200 µl of deionized water and centrifuged at 2000 rev min −1 for 5 min to pellet bacteroides. Total RNA was isolated using the RNeasy Mini Kit (Qiagen, Netherlands) according to the manufacturer's instructions. 1-2.5 µg of DNA-free RNA was used with random hexamers for first-strand cDNA synthesis using the avian myeloblastosis Reverse Transcriptase for RT-PCR (Reverse Transcriptase, AMV-Roche®) according to the manufacturer's instruction. qPCR was conducted using a set of specific primers amplifying two regulatory nitrogen fixation genes nifA and fixL, the ACC deaminase coding gene (acdS), a constitutive and an inducible type III secretion system gene, rhcC1 and nopP, respectively, and two transcriptional regulators nodW and nodD1 (Table 1). Each reaction was performed in a final volume of 10 µl, containing 0.2 mM of the primers pair, one microliter of a 1:10 dilution of the previously synthesized cDNA and the corresponding volume of iQ™ SYBR® Green Supermix (Bio-Rad) using the iCycler iQ system (Bio-Rad). The reaction conditions were 95 °C for 3 min followed by 40 cycles of 95 °C for 15 s, 56 °C for 15 s, and 72 °C for 15 s and one cycle of final extension of 72 °C for 3 min. Melting curve analysis was performed. Data were analyzed using the iQ5 software. Gene expression of the above described genes was normalized according to the expression levels of 16S rRNA gene and expressed as the relative fold change compared to control strain B. japonicum E109, following the formula

Leghemoglobin and Ammonia Content in Nodules
Fresh nodules in an amount of 0.5 g were homogenized in 3 ml of an extraction buffer containing 0.02% (w/v) potassium ferricyanide and 0.1% sodium bicarbonate. Homogenates were centrifuged at 13,000 rev min −1 for 30 min and Table 1 Primers synthesized with DNA Synthesizer® 3400 (Applied Biosystem) at the Institute of Parasitology and Biomedicine "López-Neyra" (C.S.I.C., Granada) CGT CTC GCT CAT TCT GAT  SL15  GCG ATG AAC TCT TTA CTA TGA  nifA  SL16  CTG AGA GAA TGA CCC TGA A  SL17  TGA TGA ACT GTT TGG AAT GAG  rhcC1  SL18 TGT TAC GAA GTC AGA AAG GA leghemoglobin was measured fluorometrically in the red supernatant obtained [24]. Bovine hemoglobin was used as a standard. Nodule samples were collected five, six, and seven 7 weeks after inoculation. The ammonia content was determined by means of the phenol-hypochlorite method [4]. 0.5 g of nodules was homogenized in 2.5 ml of 0.3 mM H 2 SO 4 and centrifuged at 13,000 rev min −1 for 15 min. A calibration curve with NH 4 Cl was used as standard. Nodule samples were taken six and seven 7 weeks after inoculation.

B. japonicum Isolates 163 and 366 Induce Nitrogen-Fixing Nodules in Soybean Plants
We estimated the nodulation capacity of the selected isolates by measuring nodule number and dry weight. In addition, we determined the nitrogen fixation capacity indirectly by measuring plant biomass and by estimating the activity of nitrogenase through the acetylene reduction assay. Regarding nodulation, soybean plants inoculated with E109 and those inoculated with isolate 163 developed a similar number of nodules 24 per plant that were of approximately the same size, the average weight per nodule was 12 mg (Table 2). Soybeans inoculated with isolate 366 developed a significantly higher number of nodules per plant that also were larger (Table 2).
It is well known that growth depends on available N, so plant dry weight can be used to estimate nitrogen fixation of legume-rhizobia interactions along plant growth. On the other hand, the acetylene reduction assay reflects nitrogenase activity at a moment (sampling time) in plant growth. Plant biomass of soybeans inoculated with isolate 366 or 163 was larger than that of plants inoculated with E109, which indicates that most probably isolates 366 and 163 provided more nitrogen than E109 (Table 2). Interestingly, nitrogenase activity estimated based on the acetylene reduction assay 6 weeks after inoculation was similar among treatments. While doing this analysis, we observed that some nodules induced by control strain E109 as well as 366 had interiors with a greenish/brownish appearance compared to those induced by isolate 163, which had interiors with red color. All this together suggested that nodules induced by these two isolates probably were at a different developmental stage. While those induced by E109 and isolate 366 were probably at the beginning of senescence, those induced by 163 appeared to be actively fixing nodules.

ACC Deaminase Expression in B. japonicum 163 Bacteroides Suggested a Delay in Nodule Senescence
The level of expression of genes coding for proteins involved in Type III secretion system, nodulation, and nitrogen fixation, was evaluated. The activity of these genes might be considered indexes of highly efficient nitrogen-fixing strains. We evaluated by means of RT-PCR the expression of (a) nifA and fixL, genes that encode transcriptional regulators of nitrogen fixation; (b) rhcC1 and nopP that encode a constitutive and an inducible gene of a type III secretion system, respectively; (c) nodW and nodD1, two transcriptional regulators controlling the nodulation process; and (d) acdS (blr0241), a gene that encodes for ACC deaminase. This was done by using the sets of primers presented in Table 1.
In liquid cultures of isolates 163 and 366, genes were expressed at low levels compared to control strain E109 (Table 3). In nodules, expression of nodulation and nitrogen fixation genes was different in plants inoculated with isolate 163 or 366 than on nodules of plants inoculated with E109 (Fig. 1). Regulatory genes of nodulation had a similar transcriptional pattern in nodules induced either by isolates 163 or 366, though higher levels of expression were found in nodule samples induced by 163. Regulatory genes of nitrogen fixation were expressed at different levels in nodules induced by any of the three isolates. In nodules developed by B. japonicum 163, the level of expression of fixL, acdS, and rhcC1 was higher than that of nifA and nopP. Interestingly, most of these genes, except rhcC1, were slightly

Leghemoglobin and Ammonia Content Indicates Different Rates of Nodule Senescence
In preliminary experiments nodules of soybean plants inoculated with isolates 163, 366, or control strain E109 differed in the color of their interior, which was confirmed in the experiments described above. Therefore, we cultivated soybeans inoculated with each of the isolates and determined the leghemoglobin and ammonia content in 5-, 6-, and 7-week-old nodules. Five weeks after inoculation, nodules of plants inoculated with B. japonicum isolates 163 and 366 had significantly higher levels of leghemoglobin than those induced by control strain E109. The content of leghemoglobin decreased substantially in nodules 6 weeks after inoculation. As shown in Fig. 2 , primarily the content of leghemoglobin decreased by 15.45, 13.40, and 15.95% in nodules induced by E109, 163, and 366, respectively. Such reduction in leghemoglobin continued and 7-week-old nodules had less protein; such reduction consisted in an additional loss of leghemoglobin of 6.00, 5.85, and 17.40% in nodules induced by E109, 163, and 366, respectively. As a whole, the reduction in leghemoglobin content in nodules along the period was of 20.50, 18.50, and 30.50% in nodules induced by E109, 163, and 366, respectively. The content of ammonium was indirectly related with that of leghemoglobin. The largest quantity of ammonium was detected in B. japonicum 366-induced nodules, then in nodules induced by E109 and the lower amount was detected in nodules induced by B .japonicum 163. Interestingly, the latter were those where senescence was delayed, as estimated based on the leghemoglobin content.

Discussion
The use of plant growth-promoting bacteria is crucial to produce crops and vegetables in a sustainable management system of production. Rhizobia are probably the most important and frequently used PGPR Gram (−) bacteria. They establish interactions either with legumes, a symbiotic one at the root level that fixes nitrogen, or with non-legumes. In such interactions, alternative mechanisms of growth promotion are phosphate solubilization and siderophore production [9], systemic resistance induction [40], and biosynthesis of hormones [3,7,8]. In both cases, interactions with legumes or non-legumes, bradyrhizobia might contribute to improve soil fertility and N availability [2]. The technology to produce soybean includes the addition of a bacterial suspension of N-fixing bacteria at seeding, a process known as inoculation. Soybean was introduced in Argentina in the early 1970s and since then, the cultivated area with it, along the country, has increased substantially. Because of this, the soils of Argentina nowadays contain naturalized populations of rhizobia. One of the first strains used as inoculant was USDA138 and a re-isolation of it, which is the most efficient strain used in Argentina, has been identified as E109 and has been and still is recommended by INTA to formulate commercial inoculants. It has an outstanding ability to fix nitrogen, compete, and survive [17,43,54]. Recently Torres et al. [54] sequenced the genome of E109, which has characteristic features already described in other bradyrhizobia.
In the fields of Argentina, even if you use high-quality inoculants, soybean develops nodules that are frequently occupied by naturalized isolates of rhizobia that are mostly adapted to the soil rather than fixing nitrogen efficiently [38]. Soils in Argentina were initially devoid of soybean rhizobia; all naturalized populations might have their origin in commercial inoculants. These populations quite often have an outstanding competitive ability but low nitrogenfixing capacity [6,12]. The soils of Argentina contain highly diverse populations of bradyrhizobia [38], which might be the result of natural mutations and/or horizontal gene transfer events that occur in nature, an adaptation process of the organisms to improve their biological fitness [12,19]. However, despite considerable efforts, it has been and still is particularly difficult to identify more effective N 2 -fixing strains of B. japonicum [30]. Regarding this, we identified, among two hundred isolates, two B. japonicum isolates, 163 and 366 that based on biomass production, an indirect index of N availability, fix more N than B. japonicum E109.
Nitrogenase activity remained the same whether the plants were inoculated with control strain E109 or any of the collected isolates. However, isolate 366 developed more and larger nodules and soybeans inoculated with this strain presented a larger biomass, probably due to a higher amount of available N. The other isolate 163 might have a different strategy, since nodulation was not different from that induced by E109 but still soybean plants have a larger biomass. Evidently, these two isolates 163 and 366 are potential resources to develop new inoculants since plants inoculated with any of the isolates performed better than E109. Interestingly, it appears that the N-fixing ability of each isolate relies on different mechanisms or strategy.
Young non-fixing nodules have white interiors that later evolve to mature nitrogen-fixing nodules with red interiors that after 20-30 days senesce developed a brownish color in Fig. 2 Leghemoglobin and ammonia content of soybean nodules obtained from plants inoculated with E109, 163, or 366, respectively. Bars indicate the level of leghemoglobin in nodules induced by each isolate 5, 6, and 7 weeks after inoculation. Lines correspond to the ammonia content found in nodules of plants inoculated with the B. japonicum isolates under study 6 and 7 weeks after inoculation the interior [13,47], typical changes that occur in senescing nodules. Senescence is a complex and programmed process that leads to a reduction in N 2 -fixing activity and leghemoglobin content in aging nodules [4]. The interior of nodules induced by isolate 163, 366, and E109 6 weeks after inoculation differed in the color of their interiors suggesting that they might be in a different developmental stage. The amounts of leghemoglobin and ammonia in 6-7-weekold nodules formed by B. japonicum 163 also suggest that these nodules were at a different developmental stage than those induced by B. japonicum 366 or E109. While the latter ones are senescing, those induced by isolate 163, are still active. These changes in color of nodules interiors have been attributed to the activity of a plethora of proteases [46,56]. Federova [14] found that a cluster of Cys-proteins are induced in nodules before N fixation commences activity that is extended throughout nodule senescence. So, the larger biomass of plants inoculated with isolate 163 might be due to the fact that more N was provided by nodules that remain actively fixing nitrogen for a longer period of time.
Ethylene is the plant hormone related with senescence that might inhibit nodulation but it might also play a role in nitrogen-fixing nodules and nodule senescence. It has been proposed that nodulation of soybean might be improved by inhibiting ACC synthase and because of this nitrogen fixation as well [16,34]. Alternatively, a similar effect might be achieved by inoculating rhizobia with a highly active ACC deaminase activity, which might either increase nodulation capacity and/or delay senescence, since the enzyme prevents ethylene synthesis and accumulation [53]. We measured the expression of nodulation and nitrogen fixation genes in aerated liquid cultures of control strain B. japonicum E109 and B. japonicum 163 and 366. All the genes were hardly expressed in culture. Interestingly, it has been found that expression of nifA and acdS depended on the oxygen-level. acdS was upregulated during free-living microoxic (4.8-fold change) and also in anoxic conditions (12-fold change) as well as in symbiosis [21,41]. In addition, Murset et al. [32] found that acdS was expressed at high levels in symbiosis compared to cells grown in aerobic cultures. Furthermore, they showed that acdS expression was dependent on the expression of transcription factor genes nifA and rpoN, as it was described for Mesorhizobium [37,55]. In agreement with this we found that nifA and fixL as well as acdS were expressed at high levels in symbiosis but not under oxic environments. The dependence of acdS expression on the transcription factor NifA explains the relationship of the oxic conditions and acdS expression, and so there might not be a straight relationship between nitrogen fixation and acdS expression. Interestingly, a proteomic study of B. japonicum revealed that ACC deaminase is abundant in root-nodule bacteroides of soybean, siratro, and cowpea [11,23]. Furthermore, Murset et al. [32] found high levels of ACC activity in soybean nodules induced by ACC. In our view, in nodules induced by B. japonicum 163 the anoxic conditions lead to the expression of nitrogen-fixing regulatory genes, whose presence is crucial for the expression of acdS. The latter one is expressed at high levels in the nodules, and most probably because of this degradation of the enzymes involved in nitrogen fixation including all the necessary accessory proteins needed for the process is delayed.

Conclusions
B. japonicum isolates 163 and 366 were more efficient in fixing nitrogen than the highly proficient strain E109. While isolate 366 induced a larger nodule biomass, nodules induced by isolate 163 remained with red interiors due to leghemoglobin for a longer period. acdS, an ACC deaminase coding gene, was expressed only in nitrogen-fixing nodules and at higher levels in nodules induced by isolate 163, this probably reduced the amount of ethylene and delayed nodule senescence. We confirmed that ACC expression is dependent on nifA, whose expression is a function of the anoxic conditions. To our knowledge this is the first natural isolate described with a delayed senescence phenotype that leads to higher nitrogen fixation in plants. This study suggests that diverse naturalized rhizobia develop in the soil as a result of genetic changes that occur during adaptation. Along the process, changes generate alternative mechanisms that might make organisms more efficient nitrogen fixers that might lead to increase crop yield. So evidently, the soil is an enormous source of organisms that differ not only in their biological proficiency but also in the mechanisms that lead to their outstanding competitiveness and ability to fix nitrogen. The question arises regarding the fact that selected strains have been and still are on the market and have not been displaced by new isolates that establish more proficient symbiotic interactions.