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Article

Metabolic Adjustment of Glycine max (L.) Merril in the Presence of Nitrate and Bradyrhizobium japonicum

by
Alberto Mongolo Júnior
1,
Felipe Girotto Campos
1,*,
Gustavo Ribeiro Barzotto
2,
Jonas Akenaton Venturineli Pagassini
2,
Maria Aparecida Ribeiro Vieira
1 and
Carmen Sílvia Fernandes Boaro
1
1
Biostatistics, Plant Biology, Parasitology and Zoology Department, Campus Botucatu, Institute of Biosciences, UNESP, São Paulo State University, District de Rubião Junior, Botucatu, São Paulo 01049-010, SP, Brazil
2
Plant Production Department, School of Agriculture, UNESP, Campus Botucatu, São Paulo State University, Botucatu, São Paulo 01049-010, SP, Brazil
*
Author to whom correspondence should be addressed.
Agronomy 2021, 11(8), 1518; https://doi.org/10.3390/agronomy11081518
Submission received: 17 June 2021 / Revised: 14 July 2021 / Accepted: 23 July 2021 / Published: 29 July 2021
(This article belongs to the Special Issue Rhizobia and Stress)
Browse Figures
Versions Notes

Abstract

:
Reactive oxygen species are generated during the processes of photosynthesis and nitrate reduction, which can compromise the integrity of biomolecules and membranes. During the vegetative phase of Fabaceae species, around half of translocated carbohydrate is used for nodule growth, while the other half returns to the aerial part with nitrogen incorporated. These sugars may be yet involved with membrane stabilization, signaling, and activation of important genetic pathways for plant development. Thus, the aim was to study the adjustments of the photosynthetic and antioxidant systems and the accumulation of carbohydrates and biomass in Glycine–Bradyrhizobium cultivated with nitrate (NO 3 − ). Four treatments were evaluated in completely randomized blocks. Glycine–Bradyrhizobium was grown with 1.7 mM of NO 3 − (GB: 1.7 mM NO 3 − ) and without NO 3 − (GB: 0 mM NO 3 − ), and Glycine was grown with 1.7 mM of NO 3 − (G: 1.7 mM NO 3 − ) and without NO 3 − (G: 0 mM NO 3 − ). Glycine–Bradyrhizobium symbiosis contributes to photosynthetic metabolism and total sugars, reduces the action of antioxidant enzymes, and minimizes the use of nitrate in soybean cultivation.; Glycine–Bradyrhizobium with nitrate provided greater plant dry mass in the vegetative phase, along with increased enzymatic activity and reduced nodule mass.

1. Introduction

Symbiosis with rhizobia, bacteria present in the soil that can infect plants, forming root nodules and providing atmospheric nitrogen (N) in the form of NH 3 + , is an important ecophysiological characteristic of soy (Glycine max (L.) Merrill) and other species of Fabaceae. Rhizobium and Bradyrhizobium are the most common genera in symbiotic association with Fabaceae crops [1].
The conversion of nitrogen gas via symbiotic fixation is catalyzed by the nitrogenase enzyme [2]. Biomolecules are exchanged in symbiosis, with the plant providing, via phloem, essential carbohydrates for the development and metabolism of rhizobia, which in turn provide, via the xylem, nitrogen compounds for plant protein synthesis [3].
The processes of nitrogen fixation and assimilation demand chemical energy in the form of adenosine triphosphate (ATP) and nicotinamide adenine dinucleotide phosphate (NADPH + H + ). Carbonic skeletons, derived from photosynthesis, are important in nitrogen assimilation since, when incorporating NH 3 + , they form amino acids [4]. Glyceraldehyde-3-phosphate synthesis and the fixation and assimilation of nitrogen require ATP and NADPH + H + , which implies competition for these resources between carbon and nitrogen metabolisms. In addition, for legumes associated with rhizobia, the carbohydrates produced by photosynthesis are also used by bacteria [5].
Potentially harmful reactive oxygen species (ROS) are generated during the processes of photosynthesis and nitrate reduction, which can compromise the integrity of biomolecules and membranes. Plants have a defense system, composed of antioxidant enzymes, to neutralize the action of these ROS, mainly superoxide dismutase (SOD), peroxidases (POD), and catalases (CAT) [6].
During the vegetative phase of some species of Fabaceae, such as Pisum sativum, Lupinus spp., Glycine spp. and Vigna sinensis, most of the carbohydrates assimilated in the leaves (sources) are displaced to the roots and nodules (drains). Around half of the carbohydrate translocated is used for aerobic respiration by rhizobia and nodule growth [7], while the other half returns to the aerial part with nitrogen incorporated [3]. The concentration of total soluble sugars can indicate the supply capacity of the leaves and the ability to transform and use part of the photoassimilates for filling grains. These sugars may be involved with membrane stabilization, signaling, and activation of important genetic pathways for plant development [8].
Recommendations for soy cultivation in Brazil include the use of Bradyrhizobium inoculants without the addition of N fertilizer, or up to 20 kg ha − 1 of N when sowing in soil that is poor in organic matter [9]. Soy cultivation with urea supply revealed changes in nodulation and grain yield [1]. Heckmann and Drevon [10] found that the supply of 3 mM nitrate reduces the activity of the nitrogenase enzyme, which is essential for the biological fixation process. Therefore, we suspect that doses lower than 20 kg ha − 1 of N in poor soils, as well as concentrations below 3 mM of nitrate in the nutrient solution, are capable of causing metabolic changes in plants, impairing the process of nodulation and biological N fixation.
Understanding the interaction between inoculation with Bradyrhizobium japonicum and nitrogen fertilization in photosynthetic metabolism, the translocation of photoassimilates, the antioxidant system, and biomass accumulation can help soybean management. Thus, the aim was to study the adjustments of photosynthetic and antioxidant systems and accumulation of carbohydrates and biomass in Glycine–Bradyrhizobium cultivated with nitrate.

2. Materials and Methods

The experiment was carried out in a “Paddy-Fan” type greenhouse with temperature control at the Biostatistics, Plant Biology, Parasitology and Zoology Department, of the Biosciences Institute, UNESP, Botucatu, São Paulo (22°49 ′ 10 ″ S, 48°24 ′ 35 ″ W; 800 m above sea level).
Soybean seeds (Glycine max L.) of the BMX Active RR variety, supplied by the Department of Plant Production, School of Agriculture, UNESP, Botucatu, were submitted to phytosanitary treatment. The seeds subsequently received commercial inoculants ofBradyrhizobium japonicum (Total Nitro Full®) using 6.0 mL of solution per Kg of seeds.
Glycine–Bradyrhizobium, in symbiosis or not, and Glycine were sown in a greenhouse, two seeds per plastic pot containing washed and sterilized sand. The seeds were irrigated in the initial stage with a 50% nutrient solution without nitrogen [11], as shown in Table 1. Irrigation with the solution was intercalated with deionized water for 28 days after sowing (DAS), during which 40 mL was used for the first five days, and 70 to 80 mL of solution or water was used thereafter. The use of 1.52 L of nutrient solution with 1.7 mM NO 3 − per plant provided the equivalent of 10 Kg ha − 1 of nitrogen applied at sowing. The pH of the nutrient solution was 6.67, which is a favorable condition for the development of B. japonicum.
Hoagland and Arnon’s nº 1 nutritional solution [11] (supply of nitrogen only as nitrate) was modified to provide 50% of the calcium concentration [Ca (H 2 PO 4 ) 2 ] and 25% of the other elements (Table 1), with calcium being essential in the period of infection and formation of nodules. At 28 DAS, one plant per pot was maintained by thinning with a nutrient solution with or without nitrate (Table 1).
Four treatments were evaluated, with five repetitions in completely randomized blocks. The treatments consisted of soybean inoculated with B. japonicum grown in the presence (GB: 1.7 mM NO 3 − ) or absence (GB: 0 mM NO 3 − ) of nitrate and uninoculated soybean in the presence (G: 1.7 mM NO 3 − ) or absence (G: 0 mM NO 3 − ) of nitrate.
Chlorophyll a fluorescence and gas exchange were measured between 09:00 and 11:00 hours, in fully expanded leaves, at 32, 35 and 41 DAS, using open photosynthesis system equipment with a CO 2 analyzer and water vapor analyzed by infrared radiation (“Infra-Red Gas Analyzer - IRGA”, model GSF 3000 Fl WALZ), under saturating irradiance of 1200 μ mol m − 2 s − 1 , with a coupled modulated light fluorometer (LED-Array/PAM-Fluorometer 3055-FL). Effective quantum yield ( ϕ PSII) to estimate the production of NADPH + H + , electron transport rate (ETR), photosystem II efficiency (Fv’/Fm’) in capturing light, light fraction absorbed by the PSII antenna that is dissipated as heat (D), the fraction of dissipated excitation energy in the antenna that cannot be used for photochemical phase (Ex), and photochemical quenching (qL) were measured [12,13].
The measured gas exchange variables were net CO 2 assimilation rate (A, μ mol CO 2 m − 2 s − 1 ), transpiration rate (E, mmol water vapor m − 2 s − 1 ), stomatal conductance (Gs, mmol m − 2 s − 1 ) and internal leaf CO 2 concentration (Ci, μ mol CO 2 mol − 1 ). Water use efficiency (WUE, μ mol CO 2 mmol H 2 O − 1 ) was determined as the ratio between net CO 2 assimilation rate and transpiration rate (Anet/E), while instant carboxylation efficiency of ribulose enzyme 1,5-diphosphate carboxylase (RuBisCO) was calculated as the ratio between net CO 2 assimilation rate and internal leaf C O 2 concentration (Anet/Ci, μ mol m − 2 s − 1 Pa − 1 ) [14], with five biological repetitions for each treatment. After harvesting the plants at 41 DAS, the dry masses of the leaf blade, stem + petiole, root, nodule, root + nodule, and total were determined.
To determine antioxidant enzyme and lipid peroxidation activities, leaves were collected at 41 DAS, with the necessary care to minimize enzyme degradation by light. The samples were wrapped in aluminum foil, packed in plastic bags, and frozen in liquid nitrogen to stop all reactions. The total soluble protein content was determined by the method proposed by Bradford (1976) [15]. Readings were taken with a spectrophotometer at 595 nm and compared with the standard curve of 1 % casein. Sample protein content was expressed in mg of protein g − 1 of fresh matter.
For determining peroxidase activity (POD, EC 1.11.1.7), the collected vegetal material was weighed and macerated in 5 mL of phosphate buffer (0.2 M, pH 6.7) in an ice bath to obtain crude extract. Peroxidase activity was determined according to the spectrophotometric method proposed by Teisseire and Guy (2000) [16] using the obtained crude extract. Specific peroxidase activity was expressed in μ moles of H 2 O 2 decomposed min − 1 mg − 1 of protein.
Catalase activity (CAT, EC 1.11.1.6) was determined by adding 100 μ L of the enzyme extract to 1900 μ L of 50 mmol L − 1 potassium phosphate buffer solution, pH 7.0, supplemented with hydrogen peroxide (H 2 O 2 , 12.5 mmol L − 1 ), for a total of 2000 μ L. The reaction was maintained for 80 s, with readings being made from the beginning to the end of this interval using a spectrophotometer at 240 nm to observe the decrease in absorbance. Calculation of activity was performed using the H 2 O 2 molar extinction coefficient (39.4 mmol L − 1 cm − 1 ) and expressed in nmol of H 2 O 2 consumed min − 1 mg − 1 protein [17].
Superoxide dismutase activity (SOD, EC 1.15.1.1) was determined by adding 50 μ L of crude extract to a solution containing 13 mM methionine, 75 μ L NBT, 100 nM EDTA and 2 μ M riboflavin in 3.0 mL of 50 mM potassium phosphate buffer, pH 7.8. The reaction was initiated by exposing the tubes to light. After 5 min of incubation [18], the absorbance of the bluish compound formed (formazan) by photoreduction of NBT was measured by reading on a spectrophotometer at 560 nm. A SOD unit is defined as the enzyme activity necessary for 50 % inhibition of NBT photoreduction. Enzyme-specific activity was calculated considering the obtained inhibition percentage, sample volume, and protein concentration in the sample ( μ g μ L − 1 ). Then, 50 μ L of crude extract was added in 950 μ L of 50 mM potassium phosphate buffer, pH 7.0, supplemented with hydrogen peroxide at a final concentration of 12.5 mM. Variation in absorption was calculated for an interval of 80 s, and enzyme activity was calculated using a molar extinction coefficient of 39.4 mM cm − 1 .
Lipid peroxidation activity was assessed using the methodology proposed by Heath and Packer (1968) [19]. For this, 200 mg of fresh material was macerated in a mortar, to which 5 mL of a reaction medium was added: 0.25 %  w/v thiobarbituric acid and 10 %  w/v trichloroacetic acid. The solution was placed in sealed test tubes and incubated in a water bath at 95 ° C for 1 h. The resulting solution was centrifuged at 12,000 rpm for 40 min at 25 ° C, and the collected supernatant was read with a spectrophotometer at wavelengths of 532 and 600 nm. The amount of malondialdehyde (MDA) formed was calculated using an extinction coefficient of 155 mM − 1 cm − 1 . Total soluble sugars were obtained by triple extraction with 80 % ethanol, with the supernatants being combined and the pellet frozen [20].
Starch was extracted from the pellet by triple extraction with 52 % chilled perchloric acid, and the supernatants were combined in a falcon tube until reading [20]. The anthrone method was used to quantify total soluble sugars and starch, with spectrophotometer readings made at 620 nm being expressed in a standard glucose curve [21,22]. Reducing sugars were quantified with the use of dinitrosalicylic acid (DNS), with a reading at 540 nm, and the curve expressed in a standard glucose curve [23].
The results were subjected to factorial analysis of variance at 5 % significance. Homogeneity of variances was tested using Levene’s test, and means were compared using Tukey’s test at the level of 5 % probability [24].

3. Results

3.1. Chlorophyll a Fluorescence

Glycine–Bradyrhizobium with 1.7 mM NO 3 − or without nitrate and Glycine with 1.7 mM NO 3 − showed, in general, higher photosystem II efficiency (Fv’/Fm’), excitation energy fraction not dissipated in the antenna that cannot be used for the photochemical phase (Ex), photochemical quenching (qL), effective quantum yield ( ϕ PSII) and electron transport rate (ETR), and lower light fraction absorbed by the PSII antenna dissipated as heat (D), compared to Glycine grown without nitrate (G: 0 mM NO 3 − ) (Figure 1).

3.2. Gas Exchange

Carbon assimilation and carboxylation efficiency of the RuBisCO enzyme (Anet/Ci) did not differ significantly between Glycine–Bradyrhizobium symbiotic systems with 1.7 mM NO 3 − and without nitrate, at different times (Figure 2). Glycine without nitrate showed low Anet and Anet/Ci and was stable over the seasons when compared to the other treatments (Figure 2). The Glycine–Bradyrhizobium symbiotic system grown without or with 1.7 mM NO 3 − showed a higher stomatal conductance (Gs) and water use efficiency (WUE) relative to uninoculated plants (Glycine). Glycine cultivated with nitrate showed a higher transpiration rate (E) and low water use efficiency and, at 35 DAS, revealed low carbon assimilation relative to inoculated plants. Glycine grown without nitrate showed lower transpiration and stomatal conductance, compared to other treatments. These plants also showed less water use efficiency, compared to Glycine–Bradyrhizobium without nitrate (GB: 0 mM NO 3 − ) (Figure 2).

3.3. Dry Mass Production

Glycine–Bradyrhizobium with a supply of 1.7 mM NO 3 − showed an increase in the dry mass of leaf blade, stem + petiole, root, root + nodule, and total, relative to other treatments. Glycine–Bradyrhizobium without nitrate supply had higher nodule biomass production than Glycine–Bradyrhizobium with 1.7 mM NO 3 − . Glycine without nitrate showed a lower dry mass of leaf blade, stem + petiole, and total, compared to other treatments (Table 2).

3.4. Antioxidant Enzymes and Lipid Peroxidation

Glycine–Bradyrhizobium grown without and with 1.7 mM NO 3 − showed higher POD activity relative to uninoculated plants (Glycine). Glycine–Bradyrhizobium with 1.7 mM NO 3 − showed greater activities of SOD and CAT and lipid peroxidation was lower in the symbiotic systems, compared to uninoculated plants (Glycine) (Figure 3).

3.5. Quantification of Carbohydrates

Glycine–Bradyrhizobium without nitrate showed a higher total soluble sugars concentration than other treatments and a concentration of reducing sugars not significantly different from Glycine–Bradyrhizobium with 1.7 mM NO 3 − (Figure 4). Glycine with 1.7 mM NO 3 − and without nitrate showed a lower total soluble sugars concentration than inoculated plants. The reducing sugars concentration was higher in Glycine with 1.7 mM NO 3 − relative to Glycine without nitrate (Figure 4). Glycine without nitrate showed a higher starch concentration, compared to other treatments (Figure 4).

4. Discussion

Chlorophyll a fluorescence, in general, was influenced by the lack of nitrogen supply with photochemical limitation. These results agree with a study that refers to the use of photochemical performance as a tool for monitoring plants subjected to stress [25]. Photosystem II efficiency (Fv’/Fm’), photochemical quenching (qL), effective quantum yield ( ϕ PSII), and electron transport rate (ETR), which characterize the photochemical phase, must have contributed to the metabolic adjustment of the Glycine–Bradyrhizobium system with nitrate, since less energy dissipation in the form of heat (D) and a greater fraction of excitation energy not dissipated in the antenna and not used in the photochemical phase (Ex) may have been directed to nitrogen metabolism as a form of photo-protection, as mentioned in a review of ways of dissipating energy [26]. The greater dry mass production obtained by the Glycine–Bradyrhizobium system with nitrate is in accordance with the directing of energy toward nitrogen metabolism.
The greater carbon assimilation (Anet), carboxylation efficiency of the RuBisCO enzyme (Anet/Ci), and the concentration of reducing sugar and biomass for Glycine–Bradyrhizobium with nitrate suggest that the carbon skeletons were used for growth, which is in accordance with the literature [27].
Nitrate supply for Glycine–Bradyrhizobium resulted in a lower total soluble sugars concentration, suggesting a lower sucrose concentration since the reducing sugars did not vary in the symbiosis without nitrate. The 50% decrease in nodule biomass, compared to Glycine–Bradyrhizobium without nitrate, can be explained by the translocation of sucrose and the action of sucrose synthase in the root system, which is important for nodule formation [7]. In addition, the presence of nitrate influences leghemoglobin by limiting its diffusion to the nodule and inhibiting the enzyme nitrogenase [27,28], which impairs nodulation and may explain the reduced nodule biomass.
Supply of 3 mM NO 3 − caused a reduction of approximately 55% of nitrogenase activity in B. japonicum bacteroids, on the account of the accumulation of nitrite resulting from the action of nitrate reductase and competition for the reducing power necessary for nitrogen metabolism [10]. In the present study, we verified that the supply of 1.7 mM of NO 3 − was enough to damage the nodulation. Nishida and Suzaki [29] also showed that the presence of nitrate promotes signaling similar to nodulation self-regulation, with the expression of genes that regulate nodule formation.
Glycine–Bradyrhizobium showed high water use efficiency, indicating compartmentalized metabolic adjustment between the two organisms. Association with Bradyrhizobium allows plants to direct energy resources towards carbon assimilation since it reduces the need for nitrate reduction [30]. Nitrate supply to Glycine resulted in decreased water use efficiency, which can be explained by greater transpiration (E), indicating greater water consumption, as already observed in Manihot esculenta [31]. In addition, Glycine with nitrate had reduced carbon assimilation rate (Anet), carboxylation efficiency of the RuBisCO enzyme (Anet/Ci), and concentrations of total soluble sugars, reducing sugars, and starch, indicative of competition between nitrogen and carbon metabolisms, as verified in previous studies [32]. Less photosynthetic activity in the absence of nitrogen and greater starch accumulation suggest that energy resources were used for the synthesis of reserve carbohydrates; these results are in agreement with those in the literature that report that nitrogen metabolism works with a carbon skeleton drain [7].
High SOD and CAT activities in Glycine–Bradyrhizobium with 1.7 mM NO 3 − suggest that the presence of this ion promoted metabolic changes, resulting from the production of reactive oxygen species, which were efficient in maintaining the low lipid peroxidation level. These results are supported in the literature since the nitrate ion requires reduction for assimilation and requires reducing agents, which are also used in photosynthesis [30]. These two energy drains allow a high rate of electron transport and generation of reactive oxygen species to be sustained, which are neutralized with the activity of antioxidant enzymes [26]. Glycine with 1.7 mM NO 3 − revealed low enzyme activity and greater lipid peroxidation, suggesting a greater benefit of the presence of the bacterium for the Glycine–Bradyrhizobium antioxidant system than only nitrate, as shown in the literature [1]. Nitrate supply to the symbiotic system may have promoted a signal in the root system that was conducted to the aerial part via the xylem, promoting greater activity of the enzymatic antioxidant system [3].
Glycine–Bradyrhizobium without nitrate showed low SOD activity, responsible by dismutase of superoxide (O 2 − ) and CAT, the main hydrogen peroxide neutralizing enzyme, which indicates the absence of stress. In this situation, POD activity was sufficient to control lipid peroxidation levels, and these results agree with studies carried out with growth-promoting rhizobacteria [33].
Soybean plants that were not inoculated and cultivated without nitrogen supply showed hydrogen peroxide accumulation, which is responsible for the higher lipid peroxidation and can be explained by the low CAT and POD activities, despite the high SOD activity. This condition indicates the need for nutrient availability.
Glycine–Bradyrhizobium without nitrate showed high photosynthetic activity, a higher concentration of total sugars, and less enzymatic activity, with efficient control of lipid peroxidation, which suggests a metabolic adjustment between the plant and the symbiote. Glycine–Bradyrhizobium with 1.7 mM NO 3 − , on the other hand, showed high photosynthetic activity with no increase in total sugar concentration, yet with greater enzymatic activity it kept the level of lipid peroxidation low. The comparison between the two conditions suggests that inoculated plants with nitrate provision had higher energy expenditure to control lipid peroxidation with the consumption of a part of the total sugars.
Glycine–Bradyrhizobium with nitrate promoted greater accumulation of dry matter since this nitrogen needs to be reduced. As this process occurs mainly in leaves, it can stimulate the production of leaf mass [34]. The presence of nitrate also induced greater root dry mass, which, according to the literature, can cause the accumulation of auxin in lateral roots, stimulating their growth [35]. It is important to note that greater vegetative mass may not result in greater grain yield. There was no gain in productivity of soybeans inoculated and cultivated in medium-to-high soil fertility, with the initial or late supply of nitrogen, although they presented greater vegetative development [1].
In the present work, soybeans were grown in washed and autoclaved sand, with an equivalent supply of 10 Kg ha − 1 of N, half the maximum recommended dose for soils poor in the organic matter [9]. The supply of nitrate can reduce nodulation [27,28], as verified in the present study. In addition to this reduction, the main sources of nitrogen are easily leached, which can lead to the non-availability of the element in moments of greatest demand [36,37].
Glycine–Bradyrhizobium without nitrate accumulated a higher concentration of total sugars, although with less growth, which contrasts to what occurred with Glycine–Bradyrhizobium with 1.7 mM NO 3 − . The relationship between total sugars and productivity has been assessed in other studies. A positive correlation between grain yield and accumulation of total sugars was observed in peanuts [38]. Low levels of total soluble sugars in the vegetative parts of soybean plants in shade were responsible for a lower number of pods formed and high percentages of abscission of reproductive structures [8].
In the absence of nitrate supply, the symbiotic system showed greater water use efficiency and greater concentration of total sugar and lowered enzyme activity and lipid peroxidation, which may support future studies for water conservation and stress prevention in soy cultivation. In addition, lessening the supply of nitrate can reduce the cost of nitrogen fertilizer and contribute to the conservation of natural resources.

5. Conclusions

Glycine–Bradyrhizobium symbiosis contributes to photosynthetic metabolism and total sugars, reduces the action of antioxidant enzymes, and minimizes the use of nitrate in soybean cultivation.
Glycine–Bradyrhizobium with nitrate provided greater plant dry mass in the vegetative phase, along with increased enzymatic activity and reduced nodule mass.

Author Contributions

Conceptualization, A.M.J., F.G.C., M.A.R.V., and C.S.F.B.; methodology, F.G.C., M.A.R.V., and C.S.F.B.; validation, F.G.C., G.R.B., J.A.V.P., and C.S.F.B.; formal analysis, F.G.C., G.R.B., and J.A.V.P.; investigation, A.M.J., F.G.C., G.R.B., and J.A.V.P.; data curation, F.G.C. and C.S.F.B.; writing—original draft preparation, A.M.J., F.G.C., G.R.B., J.A.V.P., and M.A.R.V.; writing—review and editing, F.G.C., G.R.B., J.A.V.P., and C.S.F.B.; supervision, C.S.F.B.; project administration, F.G.C. and C.S.F.B. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

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Agronomy 11 01518 g001 550
Figure 1. Chlorophyll a fluorescence, photosystem II efficiency (Fv’/ Fm’), light fraction absorbed by the PSII antenna dissipated as heat (D), excitation energy fraction not dissipated in the antenna that cannot be used for the photochemical phase (Ex), photochemical quenching (qL), electron transport rate (ETR), effective quantum yield ( ϕ PSII) of Glycine–Bradyrhizobium and Glycine cultivated with 0 mM and 1.7 mM nitrate. Uppercase letters indicate significant differences between seasons, and lowercase letters indicate significant differences between treatments (p < 0.05). G, Glycine; GB, Glycine–Bradyrhizobium.
Figure 1. Chlorophyll a fluorescence, photosystem II efficiency (Fv’/ Fm’), light fraction absorbed by the PSII antenna dissipated as heat (D), excitation energy fraction not dissipated in the antenna that cannot be used for the photochemical phase (Ex), photochemical quenching (qL), electron transport rate (ETR), effective quantum yield ( ϕ PSII) of Glycine–Bradyrhizobium and Glycine cultivated with 0 mM and 1.7 mM nitrate. Uppercase letters indicate significant differences between seasons, and lowercase letters indicate significant differences between treatments (p < 0.05). G, Glycine; GB, Glycine–Bradyrhizobium.
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Agronomy 11 01518 g002 550
Figure 2. Gas exchange, stomatal conductance (Gs), CO 2 assimilation rate (Anet), transpiration rate (E), carboxylation efficiency of the RuBisCO enzyme (Anet/Ci) and water use efficiency (WUE) of Glycine–Bradyrhizobium and Glycine cultivated with 0 mM and 1.7 mM nitrate. Uppercase letters indicate significant differences between seasons, and lowercase letters indicate significant differences between treatments (p < 0.05). G, Glycine; GB, Glycine–Bradyrhizobium.
Figure 2. Gas exchange, stomatal conductance (Gs), CO 2 assimilation rate (Anet), transpiration rate (E), carboxylation efficiency of the RuBisCO enzyme (Anet/Ci) and water use efficiency (WUE) of Glycine–Bradyrhizobium and Glycine cultivated with 0 mM and 1.7 mM nitrate. Uppercase letters indicate significant differences between seasons, and lowercase letters indicate significant differences between treatments (p < 0.05). G, Glycine; GB, Glycine–Bradyrhizobium.
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Agronomy 11 01518 g003 550
Figure 3. Antioxidant enzymes, superoxide dismutase (SOD, mg U protein − 1 ), peroxidase (POD, μ mol of purpurogallin min − 1 mg protein − 1 ), catalase (CAT, μ Kat μ g protein − 1 ), and lipid peroxidation (MDA, malondialdehyde nmol g FW − 1 ) of Glycine–Bradyrhizobium and Glycine cultivated with 0 mM and 1.7 mM nitrate at 41 days after sowing. G, Glycine; GB, Glycine–Bradyrhizobium.
Figure 3. Antioxidant enzymes, superoxide dismutase (SOD, mg U protein − 1 ), peroxidase (POD, μ mol of purpurogallin min − 1 mg protein − 1 ), catalase (CAT, μ Kat μ g protein − 1 ), and lipid peroxidation (MDA, malondialdehyde nmol g FW − 1 ) of Glycine–Bradyrhizobium and Glycine cultivated with 0 mM and 1.7 mM nitrate at 41 days after sowing. G, Glycine; GB, Glycine–Bradyrhizobium.
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Figure 4. Carbohydrates, total soluble sugars ( μ g g FW − 1 ); starch ( μ g g FW − 1 ) and reducing sugar ( μ g g FW − 1 ) of Glycine–Bradyrhizobium and Glycine cultivated with 0 mM and 1.7 mM nitrate at 41 days after sowing. G, Glycine; GB, Glycine–Bradyrhizobium.
Figure 4. Carbohydrates, total soluble sugars ( μ g g FW − 1 ); starch ( μ g g FW − 1 ) and reducing sugar ( μ g g FW − 1 ) of Glycine–Bradyrhizobium and Glycine cultivated with 0 mM and 1.7 mM nitrate at 41 days after sowing. G, Glycine; GB, Glycine–Bradyrhizobium.
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Table
Table 1. Composition of Hoagland and Arnon (1950) nutrient solution in the initial stage and with and without nitrate for cultivation of Glycine max L. in symbiosis or not with Bradyrhizobium japonicum.
Table 1. Composition of Hoagland and Arnon (1950) nutrient solution in the initial stage and with and without nitrate for cultivation of Glycine max L. in symbiosis or not with Bradyrhizobium japonicum.
Solution MolarInitial StepWith NitrateWithout Nitrate
Nutritional Solution (mL L − 1 )
Macronutrients
KH 2 PO 4 1.00-25-
K2SO 4 0.50125-125
KNO 3 1.00-125-
Ca(NO 3 ) 2 1.00-125-
Ca(H 2 PO 4 ) 2 0.05500-250
MgSO 4 1.00505025
CaSO 4 0.01--5000
Micronutrients-252525
Fe EDTA-252525
Table
Table 2. Production of dry leaf blade mass (DLBM), stem + petiole (DSM + P), nodule (DNM), root (DRM), root + nodule (DRM + N) and total dry mass (TDM) in g Glycine–Bradyrhizobium and Glycine grown with 1.7 mM NO 3 − and without (0 mM) at 41 days after sowing. Values corresponding to the means ± SE (n = 5).
Table 2. Production of dry leaf blade mass (DLBM), stem + petiole (DSM + P), nodule (DNM), root (DRM), root + nodule (DRM + N) and total dry mass (TDM) in g Glycine–Bradyrhizobium and Glycine grown with 1.7 mM NO 3 − and without (0 mM) at 41 days after sowing. Values corresponding to the means ± SE (n = 5).
TreatmentsDLBMDSM + PDNMDRMDRM + NTDM
GB: 1.7 mM1.45 ± 0.06 a1.16 ± 0.02 a0.08 ± 0.01 b1.00 ± 0.02 a1.09 ± 0.05 a3.73 ± 0.01 a
GB: 0 mM0.78 ± 0.03 b0.76 ± 0.04 b0.15 ± 0.016 a0.60 ± 0.03 b0.75 ± 0.04 b2.32 ± 0.09 c
G: 1.7 mM0.92 ± 0.04 b0.85 ± 0.01 b-0.90 ± 0.02 a0.90 ± 0.02 b2.72 ± 0.05 b
G: 0 mM0.32 ± 0.01 c0.40 ± 0.01 c-0.46 ± 0.02 b0.46 ± 0.02 c1.17 ± 0.02 d
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Júnior, A.M.; Campos, F.G.; Barzotto, G.R.; Pagassini, J.A.V.; Vieira, M.A.R.; Boaro, C.S.F. Metabolic Adjustment of Glycine max (L.) Merril in the Presence of Nitrate and Bradyrhizobium japonicum. Agronomy 2021, 11, 1518. https://doi.org/10.3390/agronomy11081518

AMA Style

Júnior AM, Campos FG, Barzotto GR, Pagassini JAV, Vieira MAR, Boaro CSF. Metabolic Adjustment of Glycine max (L.) Merril in the Presence of Nitrate and Bradyrhizobium japonicum. Agronomy. 2021; 11(8):1518. https://doi.org/10.3390/agronomy11081518

Chicago/Turabian Style

Júnior, Alberto Mongolo, Felipe Girotto Campos, Gustavo Ribeiro Barzotto, Jonas Akenaton Venturineli Pagassini, Maria Aparecida Ribeiro Vieira, and Carmen Sílvia Fernandes Boaro. 2021. "Metabolic Adjustment of Glycine max (L.) Merril in the Presence of Nitrate and Bradyrhizobium japonicum" Agronomy 11, no. 8: 1518. https://doi.org/10.3390/agronomy11081518

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