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anticatabolic and adaptogenic effects of 24-epibrassinolide on Lupinus angustifolius: Causes and consequences
Steroids 154 (2020) 108545
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Anabolic/anticatabolic and adaptogenic effects of 24-epibrassinolide on Lupinus angustifolius: Causes and consequences
T
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Olga L. Kandelinskayaa, , Helena R. Grischenkoa, Vladimir A. Кhripachb, Vladimir N. Zhabinskiib, Lylia E. Kartizhovac, Yuriy K. Shashkod, Olga V. Kosmachevskayae, Elvira I. Nasybullinae, Alexey F. Topunove a
Kuprevich Institute of Experimental Botany of the National Academy of Sciences of Belarus, 220072 Minsk, Akademicheskaya, 27, Belarus Institute of Bioorganic Chemistry of the National Academy of Sciences of Belarus, 2200141 Minsk, Kuprevich st, 5/2, Belarus c Institute of Microbiology of the National Academy of Sciences of Belarus, 2200141 Minsk, Kuprevich st, 2, Belarus d Research and Practical Center of the National Academy of Sciences of Belarus for Arable Farming, 222160 Zhodino, Timiriyazeva, 1, Belarus e Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences, Leninsky pr. 33, Moscow 119071, Russia b
A R T I C LE I N FO
A B S T R A C T
Keywords: Brassinosteroids Anabolic/anticatabolic action Lupine N2-fixing system Protein biosynthesis Environmental sustainability
Lupinus angustifolius L. is a legume culture known as a source of valuable feed protein and the N2-fixator for improving soil fertility. However, its low ecological resistance does not allow for a stable yield of the crop. Earlier, we have shown that steroid phytohormone 24-epibrassinolide (EBR) increases the tolerance of lupine to chlorine ions by activating the protective proteins in ripening seeds (such as proteinase inhibitors that prevent protein breakdown) and lectins. Here we investigated the effect of EBR on the functional status of the N2-fixing system in root nodules, protein synthesis in ripening seeds and the resistance of lupine plants to various pathogens. It was found that EBR enhanced the nodulation process, N2-fixing activity of nitrogenase and the accumulation of poly-β-hydroxybutirate in the bacteroides, increased the leghemoglobin content in the nodules as well as the metabolic activity of bacteroides. According to data on the inclusion of 14C-leucine in maturing seed proteins, EBR increased the accumulation of protein in them against the background of a short-term decrease in protein synthesis and its subsequent regeneration to the control level. Gradual inhibition of protein synthesis, characteristic of other legumes, was observed in control variants of lupine. EBR increased lupine resistance to phytopathogenic fungi of Colletotrichum genus and insects of Noctuidae and Scarabaeidae families. We concluded that a more complete implementation of the potential productivity and sustainability of lupine under the action of EBR was achieved due to the anabolic/anti-catabolic effect on the N2 fixation system in root nodules, as well as on protein synthesis in ripening seeds.
1. Introduction Brassinosteroids (BRs) have been revealed to be vital for providing plant integrity accounting for a subtle regulation of plant cell metabolism and processes of growth and development. BRs have an impact on the reception and transduction of signals thus being similar to steroid hormones in animals and humans. The receptor kinases (BRI1 and BRI1 associated receptor kinase 1, BAK1) ensure the flow of signals taking place through a cascade mechanism of protein-protein interactions. BRs also induce and regulate the level of gene expression, which in turn is responsible for implementing and developing the plant
growth programs [1–3]. Numerous data have led to the conclusion that BRs are bioregulators, which are believed to be synergists of other “classical” phytohormones such as auxins, gibberellins, cytokinins and ethylene [4]. BRs regulate hormone balance, synthesis of nucleic acids and proteins, cell division, biosynthesis of the cell wall components and affect seed germination, flowering and donor-acceptor relations, aging, photomorphogenesis, and adaptation [4–9]. BRs control plant-microbial symbiotic relationships, in particular, the formation of symbiosis between legumes and soil symbiotic bacteria of Rhizobia genus [10–12]. However, the results obtained using BRs on
Abbreviations: BRs, brassinosteroids; EBR, 24-epibrassinolide; N2-fixation, nitrogen fixation; Lb, leghemoglobin; PBS, phosphate buffered saline; PHB, poly-βhydroxybutyrate; TCA, tricarboxylic acid ⁎ Corresponding author. E-mail address: [email protected] (O.L. Kandelinskaya). https://doi.org/10.1016/j.steroids.2019.108545 Received 17 July 2019; Received in revised form 19 October 2019; Accepted 14 November 2019 Available online 21 November 2019 0039-128X/ © 2019 Elsevier Inc. All rights reserved.
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of a specialized nursery of Research and Practical Center of National Academy of Sciences of Belarus for Arable Farming (Zhodino, Belarus, 54°06′08.8″ N – 28°18′29.1″ E). 2.3. Isolation of bacteroides The bacteroides from the lupine root nodules were isolated by the method of Romanov et al. [22]. 2.4. Nodulation process in lupine roots and nitrogen-fixing activity in the nodule bacteroides Fig. 1. Structure of the steroid phytohormone 24-epibrassinolide.
This series of experiments was carried out at the stage of flowering of lupine plants (the 2d-3d decades of June), when N2-fixing activity in the nodule bacteroides is maximal. To assess the process of nodule formation, the number and mass of nodules per plant were estimated. The N2 fixing activity of nitrogenase enzyme complex in the nodule bacteroides was determined using the acetylene method, since nitrogenase has a low substrate specificity and, instead of nitrogen, can reduce other compounds with a triple bond, for example, acetylene to ethylene. This indirect method of assessing the activity of nitrogenase by a gas chromatograph is widely used to determine N2-fixation [23]. The activity index of nitrogenase was measured by calculating the nMproduced C2H4 per 1 g of roots with nodules within 30 min.
legumes are very controversial [13–18]. It is still unclear what impact BRs have on the functional status of the nitrogen-fixing system in legumes (N2-fixing system), which plays a crucial role in ensuring plant productivity and their resistance to adverse effects. In addition, the relationship between the high yield and the increase of sustainability of legumes under the influence of BR also remains unclear, since the usually stable productivity of legumes is limited by their poor environmental resistance [19]. It is not excluded that BRs play a coordinating role in the integration mechanisms of such fundamental processes in legumes as the functioning of the nitrogen-fixing system and processes of anabolism and catabolism. The purpose of this work was to study the influence of steroid phytohormone 24-epibrassinolide (EBR) (Fig. 1), one of the most active BRs [7], on the functional status of nitrogen-fixing system in the nodules; protein synthesis in ripening seeds and resistance of Lupinus angustifolius L. to diseases and pests.
2.5. Leghemoglobin content assay The content of leghemoglobin (Lb) in nodules was determined by measuring the fluorescence intensity of porphyrin produced by the interaction of hot saturated oxalic acid with a heme protein. Fluorescence makes it possible to estimate the total amount of Lb in the system, regardless of whether it is in its ligated or conformational form and to exclude other impurities contained in the solution [24].
2. Experimental 2.1. Chemicals and reagents
2.6. Metleghemoglobin reductase (met-Lb-reductase) activity assay
Steroid phytohormone 24-epibrassinolide (EBR, (22R,23R,24R)2α,3α,22,23-tetrahydroxy-24-methyl-B-homo-7-oxa-5α-cholestan-6one) was synthesized in the Laboratory of Steroid Chemistry of the Institute of Bioorganic Chemistry, the National Academy of Sciences of Belarus (Minsk, Belarus) [20]. The amino acid 14C-leucine was produced by PerkinElmer (USA). The remaining reagents were manufactured by Sigma (USA).
This indicator was estimated by the method of Golubeva et al. [25]. 2.7. Poly-β-hydroxybutyrate content assay The content of poly-β-hydroxybutyrate (PHB) in bacteroides was determined using Nile Red lipophilic fluorescent dye [22,26]. For this purpose, 500 μl of bacteroid suspension (A600 = 0.25 U) in 0.1 M PBS (pH 7.4) after adding 20 μl Nile Red (10 mg/ml in DMSO) were incubated for 1 h at room temperature in the dark. Then the suspension was washed twice with distilled water and centrifuged for 2 min at 13400 rpm. The pellet was resuspended in 500 μl of 0.1 M PBS. For measurements, the samples were diluted 10 times with 0.1 M PBS. Fluorescence was recorded on a spectrofluorimeter Shimadzu RF-5301 PC (Japan); λex = 550 nm; λem = 635 nm; Slit width: Ex = 3 nm, Em = 3 nm. Results are reported in relative fluorescence units.
2.2. Plant materials Seeds of narrow-leaved lupine (Lupinus angustifolius L., variety Mirtan) were provided by Research and Practical Center of the National Academy of Sciences of Belarus for Arable Farming (Zhodino, Belarus). Before sowing, lupine seeds were sterilized in 96% ethanol, dried and soaked for 1 day either in 1 nM EBR solution or without EBR (control group). This concentration of EBR was previously identified as optimal for lupine plants [21]. Test solutions of the chosen concentration were prepared by dilution of a stock solution of EBR (10−6 M) in ethanol with distilled water. Since the nitrogen-fixing nodules on the lupine roots were formed due to the active aboriginal rhizobia strains, the seeds were not inoculated with any Rhizobium lupini strains before sowing. In the course of the study, a series of field experiments were carried out on the plots of 1 m2 with the sod-podzolic soil in 4-fold repetitions. Experiments to assess the effect of EBR on the process of nodulation and N2 fixation in nodule bacteroides; on protein synthesis in ripening seeds; on lupine resistance to insect pests were carried out at the experimental station of the Institute of Genetics and Cytology of the National Academy of Sciences of Belarus, Minsk, Belarus (53°55′28″ N – 27°41′44″ E). To study the effect of EBR on tolerance of lupine to pathogenic fungi, the field experiments were carried out on the territory
2.8. Metabolic activity of bacteroides The metabolic activity of bacteroides was determined in vitro using an MTT test based on the ability of NAD(P)H-dependent cellular dehydrogenases to reduce the yellow tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT reagent) to dark purple formazan. To do this, the bacteroides, obtained as described above, were incubated in a nutrient medium with a tetrazolium dye at 37 °C for 20 h. The metabolic activity of bacteroides was evaluated by the intensity of formazan accumulation. In fact, the amount of formazan formed in the system corresponds to the number of the reducing equivalents NADP(H) and reflects the number of viable cells. The results are presented in mM of formazan. 2
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Fig. 2. The effect of EBR on the number of nodules (A) and the N2 fixing activity of nodules (B) in the narrow-leaved lupine plants. There were 10 plants in the 4-fold replications in each variant of the experiment. The differences are significant at p < 0.05.
2.9. Intensity of protein synthesis
2.12. Statistical processing
The intensity of protein synthesis in the lupine ripening seeds was determined by the incorporation of amino acid 14C-leucine into the seed total proteins. Samples of ripening lupine seeds for analysis were taken through 26 and 37 days after flowering (the 1st – 3d decades of July and 1st – 2d decades of August) [27]. According to various authors, the most significant changes in the protein content in ripening legume seeds were observed, on average, between the 21st–40th days after flowering [28,29]. The seeds of control group (without EBR) and those of the experimental group (with EBR) were placed into the solution of 14Cleucine (5 µCi/ml) for 2 h, then were washed with distilled water to get rid of the unbound label and stored at −18 °C. The intensity of protein synthesis was determined by 14C-leucine incorporation into the trichloracetic acid insoluble material according to the method [30], using a Wallac scintillation counter (Turku, Finland). Results were expressed in impulses per mg of total protein. The protein content in ripening seeds was determined by the method of Lowry [31]. The results are presented in mg of protein per 1 seed.
All the presented results are mean values ± a standard deviation. The significance of differences between the mean values was estimated using Mann–Whitney test (U test) at p < 0.05. The calculations were performed in AnalystSoft, StatPlus, Excel. 3. Results and discussion 3.1. Influence of EBR on the nodulation and N2-fixation in the bacteroides of lupine root nodules, metabolic activity of bacteroides and accumulation of poly-β-hydroxybutirate in them Legumes have the unique ability to capture inert, atmospheric N2 and convert it into bioavailable NH3 due to symbiosis with soil nitrogen-fixing bacteria of the genus Rhizobium. Symbiotic rhizobacteria invade the roots of legumes and multiply in the cells of the cortex, forming de novo organs called nodules. Inside the nodule, rhizobia cells differentiate into an endosymbiotic form, called bacteroides, which fix N2 with the enzyme complex of nitrogenase, which catalyzes the reduction of N2 to ammonia. In general, the establishment of successful symbiotic relations between host plants and their rhizobacteria, as well as the formation of nitrogen-fixing nodules, are very complex multistep processes that are accompanied by an exchange of molecular signals between symbiotic partners [32–38]. BRs are known to be involved in the regulation of nodulation and N2 fixation [10–14]. It should be mentioned that this influence can be both positive and negative [15–18]. Perhaps this is due to the different uses of BRs in plants, or with species/varietal characteristics of the host plant, or with the physiology of root nodules formed by the host plant, etc. In our experiments, we have observed that EBR produced a stimulating effect on the nodulation process and activity of the N2 fixing system in lupine. According to the data in Fig. 2 and Table 1, EBR triggered an increase in both the number and fresh mass of nodules per plant, as well as the N2 fixing activity of nitrogenase enzyme in the bacteroides. For normal functioning of the nitrogenase complex, it is very important to maintain an optimal O2 regime in the nodule. However, on the one hand, nitrogenase enzyme is irreversibly inactivated by the free O2, and, on the other hand, O2 is necessary for metabolism of the bacteroides themselves. Therefore, aerobic N2 fixation requires a minimal concentration of O2. This O2 paradox have been resolved in the rhizobial bacteroides by O2-delivering hemoprotein leghaemoglobin combining the regulation of microaerobic environment of the nodule with respiration. Consequently, Lb plays a dual role in legumes,
2.10. Influence of EBR on the survival of infected lupine plants To estimate the effect of EBR on the resistance of lupine plants to phytopathogenic fungi of the genus Colletotrichum, lupine seeds were pretreated using the above method and then placed in Colletotrichum lupini Bond sp. spore suspension with a spore load of 4.2 × 106 for 4 h. After these manipulations, the lupine seeds were planted. The following groups were used in this experiment: intact lupine seeds (control variant); the lupine seeds placed to Colletotrichum lupini Bond spore suspension (variant 1); the EBR-pretreated lupine seeds + Colletotrichum lupini Bond spore suspension (variant 2). Plant survival in the field was determined as a percentage of the survived plants before the seeds mature and the number of plants grown.
2.11. The effect of EBR on the resistance of lupine plants to insect pests The effect EBR-pretreatment of the lupine seeds on the tolerance of the crops to insects of the Noctuidae family was assessed through the number of the infected seeds in the control (control variant) compared with experimental groups (EBR). The result was calculated as a percentage. To assess the influence of EBR on the productivity of lupine when being hampered by insects of Scarabaeidae family, the number of beans per plant (% of control) and yield (% of control) was evaluated. 3
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as metabolic activity of bacteroides, initiates an even more pronounced oxygen limitation and a simultaneous increase of NAD(P)H amounts in them compared to the control variants. In turn, this causes the inhibition of crucial enzymes of the TCA cycle such as citrate synthase (EC 2.3.3.1) and isocitrate dehydrogenase (NADP+) (EC 1.1.1.42) as well as TCA. However, to maintain a high level of O2-labile nitrogenase activity, a high level of respiration is required. Under such conditions of O2 limitation, PHB plays the role of an alternative electron acceptor regenerating NAD(P)+, and thus prevents the inhibition of enzymes mentioned above to continue the operation of the TCA cycle [50,54,56,57]. It is necessary to take into account that the processes of PHB biosynthesis/accumulation and N2 fixing compete for the same sources of energy and carbon, and there is an inverse correlation between them [22]. In this regard, it would be suggested that EBR in lupine plants can cause the nutrient imbalance (for example, the carbon source excess and limitation of oxygen). In addition, EBR can stimulate the hyperproduction of PHB by the bacteroides as observed during the exponential growth phase. As a result, under the action of the EBR, the reserves of cellular PHB in bacteroides are significantly higher than the rate of its utilization in the process of N2-fixation [57,58].
Table 1 The influence of EBR on the functional state of the nitrogen-fixing system in lupine nodules. Indicator
Control
EBR
Fresh mass of nodules per 1 plant, g Lb, μg per g of fresh mass of nodules MeтLb-Red, μM per 1 min Poly-β-hydroxybutyrate (PHB), relative units of fluorescence Metabolic activity of bacteroides, amount of formazan, мM (MTT-test)
1.78 ± 0.15 43.26 ± 1.25 0.10 ± 0.01 88.35 ± 3.00
2.16 ± 0.30* 60.19 ± 2.35* 0.13 ± 0.01* 97.40 ± 3.22*
31.39 ± 1.58
71.86 ± 3.46*
*The differences are significant at p < 0.05.
protecting them from excess free oxygen and supplying the microsymbiont fixing nitrogen with bound oxygen. The quantity of Lb in the nodules serve as a biochemical marker indicating the state of the entire N2-fixing system of a legume plant [39–43]. Reversible binding of oxygen provides only Lb reduced form by metLb reductase. The function of this enzyme, which uses NADH as an electron donor, is to maintain heme iron Lb in a functionally active reduced state [44–46]. It was shown that met-Lb-reductase activity correlates with N2-fixing activity of the nodules [45,46]. According to our data, under the action of EBR the met-Lb-reductase activity was increased up to 30% as compared with the control variants (Table 1). It was suggested that the functional activity of the nitrogenase complex and the Lb content are directly related [47,48]. It is believed that the higher the leghemoglobin content in the nodules, the lower concentration of free oxygen and the more efficient the fixation of N2. For this reason, the level of Lb in bacteroides is considered a biochemical marker of the state of the entire N2-fixing system of a legume plant [39,41]. Our results confirm this. Moreover, the data presented here shows that EBR increased not only Lb accumulation and nitrogenase activity, but also the metabolic activity of bacteroides by more than 2 times according to the MTT test, which correlates with high levels of cellular reducing equivalents NAD(P)H. In our experiments we also observed that EBR enhanced PHB accumulation in the bacteroides of the experimental plants up to 10% as compared with the control variant (Table 1). Poly-β-hydroxybutyrate (PHB) is a microbial storage biopolymer with a high molecular mass. It is accumulated by different bacteria including Rhizobia sp. in the form of insoluble granules in the cytoplasm. PHB performs not only the functions of carbon and energy reserve, but is also important for maintaining survival and also for rhizobia reproduction during starvation [49–51]. PHB was shown to protect the microbial cell from different stress factors, such as osmotic, high temperature, UV radiation, oxidizing agents, saline, etc. [50,52–54]. This biopolymer is involved in maintaining the specific interactions of plants and bacteria, N2 fixing activity of nitrogenase in the bacteroides and the capacity of symbiotic bacteria to stimulate plant growth of different crops and their yields [49,53,55]. PHB metabolism is associated with tricarboxylic acid (TCA) cycle intermediates. The accumulation of PHB depends on the cellular redox potential and is supposed to be induced by the conditions of low oxygen content, which can be found inside the bacteroides of root nodules [50,54,56]. It can be assumed that the synthesis of PHB is controlled by many different factors. Some of them are the partitioning of acetyl-CoA between synthesis of PHB and the TCA cycle; the cellular redox state and TCA cycle activity under microaerobic conditions, etc. It has been shown that PHB plays the role of an alternative electron sink, contributing to the regeneration of NAD(P) + and the functioning of the TCA cycle under conditions of oxygen limitation [50,54,56–58]. Based on our present results and literature data, we assume that EBR, contributing to an increase in the N2-fixing activity of nitrogenase enzyme complex and the leghemoglobin content in the nodules, as well
3.2. The effect of EBR on biosynthesis and accumulation of proteins in the ripening lupine seeds The process of symbiotic N2 fixation in the nodules is closely connected with nitrogen metabolism in the ripening seeds. The latter become a powerful center of attraction for nutrients, including nitrogen. As seeds mature, their metabolic activity gradually decreases, whereas the accumulation of protein in them gradually increases due to a decrease in the moisture content [28,29,59,60]. Here we have shown that protein synthesis in ripening lupine seeds of intact plants (control variants) gradually decreases, but the accumulation of protein in seeds increases (Fig. 3A, B), as it was established earlier in the relation to Phaseolus vulgaris and Pisum sativum seeds [28,29]. It should be noted that a decrease in the water content of ripening seeds and, consequently, a decrease in respiration rate and metabolic rate in them are really significant for this phenomenon [28,29]. In addition, in the process of seed ripening, the activity of hydrolytic enzymes in them, such as proteinases of various classes (neutral, acid, and BAPA-ase), decreases, and the activity of proteinase inhibitors increases. The latter complicates the process of intracellular proteolysis of proteins and contributes to their accumulation. Activated inhibitors bind proteinases to form a proteinase-inhibitor complex, which leads to a decrease in metabolic activity and can be considered as the most important mechanism limiting the intracellular proteolysis of proteins in ripening seeds. Thus, the process of seed ripening is associated with an increase in the activity of proteinase inhibitors and a decrease activity of proteolytic enzymes [61]. Our previous results and literature data have shown that BRs can vary the intensity of metabolic processes, and not their direction [4,7,21,27]. Here we found that, in accordance with the data on the inclusion of 14C-leucine in the proteins of ripening seeds, EBR clearly increased the accumulation of protein in them against the background of a transient decrease in protein synthesis and its subsequent regeneration to the control level. Gradual inhibition of protein synthesis, characteristic of other legumes, was observed in control variants of lupine. A similar pattern was observed in yellow lupine plants (data not shown). Whether the presented results contradict the previously established fact that BRs produce a stimulating anabolic effect on protein synthesis in different plants [21,62]? In our opinion, there is no contradiction in this, since the anabolic action of any bioregulators, as well as BRs, include an anticatabolic effect, which can be associated not only with stimulation of protein synthesis, but also with a temporary protective 4
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Fig. 3. The effect of EBR on protein synthesis (A) and its accumulation (B) in the ripening lupine seeds: A – the intensity of protein synthesis by 14Cleucine incorporation into the seed proteins; B – protein content in the ripening seeds. There were 10 seeds in 4 replications in each variant of the experiment. The differences are significant at p < 0, 05.
reduction of metabolic rate and activation of mechanisms preventing protein degradation [63,64]. According to our previous data, we believe that the observed effects in ripening seeds induced by EBR are associated with the temporary activation of protective proteins, such as inhibitors of proteolytic enzymes, as well as the carbohydrate-binding proteins of the lectin family [27]. The consequence of these EBR-induced effects is an increase in the protein accumulation in mature seeds and seed productivity of lupine, as it was shown earlier [7].
Table 3 The effect of EBR-pretreatment on lupine plants affected by insect pests of Noctuidae.
In fact, the observed combination of anabolic and anticatabolic effects of the EBR helps to increase the threshold sensitivity of lupine plants to subsequent stress effects [63]. This assumption is supported by data on the increase in lupine tolerance to phytopathogenic fungi of Colletotrichum genus (Table 2), insect pests of Noctuidae (Table 3) and Scarabaeidae families under the influence of EBR. According to the Table 2, the development of Colletotrichum lupini Bond was decreased by 10.3% under the influence of EBR. Judging from the data in Table 3, the pretreatment of lupine seeds by EBR contributed to the reduction of the infestation of lupine plants by pests of the Noctuidae by more than 30%. The protective effect of EBR was also manifested when lupine flowers were damaged by Oxythyrea funesta of Scarabaeidae family (Table 4). According to Table 4, the EBR pretreatment of lupine seeds reduced the yield loss by almost 30%, since the buds were preserved and, accordingly, to increase the number of beans per 1 plant. Taking into account our present results on the pesticide action of EBR and the results of other authors [4,65,66], we cannot exclude the direct pesticidal effect of EBR. However, the most likely is a mediated mechanism of EBR protective action by molecules with pesticidal effects. Apart from such effects of secondary metabolites [67,68], a crucial role in the plant metabolism, as well as in the mechanisms of
Anthracnose development, %
1 2
Control (without infection and EBR) Variant 1 (Colletotrichum lupini Bond without EBR) Variant 2 (Colletotrichum lupini Bond + EBR)
6.4 ± 1.2 31.6 ± 2.7
3
Crop damage, %
1 2
Control EBR
10.0 ± 0.1 3.3 ± 0.3
Parameter
EBR
Quantity of beans per 1 plant, % to control Seed production, % to control
133.3 ± 2.2 137.2 ± 1.2
resistance to the adverse effects of abiotic and biotic nature play the activation of lectins [69–71] and proteinase inhibitors [72,73]. We also assume that increasing the stability of lupine under the influence of EBR is related to the fact that BRs are able to regulate the biosynthesis of ethylene, a stress hormone that is involved in protection against pathogens [74–76]. This can be realized through interaction with other phytohormones, such as auxins, as well as through regulation of the stability of aminocyclopropane-1-carboxylic acid synthase (ACS), the enzyme responsible for ethylene production [6,74–76]. Perhaps, literature data on the participation of ethylene in BRs-induced activation of an alternative respiratory pathway are quite relevant [77]. 4. Conclusion As is known, adaptation processes are inevitably accompanied by the activation of catabolic reactions that cause a decrease in crop yields. Here we sought to understand how EBR increase the productivity of legumes and their threshold of sensitivity to the adverse effects of biotic nature. In this context, we found three interrelated aspects of the action of EBR on lupine plants:
Table 2 The effect of EBR-pretreatment of lupine seeds on the development of Colletotrichum lupini Bond. Variant
Variant
Table 4 Influence of EBR on lupine seed production under the action of Oxythyrea funesta (% to control).
3.3. Influence of EBR on lupine tolerance to unfavorable factors of environment of biotic nature
№
№
(i) EBR had an anabolic effect on lupine N2-fixing system, including the stimulation of nodulation process (up to 25%), increasing the activity of the nitrogenase enzyme in root nodules (up to 78%) and the metabolic activity of bacteroides (2–3 times), as well as the amount of leghemoglobin (up to 39%) and poly-β-hydroxybutirate (up to 10,2%) in bacteroides; (ii) EBR had an anti-catabolic effect, which was associated with an
21.3 ± 1.5
5
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increase in protein accumulation in ripening lupine seeds (2.7–3 times) against the background of a transient decrease in its synthesis rate in ripening seeds in accordance with the results on the inclusion of 14C-leucine in seed proteins; (iii) EBR reduced the extent of damage of lupine plants by phytopathogenic fungi of the genus Colletotrichum (by 10%) and insects of the families Noctuidae and Scarabaeidae (more than 30%).
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Taken together, the results obtained in this study, our previous data and literature data suggest that BRs are plant anabolic/anticatabolic agents. They can not only stimulate the protein synthesis and nitrogen fixation in lupine, but also prevent the protein degradation in them by activating of protective proteins, such as lectins and proteinase inhibitors, despite a temporary decrease of protein synthesis in ripening seeds. The consequence of these events and, perhaps, the change of balance of some phytohormones, such as auxin and ethylene, is the BRsenhanced accumulation of proteins in mature seeds, more efficient realization of the lupine productivity potential and its resistance to biotic stresses. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments The authors thanks Mr. Aliaksandr A. Kazlouski (Research and Practical Center of National Academy of Sciences of Belarus for Arable Farming) for technical help. This work was supported by Government Programme of Scientific Investigations “Bioregulators” (Belarus), grant N 3.6, and by the Ministry of Science and Higher Education of the Russian Federation. References [1] S.D. Clouse, Recent advances in brassinosteroid research: From molecular mechanisms to practical applications, J. Plant Growth Regul. 22 (4) (2003) 273–275, https://doi.org/10.1007/s00344-003-0067-y. [2] G.J. Bishop, C. Koncz, Brassinosteroids and plant steroid hormone signaling, Plant Cell 14 (2002) S97–S110, https://doi.org/10.1105/tpc.001461. [3] C.J. Yang, C. Zhang, Y.N. Lu, J.Q. Jin, X.L. Wang, The mechanisms of brassinosteroids' action: from signal transduction to plant development, Mol. Plant 4 (2011) 588–600, https://doi.org/10.1093/mp/ssr020. [4] V.A. Khripach, V.N. Zhabinskii, A. de Groot, Brassinosteroids. A New Class of Plant Hormones, Academic Press, San Diego, 1999, , https://doi.org/10.1016/B978-0-12406360-0.X5000-X. [5] S. Hayat, A. Ahmad, Brassinosteroids: A Class of Plant Hormone, Springer, Dordrecht, 2011, p. 462. [6] S. Zhang, Y. Wei, Y. Lu, X. Wang, Mechanisms of brassinosteroids interacting with multiple hormones, Plant Signal. Behav. 4 (12) (2009) 1117–1120. [7] O.L. Kandelinskaya, A.F. Topunov, E.R. Grishchenko, Biochemical aspects of growth-stimulating effects of steroid phytohormones on lupine plants, Appl. Biochem. Microbiol. 43 (3) (2007) 324–331, https://doi.org/10.1134/ S0003683807030155. [8] A. Bajguz, S. Hayat, Effects of brassinosteroids on the plant responses to environmental stresses, Plant Physiol. Biochem. 47 (1) (2009) 1–8, https://doi.org/10. 1016/j.plaphy.2008.10.002. [9] U. Kutschera, Z.-Y. Wang, Brassinosteroid action in flowering plants: a Darwinian perspective, J. Exp. Bot. 63 (10) (2012) 3511–3522, https://doi.org/10.1093/jxb/ ers065. [10] P.N. McGuiness, J.B. Reid, E. Foo, The role of gibberellins and brassinosteroids in nodulation and arbuscular Mycorrhizal associations, Front. Plant Sci. 10 (2019) 269, https://doi.org/10.3389/fpls.2019.00269. [11] H. Liu, C. Zhang, J. Yang, N. Yu, E. Wang, Hormone modulation of legume-rhizobial symbiosis, J. Integr. Plant Biol. 60 (8) (2018) 632–648, https://doi.org/10.1111/ jipb.12653. [12] E. Foo, E.L. McAdam, J.L. Weller, J.B. Reid, Interactions between ethylene, gibberellins, and brassinosteroids in the development of rhizobial and mycorrhizal symbioses of pea, J. Exp. Bot. 67 (8) (2016) 2413–2424, https://doi.org/10.1093/ jxb/erw047. [13] Z. Wei, J. Li, Brassinosteroids regulate root growth, development, and symbiosis, Mol. Plant 9 (1) (2016) 86–100, https://doi.org/10.1016/j.molp.2015.12.003.
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Anabolic/anticatabolic and adaptogenic effects of 24-epibrassinolide on Lupinus angustifolius: Causes and consequences
T
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Olga L. Kandelinskayaa, , Helena R. Grischenkoa, Vladimir A. Кhripachb, Vladimir N. Zhabinskiib, Lylia E. Kartizhovac, Yuriy K. Shashkod, Olga V. Kosmachevskayae, Elvira I. Nasybullinae, Alexey F. Topunove a
Kuprevich Institute of Experimental Botany of the National Academy of Sciences of Belarus, 220072 Minsk, Akademicheskaya, 27, Belarus Institute of Bioorganic Chemistry of the National Academy of Sciences of Belarus, 2200141 Minsk, Kuprevich st, 5/2, Belarus c Institute of Microbiology of the National Academy of Sciences of Belarus, 2200141 Minsk, Kuprevich st, 2, Belarus d Research and Practical Center of the National Academy of Sciences of Belarus for Arable Farming, 222160 Zhodino, Timiriyazeva, 1, Belarus e Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences, Leninsky pr. 33, Moscow 119071, Russia b
A R T I C LE I N FO
A B S T R A C T
Keywords: Brassinosteroids Anabolic/anticatabolic action Lupine N2-fixing system Protein biosynthesis Environmental sustainability
Lupinus angustifolius L. is a legume culture known as a source of valuable feed protein and the N2-fixator for improving soil fertility. However, its low ecological resistance does not allow for a stable yield of the crop. Earlier, we have shown that steroid phytohormone 24-epibrassinolide (EBR) increases the tolerance of lupine to chlorine ions by activating the protective proteins in ripening seeds (such as proteinase inhibitors that prevent protein breakdown) and lectins. Here we investigated the effect of EBR on the functional status of the N2-fixing system in root nodules, protein synthesis in ripening seeds and the resistance of lupine plants to various pathogens. It was found that EBR enhanced the nodulation process, N2-fixing activity of nitrogenase and the accumulation of poly-β-hydroxybutirate in the bacteroides, increased the leghemoglobin content in the nodules as well as the metabolic activity of bacteroides. According to data on the inclusion of 14C-leucine in maturing seed proteins, EBR increased the accumulation of protein in them against the background of a short-term decrease in protein synthesis and its subsequent regeneration to the control level. Gradual inhibition of protein synthesis, characteristic of other legumes, was observed in control variants of lupine. EBR increased lupine resistance to phytopathogenic fungi of Colletotrichum genus and insects of Noctuidae and Scarabaeidae families. We concluded that a more complete implementation of the potential productivity and sustainability of lupine under the action of EBR was achieved due to the anabolic/anti-catabolic effect on the N2 fixation system in root nodules, as well as on protein synthesis in ripening seeds.
1. Introduction Brassinosteroids (BRs) have been revealed to be vital for providing plant integrity accounting for a subtle regulation of plant cell metabolism and processes of growth and development. BRs have an impact on the reception and transduction of signals thus being similar to steroid hormones in animals and humans. The receptor kinases (BRI1 and BRI1 associated receptor kinase 1, BAK1) ensure the flow of signals taking place through a cascade mechanism of protein-protein interactions. BRs also induce and regulate the level of gene expression, which in turn is responsible for implementing and developing the plant
growth programs [1–3]. Numerous data have led to the conclusion that BRs are bioregulators, which are believed to be synergists of other “classical” phytohormones such as auxins, gibberellins, cytokinins and ethylene [4]. BRs regulate hormone balance, synthesis of nucleic acids and proteins, cell division, biosynthesis of the cell wall components and affect seed germination, flowering and donor-acceptor relations, aging, photomorphogenesis, and adaptation [4–9]. BRs control plant-microbial symbiotic relationships, in particular, the formation of symbiosis between legumes and soil symbiotic bacteria of Rhizobia genus [10–12]. However, the results obtained using BRs on
Abbreviations: BRs, brassinosteroids; EBR, 24-epibrassinolide; N2-fixation, nitrogen fixation; Lb, leghemoglobin; PBS, phosphate buffered saline; PHB, poly-βhydroxybutyrate; TCA, tricarboxylic acid ⁎ Corresponding author. E-mail address: [email protected] (O.L. Kandelinskaya). https://doi.org/10.1016/j.steroids.2019.108545 Received 17 July 2019; Received in revised form 19 October 2019; Accepted 14 November 2019 Available online 21 November 2019 0039-128X/ © 2019 Elsevier Inc. All rights reserved.
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of a specialized nursery of Research and Practical Center of National Academy of Sciences of Belarus for Arable Farming (Zhodino, Belarus, 54°06′08.8″ N – 28°18′29.1″ E). 2.3. Isolation of bacteroides The bacteroides from the lupine root nodules were isolated by the method of Romanov et al. [22]. 2.4. Nodulation process in lupine roots and nitrogen-fixing activity in the nodule bacteroides Fig. 1. Structure of the steroid phytohormone 24-epibrassinolide.
This series of experiments was carried out at the stage of flowering of lupine plants (the 2d-3d decades of June), when N2-fixing activity in the nodule bacteroides is maximal. To assess the process of nodule formation, the number and mass of nodules per plant were estimated. The N2 fixing activity of nitrogenase enzyme complex in the nodule bacteroides was determined using the acetylene method, since nitrogenase has a low substrate specificity and, instead of nitrogen, can reduce other compounds with a triple bond, for example, acetylene to ethylene. This indirect method of assessing the activity of nitrogenase by a gas chromatograph is widely used to determine N2-fixation [23]. The activity index of nitrogenase was measured by calculating the nMproduced C2H4 per 1 g of roots with nodules within 30 min.
legumes are very controversial [13–18]. It is still unclear what impact BRs have on the functional status of the nitrogen-fixing system in legumes (N2-fixing system), which plays a crucial role in ensuring plant productivity and their resistance to adverse effects. In addition, the relationship between the high yield and the increase of sustainability of legumes under the influence of BR also remains unclear, since the usually stable productivity of legumes is limited by their poor environmental resistance [19]. It is not excluded that BRs play a coordinating role in the integration mechanisms of such fundamental processes in legumes as the functioning of the nitrogen-fixing system and processes of anabolism and catabolism. The purpose of this work was to study the influence of steroid phytohormone 24-epibrassinolide (EBR) (Fig. 1), one of the most active BRs [7], on the functional status of nitrogen-fixing system in the nodules; protein synthesis in ripening seeds and resistance of Lupinus angustifolius L. to diseases and pests.
2.5. Leghemoglobin content assay The content of leghemoglobin (Lb) in nodules was determined by measuring the fluorescence intensity of porphyrin produced by the interaction of hot saturated oxalic acid with a heme protein. Fluorescence makes it possible to estimate the total amount of Lb in the system, regardless of whether it is in its ligated or conformational form and to exclude other impurities contained in the solution [24].
2. Experimental 2.1. Chemicals and reagents
2.6. Metleghemoglobin reductase (met-Lb-reductase) activity assay
Steroid phytohormone 24-epibrassinolide (EBR, (22R,23R,24R)2α,3α,22,23-tetrahydroxy-24-methyl-B-homo-7-oxa-5α-cholestan-6one) was synthesized in the Laboratory of Steroid Chemistry of the Institute of Bioorganic Chemistry, the National Academy of Sciences of Belarus (Minsk, Belarus) [20]. The amino acid 14C-leucine was produced by PerkinElmer (USA). The remaining reagents were manufactured by Sigma (USA).
This indicator was estimated by the method of Golubeva et al. [25]. 2.7. Poly-β-hydroxybutyrate content assay The content of poly-β-hydroxybutyrate (PHB) in bacteroides was determined using Nile Red lipophilic fluorescent dye [22,26]. For this purpose, 500 μl of bacteroid suspension (A600 = 0.25 U) in 0.1 M PBS (pH 7.4) after adding 20 μl Nile Red (10 mg/ml in DMSO) were incubated for 1 h at room temperature in the dark. Then the suspension was washed twice with distilled water and centrifuged for 2 min at 13400 rpm. The pellet was resuspended in 500 μl of 0.1 M PBS. For measurements, the samples were diluted 10 times with 0.1 M PBS. Fluorescence was recorded on a spectrofluorimeter Shimadzu RF-5301 PC (Japan); λex = 550 nm; λem = 635 nm; Slit width: Ex = 3 nm, Em = 3 nm. Results are reported in relative fluorescence units.
2.2. Plant materials Seeds of narrow-leaved lupine (Lupinus angustifolius L., variety Mirtan) were provided by Research and Practical Center of the National Academy of Sciences of Belarus for Arable Farming (Zhodino, Belarus). Before sowing, lupine seeds were sterilized in 96% ethanol, dried and soaked for 1 day either in 1 nM EBR solution or without EBR (control group). This concentration of EBR was previously identified as optimal for lupine plants [21]. Test solutions of the chosen concentration were prepared by dilution of a stock solution of EBR (10−6 M) in ethanol with distilled water. Since the nitrogen-fixing nodules on the lupine roots were formed due to the active aboriginal rhizobia strains, the seeds were not inoculated with any Rhizobium lupini strains before sowing. In the course of the study, a series of field experiments were carried out on the plots of 1 m2 with the sod-podzolic soil in 4-fold repetitions. Experiments to assess the effect of EBR on the process of nodulation and N2 fixation in nodule bacteroides; on protein synthesis in ripening seeds; on lupine resistance to insect pests were carried out at the experimental station of the Institute of Genetics and Cytology of the National Academy of Sciences of Belarus, Minsk, Belarus (53°55′28″ N – 27°41′44″ E). To study the effect of EBR on tolerance of lupine to pathogenic fungi, the field experiments were carried out on the territory
2.8. Metabolic activity of bacteroides The metabolic activity of bacteroides was determined in vitro using an MTT test based on the ability of NAD(P)H-dependent cellular dehydrogenases to reduce the yellow tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT reagent) to dark purple formazan. To do this, the bacteroides, obtained as described above, were incubated in a nutrient medium with a tetrazolium dye at 37 °C for 20 h. The metabolic activity of bacteroides was evaluated by the intensity of formazan accumulation. In fact, the amount of formazan formed in the system corresponds to the number of the reducing equivalents NADP(H) and reflects the number of viable cells. The results are presented in mM of formazan. 2
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Fig. 2. The effect of EBR on the number of nodules (A) and the N2 fixing activity of nodules (B) in the narrow-leaved lupine plants. There were 10 plants in the 4-fold replications in each variant of the experiment. The differences are significant at p < 0.05.
2.9. Intensity of protein synthesis
2.12. Statistical processing
The intensity of protein synthesis in the lupine ripening seeds was determined by the incorporation of amino acid 14C-leucine into the seed total proteins. Samples of ripening lupine seeds for analysis were taken through 26 and 37 days after flowering (the 1st – 3d decades of July and 1st – 2d decades of August) [27]. According to various authors, the most significant changes in the protein content in ripening legume seeds were observed, on average, between the 21st–40th days after flowering [28,29]. The seeds of control group (without EBR) and those of the experimental group (with EBR) were placed into the solution of 14Cleucine (5 µCi/ml) for 2 h, then were washed with distilled water to get rid of the unbound label and stored at −18 °C. The intensity of protein synthesis was determined by 14C-leucine incorporation into the trichloracetic acid insoluble material according to the method [30], using a Wallac scintillation counter (Turku, Finland). Results were expressed in impulses per mg of total protein. The protein content in ripening seeds was determined by the method of Lowry [31]. The results are presented in mg of protein per 1 seed.
All the presented results are mean values ± a standard deviation. The significance of differences between the mean values was estimated using Mann–Whitney test (U test) at p < 0.05. The calculations were performed in AnalystSoft, StatPlus, Excel. 3. Results and discussion 3.1. Influence of EBR on the nodulation and N2-fixation in the bacteroides of lupine root nodules, metabolic activity of bacteroides and accumulation of poly-β-hydroxybutirate in them Legumes have the unique ability to capture inert, atmospheric N2 and convert it into bioavailable NH3 due to symbiosis with soil nitrogen-fixing bacteria of the genus Rhizobium. Symbiotic rhizobacteria invade the roots of legumes and multiply in the cells of the cortex, forming de novo organs called nodules. Inside the nodule, rhizobia cells differentiate into an endosymbiotic form, called bacteroides, which fix N2 with the enzyme complex of nitrogenase, which catalyzes the reduction of N2 to ammonia. In general, the establishment of successful symbiotic relations between host plants and their rhizobacteria, as well as the formation of nitrogen-fixing nodules, are very complex multistep processes that are accompanied by an exchange of molecular signals between symbiotic partners [32–38]. BRs are known to be involved in the regulation of nodulation and N2 fixation [10–14]. It should be mentioned that this influence can be both positive and negative [15–18]. Perhaps this is due to the different uses of BRs in plants, or with species/varietal characteristics of the host plant, or with the physiology of root nodules formed by the host plant, etc. In our experiments, we have observed that EBR produced a stimulating effect on the nodulation process and activity of the N2 fixing system in lupine. According to the data in Fig. 2 and Table 1, EBR triggered an increase in both the number and fresh mass of nodules per plant, as well as the N2 fixing activity of nitrogenase enzyme in the bacteroides. For normal functioning of the nitrogenase complex, it is very important to maintain an optimal O2 regime in the nodule. However, on the one hand, nitrogenase enzyme is irreversibly inactivated by the free O2, and, on the other hand, O2 is necessary for metabolism of the bacteroides themselves. Therefore, aerobic N2 fixation requires a minimal concentration of O2. This O2 paradox have been resolved in the rhizobial bacteroides by O2-delivering hemoprotein leghaemoglobin combining the regulation of microaerobic environment of the nodule with respiration. Consequently, Lb plays a dual role in legumes,
2.10. Influence of EBR on the survival of infected lupine plants To estimate the effect of EBR on the resistance of lupine plants to phytopathogenic fungi of the genus Colletotrichum, lupine seeds were pretreated using the above method and then placed in Colletotrichum lupini Bond sp. spore suspension with a spore load of 4.2 × 106 for 4 h. After these manipulations, the lupine seeds were planted. The following groups were used in this experiment: intact lupine seeds (control variant); the lupine seeds placed to Colletotrichum lupini Bond spore suspension (variant 1); the EBR-pretreated lupine seeds + Colletotrichum lupini Bond spore suspension (variant 2). Plant survival in the field was determined as a percentage of the survived plants before the seeds mature and the number of plants grown.
2.11. The effect of EBR on the resistance of lupine plants to insect pests The effect EBR-pretreatment of the lupine seeds on the tolerance of the crops to insects of the Noctuidae family was assessed through the number of the infected seeds in the control (control variant) compared with experimental groups (EBR). The result was calculated as a percentage. To assess the influence of EBR on the productivity of lupine when being hampered by insects of Scarabaeidae family, the number of beans per plant (% of control) and yield (% of control) was evaluated. 3
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as metabolic activity of bacteroides, initiates an even more pronounced oxygen limitation and a simultaneous increase of NAD(P)H amounts in them compared to the control variants. In turn, this causes the inhibition of crucial enzymes of the TCA cycle such as citrate synthase (EC 2.3.3.1) and isocitrate dehydrogenase (NADP+) (EC 1.1.1.42) as well as TCA. However, to maintain a high level of O2-labile nitrogenase activity, a high level of respiration is required. Under such conditions of O2 limitation, PHB plays the role of an alternative electron acceptor regenerating NAD(P)+, and thus prevents the inhibition of enzymes mentioned above to continue the operation of the TCA cycle [50,54,56,57]. It is necessary to take into account that the processes of PHB biosynthesis/accumulation and N2 fixing compete for the same sources of energy and carbon, and there is an inverse correlation between them [22]. In this regard, it would be suggested that EBR in lupine plants can cause the nutrient imbalance (for example, the carbon source excess and limitation of oxygen). In addition, EBR can stimulate the hyperproduction of PHB by the bacteroides as observed during the exponential growth phase. As a result, under the action of the EBR, the reserves of cellular PHB in bacteroides are significantly higher than the rate of its utilization in the process of N2-fixation [57,58].
Table 1 The influence of EBR on the functional state of the nitrogen-fixing system in lupine nodules. Indicator
Control
EBR
Fresh mass of nodules per 1 plant, g Lb, μg per g of fresh mass of nodules MeтLb-Red, μM per 1 min Poly-β-hydroxybutyrate (PHB), relative units of fluorescence Metabolic activity of bacteroides, amount of formazan, мM (MTT-test)
1.78 ± 0.15 43.26 ± 1.25 0.10 ± 0.01 88.35 ± 3.00
2.16 ± 0.30* 60.19 ± 2.35* 0.13 ± 0.01* 97.40 ± 3.22*
31.39 ± 1.58
71.86 ± 3.46*
*The differences are significant at p < 0.05.
protecting them from excess free oxygen and supplying the microsymbiont fixing nitrogen with bound oxygen. The quantity of Lb in the nodules serve as a biochemical marker indicating the state of the entire N2-fixing system of a legume plant [39–43]. Reversible binding of oxygen provides only Lb reduced form by metLb reductase. The function of this enzyme, which uses NADH as an electron donor, is to maintain heme iron Lb in a functionally active reduced state [44–46]. It was shown that met-Lb-reductase activity correlates with N2-fixing activity of the nodules [45,46]. According to our data, under the action of EBR the met-Lb-reductase activity was increased up to 30% as compared with the control variants (Table 1). It was suggested that the functional activity of the nitrogenase complex and the Lb content are directly related [47,48]. It is believed that the higher the leghemoglobin content in the nodules, the lower concentration of free oxygen and the more efficient the fixation of N2. For this reason, the level of Lb in bacteroides is considered a biochemical marker of the state of the entire N2-fixing system of a legume plant [39,41]. Our results confirm this. Moreover, the data presented here shows that EBR increased not only Lb accumulation and nitrogenase activity, but also the metabolic activity of bacteroides by more than 2 times according to the MTT test, which correlates with high levels of cellular reducing equivalents NAD(P)H. In our experiments we also observed that EBR enhanced PHB accumulation in the bacteroides of the experimental plants up to 10% as compared with the control variant (Table 1). Poly-β-hydroxybutyrate (PHB) is a microbial storage biopolymer with a high molecular mass. It is accumulated by different bacteria including Rhizobia sp. in the form of insoluble granules in the cytoplasm. PHB performs not only the functions of carbon and energy reserve, but is also important for maintaining survival and also for rhizobia reproduction during starvation [49–51]. PHB was shown to protect the microbial cell from different stress factors, such as osmotic, high temperature, UV radiation, oxidizing agents, saline, etc. [50,52–54]. This biopolymer is involved in maintaining the specific interactions of plants and bacteria, N2 fixing activity of nitrogenase in the bacteroides and the capacity of symbiotic bacteria to stimulate plant growth of different crops and their yields [49,53,55]. PHB metabolism is associated with tricarboxylic acid (TCA) cycle intermediates. The accumulation of PHB depends on the cellular redox potential and is supposed to be induced by the conditions of low oxygen content, which can be found inside the bacteroides of root nodules [50,54,56]. It can be assumed that the synthesis of PHB is controlled by many different factors. Some of them are the partitioning of acetyl-CoA between synthesis of PHB and the TCA cycle; the cellular redox state and TCA cycle activity under microaerobic conditions, etc. It has been shown that PHB plays the role of an alternative electron sink, contributing to the regeneration of NAD(P) + and the functioning of the TCA cycle under conditions of oxygen limitation [50,54,56–58]. Based on our present results and literature data, we assume that EBR, contributing to an increase in the N2-fixing activity of nitrogenase enzyme complex and the leghemoglobin content in the nodules, as well
3.2. The effect of EBR on biosynthesis and accumulation of proteins in the ripening lupine seeds The process of symbiotic N2 fixation in the nodules is closely connected with nitrogen metabolism in the ripening seeds. The latter become a powerful center of attraction for nutrients, including nitrogen. As seeds mature, their metabolic activity gradually decreases, whereas the accumulation of protein in them gradually increases due to a decrease in the moisture content [28,29,59,60]. Here we have shown that protein synthesis in ripening lupine seeds of intact plants (control variants) gradually decreases, but the accumulation of protein in seeds increases (Fig. 3A, B), as it was established earlier in the relation to Phaseolus vulgaris and Pisum sativum seeds [28,29]. It should be noted that a decrease in the water content of ripening seeds and, consequently, a decrease in respiration rate and metabolic rate in them are really significant for this phenomenon [28,29]. In addition, in the process of seed ripening, the activity of hydrolytic enzymes in them, such as proteinases of various classes (neutral, acid, and BAPA-ase), decreases, and the activity of proteinase inhibitors increases. The latter complicates the process of intracellular proteolysis of proteins and contributes to their accumulation. Activated inhibitors bind proteinases to form a proteinase-inhibitor complex, which leads to a decrease in metabolic activity and can be considered as the most important mechanism limiting the intracellular proteolysis of proteins in ripening seeds. Thus, the process of seed ripening is associated with an increase in the activity of proteinase inhibitors and a decrease activity of proteolytic enzymes [61]. Our previous results and literature data have shown that BRs can vary the intensity of metabolic processes, and not their direction [4,7,21,27]. Here we found that, in accordance with the data on the inclusion of 14C-leucine in the proteins of ripening seeds, EBR clearly increased the accumulation of protein in them against the background of a transient decrease in protein synthesis and its subsequent regeneration to the control level. Gradual inhibition of protein synthesis, characteristic of other legumes, was observed in control variants of lupine. A similar pattern was observed in yellow lupine plants (data not shown). Whether the presented results contradict the previously established fact that BRs produce a stimulating anabolic effect on protein synthesis in different plants [21,62]? In our opinion, there is no contradiction in this, since the anabolic action of any bioregulators, as well as BRs, include an anticatabolic effect, which can be associated not only with stimulation of protein synthesis, but also with a temporary protective 4
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Fig. 3. The effect of EBR on protein synthesis (A) and its accumulation (B) in the ripening lupine seeds: A – the intensity of protein synthesis by 14Cleucine incorporation into the seed proteins; B – protein content in the ripening seeds. There were 10 seeds in 4 replications in each variant of the experiment. The differences are significant at p < 0, 05.
reduction of metabolic rate and activation of mechanisms preventing protein degradation [63,64]. According to our previous data, we believe that the observed effects in ripening seeds induced by EBR are associated with the temporary activation of protective proteins, such as inhibitors of proteolytic enzymes, as well as the carbohydrate-binding proteins of the lectin family [27]. The consequence of these EBR-induced effects is an increase in the protein accumulation in mature seeds and seed productivity of lupine, as it was shown earlier [7].
Table 3 The effect of EBR-pretreatment on lupine plants affected by insect pests of Noctuidae.
In fact, the observed combination of anabolic and anticatabolic effects of the EBR helps to increase the threshold sensitivity of lupine plants to subsequent stress effects [63]. This assumption is supported by data on the increase in lupine tolerance to phytopathogenic fungi of Colletotrichum genus (Table 2), insect pests of Noctuidae (Table 3) and Scarabaeidae families under the influence of EBR. According to the Table 2, the development of Colletotrichum lupini Bond was decreased by 10.3% under the influence of EBR. Judging from the data in Table 3, the pretreatment of lupine seeds by EBR contributed to the reduction of the infestation of lupine plants by pests of the Noctuidae by more than 30%. The protective effect of EBR was also manifested when lupine flowers were damaged by Oxythyrea funesta of Scarabaeidae family (Table 4). According to Table 4, the EBR pretreatment of lupine seeds reduced the yield loss by almost 30%, since the buds were preserved and, accordingly, to increase the number of beans per 1 plant. Taking into account our present results on the pesticide action of EBR and the results of other authors [4,65,66], we cannot exclude the direct pesticidal effect of EBR. However, the most likely is a mediated mechanism of EBR protective action by molecules with pesticidal effects. Apart from such effects of secondary metabolites [67,68], a crucial role in the plant metabolism, as well as in the mechanisms of
Anthracnose development, %
1 2
Control (without infection and EBR) Variant 1 (Colletotrichum lupini Bond without EBR) Variant 2 (Colletotrichum lupini Bond + EBR)
6.4 ± 1.2 31.6 ± 2.7
3
Crop damage, %
1 2
Control EBR
10.0 ± 0.1 3.3 ± 0.3
Parameter
EBR
Quantity of beans per 1 plant, % to control Seed production, % to control
133.3 ± 2.2 137.2 ± 1.2
resistance to the adverse effects of abiotic and biotic nature play the activation of lectins [69–71] and proteinase inhibitors [72,73]. We also assume that increasing the stability of lupine under the influence of EBR is related to the fact that BRs are able to regulate the biosynthesis of ethylene, a stress hormone that is involved in protection against pathogens [74–76]. This can be realized through interaction with other phytohormones, such as auxins, as well as through regulation of the stability of aminocyclopropane-1-carboxylic acid synthase (ACS), the enzyme responsible for ethylene production [6,74–76]. Perhaps, literature data on the participation of ethylene in BRs-induced activation of an alternative respiratory pathway are quite relevant [77]. 4. Conclusion As is known, adaptation processes are inevitably accompanied by the activation of catabolic reactions that cause a decrease in crop yields. Here we sought to understand how EBR increase the productivity of legumes and their threshold of sensitivity to the adverse effects of biotic nature. In this context, we found three interrelated aspects of the action of EBR on lupine plants:
Table 2 The effect of EBR-pretreatment of lupine seeds on the development of Colletotrichum lupini Bond. Variant
Variant
Table 4 Influence of EBR on lupine seed production under the action of Oxythyrea funesta (% to control).
3.3. Influence of EBR on lupine tolerance to unfavorable factors of environment of biotic nature
№
№
(i) EBR had an anabolic effect on lupine N2-fixing system, including the stimulation of nodulation process (up to 25%), increasing the activity of the nitrogenase enzyme in root nodules (up to 78%) and the metabolic activity of bacteroides (2–3 times), as well as the amount of leghemoglobin (up to 39%) and poly-β-hydroxybutirate (up to 10,2%) in bacteroides; (ii) EBR had an anti-catabolic effect, which was associated with an
21.3 ± 1.5
5
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increase in protein accumulation in ripening lupine seeds (2.7–3 times) against the background of a transient decrease in its synthesis rate in ripening seeds in accordance with the results on the inclusion of 14C-leucine in seed proteins; (iii) EBR reduced the extent of damage of lupine plants by phytopathogenic fungi of the genus Colletotrichum (by 10%) and insects of the families Noctuidae and Scarabaeidae (more than 30%).
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Taken together, the results obtained in this study, our previous data and literature data suggest that BRs are plant anabolic/anticatabolic agents. They can not only stimulate the protein synthesis and nitrogen fixation in lupine, but also prevent the protein degradation in them by activating of protective proteins, such as lectins and proteinase inhibitors, despite a temporary decrease of protein synthesis in ripening seeds. The consequence of these events and, perhaps, the change of balance of some phytohormones, such as auxin and ethylene, is the BRsenhanced accumulation of proteins in mature seeds, more efficient realization of the lupine productivity potential and its resistance to biotic stresses. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments The authors thanks Mr. Aliaksandr A. Kazlouski (Research and Practical Center of National Academy of Sciences of Belarus for Arable Farming) for technical help. This work was supported by Government Programme of Scientific Investigations “Bioregulators” (Belarus), grant N 3.6, and by the Ministry of Science and Higher Education of the Russian Federation. References [1] S.D. Clouse, Recent advances in brassinosteroid research: From molecular mechanisms to practical applications, J. Plant Growth Regul. 22 (4) (2003) 273–275, https://doi.org/10.1007/s00344-003-0067-y. [2] G.J. Bishop, C. Koncz, Brassinosteroids and plant steroid hormone signaling, Plant Cell 14 (2002) S97–S110, https://doi.org/10.1105/tpc.001461. [3] C.J. Yang, C. Zhang, Y.N. Lu, J.Q. Jin, X.L. Wang, The mechanisms of brassinosteroids' action: from signal transduction to plant development, Mol. Plant 4 (2011) 588–600, https://doi.org/10.1093/mp/ssr020. [4] V.A. Khripach, V.N. Zhabinskii, A. de Groot, Brassinosteroids. A New Class of Plant Hormones, Academic Press, San Diego, 1999, , https://doi.org/10.1016/B978-0-12406360-0.X5000-X. [5] S. Hayat, A. Ahmad, Brassinosteroids: A Class of Plant Hormone, Springer, Dordrecht, 2011, p. 462. [6] S. Zhang, Y. Wei, Y. Lu, X. Wang, Mechanisms of brassinosteroids interacting with multiple hormones, Plant Signal. Behav. 4 (12) (2009) 1117–1120. [7] O.L. Kandelinskaya, A.F. Topunov, E.R. Grishchenko, Biochemical aspects of growth-stimulating effects of steroid phytohormones on lupine plants, Appl. Biochem. Microbiol. 43 (3) (2007) 324–331, https://doi.org/10.1134/ S0003683807030155. [8] A. Bajguz, S. Hayat, Effects of brassinosteroids on the plant responses to environmental stresses, Plant Physiol. Biochem. 47 (1) (2009) 1–8, https://doi.org/10. 1016/j.plaphy.2008.10.002. [9] U. Kutschera, Z.-Y. Wang, Brassinosteroid action in flowering plants: a Darwinian perspective, J. Exp. Bot. 63 (10) (2012) 3511–3522, https://doi.org/10.1093/jxb/ ers065. [10] P.N. McGuiness, J.B. Reid, E. Foo, The role of gibberellins and brassinosteroids in nodulation and arbuscular Mycorrhizal associations, Front. Plant Sci. 10 (2019) 269, https://doi.org/10.3389/fpls.2019.00269. [11] H. Liu, C. Zhang, J. Yang, N. Yu, E. Wang, Hormone modulation of legume-rhizobial symbiosis, J. Integr. Plant Biol. 60 (8) (2018) 632–648, https://doi.org/10.1111/ jipb.12653. [12] E. Foo, E.L. McAdam, J.L. Weller, J.B. Reid, Interactions between ethylene, gibberellins, and brassinosteroids in the development of rhizobial and mycorrhizal symbioses of pea, J. Exp. Bot. 67 (8) (2016) 2413–2424, https://doi.org/10.1093/ jxb/erw047. [13] Z. Wei, J. Li, Brassinosteroids regulate root growth, development, and symbiosis, Mol. Plant 9 (1) (2016) 86–100, https://doi.org/10.1016/j.molp.2015.12.003.
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