Nitric oxide modulates polyamine homeostasis in sunflower seedling cotyledons under salt stress

ABSTRACT

Free polyamine (PA) titers in plants may be regulated through reversible conjugate formation and/or through modulation of their synthesis, transport and degradation. PA signaling involves the well-acknowledged signaling molecule, nitric oxide (NO), which functions in diverse biological processes. Present investigations demonstrate the influence of salt stress (120 mM NaCl) and exogenous NO donor (250 µM Diethylenetriamine, DETA) on PA homeostasis of 2 d old, etiolated sunflower (Helianthus annuus L.) seedling cotyledons as a long-distance signaling response. Significantly enhanced intracellular spermine (Spm) accumulation was observed in seedling cotyledons under salt stress and in response to NO donor, the increase being more pronounced in seedlings treated with NO, evidently as a result of upregulation of the PA biosynthetic enzymes – arginine decarboxylase (ADC) and S-adenosylmethionine decarboxylase (SAMDC) – as revealed by Western blot and confocal imaging (CLSM). Moreover, salt stress induced the activity of polyamine oxidase (PAO), a PA catabolic enzyme, while NO lowered its activity in salt-stressed seedling cotyledons. NO, thus, appears to assist the seedlings in adapting to salt stress by positively regulating PA homeostasis through regulation of PA distribution between free, conjugated and bound forms, increased accumulation of PA biosynthetic enzymes and lowering the rate of PA catabolism.

Polyamines (PAs) are key regulators of an array of biological processes crucial for growth and development as well as for survival and defense against stressful environmental conditions.¹⁻⁴ PAs constitute an integral component of plant defense responses to both biotic and abiotic stress conditions.^1,3-7 PA metabolic pathways are carefully regulated in plants and the dynamic balance between their synthesis and degradation is crucial in conferring these molecules the ability to combat stress-induced adversities. PAs are basic in nature and exist in polycationic forms at physiological pH due to the presence of two (diamine) or more (polyamine) amino groups, a property by virtue of which they are able to bind to and exert stabilizing effects on the negatively charged biological macromolecules, such as nucleic acids, phospholipids in the membranes, and proteins. Free PA titers in plants may be regulated through reversible conjugate formation and/or through modulation of their synthesis, transport and degradation. Out of several PAs present in plants, diamine putrescine [Put, NH₂(CH₂)₄NH₂], triamine spermidine [Spd, NH₂(CH₂)₃NH(CH₂)₄NH₂ and tetraamine spermine [Spm, NH₂(CH₂)₃NH(CH₂)₄NH(CH₂)₃NH₂] account for the major components of cellular PA pool, and they can be present in free, conjugated and bound forms. Cytoplasmic levels of PAs are also modulated by compartmentalization within different organelles, conjugation and/or interaction with cell wall. The conjugated forms are mostly found associated with hydroxycinnamic acids. PAs bound to various biomolecules, such as nucleic acids and proteins (bound forms), constitute a significant portion of cellular PA pool in plants and may serve as storage forms of PAs, from which free forms may be released when required.⁸ Therefore, an equilibrium is maintained between the free and bound PAs. A growing body of evidence suggests that conjugated PAs are essential for the development and biotic interactions and in stress responses, regulating free PA titers and detoxification of phenolic compounds.^8-10 Two main pathways of PA biosynthesis exist in plants wherein Put serves as the primer moiety for the production of higher PAs (Spd and Spm). Put can be produced either from ornithine in a reaction catalyzed by ornithine decarboxylase (ODC; EC 4.1.1.17) or from arginine via agmantine in an alternate multi-step pathway involving arginine decarboxylase (ADC; EC 4.1.1.19). Spd and Spm are synthesized from Put by sequential transfer of aminopropyl moieties from decarboxylated S-adenosylmethionine (dcSAM) produced through the action of S-adenosylmethionine decarboxylase (SAMDC; EC 4.1.1.50) on S-adenosylmethionine (SAM).^11,12 PA degradation is mediated by a heterogeneous class of enzymes – amine oxidases (AOs). These include diamine oxidases/copper-containing amine oxidases (DAOs/CuAOs) and Flavin-containing (FAD-dependent) polyamine oxidases (PAOs) which catalyze the oxidative deamination of diamines and polyamines at the primary and secondary amino groups, respectively, to generate H₂O_2, pyrrolidone and respective aminoaldehyde. A precise regulation of PA biosynthesis and PA catabolism is important in the maintenance of cellular PA homeostasis.^12,13 This dynamic regulation of cellular PA levels is important for eliciting responses to specific developmental or environmental cues, including stress, through a complex interplay among signaling molecules such as H₂O₂, NO, Ca²⁺ and ABA, which have been revealed to be essential components of PA signaling.¹⁴ NO, a well-known signaling molecule, implicated in a wide array of biological processes and stress responses, is a component of PA signaling.^14,15 The objective of the present investigations was to examine the modulation of the expression/activity of the PA biosynthetic (ODC, ADC and SAMDC) and catabolic enzymes (DAO and PAO) by salt stress (120 mM NaCl), and the impact of NO provided as NO donor (DETA, 250 µM) as long-distance signaling response in dark-grown, 2 dold sunflower (Helianthus annuus L.) seedling cotyledons. The concentrations of NaCl and DETA have been selected on the basis of already established and recently published work from the author’s laboratory.^16,17

Salt stress and NO enhance Spm accumulation and abundance of PA biosynthetic enzymes, and differentially regulate PA catabolism

Spm was the major form of PA in cotyledons obtained from 2 d-old dark-grown sunflower seedlings raised in the absence or presence of salt (120 mM NaCl) or 250 µM of NO donor, DETA in the absence or presence of 120 mM NaCl (Figure 1). Salt stress is reported to induce Spm accumulation with a simultaneous decrease in the lower forms of PAs, Put and Spd or Put alone.^18-21 Significant increment in the conjugated as well as bound forms of Spm, represented by supernatant hydrolysate and pellet hydrolysate fractions, respectively, was evident in response to salt stress. NO donor application significantly increased the production of Spm. However, it did not alter the level of free Spm (supernatant fraction) but enhanced the proportion of conjugated and bound Spm, irrespective of salt stress. Hence, a positive regulation of the PA metabolism by NO was revealed. Spm, in particular, has been proposed to play versatile roles in plant responses to stress.^22,23 It has been proposed that the presence of two additional primary amino groups (-NH₂) in Spm as compared to Put endows it with a greater, more efficient protective capacity.²⁴ Spm has been suggested to act as a signaling molecule in the cascade leading to activation of the antioxidant enzymes. Another important role ascribed to Spm is the protection of DNA from free radical attack since it is present in nucleus in large quantities.²² It has also been implicated in plant defense responses against biotic threats by triggering defense-related genes and causing hypersensitive response (HR). Thus, increased accumulation of Spm in seedlings reported in the present work suggests its predominant protective role in counteracting salt stress in sunflower seedlings. Synergistic interplay between NO and PAs confers tolerance to abiotic as well as biotic stresses.^25-28 NO promotes an increase in contents of PAs and protective metabolites, like γ-aminobutyric acid (GABA) and proline.^27,29,30 Increase in the conjugated form of Spm in response to NO could possibly strengthen the antioxidant machinery of plant cells. Based on earlier reports, conjugation of PAs to phenolic acids could serve as an effective non-enzymatic antioxidant mechanism and, thus, improve the cellular antioxidant capacity.^8,31,32 NO also promoted an increase in the insoluble bound fraction of Spm under both saline and non-saline conditions. However, this increment in insoluble Spm fraction was more pronounced in seedlings subjected to NO donor in the absence of salt. It implies that NO is likely promoting PAs to interact with biological macromolecules. PAs, when bound to these macromolecules, exert stabilizing effects on them, protect them from ROS attack and maintain their structural and functional integrity.^4,8 Thus, a possible protective role for NO in the protection of macromolecules is suggested. Bound PAs also serve as storage forms of PAs from where they can be released in a regulated manner whenever needed by the cell or upon encountering specific conditions.⁸ Altogether, these findings indicate that NO may facilitate de novo synthesis of Spm (by facilitating the conversion from Put) and promote its conjugation to phenolic acids as well as biological macromolecules and is likely to aid the plant to cope with the stressful conditions and minimize the salt-induced damage. These observations, however, need further investigations.

Figure 1.

(a,b) Modulation of spermine content in various fractions obtained from sunflower seedling (2 d old) cotyledons (as estimated post-dansylation followed by quantification of TLC fractions by spectrofluorometry). Statistical significance of changes was analyzed by one-way ANOVA using SPSS 22.0 and changes were found to be significant (*P < .05, **P < .001, ***P < .0001) in comparison to the control. Data represent mean values from triplicates. (c–f) Modulation of expression of PA biosynthetic enzymes [ADC (Arginine decarboxylase), ODC (Ornithine decarboxylase) and SAMDC (S-adenosylmethionine decarboxylase)] in seedling cotyledons by NaCl (120 mM) and NO donor (DETA, 250 μM) application.

Accumulating evidence report upregulation of most of the genes encoding PA biosynthetic enzymes, including Arginine Decarboxylase (ADC), Spermidine Synthase (SPDS), Spermine synthase (SPMS) and S-adenosylmethionine Decarboxylase (SAMDC) under various stress conditions, although the timing and degree of induction may differ in different species.^33,34 Salt-induced upregulation of the genes of PA metabolic pathway enzymes has been well-documented.^1,20,35,36 Transgenic plants engineered to overexpress or those introduced with genes of enzymes catalyzing PA biosynthesis exhibit enhanced tolerance to both biotic and abiotic stresses and as well as improved yield.^6,37-39 Elevated PA contents in the plants are attributed in part to their increased de novo synthesis as a result of enhanced accumulation of PA biosynthetic enzymes ODC, ADC and SAMDC. Same is evident from the present investigations (Western blot analysis and CLSM imaging), both under salt-stress and in response to NO application, with effect of NO being more pronounced (Figures 1 and 2). Salt stress enhanced the abundance of ADC and SAMDC, the key enzymes of PA biosynthesis. No noteworthy influence was observed on ODC. NO significantly increased the accumulation of enzymes ADC and SAMDC both under control and saline conditions. The expression of these enzymes is, thus, positively affected by NaCl stress as well as NO.

Figure 2.

Immunolocalization of the three PA biosynthetic enzymes ADC, ODC and SAMDC using CLSM. (a) Immunolocalization of the three PA biosynthetic enzymes was performed by CLSM imaging of 7 µm thick transverse sections of cotyledons harvested from 2 d-old-dark-grown seedlings raised in the absence or presence of salt stress (120 mM NaCl) or those raised in Hoagland nutrient medium supplemented with 250 µM of DETA in the absence or presence of 120 mM NaCl. (b–d) Quantification of fluorescence units for ADC, ODC and SAMDC, respectively. Statistical significance of changes was analyzed by one-way ANOVA using SPSS 22.0 and changes were found to be significant (*P < .05, **P < .001) in comparison to control. Data represent mean values from triplicates [Magnification: 200×; Scale bar: 60 µm].

The method of estimation of PA catabolic enzyme activities was based on the estimation of H₂O₂ produced.⁴⁰ No significant modulation of PA catabolic enzyme DAO was observed by 120 mM NaCl stress or NO donor in terms of its activity (Table 1). In contrast, significant increase in PAO activity, another PA catabolic enzyme, was revealed both under salt stress and in response to NO treatment. DETA application, however, reduced the salt-induced increase in enzyme activity, which possibly helps in maintaining PA levels in the tissue by retarding their catabolic rate. This suggested the regulatory role played by PAO in PA catabolism and, therefore, overall PA homeostasis, in response to both salt stress and NO. Salt stress-induced PAO activity has been reported to be important in regulating salt tolerance in plants.^41,42 Moreover, loss of PAO activity induces tolerance to salt stress.^43,44 Irrespective of the pathways involved, oxidative catabolism of PAs by DAO and PAO is crucial in PA-mediated stress tolerance as it leads to the generation of H₂O₂, a signaling molecule which regulates downstream components in signal transduction pathways leading to triggering of various stress responses and GABA which serves as an osmoprotectant and has important protective roles in plant growth.^45,46

Table 1.

Alteration in the activity of the two polyamine catabolic enzymes, diamine oxidase (DAO) and polyamine oxidase (PAO) in sunflower seedling cotyledons in response to salt stress and treatment with NO donor (DETA). Data represent mean values from triplicates and the standard errors. Statistical significance of changes was analyzed by one-way ANOVA using SPSS 22.0 and changes were found to be significant (*P < .05, **P < .001) in comparison with control.

	Treatments		Specific activity (mol.µg−1 protein.min−1 × 10−10)
			Diamine oxidase	Polyamine oxidase
	Control	(−) NaCl	79.85 (± 5.26)	27.46 (± 4.38)
		(+) NaCl	86.71 (± 4.06)	55.38 (± 0.67)**
	DETA	(−) NaCl	87.32 (± 5.93)	45.91 (± 3.49)*
		(+) NaCl	86.56 (± 8.38)	39.26 (± 2.26)*

Conclusions

Maintenance of balance between PA biosynthesis and catabolism and their careful regulation by salt stress and NO appear to be crucial in acclimatization to salt stress (Figure 3). Salt stress leads to elevated PA catabolic enzyme (PAO) activity. NO reduces this salt-induced increase in the enzyme activity and thus seems to assist plants in adapting to salt stress by positively regulating overall intracellular PA homeostasis by increasing the abundance of PA biosynthetic enzymes (especially ADC and SAMDC) and lowering the rate of PA catabolism by PAO. NO also regulates spatial distribution of Spm in affected cells. The exact mechanism underlying the modulation of PA metabolic pathways by NO, however, needs to be further investigated in order to know the precise means by which it participates in PA signaling routes, particularly under salt stress.

Figure 3.

Nitric oxide and salt stress-mediated modulation of polyamine biosynthetic and catabolic routes.

Funding Statement

This work was supported by the UGC-ISF Joint Research Grant [Grant No. F. No. 6-9/2017 (IC)].

Abbreviations

ABA	abscisic acid
ADC	arginine decarboxylase
DAO	diamine oxidase
dcSAM	decarboxylated S-adenosylmethionine
DETA	diethylenetriamine
GABA	γ-aminobutyric acid
NO	nitric oxide
ODC	ornithine decarboxylase
PA	polyamine
PAO	polyamine oxidase
Put	putrescine
SAM	S-adenosylmethionine
SAMDC	S-adenosylmethionine decarboxylase
Spd	spermidine
SPDS	spermidine synthase
Spm	spermine
SPMS	spermine synthase

Acknowledgments

AT is grateful to University Grants Commission (UGC) for Research Fellowship awarded to her vide sanction no. 2061530546; Ref. No. 21/06/2015(i)EU-V dated 27 July 27 2016. Funding source for the present work is UGC-ISF Joint Research Grant [Grant No. F. No. 6-9/2017 (IC)].