Nitric oxide and light co-regulate glycine betaine homeostasis in sunflower seedling cotyledons by modulating betaine aldehyde dehydrogenase transcript levels and activity

ABSTRACT

Glycine betaine (GB), an osmolyte, is produced in chloroplasts by the action of betaine aldehyde dehydrogenase (BADH) on its precursor betaine aldehyde. The present work highlights the significance of nitric oxide (NO) in GB homeostasis as a long-distance salt (120 mM NaCl) stress-elicited response. In light-grown seedling cotyledons, both the activity and transcript levels of BADH are much higher than in dark-grown seedlings irrespective of salt stress. Significantly high accumulation of GB in dark-grown seedling cotyledons indicates its preferential mobilization from cotyledons to other plant parts in light-grown seedlings. NO donor application (diethylenetriamine) maintains high BADH activity in light, although in dark it is brought down marginally. BADH levels are maintained high in light than in dark in respective treatments. Reversal of the effect of NO donor on age-dependent GB content, BADH activity, and transcript levels by NO scavenger (diethyldithiocarbamate) further demonstrates the impact of NO on GB homeostasis in light- and dark-grown seedlings in an age-dependent manner, major modulation being observed in 4-d-old seedlings. The present work, thus, provides new information on co-regulation of GB homeostasis by NO and light. It also puts forward new information of GB–NO crosstalk in maneuvering salt stress sensing as a long-distance response in seedlings.

KEYWORDS:

• Salt stress
• glycine betaine
• nitric oxide
• betaine aldehyde dehydrogenase
• photomodulation

Introduction

Plants sense and modify their metabolism and activate a variety of defense mechanisms during stress conditions in order to get acclimatized.^1,2 Adjustment of osmotic balance, accumulation of reactive oxygen species (ROS), and activation of antioxidant enzymes are the primary and major biochemical events taking place in plants during stress conditions.^3,4 Accumulation of “compatible solutes” confers stress tolerance to plants through regulation of cellular osmotic balance, detoxification of ROS, and maintenance of plasma membrane integrity.⁵ Glycine betaine (GB) is one of the most cosmopolitan osmolytes in plants, animals, and bacteria. GB (N, N, N-trimethylglycine) is zwitterionic in nature, and its nonpolar hydrocarbon moiety enables it to easily communicate with all the domains of biomolecules, such as proteins and enzymes.^6,7 Enhanced accumulation of GB in plants directly signifies the degree of stress tolerance in plants.^8–10 Predominantly, GB synthesis occurs in chloroplasts through the action of enzymes such as choline monooxygenase (CMO) and betaine aldehyde dehydrogenase (BADH) on choline and betaine aldehyde (BA), respectively.¹¹ In some plants such as barley, however, CMO and BADH have also been reported to be localized in peroxisomes in addition to chloroplasts.^12,13 GB uses multifaceted defense mechanisms to protect plants from the deleterious effect of various stresses^14–23 (Figure 1). It reduces the loss of K⁺ ions and maintains high K⁺/Na⁺ ratio in cells during salinity stress. GB maintains the equilibrium of K⁺ ions by suppressing Na⁺ uptake through apoplastic route and thus, alleviating stress.^24–26 Based on already standardized concentrations of NaCl (120 mM), diethylenetriamine (DETA; 250 μM), and diethyldithiocarbamate (DETC; 250 μM) in the author’s laboratory,²⁷ the present work focuses on examining photomodulation of GB homeostasis,^28,29 as a long-distance signaling response to salt stress in sunflower seedling cotyledons. Modulation of BADH activity^30,31 and transcript³¹ levels by nitric oxide (NO) has been proposed to be differentially regulated in light- and dark-grown seedlings, thereby altering GB homeostasis. Furthermore, GB's interaction with NO has been demonstrated to highlight its role as cross-talking molecule.

Figure 1. Various roles of glycine betaine (GB) in plants exposed to abiotic stress conditions.

Results

Tissue sensitivity (age), growth conditions (light or dark), and NO modulate GB homeostasis in response to salt stress

Accumulation of GB in dark-grown seedling cotyledons of sunflower (Helianthus annuus L.) varied from 9.45 μg.mg⁻¹ Fresh weight (FW) (in salt-stressed 6-d-old seedling) to 35.61 μg.mg⁻¹ FW (in salt-stressed 4-d-old seedling). Maximum GB accumulation occurred in 4-d-old NaCl-treated (120 mM NaCl) seedling cotyledons. NaCl treatment resulted in enhanced GB content in cotyledons of 2- and 4-d-old seedlings by approximately 25% and 32%, respectively, with respect to control plants at their respective developmental ages. However, prolonged NaCl treatment (up to 6-d stage) decreased the accumulation of GB by ~23% compared to the respective content in plants raised without NaCl stress. This indicates that NaCl stress stimulates GB accumulation, and its content is further modulated by the developmental age of seedlings. In light-grown seedling cotyledons, GB content remained relatively moderate, though salt stress treatment did enhance the level in 4- and 6-d-old stage (Figure 2). There was no significant difference in the level of GB content in seedling cotyledons raised in the presence of NO donor, DETA in 2- and 4-d-old stage. NO scavenger, DETC treatment, however, resulted in variable response in GB accumulation in seedling cotyledons depending on seedling age. Light downregulated the impact of NO donor treatment on GB content up to 4-d stage (Figure 2). Thus, seedling age, in other words, tissue sensitivity to NO donor (DETA) and NO scavenger (DETC), was significant and variable with respect to GB accumulation (Figure 2).

Figure 2. (a) Impact of nitric oxide (NO) on glycine betaine (GB) accumulation in dark-grown sunflower seedling cotyledons raised in the absence or presence of salt (120 mM), NO donor (DETA; 250 μM), and NO scavenger (DETC; 250 μM). (b) BADH activity in seedling cotyledons as affected by NO. (c) Zymographic analysis of BADH activity. (d) Abundance of BADH transcript. Data represent the mean and standard deviation from three replicates (n = 3). NaCl and treatment-induced changes in GB accumulation and BADH abundance and activity were analyzed by one-way ANOVA using SPSS 22.0 and were statistically significant (*p < .05, **p < .01, ***p < .001).

Betaine aldehyde dehydrogenase transcript levels and activity are differentially modulated by NO in light and dark growth conditions

BADH activity, analyzed by NAD⁺-dependent oxidation of BA, was high in 2- and 4-d-old control seedlings but declined at 6-d stage of seedling growth in sunflower, more so in NO-treated seedlings. Salt stress reduced BADH activity in 2-d-old seedlings by about 32% approximately but increased in 4- and 6-d-old seedling cotyledons to approximately 103% and 62%, respectively, in control conditions. Therefore, positive modulation of BADH activity by salt stress is presumably dependent on seedling age. The effect of NO was evident on BADH activity, and its exogenous application (as DETA) resulted in increased activity in 2- and 4-d-old seedlings. However, the enhancement in activity was higher in seedlings raised without NaCl stress compared to seedling raised in presence of NaCl stress. This indicates that NO might be involved in the regulation of GB accumulation in salt stress through modulation of its biosynthetic enzyme, i.e., BADH. BADH activity was highest in light-grown seedling cotyledons at 4-d stage, and it was not significantly affected by NO or salt stress (Figure 3).

Figure 3. (a–d) Glycine betaine content and BADH analysis in light-grown seedlings. Other details as in Figure 2. Data represent the mean and standard deviation from three replicates (n = 3). NaCl and treatment-induced changes in GB accumulation and BADH abundance and activity were analyzed by one-way ANOVA using SPSS 22.0 and were statistically significant (*p < .05, **p < .01, ***p < .001).

Zymographic analysis of BADH activity highlighted the presence of two BADH isoforms in sunflower seedling cotyledons. The level of expression of BADH transcripts decreased with advancing age and was highest and lowest in 4-d and 6-d-old seedlings, respectively, irrespective of light or dark growth conditions. The activity of both the isoforms was suppressed by salt stress. In 2-d-old, dark-grown seedlings, salt treatment resulted in decreased BADH activity. Zymographically detectable BADH activity was consistently low in 6-d-old seedlings irrespective of NO and salt stress. However, in 6-d-old salt-treated seedlings, BADH activity was enhanced relative to the 2-d-old seedlings grown under salt conditions. The protein extracts of seedling cotyledons showed the presence of single distinctive ~61–63 kDa isozyme of BADH when resolved through the electrophoretic method and detected using hybridized ALDH7A1 antibody (polyclonal ALDH7A1 raised in rabbit/IgG, ThermoFisher Scientific, USA). Salt stress apparently reduced the abundance of BADH enzyme. Treatment of NO donor (DETA) resulted in a marked improvement in BADH abundance at all growth stages in control conditions. The reverse response was evident with NO scavenger treatment (DETC), which reduced the BADH transcript level in the seedling cotyledons at each growth stage, more so under salt stress conditions. These results are concurrent with the high BADH activity observed spectrophotometrically in 4 day old light-grown seedling cotyledons. DETA leads to increase BADH abundance in 6 day old seedlings.

Discussion

The present work highlights that a reasonable amount of GB is being constitutively synthesized in sunflower seedling cotyledons. Sensing of NaCl stress, however, enhances the GB concentration by several folds. Analysis of GB accumulation at three different growth stages of seedling cotyledons (2, 4, and 6 days old) showed that accumulation is age dependent. This is in congruence with the fact that primary synthesis and accumulation of GB occur in young parts of plants, and it is then transported to the older regions/tissues through phloem.^32–34 Significant modulation of GB accumulation by salt stress is evident in seedling cotyledons. Independent roles of NO and GB in abiotic stress are known.^35–38 Earlier investigations have revealed that endogenous NO derived from nitric oxide synthase (NOS)-like activity in plants can have a possible role in maintaining osmolyte accumulation.^16,39 Inhibition of NOS activity affects GB accumulation in maize.³⁵ The present work provides evidence for quenching of NO in solution by GB, indicating a possible direct interaction between the two molecules in plant tissues as well (Figure 4). The necessary role of NO in maintaining endogenous levels of GB has also been confirmed in the present work by the reduction in GB content caused by NO scavenger (DETC) treatment. NaCl stress and availability of NO regulate BADH activity and, therefore, accumulation of GB. The role of NO in enhancing GB accumulation is maintained through the upsurge in abundance and activity of enzyme BADH. Therefore, it is likely that NO’s involvement in the biosynthetic pathway of GB production in plants takes place prior to the involvement of BADH in converting the toxic intermediate (BA) to GB. Modulation of BADH transcripts and its activity by NO could possibly be an outcome of transcriptional-level regulation of BADH transcripts or a consequence of post translational modification of BADH protein by NO. This aspect needs to be addressed to fully understand the amelioration effects of GB in plants under different abiotic stress. Sunflower seedling cotyledons respond differentially in accumulating GB in the presence and absence of light during the germination growth period of seedlings. Exposing seedling to 16-hr light cycle during their growth resulted in the accumulation of GB in reverse pattern as compared to the seedling grown in dark. This highlights the involvement of a mechanism of active transportation of GB to other plant parts that might be initiated by light. The extent of the impact of NO on GB content and BADH activity also varied with light exposure, but the pattern observed was similar to that observed in dark-grown seedlings. The activity and abundance of BADH were enhanced in response to NO availability, whereas NaCl stress resulted in decreased activity and abundance of BADH. This suggests that light is likely to be involved in initiating the transportation of GB across different parts of the seedling, thereby modulating the extent of accumulation under salt stress.

Figure 4. NO quenching ability of GB. NO released from NO donor (DETA) reacts with NO probe (MNIP-Cu) yielding an excitation peak at 417 nm (ex. 365 nm; em. 420 nm). The peak is, however, completely abolished upon co-addition of GB. DETA and GB were used at 100 μM, whereas MNIP-Cu was used at 2.5 μM as per established procedure⁴¹ (MNIP-Cu: a copper derivative 4-mrthoxy-2-(1H-naptho (2, 3-d) imidazol-2-yl) phenol).

To sum up, sunflower seedling cotyledons constitutively accumulate a moderate amount of GB. Its level increases several folds in response to salt stress or NO availability. Levels of both BADH transcripts and enzyme activity seem to be differentially modulated by salt stress and age of seedlings. NO seems to be a prime regulator of GB accumulation through modulation of abundance and activity of its biosynthetic enzyme, BADH. It seems likely that GB complexed with NO could be the key molecule in eliciting a response. A model proposed in Figure 5 suggests three actions of NO in maintaining GB homeostasis: (1) Preferential mobilization of S-adenosylmethionine (SAM) in the biosynthesis of choline is due to inhibition of aminocyclopropanecarboxylate oxidase (ACC oxidase) activity by NO binding in the cytosol;⁴⁰ (2) NO further modulates BADH transcript levels and activity; and (3) GB interacts with NO and their complex (GB-NO) could be a key molecule in regulating plant growth under salt stress. These findings hold promise for further interesting work.

Figure 5. Probable mode of NO action on GB accumulation and GB–NO crosstalk in sunflower seedling cotyledons (ACC: 1-aminocyclopropane-1-carboxylic acid; SAM: S-adenosylmethionine).

Abbreviations

ACC oxidase	=	Aminocyclopropanecarboxylate oxidase
BA	=	Betaine aldehyde
BADH	=	Betaine aldehyde dehydrogenase
CMO	=	Choline monooxygenase
DETA	=	Diethylenetriamine
DETC	=	Diethyldithiocarbamate
GB	=	Glycine betaine
NAD+	=	Nicotinamide adenine dinucleotide (oxidized form)
NO	=	Nitric oxide
NOS	=	Nitric oxide synthase
ROS	=	Reactive oxygen species
SAM	=	S-adenosylmethionine

Acknowledgments

This work was supported by the Joint University Grants Commission–Israel Science Foundation Research Project (UGC-ISF); [F. No. 6-9/2017(IC)]. Financial assistance to the authors in the form of fellowship was provided by University Grants Commission (UGC), New Delhi, India (Ref No. 21/12/2014(ii)EU-V).

Disclosure of Conflict of Interest

No potential conflict of interest was disclosed.

Additional information

Funding

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