Influence of calcium channel modulators on the production of serotonin, gentisic acid, and a few other biosynthetically related phenolic metabolites in seedling leaves of salt tolerant rice variety Nonabokra

ABSTRACT

Rice, a most salt-sensitive cereal plant, adopts diverse pathways to withstand sodium chloride–induced salinity-related adversities. During the present study, attempt was made to understand the role of calcium on metabolite profile of the leaves of salt tolerant rice seedlings of variety of Nonabokra under sodium chloride induced salinity, by Gas Chromatography–Mass Spectrometry-based metabolomics approach. Calcium availability in the seedlings was reduced or enhanced applying inhibitors (vanadyl sulfate, lanthanum chloride, and verapamil) or promoters of calcium influx (calcimycin also known as calcium ionophore A23187) in the sodium chloride (100 mM) supplemented growth medium. Growth medium of ten-day-old seedlings was replaced by sodium chloride supplemented hydroponic solution with promotor or inhibitors of calcium channel. Fifteen days old seedlings were harvested. It was observed that depletion of calcium availability increased the level of serotonin and gentisic acid whereas increased calcium level decreased these metabolites. It was concluded from the results that production of the signaling molecules serotonin and gentisic acids was elevated in calcium-deficient seedlings under salt stress the condition that was considered as control during the experiment. The two signaling molecules probably help this tolerant rice variety Nonabokra to withstand the salt-induced adversities.

Excess sodium chloride (NaCl) induced salinity in the soil, one of the main abiotic stresses, adversely affects plant growth. Calcium deficiency symptoms are common when the sodium/calcium ratio is high in soil water. Excess sodium in the soil causes development of osmotic stress and disrupts cell ion homeostasis by inducing accumulation of sodium and inhibiting uptake of calcium and potassium.¹ This leads to oxidative stress, nutrition deficiency, hindrance of enzymatic activities, instability of cell membrane, retarded growth, and cell death.² Thus, high levels of sodium in the soil affect plant growth in salt-sensitive plants. On the contrary, many studies revealed a rapid increase of calcium in the cytosol on exposure to sodium chloride.³ However, from different experimental results, it was also observed that the change of calcium in the cytosol is not uniform and varies with species, the plant organs where they are measured such as root, shoot tissues, or cell types.⁴ Sodium toxicity may induce different signals in roots and shoots. It is likely that salinity stress elicits differential calcium signatures that vary between salinity tolerant and sensitive plant species.⁴

Rice (Oryza sativa L.) is extremely sensitive to salinity and most salt-sensitive cereal crop.⁵ Salt tolerant genotypes of rice use mechanisms in sensing and signaling salt stress, regulating transcription, ionic, and osmotic homeostasis and inhibiting reactive oxygen species (ROS).⁶ Exogenous application of calcium enhanced photosynthetic abilities, overall growth performances in salt-stressed rice seedlings in Pokkali (a salt tolerant variety) and IR29 (a susceptible variety).⁷ Exogenous supplementation of calcium improved ion homeostasis, ROS production and methylglyoxal detoxification in salt stressed rice BRRI dhan 47, a salt tolerant rice.⁸ Studies with salt tolerant varieties, for example, Binadhan-10 and Binadhan-7 showed that application of calcium-rich fertilizers in saline soil was effective to cultivate these rice varieties enhancing shoot growth and oppressing ROS.⁹ Exogenous calcium application improved salinity tolerance in BRRI Dhan 28 (a salt susceptible cultivar) in sandoponic culture with Hoagland’s nutrient solution improved germination indices, growth, and biomass-related parameters.¹⁰

Our study intended to understand the effect of NaCl-induced stress on plant metabolism in salt tolerant and sensitive varieties. Earlier work revealed elevation of signaling molecules serotonin and gentisic acid in the seedling leaves of the salt-tolerant rice varieties Nonabokra and Bhutnath growing under saline condition. But in salt-sensitive varieties, these signaling molecules were not biosynthesized.¹¹ In the present study, attempt was made to understand the role of calcium on metabolite profile of the leaves of a salt tolerant rice variety Nonabokra under sodium chloride–induced salinity, by GC-MS-based metabolomics approach.

1. Materials and methods

1.1. Seedling growth

The seeds of a salt tolerant variety Nonabokra were collected from Rice Research Station Chinsurah, Directorate of Agriculture, Government of West Bengal, India. The seeds, after surface sterilization with 5% sodium hypochlorite followed by washing with distilled water, were germinated in dark on moistened filter paper in petridishes at 32 ± 2°C for 3 days. Then, the seedlings were transferred to hydroponic solution and grown in photoperiodic condition of 12 h light and 12 h darkness with 135 μmol m-² s-¹ proton flux intensity and 32 ± 2°C temperature as mentioned previously.¹¹ The hydroponic solution consisted of 2 mM Ca(NO₃)₂, 5 mM KNO₃, 5 mM NH₄NO₃, 2 mM MgSO₄, 0.1 mM KH₂PO₄, 0.5 mM Na₂SiO₃, 0.05 mM NaFe(III)EDTA, 5 μM MnCl₂, 5 μM ZnSO₄, 0.5 μM CuSO₄, 0.1 μM NaMoO₃, and 5 μM H₃BO₃. The germinated seedlings were placed near the rim of the culture tubes (inside layered with blotting paper) containing 40-ml hydroponic solution to allow growth. Two seedlings were grown in each culture tube. The growth medium of 10 days old seedlings was replaced by hydroponic solution containing 100 mM NaCl (control). To study the role of calcium depletion on salinity stress, medium considered as control was supplemented with 10 mM of vanadyl sulphate (VOSO₄) or 3 mM of lanthanum chloride (LaCl₃) or verapamil (1 mM, 3 mM, and 5 mM). To study the role of increased calcium, nutrient medium supplemented with NaCl was added either with CaCl₂ (10 mM, 20 mM, and 40 mM) or calcimycin (calcium ionophore A23187). In all the treatment sets, along with control, seedlings were grown for five more days. Leaves of 15 days old seedlings were harvested, and crushed in liquid nitrogen.

1.2. Extraction

Leaf tissues (120 ± 20 mg) homogenized in liquid nitrogen with added internal standard ribitol (20 μl of 0.2% aqueous solution) was extracted with 50% aqueous methanol at 60°C for 30 min. Extracts were centrifuged at 12,000 rpm for 10 min, and then 150 μl of aliquots was distributed to tubes and evaporated to dryness. The process was repeated for five biological replicates of each treatment.

1.3. Derivatization

Each extract was derivatized for 90 min with 5 μl of 20 mg/ml methoxyamine hydrochloride in pyridine, and then with 45 μl of N-Methyl-N-(trimethylsilyl)trifluoroacetamide for trimethylsilylation of acidic protons to volatilize the metabolites. Then, 1 μl of fatty acid methyl ester (FAME) markers [a mixture of internal retention index (RI) markers was prepared by adding fatty acid esters of C8, C10, C12, C14, C16, C18, C20, C22, C24 and C26 linear chain length] dissolved in chloroform was added.¹² Agilent 7890 gas chromatography [software driver version 4.01(054)] equipped with 5975 inert MSD with triple axis detector with HP-5 MS capillary column [Agilent J & W; GC column (USA) of dimensions 30 m × 0.25 mm × 0.25 μm] was used for GC-MS analysis. Standardized method used previously¹¹ was followed for analysis of a 37.5 min run. Mass spectra were recorded from 50 to 500 m/z. The analysis for compound identification was done following users’ guide using automated mass spectral deconvolution and identification system (AMDIS) calibrated with the method available in Fiehns library to deconvolute the results for identifying chromatographic peaks. Retention time (RT) in Agilent Fiehn GC-MS metabolomics RTL library (Agilent Technologies, USA, 2008) and mass spectral fragmentation pattern was compared for identification of peaks.

1.4. Statistical analyses

For normalization, compound peak areas were divided by the peak area of ribitol (internal standard) and sample fresh weight (g) to obtain relative response ratio. Missing values were replaced by half of the detection limit before normalization.¹³ The data obtained were exported to Metaboanalyst 3.6 for different multivariate analysis such as Partial Least Squares Discriminant Analysis (PLS-DA). Other calculations were made using Xcel sheet.

2. Results and discussion

In continuation to our previous work,¹¹ we tried to understand the role of calcium in production of serotonin, gentisic acid and other salt-stress-induced metabolites. We considered Nonabokra variety of rice as experimental material as it is one of the most tolerant varieties of indica rice¹⁴ and a donor of salinity tolerance in classical breeding.¹⁵ As serotonin was detected only after NaCl induced stress in the tolerant varieties (Nonabokra and Bhutnath), the experimental control set contained NaCl (100 mM). CaNO₃ was the source of calcium in the hydroponic solution. Calcium levels were modulated by adding vanadyl sulfate (VOSO₄), lanthanum chloride (LaCl₃), verapamil, different concentrations of calcium chloride (CaCl₂), and calcimycin (calcium ionophore A23187). Different concentrations of CaCl₂ (10 mM, 20 mM, and 40 mM) were added to the medium (control) to increase the level of calcium. Calcium influx was induced by applying the antibiotic calcimycin that increases the concentration of cytosolic calcium.¹⁶ Availability of calcium to the seedlings was reduced by adding the calcium channel blockers LaCl₃¹⁷ and verapamil.¹⁸ During the experiment VOSO₄ was used for reducing the level of cytosolic calcium as vanadium is known to inhibit plasma membrane Ca2+-ATPase, a calcium pump.¹⁹ Leaf samples were harvested from differently treated plants and the metabolites were analyzed after extraction and derivatization. The metabolic data (relative response ratio per g fresh weight of leaf) were then analyzed using PLS-DA method to extract information regarding the changes happening in response to components used for depletion of cytosolic calcium, increasing the level of calcium, and compared with that of control. PLS-DA (Figure 1a) distinctly separated three groups, that is, control, calcium depleted sets, and samples with increased levels of calcium. Out of total 87 metabolites detected, the top 10 contributory metabolites responsible for the separation of the three clusters were identified (Figure 1b) from weighted sum of absolute regression coefficients (coeff.), an important measure of PLS-DA. Interestingly, the metabolites of importance were mostly shikimic acid pathway-derived phenolic metabolites such as serotonin, gentisic acid, phloroglucinol, benzoic acid, and 4-hydroxy-3-methoxybenzoic acid (vanillic acid). Other important metabolites were sugars (lactobionic acid, maltose, and glycerol 1-P), organic acid (nicotinic acid), and fatty acid (myristic acid).

Figure 1.

(a): PLS-DA score plot; (b): Weighted sum of absolute regression coefficients

The increased or decreased levels of the metabolites of importance in the three sets of experiments were compared graphically (Figure 2, Figure 3). The level of glycerol-1-phosphate either decreased significantly (with VOSO₄) or did not show significant changes with the inhibitors of cytosolic calcium. But increased level of calcium showed a trend to increase this metabolite. Other non-phenolic metabolites did not show any correlation in their level based on increased or decreased levels of calcium (Figure 2). However, all the important phenolic metabolites identified from such analysis were those previously reported to be increased by salt stress in Nonabokra.¹¹ The metabolites were shikimic acid pathway-derived metabolites. The metabolites were mapped on to the metabolic pathway (Figure 3). It was observed that the levels of all those phenolic metabolites, including serotonin and gentisic acid, increased with decreasing level of calcium using different types of inhibitors affecting calcium influx with respect to control. On the contrary, such metabolite contents decreased or showed no significant changes with increased level of calcium with respect to control. The two signaling molecules serotonin and gentisic acid also showed the same trend. Increased level of calcium significantly decreased the biosynthesis of the signaling molecule serotonin. Biosynthesis of gentisic acid and other related phenols either decreased or showed no significant changes when calcium level was increased. It was also noticed that the calcium channel modulators did not affect the primary metabolites adversely (Supplementary Table 1). Vanadium sulfate increased organic acids of the tricarboxylic acid cycle, and most of the amino acids statistically significantly over that of control. LaCl₃ also increased the metabolites (not statistically significant). Lower concentrations of verapamil (1 mM, 3 mM) either increased (not significant) or did not show any change in the levels of primary metabolites. However, verapamil, at 5 mM concentration, increased many of the metabolites significantly. Calcimycin, applied to increase the cytosolic calcium, either showed no change in metabolite level or did not show significant increase or decrease of the metabolites over that of control. Exogenous application of CaCl₂, at concentration 20 mM and 40 mM, showed some significant increases in metabolite levels. Otherwise, the changes were statistically insignificant. From the supplementary Table 1, it is also apparent that in the presence of the calcium channel inhibitors vanadium, LaCl₃ and verapamil (particularly at 5 mM concentration) the levels of metabolites were higher than that of control, indicating better primary metabolism. Under these circumstances the levels of serotonin and gentisic acid also increased. The two signaling molecules were not formed in the presence of calcium chloride or calcimycin when the level of primary metabolites were also low.

Figure 2.

Changes in non-phenolic metabolite levels with inhibitors and promoters of cytosolic calcium

*Significant increase, # Significant decrease

Figure 3.

Mapping the important phenolic metabolites on the metabolite pathway

* Significant increase, # Significant decrease

Serotonin (5-hydroxy tryptamine), a conserved indoleamine derived from tryptophan, is a signaling molecule that mediates stress tolerance in plants. Serotonin and the auxin indole acetic acid share common biosynthetic precursor. Serotonin regulates gene expression partially inhibiting auxin biosynthesis and transport.²⁰ Serotonin accumulated in senescing rice leaves to attenuate the process. Its abundance in vascular parenchyma, including companion cells and xylem parenchyma, suggests its role in facilitating nutrient recycling and maintaining cellular integrity.²¹ Serotonin attenuated biotic stress acting as a scavenger of oxygen radicals to protect uninfected cells against damage caused by hypersensitive response in rice K59, transformants of Nipponbare cultivar of rice infected by Magnaporthe oryzae.²² In Heliathus annuus L. seedlings, differential spatial distribution of serotonin in roots and cotyledons during salt stress was reported. Root growth inhibition induced by salt stress was caused by increased serotonin biosynthesis, impairing auxin functions.²³ Exogenous application of serotonin ameliorated salinity induced toxicity by scavenging ROS, regulating osmotic pressure in Brassica napus L. seedlings.²⁴ It was previously reported in Nonabokra variety of rice that the leaf length and leaf fresh weight decreased with increase in NaCl concentration in the growth medium.¹¹ Present results also show that leaf length decreased and serotonin level increased when inhibitors of calcium uptake were used in the growth medium. Leaf length increased (not significantly) (Table 1) and serotonin level decreased with use of calcimycin, to increase cytosolic calcium. Decrease in leaf length with increase in serotonin level may be due to suppressed auxin formation. Increased serotonin level plausibly helped reducing oxidative stress in Nonabokra. Further studies, in this regard, are necessary.

Table 1.

Changes in leaf length in response to treatment

Treatment	Leaf length (cm)
	Mean	±	se	p value
Control	20.88	±	0.79
VOSO4 (10 mM)	13.75	±	0.53 ↓	0.00
LaCl3 (3 mM)	17.50	±	1.01 ↓	0.02
Verapamil (1 mM)	18.50	±	1.17 ↓	0.13
Verapamil (3 mM)	16.38	±	1.18 ↓	0.01
Verapamil (5 mM)	15.50	±	0.70 ↓	0.00
CaCl2 (10 mM)	15.92	±	0.72 ↓	0.00
CaCl2 (20 mM)	15.75	±	0.94 ↓	0.00
CaCl2 (40 mM)	17.27	±	0.81 ↓	0.01
Calcimycin (0.38 µM)	22.06	±	0.26 ↑	0.17

↓: Decrease; ↑: Increase.

Gentisic acid (2,5-dihydroxybenzoic acid), a common aromatic acid in green plants and its biosynthetic precursor salicylic acid are defense-signaling molecules.²⁵ Gentisic acid accumulated in Lycopersicon esculentum cv. Rutgers in response to CEVd and ToMV infections, as a pathogen, induced signal for activation of plant defense genes.²⁶ Gentisic acid (found mostly in a conjugated β-glucoside form) could also be detected in Cucumis sativus L. and Gynura auriantiaca infected with prunus necrotic ring spot virus (PNRSV) and exocortis viroid (CEVd), respectively.²⁷ Gentisic acid 5-O-β-D-xylopyranoside was also detected in Cucumis sativus and Lycopersicon esculentum plants infected with different pathogens.²⁸ Both gentisic acid and salicylic acid accumulated in Lycopersicon esculentum and Gynura auriantiaca infected by RNA pathogens and induced resistance to the pathogens by pre-induction of RNA-silencing-related genes.²⁹ Salt stress also induced production of gentisic acid in salt-tolerant varieties (Nonabokra and Bhutnath) of rice.¹¹ However, their role in salt tolerance is still unknown. The present study finds a correlation between increase in gentisic acid level with application of calcium channel blockers. Further experiments using calcium chelators would give us some more insights into this regard.

3. Conclusion

From the findings of the present study, it could be concluded that sodium chloride–-induced depletion of calcium level is probably responsible for the changes in phenolic metabolism with elevated production of the signaling molecules gentisic acid and serotonin and some other phenolic compounds in salt tolerant rice Nonabokra. We came to this conclusion because further depletion of calcium by using inhibitors of calcium influx significantly increased the level of these two signaling molecules. These two signaling molecules probably helped this tolerant rice variety Nonabokra to withstand the salt induced adversities and confer stress tolerance to a certain extent. Future research should emphasize to explore the role of serotonin and gentisic acid in the varieties of tolerant rice to salt stress. As the percentage of salt affected irrigating land areas are increasing,³⁰ this salt tolerant variety could be used to develop other salt-tolerant varieties through plant breeding for sustainable agriculture and food security.

Funding Statement

This work was supported by West Bengal State Council of Science and Technology (grant number 104/WBSCST/F/0520/14) and Department of Science and Technology, Government of India (FIST programme) for instrumental facility.

Disclosure Statement

No potential conflicts of interest were disclosed.

Supplementary material

Supplemental data for this article can be accessed on the publisher’s website