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Differential regulation of defence pathways in aromatic and non-aromatic indica rice cultivars towards fluoride toxicity

  • Aditya Banerjee
  • Aryadeep RoychoudhuryEmail author
Original Article

Abstract

Key message

Excessive bioaccumulation of fluoride in IR-64 caused low abscisic acid level, inhibition of polyamine biosynthesis and ascorbate–glutathione cycle but not in Gobindobhog which had higher antioxidant activity.

Abstract

The current study presents regulation of diverse metabolic and molecular defence pathways during fluoride stress in non-aromatic rice variety, IR-64 and aromatic rice variety, Gobindobhog (GB). Increasing concentration of fluoride affected fresh weight, dry weight, vigour index and relative water content to a lesser extent in GB compared to IR-64. GB exhibited lower methylglyoxal accumulation and lipoxygenase activity compared to IR-64 during stress. The level of osmolytes (proline, amino acids and glycine-betaine) increased in both the stressed varieties. The biosynthesis of higher polyamines was stimulated in stressed GB. IR-64 accumulated higher amount of putrescine due to degradation of higher polyamines as supported by gene expression analysis. Unlike IR-64, GB efficiently maintained the ascorbate–glutathione cycle due to much lower fluoride bioaccumulation, compared to IR-64. GB adapted to fluoride stress by strongly inducing guaiacol peroxidase, phenylalanine ammonia lyase and a novel isozyme of superoxide dismutase. While GB accumulated higher abscisic acid (ABA) level during stress, IR-64 exhibited slow ABA degradation which enabled induction of associated osmotic stress-responsive genes. Unlike GB, ABA-independent DREB2A was downregulated in stressed IR-64. The research illustrates varietal differences in the defence machinery of the susceptible variety, IR-64, and the well adapted cultivar, GB, on prolonged exposure to increasing concentrations of fluoride.

Keywords

Fluoride Injuries Osmolytes Antioxidants Abscisic acid Defence mechanism Rice 

Introduction

The World Health Organization (WHO) has prohibited fluoride ingestion beyond the safe limit of 1.5 mg L−1. Higher doses of fluoride cause fluorosis and neurological disorders in animals and humans (Choubisa 2013). As high as 48 mg L−1 of fluoride has been detected across Indian states (Susheela 1999). Fluoride is an accumulative poison in plants and reduces the overall biomass by triggering oxidative stress (Banerjee and Roychoudhury 2019a). Recent reports have verified the toxic effects of fluoride in plant species such as Cajanus cajan, Carthamus tinctorius, Camellia sinensis and Pteris ensiformis (Yadu et al. 2017, 2019; Ghassemi-Golezani and Farhangi-Abriz 2019; Das et al. 2017). Oxidative stress usually stimulates uncontrolled production of reactive oxygen species (ROS) which along with increased lipoxygenase (LOX) activity triggers membrane lipid peroxidation (Banerjee and Roychoudhury 2019b; Che-Othman et al. 2017). The cytotoxic reactive compound methylglyoxal (MG) is produced abundantly during abiotic stresses (Banerjee and Roychoudhury 2018). Plants recruit elaborate antioxidant machinery to counteract the overproduction of ROS. Such machinery may involve enzymes such as superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (GPOX), glutathione peroxidase (GPX), ascorbate peroxidase (APX), glutathione-S-transferase (GST), enzymes of ascorbate–glutathione cycle [monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR) and glutathione reductase (GR)], and non-enzymatic antioxidants such as anthocyanin, phenolics, flavonoids, β-carotene, xanthophylls, ascorbic acid (AsA), and glutathione (GSH) (Roychoudhury and Basu 2012; Das and Roychoudhury 2014). Methylglyoxal is detoxified via glyoxalase system, which constitutes two enzymes, glyoxalase I (gly I) and glyoxalase II (gly II) (Hong et al. 2016). In addition, plants are also able to synthesize certain compatible solutes such as proline (Pro), amino acids, reducing sugars, glycine-betaine (gly-bet) and polyamines (PAs) such as putrescine (Put), spermidine (Spd) and spermine (Spm), the accumulation of which has been shown to confer stress resistance to the plants by serving as a membrane stabilizer, transient source of both carbon and nitrogen, and direct quencher of free radicals (Paul et al. 2017). The enzymes such as Δ1-pyrroline-5-carboxylate synthetase (P5CS) and proline dehydrogenase (PDH), responsible for Pro metabolism, betaine aldehyde dehydrogenase 1 (BADH1) for gly-bet production, spermidine synthase (SPDS), spermine synthase (SPMS), diamine oxidase (DAO) and polyamine oxidase (PAO), involved in polyamine metabolism have been reported to play major roles in regulating tolerance against multiple environmental stresses (Paul et al. 2017). The transcript level as well as the activity of phenylalanine ammonia lyase (PAL), producing flavonoids from phenylalanine, is increased in plants exposed to varied abiotic stress conditions (Wada et al. 2014).

Any form of abiotic stress also triggers the accumulation of universal stress phytohormone abscisic acid (ABA) in plants. The 9-cis-epoxycarotenoid dioxygenase 3 (NCED3) is the rate-limiting enzyme of ABA synthesis, whereas ABA-8-oxidase1 (ABA8ox1) is involved in ABA catabolism (Roychoudhury and Banerjee 2017). The osmotic stress-responsive genes such as Responsive to ABA 16A (Rab16A), Oryza sativa embryonic abundant (Osem) [encoding dehydrins], TRAB1, WRKY71 and dehydration-responsive element binding 2A (DREB2A) [encoding transcription factors] are predominantly upregulated in rice exposed to different environmental stresses (Roychoudhury et al. 2013).

Rice is a water-intensive staple food crop largely cultivated across the fluoride-infested states of India and Bangladesh. Based on the metabolites and genes discussed above, the aim of the manuscript was to characterize the contrasting defence mechanism in two varieties of indica rice, viz., IR-64 (non-aromatic) and Gobindobhog (GB) (aromatic), exposed to long-term fluoride stress for 20 days, using two different NaF concentrations, viz., 15 mg L−1 and 25 mg L−1.

Materials and methods

Plant materials, growth conditions and stress treatment

The seeds of Oryza sativa L. cv. IR-64 and aromatic cv. GB were procured from Chinsurah Rice Research Station (Hooghly, West Bengal, India) and Bidhan Chandra Krishi Viswavidyalaya (Nadia, West Bengal, India), respectively. The sterilized seeds of IR-64 and GB were placed over sterile gauge in Petri dishes (three sets for each variety). One set was treated with double-distilled water. This was considered as the control. Two other sets for each variety were treated with different concentrations of NaF, i.e., 15 mg L−1 and 25 mg L−1. These seedlings were regarded to be under fluoride stress. The seedlings of both the varieties (in three sets) were maintained and grown for a total period of 20 days at 32 °C under 16-h light and 8-h dark photoperiodic cycle with 50% relative humidity and 700 μmol photons m−2 s−1 in a plant growth chamber (NIPPON, LHP-100-RDS, Tokyo, Japan) as previously standardized (Roychoudhury et al. 2008).

Analysis of physiological and biochemical parameters: seedling biomass, vigour index, relative water content (RWC), methylglyoxal (MG), protein carbonylation, lipoxygenase (LOX, EC 1.13.11) and ascorbic acid oxidase (AAO, EC 1.10.3.3) activity

Fresh weight (FW) was measured separately from 50 seedlings of each set. The tissues were thereafter dried at 80 °C for two days and the dry weight (DW) was documented. Vigour index was calculated as: [(root length + shoot length) × percentage of germination] (Vijaya Geetha et al. 2014). RWC was calculated from freshly harvested leaves as [{(FW-DW)/(turgid weight-DW)} × 100] (Barr and Weatherley 1962). MG content was measured spectrophotometrically according to Banerjee et al. (2019). Protein carbonylation was measured spectrophotometrically by reaction with dinitrophenyl hydrazine following Levine et al. (1994) and Reznick and Packer (1994). Lipoxygenase activity was estimated by observing the increase in absorbance at 234 nm after incubating the extract with linoleic acid following Basu et al. (2012). The AAO activity was recorded according to Pignocchi et al. (2003). Oxidation of one μmol l-ascorbic acid min−1 at 265 nm was considered as one unit of AAO activity.

Estimation of fluoride content in rice seedlings

0.2 g of total seedlings was dried at 80 °C overnight. The dried tissue (devoid of water) was homogenized in TISAB buffer and the homogenate was mineralized by boiling on a water bath for 30 min. The mineralized solution was then centrifuged at 12,000×g for 20 min and the supernatant was carefully extracted and filtered through sterile Miracloth (Merck Millipore USA). The filtered extract was used for analyzing fluoride bioaccumulation. Fluoride content in total seedlings was measured using a fluoride-sensitive electrode (Cole-Parmer, USA).

Analysis of osmolyte contents: proline (Pro), glycine-betaine (gly-bet), total amino acids and polyamines (PAs)

For measuring Pro content, 0.5 g of freshly harvested seedlings was crushed in 3% (w/v) aqueous sulfosalicylic acid. The filtrate was mixed with freshly prepared acid ninhydrin and glacial acetic acid, and the absorbance was measured at 520 nm (Roychoudhury et al. 2011). Pro content was determined from a standard curve generated using known concentrations of Pro. For measuring gly-bet, the aqueous extract of 0.5 g freshly harvested tissue was diluted with equal volume 2 N sulphuric acid and cooled on ice for 1 h. Next, ice cold Lugol’s iodine was added and the mixture was incubated overnight at 4 °C to allow the periodide crystals to precipitate. The crystals were dissolved in 1, 2-dichloroethane and the absorbance was determined at 365 nm (Grieve and Grattan 1983). Gly-bet content was determined from a standard curve generated using known concentrations of gly-bet. Total amino acid level was quantified from 0.5 g of freshly harvested tissue using freshly prepared ninhydrin according to Moore (1968). Total amino acid content was determined from a standard curve generated using known concentrations of Pro. For the estimation of PAs, the freshly harvested seedlings were crushed in ice cold 10% (v/v) perchloric acid and the homogenate was allowed to react with dansyl chloride overnight in dark for efficient dansylation of the amines. After extraction of the dansylated amines using toluene, putrescine (Put), spermidine (Spd) and spermine (Spm) levels were detected by running known standards on silica gel thin-layer chromatography plate (Merck Millipore USA). The fluorescent spots corresponding to the above PAs were quantified by fluorescence spectrophotometry using 360 nm as the excitation wavelength and 506 nm as the emission wavelength (Roychoudhury et al. 2008).

Analysis of non-enzymatic antioxidants

Estimation of anthocyanin, flavonoids, carotene, xanthophyll, thiamine, riboflavin and phenolic content

The anthocyanin content was recorded at 525 nm following Roychoudhury et al. (2007). Determination of flavonoids was performed according to Quettier et al. (2000) and assayed at 415 nm. Flavonoid content was derived from a standard curve generated with increasing concentrations of rutin. The carotene and xanthophyll were separated from the extract crushed in 80% (v/v) acetone following Davies (1965). For thiamine estimation, 0.2 g of freshly harvested tissue was crushed in 2 ml of 0.1 N sulphuric acid. 0.5 ml of the extract was mixed with 0.5 ml of 15% (w/v) sodium hydroxide and 0.1 ml of 1% (w/v) potassium ferricyanide, and was assayed spectrofluorometrically with an excitation wavelength of 310 nm and emission wavelength of 475 nm. Riboflavin content was estimated spectrofluorometrically according to Radzuan and Sulaiman (2017). Total phenolic content (TPC) of the samples was determined according to Basu et al. (2012) and the absorbance was measured at 760 nm.

Estimation of ascorbic acid (AsA) content, glutathione redox state and DPPH radical scavenging activity

The AsA content was determined spectrophotometrically according to Khan and Panda (2008) with minor modifications at 660 nm. Reduced glutathione (GSH) and total glutathione contents were derived spectrophotometrically using Ellman’s reagent according to Bonifacio et al. (2011) and Moron et al. (1979), respectively. Glutathione redox state was determined as [(GSH/total glutathione) × 100]. The DPPH radical scavenging activity was performed using 0.004% (v/v) methanolic solution of DPPH according to Basu et al. (2012).

Analysis of antioxidative enzyme activity: superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), guaiacol peroxidase (GPOX, EC 1.11.1.7), ascorbate peroxidase (APX, EC 1.11.1.11), monodehydroascorbate reductase (MDHAR, EC 1.6.5.4), dehydroascorbate reductase (DHAR, EC 1.8.5.1), glutathione reductase (GR, EC 1.8.1.7), glutathione peroxidase (GPX, 1.11.1.9) and phenylalanine ammonia lyase (PAL, EC 4.3.1.24)

The SOD activity was determined from 0.5 g freshly harvested tissue according to Alonso et al. (2001). The amount of enzyme which inhibited 50% of the initial rate of reaction in the absence of enzyme was considered as 1 U of SOD activity. The CAT activity was measured by assaying the decomposition of H2O2 at 240 nm (Velikova et al. 2000). The GPOX activity was determined on the basis of the formation of tetraguaiacol at 470 nm (Srinivas et al. 1999). The APX activity was measured by observing the oxidation of ascorbate (Nakano and Asada 1981). The MDHAR, DHAR and GR activities were performed according to Colville and Smirnoff (2008) with minor modifications. The GPX assay was performed by documenting the decrease in NADPH at 340 nm (Awasthi et al. 1975). The PAL activity was measured by recording the formation of trans-cinnamic acid at 290 nm (Cheng and Breen 1991).

In-gel analysis of isozymes for SOD, GPOX, APX and CAT

Isozyme analysis of antioxidative enzymes was performed through electrophoresis in native polyacrylamide gel. The SOD isozymes were detected by incubating the gel in freshly prepared staining buffer containing 50 mM phosphate buffer (pH 7.0), 0.1 mM EDTA, 28 mM TEMED, 3 μM riboflavin and 0.25 mM NBT under dark conditions. The bands were visualized by keeping the gel submerged in staining buffer under white light as standardized by Roychoudhury et al. (2016). The GPOX isozymes were visualized by staining the gel in 50 mM potassium phosphate buffer (pH 7.0) containing 0.46% (v/v) guaiacol and 13 mM H2O2. Upon gentle shaking in dark, the GPOX isoforms appeared as dark brown bands on a colourless background. For detection of APX isozymes, the gel was equilibrated in 50 mM phosphate buffer (pH 7.0) and 2 mM sodium ascorbate for 30 min after which the gel was incubated in 50 mM phosphate buffer (pH 7.0) containing 4 mM ascorbate and 2 mM H2O2 for 20 min. Next, the gel was washed in 50 mM phosphate buffer (pH 7.0) for 1 min and incubated under white light in 50 mM phosphate buffer (pH 7.8), 28 mM TEMED and 2.45 mM NBT. The APX isozymes appeared as achromatic bands on a purple background (Roychoudhury et al. 2016). The isozymes of CAT were detected by incubating the gel in 0.05% (v/v) H2O2, 2% (w/v) ferric chloride and 2% (w/v) potassium hexacyanoferrate (III) as previously standardized (Roychoudhury et al. 2016).

Measurement of endogenous abscisic acid (ABA) content

0.1 g of freshly harvested tissue was crushed in 4 ml of lysis buffer, containing potassium dihydrogen phosphate and dipotassium hydrogen phosphate as the solution stabilizers. The homogenate was centrifuged at 12,000×g for 10 min and the supernatant was carefully retrieved in a fresh sterile tube. The extract was filtered using sterile Miracloth (Merck Millipore USA). The filtered extract was diluted with phosphate buffer saline, and ABA content was measured by competitive enzyme-linked immunosorbent assay (ELISA) at 405 nm using ABA immunodetection kit (Sigma-Aldrich, USA) following the manufacturer’s protocol. The colour developed was due to the formation of p-nitrophenol from the substrate p-nitrophenyl phosphate and intensity of the colour was inversely proportional to the amount of endogenous ABA. The actual concentration of ABA was determined from a standard curve generated using known concentrations of the ABA standard provided in the kit.

Expression analysis of genes

The seedlings (100 mg) from each of the experimental sets were used for RNA isolation. About 5 μg of total RNA was reverse transcribed and comparative RT-PCR analysis was performed using gene-specific primers and standard reagents, with β-actin as internal control as described earlier (Paul and Roychoudhury 2018).

Protein estimation and statistical analysis

Equal amount of total protein was loaded for all the experiments following Bradford (1976). Statistical analyses were performed in completely randomized design using three replicates (n = 3). Each replication contained average of 50 seeds. Standard error (SE) and statistical significance were calculated using two-way analysis of variance (ANOVA) at p ≤ 0.05 in XLSTAT 2018.

Results

Analysis of damage indices and associated gene expression

Higher concentrations of fluoride triggered prominent shoot tip burn and chlorosis in the seedlings of IR-64 (Fig. 1a). In case of GB seedlings, the extent of such visual damages during exposure to high fluoride stress was observed to be lower compared to that in IR-64 (Fig. 1a). Higher concentrations of NaF triggered secondary root growth in both the cultivars compared to the control sets. Secondary root growth was maximum and most vigorous in case of IR-64 seedlings supplemented with 25 mg L−1 NaF (Fig. 1b). The FW and DW of seedlings decreased with increasing concentration of NaF in IR-64, whereas these remained almost constant in case of control and treated sets of GB (Table 1). The vigour index decreased in both the varieties during 15 mg L−1 and 25 mg L−1 fluoride stress (Table 1). The reduction in RWC was less predominant in GB compared to IR-64 (Table 1).
Fig. 1

Photographs showing the visual effects of increasing fluoride stress on seedling leaf tip burn and chlorosis (a) and root morphology (b) in IR-64 and GB seedlings grown with double-distilled water (control), 15 mg L−1 NaF and 25 mg L−1 NaF

Table 1

Physiological and molecular damage indices, level of osmolytes and non-enzymatic antioxidants, antioxidant enzyme activity, and ABA

 

Parameters

IR-64 Control

IR-64 (15 mg L−1 NaF)

IR-64 (25 mg L−1 NaF)

GB Control

GB (15 mg L−1 NaF)

GB (25 mg L−1 NaF)

Physiological parameters

Fresh weight (mg)

122.5 ± 2.5

103.0 ± 2.0*

91.0 ± 2.0*

97.0 ± 1.7#

94.0 ± 1.5

93.5 ± 1.0

Dry weight (mg)

51.5 ± 1.5

41.5 ± 0.5*

37.0 ± 2.0*

39.5 ± 0.5#

38.5 ± 0.5

38.5 ± 1.7

Vigour index

1920.7 ± 9.8

1441.5 ± 15.5*

1229.8 ± 14.3*

1911.0 ± 19.5

1464.1 ± 7.8

1308.0 ± 21.4*

Relative water content

97.9 ± 2.5

82.9 ± 1.3*

73.9 ± 4.3*

94.7 ± 4.5

91.0 ± 3.1#

89.1 ± 2.2*#

Molecular damage indices

Fluoride accumulation (mg g−1 FW)

0.7 ± 0.1

3.8 ± 1.4

39.5 ± 5.6*

0.25 ± 0.1

6.1 ± 1.8*#

6.9 ± 1.5*#

Methyl-glyoxal (mg g−1 FW)

121.7 ± 1.2

131.0 ± 3.4*

152.8 ± 8.3*

73.9 ± 1.5#

77.8 ± 0.5#

104.1 ± 5.4*#

Protein carbonylation (mmol carbonyl mol−1 BSA)

301.2 ± 20.0

1812.3 ± 80.1*

1903.2 ± 10.4

209.5 ± 30.8

2501.9 ± 30.3*#

2418.6 ± 37.6

Lipoxygenase activity (U μg−1 FW)

12.4 ± 0.3

12.6 ± 1.1

13.4 ± 2.3

7.9 ± 1.2#

10.2 ± 0.5

12.8 ± 1.1*

Ascorbic acid oxidase activity (mU g−1 FW)

16.4 ± 2.7

5.3 ± 2.1*

6.7 ± 1.9

5.8 ± 0.9#

7.8 ± 2.3

9.1 ± 1.5*

Osmolytes

Proline (μg g−1 FW)

9.8 ± 0.6

12.1 ± 1.5

13.7 ± 0.7*

9.9 ± 0.8

16.5 ± 2.3*#

17.2 ± 1.2#

Glycine-betaine (mg g−1 FW)

30.9 ± 1.3

23.5 ± 1.8*

28.7 ± 0.8*

19.4 ± 2.1#

17.1 ± 3.4#

23.2 ± 2.1*#

Total amino acids (μg g−1 FW)

31.5 ± 2.3

33.4 ± 1.4

34.9 ± 0.7*

18.6 ± 0.6#

28.7 ± 4.2*

24.3 ± 2.4#

Polyamines

Putrescine (nmol g−1 FW)

1.6 ± 0.2

1.7 ± 0.5

2.9 ± 0.4*

2.9 ± 0.1#

2.4 ± 0.3

1.9 ± 0.1*#

Spermidine (nmol g−1 FW)

3.1 ± 0.9

1.8 ± 0.3*

1.7 ± 0.8

2.1 ± 0.2#

2.8 ± 0.7#

5.2 ± 1.3*#

Spermine (nmol g−1 FW)

1.7 ± 0.2

1.4 ± 0.2

1.1 ± 0.1*

1.4 ± 0.1

1.6 ± 0.2

1.9 ± 0.4*#

Non-enzymatic antioxidants

Anthocyanin (mM g−1 FW)

192.5 ± 10.5

172.6 ± 5.8*

218.7 ± 9.8*

65.8 ± 8.1#

237.3 ± 10.6*#

294.5 ± 11.8*#

Flavonoids (μg g−1 FW)

210.1 ± 4.8

193.8 ± 8.2

165.6 ± 12.2*

71.8 ± 5.1#

103.1 ± 2.8*#

117.2 ± 8.9#

β-carotene (μM g−1 FW)

19.4 ± 2.3

54.1 ± 3.4*

102.5 ± 10.1*

89.1 ± 8.7#

114.1 ± 12.2*#

150.5 ± 9.1*#

Xanthophyll (μM g−1 FW)

17.8 ± 2.1

25.2 ± 3.2*

23.9 ± 1.8

9.6 ± 1.5#

14.9 ± 2.2*#

37.1 ± 3.4*#

Thiamine (mg g−1 FW)

0.73 ± 0.03

0.61 ± 0.08

0.56 ± 0.04

0.43 ± 0.04

1.16 ± 0.08*#

0.81 ± 0.03*#

Riboflavin (μg g−1 FW)

26.9 ± 2.3

22.2 ± 1.2

21.2 ± 0.9*

32.8 ± 6.7

24.1 ± 2.5*

20.3 ± 3.1*

Phenolic content

(μg g−1 FW)

157.2 ± 5.1

154.1 ± 3.2

150.1 ± 7.2

71.9 ± 8.8#

97.5 ± 7.6*#

129.5 ± 10.9*#

Ascorbic acid (μg g−1 FW)

1803.3 ± 232.2

2103.7 ± 301.7

2428.3 ± 30.3*

2337.3 ± 104.5

1508.1 ± 70.1*#

1103.9 ± 20.7*#

Glutathione redox state (%)

50.8 ± 3.8

57.1 ± 0.7*

43.7 ± 0.5

26.8 ± 1.7#

38.5 ± 5.0*#

61.9 ± 3.2*#

DPPH radical scavenging activity

15.2 ± 3.8

5.7 ± 2.4*

1.6 ± 1.1*

4.6 ± 1.4#

17.8 ± 2.6*#

23.5 ± 5.2*#

Activity of enzymatic antioxidants

SOD (U g−1 FW)

4.12 ± 0.06

4.34 ± 0.08

5.10 ± 0.05*

3.91 ± 0.08

5.23 ± 0.35*#

4.10 ± 0.07*#

CAT (mmol min−1 g−1 FW)

108.8 ± 11.1

129.6 ± 4.3*

150.2 ± 9.8*

135.3 ± 7.3#

130.0 ± 3.0

120.2 ± 7.7*#

GPOX (nmol min−1 g−1 FW)

30.1 ± 1.9

38.7 ± 2.6*

44.9 ± 1.7*

35.9 ± 1.9#

36.3 ± 2.1

83.5 ± 4.2*#

APX (mmol min−1 g−1 FW)

450.9 ± 17.8

716.1 ± 33.9*

864.2 ± 32.1*

530.3 ± 8.9#

170.6 ± 12.5*#

142.8 ± 7.1*#

MDHAR (μmol min−1 g−1 FW)

893.1 ± 191.3

546.2 ± 39.9*

382.0 ± 63.6*

30.6 ± 7.6#

244.9 ± 61.2*#

403.4 ± 124.1*#

DHAR (μmol min−1 g−1 FW)

16.5 ± 0.6

10.7 ± 0.4*

6.9 ± 0.4*

7.5 ± 0.3#

6.6 ± 1.2#

10.1 ± 0.8*#

GR (μmol min−1 g−1 FW)

52.6 ± 3.1

28.6 ± 3.3*

17.5 ± 3.1*

14.3 ± 2.9#

16.8 ± 1.5#

26.4 ± 1.6*#

GPX (μmol min−1 g−1 FW)

584.6 ± 14.9

232.5 ± 2.6*

133.1 ± 9.5*

587.7 ± 10.5

221.8 ± 17.5*

133.8 ± 1.2*

PAL (μmol min−1 g−1 FW)

9.7 ± 0.9

11.9 ± 0.1

12.1 ± 0.1*

9.3 ± 0.3

11.5 ± 0.7

12.6 ± 0.6*

Universal stress hormone

Abscisic acid content (pmoles g−1 FW)

119.6 ± 3.4

96.1 ± 5.7*

83.5 ± 7.1*

115.1 ± 0.5

118.5 ± 3.2#

132.0 ± 6.3*#

The data are the mean values (n = 3) ± SE

SE (p ≤ 0.05) is represented as ‘*’ (for comparison within treatments) and ‘#’ (for comparison between cultivars)

Accumulation of MG increased in IR-64 during fluoride stress. The MG content remained unchanged compared to the control in GB during 15 mg L−1 stress, but increased during 25 mg L−1 stress. The MG accumulation was much lower in GB compared to IR-64 under all the conditions (Table 1). The gly II expression was gradually triggered and significantly suppressed in stressed IR-64 and GB respectively (Fig. 2a).
Fig. 2

Expression of gly II (a), hsp 101 (b), PAL (c), SOD (D), CAT (e), APX (f), GPX (g), GST (h), MDHAR (i), DHAR (j) and GR (k) in IR-64 and GB seedlings grown with double-distilled water (control), 15 mg L−1 NaF and 25 mg L−1 NaF. The data are the mean values (n = 3) ± SE. ‘asterisk’ (for comparison within treatments) and ‘ash’ (for comparison between cultivars) designated on top of the error bars represent significance at p ≤ 0.05

The extent of protein carbonylation was significantly increased in both the cultivars during fluoride stress (Table 1). The carbonylation was higher in GB compared to IR-64 under both the concentrations of NaF, being about 6-fold and 11-fold higher in NaF (25 mg L−1)-stressed IR-64 and GB seedlings respectively, compared to their control counterparts. GB maintained higher hsp 101 expression compared to IR-64 under both concentrations of NaF (Fig. 2b).

The LOX activity gradually increased in IR-64 and GB upon exposure to increasing fluoride stress (Table 1). Compared to GB, IR-64 maintained higher LOX activity under all the conditions, illustrating lower membrane peroxidation in stressed GB compared to IR-64. The AAO activity decreased in the stressed sets of IR-64 (2.5-fold) compared to the control. Increasing fluoride concentrations triggered higher AAO activity in the stressed GB seedlings (Table 1).

Fluoride bioaccumulation in total seedlings

Content of fluoride drastically increased (10.4-fold) in NaF (25 mg L−1)-stressed IR-64 seedlings compared to 15 mg L−1 fluoride stress, accounting for high fluoride susceptibility of IR-64. Fluoride accumulation in NaF (25 mg L−1)-stressed GB seedlings was much lower (Table 1) than stressed IR-64.

Osmolyte levels and associated gene expression

Fluoride stress triggered Pro accumulation in the cultivars. Pro production was significantly higher in GB compared to IR-64 under both concentrations of NaF (Table 1). Pro content was increased by 1.7-fold in NaF (25 mg L−1)-stressed GB seedlings compared to the control set. P5CS expression was triggered in IR-64 plants exposed to 15 mg L−1 fluoride, whereas during 25 mg L−1 fluoride stress, the transcript level was marginally reduced, yet was maintained higher than the control. In case of GB, P5CS was suppressed and then induced during 15 mg L−1 and 25 mg L−1 of NaF respectively (Fig. 3a). The PDH expression was induced by fluoride in both the cultivars (Fig. 3b).
Fig. 3

Expression of P5CS (a), PDH (b), BADH1 (c), SAMDC (d), SPDS (e), SPMS (f), DAO (g) and PAO (h) in IR-64 and GB seedlings grown with double-distilled water (control), 15 mg L−1 NaF and 25 mg L−1 NaF. The data are the mean values (n = 3) ± SE. ‘asterisk’ (for comparison within treatments) and ‘ash’ (for comparison between cultivars) designated on top of the error bars represent significance at p ≤ 0.05

Under all the conditions, IR-64 maintained higher gly-bet level than GB. The gly-bet content was increased in GB during 25 mg L−1 of fluoride stress compared to the control. Though gly-bet accumulation in IR-64 was higher in 25 mg L−1 of stress compared to 15 mg L−1 of stress, it was lower than that in the control (Table 1). The BADH1 expression was also higher in IR-64 compared to GB (Fig. 3c). The total amino acid content was gradually increased in IR-64 upon increasing fluoride concentration (Table 1). This parameter increased in NaF (15 mg L−1)-stressed GB compared to the control, whereas it decreased during 25 mg L−1 NaF stress.

Put content remained unchanged in IR-64 during 15 mg L−1 stress compared to the control, but increased during 25 mg L−1 NaF stress. The content of higher PAs such as Spd and Spm decreased upon exposure to fluoride stress in IR-64. On the contrary, GB exhibited gradually lower Put and higher Spd and Spm accumulation during fluoride stress. The Spd accumulation in GB increased by 2.5-fold during 25 mg L−1 of fluoride stress compared to the control (Table 1). The SAMDC (encoding the rate-limiting enzyme) expression marginally decreased in IR-64 with increase in fluoride concentration. This gene was induced in GB during both concentrations of fluoride stress, thus verifying the enhanced accumulation of higher PAs during stress (Fig. 3d). The SPDS expression was slightly triggered in IR-64 during 15 mg L−1 of fluoride stress. The transcript level was reduced (close to control IR-64) during higher fluoride stress. The SPDS was gradually induced in GB with increasing fluoride stress (Fig. 3e). The SPMS was steadily induced in IR-64 with increasing concentration of fluoride, whereas it remained close to that in control during 15 mg L−1 fluoride stress, but significantly increased in NaF (25 mg L−1)-stressed GB (Fig. 3f). Increasing concentrations of fluoride steadily triggered DAO expression in IR-64. Induction of DAO in GB was strikingly high during 15 mg L−1 and the elevated expression was maintained even during 25 mg L−1 of NaF stress (Fig. 3g). The PAO was highly induced in IR-64 exposed to 25 mg L−1 of fluoride, whereas it was increased during 15 mg L−1 of NaF treatment and was significantly decreased during 25 mg L−1 NaF stress in GB (Fig. 3h).

Non-enzymatic antioxidants and associated gene expression

Increase in anthocyanin content was more pronounced in stressed GB compared to the stressed IR-64 seedlings. Whereas the pigment content was first decreased and then increased in stressed IR-64, it steeply increased in GB seedlings at 15 mg L−1 (3.6-fold) and 25 mg L−1 (4.5-fold) of NaF treatment compared to the control, thus depicting the fluoride-adaptable nature of GB (Table 1). The endogenous flavonoid content was decreased and increased in IR-64 and GB, respectively, on exposure to increasing fluoride concentration (Table 1).

The β-carotene content was increased by about 5-fold and 1.8-fold in NaF (25 mg L−1)-stressed IR-64 and GB seedlings respectively, compared to their control counterparts. GB maintained higher β-carotene level compared to IR-64 under all the conditions (Table 1). The xanthophyll content was first increased and then slightly decreased in IR-64 during 15 mg L−1 and 25 mg L−1 of fluoride stress, respectively, whereas it was concurrently increased (1.6-fold in 15 mg L−1 NaF and 3.8-fold in 25 mg L−1 NaF) in GB compared to the control. The xanthophyll level in GB seedlings at 25 mg L−1 of NaF was much higher compared to that in IR-64 seedlings (Table 1).

The thiamine content was gradually decreased in stressed seedlings of IR-64, whereas it was significantly increased in GB during 15 mg L−1 of fluoride stress, but was again decreased during 25 mg L−1 stress. The thiamine content in stressed GB was maintained at higher levels than that of control GB and stressed IR-64 seedlings, again highlighting the fluoride-adaptive character of GB (Table 1). The riboflavin content was decreased steadily in both the cultivars exposed to increasing fluoride stress (Table 1).

Total phenolic content (TPC) decreased in the fluoride-stressed IR-64 seedlings compared to the control. The PAL activity and gene expression increased in the fluoride-stressed IR-64 seedlings. TPC along with PAL activity and corresponding gene expression increased in GB grown under increasing fluoride stress (Table 1, Fig. 2c).

The AsA content was significantly increased in IR-64 (due to lowered AAO activity) and decreased in GB on exposure to increasing concentrations of NaF (Table 1). The endogenous AsA level was increased by approximately 1.4-fold and decreased by about 2.2-fold in NaF (25 mg L−1)-stressed IR-64 and GB, respectively, compared to their control counterparts. The GSH redox state was first increased and then decreased in IR-64 during 15 mg L−1 and 25 mg L−1 fluoride stress, respectively. However, a significant increase (1.4-fold during 15 mg L−1 NaF and 2.3-fold during 25 mg L−1 NaF stress) was noted in GB compared to the control. The GSH redox state was higher in GB seedlings at 25 mg L−1 NaF, compared to the IR-64 seedlings (Table 1).

DPPH radical scavenging activity was drastically decreased in IR-64 during fluoride stress (9.5-fold in 25 mg L−1 NaF compared to the control), whereas it significantly increased in fluoride-stressed GB (5.1-fold in 25 mg L−1 NaF, compared to the control) (Table 1).

Enzymatic antioxidants and associated gene expression

The SOD activity was steadily increased in stressed IR-64. In GB, a significant increase and then a decrease (reverting back to control) were observed during 15 mg L−1 and 25 mg L−1 of fluoride stresses respectively (Table 1). The stressed IR-64 seedlings exhibited significant increase in CAT activity, whereas it decreased in stressed GB (Table 1). The SOD expression decreased in both the cultivars during 15 mg L−1 NaF stress compared to their control counterparts. The gene was, however, induced during 25 mg L−1 fluoride stress in IR-64 and GB (Fig. 2d). The trend for the expression of CAT in both the varieties resembled that of SOD (Fig. 2e). Though GPOX activity increased in both the stressed cultivars, the maximum activity (2.3-fold higher than control) was observed during 25 mg L−1 of NaF in GB seedlings (Table 1). The APX activity increased significantly in stressed IR-64 (1.9-fold in 25 mg L−1 NaF compared to the control), but steadily decreased in fluoride-stressed GB plants (3.7-fold in 25 mg L−1 NaF compared to the control) (Table 1). The APX was gradually induced in stressed IR-64, whereas the expression decreased significantly in GB with increasing fluoride stress (Fig. 2f).

The GPX activity was concurrently inhibited by both the concentrations of fluoride in IR-64 and GB (4.4-fold in 25 mg L−1 NaF compared to the control in both the varieties) (Table 1). Induction of GPX was strongly triggered during 25 mg L−1 fluoride treatment in IR-64 and GB (Fig. 2g). The GST was significantly induced by 25 mg L−1 of NaF in both the cultivars (Fig. 2h).

The AsA–GSH cycle was inhibited by fluoride stress in IR-64, whereas it was efficiently maintained in GB. The MDHAR activity steadily decreased in stressed IR-64 (2.3-fold in 25 mg L−1 NaF compared to the control), whereas it was increased significantly in the stressed GB sets (13-fold in 25 mg L−1 NaF compared to the control) (Table 1). The MDHAR was significantly suppressed in IR-64 with increasing concentrations of fluoride. The expression of this gene remained almost unchanged in GB seedlings under 15 mg L−1 NaF and was slightly induced during 25 mg L−1 fluoride stress (Fig. 2i). The DHAR activity was inhibited in IR-64 seedlings exposed to increasing fluoride concentrations. The activity of this enzyme was decreased by 2.4-fold in NaF (25 mg L−1)-stressed IR-64 compared to that in control. In case of GB, the DHAR activity was decreased (not significant) during 15 mg L−1 of stress. The DHAR activity was increased significantly during 25 mg L−1 NaF stress in GB (Table 1). The DHAR was upregulated in IR-64 exposed to increasing concentrations of NaF, whereas strong induction of this gene was observed in GB seedlings at 25 mg L−1 NaF (Fig. 2j). The GR activity was significantly reduced in the stressed seedlings of IR-64 (3-fold in 25 mg L−1 NaF compared to the control), though GR was upregulated during stress. The activity of GR was steadily increased in the fluoride-stressed GB plants (1.8-fold in 25 mg L−1 NaF compared to the control) (Table 1). The GR expression was stably maintained in GB under all the conditions (Fig. 2k).

In-gel isozyme profiling

Four SOD isozymes were identified in IR-64 and GB. SOD1 was identified in the control IR-64 seedlings only. SOD2 and SOD3 could be detected for all the samples in both the cultivars. In case of IR-64, the intensity of SOD2 and SOD3 was gradually increased when the fluoride concentration was increased to 15 mg L−1 and then 25 mg L−1. However, the intensity of these isozymes was gradually decreased in GB during fluoride stress. SOD4 was found to be a novel isoform activated only during 25 mg L−1 fluoride stress in both IR-64 and GB (Fig. 4).
Fig. 4

Zymogram of SOD (a), GPOX (b), APX (c) and CAT (d) in IR-64 and GB seedlings grown with double-distilled water (control), 15 mg L−1 NaF and 25 mg L−1 NaF

Three GPOX isozymes were identified among IR-64 and GB. Intensity of GPOX1 and GPOX2 was gradually decreased in IR-64 when the fluoride concentration was increased. On the contrary, the band intensity of GPOX1 and GPOX2 was steadily increased in fluoride-stressed GB seedlings. GPOX3 was clearly visible only under 25 mg L−1 NaF stress in GB and can be regarded as a fluoride-responsive isoform (Fig. 4).

Three APX isozymes were identified among IR-64 and GB. Intensity of APX1 and APX2 was almost unchanged in the control and treated sets of IR-64 and GB. The intensity of the band corresponding to APX3 was found to increase in IR-64 during 25 mg L−1 fluoride stress. However, the band intensity of this isozyme was gradually decreased in GB upon increasing fluoride concentrations (Fig. 4).

Two CAT isozymes were identified in IR-64 and GB. Intensity of CAT1 was almost unchanged for control and treated IR-64. In case of GB, the CAT1 intensity was decreased during 25 mg L−1 of fluoride stress. The intensity of the band corresponding to CAT2 was increased during 25 mg L−1 of NaF stress in IR-64, but gradually decreased in GB with increasing fluoride concentration (Fig. 4).

ABA content and associated gene expression

The ABA level was decreased in IR-64 exposed to 15 mg L−1 (1.24-fold) and 25 mg L−1 (1.43-fold) of NaF stress, compared to the control. A marginal increase and a significant enhancement in ABA content were observed in NaF (both 15 mg L−1 and 25 mg L−1)-stressed GB respectively. ABA level was increased by about 1.2-fold in NaF (25 mg L−1)-stressed GB compared to that in control (Table 1). The NCED3 (ABA biosynthetic gene) expression was almost unchanged in IR-64 on exposure to fluoride stress, though it was significantly increased in GB exposed to stress (Fig. 5a). ABA8ox1 (ABA catabolic gene) was suppressed during fluoride stress in both the cultivars, with GB maintaining much lower expression of this gene compared to IR-64 under all the conditions (Fig. 5b). Rab16A was strongly induced in IR-64 on exposure to fluoride stress, whereas it was stably maintained in stressed GB (Fig. 5c). The expression of Osem was stably maintained in stressed IR-64 compared to the control with no significant change, whereas it was induced during 25 mg L−1 of NaF in GB seedlings (Fig. 5d). TRAB1 expression was triggered in IR-64 during both the concentrations of fluoride. No change was observed in TRAB1 expression between control and 15 mg L−1 of NaF in GB seedlings. The transcript level of TRAB1, however, significantly increased in GB exposed to the higher fluoride concentration (Fig. 5e). WRKY71 expression decreased (not statistically significant) during 15 mg L−1 of NaF stress in IR-64, but strongly increased during 25 mg L−1 fluoride stress. WRKY71 in GB was significantly induced during increasing concentrations of fluoride exposure (Fig. 5g). ABA-independent DREB2A was gradually suppressed in IR-64 and rapidly induced in GB during increasing concentrations of fluoride stress (Fig. 5f).
Fig. 5

Expression of NCED3 (a), ABA8ox1 (b), Rab16A (c), Osem (d), TRAB1 (e), DREB2A (f) and WRKY71 (g) in IR-64 and GB seedlings grown with double-distilled water (control), 15 mg L−1 NaF and 25 mg L−1 NaF. The data are the mean values (n = 3) ± SE. ‘asterisk’ (for comparison within treatments) and ‘ash’ (for comparison between cultivars) designated on top of the error bars represent significance at p ≤ 0.05

Discussion

Fluorine is the 13th most abundant element found on the earth’s crust and is a toxic environmental xenobiotic. Severely contaminated areas have been reported to contain as high as 189 mg kg−1 fluoride in the upper soil layer and about 139 mg kg−1 fluoride in the lower soil layer (Bhat et al. 2015). Rice is a water-intensive plant and the staple food crop in several parts of the world. Preliminary reports have shown that irrigation of rice seedlings with fluoride-contaminated groundwater reduces growth and physiological development (Gupta et al. 2009; Mondal 2017).

In the present study, visual observations and the analysis of physiological parameters such as FW, DW and RWC indicated that unlike IR-64, GB endured the increasing concentrations of NaF-mediated stress. Low relative water content (RWC) was detected in rice varieties irrigated with fluoride-contaminated groundwater (Mondal and Gupta 2015). Fluoride bioaccumulation beyond 1.5 μg g−1 tissue could be detrimental for the grazing livestock (Banerjee and Roychoudhury 2019a). Both IR-64 and GB showed fluoride bioaccumulation within their tissues. Such accumulation surpassed the prescribed safe limit of 1.5 µg g−1, signifying a potential biohazard. Cattle consuming such contaminated vegetative seedlings will directly face the severe consequences of fluorosis. The consumed fluoride would then accumulate in the dairy products and further biomagnify within the food chain.

The oxidative stress triggered by uncontrolled fluoride bioaccumulation in IR-64 increased the LOX activity and subsequent membrane damage in IR-64 seedlings. The fluoride-stressed GB seedlings bioaccumulated lower level of fluoride and hence was able to maintain lower LOX activity compared to that in IR-64. Oxidative stress also triggers covalent carbonylation of arginine, lysine, threonine and proline residues within protein tertiary structures (Debska et al. 2012). We observed a significant increase in overall protein carbonylation in both the fluoride-stressed varieties. Fluoride stress strongly induced the expression of hsp 101 gene that encodes a potential chaperone, preventing protein misfolding and aggregation in rice (Gurley 2000). Increased hsp 101 transcript accumulation in fluoride-stressed GB compared to fluoride-stressed IR-64 might have ensured efficient refolding of denatured, misfolded and aggregated proteins.

Both the rice varieties showed increase in the level of endogenous osmolytes during fluoride stress. GB exhibited a reduced pool of total amino acids possibly due to efficient utilization of the resources in producing novel proteins involved in protection. The accumulation of different osmolytes have been reported in rice cultivars exposed to salinity, drought, cadmium-induced toxicity, UV-B mediated stress, etc. (Roychoudhury and Chakraborty 2013; Roychoudhury and Banerjee 2016). P5CS encodes the key enzyme involved in Pro production from glutamate during abiotic stress (Guan et al. 2018). The enzyme encoded by PDH promotes Pro catabolism and such degradation often provides energy resource for the plants during abiotic stress (Liang et al. 2013). The high transcript level of P5CS and PDH corresponded with high Pro level in GB exposed to 25 mg L−1 NaF stress. This evidently illustrated that during fluoride stress, GB seedlings maintained an extensively large pool of Pro via efficient metabolite cycling. High P5CS ensured Pro formation from glutamate, whereas high PDH ensured conversion of Pro to Δ1-pyrroline-5-carboxylate (P5C) which could again be converted to glutamate and catalyzed by P5CS to form Pro. IR-64 accumulated low amount of Pro compared to GB during 25 mg L−1 of NaF stress due to lower P5CS expression than in GB. Gly-bet accumulation is catalyzed by BADH1. The transcript level of this enzyme has been reported to steeply increase during multiple abiotic stresses (Roychoudhury and Banerjee 2016). Lower level of gly-bet, in spite of induced BADH1, indicated towards efficient utilization of this metabolite for stress amelioration in IR-64 exposed to 25 mg L−1 NaF. The SPDS and SPMS encode enzymes which catalyze the formation of Spd and Spm, respectively. The enzyme encoded by DAO breaks down the lower PA, Put, whereas that encoded by PAO promotes the catabolism of Spd and Spm (Gill and Tuteja 2010; Roychoudhury and Das 2014). Distinct varietal difference was evident in PA regulation during fluoride stress. The expression of SAMDC (which encodes the rate-limiting enzyme of PA biosynthesis) was inhibited in IR-64 so as to limit PA biosynthesis in case of NaF-stressed IR-64, though SPMS expression was increased under the same conditions. In addition to such inhibited PA production, high PAO expression ensured increased degradation of higher PAs like Spd and Spm to Put in IR-64. The increase in Put content due to the breakdown of higher PAs has been suggested as the prominent character of any susceptible variety during salt stress (Ndayiragije and Lutts 2006). Fluoride-stressed GB exhibited enhanced SAMDC, SPDS and SPMS expressions, and decreased PAO expression during 25 mg L−1 of NaF stress. As a result, most of the Put was converted to Spd and Spm in GB. The higher PAs, with more positive charges, contribute better stability to membranes and nucleic acids during abiotic stress (Roychoudhury et al. 2012). Enhanced DAO expression during stress pointed towards the possible utilization of a part of Put in mitigating stress in IR-64 and GB. Such observations have also been reported earlier in Arabidopsis thaliana exposed to dehydration stress (Alet et al. 2011).

The non-enzymatic antioxidants such as anthocyanin, phenolics, flavonoids, β-carotene and xanthophyll act as effective protective agents during oxidative stress in plants (Roychoudhury et al. 2012; Du et al. 2010). Both IR-64 and GB accumulated high amounts of non-enzymatic antioxidants required to scavenge the excess ROS produced during fluoride-mediated oxidative stress. Flavonoid level decreased in IR-64 during stress either due to inability to accumulate the antioxidant by virtue of being a susceptible variety or due to efficient utilization towards the mitigation of fluoride stress. However, the decrease in TPC in 25 mg L−1 NaF-stressed IR-64 was possibly due to efficient utilization of these compounds towards stress protection. This is further supported by the increase in PAL activity as well as PAL expression in IR-64 similar to GB during 25 mg L−1 fluoride exposure. The action of the vitamins, riboflavin and thiamine has not been well characterized under any abiotic stress. However, it is understood that riboflavin acts as an antioxidant during salt stress. Thiamine being the most metabolically labile vitamin-B is less maintained in the stress-susceptible variety (Hanson et al. 2016). Compared to IR-64, GB could maintain higher reserves of thiamine during high fluoride stress, establishing once more its adaptive role. Riboflavin also appeared to be a reliable marker of fluoride stress in both the varieties, since its content steadily decreased during stress. Due to an evolved endogenous capacity to tackle fluoride stress, GB accumulated much lower level of cytotoxic MG compared to IR-64 in spite of low gly II expression. MG is a short oxygenated aldehyde produced as a by-product of varied metabolic reactions including glycolysis. Abiotic stress triggers the cellular accumulation of MG leading to necrosis (Hoque et al. 2016). Stress-tolerant plants effectively detoxify the excess MG via the glyoxylase system (Hoque et al. 2016). Rice gly II is crucially involved in ameliorating salt and dicarbonyl stress in transgenic tobacco (Ghosh et al. 2014). The activity of enzymatic antioxidants such as SOD, CAT, APX, GPOX and GPX has been found to increase in rice seedlings exposed to salt stress (Paul et al. 2017; Paiva et al. 2019). Among the studied antioxidant enzymes, GPOX, GR and PAL activities were significantly triggered in NaF (25 mg L−1)-stressed GB seedling compared to its control counterpart and also in the stressed IR-64 plants. Paul et al. (2017) reported that all isozymes of SOD, CAT, APX and peroxidase are not responsive to salt stress in rice. Isozyme analysis identified novel SOD and GPOX isoforms in fluoride (25 mg L−1)-stressed GB. Taking into account the high transcript level of SOD and CAT and low activity of the corresponding enzymes, it can be said that fluoride inhibited these enzymes in GB.

The GSH redox state indicated the reduced glutathione:oxidized glutathione (GSH:GSSG) ratio, which crucially dictates plant tolerance against abiotic stress. Under oxidative stress, a high GSH:GSSG ratio is essential to effectively reduce the widespread oxidation triggered by ROS and maintain the cellular equilibrium (Chin et al. 2016). Maintenance of high GSH within the cell is chiefly regulated by the enzymes belonging to the ascorbate–glutathione (AsA–GSH) cycle (Banerjee and Roychoudhury 2018). These enzymes exhibit variable activity and differential transcript level during several environmental stresses (Akram et al. 2017). The closely associated metabolite, AsA, acts as a potent antioxidant and is degraded in stress-susceptible species by the action of ascorbic acid oxidase (AAO) (Akram et al. 2017). Distinct varietal difference was observed in the operation of the AsA–GSH cycle in our study. It appeared from the transcript analysis data that fluoride stress significantly inhibited the chief enzymes of the AsA–GSH cycle such as MDHAR, DHAR and GR in IR-64, though APX activity was enhanced. Compared to GSH, AsA was maintained at higher level in IR-64 during high fluoride stress due to reduced AAO activity. The AsA–GSH cycle in GB was triggered by fluoride exposure leading to higher GSH redox state compared to AsA. The AAO activity was stimulated and APX activity was suppressed in fluoride-stressed GB seedlings. Due to efficient maintenance of the chief redox stabilizer, viz., GSH, the fluoride-stressed GB seedlings exhibited higher DPPH radical scavenging activity compared to their control counterparts. On the contrary, the DPPH radical scavenging activity was greatly suppressed in the fluoride-stressed IR-64 seedlings due to inhibition of the crucial ROS-detoxifying AsA–GSH cycle. The GPX enzyme was found to be fluoride sensitive in both the varieties. Induction of GST indicated towards the possible role of the enzyme in chelating toxic fluoride ions. GST encodes an antioxidant enzyme which quenches the reactive oxygen molecules in association with GSH and protects the cell from oxidative damages (Kumar and Trivedi 2018). Thus, it appeared that due to low bioaccumulation of fluoride, GB could mitigate the injuries by maintaining enhanced activity of GPOX, GST, GR and PAL. Due to low endogenous radical scavenging potential, the fluoride-stressed IR-64 seedlings had to maintain elevated SOD, CAT, APX, PAL, GPOX and GST activity to ameliorate the damages.

ABA is a diverse signalling molecule which upregulates a set of osmotic stress-responsive genes against multiple abiotic stresses (Banerjee and Roychoudhury 2016). We observed notable differences in ABA accumulation and utilization in the two cultivars during fluoride stress. High level of endogenous ABA content in stressed GB was supported by induced NCED3 and downregulated ABA8ox1 expression. ABA-inducible genes such as Rab16A, Osem, TRAB1 and WRKY71 are all predominantly upregulated in rice exposed to salt stress (Paul and Roychoudhury 2019). Rab16A and Osem are late embryogenesis-abundant (LEA) proteins, which are unstructured and exhibit moonlighting activity. The LEA proteins contain positively charged residues by virtue of which they interact with water molecules and conserve them during osmotic and desiccation stress (Banerjee and Roychoudhury 2016). TRAB1 is an important basic leucine zipper (bZIP) transcription factor (TF) which was shown to accumulate during multiple abiotic stresses in rice (Kagaya et al. 2002). WRKY71 is a crucial TF operative during salinity stress and cadmium-induced toxicity in rice (Banerjee and Roychoudhury 2015). The DREB2A gene has been reported to be induced by salt and drought stress via an ABA-independent pathway (Sakuma et al. 2006). The accumulation of ABA in fluoride-stressed GB led to the induction of Rab16A, Osem, TRAB1 and WRKY71. The expression of the ABA-independent DREB2A was stimulated in stressed GB seedlings compared to the control sets. On the contrary, increasing concentration of NaF reduced the endogenous ABA content in IR-64. Though NCED3 expression was almost unchanged during stress, ABA8ox1 was gradually downregulated in IR-64, indicating towards slower degradation of ABA, in spite of its low production in IR-64. The suppressed rate of degradation might increase the mean half life of this phytohormone, allowing conservation of ABA, which in turn might have accounted for the induction of ABA-responsive genes such as Rab16A, TRAB1 and WRKY71 in IR-64 during stress. However, downregulated expression of DREB2A leads to the hypothesis that unlike GB, IR-64 tried to acclimatize to increasing fluoride concentrations only via a compromised ABA-dependent pathway. Paul and Roychoudhury (2019) established a positive correlation between ABA content and PA biosynthesis in rice. In line with the observation, we found that SAMDC (encoding the PA rate-limiting enzyme) was downregulated in IR-64 during fluoride stress in accordance with low ABA accumulation. On the contrary, high induction of SAMDC along with high accumulation of higher PAs corresponded with enhanced ABA accumulation in GB.

Thus, fluoride stress variably affected the defence machineries of the fluoride-susceptible non-aromatic cultivar IR-64 and the fluoride-adapted aromatic variety GB. PA and ABA biosyntheses as well as AsA–GSH cycle were inhibited in the fluoride-stressed IR-64 seedlings due to high bioaccumulation of fluoride. GB, on the contrary, accumulated lower amount of the toxic ions and showed potential maintenance of the aforementioned metabolic pathways. GB efficiently tackled the sub-optimal condition by maintaining a high reservoir of the majority of osmolytes and antioxidants as well as endogenous ABA level. The manuscript exhaustively establishes the clear-cut differential defence mechanism adopted by the two rice cultivars and manifests varietal differences to fluoride toxicity both at the biochemical and molecular level.

Notes

Acknowledgements

Financial assistance from Science and Engineering Research Board, Government of India through the grant [EMR/2016/004799] and Department of Higher Education, Science and Technology and Biotechnology, Government of West Bengal, through the Grant [264(Sanc.)/ST/P/S&T/1G-80/2017] to Dr. Aryadeep Roychoudhury is gratefully acknowledged. Mr. Aditya Banerjee is thankful to University Grants Commission, Government of India, for providing Junior Research Fellowship in course of this work.

Author contribution statement

AB performed the experiments and generated data. AB and ARC drafted the manuscript. ARC supervised the entire work, provided critical comments and suggested and incorporated necessary corrections or modifications within the manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest in publication of the manuscript.

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© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Post Graduate Department of BiotechnologySt. Xavier’s College (Autonomous)KolkataIndia

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