Introduction
Fluoride is an emerging toxicant in the face of global groundwater pollution (Baunthiyal and Sharma 2014). The World Health Organization (WHO) has set 1.5 mg L−1 as the upper limit of fluoride ingestion. Beyond this level, fluoride causes severe fluorosis and associated skeletal deformities in humans and animals (Mondal and Gupta 2015). Irrigation of crops with fluoride-infested groundwater can be detrimental to the health of the consumers, since plants bioaccumulate toxic fluoride ions within their tissues. High accumulation
of fluoride within plant tissues also results in physiological anomalies and reduction in crop yield (Yadu et al. 2018a, b). Our previous studies have established that due to extensive cultivation in areas of West Bengal (India), China, Bangla- desh and Pakistan which experience acute endemic fluorosis, rice should be regarded as one of the focus crops for fluoride amelioration (Banerjee et al. 2019a; Singh et al. 2019; Gao et al. 2019; Rashid et al. 2020; Yuan et al. 2020). Design- ing of strategies facilitating lowered fluoride accumulation in rice will not only increase the crop yield, but will also restrict the entry of the xenobiotic within the food chain.
Fluoride behaves as a slow poison for susceptible plant
species (Hong et al. 2016). We previously showed that the
* Aryadeep Roychoudhury aryadeep.rc@gmail.com
1 Department of Biotechnology, St. Xavier’s College (Autonomous), 30, Mother Teresa Sarani, Kolkata, West Bengal 700016, India
indica rice cultivar, IR-64 widely cultivated in India and known for superior yields, is sensitive to fluoride toxicity (Banerjee and Roychoudhury 2019a). Fluoride imposed oxi- dative stress in the seedlings due to elevated production of hydrogen peroxide (H2O2) (Kubi 2005). Increased oxidative
1 3
stress negatively affected membrane stability and integrity, resulting in the formation of malondialdehyde (MDA) which signified severe membrane damage (Banerjee and Roychoudhury 2018). The photosynthetic process was hin- dered due to increased chlorophyll (Chl) degradation. The accessory pigments like β-carotene and xanthophyll aid in the maintenance of cellular redox homeostasis (Alonso et al. 2001). Oxidative stress also affected the content of these pigments. In order to balance such disruption in the cellular equilibrium, plants increase the synthesis of non-enzymatic antioxidants like proline (Pro), anthocyanin, flavonoids and phenolics (Che-Othman et al. 2017). Apart from these pro- tective metabolites, plants also alter the activity of enzy- matic antioxidants like catalase (CAT), ascorbate peroxidase (APX) and guaiacol peroxidase (GPOX) in order to reduce oxidative load within the cells (Colville and Smirnoff 2008). Gibberellic acid (a pentacyclic diterpene) is a crucial phy- tohormone and an important plant growth regulator which dictates internodal growth (Chrispeels and Varner 1967). We previously showed that the endogenous GA content is decreased during fluoride stress in IR-64 (Banerjee and Roy- choudhury 2019b). We also showed higher conservation of abscisic acid (ABA) due to lowered ABA catabolism in rice seedlings exposed to fluoride stress. Since it is well known that ABA acts antagonistically with the GA pathway (Shu et al. 2018), more ABA conservation is correlated with low- ered GA level. It is possible that as a result of such lowered GA content, the seedlings exhibited higher level of suscepti- bility to fluoride toxicity. Hence, it can be hypothesized that high GA accumulation could be associated with the fluo- ride tolerance pathways. Indole-3-acetic acid (IAA) is the most abundant auxin and is a monocarboxylic acid hormone (Chen et al. 2009). Salt stress reduced IAA accumulation in tomato seedlings as a result of which the overall growth was hindered (Dunlap and Binzel 1996). Thus, IAA acts as a stress marker. The interaction between GA and IAA during fluoride stress has not been reported yet and hence requires elucidation in order to understand the underlying physiology
of growth of rice plants exposed to fluoride.
Gibberellins have been used as an exogenous protective chemical agent in crops like rice, maize, oat, wheat and sugar beet to develop salt tolerance (Chauhan et al. 2019). Therefore, in this study, we primed the seeds of the fluoride- susceptible cultivar, IR-64 with GA to study the physiologi- cal behaviour of the seedlings under fluoride stress. The advantages of priming compared to traditional breeding and transgenic technologies are its efficiency, biosafety and easy execution at the field level (Paul et al. 2017). Chan and Arora (2013) reviewed that priming generates ‘stress memory’ in plants which enables faster post-stress recovery and also bet- ter systemic response upon later stress exposures. Thus, the aim of the study was to decipher the physiological and bio- chemical mechanism of GA as a protective priming agent in
the soil-grown rice seedlings exposed to prolonged fluoride stress. The manuscript illustrates the effects of GA-priming in: (i) mediating the homeostasis between the endogenous gibberellins and IAA; (ii) abrogating fluoride accumula- tion and injuries; and (iii) stimulating the defence system to reduce the damages caused by prolonged fluoride stress.
Materials and Methods
Seed Priming and Seedling Growth Conditions
Freshly harvested seeds of Oryza sativa cv. IR-64 were col- lected from Chinsurah Rice Research Station (West Bengal, India). For priming, the seeds were surface sterilized and imbibed in 150 mg L−1 gibberellic acid 3 (GA) (Sigma- Aldrich, USA) for 24 h at 25 °C. Iqbal and Ashraf (2013) showed earlier that seed priming with 150 mg L−1 GA ame- liorated the physiological injuries imposed by salt stress in wheat seedlings. The seeds were then dried for 24 h at 25 °C till they reached their initial moisture content. The non-primed and the primed seeds were equally sowed in pots containing soil in two sets (i.e., four sets in total) in completely randomized design (CRD). The pots were then maintained in the experimental garden under normal photo- period. The composition of the soil was prepared following Banerjee et al. (2020a). The four sets of samples that were maintained included the following:
Set 1: Non-primed seeds irrigated only with double dis- tilled water, i.e., control (Cont).
Set 2: Non-primed seeds irrigated with 25 mg L−1 NaF (NaF).
Set 3: GA-primed seeds irrigated only with double dis- tilled water (GA).
Set 4: GA-primed seeds irrigated with 25 mg L−1 NaF (GA + NaF).
After 20 days of growth, the seedlings were harvested and frozen in liquid nitrogen for further experiments. The extent of fluoride stress was decided following our previous report (Banerjee and Roychoudhury 2019a).
Measurement of Basic Physiological Parameters
The mean lengths of the primary shoot (SL) and primary root (RL) were measured from an average of 50 seedlings using a steel ruler (Banerjee et al. 2019a). The weight of the newly harvested seedlings measured in an analyti- cal balance was documented as fresh weight (FW). These seedlings were then dried in hot air oven at 80 °C for three days. The weight of the dried samples was regis- tered as the dry weight (DW) (Banerjee et al. 2020b). The
relative water content (RWC) was measured by imbibing the freshly harvested seedlings in 5 mM CaCl2 for 8 h in order to note the turgid weight (TW). The RWC was calculated as: [(FW−DW)/ (TW−DW)] × 100 (Gonzalez and Gonzalez-Vilar 2001). The freshly harvested seed- lings were incubated in double distilled water for 22 h. The initial conductivity was measured using a conductivity meter (Digital Instruments Corporation, India). The total conductivity was measured upon boiling the seedlings for 10 min. The electrolyte leakage was measured as the per- centage of total conductivity (Banerjee et al. 2019a).
Measurement of Endogenous Fluoride, H2O2 and MDA Contents
0.2 g of freshly harvested seedlings was mineralized in 4 ml of TISAB buffer. The mixture was centrifuged and the super- natant was collected. The fluoride content was measured using a fluoride-sensitive electrode (Cole-Palmer, USA) (Banerjee and Roychoudhury 2019b). For measuring H2O2 level, 0.5 g of freshly harvested tissue was homogenized in 3 ml of 0.1% (w/v) trichloroacetic acid and the extract was collected. To 0.5 ml of the extract, 0.5 ml of 10 mM potas- sium phosphate buffer (pH 7.0) and 1 ml of 1 M potassium iodide (KI) were added. The mixture was incubated in dark for 1 h and the H2O2 content was measured spectrophotomet- rically by recording the absorbance of the H2O2–KI complex at 390 nm (Velikova et al. 2000). For measuring the MDA content, 0.5 g of tissue was homogenized in 5 ml of 50 mM sodium phosphate buffer (pH 7.0) and the supernatant was collected after centrifugation. 0.5 ml of the supernatant was mixed with 1 ml of 0.5% (w/v) thiobarbituric acid solution prepared in 20% (w/v) trichloroacetic acid and the mixture was heated at 95 °C for 30 min. The absorbance of the result- ing complex was recorded at 532 nm and 600 nm (Banerjee et al. 2019b).
Measurement of Endogenous Gibberellic Acid and IAA Contents
The gibberellic acid content was measured following the technique described by Graham and Thomas (1961) with minor modifications. A standard curve was prepared using known concentrations of GA and the actual endogenous gib- berellic acid level was calculated by plotting the absorbance values in the derived standard curve. For extraction of IAA, the seedlings were homogenized in NaH2PO4–NaOH buffer. The IAA content was detected following the protocol by Liu and Wan (2013) using first derivation synchronous fluores- cence spectroscopy.
Measurement of Chl and Accessory Pigments Like
β‑carotene and Xanthophyll
For measurement of Chl, the leaves were homogenized in chilled 80% (v/v) acetone and the absorbance was meas- ured at 645 nm and 663 nm (Arnon 1949). One millilitre of the above extract was mixed with 1 ml of n-hexane in order to separate Chl from β-carotene and xanthophyll. After centrifugation, the supernatant was collected. The β-carotene and xanthophyll were separated by adding 1 ml of 90% (v/v) methanol to the supernatant following Baner- jee and Roychoudhury (2019a).
Measurement of Non‑enzymatic Antioxidants like Pro, Anthocyanin, Flavonoids and Total Phenolics
For measuring the Pro level, 0.5 g of tissue was crushed in 5 ml of 3% (w/v) sulfosalicylic acid and the superna- tant was collected after centrifugation. 2 ml of extract was added to 2 ml of freshly prepared acid ninhydrin and 2 ml of glacial acetic acid, followed by boiling the mixture for 30 min. After cooling the reaction mixture on ice, Pro con- tent was measured by recording the absorbance of the red- dish complex at 520 nm. Actual Pro level was deciphered by plotting the absorbance values on a standard curve prepared against known concentrations of Pro (Banerjee et al. 2019c). For measuring the anthocyanin content, 0.5 g of freshly harvested tissue was crushed in 5 ml of abso- lute methanol [acidified with 1% (v/v) hydrochloric acid]. The mixture was incubated overnight at 4 °C for proper
Fig. 1 Assessment of growth of seedlings raised from non-primed IR-64 seeds grown in distilled water, i.e., control (Cont) or 25 mg L−1 NaF (NaF), and seedlings raised from seeds primed with 150 mg L−1 GA and grown in either distilled water (GA) or 25 mg L−1 NaF (GA + NaF)
Table 1 Physiological parameters associated with growth and oxi- dative injuries in the seedlings raised from non-primed IR-64 seeds grown in distilled water, i.e., control (Cont) or 25 mg L−1 NaF (NaF),
and seedlings raised from seeds primed with 150 mg L−1 GA and grown in either distilled water (GA) or 25 mg L−1 NaF (GA + NaF)
|
Parameters |
Cont |
NaF |
GA |
GA + NaF |
|
|
Physiological parameters |
Shoot length (cm) |
12.1 ± 1.6 |
8.2 ± 1.6* |
14.2 ± 1.4* |
13.1 ± 1.7 |
|
Root length (cm) |
5.1 ± 1.4 |
2.7 ± 0.5* |
6.2 ± 1.3* |
4.3 ± 0.3* |
|
|
Fresh weight (mg) |
75.1 ± 3.4 |
61.3 ± 2.7* |
79.5 ± 3.1* |
70.4 ± 3.8 |
|
|
Dry weight (mg) |
12.1 ± 3.1 |
8.9 ± 1.1* |
12.6 ± 1.2* |
10.7 ± 0.5 |
|
|
Relative water content |
97.4 ± 3.1 |
71.3 ± 2.5* |
97.1 ± 2.9* |
85.3 ± 2.9* |
|
|
Electrolyte leakage (%) |
16.8 ± 1.8 |
35.7 ± 1.7* |
16.1 ± 2.9* |
27.9 ± 2.1* |
The data are the mean values (n = 3) ± standard error (SE). The SE (p ≤ 0.05) in each case is represented as ‘*’ (for comparison within treat- ments). The symbols represent significance at p ≤ 0.05
extraction of anthocyanin. The anthocyanin content was determined spectrophotometrically at 425 nm using the supernatant derived after centrifugation of the reaction mixture post-incubation (Banerjee and Roychoudhury 2019a). For measuring the flavonoid content, 0.5 g of seedlings was homogenized in absolute methanol. 0.2 ml of the methanolic extract was incubated with 1 ml of 2% (w/v) aluminium chloride and incubated at room tempera- ture (25 °C) for 1 h. The absorbance of the mixture was measured spectrophotometrically at 415 nm. The actual flavonoid content was determined from a standard curve prepared using known concentrations of rutin (Quettier et al. 2000). The total phenolic content (TPC) was meas- ured by incubating 0.2 ml of the methanolic extract with 2 ml of 7.5% (w/v) sodium carbonate and 2.5 ml of Folin Ciocalteau reagent (diluted tenfold in water) for 20 min in dark. The absorbance of the mixture was measured at 760 nm (Basu et al. 2012). The actual TPC was calculated by plotting the absorbance values in a standard curve pre- pared using known concentrations of tannic acid.
Measurement of Cumulative Antioxidant Responses Like Reducing Power, Total Antioxidant Capacity and 2,2‑diphenyl‑1‑picrylhydrazyl (DPPH) Radical Scavenging Activity
The methanolic extract of the seedlings was incubated with ferric chloride and the reducing power was spectrophotometri- cally analysed at 700 nm. The known concentrations of ascor- bic acid were used to prepare a standard curve from which the actual reducing power was determined (Banerjee et al. 2019b). For the calculation of total antioxidant capacity, the capacity of the methanolic extracts to reduce Mo (VI) to Mo (V) was analysed and the total antioxidant capacity was detected from the above standard curve prepared using ascorbic acid (Basu et al. 2012). The aqueous extract of the seedlings was mixed with 0.004% (v/v) DPPH solution to calculate the DPPH radi- cal scavenging activity (Banerjee and Roychoudhury 2019a).
Measurement of Enzymatic Antioxidants, CAT (EC 1.11.1.6), APX (EC 1.11.1.11) and GPOX (EC 1.11.1.7)
For assessment of the activity of the enzymatic antioxidants, freshly harvested seedlings were homogenized in sodium phos- phate buffer (pH 7.0) and the supernatant was collected. The supernatant was incubated with freshly prepared H2O2 solution and the reduction in absorbance at 240 nm was recorded to calculate the CAT activity (Velikova et al. 2000). The reduced absorbance at 290 nm due to the decreasing content of sodium ascorbate in the presence of the enzyme extract was recorded to calculate the APX activity (Basu et al. 2012). The rate of conversion of guaiacol to the brown-coloured tetraguaiacol in the presence of enzyme extract was recorded at 470 nm in order to calculate the GPOX activity (Banerjee et al. 2020a).
Measurement of Total Protein Content and Analysing the Statistical Significance
Using bovine serum albumin (BSA) as the standard, total protein content was estimated following Bradford (1976) in order to use equal concentration of total protein for the dif- ferent enzyme assays. Three replicates (n = 3) for the experi- ments were performed, and the standard error and statistical significance were calculated using one way analysis of vari- ance (ANOVA) at p ≤ 0.05 in XLSTAT 2018.
Results and Discussion
Assessment of Seedling Growth and Physiological Parameters
The growth of IR-64 seedlings was suppressed under fluo- ride stress. However, the growth of the plants which germi- nated from the GA-primed seeds under fluoride stress was found to be improved compared to that in the non-primed, stressed seedlings (Fig. 1). Overall, NaF treatment sig- nificantly reduced the SL, RL, FW, DW and RWC in the
A 4 A
*
*
*
*
0.9
*
Gibberellin content (µg g-1 FW)
0.6
2
Fluoride content (mg g-1 FW)
0.3
0
Cont NaF GA GA+NaF
Shoot Root
B
*
*
16
H O content
(nmol g-1 FW)
8
2 2
0
B 25
IAA content (µg g-1 FW)
20
15
10
5
0
Cont NaF GA GA+NaF
*
*
Cont NaF GA GA+NaF
0
C 1.8
*
*
MDA content (µM g-1 FW)
0.9
0
Cont NaF GA GA+NaF
Cont NaF GA GA+NaF
Fig. 3 Endogenous level of gibberellic acid (a) and IAA (b) in the seedlings raised from non-primed IR-64 seeds grown in distilled water, i.e., control (Cont) or 25 mg L−1 NaF (NaF), and seedlings raised from seeds primed with 150 mg L−1 GA and grown in either distilled water (GA) or 25 mg L−1 NaF (GA + NaF). The data are the mean values (n = 3) ± standard error (SE). The SE (p ≤ 0.05) in each case is represented as ‘*’ (for comparison within treatments). The symbols represent significance at p ≤ 0.05
seedlings germinated from the non-primed seeds. Such flu- oride-mediated inhibition in growth and development has been previously reported in IR-64 (Banerjee et al. 2020a). The GA-priming increased plant growth exposed to stress by stimulating the SL, RL, FW, DW and RWC by 1.6-, 1.6-, 1.1-, 1.2- and 1.2-fold, respectively, compared to those in the non-primed, stressed seedlings (Table 1). Alhadi et al. (1999) also showed improved growth physiology and leaf water relation in the GA-primed fenugreek plants exposed to desiccation. Oxidative stress induces the loss of crucial elec-
Fig. 2 Measurement of damage indices like fluoride bioaccumula- tion (a), H2O2 (b) and MDA (c) in the seedlings raised from non- primed IR-64 seeds grown in distilled water, i.e., control (Cont) or 25 mg L−1 NaF (NaF), and seedlings raised from seeds primed with 150 mg L−1 GA and grown in either distilled water (GA) or 25 mg L−1 NaF (GA + NaF). The data are the mean values (n = 3) ± standard error (SE). The SE (p ≤ 0.05) in each case is represented as ‘*’ (for comparison within treatments). The symbols represent significance at p ≤ 0.05
trolytes through membrane disintegration, ultimately lead- ing to cellular necrosis and tissue damage (Demidchik et al. 2014). Priming the seeds with GA decreased the percent of electrolyte leakage by 1.3-fold compared to that in the non- primed, stressed plants where the rate of electrolyte loss was significantly increased (Table 1). Siddiqui et al. (2011) also reported reduced electrolyte leakage due to lesser membrane damage in the GA-primed wheat seedlings exposed to nickel toxicity.
A 60
30
Chl content (µg g-1 FW)
0
*
*
Cont NaF GA GA+NaF
B 150
100
β-carotene content (µM g-1 FW)
50
0
*
Cont NaF GA GA+NaF
C 25
20
Xanthophyll content (µM g-1 FW)
15
10
5
0
*
*
Cont NaF GA GA+NaF
Fig. 4 Measurement of Chl (a), β-carotene (b) and xanthophyll (c) contents in the seedlings raised from non-primed IR-64 seeds grown in distilled water, i.e., control (Cont) or 25 mg L−1 NaF (NaF), and seedlings raised from seeds primed with 150 mg L−1 GA and
grown in either distilled water (GA) or 25 mg L−1 NaF (GA + NaF). The data are the mean values (n = 3) ± standard error (SE). The SE (p ≤ 0.05) in each case is represented as ‘*’ (for comparison within treatments). The symbols represent significance at p ≤ 0.05
Assessment of Fluoride Accumulation and Damage Indices
The significantly high bioaccumulation of fluoride in the shoot and root tissues of the non-primed, stressed plants was reduced by 1.5-fold in both the shoot and root of the primed seedlings exposed to stress (Fig. 2a). Thus, application of GA reduced fluoride uptake and accumulation in rice seed- lings which accumulated high level of the xenobiotic during stress exposure (Banerjee and Roychoudhury 2019a). Due to reduced fluoride content within the tissues, the primed seedlings experienced lower level of oxidative stress which was evident from the decreased accumulation of H2O2 and the stress marker MDA (Fig. 2b, c). While the H2O2 content was restored close to that in the control seedlings, the MDA level was also significantly reduced (compared to the non- primed, stressed seedlings) in the GA-primed plants grown under stress conditions. Chauhan et al. (2019) reported the positive effects of GA in ameliorating oxidative injuries and membrane damage in oat seedlings exposed to higher con- centrations of salt. The wheat seedlings germinated from GA-primed seeds also exhibited lower extent of oxidative
injuries in response to high nickel toxicity (Siddiqui et al. 2011).
Assessment of Endogenous Gibberellin and IAA Content
The gibberellin content in the seedlings germinated from the GA-primed seeds was increased by 1.3-fold under fluoride stress with respect to that in the non-primed sets irrigated with NaF alone (Fig. 3a). Prakash and Prathapasenan (1990) also showed the increased accumulation of gibberellin-like metabolites in the salt-stressed rice seedlings treated with GA. Increased gibberellic acid accumulation was found to be associated with lower oxidative injury in wheat seedlings exposed to toxicity mediated by zinc oxide nanoparticles (Iftikhar et al. 2019). In line with our previous report, the IAA level significantly increased during fluoride stress with respect to the control plants (Banerjee and Roychoudhury 2019b). However, the IAA content marginally decreased in the GA-primed seedlings exposed to fluoride toxicity (though higher than that in the control plants) with respect to that in the non-primed, stressed plants (Fig. 3b). The
A 20 B 180
*
*
*
*
Pro content (µg g-1 FW)
10 90
Anthocyanin content (mM g-1 FW)
0
C 500
Cont NaF GA GA+NaF
0
D 200
*
*
Cont NaF GA GA+NaF
*
*
250
Flavonoid content (µg g-1 FW)
100
0
TPC (µg g-1 FW)
Cont NaF GA GA+NaF
0
Cont NaF GA GA+NaF
Fig. 5 Measurement of non-enzymatic antioxidants like Pro (a), anthocyanin (b), flavonoids (c) and TPC (d) in the seedlings raised from non-primed IR-64 seeds grown in distilled water, i.e., control (Cont) or 25 mg L−1 NaF (NaF), and seedlings raised from seeds primed with 150 mg L−1 GA and grown in either distilled water
(GA) or 25 mg L−1 NaF (GA + NaF). The data are the mean values (n = 3) ± standard error (SE). The SE (p ≤ 0.05) in each case is repre- sented as ‘*’ (for comparison within treatments). The symbols repre- sent significance at p ≤ 0.05
synergistic roles of GA and IAA have been reported to stim- ulate the growth of Balanites aegyptiaca plants (Mostafa and Alhamd 2011). Nakamura et al. (1975) reported the synchro- nized roles of GA and IAA in mediating stress-relaxation in pea hook cell walls. Thus, increased GA and IAA accumula- tion in the primed seedlings compared to that in the control plants promoted overall tolerance to fluoride stress. It is evi- dent that such alteration in the phytohormonal homeostasis aided the primed seedlings to restrict fluoride uptake from the contaminated soil and to exhibit high survival under the challenging situations. Increased abundance of GA ensures its interaction with the DELLA proteins and the nuclear receptor, GID1 resulting in the activation of downstream stress-responsive cascades. This type of GA-mediated sig- nalling has been reported to mediate tolerance against salt, drought and cold in several plant species (Colebrook et al. 2014).
Assessment of the Levels of Chl and Accessory Pigments
Fluoride stress reduced Chl content in the non-primed plants. However, the seedlings germinated from the GA- primed seeds were able to maintain significantly higher level of Chl during fluoride toxicity. The Chl level in the GA- primed stressed seedlings was even higher than that in the control plants (Fig. 4a). Endogenous Chl is a typical marker of plant susceptibility to abiotic stress. High Chl level cor- responds to normal photosynthesis, and it has been observed that the tolerant plant species experience lower extent of chlorosis compared to the susceptible species (Pinto-Mari- juan and Munne-Bosch 2014). Exogenous application of GA increased the Chl fluorescence in the seedlings of Syzygium samarangense (Khandaker et al. 2015). Fluoride toxicity drastically reduced the content of the accessory pigments like β-carotene and xanthophyll. The GA-priming did not affect the β-carotene content during stress with respect to that in the non-primed, stressed plants, but significantly increased the xanthophyll level by 1.8-fold during stress with respect to that in the non-primed, stressed seedlings
A 0.9 B 4
*
*
*
0.6
Reducing power (mg g-1 FW)
0.3
0
Cont NaF GA GA+NaF
2
0
Total antioxidant capacity (mg g-1 FW)
Cont NaF GA GA+NaF
C 16
*
*
Percent of DPPH radical scavenging activity
8
0
Cont NaF GA GA+NaF
Fig. 6 Measurement of cumulative antioxidant responses like reduc- ing power (a), total antioxidant capacity (b) and DPPH radical scav- enging activity (c) in the seedlings raised from non-primed IR-64 seeds grown in distilled water, i.e., control (Cont) or 25 mg L−1 NaF (NaF), and seedlings raised from seeds primed with 150 mg
L−1 GA and grown in either distilled water (GA) or 25 mg L−1 NaF (GA + NaF). The data are the mean values (n = 3) ± standard error (SE). The SE (p ≤ 0.05) in each case is represented as ‘*’ (for com- parison within treatments). The symbols represent significance at p ≤ 0.05
(Fig. 4b, c). Kadioglu (1992) also observed that GA did not increase the carotene content in the treated cells of Chla- mydomonas reinhardtii and Anacystis nidulans, though the endogenous Chl accumulation was stimulated. The increase in the xanthophyll content in the GA-primed stressed plants corroborated with the fact that apart from acting as an acces- sory pigment, xanthophyll also behaves as a non-enzymatic antioxidant and protects the cells against oxidative stress (Miller et al. 1996).
Assessment of the Non‑enzymatic Antioxidants and Cumulative Antioxidant Responses
Priming the seeds with GA stimulated the level of non- enzymatic antioxidants like Pro, anthocyanin, flavonoids and TPC during fluoride stress by 1.3-, 1.5-, 1.5- and 1.4-fold with respect to those in the non-primed, stressed seedlings (Fig. 5a–d). Accumulation of Pro has been asso- ciated with the generation of stress memory along with efficient protection of the cellular osmoticum, since Pro is one of the most potent endogenous compatible solutes (Roychoudhury et al. 2015). In line with our observation,
Khandaker et al. (2015) observed that the anthocyanin con- tent was increased in the fruits of Syzygium samarangense exogenously treated with GA. Park et al. (2017) observed increased accumulation of flavonoids and phenolic com- pounds in the sprouts of Fagopyrum esculentum treated with gibberellic acid and IAA. Thus, the overall accumula- tion of the non-enzymatic antioxidants enhanced the abil- ity of the susceptible seedlings to tolerate the oxidative stress imposed by fluoride within the tissue biomass. As a result of high synthesis of these osmoprotectants, the H2O2 content was reduced and the cumulative antioxidant responses like reducing power, total antioxidant capacity and the DPPH radical scavenging activity were stimulated in the GA-primed, stressed seedlings (Fig. 6a–c). The high accumulation of the fluoride negatively regulated the abil- ity of the seedlings to chelate the toxic reactive oxygen species (ROS), as evident from the lowered DPPH radi- cal scavenging activity in the non-primed, stressed plants (Fig. 6c). However, GA priming activated the endogenous scavenging ability of the stressed seedlings and enabled them to exhibit tolerant phenotype even under prolonged fluoride toxicity (Fig. 6c).
A 14 B 0.5
*
*
*
*
*
7 0.25
CAT activity (µmol min-1 g-1 FW)
APX activity (mmol min-1 g-1 FW)
0
Cont NaF GA GA+NaF
0
Cont NaF GA GA+NaF
C 20
*
*
10
GPOX activity (µmol min-1 g-1 FW)
0
Cont NaF GA GA+NaF
Fig. 7 Measurement of the activity of the enzymatic antioxidants like CAT (a), APX (b) and GPOX (c) in the seedlings raised from non- primed IR-64 seeds grown in distilled water, i.e., control (Cont) or 25 mg L−1 NaF (NaF), and seedlings raised from seeds primed with 150 mg L−1 GA and grown in either distilled water (GA) or 25 mg
L−1 NaF (GA + NaF). The data are the mean values (n = 3) ± standard error (SE). The SE (p ≤ 0.05) in each case is represented as ‘*’ (for comparison within treatments). The symbols represent significance at p ≤ 0.05
Assessment of the Enzymatic Antioxidants
In order to tackle the excess H2O2 produced during fluo- ride stress, the activity of the enzymatic antioxidants like
CAT, APX and GPOX was significantly increased in the non-primed, stressed seedlings with respect to that in the control plants (Fig. 7a–c). This was in line with our previous observation where enhanced activity of these enzymes was observed during fluoride toxicity (Banerjee et al. 2020a). The GA-priming further stimulated the activity of CAT and APX by 1.7- and 1.4-fold, respectively, during stress with respect to those in the non-primed, stressed plants (Fig. 7a, b). However, the GPOX activity was decreased by 1.5-fold in the primed seedlings exposed to stress with respect to that in the non-primed seedlings treated with NaF (Fig. 7c). The activity of GPOX in the GA-primed stressed plants was maintained at a higher level with respect to that in the con- trol seedlings. Jaleel et al. (2010) also reported increased CAT and APX activity in the leaves and roots of Catharan- thus roseus seedlings treated with GA. The enzymes, viz.,
CAT and APX both scavenge the toxic H2O2
GA
Rice
Fluoride stress
Growth
GA
IAA
Endogenous levels
Weight
Relative water content
Reduced fluoride bioaccumulation
Increased photosynthesis and accessory pigments
Electrolyte leakage
Increased accumulation of non-enzymatic antioxidants
Increased activity of enzymatic antioxidants
molecules and
Fig. 8 Model illustrating the mode of protection mediated by GA- priming during prolonged fluoride toxicity in rice
detoxify them to produce water molecules (Banerjee et al.
2019b). Thus, increased CAT and APX activity corrobo- rated with the low H2O2 level in the GA-primed, stressed plants, as revealed from our current study. Probably due to such reduced H2O2 content, the primed seedlings did not require the necessity of further stimulating the GPOX
activity during stress, since mitigation of oxidative stress was sufficiently carried out via the enhanced activity of CAT and APX. As a result, the metabolic equivalents required to maintain the high GPOX activity could be channelized towards the growth and development of the seedlings.
Conclusion
The present manuscript established the ameliorative effects of GA-priming during fluoride stress in the sensitive rice cultivar IR-64. Pre-treatment of the seeds with GA prior to sowing led to elaborate re- organization of the cellular metabolism. As a result, the seedlings germinated from these primed seeds exhibited appreciable growth under fluoride stress along with significantly reduced symptoms of toxic-ity. This was primarily achieved due to restricted fluoride accumulation within the tissue biomass, due to which lower amount of fluoride was accumulated, thus accounting for lesser oxidative injuries. Reduced production of H2O2 ena-bled efficient maintenance of the elaborate membrane sys-tems which was evident from the low accumulation of MDA. Amelioration of the fluoride-mediated oxidative stress was also due to the high accumulation of the non-enzymatic antioxidants like Pro, anthocyanin, flavonoids, TPC, xantho-phyll, reducing power and total antioxidant capacity. Along with these osmoprotectants, the activity of the enzymatic antioxidants like CAT, APX and GPOX was also stimulated upon GA-priming during fluoride stress. The enzymatic as well as the non- enzymatic antioxidants worked in synchro-nization to scavenge and detoxify the H2O2 molecules pro-duced in the presence of the xenobiotic within the tissues. Such activation of the antioxidant machinery was regulated by increased accumulation of GA (compared to that in the non-primed, stressed plants) and IAA (compared to that in the control seedlings) in the GA-primed plants exposed to fluoride stress. As a result of such altered phytohormonal homeostasis, the plants exhibited lowered level of chlorosis and maintained higher photosynthetic efficiency. Overall, the current manuscript characterized the biochemical and metabolic basis of the fluoride stress-abrogating properties of the crucial plant growth regulator, gibberellin in rice. A model illustrating the protective roles of GA during fluoride toxicity in rice is presented in Fig. 8 on the basis of our observations. Seed priming with protective chemical agents has been widely regarded to be more economically sustaina-ble compared to the traditional exogenous spraying strategy. This is because priming requires lower volumes of solution to imbibe the seeds compared to that required for exogenous spraying on crops, which demands substantial quantity of the chemical agents. Seed priming is an easy scientific method to accelerate crop performance under sub-optimal condi-tions and use of this strategy requires no intense technical
knowledge due to which it is likely to be more acceptable and popular among the farmers. Thus, GA-priming could be used as an economic and eco-friendly strategy to cultivate low fluoride-accumulating rice in the fields infested with endemic fluorosis.
Acknowledgements Financial assistance from Science and Engi- neering 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. Ary- adeep Roychoudhury is gratefully acknowledged. Mr. Aditya Banerjee is thankful to University Grants Commission, Government of India, for providing Senior Research Fellowship in course of this work.
Compliance with Ethical Standards
Conflict of interest The authors declare that there is no conflict of in- terest in publishing this manuscript.
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