Herbal and chemical seed potentiations improve the redox health of aged seeds of indigenous aromatic rice cultivars through regulation of oxidative window, gene expression, and restoration of hormonal homeostasis

Abstract

Previous studies associated with seed potentiation support the critical role of metabolic readjustment in restricting the loss of seed vigor and viability of aged seeds. However, their exact role in the regulation of ‘oxidative windows’ of potentiated seeds is rarely studied and hence is the subject of the present investigation. Seed potentiation of two contrasting indigenous aromatic rice cultivars, differing in sensitivity towards redox attributes (Oryza sativa L., Cultivars Tulaipanji and Jamainadu), with standardized doses of hydrogen peroxide (20 mM), triadimefon (250 μM), herbal extract (1% aqueous extract of Lantana camara flower) and distilled water before accelerated aging (RH 92% and 41 °C for 24 h) found to have significant impact on redox regulation of aged seeds and improvement of germination phenotypes. The efficacy of integrated RBOH-ascorbate–glutathione/catalase pathway, redox status and other redox fingerprints in the metabolic landscape of potentiated-aged seeds vis-a-vis non-potentiated-aged seeds corroborate the impact of seed potentiation on the regulation of ‘oxidative window’ of experimental rice seeds. Gene expression analysis of central redox hub enzymes (Osrboh, OsAPx2, OsGRase, OsCatA) strongly substantiates the impact of seed potentiation on transcriptional regulation of genes for redox homeostasis in accelerated aged seeds. The novelty of the current effort is that it suggests a positive nexus between seed potentiation-induced redox regulation and hormonal homeostasis. The efficacy of seed potentiation on the redox regulation of experimental accelerated aged seeds is found to be cultivar-specific and comparatively better in the cultivar Tulaipanji as compared to the cultivar Jamainadu and in the order herbal extract, hydrogen peroxide, hydropriming and triadimefon.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12298-023-01375-9.

Introduction

Seed aging during storage is a serious concern to agriculture as it is projected that about one-fourth of the seeds harvested lose their vigor and viability annually causing grave economic losses (Zhang et al. 2020). Seed aging during storage, which suffers cumulative irreversible and relentless oxidative changes, causes ill health of the seed which is exhibited by altered germination phenotypes and reduced environmental stress resilience (Zhang et al. 2020; Xu et al. 2020). The concept of the ‘oxidative window’ of the aging seeds hypothesizes that both higher and lower endogenous titer of reactive oxygen species (ROS) have significantly negative effects whereas maintenance of the critical range of ROS titer has a positive effect on seed health (Li et al. 2022; Ratajczak et al. 2015; Kurek et al. 2019). Therefore, the concept of regulation of the ‘oxidative window’ of aging seeds was found to have tremendous significance in the maintenance of redox health in aging seeds for regulation of their vigor and viability (Li et al. 2022). The altered ‘oxidative windows’ of aged seeds primarily due to over-accumulation of prooxidants, instigate diverse nonspecific oxidative damages, resulting in loss of vigor and viability. The central role of a redox hub consisting of ROS generating system, antioxidative defense mechanism at the metabolic interface, redox sensors, and signaling system that play a pivotal role in determining seed aging is yet to be elucidated (Kurek et al. 2019; Ebone et al. 2019; Zhang et al. 2020; Xu et al. 2020).

The ROS-antioxidant interaction at the metabolic interface during seed aging plays a central role in determining the status of seed health and viability (Ratajczak et al. 2015; Kurek et al. 2019). A major tilt in redox homeostasis towards prooxidant combined with reduced efficacy of antioxidative defense, and lack of proper redox signaling for oxidative stress amelioration always instigate oxidative damage that accumulate overtime during aging causing loss of vigor and viability (Ratajczak et al. 2015; Kurek et al. 2019). Aggravated production of prooxidants instigate oxidative damages to membrane lipid, protein, enzymes, and nucleic acids, causing metabolic dysfunction, impacting the redox health of aging seeds, and if allowed, reducing the seed viability (Ratajczak et al. 2015).

The role of ROS signaling is found to be extremely significant in regulating dormancy and germination of seeds (Li et al. 2022; Liu et al. 2010; Wang et al. 2015a), probably through cross-talking with germination regulating hormones [like gibberellic acid (GA), abscisic acid (ABA), jasmonic acid (JA)] and utilization of storage proteins through oxidative degradation (Gong et al. 2022; Kurek et al. 2019). Therefore, a balanced level of prooxidants that determine the sound redox health of the seed while aging is taking place, is a pre-requisite for the maintenance of viability, vigor, and germinability of seeds (Li et al. 2022). Central antioxidant redox hub consisting of respiratory burst oxidase homolog (RBOH)–superoxide dismutase (SOD)–ascorbate–glutathione (ASC-GSH)/catalase (CAT) pathway, plays an important role in redox regulation by integrating both primary ROS-generating system with its counterpart that is antioxidative processing of ROS involving ascorbate–glutathione pathway (Foyer and Noctor 2016; Kurek et al. 2019).

In the recent past, there has been several evidence of herbal seed potentiation for restoring seed viability and vigor under aging conditions. The profuse leakage of amino acids from aged seeds is prohibited significantly by the application of bioactive chemicals present in herbal extracts (Basra et al. 2011; Kumar et al. 2020). In fact, it has been noticed that seeds pretreated with herbal extracts significantly reduce the loss of protein, nucleic acids, and insoluble carbohydrates and restore the activity of the antioxidative defense enzyme catalase during accelerated or forced aging (Pawar and Laware 2018). Though the usage of obnoxious weed Lantana camara as an herbal seed potentiating agent is not recorded in literature, however previous studies exhibited rich sources of bioactive compounds and phytoconstituents like fatty acid esters, fatty alcohols, aromatic acids, ethers, essential oils, etc. in different plant parts that support their potential uses in seed invigoration while aging (Saravanan 2017; Ved et al. 2018).

It has been reported that hydrogen peroxide primarily can affect seed aging by reverting the oxidative deterioration (Hossain et al. 2015). Hydrogen peroxide (H₂O₂) application might function in the redox-regulatory event in aging seeds. It has been noticed that hydrogen peroxide pretreatment in aging seeds might influence cross-talking with several other hormonal systems and signaling molecules, like GA, ABA, ethylene, NO, etc., and regulate defense processes during germination (Hossain et al. 2015; Farooq et al. 2020; Yan et al. 2020; Chen et al. 2021) and hence was explored in the present investigation as seed potentiating agent. Previous works suggest that pretreatment of seeds with triazole-type growth retardant significantly reduces the deleterious effects of aging on germination performance and field emergence (Sridharan et al. 2015; Pawar and Laware 2018; Chen et al. 2021). Further, in these potentiated seeds, an upregulated antioxidative defense system represents one of the most important defense strategies to counter oxidative threats promoted by accelerated aging conditions (Yang et al. 2014). So, in the present study one triazole compound, triadimefon has been used to explore further its role as seed potentiating chemical based on its established antioxidant properties.

The storage of seeds of IARCs in the tropical climate of Rarh West Bengal (phytogeographical regions with alluvial soil and an average rainfall of 135 cm) poses serious concern because high temperature and RH (relative humidity) induced loss of seed quality and enhancement of the phenomenon of oxidative damages during seed aging (Zhang et al. 2020; Chen et al. 2021). Due to this kind of environment in Rarh Bengal, long-term storage of the seeds of IARCs became not only problematic but also demands proper physiological maintenance for restricting oxidative deterioration while regulating the redox health of aged seeds (Zhang et al. 2020; Chen et al. 2021). So, in this backdrop of a lack of systematic study on the exact role of seed potentiation in relation to redox regulation, gene expression, and hormonal homeostasis of IARCs, the present study was rationally framed to unfold the impact of seed potentiation on ‘oxidative window’ of seeds of IARCs to elucidate the role of central redox hub in calibrating the loss of seed vigor and viability.

Materials and methods

Seed potentiation, accelerated aging, and seedling growth

Seeds of two IARCs (Oryza sativa L., Cultivars Tulaipanji, and Jamainadu) were selected as experimental materials based on their contrasting redox attributes under drought stress (Bhattacharjee and Dey 2018). Viable seed lots were procured from Chinsurah Rice Research Station, Government of West Bengal, India, and subsequently cultivated at CRSMF (Crop Research and Seed Multiplication Farm), University of Burdwan, West Bengal, India, for maintenance and uses for experimental purpose. The treatment conditions for accelerated aging and seed potentiation were standardized based on the results of a pilot experiment with IC₅₀ value of germination (supplementary tables 1 and 2). The seed potentiation and accelerated aging, treatment conditions were standardized to investigate the biology of seed potentiation on accelerated aging, based on the results of a pilot experiment as given in Table 1.

Different seed potentiation and accelerated aging conditions standardized for investigating the impact of potentiation of seeds of experimental indigenous aromatic rice cultivars (Oryza sativa L., Cultivars Tulaipanji and Jamainadu)

Serial no.	Seed potentiating condition	Accelerated aging condition
1	Untreated control (No potentiating treatment)	Untreated control (No aging treatment) (RH 65%)
2	Nil (No potentiating treatment)	4.5% KOH solution (RH 92% and 41 °C for 24 h)
3	Hydro-priming (distilled H2O) (for 18 h and then air dried for 24 h)	4.5% KOH solution (RH 92% and 41 °C for 24 h)
4	Herbal extract (1% aqueous extract of Lantana camara flower) (for 18 h and then air dried for 24 h)	4.5% KOH solution (RH 92% and 41 °C for 24 h)
5	20 mM H2O2 solution (for 18 h and then air dried for 24 h)	4.5% KOH solution (RH 92% and 41 °C for 24 h)
6	250 μM Triadimefon [1-(4-chlorophenoxy)-3,3-dimethyl-1-(1H-1,2,4-triazole-1-yl)-2-butanone] solution (for 18 h and then air dried for 24 h)	4.5% KOH solution (RH 92% and 41 °C for 24 h)

The accelerated-aged and potentiated-aged seeds were air dried to make the seed moisture content like the previous one (for Tulaipanji 9.7% and Jamainadu 10.5%). Seeds were then surface sterilized for five minutes with 0.1% HgCl₂ and subsequently washed thoroughly with Milli Q water. Surface sterilized seeds were imbibed in Milli Q water for 24 h in darkness at 25 ± 2 °C. For all the biochemical analyses, water-imbibed seeds were used and for physiological phenotyping, the imbibed seeds were grown on moist filter paper on petri plates and were kept in a seed germinator cum plant growth chamber (LAB-X-India) for up to 7 days. For the untreated control set, water-imbibed seeds were sown directly on Petri plates and exposed to the same condition. All the seed lots were allowed to grow in a condition set at a temperature of 25 ± 2 °C with 78 ± 2% relative humidity and a photocycle having 14 h photoperiod with 270 µmol m⁻² s⁻¹ light intensity.

Spectrofluorometric estimation of “total ROS” generation

30 mg of de-husked seeds were placed in 8 mL 40 mM TRIS–HCl buffer (pH-7.0) in the presence of 100 μM 2,7-dichlorofluorescein diacetate (DCFDA, Sigma-Aldrich) at 30 °C. After 60 min, supernatants were taken and the fluorescence was monitored with excitation at 504 nm and emission at 525 nm in a spectrofluorometer (Hitachi, Model F-4500 FL Spectrofluorometer) according to the procedure of Simontacchi et al. (1993).

In situ localization of H₂O₂ by laser confocal microscopy

The sample preparation for in situ localization of H₂O₂ was done by following the method of Kaur et al. (2016). Experimental seeds were dipped in 10 μM H₂DCFDA (Sigma-Aldrich) solution and incubated at room temperature for 1 h. Treated seeds were washed thoroughly with autoclaved Milli-Q water and mounted with 20% glycerol in the slide for observation. Histochemical localization of H₂O₂ in the seed embryonic axis was monitored by dichlorofluorescein diacetate staining followed by laser confocal microscopy with attached Leica application suite X software (Leica TCS SP8, Objective 40X), in scanning excitation mode at 488 nm and emission at 505–530 nm. Detection of H₂O₂ accumulation was done by identifying green fluorescence in the seed embryo axis.

Estimation of “H₂O₂ and O₂^·−” generation

The procedure of MacNevin and Uron (1953) was explored to estimate hydrogen peroxide using titanic sulfate. Hydrogen peroxide was extracted in acetone using 5% titanium sulphate after that washed in acetone and finally, the absorbance was taken at 420 nm in UV–VIS spectrophotometer (Shimadzu, Japan). For the estimation of superoxide anion, 500 mg of tissues were extracted in cold phosphate buffer (0.2 M, pH-7.2), and then diethyldithiocarbamate (10^–3 M) was added. The homogenate was centrifuged immediately at 3000 g at 4 °C for 15 min. Reduction of NBT (nitro blue tetrazolium, 2.5 × 10^–4 M) was used to assess superoxide anion content. The absorbance was measured at 540 nm in UV–VIS spectrophotometer (Shimadzu, Japan), and the formation was expressed in terms of ΔA₅₄₀ g⁻¹ dm min⁻¹ (Chaitanya and Naithani 1994).

DAB assay for in situ localization of hydrogen peroxide

For the DAB assay, the de-husked experimental seed was immersed into 2 mL of DAB (3,3′-Diaminobenzidine) (Sigma-Aldrich) stain (dissolved in Milli-Q water, pH-3.8) in a Petri plate and kept for 8 h at room temperature under the bright artificial light. Then, the sample was immersed in absolute ethanol for half an hour. Treated seeds were then mounted with 20% glycerol on a slide, as per the procedure of He et al. (2009). Finally, the image of the stained embryo axis was captured using a stereo microscope. Detection of H₂O₂ accumulation was done by identifying brown spots.

Assay for DPPH (2,2′-diphenyl-1-1-pycryl hydrazyl) free radical scavenging property

For the determination of DPPH radical scavenging activity, the process of Mensor et al. (2001) was followed. 1.5 g dry experimental seeds (kept at 45 °C, 2 days) were taken and homogenized in 30 mL 80% methanol in a shaking incubator for a day. The homogenate was then centrifuged at 3500 rpm for 20 min at 4 °C. The supernatant was used to determine the DPPH radical scavenging activity.

Total non-protein thiol content

For the extraction and estimation of total thiol content, 500 mg germinating seeds (500 mg) were homogenized in 3% trichloroacetic acid (TCA) and the homogenate was centrifuged at 8000 rpm for 15 min. The supernatant was collected and then diluted with 100 mM sodium phosphate buffer, pH 7.5 (tenfold dilution). Subsequently, 0.5 U/mL glutathione reductase, 0.2 mM NADPH (reduced nicotinamide adenine dinucleotide phosphate), 0.5 mM DTNB (5, 5′-dithiobis-2-nitrobenzoic acid) and were added. Finally, the total thiol content was measured by the absorbance taken at 412 nm in a UV–VIS spectrophotometer (Shimadzu, Japan) (Tietze 1969).

Total phenol content

For the extraction and estimation of the total phenol pool, the process of Chang et al. (2002) was followed. Experimental seeds were dried in a hot air oven at 45 °C for 48 h and then crumpled to powder. 5 g of powdered plant sample was homogenised in 50 mL methanol by recurrent maceration for 48 h. Then, the solvent was evaporated in a rotary vacuum evaporator reduced to 5 mL volume, and centrifuged at 10000 rpm for 15 min. The supernatant was collected for quantification of the phenol pool. To 0.5 mL of supernatant, 2 mL Folin ciocalteu reagent (1N) and 2 mL saturated sodium bicarbonate solution were added and incubated for 45 min at room temperature and the absorbance was taken at 765 nm by UV–VIS spectrophotometer (Shimadzu, Japan). The standard curve was prepared using Gallic acid.

Total flavonoid content

For the extraction and estimation of the total pool of flavonoids, the process of Chang et al. (2002) was followed. For this, 0.1 mL plant extract (extraction same as total phenol) was added to 0.1 mL 10% AlCl₂ and 0.1 mL 1(N) potassium acetate followed by the addition of 2.7 mL Milli-Q water. Then the assay mixture was incubated for 30 min at 37 °C and the absorbance was taken at 415 nm by UV–VIS spectrophotometer (Shimadzu, Japan). The standard curve was prepared using quercetin solutions in methanol.

Extraction and estimation of enzymes of central redox hub

Extraction and estimation of NADPH oxidase (RBOH) (EC 1.6.3.1) activity was done following the procedure of Dey and Bhattacharjee (2022). Experimental seeds were extracted with 25 mM potassium phosphate buffer (pH-7.0) containing 5 mM Na-ascorbate and then subsequently centrifuged at 20000 rpm for 30 min at 4 °C. Supernatant was used as enzyme source. The assay mixture contained 0.5 mM paracaumaric acid containing 0.2 mM NADPH, 20 μL enzyme extract, 100 mM Na-acetate, 1 mM MnCl₂ (manganese chloride), and the enzyme activity was measured by monitoring the reduction in absorbance at 340 nm in UV–VIS spectrophotometer (Shimadzu, Japan).

For the extraction and estimation of the enzyme ascorbate peroxidase (APX) (EC 1.11.1.11), the procedure of Nakano and Asada (1981) was followed. Enzyme activity was estimated following the reduction in absorbance at 290 nm in a UV–VIS spectrophotometer (Shimadzu, Japan)] in 1 mL reaction mixture containing 0.1 mM H₂O₂ as substrate and 0.5 mM ascorbate and 200 μL enzyme extract. The reaction was started by adding the enzyme extract. Absorbance was taken before and after (1 min) the addition of the enzyme source.

The extraction and estimation of the enzyme dehydroascorbate reductase (DHAR) (EC 1.8.5.1), was done following the process of Nakano and Asada (1981). The enzyme extract was mixed with 50 mM potassium phosphate buffer (pH-7.8), 2.5 mM reduced glutathione, 0.2 mM dehydro ascorbate, 0.1 mM ethylenediamine tetraacetic acid, and reaction rates were assessed by monitoring the increase in absorbance at 265 nm in 10 s and 30 s after addition of enzyme source.

For the extraction and estimation of glutathione reductase (GR) (EC 1.6.4.2) activity, experimental seeds were homogenized with 50 mM potassium phosphate buffer (pH 7.2) and centrifuged at 5000 rpm for 15 min. The reaction mixture containing 0.5 mM GSSG (glutathione disulfide), 4 mL 50 mM potassium phosphate buffer (pH-7.0) containing 2 mM Na₂EDTA, 1 mL enzyme extract, 0.15 mM NADPH was incubated for 30 min, and then absorbance was taken at 340 nm (Δ₁). For determining the correction factor, the absorbance of the same composition of the assay mixture without NADPH was taken (Δ₂). Finally, Δ₁–Δ₂ was determined for the estimation of the activity of GR (Nakano and Asada 1981).

For the extraction and estimation of the activity of catalase (CAT) (EC 1.11.1.6) the process of Snell and Snell (1971) was followed. The procedure based on the consumption of H₂O₂ by the extracted enzyme catalase was used with an extinction coefficient of 39.4 mM⁻¹ cm⁻¹ at 240 nm [absorbance measured by UV–VIS spectrophotometer (Shimadzu, Japan)] for 3 min. The assay mixture contained 50 mM potassium phosphate buffer (pH-7), 10 mM H₂O₂ and 500 μL enzyme extract in a total volume of 3 mL.

Extraction and estimation of soluble components of the ascorbate–glutathione pathway

The procedure of Law et al. (1983) was followed for the extraction and estimation of total and reduced ascorbate content. 1 g of experimental seed sample was extracted with 10 mL cold 5% metaphosphoric acid and then centrifuged at 15000 rpm for 30 min at 4 °C. The supernatant was collected for quantitative estimation of ascorbate and glutathione. The assay mixture consists of 10 mM DDT (dichlorodiphenyltrichloroethane) and 150 mM phosphate buffer (pH 7.4) which contains 5 mM EDTA and the enzyme extract. The mixture was then incubated at room temperature for 10 min and subsequently, 0.5% N-ethylmaleimide was added to the assay mixture. After that, 44% orthophosphoric acid, 10% TCA, and 4% of α,α′-bipyridyl were added one after another. Finally, 3% FeCl₃ was supplanted to the mixture and the mixture was incubated for 40 min at 40 °C, and absorbance was taken at 525 nm. The dehydroascorbate (DHA) content was assessed by the deduction of reduced ascorbate content from the total ascorbic acid. The total glutathione content was assessed using the absorbance at 412 nm in a UV–VIS spectrophotometer (Shimadzu, Japan) based on the procedure of Zhang and Kirkham (1996). The reduced glutathione content was estimated from the standard curve of 0–30 µmol mL⁻¹ standard glutathione. Oxidized glutathione (GSSG) contents were determined following the removal of glutathione (GSH) by the addition of 2-vinylpyridine derivative. Finally, the glutathione (GSH) contents were determined by the subtraction of oxidized glutathione (GSSG) contents from total glutathione contents.

RT-qPCR study of differential expression of central redox hub genes (Osrboh, OsAPx2, OsGRase, OsCatA) of potentiated-accelerated aged seeds and non-potentiated-accelerated aged seeds

Guanidium isothiocyanate-phenol based reagent (RNA- XPress™ reagent, HiMedia) was used for the extraction of the total RNA pool from experimental seeds as per the manufacturer’s instructions. 100 mg of liquid nitrogen frozen experimental seed tissue was homogenized in 1 mL of RNA express reagent (RNA-XPress™ reagent, HiMedia) and then 200 μL chloroform was added. The extract was incubated for 5 min at room temperature and centrifuged at 12000 rpm at 4 °C for 15 min. The topmost aqueous layer was collected, and chilled isopropyl alcohol (500 μL) was added. The mixture was incubated for 10 min and then centrifuged at 12,000 rpm for 10 min in cold conditions. The precipitated RNA pellet was collected and washed with 75% cold ethanol. The washed pellet was air-dried and stored at − 80 °C for further use. The authenticity of RNA and quantification was performed using a NanoDrop spectrophotometer (ND1000, NanoDrop Technologies, South Korea).

In the next step, first-strand cDNA was synthesized with a reverse transcriptase system (Superscript III, Invitrogen) according to the manufacturer’s protocol. Then, quantification of cDNA was done using Nano Drop spectrophotometer (ND1000, NanoDrop Technologies, South Korea) at 260 nm and the cDNA was stored in an ultra freezer at − 80 °C for further use. The primers for the genes redox genes Osrboh, OsAPx2, OsGRase, OsCatA, and the housekeeping gene 18S rRNA (Hsk) were constructed using NCBI-Primer blast (Supplementary table 3).

RT-qPCR analysis was done for the assessment of transcript abundance of the above-mentioned genes using quantitative Real-time PCR (CFX Connect Real-Time PCR Detection System from Bio-Rad) following the procedure of Burch-Smith et al. (2006). Reactions were accomplished using Fast Start Universal SYBR Green Master Mix (Invitrogen, Applied Biosystems) following the manufacturer’s instructions. PCR reactions were performed by making an assay mixture consisting of 10 μL SYBR Green Master mix, 1 μL of each forward and reverse primer, 1 μL cDNA, and ultrapure Milli-Q water to make the final volume 20–25 μL. Then, the reaction mixture prepared was subjected to denaturation at 95 °C for 10 min subsequently 40 cycles at 95 °C for 15 s, and finally annealing at 60 °C for 60 s. The relative expression levels of each gene were calculated based on the ΔΔCt method (Pfaffl 2001) and then normalized to the Ct data relative to the transcript level of the internal control, i.e. 18S rRNA (Hsk) gene.

RP-HPLC-based quantification of ABA, GA, and JA in germinating seeds

RP-HPLC coupled Photodiode assay was explored for quantification of GA, ABA, and JA following the procedure of Dey and Bhattacharjee (2022) using Dionex UltiMate 3000 liquid Chromatograph with a 5 cm flow cell and Chromeleon system manager as the data processor. Experimental seeds were crushed to powder using liquid nitrogen and stored for further use. Seed powder was homogenized with 2 mL of cold acetonitrile and after 12 h centrifuged at 20,000 rpm for 10 min at 4 °C. The supernatant collected was mixed with 1.5 mL of 0.1 M phosphate buffer (pH-7.1) and kept at − 80 °C for 30 min. The mixture was thawed at 4 °C and extracted three times ethyl acetate, after the addition of HCl. Then the mixture was subjected to centrifugation for 10 min at 15,000 g in cold and the ethyl acetate phase was collected. The sample was dried in a Rotary vacuum evaporator (Eyela) and then finally dissolved in 1 mL of mobile phase (methanol and H₂O in a 1:1 ratio). The extract was filtered through a millipore membrane filter (0.22 mm) for HPLC in C₁₈ Column (5-micron particle size, 250 × 4.6 mm) and UV detector. Absorbance was taken at 254 nm for the detection of hormone peaks. Standard GA, ABA, and JA (Sigma Alrich) are used for the development of the standard curve.

Extraction and estimation of free carbonyl content

For the extraction and estimation of free carbonyl content (index of protein oxidation), the process of Jiang and Zhang (2001) was followed. Experimental seeds were extracted with 5 mL of 50 mM potassium phosphate buffer (pH 7.0) containing 1 mM EDTA (ethylene diamine tetra acetic acid), 1 mM PMSF (phenyl methyl sulfonyl fluoride) 10 mM DTT (dithiothreitol) and 5 µg mL⁻¹ leupeptin (protease inhibitor). Centrifugation was done at 14000 × g for 30 min and 10 mM DNPH (Dinitrophenylhydrazine) dissolved in 2 M HCl was added to the supernatant containing oxidized protein. Subsequently, the samples were treated with TCA (trichloroacetic acid) and centrifuged in a microfuge for 5 min. The supernatant was collected and absorbance was taken at 370 nm in a UV–VIS spectrophotometer (Shimadzu, Japan). Free carbonyl contents were quantified based on the molar absorption coefficient of 22 mM cm⁻¹.

Extraction and estimation of hydroperoxide

500 mg of experimental seed was extracted with Tris HCl (150 mM, pH-6.8). The homogenate was centrifuged at 5000 rpm and assessed for hydroperoxide content according to Devasagayam et al. (2003). The assay mixture consists of an aliquot of sample, 4 mM BHT in 90% methanol (v/v), 100 mM xylenol orange 250 mM ammonium ferrous sulfate, 0.25 mM H₂SO₄. After incubation for 30 min, 100 mM triphenyl phosphine was added to reduce only hydroperoxide to distinguish from H₂O₂, and the absorbance was taken at 560 nm.

Extraction and estimation of conjugated diene

The procedure of Buege and Aust (1978) was followed for the estimation of conjugated diene. Shortly, seeds were homogenized with chloroform: methanol mixture (2:1) and subjected to vertexing for some time. Then the extract was centrifuged for 10 min at 2000 rpm. The lower chloroform layer was collected and dried in a rotary vacuum evaporator with a continuous flow of nitrogen at 45 °C. The residue thus found was dissolved in cyclohexane and absorbance was measured at 230 nm in an UV–VIS spectrophotometer (Shimadzu, Japan) against cyclohexane.

Extraction and estimation of thiobarbituric acid reactive substances

For the estimation of oxidative membrane lipid peroxidation, the accumulation of thiobarbituric acid reactive substances (TBARS) was assessed as per the procedure of Heath and Packer (1968). Shortly, seeds were homogenized in trichloroacetic acid (0.1%) and centrifuged for 15 min at 10000 rpm. Now, in 5% TCA containing 1% thiobarbituric acid (TBA), 1 mL of supernatant was added and subsequently placed in a hot water bath for 30 min and then cooled rapidly. Now, the cooled mixture was centrifuged at 10000 rpm for 10 min in cold conditions. Finally, the absorbance of the supernatant was taken at 530 nm in a UV–VIS spectrophotometer (Shimadzu, Japan). The TBARS concentration was measured from its extinction coefficient of 155 µM cm⁻¹.

Determination of germination and early growth phenotypes

For investigating germination and early growth phenotypes, T₅₀ value, GR (germination rate), CVG (coefficient of velocity of germination), MDG (mean daily germination), GRI (germination rate index), RGI (relative growth index), and VI (vigor index) were calculated based on the formulae of Rubio-Casal et al. (2003) and Bhattacharjee (2008).

Statistical analysis

All individual experiments were carried out twice and for imposing different treatment conditions three replicates at diverse times were used. Results presented in each case are the mean of three replicates ± standard error (SE). ANOVA was performed and the means of the significant differences were analyzed for statistical analysis of the data for significance, using Fisher’s least significant test with difference at the 0.05 level of probability.

Result

Seed potentiation caused a shift in endogenous redox cues in the metabolic landscape of accelerated aged seeds of two indigenous aromatic rice cultivars (IARCs)

Results at large exhibited significant suppression in the accumulation of prooxidants both at the individual and total level in differently potentiated-accelerated aged seed lots over the non-potentiated and hydro-primed-accelerated aged seed lots in both the experimental IARCs (Fig. 1a–c). However, when compared with freshly harvested seed lots, the differently potentiated seed lots exhibited marginal but significant increments of both O₂^·−, H₂O_2, and total ROS (Fig. 1a–c). When compared between the impact of different potentiating agents, herbal seed potentiation with aqueous extract of the flower of Lantana camara was found to be more effective in restricting the accumulation of ROS followed by H₂O₂ and triadimefon (Fig. 1a–c). The cultivar Tulaipanji, on the other hand, seems to be more responsive in down-regulating the accumulation of H₂O₂ (Fig. 1b) and total ROS (Fig. 1c) as compared to the cultivar Jamainadu. Hydro-priming prior to accelerated aging caused only a marginal impact on restricting the accumulation of pro-oxidants, which is even comparable with the impact of triadimefon (Fig. 1a–c). Histochemical study for in-situ histochemical localization of reactive oxygen species (ROS) through DAB staining (Fig. 2a–l) and laser confocal microscopy (Fig. 3a–l) also substantiates the data of pro-oxidants accumulation at the metabolic interface. Both DAB (3, 3′-Diaminobenzidine) staining (Fig. 2a–l) and laser confocal microscopic investigation (Fig. 3a–l) of in-situ localization of ROS in the embryo of the potentiated-accelerated aged seed, hydro-primed-accelerated aged, accelerated aged and freshly harvested seeds revealed the same trend of ROS accumulation. H₂O₂ accumulation was found to be maximum in the embryo of accelerated aged seeds of both the investigational IARCs as detected through laser confocal microscopy (Fig. 3b, h) and DAB staining (Fig. 2b, h) over potentiated-accelerated aged seeds. The staining intensity of the embryo of accelerated aged seed was found to be significantly more intense with greater surface area (Fig. 2b, h) as compared to freshly harvested seeds (Fig. 2a, g). The differently potentiated-accelerated aged seed lots exhibited a significant decline in the intensity and the surface area of staining of the embryo as detected by laser confocal microscopy (Fig. 3) and stereo zoom microscopy (Fig. 2) over non-potentiated and accelerated aged seeds, confirming the impact of seed potentiation on regulatory redox metabolic shift. The result in general exhibited a substantial reduction in the accretion of non-protein thiol compounds (Fig. 4c) as well as radical scavenging property (Fig. 4d) of accelerated aged seeds of both the investigational IARCS. When potentiated with herbal extract, H₂O₂ and triadimefon and subsequently aged, we found a significant upregulation in the accumulation of total non-protein thiol compound (Fig. 4c) as well as radical scavenging property (Fig. 4d) of seeds of both the experimental rice cultivars over non-potentiated accelerated aged counterparts. When compared, the accelerated aged seed lots of both the rice cultivars showed significant inhibition in the competence of the cumulative antioxidant properties and non-protein thiol dependent antioxidant competence hinting at a significant loss of the redox health of accelerated aged seeds. Potentiation with herbal extract as well as hormone like substances (H₂O₂ and triadimefon), prior to accelerated aging significantly up-regulates the non-enzymatic thiol dependent (Fig. 4c) and cumulative antioxidant competence (Fig. 4a, b, d) of the aged seeds, confirming their impact on antioxidant coupled regulation of redox health at the metabolic interface. Moreover, when compared, the herbal extract proves to be more potent in potentiating accelerated aging seeds followed by H₂O₂ and triadimefon. Hydro-priming prior to accelerated aging though marginally restored cumulative antioxidants properties and non-protein thiol level, but when compared, the impact seems to be significantly lesser than herbal potentiation and redox (H₂O₂) potentiation (Fig. 4a–d).

Fig. 1.

Seed potentiation impact (Herbal, H₂O₂, Triadimefon, and hydroprimed) of accelerated aged seeds on the accumulation of prooxidants [superoxide (a), H₂O₂ (b) and total ROS (c)] during early germination of two experimental IARCs (Oryza sativa L., Cultivars Tulaipanji and Jamainadu). Results are the mean of three replicates ± standard error. Different alphabets represent significant differences, using Fisher’s least significant difference test (p < 0.05)

Fig. 2.

In situ localization of H₂O₂ by DAB staining in the embryo of different types of potentiated-accelerated aged seeds [Hydro-primed (c, i), Herbal (d, j), H₂O₂ (e, k) and Triadimefon (f, l)], non-potentiated-accelerated aged seeds (b, h) vis-a-vis untreated control (a, g) during early germination of two experimental IARCs (Oryza sativa L., Cultivars Tulaipanji and Jamainadu)

Fig. 3.

Laser confocal microscopy for subcellular localization of ROS in the embryo of different types of potentiated-accelerated aged seeds [Hydro-primed (c, i), Herbal (d, j), H₂O₂ (e, k) and Triadimefon (f, l)] and non-potentiated accelerated aged seeds (b, h) vis-a-vis untreated control (a, g) during early germination of two experimental IARCs (Oryza sativa L., Cultivars Tulaipanji and Jamainadu). Green fluorescence indicates the presence of ROS

Fig. 4.

Seed potentiation impact (Hydro-primed, Herbal, H₂O₂ and Triadimefon) of accelerated aged seeds on the accumulation of non-enzymatic antioxidant [total phenol (a), flavonoids (b) and total thiol content (c)] and radical scavenging property (by DPPH assay) (d) during early germination of two experimental IARCs (Oryza sativa L., Cultivars Tulaipanji and Jamainadu). Results are the mean of three replicates ± standard error. Different alphabets represent significant differences, using Fisher’s least significant difference test (p < 0.05)

Seed potentiation modulates the efficiency of the integrated RBOH-ASC-GSH/CAT pathway in accelerated-aged seeds of two IARCs

Comparative evaluation of the activities of RBOH (NADPH oxidase) in differently potentiated-accelerated aged seed lots vis-a-vis to non-potentiated-accelerated aged, hydro-primed-accelerated aged and freshly harvested seed lots, showed a substantial down-regulation in the efficiency of the enzyme under herbal and redox potentiation for both the experimental IARCs (Fig. 5a). Seed potentiation particularly with herbal extract found to have significant impact in down-regulating the activity of NADPH oxidase (Fig. 5a) for the cultivar Tulaipanji over non-potentiated-accelerated aged seed lots. The extent of down-regulation in H₂O₂ and triadimefon potentiated and accelerated aged seed lots of the cultivar Tulaipanji was found to be suggestively minor compared to the herbal extract potentiated seed lots (Fig. 5a). On the contrary, the cultivar Jamainadu exhibited only marginal and inconsequential impact in down-regulating the activity of NADPH oxidase (Fig. 5a) over the non-potentiated-accelerated aged seed lots, therefore a clear cultivar specific difference in the impact of seed potentiation on NADPH oxidase activities pertaining to the generation of ROSwas observed. Hydro-priming shows a comparable impact to that of triadimefon potentiated seeds, in restricting the activity of NADPH oxidase (Fig. 5a).

Fig. 5.

Seed potentiation impact (Hydro-primed, Herbal, H₂O_2, and Triadimefon) of accelerated aged seeds on the activities of the enzymes of central redox hub [RBOH (a), Ascorbate peroxidase (b), Dehydroascorbate reductase (c), Glutathione reductase (d) and Catalase (e)] during early germination of two experimental IARCs (Oryza sativa L., Cultivars Tulaipanji and Jamainadu). Results are the mean of three replicates ± standard error. Different alphabets represent significant differences, using Fisher’s least significant difference test (p < 0.05)

Seed potentiation with herbal extract and H₂O₂ was found to have a significant impact in upregulating the activities of ascorbate peroxidase (APOX), dehydroascorbate reductase (DHAR), and glutathione reductase(GR) (Fig. 5b–d) in accelerated aged seeds as compared to non-potentiated-accelerated aged seeds, substantiating the role of seed potentiation through herbal agent and H₂O₂ in fine-tuning the redox status through efficient ROS management system necessary for the maintenance of redox homeostasis. Seed potentiation with triadimefon and hydro-priming was found to have only a minor insignificant effect in up-regulating the effectiveness of all the enzymes of the H₂O₂ processing pathway (Fig. 5b–e). Moreover, the drought-resistant redox competent cultivar Tulaipanji was found to be better responsive in coordinating RBOH-ASC-GSH/CAT pathway, when seed lots were potentiated prior to accelerated aging as compared to its counterpart drought-susceptible redox incompetent cultivar Jamainadu.

In the present study, a significant up-regulation in the accumulation of overall and reduced pool of ascorbate and glutathione has been noticed for differently potentiated-accelerated aged seed lots of the cultivar Tulaipanji over their non-potentiated-accelerated aged and hydro-primed-accelerated aged seed lots (Fig. 6a, c, d, f). On the contrary, there was the least impact on the accumulation of oxidized form of ascorbate (Fig. 6b) under different potentiation in the cultivar Tulaipanji, confirming seed potentiation induced shift in redox turnover dynamics towards reduced state through Ascorbate–glutathione pathway for restoration of redox health of the accelerated aged seeds. The cultivar Jamainadu though exhibited the same pattern of response of seed potentiation the cultivar Tulaipanji, but when compared, the extent of activation of ascorbate–glutathione pathway seems to be significantly lower under all potentiating conditions in the cultivar Jamainadu (Fig. 6a–f). Hydro-priming prior to accelerated aging though seems to have an impact on the ascorbate–glutathione pathway over non-potentiated-accelerated aged seed lots, when compared with other types of potentiation, particularly herbal (1% aqueous extract of Lantana camara flower) and redox (H₂O₂), the impact seems to be significantly lower (Fig. 6a–f).

Fig. 6.

Seed potentiation impact (Hydro-primed, Herbal, H₂O₂ and Triadimefon) of accelerated aged seeds on the redox turnover of soluble compounds of Ascorbate–Glutathione pathway [total ascorbate (a), oxidized ascorbate (b), reduced ascorbate (c), total glutathione (d), oxidised glutathione (e) and reduced glutathione (f)] during early germination of two experimental IARCs (Oryza sativa L., Cultivars Tulaipanji and Jamainadu). Results are the mean of three replicates ± standard error. Different alphabets represent significant differences, using Fisher’s least significant difference test (p < 0.05)

Seed potentiation triggered changes in transcriptional modulation of genes of integrated NADPH oxidase-ascorbate–glutathione pathway in accelerated aged seeds of two IARCs

The present study also explored the seed potentiation-induced changes in the transcript level of vital genes (Osrboh, OsAPx2, OsGRase, OsCatA) (Fig. 7a–d) of the enzymes of combined NADPH oxidase-ascorbate–glutathione/catalase (RBOH-ASC-GSH/CAT) pathway under accelerated aging. Imposition of accelerated aging to the seed lots of the experimental rice cultivars instigated substantial transcriptional upregulation of the Osrboh (Fig. 7a) gene in both the experimental IARCs over the freshly harvested seed lots. Seed potentiation with herbal extract, H₂O₂, triadimefon and even hydro-priming followed by accelerated aging significantly down-regulates the transcript level of the Osrboh gene (Fig. 7a) of both the investigational cultivars of rice as compared to their non-potentiated-accelerated aged counterparts, hinting at the role of seed potentiation on restricting the genesis of ROS H₂O₂. When comparing between the efficacy of seed potentiation in terms of expression of Osrboh under accelerated aging, we find herbal extract to be the most potent one followed by H₂O₂ and triadimefon (Fig. 7a). Hydro-priming, though caused significant down regulation in the expression of Osrboh gene over non-potentiated-accelerated aged seed lots, but when compared with other kinds of potentiation, it showed similarity with triadimefon potentiated seed lots (Fig. 7a). When we assessed the transcript level of two important genes of the enzymes of ASC-GSH (ascorbate–glutathione) pathway (OsAPx2 and OsGRase) from differently potentiated and accelerated aged seed lots of both the cultivars of experimental rice and compared with non-potentiated-accelerated aged seed lots, we found significant transcriptional upregulation of both the genes under potentiating conditions (Fig. 7b, c). The significant upregulation of transcript level of the gene OsAPx2 and OsGRase, particularly in herbal and H₂O₂ mediated potentiated seeds over their corresponding non-potentiated seeds under accelerated aging unequivocally substantiate the transcriptional regulation of antioxidative defense mechanism or antioxidant coupled redox buffering essential for the conservation of redox homeostasis under accelerated aging (Fig. 7b, c). The impact of triadimefon and hydro-priming as seed potentiating agents on transcriptional regulation of both the ascorbate–glutathione pathway genes are found to be quite similar but significantly lesser, when compared with herbal and H₂O₂-mediated potentiation (Fig. 7b, c). Moreover, the cultivar Tulaipanji seems to be better responsive to all seed potentiating conditions as compared to the cultivar Jamainadu, hinting at germplasm-specific responsiveness to seed potentiation. qRT-PCR data of transcript level of the gene OsCatA analyzed from differently potentiated-accelerated aged seeds vis-à-vis non-potentiated-accelerated aged seeds also revealed almost the same trend except the fact that the efficacy of H₂O₂ as seed potentiating agent was found to be the higher as compared to herbal and triadimefon (Fig. 7d).

Fig. 7.

Transcript abundance of genes of central redox hub [Osrboh (NADPH-Oxidase), OsAPx2 (Ascorbate peroxidase), OsGRase (Glutathione reductase) and OsCatA (Catalase)] in different types of potentiated-accelerated aged seeds (Hydro-primed, Herbal, H₂O₂ and Triadimefon) vis-a-vis non-potentiated-accelerated aged seeds of two experimental IARCs (Oryza sativa L., Cultivars Tulaipanji and Jamainadu). a–d Bar diagrams represent relative fold gene expression of NADPH-oxidase, Ascorbate peroxidase, Glutathione reductase and Catalase in different treating conditions. Antioxidative genes are amplified for 40 cycles. Error bars represent SE of means (n = 3). Different alphabets represent significant differences, using Fisher’s least significant difference test (p < 0.05)

Seed potentiation down-regulate protein carbonylation and membrane lipid peroxidation in accelerated aged seeds of two experimental IARCs

Accelerated aging of the seed lots to the investigational rice cultivars aggravates the accumulation of all the oxidative deterioration products i.e. free carbonyl (RC=O), hydroperoxide (HPOX), conjugated diene and TBARS content (Fig. 8a–d). When compared, the impact of accelerated aging of seeds seems to be suggestively more for the IARC Jamainadu as compared to Tulaipanji (Fig. 8a–d). Seed potentiation particularly with herbal extract and H₂O₂ significantly restricts both protein oxidation and lipid peroxidation in accelerated aged seeds of both the IARCs which can be substantiated from the data of accumulation of RC=O (free carbonyl), HPOX (hydroperoxide), conjugated diene and TBARS (thiobarbituric acid reactive substances) content (Fig. 8a–d). When comparing the efficacy of seed potentiating agents, we found it in the order herbal extract, H₂O₂, triadimefon, hydro-priming, corroborating the data of redox metabolic shift, efficacy of dominant redox hub studied and transcriptional regulation of NADPH oxidase-ascorbate–glutathione pathway.

Fig. 8.

Seed potentiation impact (Hydro-primed, Herbal, H₂O₂ and Triadimefon) of accelerated aged seeds on the accumulation of oxidative deterioration product or redox biomarkers [free carbonyl content (a), hydroperoxide (b), conjugated diene (c), TBARS content (d)] during early germination of two experimental IARCs (Oryza sativa L., Cultivars Tulaipanji and Jamainadu). Results are the mean of three replicates ± standard error. Different alphabets represent significant differences, using Fisher’s least significant difference test (p < 0.05)

Seed potentiation modulates endogenous levels of phytohormones (GA, ABA, and JA) in accelerated-aged seeds of two experimental IARCs

Reverse phase high-performance liquid chromatography, coupled with photodiode assay exhibited a significant reduction in accumulation of GA and JA under accelerated aging (Supplementary table 4) (Supplementary Fig. 1). Seed potentiation with herbal extract not only restores the level of GA under accelerated aging but also found to upregulate their upregulation in both the experimental rice cultivars. H₂O₂ mediated seed potentiation on the other hand restricts the loss of GA in both the cultivars as compared to the non-potentiated-accelerated aged seed lots. On the contrary, triadimefon-mediated seed potentiation showed significant down-regulation of GA accumulation in both the rice cultivars under accelerated aging (Supplementary table 4) (Supplementary Fig. 1). Extraction and comparative estimation of ABA instead showed a significant upregulation in accelerated aged seed which was otherwise reduced significantly in herbal extract mediated potentiated seeds. Both H₂O₂ and triadimefon-mediated potentiated seeds found to be significantly reduced the ABA level for the cultivar Tulaipanji under accelerated aging which was otherwise enhanced for the cultivar Jamainadu. Seed potentiation with herbal extract and H₂O₂ was found to restrict the loss of JA in both the experimental IARCs (Supplementary table 4) (Supplementary Fig. 1).

Seed potentiation improves germination phenotypes of accelerated aged seeds of two experimental IARCs

In order to ascertain the efficacy of seed potentiating agents studied on the germination phenotypes and early growth performances, we have assessed and compared the standardized parameters of ISTA like T₅₀ value, CVG, MDG, GR, GRI, VI, RGI, R/S length etc. (Supplementary table 5) (Supplementary Fig. 2). A comparative evaluation of all the of germination phenotypes and initial growth performances revealed differential efficacy in combating the accelerated aging effect which is in the order herbal extract, H₂O₂, hydro-priming, triadimefon (Supplementary table 5) (Supplementary Fig. 2), corroborating strongly the essential contribution of redox regulation and the involvement of the central redox hub in regulating the oxidative windows of potentiated seeds under accelerated aging.

Discussion

In the present investigation, we compared the impact of accelerated aging of two different seed lots of aromatic rice of West Bengal (Oryza sativa L., Cultivars Tulaipanji and Jamainadu), previously screened by this laboratory based on their differential redox responsiveness (Bhattacharjee and Dey 2018; Dey and Bhattacharjee 2020). Accelerated aging (RH 92% and 41 °C for 24 h) imposed on the experimental seed lots, left imprints of oxidative deteriorative events that reflect the redox health of the seeds (Ebone et al. 2019; Xu et al. 2020; Zhang et al. 2020). The differential impact of the accelerated aging of the same magnitude on the two experimental IARCs was evident when we compared the ‘oxidative window’ of the aged seeds in terms of a shift in the redox status and redox markers [evaluated in terms of oxidative membrane deterioration (Fig. 8), efficacy of central redox hub (Fig. 5, 6)] along with the changes in hormonal profile (Supplementary table 4) (Supplementary Fig. 1) and transcriptional regulation of important redox-regulatory genes (Fig. 7).

The regulation of ‘oxidative window’ of accelerated aged seeds of the cultivar Tulaipanji towards a balanced generation of prooxidants for the maintenance of critical level of ROS, neither too high nor too low, found to be the determinant in regulating the redox health of aging seeds for the maintenance of their viability and vigor (Bailly et al. 2008; El-Maarouf-Bouteau and Bailly 2008; Wang et al. 2015b). Thus, the cultivar Tulaipanji exhibited significant down-regulation of the oxidative deterioration event (Figs. 1, 8), maintaining better redox health under aging as compared to the other IARC Jamainadu. It was proposed that the aging of the seed is intimately connected to the ROS management system of the quiescent embryo and other viable parts of the seed to deter oxidative deterioration events (Kurek et al. 2019; Adetunji et al. 2021). Biomolecules like proteins, genomic nucleic acids, stored mRNA, membrane lipids, and enzymes are the major targets of mismanagement of ROS at the metabolic interface, causing poor seed health during aging (Kurek et al. 2019; Adetunji et al. 2021). Thus, our result strongly supports the genotype-specific differences in the ROS management system of the quiescent embryo as the basis of differential response towards accelerated aging of the experimental seeds of IARCs.

The significant differences in the competence of redox hub (RBOH-ASC-GSH/CAT pathway) between accelerated aged seed lots of the experimental IARCs also kept the IARC Tulaipanji in a redox competent state to combat the oxidative threat that originates due to accelerated aging (Foyer and Noctor 2016; Kurek et al. 2019). The previous worker (Kurek et al. 2019) also noticed the role of antioxidative defense enzyme associated with the central redox hub for restoring redox homeostasis in aged seeds. The transcript abundance data of antioxidative defense genes, studied in the present investigation (OsAPx2, OsGRase, OsCatA), which showed a general trend of down-regulation (Fig. 7b–d) in accelerated aging, corroborates well with the declining trend of the activity of the corresponding enzymes for both the experimental seed lots (Fig. 5b, d, e). The marginally greater extent of transcriptional down-regulation of genes of enzymatic antioxidants and their activities for the IARC Tulaipanji compared to Jamainadu, might be due to the germplasm-specific regulatory maintenance of H₂O₂ necessary for redox signaling and keeping the accelerated aged seeds viable. Results strongly corroborate the concept of regulation of the ‘oxidative window’ for the maintenance of the viability of seeds (Bailly et al. 2008; El-Maarouf-Bouteau and Bailly 2008). Accelerated aging in general, caused transcriptional up-regulation of RBOH gene (Fig. 7a) and corresponding enhancement of the activities of the enzyme NADPH oxidase (Fig. 5a) to both the experimental IARCs, confirming the balancing of steady state level of ROS through ‘oxidative window’ consisting of ROS-generating and ROS-quenching systems (Adetunji et al. 2021).

The result also confirmed, the overall up-regulation in the antioxidant competence in the potentiated-aged seeds over non-potentiated aged seeds, corroborating strong potentiation induced antioxidant-coupled redox regulation of seed aging (Forti et al. 2020; Adetunji et al. 2021). The controlled generation of ROS through antioxidant-coupled calibration also showed a differential response pattern for mitigating seed aging and it was in the order, herbal extract, H₂O₂, hydro-priming, and triadimefon (Bailly et al. 2008; Chen and Arora 2013).

The antioxidative protective function through the central redox hub under potentiation to the seeds of investigational cultivars of rice might allow the seeds to maintain redox and metabolic homeostasis during the pre-germinative phase, thereby, paving the way to maintain viability, vigor, and germinability of the seeds (Chen and Arora 2013; Forti et al. 2020; Adetunji et al. 2021). Several workers highlighted the significance of the antioxidant-coupled ameliorative effect of differently primed seeds on subsequent exposure to stressors (Kibinza et al. 2011; Hossain et al. 2015; Ma et al. 2016).

Numerous earlier studies showed the significant impact of redox modulation of seeds through priming experiments on the maintenance and regulation of potency and viability of seeds during aging (Yasmeen et al. 2012). Further, there are several reports of seed invigoration through antioxidative compounds for regulating stress tolerance (Jisha et al. 2013). Previous works also suggest the prominent role of soluble low molecular weight antioxidants like ascorbate and glutathione, other thiol-containing small peptides, and amino acid in maintaining the redox health of the seeds during aging. Draganic and Lekic (2012) and Adetunji et al. (2021) proposed the role of glutathione and tocopherol as significant seed priming agents for the maintenance of competitive antioxidant competence and regulation of seed vigor during aging. The role of ascorbate is also being proposed as the most prospective redox modulatory treatment for augmenting the antioxidative defense of aged seeds (Adetunji et al. 2021).

Some works support the existence of cross-talk between endogenous redox cue (the level of prooxidants) and phytohormone signaling, which even found to modulate the expression of genes and cellular redox state and homeostasis (Xia et al. 2015; Wahid and Khaliq 2015). So, in the present case, seed priming with H₂O₂ and triadimefon might influence the balance of GA and ABA (Supplementary table 4) (Supplementary Fig. 1), which is crucial for determining the performance of seeds under accelerated aging (Wahid and Khaliq 2015). As the hormones ABA and GA are antagonistic in their role in determining the biology of seeds, their manipulation through seed potentiation towards the restoration of promoter and reduction of inhibitor might influence the germinability and vigor of accelerated aged seeds (Rajjou et al. 2012; Golldack et al. 2013; Wojtyla et al. 2016).

Priming seeds with diverse herbal extracts has been used as a common strategy to enhance the vigor and viability of seeds (Basra et al. 2011; Yasmeen et al. 2012). In most of the cases, bio priming for enhancement of seed vigor, viability and stress protectant is found to be related to modulation of antioxidative defense and metabolic adjustment (El-Maarouf-Bouteau and Bailly 2008; Kibinza et al. 2011; Hossain et al. 2015; Wang et al. 2015b). The allelochemicals (flavonoids, phenolic acids, tannins, lantadene terpenoids, triterpenoids, glycosides, etc.) present in the floral extract of Lantana camara, might also cause a chemical stress to the embryo of the seed, causing up-regulation of antioxidative defense to prevent a subsequent episode of accelerated aging induced oxidative deterioration (Ved et al. 2018).

The present study revealed a marginal but significant increment of the ascorbate–glutathione cycle coupled with redox management and mitigation of oxidative deterioration (Figs. 5, 6) in both the investigational rice cultivars when potentiated with triadimefon. The application of triadimefon, prior to the accelerated aging of seeds, might interfere with isoprenoid metabolism and alter the balance of important plant growth regulators like abscisic acid, gibberellic acid, and cytokinin (Arab and Ehsanpour 2012). Jaleel and Gopi (2007) through their study, exhibited the role of triadimefon-mediated seed priming in salinity tolerant, which involves largely the augmentation of antioxidative defense (both enzymatic and non-enzymatic). Therefore, the impact of triadimefon-induced seed potentiation of two IARCs might be due to a shift in hormonal signaling (ABA, GA, and JA) and associated elevation of antioxidative defense.

Several workers also noticed enhanced efficacy of enzymatic antioxidant competence (SOD, APOX, CAT, and GR) that tend to have an impact on restoration of redox homeostasis of aged seeds on hydro-priming (Adetunji et al. 2021). These redox-regulatory protective functions are stimulated throughout the process of hydro-priming, which might activate dynamic vicissitudes at the metabolic interface during succeeding aging and metabolic reactivation phase of germination, leading to enhanced and improved germination and seedling vigor (Adetunji et al. 2021).

When we compared the overall impact of seed potentiation on accelerated aged seed lots of two experimental IARCs, the cultivar Tulaipanji showed better responsiveness towards regulation of essential redox hub in addition to maintenance of hormonal homeostasis. This result strongly conveys a genotype-specific response of seed potentiation, substantiating the role of the capability of experimental seeds to respond to herbal redox and PGR mediated potentiation by orchestrating defense signaling mechanisms and restricting oxidative deterioration (Ella et al. 2011; Illangakoon et al. 2016).

Conclusion

Overall, the present work proposes critical role of central redox hub in regulating ‘oxidative windows’ of potentiated-aged seeds of experimental IARCs. Further, the efficacy of seed potentiation found to be dependent on redox calibration towards origin of favorable redox cue necessary for maintaining health of deteriorating seeds. Finally, unfolding regulatory events in the redox metabolic landscape of aging seeds of IARCs under potentiation by specific herbal and PGR like chemicals would help us in future to explore this simple and cost-effective technology in more effective manner.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

Study conception and design was done by SB. Material preparation, data collection and analysis were performed by BP, SB. The first draft of the manuscript was written by SB and all authors commented on previous versions of the manuscript. All authors approved the final manuscript.

Declarations

Conflict of interest

There is no conflict of interest.