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. 2023 Nov 30;13(12):422. doi: 10.1007/s13205-023-03840-4

Biochemical and molecular properties of Boro rice (Oryza sativa L.) cultivars under abiotic stresses

Mehzabin Rehman 1,2, Bikash Kundu 1, Preetom Regon 1, Bhaben Tanti 1,✉
PMCID: PMC10689613  PMID: 38047036

Abstract

The present investigation was conducted so as to unravel the various underlying antioxidant enzyme and non-enzyme defence mechanisms in some selected Boro rice cultivars that differ in temperature stress tolerance. Oxidative injury under heat and cold stress, H2O2 level showed a decline in roots and shoots of Boro in stressed condition whilst significant rise in the susceptible varieties was observed under both the stresses. However, susceptible varieties, such as Disang (shoots), Moricha (shoots) and China Boro (roots), showed a decreased H2O2 content at recovery. Under cold stress, roots and shoots of Boro and Laal Bihari showed a decreased level of lipid peroxidises and Boro and Kolong under heat stress. In contrast, significant enhancement of lipid peroxidase was revealed in the susceptible varieties. Remarkable increase in non-enzymatic antioxidants like proline, glutathione and ascorbate content was seen in the shoots of Boro in the treated and the recovery conditions. On the other hand, in enzymatic antioxidants like ascorbate peroxidase, guaiacol peroxidase, superoxide dismutase, catalase, and glutathione reductase activity, marked enhancement in ascorbate peroxidase activity was seen in the roots and the shoots of Boro and Kolong in treated and recovery samples and decreased in Swarnabh under heat stress. The guaiacol peroxidase activity of roots and shoots increased in Boro and Kolong under heat stress, and decreased in China boro and Swarnabh. The superoxide dismutase activity in the roots and shoot of Boro increased significantly under both the stress conditions in treated and recovery. Root and shoots of Swarnabh and Moricha showed decline in SOD activity in stressed conditions. The catalase activity in the case of Boro, showed a significant increase in both its roots and shoots under cold and heat stresses in the treated and the recovery samples. Moreover, under heat stress, the root and the shoots of Boro and Kolong showed the maximum glutathione activity, whilst Swarnabh and China Boro showed reduced glutathione activity at 96 h and recovery. On the other hand, the gene expression pattern of the cold-responsive genes (OsHAN1/OsCYP9B4 and FeSOD1) showed significant upregulation in the tolerant than the sensitive cultivars. Similarly, heat-responsive genes (OsTT1/OsPAB1 and OsHsfC1b) are also highly upregulated in the tolerant than the susceptible ones. Thus, the findings would provide a thorough insight into various non-enzymatic and enzymatic antioxidants and stress-responsive genes of Boro rice that could help in the future rice breeding programmes for cold and heat stresses.

Keywords: Antioxidant defence mechanism, Boro rice, Cold and heat stresses, Gene expression analysis

Introduction

Rice (Oryza sativa L.) is the staple food crop feeding about more than 2.7 billion people globally. About 90% of the rice consumption comes from Asia alone (Kalita et al. 2018; Lahkar and Tanti 2018). Rice cultivation is however under the severe threat of temperature stresses. When temperature deviates from optimal, alterations in physio-biochemical and molecular levels are observed in response to either increase or decrease of temperature leading to create stresses on the plant health. This leads to reduced growth and development in plants, thereby affecting production/yield. Temperature stress includes both cold stress and heat stress. However, cold stress may be categorised as freezing (< 0 °C) and cold (0–15 °C). Whilst temperature ranging between 35 and 40 °C can be termed as heat stress (Sanghera et al. 2011; Zhang et al. 2014).

Boro is summer rice, traditionally grown in low-lying areas during November–May. Major areas growing Boro rice traditionally include Assam, Bihar, eastern Uttar Pradesh, West Bengal and Orissa in India. In these regions, the water accumulated during monsoon having the ability to withstand flood (Rehman and Tanti 2021; Rehman et al. 2021). Other rice productions like ‘kharif’ (monsoon or autumn rice) or ‘ahu’ (autumn rice) in these areas are traditionally low. However, Boro rice showed improved productivity in the regions. Due to lower temperature at the early growth period, there is higher accumulation of photosynthates that facilitates the nitrogen:carbon ratio (You et al. 2015; Fahad et al. 2017; Rehman and Tanti 2020; Pradhan et al. 2020), consequently responding to higher amount of fertilisers that results in increased production. During 2007–2019, Assam in overall crop growing seasons received total actual rainfall of 2650.01 mm with a deviation of ( – )13 from the total normal rainfall of 3035.67 mm indicating a normal amount of rainfall (Choudhury et al. 2013; de Freitas et al. 2019). However, the erratic rainfall pattern with high fluctuations in precipitation time series led to high unpredictable floods and drought conditions. Exposure to low temperature at the germination and seedling development stages is detrimental for rice ecosystems during Boro season (Rehman and Tanti 2020). Another study on cold temperature revealed that temperatures as low as 12 °C would not induce sterility if it lasts for 2 days, but if it continues for about 6 days, there is 100% sterility (Singh et al. 2017). Furthermore, since it is grown during winter season, therefore, it is also prevented from insect and pest infestation. Due to such salient features, Boro rice has been receiving a worldwide demand (Ahmed et al. 2011; Singh et al. 2017).

Some of the major consequences of temperature stress are the excessive generation of reactive oxygen species (ROS) (Mittler 2002; Zhang et al. 2014; Regon et al. 2022). Apart from the antioxidant defence systems, stress-responsive genes play a significant role in cold and heat acclimation in plants (Pradhan et al. 2021; Agarwal et al. 2021). In the present study, coupling with comparative biochemical and gene expression analyses in Boro rice cultivars under temperature stresses would be fruitful for promoting a strong understanding of resistant mechanisms. Though lots of stress-related studies have been conducted on rice in general, but particularly, this is the first report on the Boro rice to understand the defence mechanisms under temperature stresses. The present investigation would be insightful to be a necessary step to improve tolerant/resilient Boro rice varieties in further rice breeding programme.

Materials and methods

The present investigation was conducted with seven Boro rice cultivars that are previously screened. The collected Boro rice samples were submitted to the Museum of the Department of Botany, Gauhati University, India. The vernacular names, geographical locations in addition to the Museum assigned codes are China Boro (Kamrup, Lower Brahmaputra valley; PMB/BT/R212), Swarnabh (Nagaon, Central Brahmaputra valley; PMB/BT/R216), LaalBihari (Morigaon, Central Brahmaputra valley; PMB/BT/R217), Boro (Cachar. Barak valley; PMB/BT/R235), Moricha (Cachar, Barak valley; PMB/BT/R249), Kolong (Kamrup, Lower Brahmaputra valley; PMB/BT/R256) and Disang (Kamrup, Lower Brahmaputra valley; PMB/BT/R257).

Germination of seeds was conducted on cotton beds in a controlled growth chamber at 28–30 °C for 72 h. The seedlings were then transferred for 13 days in soil until stress induction in an environmentally controlled growth chamber. For cold stress, 12 °C day/night was maintained at 11 h light and 13 h dark period. Whilst 42 °C/36 °C day/night was maintained at 13 h light and 11 h dark periods for heat stress, this was imposed for 24 and 96 h (Sanghera et al. 2011; Sailaja et al. 2014; Mangrauthia et al. 2017; Liu et al. 2018). The samples collected in these time frames were biochemically analysed. For gene expression analysis, stress was imposed for additional 12 h (cold) and 6 h (heat) followed by 96 h for recovery. The experiments were carried in a completely randomised design with three replications each treatment.

Determination of oxidative stress

The total hydrogen peroxide (H2O2) content was determined following the protocol by Sagisaka (1976) and the absorbance was recorded at 480 nm. Lipid peroxidation was determined using Heath and Packer (1968) based on the malondialdehyde (MDA) content and the optical density was recorded at 532 nm and corrected by subtracting absorbance at 600 nm.

Determination of enzymatic antioxidants

The estimation and the extraction of the enzymes were performed homogenising plant tissues in 0.1 M 1.05 mL potassium phosphate buffer (pH 7.0) carrying 0.5% Triton X-100 and 1% PVP (polyvinylpyrrolidone) in a chilled mortar–pestle. This was followed by centrifuging the homogenate at 15000 g at 4 °C for 20 min. The supernatant was utilised for the antioxidant enzymatic assays following the standard protocols, such as catalase (CAT) (Aebi 1983), guaiacol peroxidase (GPX) (Rao et al. 1999), superoxide dismutase (SOD) (Gupta et al. 1993) and glutathione reductase (GR) (Smith et al. 1988). But the extract for the determination of ascorbate peroxidase (APX) (Nakano and Asada 1987), the buffer contains 1 mM ascorbic acid additionally to restore the activeness of the enzyme.

Determination of non-enzymatic antioxidants

The ascorbate content was determined following Panda et al. (2003a, b). The glutathione content was estimated following Griffith (1980). For proline estimation, the method of Bates et al. (1973) was followed.

Quantitative real-time PCR analysis

The total RNA was isolated using the TRIzol method (RNAiso Plus, Takara). Verso cDNA synthesis kit, Thermo Scientific was used to synthesise the cDNA. The cDNA was quantified using a nanodrop and eventually diluted to approximately 50 ng/reaction. PowerUp SYBR Master Mix, Thermo Scientific was used as a master mix and PCR was performed in QuantStudio™ 5, Applied Biosystem Thermocycler system using OsActin as reference gene. Gene expression was calculated by the comparative CT method (Taylor et al. 2019). Following primer sequences were used for the specific genes – 5ʹ-CAATCTGGTGGTGTAAGACC-3ʹ and 3ʹ-TCCAGGAGAAGTATGACC-5ʹ (OsTT1); 5ʹ-TTACACGATCGGTTGTGGCA-3ʹ and 3ʹ-GAAGCAGAGGAGGCGCATAT-5ʹ (OsHsfC1b); 5ʹ-CTTGTGGTGGTGATGGTGAG-3ʹ and 3ʹ-GCATCTCCGTGTACCAGTCC-5ʹ (OsHAN1); 5ʹ-GGATGGGTTTGGCTTTGTTA-3ʹ and 3ʹAAGAGGATTGATGGCATTCG-5ʹ (FeSOD1); and 5ʹ-GACTCTGGTGATGGTGTCAGC-3ʹ and 3ʹ-GGCTGGAAGAGGACCTCAGG-5ʹ (OsActin).

Statistical analyses

The results of each experiment were calculated as mean ± SE with a minimum of three replicates. Statistical analyses were performed in R programme. In addition, significant levels were obtained by analysis of variance (ANOVA) followed by comparing the means using Tukey’s post hoc analysis (R Core Team 2013). The ggplot2 and ggpubr package of R was used to prepare the necessary graphs of the study (Wickham 2016).

Results

Determination of oxidative stress

Hydrogen peroxide and lipid peroxidation content

Oxidative injury under heat stress and cold stress, determined in terms of H2O2 level showed a decline in the roots and the shoots of Boro in stressed than in controlled condition. Being a positive control, i.e. tolerant to cold stress and heat stress, the results thus obtained were in accordance with the expectation. Boro also showed a decline at recovery. A significant rise in the susceptible varieties was also seen under both the stresses. However, susceptible varieties, such as Disang (shoots), Moricha (shoots) and China Boro (roots), showed a decreased H2O2 content at recovery, indicating that these rice cultivars might adopt an internal stress escaping mechanism (Fig. 1).

Fig. 1.

Fig. 1

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Effect of heat and cold stresses in shoot and root along with control and recovery on H2O2 production; A–B. shoot and root under heat stress, C–D. shoot and root under cold stress; and in case of MDA content, E–F. shoot and root under heat stress, G–H. shoot and root under cold stress

The effect of temperature stress on the concentration of lipid peroxides was measured based on thiobarbituric acid reactive substances. The level of lipid peroxidation was measured in terms of MDA content, as shown in Fig. 1. Under cold stress, the roots and the shoots of Boro and Laal Bihari showed a decreased level of lipid peroxidase. Similarly, under heat stress, Boro and Kolong also showed a decline in MDA content. A significant enhancement of MDA content is seen in the susceptible varieties in treated than in control. However, under heat stress, the shoots of Swarnabh (negative control) shows a decline in MDA content at recovery, which may be due to underlying of escaping mechanism adopted by the plant during the course of their life cycle.

Non-enzymatic antioxidants

Proline, glutathione and ascorbate content

Proline is one of the major osmolytes that accumulate during temperature stress. The variation in accumulation of proline in rice varieties (control, treated and recovery) is shown in Fig. 2A. Remarkable increase in the proline content was seen in the shoots of Boro in the treated and recovery samples than in control. The susceptible varieties show marked decrease in level of proline, which suggests that the results obtained from this experiment were in line with expectations.

Fig. 2.

Fig. 2

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Effect of heat and cold stresses in shoot and root along with control and recovery on proline content; A–B. shoot and root under heat stress, C–D. shoot and root under cold stress; and in case of glutathione content, E–F. shoot and root under heat stress, G–H. shoot and root under cold stress

Higher level of glutathione indicates greater resistance to the stress. Under heat stress, shoots and roots of Boro and Kolong showed significant increase in glutathione content in treatment than in control. Similarly, roots and shoots of Boro and Laal Bihari showed increased glutathione content. Moreover, the susceptible variety, Swarnabh (heat), also revealed significant reduction in glutathione content. However, it increased at recovery. Under cold stress, the roots and the shoots of Disang (negative control) showed significant increase in glutathione content in the treated samples (Fig. 2).

Plants with higher ascorbate content can scavenge ROS produced during stress efficiently and offer greater tolerance to abiotic stress. In our study, under both heat and cold stress, Boro (root and shoot) showed increased ascorbate level whilst the susceptible varieties, such as Swarnabh, China Boro, Moricha and Disang, showed a decrease in ascorbate content suggesting the experiment is in agreement with the expectation. However, the roots of negative controls, Swarnabh and Disang showed a decline in the ascorbate content at recovery (Fig. 3).

Fig. 3.

Fig. 3

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Effect of heat and cold stresses in shoot and root along with control and recovery on ascorbate content; A–B. shoot and root under heat stress, C–D. shoot and root under cold stress; and in case of APX activity, E–F. shoot and root under heat stress, G–H. shoot and root under cold stress

Enzymatic antioxidants

Ascorbate peroxidase (APX), guaiacol peroxidase (GPX), superoxide dismutase (SOD), catalase (CAT), and glutathione reductase (GR) activity

A marked enhancement in APX activity was seen in the roots and the shoots of Boro and Kolong in treated and recovery samples than in the control samples and decreased in Swarnabh under heat stress. However, both the root and the shoots of Swarnabh at recovery show an increased activity. Under cold stress, there is an increase in the level of APX activity in the shoots of Boro and Laal Bihari, whilst it decreased in Disang and Moricha. Under cold stress, the roots of the susceptible variety, Disang showed decrease in APX activity. However, it showed marked increase in Moricha (Fig. 3).

The GPX activity in roots and shoots increased in Boro and Kolong under heat stress, and decreased in China boro and Swarnabh. Under cold stress, shoot and root of Boro and Laal Bihari showed increased level of GPX activity whilst it decreased in Moricha and Disang in the treated and the recovery as compared to the controlled samples (Fig. 4).

Fig. 4.

Fig. 4

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Effect of heat and cold stresses in shoot and root along with control and recovery on GPX activity; A–B. shoot and root under heat stress, C–D. shoot and root under cold stress; and in case of SOD activity, E–F. shoot and root under heat stress, G–H. shoot and root under cold stress

The SOD activity in the roots and the shoots of Boro increased significantly under both the stress conditions in treated and recovery than in control. The root and the shoots of Swarnabh and Moricha showed decline in SOD activity in stressed than in control. However, it is seen that at recovery, the shoots of Moricha and the roots of Swarnabh show a marked rise in the activity of SOD, which might be due to certain acquired internal stress escape mechanisms. The same might be in the case of Disang under cold stress (Fig. 4).

In case of Boro, the catalase activity showed a significant increase in both its root and shoots under cold and heat stresses in the treated and recovery samples to that of control. Under heat stress, there was a gradual decline in the CAT activity of both the root and shoots of the treated sample from Laal Bihari to Swarnabh. Under heat stress, there was a significant rise in the activity of the roots and shoots of China Boro and Swarnabh at recovery which denotes that they might have adopted resistance towards the stress. However, under cold stress, susceptible cultivars, such as Moricha and Disang, showed variation in the SOD activity in roots and shoots (Fig. 5).

Fig. 5.

Fig. 5

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Effect of heat and cold stresses in shoot and root along with control and recovery on CAT activity; A–B. shoot and root under heat stress, C–D. shoot and root under cold stress; and in case of GR activity, E–F. shoot and root under heat stress, G–H. shoot and root under cold stress

Under heat stress, the root and shoots of Boro and Kolong showed the maximum GR activity, whilst Swarnabh and China Boro showed reduced GR activity at 96 h and recovery. Under cold stress, shoots of Boro showed maximum activity whilst Moricha and Disang showed decreased GR activity. In case of roots, Boro showed significant increase whilst a fall in the activity was seen in Moricha in treated than in control, which later was shown to increase at recovery (Fig. 5).

Pearson’s correlation coefficient amongst different biochemical parameters

Pearson’s correlation coefficients for the comparison amongst the different biochemical parameter under cold and heat stress are shown in Tables 1 and 2 respectively. The data generated showed significant correlations amongst all the parameters. In shoots (cold stress), they showed positive correlation with MDA P < 0.01 and with H2O2, SOD and CAT by P < 0.05. MDA showed significant correlation with CAT and GR by P < 0.05. GSH was significantly correlated with Pro and GR at the level of P < 0.05. SOD showed positive correlation with CAT, APX and GPX by P < 0.05. Further CAT and APX are also related with a significance level at P < 0.01. GR content was positively correlated with APX and GPX activity at P < 0.05. Furthermore, shoots (cold recovery) showed significant positive correlation of H2O2 and MDA with Pro, CAT, APX, GPX and GR content at significance level P < 0.05. APX showed positive correlation with GPX activity at P < 0.01. However, in case of treated roots, significant correlation at significance level P < 0.05 was seen in between H2O2 with SOD, Pro and GPX; MDA with Pro, GPX; AsA with GSH, SOD, APX; GR with CAT and GPX. And SOD-GSH with a significance level at P < 0.01.

Table 1.

Pearson’s correlation matrix showing correlation among the analyzed biochemical parameters of treated and recovery roots and shoots samples under cold stress in the experimental rice varieties

Parameters H2O2 MDA Asc Glu Pro SOD CAT APX GPX GR
Stress (Shoot)
H2O2 1
MDA 0.334 1
Asc 0.586* 0.834** 1
Glu 0.345 0.178 – 0.120 1
Pro 0.738* 0.224 0.236 0.644* 1
SOD – 0.166 – 0.144 – 0.515* 0.414 0.287 1
CAT – 0.075 – 0.608* – 0.744* 0.255 0.122 0.731* 1
APX 0.285 – 0.212 – 0.104 – 0.063 0.330 0.565* 0.454 1
GPX 0.015 – 0.406 – 0.478 0.055 0.104 0.780* 0.814** 0.844** 1
GR 0.192 – 0.528* – 0.105 – 0.540* – 0.171 – 0.099 0.262 0.647* 0.524* 1
Recovery (Shoot)
H2O2 1
MDA – 0.173 1
Asc 0.357 – 0.218 1
Glu 0.162 – 0.295 – 0.067 1
Pro – 0.513* – 0.141 0.291 0.120 1
SOD – 0.037 – 0.149 – 0.212 – 0.730* – 0.024 1
CAT 0.703* – 0.703* 0.439 0.338 – 0.132 – 0.026 1
APX – 0.721* – 0.373 0.001 – 0.400 0.559* 0.469 – 0.230 1
GPX – 0.223 – 0.667* 0.310 – 0.440 0.330 0.587* 0.235 0.832** 1
GR – 0.124 – 0.749* 0.471 0.084 0.275 – 0.047 0.277 0.557* 0.726* 1
Stress (Root)
H2O2 1
MDA 0.478 1
Asc – 0.104 – 0.306 1
Glu 0.494 0.131 0.539* 1
Pro – 0.734* – 0.728* 0.052 – 0.265 1
SOD 0.549* 0.016 0.601* 0.867** – 0.160 1
CAT 0.040 0.122 – 0.351 – 0.708* – 0.075 – 0.321 1
APX – 0.099 0.164 0.648* 0.230 – 0.141 0.412 0.169 1
GPX – 0.704* – 0.576* – 0.009 – 0.398 0.582* – 0.344 0.126 0.198 1
GR – 0.076 – 0.420 – 0.375 – 0.450 0.229 – 0.223 0.515* – 0.078 0.682* 1
Recovery (Root)
H2O2 1
MDA 0.252 1
Asc 0.394 – 0.055 1
Glu 0.149 – 0.034 0.515* 1
Pro 0.106 – 0.591* – 0.119 – 0.236 1
SOD 0.436 0.017 0.529* 0.486 0.464 1
CAT – 0.287 0.522* – 0.716* – 0.039 – 0.179 – 0.147 1
APX 0.158 0.400 – 0.410 – 0.029 0.143 0.303 0.494 1
GPX 0.056 0.540* – 0.548* – 0.762* – 0.100 – 0.350 0.340 0.539* 1
GR 0.354 0.926** – 0.070 – 0.170 – 0.656* – 0.226 0.388 0.171 0.562* 1
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Levels of significance: **P < 0.01 level (2-tailed); *P < 0.05 level (2-tailed)

H2O2 hydrogen peroxide, MDA malone dialdehyde, Asc ascorbate, Glu glutathione, Pro proline, SOD superoxide dismutase, CAT catalase, APX ascorbate peroxidase, GPX guaiacol peroxidase, GR glutathione reductase

Table 2.

Pearson’s correlation matrix showing correlation among the analyzed biochemical parameters of treated and recovery roots and shoots samples under heat stress in the experimental rice varieties

Parameters H2O2 MDA Asc Glu Pro SOD CAT APX GPX GR
Stress (Shoot)
H2O2 1
MDA – 0.003 1
Asc – 0.363 – 0.019 1
Glu – 0.037 0.681* – 0.605* 1
Pro – 0.144 – 0.100 0.324 – 0.240 1
SOD 0.595* – 0.636* – 0.269 – 0.310 – 0.158 1
CAT 0.400 – 0.177 – 0.533* – 0.072 0.283 0.137 1
APX – 0.125 0.325 – 0.341 0.318 0.263 – 0.267 0.605* 1
GPX 0.881** – 0.372 – 0.344 – 0.356 – 0.062 0.702* 0.631* – 0.012 1
GR 0.490 – 0.618* – 0.449 – 0.387 0.138 0.481 0.833** 0.117 0.792* 1
Recovery (Shoot)
H2O2 1
MDA 0.026 1
Asc – 0.320 – 0.107 1
Glu 0.319 – 0.530* – 0.180 1
Pro – 0.291 – 0.115 – 0.500* – 0.407 1
SOD – 0.198 – 0.254 0.570* 0.044 – 0.183 1
CAT 0.554* – 0.049 – 0.835** 0.324 0.335 – 0.731* 1
APX 0.710* – 0.269 – 0.776* 0.332 0.333 – 0.324 0.810** 1
GPX – 0.582* – 0.443 0.765* 0.079 – 0.342 0.277 – 0.639* – 0.692* 1
GR 0.262 – 0.779* – 0.005 0.714* 0.009 0.424 0.180 0.393 0.052 1
Stress (Root)
H2O2 1
MDA 0.198 1
Asc – 0.253 – 0.310 1
Glu – 0.246 0.168 0.372 1
Pro – 0.579* – 0.690* 0.429 – 0.016 1
SOD 0.046 – 0.048 0.563* 0.821** 0.186 1
CAT – 0.367 – 0.544* 0.856** – 0.034 0.709* 0.264 1
APX – 0.230 – 0.495 0.695* – 0.026 0.564* 0.335 0.879** 1
GPX 0.049 – 0.105 0.067 0.542* 0.358 0.768* 0.012 0.165 1
GR – 0.391 – 0.699* 0.354 0.123 0.869** 0.413 0.609* 0.697* 0.617* 1
Recovery (Root)
H2O2 1
MDA – 0.168 1
Asc 0.370 – 0.361 1
Glu – 0.669* 0.571* – 0.352 1
Pro – 0.188 – 0.760* 0.262 – 0.101 1
SOD 0.165 – 0.037 0.243 0.439 0.160 1
CAT 0.286 – 0.796* 0.348 – 0.696* 0.242 – 0.100 1
APX – 0.312 – 0.655* 0.135 0.170 0.594* 0.507* 0.509* 1
GPX 0.029 – 0.180 0.441 0.396 0.399 0.837** – 0.126 0.515* 1
GR – 0.147 – 0.699* 0.205 0.072 0.943** 0.446 0.180 0.718* 0.615* 1
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Levels of significance: **P < 0.01 level (2-tailed); *P < 0.05 level (2-tailed)

H2O2 hydrogen peroxide, MDA malone dialdehyde, Asc ascorbate, Glu glutathione, Pro proline, SOD superoxide dismutase, CAT catalase, APX ascorbate peroxidase, GPX guaiacol peroxidase, GR glutathione reductase

At recovery, MDA-GR showed significant correlation at P < 0.01. Whilst some other parameters also showed positive correlation at the level of P < 0.05 (Table 1).

In a similar manner, under heat stress, the roots and shoots showed various positive correlations amongst their parameters at P < 0.01 and P < 0.05 level during stress and recovery stages, some of which, such as in stressed shoots, H2O2-GPX and CAT-GR, showed significant correlations at level P < 0.01. Whilst in the shoots (recovery) and roots (stress), CAT activity was correlated positively at P < 0.01 with APX and AsA content. Some other positive correlations were also revealed amongst the different parameters at different levels (Table 2).

Expression profiling under cold and heat stress

Gene expression of few selected genes was studied under cold and heat stress by quantitative Real-Time PCR (qRT-PCR). Under cold stress, the OsHAN1 and OsFeSOD1 genes were analysed. Differential expression of gene expression was observed amongst the treatments as well as in cultivars. The expression of OsHAN1 was upregulated at 12 h, where it was insignificant at other treatment duration (Fig. 6). In roots, significant upregulation was observed in Boro, China Boro, Disang and Laal Bihari. In shoots, significant upregulation was observed in most cultivars, where it was in significant in Kolong and Moricha. Interestingly, the expression of OsFeSOD1 was found to be upregulated across all treatment groups of the tolerant cultivars. Briefly, it was significantly upregulated in Boro and Laal Bihari of both roots and shoot across the treatment groups (Fig. 6).

Fig. 6.

Fig. 6

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Relative expression of genes in shoots and roots under heat and cold stresses; a–b. OsHAN1 in root and shoot; c–d. OsFeSOD1 in root and shoot; e–f. OsHsfC1b in root and shoot; g. OsTT1 (shoot) determined by quantitative RT-PCR (mean ± SE; n = 3)

Under heat stress, the expressions of OsTT1 and OsHsfC1b were analysed where differential expression was observed amongst the rice cultivars. The expression of both genes was found to be maximum at the early stage of the stress, i.e. 4 h, where it was insignificant/down regulated as the stress progresses (Fig. 6). Furthermore, the expression of OsTT1 was observed only in the shoot, whereas it was undetected in roots. Significant upregulation was observed in Boro followed by Kolong, Disang, and Moricha. No significant difference was observed in the heat-sensitive cultivars i.e. Swarnabh, China Boro and the moderately tolerant Laal Bihari (Fig. 6).

However, OsHsfC1b gene was found to be expressed highly at 4 h. In roots, it was significantly upregulated only in Boro and Kolong, whereas in the shoots, it was significantly upregulated in Boro, China Boro, Kolong and Swarnabh. Interestingly, in Boro and China Boro it was upregulated considerably across all the treatment groups (Fig. 6).

Discussion

Since Boro rice receives lower winter temperature during its early growth period, this group of rice is also known to accumulate residual moisture of monsoon cultivation which makes it highly rich in photosynthates, thereby resulting in high yield. Therefore, it is very important to screen the Boro rice varieties of this region for their cold and heat stress tolerability (Zhang et al. 2014; Nahar et al. 2018b; Shandilya and Tanti 2020).

We previously screened 27 Boro rice cultivars for cold and heat stress (Rehman and Tanti 2021). In this study, seven Boro rice cultivars were selected for biochemical and gene expression study to unveil the underlying mechanism of their adaption and tolerance to stress. Rice cultivars were selected based on their tolerability, from highly tolerant/resistant to the most susceptible ones. The experiments revealed a significant decrease in the levels of lipid peroxidation and hydrogen peroxide in the roots and shoots of the tolerant variety Boro under both the stress conditions (Hasanuzzaman et al. 2013; de Freitas et al. 2019). The susceptible varieties showed variations in the root, shoot as well as treated conditions. In most cases, the susceptible showed enhanced hydrogen peroxide activity, lipid peroxidation and proline. These results were in accordance with previous works (Das and Roychoudhury 2014; Shandilya and Tanti 2019).However, some of them showed positive results at recovery, which may be due to the adaption of certain internal stress escape mechanisms by the plant in due course of their acclimation process during stress. During stress, H2O2 gets accumulated in high amounts and functions as second messengers which cause oxidative injury leading to the untimely death of the plant (Choudhury et al. 2013; Nahar et al. 2018a; Lahkar et al. 2020). In addition to this, cold and heat stress is known to cause damage to mitochondrial functionality resulting in the induction of oxidative burst that can result in peroxidation of lipids. Furthermore, rice plants subjected to cold and heat stress accumulate an amino acid, proline which can stabilise protein synthesis, consequently maintaining optimal conditions of rice cells (Suzuki and Mittler 2006; Choudhury et al. 2013; Zhang et al. 2014).

In case of non-enzymaticantioxidant(ascorbateand glutathione), tolerant cultivars i.e. Boro and Kolong under heat; and Boro and Laal Bihari under cold stress showed higher levels of ascorbate and glutathione in roots and shoots during all time frames. On the other hand, it was comparatively low in the susceptible varieties. Under heat stress, Swarnabh (negative control) showed increased ascorbate content in shoots of the treated samples than in control suggesting the variety might have acquired adaptability towards stress during their acclimation period. Higher ascorbate and glutathione productions were reported in the tolerant cultivars, which play a significant role in defence to various abiotic stresses (Choudhury et al. 2013; Lahkar and Tanti 2017). There are reports suggesting that the ratio of conversion of reduced glutathione to oxidised form during the scavenging process. On the other hand, ascorbate is utilised during the enzymatic defence system by APX to detoxify H2O2 (Das and Roychoudhury 2014; Hasanuzzaman et al. 2013).

On the other hand, the enzymatic antioxidants were known to have increased activity in the tolerant varieties than in the susceptible ones (Gill and Tuteja 2010; Hasanuzzaman et al. 2013). Boro showed increased activity of the enzymatic antioxidants in both roots and shoot under both stress conditions in almost all time frames. The susceptible varietiesshoweddecreased enzymatic activities, althoughsomeagainshowedbetter performance in a few parameters (Baek and Skinner 2012; Hasanuzzaman et al. 2013). Apart from the biochemical antioxidant activity, we have investigated the expression pattern of few selected genes under both stresses. Under cold stress, the expression of OsHAN1 was differentially expressed at the early stage (12 h) in both roots and shoots amongst the rice cultivars. In shoots, the expression was upregulated almost all the cultivars, except the Moricha and Kolong. On the other hand, in roots, a similar expression pattern was also observed except Swarnabh. TheOsHAN1 is known to confer chilling tolerance gene identified by quantitative trait locus (QTL) analysis (Mao et al. 2019). It encodes an oxidase that catalyses active jasmonoyl-L-isoleucine (JA-Ile) to the inactive 12-hydroxy-JA-Ile (12OH-JA-Ile), thus fine-tunes the JA-mediated chilling response.

Under cold stress, FeSOD1 gene was also highly upregulated in shoots in the moderate to tolerant cultivars (Boro, Laal Bihari and Swarnabh). In contrast, it was insignificant in the moderate to sensitive (Kolong, Disang and Moricha). In exception, the expression was also insignificant in China boro, tolerant cultivar in the previous study. In roots, significant expression was observed only in Boro and Laal Bihari, which are the cold-tolerant cultivars in this study. Overexpression of FeSOD1 gene has been reported to confer enhanced tolerance to cold and heat stress (Gill et al. 2015; Lal et al. 2013; Wang et al. 2021).

Under high temperature, the expression of thermo-tolerance 1/Proteasome α-2 subunit (OsTT1/OsPAB1) was highly upregulated in the tolerant cultivars (name of the cultivars) as compared to the sensitive (name of the cultivars). The OsTT1/OsPAB1 involved in the degeneration of ubiquitinated proteins, thereby protecting cells from high temperature stress through efficient removal of cytotoxic denatured proteins and effective preservation of heat-response processes in rice plants (Li et al. 2015). However, the expression of OsTT1/OsPAB1 has been observed only in shoots and undetected in roots. Thus, indicate its role in shoot-based thermo-tolerant activity in Boro rice. Furthermore, another gene, heat shock factor C1b (OsHsfC1b) was also upregulated in both roots and shoots. Comparatively, the expression of OsHsfC1b was also higher in the tolerant cultivars than in the sensitive. The Hsf genes are induced in the presence of stressful stimuli and interact with client proteins and co-chaperones to regulate cell growth and survival. The OsHsfC1b was previously reported as salt and a heat-tolerant gene in rice (Schmidt et al. 2012; Cao et al. 2020).

Conclusion

In this investigation, Boro was found to be the most tolerant and promising Boro rice cultivar under heat and cold stress conditions. This finding could be well informative for Boro rice cultivation, and the Boro cultivar could be suggested for future rice breeding programme for developing tolerant varieties under temperature stress.

Acknowledgements

Financial and infrastructural support received from UGC-SAP (DRS-I) and DST-FIST, Govt. of India is highly acknowledged.

Author contributions

All authors contributed to the study’s conception and design. MR and PR designed the research, performed the experiments, and analysed the data. BT and P R prepared the manuscript and finalised.

Declarations

Conflict of interest

No conflict of interest was reported by the authors.

Ethical statement

Ethics approval and consent to participate all experimental studies on plants have complied with relevant institutional, national, and international guidelines and legislation.

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