Integrating physiological and multi-omics methods to elucidate heat stress tolerance for sustainable rice production

Abstract

Heat stress presents unique challenges compared to other environmental stressors, as predicting crop responses and understanding the mechanisms for heat tolerance are complex tasks. The escalating impact of devastating climate changes heightens the frequency and intensity of heat stresses, posing a noteworthy threat to global agricultural productivity, especially in rice-dependent regions of the developing world. Humidity has been demonstrated to negatively affect rice yields worldwide. Plants have evolved intricate biochemical adaptations, involving intricate interactions among genes, proteins, and metabolites, to counter diverse external signals and ensure their survival. Modern-omics technologies, encompassing transcriptomics, metabolomics, and proteomics, have revolutionized our comprehension of the intricate biochemical and cellular shifts that occur in stressed agricultural plants. Integrating these multi-omics approaches offers a comprehensive view of cellular responses to heat stress and other challenges, surpassing the insights gained from multi-omics analyses. This integration becomes vital in developing heat-tolerant crop varieties, which is crucial in the face of increasingly unpredictable weather patterns. To expedite the development of heat-resistant rice varieties, aiming at sustainability in terms of food production and food security globally, this review consolidates the latest peer-reviewed research highlighting the application of multi-omics strategies.

Introduction

Rice, scientifically known as Oryza sativa L., holds the top position in terms of human consumption, particularly in Asia, where it constitutes a staple diet (Samal et al. 2020). Presently, out of the 22 indigenous rice varieties, only two are cultivated globally i.e. Japonica and Indica Rice. The rice plant, a significant model in agriculture, possesses a genome size of 430 Mb and is predominantly cultivated in Asia, which is also home to the highest number of rice-producing countries worldwide (Manavalan et al. 2012).

Abiotic stressors present significant challenges to rice cultivation and yield, as highlighted by recent studies (Badawy et al. 2021; Rasheed et al. 2021). The term “heat stress” (HS) denotes the adverse impact of prolonged exposure to temperatures exceeding a defined threshold (Kumari et al. 2020; Mukhtar et al. 2020), which has been a subject of concern (Govindaraj et al. 2018). Over the past half-century, the combination of extreme heat and arid conditions has resulted in a 9–10% reduction in cereal production (Lesk et al. 2016). Elevated daytime temperatures, especially during blooming, detrimentally affect rice yield by diminishing fecundity (Khan et al. 2019; Wang et al. 2019a, b). Additionally, heat stress-induced fertilization failure during blooming significantly diminishes output (Abd El-Daim et al. 2014; Song et al. 2014). High temperatures trigger notable declines in assimilate and enzyme synthesis, ultimately impacting harvest outcomes (Chaturvedi et al. 2017; Mukhtar et al. 2020). The consequences of rising temperatures extend to staple crops like rice, wheat, and corn, disrupting key stages such as seed establishment, growth, development, and maturity. Chronic heat stress negatively affects various aspects, including photosynthesis, water use efficiency, pollen and grain yields, and leaf area (Hassan et al. 2020). Heat resilience encompasses an array of biochemical and physical traits that enable plants to uphold normal growth and yield under heat pressure (Wahid et al. 2007; Mukhtar et al. 2020). Rice’s resilience towards high temperature is modulated by multiple genes, influencing both its physiological and morphological attributes (Impa et al. 2021).

Three primary strategies exist to address heat stress: evasion, flight, and endurance. One evasion tactic involves completing reproduction before the onset of elevated temperatures. Plants manage water conservation by tactics such as reducing leaf surface, closing stomata, and shedding older leaves (Mohamed et al. 2021). In the context of thermal stress in rice, scholars (Julia and Dingkuhn 2013) have investigated its coping mechanisms. Rice employs controlled timing of panicle emergence and spikelet expansion as a means to avert thermal stress (Julia and Dingkuhn 2012). Researchers have extensively examined sterile spikelets as a potential approach to enhance rice’s heat resistance. Variability in spikelet fertility among strains under adverse conditions significantly influences rice’s ability to endure heat (Weerakoon et al. 2008). Through evaporative cooling facilitated by panicles, the rice plant can mitigate heat stress by as much as 10 °C, a process directly linked to spikelet vigor (Matsui et al. 2015). Rice leaves which have erectile and elongated morphology play a protective role, enhancing heat resistance (Julia and Dingkuhn 2013). To enhance heat endurance, a range of physical, genetic, and metabolic attributes can be modified. The heat-tolerant wild haplotype Oryza meridionalis maintains elevated levels of the Rubisco enzyme, sustaining a high photosynthetic rate under heat stress (Qu et al. 2021). Chlorophyll capacity of leaf and root, along with the leakage of electrolytes have been heightened due to HS and serve as indicators of heat resilience for study purposes.

Enhancing the thermal resilience of rice crops necessitates a combination of agricultural and genetic interventions. Extenuating HS’s adverse effects necessitates employing various agronomic techniques, including early planting and applying signaling molecules, hormones, and Osmo protectants. Early sowing increases the chances of survival under elevated temperatures, thereby enhancing overall yield as well as quality (Krishnan et al. 2011). Compounds of plant origin, like alpha-tocopherol, ascorbic acid, auxins, brassinosteroids, methyl jasmonate as well and salicylic acid enhance plant’s resistance to HS, hence leading to heightened productivity (Khan et al. 2019). Similar to how signaling molecules improved efficiency in terms of PS II, the utility of water, as well as antioxidant potential, they also mitigated the detrimental impacts of heat stress on rice production (Chandrakala et al. 2013). Essential osmolytes such as proline, glycine-betaine, and spermidine also play a pivotal role in enhancing rice’s tolerance to HS (Sakamoto and Murata, 2000; Khan et al. 2019).

Addressing thermal stress in rice cultivation effectively in the long term involves strategic breeding techniques. Conventional methods, such as selecting heat-tolerant varieties, enhance rice plants’ resilience to high temperatures. Additionally, innovative molecular techniques utilizing omics approaches enable the creation of transgenic plants through targeted gene modifications, significantly improving rice’s resistance to heat stress (Kosová et al. 2011; Duque et al. 2013). Identifying heat-tolerant quantitative trait loci (QTLs) and applying proteomics and transcriptomics also hold promise for developing heat-resistant strains.

This comprehensive study explores various avenues, including agricultural practices, traditional methods, and genetic strategies, to bolster rice against thermal stress. It highlights prospects for improving heat tolerance and offers insights into the processes of heat stress, the role of multi-omics techniques, and the significance of cereals, especially rice. The paper encapsulates advanced insights into the molecular dimensions of heat stress, emphasizing the importance of a multi-faceted approach to understanding and mitigating abiotic stress. Given rice’s global demand and significance as a staple food, this study showcases the progress made through diverse omics methodologies in comprehending and addressing heat stress.

Physiological responses of plants under HS

Various metabolic processes, such as photosynthesis, respiration, and evaporation, as well as factors like membrane stability in response to temperature, and regulation of osmotic balance, are susceptible to heat stress (HS). Figure 1 illustrates a few general consequences of HS on a plant’s physiological functions, development, as well as, overall productivity.

Fig. 1.

Plants have developed a series of intricate morphological, physio-chemical, and molecular responses to cope with and adapt to environmental conditions that induce heat stress. These responses are vital for the survival and growth of plants in hot and challenging climates

Photosynthesis

In most scenarios, elevated temperatures (HS) have a detrimental effect on the efficiency of photosynthesis in plants. This leads to a reduced lifespan of plants and a decline in their overall productivity (Xalxo et al. 2020). Furthermore, high temperatures pose a significant threat to the process of photosynthesis. Both the photosynthesis that occurs within the thylakoid lamellae of chloroplasts and the carbon processing that happens in the stroma are susceptible to damage when exposed to high temperatures (Hasanuzzaman et al. 2013; Hu et al. 2020).

HS wallops on plants lead to degradative effects on thylakoid membranes, which disrupt the functioning of electron transporters and enzymes associated with these membranes. Among these components, the PSII complex, which is particularly sensitive to high temperatures, experiences a considerable reduction or even a complete halt in its activity under heat stress (Hasanuzzaman et al. 2013; Szymańska et al. 2017). Additionally, heat stress leads to a decrease in ribulose-1,5-bisphosphate carboxylase/oxygenase’s efficiency, a reduction in levels of photosynthetic pigments, and a decline in the potential for carbon fixation (Hasanuzzaman et al. 2013; Song et al. 2014; Perdomo et al. 2017).

Moreover, chloroplast shape, as well as content’s temperature constancy within photosynthetic systems are also affected by heat stress. These factors collectively contribute to the diminished efficiency of photosynthesis under high temperatures. As a result, a comprehensive understanding of the response of photosynthesis metabolism is essential when studying the resilience of plants to heat stress and comprehending the negative consequences of elevated temperatures on agricultural productivity (Bita and Gerats 2013).

Cell membrane thermostability

The primary metabolic response of plants to high shear forces (HS) involves a disruption in membrane functionality. The heightened kinetic energy and protein mobility induced by extreme HS led to the weakening of molecular bonds within membranes. As a result, membrane lipids degrade, causing an escalation in membrane permeability (Dhanda and Munjal 2012). When plants are subjected to HS, their cellular membranes experience enhanced permeability and an elevated loss of ions. These effects collectively hinder proper cellular operations and diminish their efficiency towards endurance at higher temperatures (Xalxo et al. 2020). Moreover, HS triggers reactive oxygen species (ROS) accumulation, which has detrimental effects on the membranes, consequently diminishing thermo-tolerance (Mohammed and Tarpley 2009). In conclusion, maintaining the stability of membranes is of utmost importance in conferring heat tolerance to plants in the face of high shear forces.

Oxidative damage

Oxidative stress arises when plants face HS, resulting in the assemblage of ROS in singlet oxygen, superoxide radical, hydrogen peroxide, and hydroxyl radical (Nosaka and Nosaka 2017). PSI and PSII are the primary sources of these ROS, because excess energy in PSII excites triplet chlorophylls, transferring their kinetic energy to oxygen molecules, generating singlet oxygen. Under significant reduction of PSI, superoxide anions are generated, further elevating H₂O₂ production (Szymańska et al. 2017).

Elevated temperatures make plant cells more vulnerable to oxidative damage due to heightened free radical activity, leading to lipid peroxidation (Hasanuzzaman et al. 2013). Lipid peroxidation is evident through increased concentrations of malondialdehyde (MAD), a lipid peroxidation biomarker, as observed in various plants, such as sorghum, following HS exposure (Djanaguiraman et al. 2010).

The presence of ROS, such as O₂ and H₂O₂, triggers oxidative stress, resulting in modifications to membrane properties, protein degradation, and enzyme inactivation, subsequently compromising plant cell survival (Wang et al. 2016). Furthermore, ROS can induce programmed cell death when plants are exposed to HS. However, plants have evolved defense mechanisms against ROS and enhanced tolerance to high temperatures. This is achieved through the engagement of antioxidant enzymes including superoxide dismutase, ascorbate peroxidase, catalase, glutathione reductase, as well as peroxidase, which collectively enhance the plant’s ability to endure elevated temperatures (Jespersen 2020).

Other physiological responses

Fluctuations in temperatures can lead to unpredictable variations in the hydration state of plants, as highlighted in the research by Xalxo et al. (2020). HS drawbacks on a plant’s growth and development encompasses dehydration, piled up by several other adverse effects. This is further explored in the work of Liu et al. (2020), which demonstrates that exposure to high temperatures significantly diminishes photosynthesis output due to reductions in water potential and relative water capacity. Nonetheless, in the face of temporary or moderate heat stress, plants regulate their heat absorption and water loss by adapting their evaporation and respiration rates.

As a molecular response towards HS conditions, plants also modify soluble carbohydrates as well as proteins concentrations. (Wang et al. 2020a) discuss how this adjustment aids in controlling cellular osmotic pressure during periods of high temperature. Notably, the consequences of heat stress extend to agricultural productivity, as indicated by Zhang et al. (2013), who emphasize that heat stress results in decreased yields of grains, beans, and oil products.

Plants’ molecular responses to heat stress

Transcriptional regulatory network of heat stress response

The regulatory network for transcriptional processes in HS stressed plants is quite complex and plant’s heat stress response (HSR) is centered around heat shock factors (HSFs), which are pivotal components. These HSFs initiate a sequence of transcriptional events that activate downstream genes encoding various key elements, such as HSR-induced transcription factors, ROS scavenging enzymes, metabolic enzymes, and Heat Shock Proteins (HSPs) (Gong et al. 2020), as depicted in Fig. 2. A substantial portion of the 25 HSFs present in rice, specifically 22 out of them, are induced by heat (Mittal et al. 2009). These HSFs can be classified into HSFA (including 1a, 2a to 2f, 3, 4b, 4d, 5, 7, 9), HSFB (comprising 1, 2a to 2c, 4a to d), and HSFC (encompassing 1a, 1b, 2a, 2b) categories. Notably, certain HSFs, such as the HSFA1s, hold the designation of “master regulators” within this regulatory network (Ohama et al. 2017).

Fig. 2.

In response to heat stress (HS), plants undergo a complex regulatory network involving various cellular processes. HS affects cell wall structure, leading to the release of apoplastic Ca²⁺. It alters membrane properties, and disrupts chloroplasts and mitochondria, increasing cytosolic Ca²⁺, ROS, and NO. These molecules act as second messengers, activating downstream regulatory networks. HSFA1 transcription factors play a central role, regulated by interactions and post-translational modifications. Protein homeostasis is disrupted, leading to unfolded protein response (UPR) through pathways like IRE1-bZIP60 and bZIP17/bZIP28. Under non-stress conditions, HSP70/90 binding represses HSFA1s, but HS triggers their dissociation, activating HSFA1s. Non-coding RNAs play roles in the HS response, with unidentified factors in some pathways. Abbreviations: HSP, heat shock protein; HSF, heat shock transcription factor; ANN, annexin; JUB1, jungbrunnen 1; MBF1c, multiprotein-bridging factor 1c; DREB2A/2C dehydration-responsive element binding protein 2A/2C; NF-Y, nuclear factor Y; DPB3-1, DNA polymerase II subunit B3-1; ROS, reactive oxygen species; BIP, binding immunoglobulin protein; bIZP, basic leucine zipper; S-bzip60, spliced bZIP60; UPR, unfolded protein response; IRT1, inositol-requiring enzyme 1; miRNA, microRNA; lncRNA, long non-coding RNA; siRNA, small interfering RNA

An illustrative example of the critical role of HSFA1s is demonstrated by hsfa1a hsfa1b hsfa1d triple deletion mutant in Arabidopsis, which profoundly impacted up-regulating as much as 65% of heat-induced transcripts. These include HSFA2, HSFBs, dehydration-responsive element binding protein 2A (DREB2A), multiprotein bridging factor 1C (MBF1c), as well as multiple HSPs genes (Liu et al. 2011). HSFA2, a gene associated with the HSR, has been well documented as the ‘direct target’ of HSFA1s (Li et al. 2018). Furthermore, heat exposure in rice prompts the enhanced production of genes such as HSP17.7, HSP18.2, HSP21, HSP83.1 and HSP101 due to alternative splicing, which activates the transcriptionally active variant of OsHSFA2d (Cheng et al. 2015). Analogously, the overexpression of OsHSFA2e in Arabidopsis results in increased thermotolerance and upregulation of various HSP genes (Yokotani et al. 2008). Notably, OsHSFA2c, through its interaction with OsHSFB4b, straightaway binds to HSP100 gene’s promoter, along with being implicated in its transcriptional control (Singh et al. 2012). The relationship between HSFs and HSFBs is further highlighted in the context of Arabidopsis and tomato, where HSFA1s are demonstrated to regulate downstream HSFB genes (Nawaz et al. 2014; Ohama et al. 2017). The importance of OsHSFB2b, which is highly induced by heat, is underscored in rice as it is known to serve the critical function of enhancing thermotolerance (Xiang et al. 2013).

In addition to heat shock factors (HSFs), DREB2A, which is from ERF/AP2 family of transcription factors, serves major function of regulating thermotolerance, as well as, is directly influenced by HSFA1, as shown by Li et al. (2018). In Arabidopsis, DREB2A interacts with the regulators of HSFA3 and triggers its expression by forming a complex involving Nuclear Factor Y, Subunit A2 (NF-YA2), nuclear factor Y, Subunit B3 (NF-YB3), and DNA Polymerase II Subunit B3-1 (DPB3-1). Dysfunction of DPB3-1, HSFA3, or DREB2A leads to heat-sensitive traits, as identified by Sato et al. (2015), Schramm et al. (2008), and Sakuma et al. (2006). Notably, the rice equivalent of DPB3-1, OsDPB3-2, associates with OsDREB2B2, which refers to the counterpart of DREB2A in rice, as reported by Sato et al. 2016. Coexpression of NF-YA2, NF-YB3, and DPB3-1 proteins significantly heightens OsDREB2B2’s transactivation effect on promoter of HSFA3, indicating the conservation of this trimer’s influence on DREB2A protein in plants, as highlighted by Sato et al. (2016). Sato et al. (2016) study also demonstrated that overexpressing DPB3-1 in rice enhances thermotolerance without compromising growth or yield, underscoring the potential of DPB3-1 proteins for plant breeding efforts. Another key target of HSFA1 is MBF1c, which swiftly binds to CTAGA motif in target genes’ promoters, including DREB2A, HSFB2a, as well as HSFB1, as elucidated by Suzuki et al. (2011). HSFA1b straightaway governs the expression of MBF1c by binding to the heat shock element within the MBF1c promoter, as reported by Bechtold et al. (2013). MBF1c’s conserved function in thermotolerance, as demonstrated by Qin et al. (2015), was validated by the observation that overexpressing TaMBF1c. in yeast as well as rice confers thermotolerance.

The heat shock response (HSR) is a complex cellular process involving multiple interactions between proteins and various post-translational modifications. In this context, protein–protein interactions and post-translational changes play crucial roles in modulating the activities of transcription factors associated with HSR. For instance, HSP70 and HSP90 were observed to interact with HSFA1, leading to the inhibition of its transactivation activity and preventing its localization to the nucleus in Arabidopsis (Ohama et al. 2016, 2017). In Arabidopsis, overexpression of rice HSF-binding proteins, OsHSBP1 and OsHSBP2, resulted in increased survival rates after heat treatment, likely due to their interaction with activated HSFs, which act as negative regulators of HSR (Rana et al. 2012). Additionally, post-translational modifications such as phosphorylation and sumoylation play essential roles in HSR regulation (Ohama et al. 2017). HSFs’ capability of binding with heat shock elements (HSEs) was found to be enhanced in AtCBK3-overexpressing lines, as AtCBK3 can phosphorylate AtHSFA1a. This phosphorylation event appears to impact HSR positively (Liu et al. 2008). Sumoylation, another post-translational modification, also affects HSR. Overexpressing AtSUMO1 in Arabidopsis led to heat-sensitive traits resembling those observed in AtHSFA2-knockout plants. This outcome was attributed to the inhibitory effect of sumoylation on the transcriptional stimulation of AtHSFA2 (Cohen-Peer et al. 2010). Similarly, rice OsSIZ1’s over-expression, a ligase gene SUMO E3, in Arabidopsis as well as cotton, improved thermotolerance, possibly by enhancing the sumoylation process (Mishra et al. 2017, 2018). Moreover, sumoylation also has a role in maintaining the DREB2A protein during heat stress, leading to improved thermotolerance in plants (Wang et al. 2020b). Overall, the intricate network of interactions among protein–protein, as well as modifications after translational, comprising phosphorylation and sumoylation, serve the major role to regulate HSR and modulating activities of key transcription factors involved in this process (Liu et al. 2008; Cohen-Peer et al. 2010; Rana et al. 2012; Mishra et al. 2017, 2018; Ohama et al. 2017; Wang et al. 2020a, b).

Furthermore, the regulation of HSR involves participation of NAC transcription factors. For instance, NAC019 was shown to have a crucial role in triggering the HSR signalling cascade via associating with promoters of HSFA1b, HSFA6b, HSFA7a, as well as HSFC1, as demonstrated by Guan et al. (2014). In a similar vein, the activation of an additional NAC transcription factor, Jungbrunnen1 (JUB1), in Arabidopsis in response to ROS was found to strongly stimulate the transactivation of DREB2A expression through direct binding to its promoter, as reported by Wu et al. (2012). This mechanism appears to extend to rice, where ONAC066, JUB1 counterpart, along with serving like the positive regulator under oxidative stress conditions (Yuan et al. 2019) discovered that ONAC066 directly interacts with the promoter of OsDREB2A, activating its transcription and thereby contributing to the rice HSR. Notably, several other transcription factors, such as OsMYB55 (El-kereamy et al. 2012), OsbZIP46 (Chang et al. 2017b), SNAC3 (Fang et al. 2015), and OsWRKY11 (Wu et al. 2009), have been identified as essential components of HSR and thermotolerance in rice, independently of the HSFA1 pathway (Fig. 2).

HSFs are primarily recognized for their roles as transcriptional, post-transcriptional, translational, and post-translational regulators in response to abiotic stressors, particularly heat stress (Yoshida et al. 2008; Fragkostefanakis et al. 2015; Hossain et al. 2016). Understanding the molecular mechanisms of OsHSF is crucial for enhancing crop tolerance to various abiotic stresses, including heat stress. Previous studies have conducted genome-wide identification of HSFs in rice and Arabidopsis (Guo et al. 2008, 2015; Wang et al. 2009; Chauhan et al. 2011; Hossain et al. 2016). However, these studies did not explore spatio-temporal expression, plant hormonal responses, molecular signaling pathways, comparative mapping, or protein feature analysis in rice, a C3 model crop with significant tolerance to both individual and combined abiotic stresses (CAbS). Recent research by Muthuramalingam et al. (2020) investigated the OsHSF transcription factor family in rice and related grass species, including C4 panicoid genomes. This study identified 25 OsHSF genes in rice, unevenly distributed across 10 of the 12 chromosomes. These genes are implicated in responses to both individual and combined abiotic stresses, demonstrating chromosomal divergence in the rice genome. Transcriptome analysis revealed varying expression levels of these 25 OsHSF genes in response to different phytohormones, such as abscisic acid (ABA), jasmonic acid (JA), auxin, cytokinin, gibberellins, and brassinosteroids, in the shoots and roots of rice at different time points. Field conditions showed low levels of ABA and JA, while stress conditions induced higher expression levels of these hormones, suggesting their critical roles in abiotic stress responses (Gupta et al. 2017; Muthuramalingam et al. 2018). These findings underscore the importance of ABA and JA in stress conditions and provide a framework for phytohormonal expression patterns in rice under abiotic stress.

Omics approaches for understanding heat tolerance

Recent advancements in biological sciences, particularly in sequencing and high-throughput omics approaches, have enabled the generation of a vast array of omics data, encompassing transcriptomics, epigenomics, proteomics, metabolomics, hormonomics, signalomics, ionomics, and phenomics at the genomic level from O. sativa cultivated under diverse climatic conditions (Muthuramalingam et al. 2019b; Pandian et al. 2020). However, the extensive range of cell and molecular data remains largely inaccessible to the public, and the molecular physiological responses of O. sativa datasets are significantly underutilized. Multi-omics approaches (Fig. 3) offer a robust means to integrate various molecular data into comprehensive repositories, facilitating an in-depth understanding of organisms at the cellular and molecular levels. This integration could herald a modern green revolution in stress biology research across multiple plant species.

Fig. 3.

The generation of transgenic rice plants involves a multi-faceted approach that integrates various OMICS techniques to identify and manipulate specific genes and proteins. Here is a diagrammatic representation of the key steps in employing OMICS approaches for the production of transgenic rice

In the early twenty-first century, genomic investigations in rice facilitated gene discovery and provided detailed insights into unique and stress-related genes and their regulatory mechanisms. Recent advancements in omics technologies have enabled researchers to explore large-scale biological systems, revealing diverse functional aspects in plants, particularly cereals. These methodologies are crucial for developing omics resources in model plant species and have accelerated translational research by integrating data across food crops, especially rice (Muthuramalingam et al. 2018; Mochida and Shinozaki 2010). Emerging sequencing technologies have propelled biological science forward, driven by groundbreaking developments in sequencing methods and their novel applications post-complete genome sequencing. Next-generation sequencing (NGS) techniques, such as RNA-Seq for transcriptomics and non-coding RNA analysis, ChIP-seq for studying protein-DNA interactions, and whole-genome sequencing (WGS) for genetic variation and epigenomic dynamics, have significantly enhanced genome sequencing projects (Lister et al. 2009). These advancements focus on signal transduction, transcriptional reprogramming, cell signaling, and transcriptional networks.

Additionally, new technologies have emerged to analyze molecular signalomes and interactomes, elucidating interactions among proteins, nucleic acids, and other proteins (Dreze et al. 2011). Hormonal profiling has provided insights into cellular signaling (Muthuramalingam et al. 2019b; Kojima et al. 2009), while metabolomics has facilitated the analysis of metabolic pathways (Saito and Matsuda 2010; Muthuramalingam et al. 2020, 2018). Phenomic approaches have been employed to study plant morpho-physiological changes (Yang et al. 2013). These omics approache treat molecular mechanisms as interconnected elements within plant cellular and molecular physiological systems.

Bioinformatics plays a crucial role in multi-omics research, managing large experimental datasets at the whole-genome level to extract meaningful information. Integrating omics results will enhance our understanding and allow data exchange with other cereals (Muthuramalingam et al. 2019b; Pandian et al. 2020; Shinozaki and Sakakibara 2009; Song et al. 2018). Thus, multi-omics analytical repositories serve as pivotal platforms for systems analyses. The field of plant stress and molecular biology has seen significant advancements due to these integrated omics approaches and computational biology. This integration has broadened research horizons, revealing new avenues for identifying abiotic stress-related factors, including those associated with heat stress, and elucidating their molecular-physiological mechanisms.

Genomic approaches for increasing heat tolerance in rice

Genomic techniques have been increasingly applied to gain deeper insights into the arrangement and roles of coding and non-coding sections within agricultural advancement. Valuable resources such as mutant collections, complementary DNAs (cDNAs), gene activity patterns, nucleotide information datasets, and locations linked to measurable traits (Quantitative Trait Loci or QTLs) play a crucial role in investigating genetic and structural aspects (Jiang et al. 2012).

Recent research on heat tolerance in rice has provided significant insights into the genetic and molecular elements that enable the plant to withstand high-temperature stress. Notably, Gupta et al. (2012) conducted extensive investigations that identified and cloned key heat-responsive genes and discovered QTLs crucial for heat tolerance mechanisms. Similarly, Das et al. (2017) emphasized the intricate nature of heat stress responses by uncovering numerous QTLs contributing collectively to the plant’s ability to mitigate elevated temperatures’ adverse effects. More recently, Zargar et al. (2022) identified specific QTLs linked to increased grain yield and secondary attributes, which enable rice plants to endure heat stress across various agroecological contexts, such as upland and lowland rainfed environments.

These combined findings provide a comprehensive understanding of the complex genetic foundation of heat tolerance in rice, offering insights into potential breeding strategies and molecular approaches for developing rice variants capable of thriving amid rising temperatures. During extreme temperatures, different behaviors of QTLs have been observed in highland and lowland environments. Ultimately, the most suitable QTLs are selected based on factors such as habitat, genetic heritage, and prevailing climatic conditions.

Marker-assisted backcrossing (MABC) has been successfully employed in crops to develop high-yielding varieties. For example, MABC was used to enhance the productivity of the KDML105 rice variety in northeastern Thailand (Vanavichit et al. 2018). Recent advancements in molecular breeding techniques, including marker-assisted selection (MAS), the utilization of SNP markers, and genome-wide association studies (GWAS) are anticipated to contribute to a deeper understanding of the molecular mechanisms that confer resilience to crop plants against a wide spectrum of environmental challenges, such as heat stress.

Furthermore, advancements in CRISPR-Cas9 gene editing have shown promise in enhancing heat tolerance by enabling precise modifications to specific genes associated with stress responses (KhokharVoytas et al. 2023). The integration of multi-omics approaches, including genomics, transcriptomics, proteomics, and metabolomics, has further enriched our understanding of the dynamic molecular networks underlying heat stress tolerance in rice (Roychowdhury et al. 2023). Table 1 provides comprehensive information on the genes responsible for imparting heat tolerance in rice. By leveraging these cutting-edge genomic techniques, researchers can develop more resilient rice varieties, ensuring stable yields and food security in the face of climate change.

Table 1.

Candidate genes identified in transgenic rice associated with heat tolerance

Gene	Protein	Promoter	Techniques used	Cellular mechanism	References
Athsp101	HSP101	CaMV 35S promotor	Agrobacterium-mediated transformation	Enhanced heat tolerance	Katiyar-Agarwal et al. (2003)
ZFP	Protein	NA	NA	Enhanced heat tolerance at seedling stage	Wei et al. (2013)
OsWRKY11	WRKY11	HSP101 promoter	Agrobacterium-mediated transformation	Increased desiccation tolerance and survival rate of green parts	Wu et al. (2009)
OsGSK1	Glycogen synthase kinase 3	NA	Spray transformation technique	Knockout mutant of OsGSK1 gene showed tolerance to heat stress	Koh et al. (2007); Wahab et al. (2020)
OsHsfA2e	Heat stress transcription factor (HSF)	CaMV 35S promoter	Floral dipping method	Over expression of OsHsfA2e improved tolerance to heat	Yokotani et al. (2008); Malumpong et al. (2019)
mtHsp70	HSP70	CaMV 35S promoter	Agrobacterium-mediated transformation	Overexpression of mthsp70 suppresses the programmed cell death and ROS	Qi et al. (2011)
sHSP17.7	HSP17.7	CaMV 35S promoter	Agrobacterium-mediated transformation	enhanced heat and UV-B tolerance	Sato and Yokoya (2008)
FAD7	omega-3, fatty acid desaturase	Ubi1 promoter	Silencing	Transgenic rice with FAD7 gene has shown heat tolerance by increasing the growth rate and chlorophyll content	Sohn and Back (2007)
SBPase	SBPase	Ubiquitin promoter	Agrobacterium-mediated transformation	Over-expression of SBPase gene confers heat tolerance by CO2 assimilation and enhancing photosynthesis	Feng et al. (2007)
Spl7 (rice spotted leaf gene)	HSFA4d (heat stress transcription factor protein)	CaMV 35S promoter	Transcription control	Sp17 mutant showed spotted leaf phenotype under high temperature	Yamanouchi et al. (2002)
OsLea14-A	OsLea14-A	CaMV 35S promoter	Agrobacterium-mediated co-cultivation transformation	Confers stress tolerance	Hu et al. (2019)
MTH1745 (MtPDI)	Isomerase-like protein	Ubiquitin promoter	Agrobacterium mediated transformation	Increase heat stress tolerance	Wang et al. (2019a)
OsPAL	OsProDH	NA	NA	Increases heat stress tolerance	Akhter et al. (2019)
TT1		CaMV 35S promoter	Agrobacterium mediated transformation	Improves heat tolerance	Li et al. (2015)
OsHSP20	HSP20	Ubiquitin promoter	Agrobacterium-mediated transformation	Improve heat stress tolerance	Guo et al. (2020a)
eIF4A1	DEAD-box helicase protein	NA	Expression profiling and structural bioinformatics approaches	Enhances temperature stress	Singha et al. (2020)
OsProDH	OsProDH	NA	CRISPR/Cas9 (CRI) system	Proline overproduction	Guo et al. (2020b)
PSL50	OsProDH	CaMV35S promoter	Map-based resequencing (IMBR) approach	Promotes heat tolerance by acting as a modulator of H2O2 signaling in response to heat stress in rice	He et al. (2021)
OsNTL3	NAC transcription factor	Ubiquitin promoter	Agro‐bacterial mediated stable transformation	Enhances heat tolerance	Liu et al. (2020a)
OsBiP2, OsMed37_1				Controls heat tolerance	Raza et al. (2020)
OsHTAS	NA	NA	NA	Controls heat tolerance	Jan et al. (2021)
PHT3		CaMV 35S promoter	Floral dip method	Improved heat tolerance	Jia et al. (2015)
DPB3-1	DPB3	CaMV 35S promoter	Agrobacterium-mediated transformation	heat stress-inducible genes were upregulated	Sato et al. (2016)
rbcS	Rubisco protein	rbcS promoter	Sense and antisense approach	Increased rubisco and photosynthesis in rbcS-sense lines compared to wild-type	Makino and Sage (2007)
AtPLC9		CaMV 35S promoter	Heterologous transformation	Expression of transcription factors	Liu et al. (2020b)
OsWRKY		NA	Microarray	Improved thermal tolerance	Jeyasri et al. (2021)
OsIAA13, OsIAA20		NA	Microarray	Improved heat tolerance	Zhang et al. (2021)
RCA	Rubisco activase	promoter of Rca-α	Agrobacterium-mediated transformation	overexpression improved growth and yield	Scafaro et al. (2018)
OsNAC127 and OsNAC129	Transcription factor	–	CRISPR/Cas9 system	The mutant exhibited heat stress susceptibility	Ren et al. (2021)
OsNAC006	Transcription factor	–	CRISPR/Cas9 system	The mutant exhibited heat stress susceptibility	Wang et al. (2020a, b)
Osheat stressA1	The gene of chloroplast development	–	CRISPR/Cas9 system	The mutant exhibited heat stress susceptibility	Qiu et al. (2018)
OsTMS5	Transcription factor	–	CRISPR/Cas9 system	The mutant exhibited heat stress tolerance	Zhou et al. (2016)

Transcriptomics approaches for increasing heat tolerance in rice

While extensive transcriptomic studies have elucidated heat-responsive pathways and genes in crops like wheat, tomato, and potato (Kosová et al. 2015; Tang et al. 2016), there is limited genetic research on rice during the flowering period under heat stress conditions (Zhang et al. 2013). Most investigations have focused on spikelets and flag leaves rather than anthers or pistils. (Liu et al. 2020) conducted a study on thermal stress in rice plants, specifically examining the SDWG005 line. They identified a robust anther structure and discovered 3,559 differentially upregulated genes in the anthers of SDWG005 plants experiencing thermal stress during anthesis. Their analysis highlighted the role of the agmatine-coumarin-acyltransferase gene in mediating the heat tolerance of SDWG005 plants.

These transcriptomic insights reveal crucial genes and pathways that can be targeted to enhance heat tolerance in rice. By focusing on differentially expressed genes during key developmental stages, such as anthesis, researchers can identify candidate genes for genetic modification or breeding programs. Integrating these findings with proteomic data can further refine strategies to develop heat-resistant rice varieties, ensuring better resilience to increasing global temperatures.

Recent advancements in transcriptomics have provided deeper insights into enhancing heat tolerance in rice, focusing on the identification and functional analysis of heat-responsive genes and pathways during critical growth stages. One notable advancement is the utilization of RNA sequencing (RNA-seq) to generate comprehensive transcriptome profiles of rice under heat stress. This approach allows for the identification of novel heat-responsive genes and the elucidation of their regulatory networks.

A study by He et al. (2019) employed RNA-seq to analyze the transcriptomic responses of rice panicles under heat stress and identified several key heat-responsive genes, including those involved in heat shock proteins (HSPs) and heat shock factors (HSFs) pathways. The study revealed that the overexpression of specific HSF could significantly enhance the heat tolerance of rice plants, suggesting a potential target for genetic manipulation.

Moreover, the integration of transcriptomics with other omics approaches, such as metabolomics and proteomics, has proven to be a powerful strategy. A recent investigation by Yu et al. (2023) combined transcriptomic and metabolomic analyses to study rice under heat stress. They identified a set of differentially expressed genes (DEGs) associated with metabolic pathways, including the synthesis of protective osmolytes and antioxidants. This multi-omics approach provided a holistic view of the cellular responses to heat stress, identifying potential biomarkers for breeding heat-tolerant rice varieties.

Additionally, advancements in single-cell RNA sequencing (scRNA-seq) have opened new avenues for understanding cell-specific responses to heat stress in rice. Zong et al. (2022) applied scRNA-seq to rice anthers under heat stress, uncovering cell-type-specific expression patterns and identifying key regulatory genes involved in pollen development and viability under high-temperature conditions. This detailed cellular resolution has the potential to pinpoint critical genes for targeted genetic interventions.

The use of CRISPR/Cas9 genome editing technology has also been integrated with transcriptomics to enhance heat tolerance in rice. Rengasamy et al. (2024) employed CRISPR/Cas9 to knock out specific heat-sensitive genes identified through transcriptomic analyses. This gene editing approach resulted in rice plants with improved heat tolerance and reduced yield loss under heat stress, demonstrating the practical applications of transcriptomic data in developing heat-resistant crops.

In conclusion, recent advancements in transcriptomics, coupled with integrative omics approaches and genome editing technologies, have significantly advanced our understanding of heat tolerance mechanisms in rice. These approaches provide valuable tools for identifying and manipulating key genes to develop rice varieties with enhanced resilience to rising global temperatures.

Proteomics approaches for increasing heat tolerance in rice

Extensive research utilizing advanced proteomic techniques, such as two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and gel-free shotgun methods, has provided critical insights into the protein composition of rice kernels. Studies have identified various proteins, including digestive enzymes, storage proteins, structural proteins, and toxins (Koller et al. 2002; Kaneko et al. 2016). Specifically, Kaneko et al. (2016) identified 4172 distinct proteins in growing and ripening rice kernels, spanning a wide range of isoelectric points (pH 2.9–12.6) and molecular weights (5.2–611 kDa). Their analysis revealed significant shifts in metabolic processes during kernel development, with alcohol fermentation replacing carbon metabolism to facilitate glucose accumulation as the kernels matured (Xalxo et al. 2020). Furthermore, mature rice kernels were found to accumulate more proteins, particularly those involved in the citric acid cycle, lipid metabolism, glycolysis, and proteolysis, compared to growing grains (Kaneko et al. 2016).

Wang et al. (2015) reported a decrease in polyamine content during rice kernel development and desiccation, while storage proteins significantly increased during early maturation. Additionally, Chang et al. (2017a) observed elevated levels of pyruvate phosphate dikinase (PPDK) and downregulation of pullulanase (PUL) during grain loading. These findings indicate potential proteomic interventions to enhance protein production in rice, potentially improving heat resistance. By manipulating the expression of specific proteins involved in metabolic pathways, it may be possible to develop rice varieties with improved tolerance to heat stress.

Recent advancements in proteomic approaches for increasing heat tolerance in rice have leveraged cutting-edge techniques and bioinformatics tools to delve deeper into the molecular mechanisms underpinning stress responses. These advancements include the integration of quantitative proteomics, high-resolution mass spectrometry (MS), and bioinformatics-driven data analysis, enabling the identification and quantification of proteins involved in heat stress response with greater precision and depth.

One significant advancement is the application of tandem mass tag (TMT) labeling in conjunction with high-resolution MS. This approach allows for simultaneous quantification of proteins across multiple samples, facilitating a comprehensive analysis of proteomic changes under heat stress conditions. Studies using TMT labeling have identified key heat shock proteins (HSPs) and other stress-responsive proteins that play crucial roles in maintaining cellular homeostasis and protecting rice plants under elevated temperatures (Xie et al. 2024).

Another notable development is the use of isobaric tags for relative and absolute quantification (iTRAQ). iTRAQ-based proteomics has enabled the discovery of novel proteins and pathways associated with heat tolerance. For example, researchers have identified proteins involved in oxidative stress response, signal transduction, and protein folding, which are upregulated in heat-tolerant rice varieties. These proteins contribute to enhanced stress resilience by mitigating the detrimental effects of reactive oxygen species (ROS) and ensuring proper protein function under heat stress (Niu et al. 2021).

Additionally, advancements in bioinformatics and computational biology have significantly enhanced the analysis and interpretation of proteomic data. Machine learning algorithms and network analysis tools are being employed to identify key regulatory proteins and their interactions, providing insights into the complex regulatory networks governing heat stress responses. This integrative approach has led to the identification of potential protein targets for genetic engineering and breeding programs aimed at developing heat-tolerant rice varieties (Jhan et al. 2023).

Furthermore, proteomic studies have also explored post-translational modifications (PTMs) such as phosphorylation, ubiquitination, and acetylation, which play critical roles in regulating protein function and stability under stress conditions. Advanced proteomic techniques have facilitated the identification of PTMs on heat-responsive proteins, revealing how these modifications modulate protein activity and contribute to heat tolerance (Han et al. 2022).

In summary, recent advancements in proteomics, including TMT and iTRAQ labeling, high-resolution MS, and sophisticated bioinformatics analyses, have significantly enhanced our understanding of rice’s protein networks and regulatory mechanisms underlying heat tolerance. These approaches hold great potential for developing heat-resistant rice varieties through targeted manipulation of key proteins and pathways involved in stress response.

Metabolomics approaches for increasing heat tolerance in rice

Significant advancements have been achieved in the realm of metabolomics, which involves a systematic and comprehensive identification of organic substances within biological substances (Ryan and Robards 2006; Kumar et al. 2017)).

Significant advancements in the field of metabolomics have shed light on the intricate metabolic responses of rice plants to heat stress between 2015 and 2021.

Yadav et al. (2022) conducted pioneering research utilizing transgenic rice lines to unravel the metabolomic changes occurring in grains under contrasting conditions of normal watering and heat-induced stress. Their work elucidated the metabolic shifts associated with heat stress and provided insights into potential mechanisms of rice heat tolerance. Similarly, Shi et al. (2018) employed a combination of transcriptomic and metabolomic studies to investigate key pathways that contribute to maintaining photosynthesis under drought-induced heat stress, ultimately enhancing drought tolerance in rice. The comprehensive analysis conducted by Khan et al. (2021) plant delved into the complexities of grain yield reduction in rice due to abrupt alternation between drought and flooding stresses, employing an integrative approach involving proteomic, metabolomic, and physiological analyses. Furthermore, Du et al. (2013) made notable strides by conducting combinatorial studies encompassing proteomics, metabolomics, and physiological assessments of rice growth and grain yield in the context of heavy nitrogen application before and after exposure to heat stress. These groundbreaking studies collectively illuminate the intricate metabolic networks that underlie rice’s responses to heat stress and offer valuable insights into strategies for bolstering heat tolerance and grain yield in this important crop.

Metabolomics research in plant systems is rapidly advancing by examining overall or grouped compounds generated in specific samples over time. However, a substantial knowledge gap exists regarding the vast array of potential compounds comprising higher plant metabolomes, with most remaining unexplored (Wang et al. 2019a). Serving as a link between genetics and traits, plant metabolomes can be both qualitatively and quantitatively assessed, unveiling how plants respond to biological events, environmental triggers, DNA, and metabolic conditions. This domain significantly enhances the understanding of stress biology by shedding light on the roles played by various substances in plant adaptation, encompassing stress-induced by-products, signalling molecules, and other compounds (Lanzinger et al. 2015; Ramalingam et al. 2015).

During heat stress in rice, cellular metabolites undergo significant changes to cope with the challenging conditions. Metabolites, including amino acids, fatty acids, soluble sugars, nucleotides, organic acids, phenolics, peptides, cofactors, and secondary metabolites, play pivotal roles as indicators of cellular responses to heat stress (Das et al. 2017; Arora et al. 2018). These metabolites are involved in various metabolic pathways and regulatory processes that contribute to the plant’s ability to withstand and adapt to elevated temperatures. The dynamic alterations in these metabolites highlight the complex biochemical adjustments that occur in rice plants under heat stress conditions, shedding light on the intricate mechanisms of heat stress tolerance and adaptation.

Numerous metabolomic techniques have proven invaluable in discerning the diverse molecular signals exhibited by plants when confronted with abiotic stresses (Ghatak et al. 2018). These identified compounds are instrumental in facilitating the plant’s defense mechanisms.

Polyamines and phenolic compounds are vital plant secondary metabolites that confer tolerance to diverse environmental stresses, including heat stress, potentially replacing biostimulants for growth maintenance (Aninbon et al. 2016; Chen et al. 2019a, b). Polyamines regulate heat stress responses and stabilize cellular structures (Aninbon et al. 2016), while phenolic compounds enhance antioxidant defenses in rice plants (Chen et al. 2019a). These metabolites hold promise for bolstering rice’s heat stress resilience and sustainable agriculture (Aninbon et al. 2016; Chen et al. 2019a, b). Changes in metabolism under stress may increase the production of some essential metabolic products, demonstrating that metabolic pathways are differentially controlled. Plants can also alter biochemical pathways to store enormous amounts of energy necessary for survival in hostile settings (Ullah et al. 2017). Metabolic studies focusing on heat stress responses in rice have revealed significant alterations in metabolite profiles. Notably, heat-tolerant rice cultivars demonstrate a distinct accumulation of key metabolites compared to heat-sensitive varieties. For instance, metabolites such as fructose, glucose, and myo-inositol, which are associated with carbohydrate metabolism, show higher levels in heat-tolerant rice under elevated temperature conditions (Barnaby et al. 2019). Additionally, analyses of amino acid intermediate from pathways including proline, glutamine, tryptophan, alanine, aspartate, ornithine, isoleucine, leucine, and valine, as well as metabolites like 2-oxoglutarate, cis-aconitate, and succinate from the TCA cycle, exhibit significant accumulation trends under heat stress, akin to their responses in drought stress (Tarazona et al. 2015; Pires et al. 2016). Furthermore, flavonoids like quercetin and cyanidin, known for their roles in stress tolerance, also exhibit dynamic changes in response to heat stress in rice.

The investigation of differential production of primary and secondary metabolites in response to biotic and abiotic stresses can be effectively explored using quantitative and qualitative studies of rice metabolomics, as noted by Khakimov et al. (2014). Key analytical methods such as nuclear magnetic resonance (NMR), gas chromatography-mass spectrometry (GC/MS), liquid chromatography-mass spectrometry (LC/MS), and Fourier transform ion cyclotron resonance (FT-ICR) have been utilized in conjunction with databases like Kyoto encyclopedia of genes and genomes (KEGG) and MetaCyc (Kanehisa et al. 2012; Morreel et al. 2014; Caspi et al. 2018; Chen et al. 2021) to unravel metabolite profiles in organisms. Metabolic networking and the utilization of MetaCyc have certain limitations when applied to rice-specific information, as indicated by Chae et al. (2007).

Dissecting the molecular machinery involved in rice plants’ response to abiotic stresses, particularly heat stress, requires a comprehensive understanding of whole genome sequences (WGS), omics technologies, and bioinformatics tools. These prerequisites are essential for gaining detailed insights into the signaling and physiological pathways, defense mechanisms, and molecular cross-talks associated with heat stress. Advances in omics technologies and computational biology have significantly contributed to rice research, enabling the delineation of molecular interactions and the utility of biological and cellular systems. One key resource is the Kyoto encyclopedia of genes and genomes (KEGG), which provides an integrated platform for analyzing high-throughput omics data, including metabolomics, computational biology, and experimental datasets (Kanehisa and Goto 2000). Utilizing these omics and bioinformatics tools has enhanced researchers’ ability to identify and understand abiotic stress responses, including heat stress, at a molecular level. These approaches facilitate gene discovery and provide annotated information about genes, proteins, and metabolites involved in stress responses. This knowledge accelerates the mapping of gene networks, signaling pathways, and cross-talk responses critical for developing heat stress resilience in rice (Kosová et al. 2015; Muthuramalingam et al. 2018; Fumagalli et al. 2009; Ma et al. 2013). These advancements are particularly useful for reverse genetics studies, linking metabolites to proteins and genes in heat-stressed rice plants.

Investigating the molecular mechanisms and regulatory pathways underlying responses to heat and other abiotic stresses involves integrated multi-omics approaches. These approaches have provided valuable insights into developmental changes, cellular differentiation, and stress responses. Through integrated omics and systems biology analyses, coupled with resources such as PubMed, researchers have identified gene families associated with stress responses and cellular functions, elucidating their molecular cross-talks and regulatory mechanisms (Muthuramalingam et al. 2019a).

For gaining insights into the underlying mechanisms of abiotic stress responses, particularly heat stress, comparative metabolomics techniques offer a solid framework to examine changes in gene expression, protein expression, and other molecular compounds over time. Employing metabolomics as a technological tool holds significant potential to enhance our understanding of the metabolic and genomic composition of organisms, thereby supporting molecular breeding efforts in arid environments, as highlighted by Bino et al. (2004), Fernie and Schauer (2009), and Kusano et al. (2011).

Epigenomics approaches for increasing heat tolerance in rice

The epigenome can be considered the total of the molecular modifications to a cell’s nucleus polymer, basic proteins, and small non-coding ribonucleic acid during its lifetime. Epigenetics is defined as the study of epigenetic alterations in and around polymers that control cellular activity, and epigenomics is the genetics subfield that focuses on epigenomic research. Plants have developed non-heritable, highly nuanced processes to survive a wide range of external stressors. Previous studies have outlined the significant progress made over the last decade in comprehending the signaling and biochemical networks that are prominent in plant reactions to stressors (Ku et al. 2018; Kumar et al. 2019). It is common for communication networks to activate transcriptional changes to start the production of stress-responsive proteins (Kim et al. 2019; Shahid 2020; Wu et al. 2019). As a defense mechanism against environmental stresses, agricultural plants have been shown to exhibit a wide variety of alterations (Thomashow 1999). Stress-induced proteins, RNA molecules, and compounds are expressed over time as part of the sequential reactions to stress. Phenological and physical changes may allow for more time before these stress adaptations wear off. Several components and mechanisms of epigenetics, including DNA methylation, protein changes, and control by non-coding RNAs, rely on the transcriptional remodelling and modulation of stress-responsive genes (Deleris et al. 2016; Kim et al. 2017; Chen et al. 2019b). Demethylation generally takes longer under circumstances of heat duress. DNA methylation is found to have tissue-specific differences as well. Across cells, organs, genotypes, and phases of the biological process, methylation can vary by as much as 1%. It has been observed that the DNA methylation amount in roots is lower than in foliage, suggesting an essential function for roots in responding to water shortage (Suji and Joel 2010). Both upland and heat-tolerant rice varieties exhibit DNA methylation patterns that are correlated with heat stress. Differential activation of stress-responsive genes was traced back to shifts in DNA methylation patterns (Gayacharan and Joel 2013). Another rice research showed that hypomethylation is crucial to the traits of rice varieties’ heat resistance. Several methods are used in epigenomic research, including chromatin immunoprecipitation (ChiP), chromatin immunoprecipitation sequencing (ChiP-seq), methylated-DNA immunoprecipitation, and shotgun bisulfite sequencing. Understanding the character and function of epigenomic profiles, such as histone changes, DNA methylation, various types of regulating non-coding RNAs, and the 3D genomic structure of rice, is important for the creation of resistant rice varieties (Lu et al. 2020). The intricacy of agricultural plant responses to various stress factors has been revealed thanks in large part to epigenomics research into the dynamic nature of expression patterns under stress. Moreover, the classification of phenotypes that are appropriate for agricultural traits in order to provide a paradigm for improving output and characteristics in rice harvests relies heavily on an in-depth analysis of rice epigenomics variants and their identity (Chen and Zhou 2013).

Phenomics approaches for increasing heat tolerance in rice

Heat stress is a significant abiotic factor adversely affecting rice productivity, especially in the context of global climate change. Understanding the phenomics of heat stress tolerance is crucial for breeding heat-tolerant rice varieties. Phenomics, which involves the study of phenotypes on a large scale, provides comprehensive insights into how rice plants respond to heat stress at various biological levels. Rice plants exhibit various physiological changes under heat stress, including alterations in photosynthesis, respiration, and water usage. High temperatures can cause a reduction in chlorophyll content and photosynthetic rate, leading to lower biomass production and grain yield (Fahad et al. 2017). Furthermore, heat stress can disrupt membrane stability and enzyme function, exacerbating cellular damage (Wahid et al. 2007). Plants that maintain higher leaf water content and stable membrane integrity under heat stress are often more tolerant, indicating the importance of these traits in phenomic studies. Morphological traits such as plant height, leaf area, and root architecture play a crucial role in heat stress tolerance. Heat-tolerant rice varieties often exhibit traits like reduced leaf area to minimize water loss and efficient root systems to enhance water uptake (Ahamed et al. 2010). These traits can be quantitatively assessed using high-throughput phenotyping platforms, which allow the measurement of growth patterns and stress responses in real time (Yang et al. 2013).

At the genetic level, heat tolerance is a complex trait controlled by multiple genes. Quantitative trait loci (QTL) mapping and genome-wide association studies (GWAS) have identified several QTLs associated with heat tolerance in rice. For instance, QTLs on chromosomes 1, 2, and 3 have been linked to traits such as spikelet fertility and grain yield under heat stress conditions (Ye et al. 2012). Molecular studies have also highlighted the role of heat shock proteins (HSPs) and transcription factors (TFs) in mediating heat stress responses. HSPs act as molecular chaperones, protecting proteins from denaturation, while TFs regulate the expression of stress-responsive genes (Wang et al. 2003).

Advancements in high-throughput phenotyping technologies have revolutionized the study of heat stress tolerance. Imaging techniques such as thermal imaging, chlorophyll fluorescence imaging, and hyperspectral imaging enable the non-invasive assessment of plant responses to heat stress (Wang et al. 2019b). These technologies can monitor changes in canopy temperature, photosynthetic efficiency, and pigment composition, providing valuable data for phenotypic selection and breeding programs. Integrating phenotypic data with genomic information through systems biology approaches offers a comprehensive understanding of heat stress tolerance. This integration facilitates the identification of key regulatory networks and metabolic pathways involved in stress responses (Wang et al. 2019b). Additionally, modeling approaches can predict plant performance under various environmental scenarios, aiding in the development of heat-resilient rice varieties (Kumar et al. 2022). The phenomics of heat stress tolerance is pivotal for breeding programs aimed at developing heat-tolerant rice varieties. Marker-assisted selection (MAS) and genomic selection (GS) are promising strategies that utilize phenotypic and genotypic data to accelerate the breeding process (Xu et al. 2020). Future research should focus on elucidating the mechanisms underlying heat stress tolerance, exploring the genetic diversity of rice germplasm, and leveraging cutting-edge phenotyping technologies to enhance breeding efficiency. Phenomics data plays a pivotal role in identifying genes, conducting genome-wide association studies, and association mapping, making it essential for advancing genomics-assisted crop improvement. Despite advancements in genomics, the growth in this field is constrained by the limited availability of comprehensive phenomics data (Muthuramalingam et al. 2019a; Ye et al. 2021). High-throughput phenomics provides an opportunity to capture complex phenotypic variations, traditionally relied upon for phenotypic selection. Integrating phenomics data with genomics, transcriptomics, and other omics approaches can yield cost-effective and comprehensive trait data, facilitating the molecular dissection of traits related to heat tolerance (Ye et al. 2021). Understanding the intricate environmental responses of plants to various abiotic stresses, particularly heat stress, remains a significant challenge for plant researchers. Developing heat-resistant rice varieties is especially complex. Therefore, multi-omics approaches, when combined with computational biology, can unravel the complexities of heat and other abiotic stresses, offering novel solutions and pathways for research.

The phenomics of heat stress tolerance in rice encompasses a broad spectrum of physiological, morphological, genetic, and molecular responses. High-throughput phenotyping and integrative approaches hold great promise in advancing our understanding of these complex traits. By leveraging these insights, breeding programs can develop heat-tolerant rice varieties, ensuring food security in the face of climate change.

Bioinformatic databases

The integration of multiple machines in omics technologies generates high-quality datasets that are subsequently analyzed, visualized, curated, and stored using a variety of in silico approaches. These approaches are intricately connected with a broad spectrum of bioinformatics tools, platforms, packages, online repositories, mathematical models, and programming languages, all of which collectively enhance data analysis, integration, and accessibility for researchers. The research community relies on these bioinformatics tools to efficiently, accurately, and reproducibly analyze a diverse range of multi-omics data (Franceschini et al. 2013; Orozco et al. 2013; Yachdav et al. 2014; Henry et al. 2014; Franz et al. 2023).

Advancements in bioinformatics tools and databases have enabled researchers to identify and annotate a wide variety of genes responsive to abiotic stresses, such as those induced by environmental changes like heat stress (Aslam et al. 2017). These tools provide comprehensive insights, allowing researchers to apply specific bioinformatics knowledge to improve crop species for enhanced production and increased tolerance to both abiotic and biotic stresses. Developing multi-omics strategies involves detailed analyses of various agricultural crop species under abiotic stress conditions, leveraging omics tools to explore novel aspects of organisms at the whole-genome level (Aslam et al. 2017). Following genome sequencing, the primary objective is to identify and annotate functionally significant elements within the genome using bioinformatics resources (Eyras et al. 2005). Consequently, bioinformatics platforms play a pivotal role in uncovering deeper molecular and biological insights.

Role of CRISPR/Cas9 in improving heat tolerance in rice

The regular, conventional mating methods introduce both harmful and helpful traits into a population (Rasheed et al. 2021). These methods are too slow to keep up with the needs of a quickly expanding global populace. Furthermore, pollination between members of the same plant species is conceivable, though this does not lead to the creation of any novel characteristics or DNA (Jiang et al. 2012). Because of this, modern genome editing methods (GET) can quickly and effectively improve desirable characteristics in any species, overcoming the drawbacks of traditional breeding (Jiang et al. 2012). However, the implementation of GET requires knowledge of sequencing the genes, functions of genes, as well as QTL accountability in implementing interested characteristics. Using DNA-cutting enzymes called target-specific nucleases, GET can be used to alter a single copy of the gene responsible for the desired characteristic. A variety of site-specific endonucleases (SSE) have been extensively used in techniques for editing genomes, over past decades. These comprise zinc finger nucleases and transcription activator-like effector nucleases (Chen and Gao 2014). Numerous experiments were carried out in which CRISPR/Cas9 method has been employed to wipe out genes with goal of increasing rice’s resistance to thermal stress. Editing genes for thermal resistance using the CRISPR/Cas system has been extensively implemented among rice (Wang et al. 2020a). The implementation of CRISPR/Cas9 has the potential which aids to develop heat-tolerant rice varieties by altering genes responsible for plant and panicle design, leaf shape, and the ABA signaling system (Jiang et al. 2012). OsNAC006 gene deletion dramatically improved rice’s ability to withstand thermal duress (Wang et al. 2020b). Another research that used CRISPR/Cas9 for modifying OsProDH gene reached the same conclusion, stating that the gene negatively regulates the heat endurance of rice, by removing ROS. Recent GET progress has included the creation of a CRISPR and Cas system. CRISPR-based genome editing has been used to enhance several characteristics in plants, also several Cas proteins have been discovered and put to use (Cebrian-Serrano and Davies 2017; Naeem et al. 2020). For many commodities, including barley, cucumber, maize, rice, soybean as well as wheat, CRISPR/Cas9 is regarded as a simpler, more dependable, and effective method used for increasing stress resilience, cereal production, pesticide resistance, and product quality (Komor et al. 2016). Figure 4 depicted CRISPR/Cas9-mediated heat stress tolerance in rice plants shows how high temperatures adversely affect plant physiology.

Fig. 4.

A schematic representation of CRISPR/Cas9-mediated heat stress tolerance in rice plants shows how high temperatures adversely affect plant physiology. By using CRISPR/Cas9 gene editing to modify stress-responsive genes and regulatory factors, it is possible to produce non-functional proteins that regulate the biochemical and physiological properties of the plants. This genetic modification ultimately enhances the plants’ tolerance to heat stress

Heat tolerance associated quantitative trait loci (QTLs)

According to studies, in rice, several heat tolerance-associated quantitative trait loci (QTLs) are documented (Cui et al. 2003; Larkindale et al. 2005; Jagadish et al. 2010; Swamy and Kumar 2013; Ye et al. 2018). These QTLs are associated with various heat tolerance traits along with serving a major role in improving heat stress tolerance in rice crops. For instance, qHTSF4.1, located on chromosome 4, influences improved pollen fertility and yield, in HS (Cui et al. 2003). Another QTL, qHTSF12.1, found on chromosome 12, contributes to increased fertility of spikelet as well as grain output in high-temperature situations (Jagadish et al. 2010). Similarly, qHTSF7.1 on chromosome 7 plays a role in maintaining the fertility of spikelets as well as grain yields in heat stress (Jagadish et al. 2010). qHTSF1.1, located on chromosome 1, affects spikelet fertility, panicle exertion, and grain outcome in scenarios of high temperature. Additionally, qHTSF9.1 on chromosome 9 contributes to improved fertility of spikelet, as well as grain in terms of yield during HS (Cui et al. 2003). It noteworthy that these QTLs represent only a subset of the known heat tolerance-associated loci in rice, and ongoing research continues to identify and characterize additional QTLs associated with heat tolerance (Swamy and Kumar 2013).

miRNA and heat responses

MicroRNAs (miRNAs) are well-studied as the major players in the case of HS responses among rice plants (Wang et al. 2020a). These small RNAs are non-coding, but engaged via gene regulations, post-translation, being capable of modulating target genes’ expression while adapting to HS. Several miRNAs like miR156, miR159, and miR319 have been reported to be upregulated in situations of HS. These miRNAs target transcription factors involved in plants’ development as well as stress responses, suggesting their potential role in regulating heat-responsive genes in rice (Guo et al. 2017). In addition, miR398 is responsive to HS in rice. This miRNA targets copper/zinc superoxide dismutase (CSD) genes, which are involved in antioxidant defense mechanisms. The upregulation of miR398 under heat stress conditions leads to the downregulation of its target genes, potentially affecting the antioxidant responses during heat stress in rice. Furthermore, miR169 is also reportedly implicated in HSRs among rice plants (Das et al. 2022). This miRNA targets the gene for nuclear factor Y subunit A (NF-YA), which plays a role in various stress responses. Heat stress-induced upregulation of miR169 leads to the downregulation of NF-YA genes, potentially affecting the plant’s ability to cope with heat stress in rice.

These findings highlight miRNA’s participation in regulating HS responses among rice by modulating the expression of target genes involved in various cellular processes. Further research is underway to elucidate the precise regulatory mechanisms and miRNA’s functional roles towards HS adaptation in rice plants.

Conclusion and future perspectives

The review comprehensively examines how plants respond to heat stress (HS) physiologically and at the molecular level. Under HS, plants face challenges like reduced photosynthesis, cell membrane damage, and oxidative harm from reactive oxygen species (ROS), leading to decreased growth and productivity. Key players like HSFs as well as HSPs coordinate among molecular responses, involving interactions, modifications, and collaboration with other transcription factor families. Biotechnological interventions, including genetic engineering, show promise in enhancing plant heat resilience and revolutionizing agriculture amidst global warming. Omics approaches (genomic, proteomic, metabolomic, epigenomic) have unveiled rice’s heat tolerance mechanisms. Genomic studies identify heat-responsive genes and QTLs, while proteomic/transcriptomic analyses show protein/gene changes during heat stress. Metabolomics reveals vital metabolite roles, and epigenomics unveils epigenetic modifications’ significance. CRISPR/Cas9 editing improves rice heat tolerance.

In the future, deeper exploration of molecular interactions can lead to targeted interventions for better thermotolerance. Applying these insights in breeding can enhance climate resilience and food security. Integrating omics datasets and advanced techniques can identify key networks/pathways. Genomic and genetic mapping can pinpoint heat tolerance QTLs. Proteomic/transcriptomic analyses can uncover novel pathways, while metabolomics and epigenomics can gain more functional insights. Integrating omics and technology may yield heat-resilient rice, ensuring global food security amidst climate challenges. Integrating physiological and multi-omics methods to elucidate heat stress tolerance in rice is critical for enhancing sustainable rice production in both current and future scenarios. By examining physiological responses such as photosynthesis rate, transpiration, and leaf temperature, researchers can identify traits associated with heat tolerance, while multi-omics approaches, including genomics, transcriptomics, proteomics, and metabolomics, allow for the identification of key genes, proteins, and metabolic pathways involved in heat stress response. This integration aids breeding programs through marker-assisted selection and genomic selection, enhancing the development of heat-resistant rice varieties and improving the accuracy of selecting heat-tolerant genotypes. Understanding the physiological and molecular responses of rice to heat stress also informs better irrigation practices, timing of planting, and other agronomic interventions to mitigate heat stress impacts. As global temperatures rise, developing rice varieties that can withstand higher temperatures will be crucial for maintaining food security, and integrating physiological and multi-omics data into predictive models can help forecast rice plant responses to future climate conditions, guiding breeding programs accordingly. Heat-tolerant rice varieties can maintain productivity with potentially less water and nutrient input, contributing to more sustainable agricultural practices, while understanding genetic diversity in heat tolerance can help conserve and utilize rice genetic resources more effectively. Ensuring stable rice yields under heat stress conditions will be critical to feeding a growing global population, and scientific insights from this study can inform agricultural policies and investment in research and development for climate-resilient crops. The integration of physiological and multi-omics approaches encourages collaboration among plant physiologists, geneticists, molecular biologists, and agronomists, leading to more comprehensive solutions and a holistic understanding of heat stress tolerance, from molecular mechanisms to field-level performance. This interdisciplinary research drives the development of high-throughput phenotyping tools and bioinformatics platforms for data integration and analysis, fostering technological innovations that can be applied to other crops and stress conditions. Ultimately, this research is pivotal for developing resilient rice varieties, improving agricultural sustainability, and ensuring food security in the face of current and future climatic challenges, with the insights gained being instrumental in guiding breeding programs, agricultural practices, and policy-making, making it a cornerstone for future-ready rice production systems.

Author contributions

All the authors contributed in some way to the manuscript preparation. SS collected data, contributed new models, analyzed data, and prepared the manuscript. This article was designed by AP. Both ND and PB analyzed the final drafts.

Funding

AP thanks Department of Biotechnology, Ministry of Science and Technology, Govt. of India for MK Bhan Young researcher fellowship program (HRD-12/4/2020-AFS-DBT-Part (1) (13815).

Data availability

Not Applicable.

Declarations

Conflict of interest

All of the authors declare that they have no competing interests.

Ethical approval

Not Applicable.