Molecular Cascades of Heat Stress Responses in Solanaceae with Emphasis on Capsicum annuum L., Integrating Heat Shock Transcription Factors and Proteins

Abstract

Plants are capable of responding to various environmental stresses by initiating the expression of genes that encode proteins involved in plant growth, fruit ripening, maintaining protein homeostasis, and combating heat stress (HS) by activating heat tolerance systems. The mechanism of resisting against HS is very intricate, and the molecular basis and involvement of the related gene network in Capsicum annuum L. are not fully understood. There are five different heat shock proteins (HSPs) reported in the literature, namely, small HSPs (sHSPs), CaHSP60s, CaHSP70s, CaHSP90s, and CaHSP100s, which play a pivotal role in heat stress response (HSR) in C. annuum. Heat shock factors (HSFs) and heat stress elements (HSEs) govern the transcriptional modifications and control the relative expression of heat shock proteins (HSPs). The heat stress response is the reprogramming of the molecular cascades involving the cell stress responses against the HSR, which is characterized by the increased production of molecular chaperones, which help the plants to counter the negative physiological impacts on proteins, induced by heat and other abiotic stresses. Therefore, understanding the detailed molecular mechanisms of C. annuum in response to extreme temperatures is critical for exploring how they will be affected by climate change and how they behave to cope with these varied climate extremes. This study is focused on providing a complete understanding of the molecular cascades in C. annuum L.’s response to HS, which starts with the sensation of HS signals and activation of the relative molecular cascades that are responsible for the activation of HSFs and initiate their primary targets, e.g., HSPs. Overall, this review provides deep insights into all the cellular responses during HS with a special focus on categorization and physiological aspects of HSPs and HSFs.

1. Introduction

The world is experiencing a shift toward a warmer climate, which poses a significant threat to agriculture and crop yields globally. The ever-rising global temperatures have imparted strong negative impacts on the agriculture sector, such as altered ambient temperatures and disturbed precipitation patterns. There is a strong correlation between climate change and agricultural output [1]. Various plant growth and developmental processes are significantly affected by the abiotic stresses, especially the varied ambient temperatures. These processes include photosynthesis, respiration, transpiration, fruit formation, and pollination. Higher temperatures have become a limiting factor that hinders the plant’s normal growth and development. Plants are especially vulnerable to heat stress (HS) during the development of reproductive organs, such as flowering, pollination, and fertilization, leading to decreased fruit yields [2,3]. As global temperatures have consistently risen over recent decades, the study of HS effects on the morpho-physiological traits and economic yields of horticultural crops has garnered increasing interest from scientists and farmers alike. C. annuum L. plants have a significant economic relevance since their fruits constitute a group of horticultural products that are among the most consumed worldwide, either fresh or processed. This relevance is related to their significant nutritional potential since pepper fruits are a source of vitamins (A, C, E, and B6), β-carotene, minerals, folic acid, and fiber [4,5].

Capsicum annuum L. is a widely cultivated vegetable and common spice worldwide and is particularly affected by HS. The frequency and occurrence of HS have significantly increased due to global warming, presenting a major challenge to the growth, development, and productivity of pepper [6,7]. The ideal temperature range for pepper growth is between 20 °C and 30 °C, but sustained temperatures above 32 °C can lead to severe growth issues, such as blossom and fruit drop and pale spots, resulting in blemished fruits and reduced quality [8]. HS disrupts carbohydrate metabolism and photosynthesis, increases oxidative damage, and alters the accumulation of compatible solutes, including sugars, amino acids, proline, glycine betaine, and gamma-aminobutyric acid, which are essential for maintaining cellular balance [9,10].

The responses of plants to HS are profoundly affected by both the intensity and duration of the stress, as well as the developmental stage of the plant. HS can inflict direct or indirect damage on plant functions, resulting in morphophysiological changes, irregular growth phases, disrupted metabolic processes, and diminished yields [11]. These growth irregularities may persist or remain undetected while plants endure heat stress, with each plant or genotype having developed its tolerance or mechanisms to cope with such conditions [12]. Notably, some heat-tolerant genotypes within various crop species have demonstrated the highest yields under elevated temperatures. Numerous studies have underscored genotypic variations in the tolerance or susceptibility of hot peppers by comparing germination rates and seedling growth [13], crop growth, physiology, yields [14], photosynthesis [13,15], and differences in pollen tube length, pollen germination, and membrane stability between heat-susceptible and heat-tolerant genotypes [16].

High temperatures constitute a significant abiotic stressor that substantially impacts C. annuum production. Flower abscission increases when daytime temperatures range from 32 °C to 38 °C, and fruit setting fails at temperatures exceeding 40 °C [17]. Such extreme temperatures adversely affect plants’ normal physiological and metabolic functions, prompting them to evolve unique mechanisms to endure high-temperature conditions. Suboptimal temperatures present the primary challenge for pepper cultivation during winter and spring, with temperatures below 10 °C causing water loss, wilting, and slow leaf growth, and plant growth ceasing at temperatures below 5 °C [18].

Global crop productivity is under unprecedented threat due to climate change, as high temperatures negatively impact plant growth and metabolism. Because plants are immobile, they have developed intricate signaling networks that enable them to detect changes in ambient temperature, triggering a series of molecular changes that support plant survival and reproduction under adverse conditions. Understanding these mechanisms is crucial, as it could lead to the development of molecular markers that could eventually be used to breed thermotolerant crop varieties. Therefore, this review seeks to explore the genetic and epigenetic foundations for managing extremely high and low temperatures in C. annuum L. In this review, the molecular processes of C. annuum L. plants in response to heat stress from the sensing of HS, the subsequent molecular cascades associated with the activation of heat shock factors (HSFs), and their primary targets, heat shock proteins (HSPs), to the cellular responses have been summarized with an emphasis on the classification and functions of HSPs, with a special focus on HSFs for the protein expressions.

2. Mechanisms Involved in the Perception and Response to HS

Climate change, characterized by long-term alterations in temperature and weather patterns, is increasingly recognized as one of the most pressing global challenges. The manifestations of these changes are becoming more apparent, evidenced by extreme droughts, heatwaves, water scarcity, rising sea levels, flooding, polar ice melting, and declines in biodiversity. These disruptions are altering the distribution of wet and dry regions, with the frequency and intensity of extreme weather events escalating annually [19]. The implications for agriculture are particularly profound, as elevated temperatures and drought represent some of the most detrimental stressors to crop productivity. The impacts of high temperatures and droughts on plant growth and development are especially harmful to sexual reproduction, a critical phase in the plant life cycle that directly influences crop yield and population renewal [20]. However, climate-induced heat and drought stress disrupt key reproductive processes, including flowering, pollen development, pollination, and seed formation. For instance, major staple crops experience reduced pollen viability and significantly lower yields under heat stress, occasionally resulting in total crop failure [21]. HS has severely impacted the pepper plant during its germination period, reducing the emergence of both the root and shoot of newly emerged seedlings, which ultimately causes deformities and stunted vigor in the seedlings [14]. The elevated temperatures impact the plants in various ways, such as reduction in plant height, abnormal stem elongation, reduced leaf numbers, shortened roots, and finally, the overall yield and quality. Thermosensitive varieties are more susceptible to these environmental contingencies, facing heavier consequences as compared to the thermotolerant varieties (Figure 1). A reduced pollen activity is observed when the pepper plant undergoes HS, which results in the flower abortion and production of flowers with higher sterility [22] and affects fruit morphology [23]. Increased catalase activity induced by heat stress (HS) was observed in C. annuum compared to Arabidopsis when subjected to HS at varying temperatures (40 °C and 32 °C). Similarly, proline content was elevated in C. annuum relative to the control throughout the HS treatment. These findings indicate negative physiological effects of HS on C. annuum seedlings, as evidenced by compromised antioxidant capacity, biochemical parameters, and overall growth [24]. During HS, proline production is significantly induced, playing a crucial role in thermotolerance by maintaining turgor pressure and osmotic balance, ensuring membrane stability, preventing electrolyte leakage, and regulating reactive oxygen species (ROS) within optimal levels, thereby protecting plants from oxidative bursts caused by excessive ROS production [25]. Heat stress directly or indirectly impairs plant function, resulting in morphophysiological alterations, abnormal growth phases, and disrupted metabolic processes. HS significantly affects the phenological development of C. annuum, leading to early flowering, shortened vegetative growth phases, and reduced fruit set, which directly compromise productivity [26]. Elevated temperatures trigger excessive generation of ROS, including hydrogen peroxide and superoxide radicals, causing oxidative damage to membranes, proteins, and DNA. To counteract this, Capsicum activates enzymatic antioxidants such as superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) [27]. HS tends to affect the formation of fruits (fruit setting rate) by significantly impacting the production of number of leaves, which is reduced as compared to the normal conditions (33/21 °C day/night). Elevated temperatures (38/30 °C day/night) negatively impacted the weight of fruit production (52% decline), the fruit diameter (25% reduction), and the fruit length (30% reduction), which indicated HS consequences in the bell pepper, as observed by [26].

HS effects are not limited to growth stages, but highly negative correlations were found during pepper fruit development in which the fruit color, its nutritional composition, productivity, and the enrichment of capsaicin, as well as vitamin C, were significantly reduced [28]. Lower temperature also imparts some serious consequences, such as reduced nutritional quality, metabolic disproportions, impairments of organ anatomy and physiology, and a fairly reduced number of pollen production per flower, leading to stunted growth, disrupted development, and reduced productivity [29].

In severe cases, when the plant is exposed to HS for extended periods, it leads to the death of the plant, which poses significant economic consequences. Even during the storage period upon transporting the peppers, cold storage conditions (10 °C) may result in developmental impairments characterized by the formation of sepia-colored calyx and dental peel [30]. HS significantly affects both physical and biochemical processes in pepper plants. It alters the concentration of substances involved in osmoregulation and affects enzyme stability. HS also increases membrane lipid peroxidation and disrupts normal photosynthesis. Notably, reduced activity of photosystem II has been observed in C. annuum under both elevated and low temperature conditions [31,32].

3. Genetic Regulation of HS Response in C. annuum L

3.1. Heat Shock Proteins (HSPs)

C. annuum is favorably grown in warmer climatic regions where the ambient temperature is normally recorded more than 15 °C. Increased growth and productivity have been recorded when it is grown under optimal conditions. But on the other hand, a disturbed membrane stability is a serious consequence of the higher temperatures, which leads to disrupting the normal exosmosis [33,34]. Plants have evolved a variety of adaptive mechanisms to cope with these inadvertent changes brought by the HS by initiating the mechanism of action by encoding the HSPs, which play a pivotal role in acquiring thermotolerance during HS exposure [35]. Furthermore, the inception of HS leads to the acquisition of hormonal-assisted repairments such as ABA, Jasmonic acid, salicylic acid, and ethylene. The production of steroids responsible for increasing the thermotolerance is the main characteristic feature of the plants during the HSR [36]. Apart from these mechanisms, plants tend to produce specific proteins that play their part during HS to gain a recovery, and these proteins are target-specific and produced as a result of maintaining the transcriptional and translational modifications of HSP genes [37,38]. Both the overexpression and downregulation of such molecular chaperones are under the control of the transcription factors, which are termed HSFs [39].

HSPs play various important roles, as they act as a molecular chaperone, mainly responsible for maintaining protein homeostasis and repairment of the partially or fully impaired proteins during HS, thus acquiring the plant thermotolerance. Based on the varied molecular weights, HSPs are grouped into five categories, namely, HSP100s, HSP90s, HSP70s, HSP60s, and HSP20s [40,41]. HSP20s, which are usually termed as small HSPs (sHSPs) due to their lower molecular weights, i.e., 15 to 42 kDa, are considered highly conserved and are found to be the most prominently expressed when plant undergoes stress initiated by elevated temperatures [41]. Additionally, they can act as a molecular chaperone even in the absence of ATP to attain membrane stability and integrity, which indicates their significance in attaining the acquired thermotolerance [40,42].

3.1.1. Role of sHSPs in HS Responses

sHSPs possess a highly conserved molecular structure, which is characterized by their possession of the most conspicuous core conserved alpha-crystallin domain (ACD, the HSP20 domain), which comprises ~90 amino acids skirted by a dynamic N-terminal domain followed by a C-terminal extension [41]. It is observed that the ACD is responsible for recognizing the substrate exchanges. In contrast, the substrate binding is initiated by the important N-terminus. Finally, the process of homo-oligomeric substances and the production of HS-related granules is aided by the C-terminus [43]. A central functionality is assigned to HSPs20 during the recovery to acquire HSR in plants, specifically in the pepper (C. annuum L.) plant [44,45]. In research [46], HSPs were identified, and their expression was recorded in terms of abiotic stress [46]. Furthermore, researchers have studied the genomic anatomy, gene localization and duplication, phylogenetic connection, and their interconnected systems in C. annuum.

Various studies have examined the expression patterns for genes of CaHSP20s under HS conditions. For example, CaHSPs16.4 shows higher expression in thermotolerant lines compared to thermosensitive lines under HS, indicating its potential role in heat tolerance [42]. Transcriptomic analysis has also identified differentially expressed genes (DEGs), including several genes of CaHSP20s, in thermotolerant versus thermosensitive lines [47,48].

Furthermore, CaHSP40s, also known as CaDnaJ, hold a central place in plant growth and its HSR and are named due to their molecular weight (40 kDa). In another investigation [49], 76 putative pepper CaDnaJ genes were reported while employing bioinformatics approaches, and they were divided into five classes based on the possession of the complete J-domain, zinc finger domain, and C-terminus domain. The results of this study implied that these genes were responsible for pepper’s normal growth and development, and most significantly, they were induced by HS, as qRT-PCR data revealed their induction (80.6%), which is indicative of their central role in plant responses to HS exposure.

On the other hand, HSFs are regarded as a master regulatory network to initiate the downstream or upstream regulation of HS-related genes. They play a pivotal role in the transcriptional modifications of the HSPs, which are regarded as essential to acquire heat tolerance in C. annuum. The whole cascades start with the binding of the HS transcription factors (HSFs) to the HS elements (HSEs) on the promoter regions of heat-responsive genes, especially the HSPs, which trigger their expression during HS exposure [40,50]. Once HS is sensed by the plant, HSFs are mobilized toward the nucleus, where they activate the expression in HSPs. The HSPs, in turn, detect protein misfolding, protein aggregation, and denaturation due to their ubiquitous properties. The perception of HS is sensed by the HSPs indirectly, which triggers the recovery mechanism, which could be either a basal or acquired response, which enables the plants to attain thermotolerance and ensure their survival upon HS exposure [51]. Basal thermotolerance was observed in pepper plants assisted by transcription factor CaHDZ15, which was due to the activation of HSF, namely, A6A [52].

CaHSP24.2 has direct or indirect interactions with other members of genes of CaHSP20s (CaHSP17.8a, CaHSP17.8b, CaHSP18.1a, CaHSP18.1b, CaHSP18.2a, and CaHSP18.2b and CaHSP22.1, CaHSP23.8, and CaHSP26.5) and comparatively contains more intermingling genes from the other such members belonging to CaHSP20s family genes, with Ca03g01490 being the one that cooperated with CaHSP25.8. Additionally, CaHSP18.1a, CaHSP18.1b, CaHSP18.2a, and CaHSP18.2b genes have their interactions with other HS-related gene families termed as CaHSP70s genes, especially with Ca03g30260 and Ca01g31330. Contrastingly, CaHSP17.7a and CaHSP17.7b genes have strong interlinks with the genes mainly responsible for heavy metal stress, including mobilization of heavy metals and their detoxification, which is carried out by Ca02g24070, which is a superfamily gene. CaHSP16.3, on the other hand, has the closest interactions with the genes that are involved in the signal transduction of the calcium-binding protein gene named Ca10g18540. Finally, CaHSP25.9, along with a transcription factor that is a member of the MADS-box transcription factor family protein gene Ca00g74550, indicates its responsiveness in HSR in pepper plants. Overall, it is believed that genes of CaHSP20s are mainly involved in initiating HSR, starting from the sensing of HS signals from HSFs, initiating the HSR by activating HSPs, and controlling both the up- and downregulation of stress-associated genes in C. annuum [25,42,46,53]. The detailed role and the pattern of expression in HSPs are given in

Table 1. Breakdown of the role of HSPs in the HSR in pepper.

HSP Family	Symbol	Expression	Description	Reference
sHSPs	CaHSP16-4	Upregulated	A lessened production of reactive oxygen species (ROS) was associated with CaHSP16.4, which is produced during the HS and drought stress in C. annuum	[42]
	CaHSP18-2a	Upregulated	Interacted with heat stress-related genes to mitigate salt and heat-induced stresses.	[53]
	CaHSP18-7	Upregulated	Induces the ROS scavenging potentials of the C. annuum plants, which result in the alleviation of ROS production by interacting with antioxidant enzymes	[45]
	CaHSP21-2	Upregulated	Overexpression resulted in increased sensitivity to heat stress in leaves and roots.	[53]
	CaHSP22-0	Up/ Downregulated	Upstream and downstream regulations of CaHSP22 played a crucial role in acquiring thermosensitivity and salt stress tolerance by inducing their relative expressions	[54]
	CaHSP25-9	Upregulated	In transgenic Arabidopsis, the upregulation of CaHSP25.9 strengthened the HS tolerance to salt- and drought-related stresses.	[40]
CaHSP40s	CaDnaJ	Upregulated	The response of the genes was upregulated threefold, which resulted in a heat stress response.	[49]
CaHSP60s	CaHSP60-3	Downregulated	Under HS, the downstream expression was observed in heat-tolerant B6 and heat-sensitive R9 lines, which resulted in a profound HSR in pepper lines.	[55]
	CaHSP60-6	Knockdown	Knockdown of CaHSP60-6 resulted in an increase in acquired thermosensitivity when plants were exposed to HS.	[55]
CaHSP70s	CaHSP70-1	Upregulated	CaHsp70-1, as a member of the cytosolic Hsp70 subgroup, may be involved in HS defense response via a signal transduction pathway containing Ca2+, H2O2, and putrescine.	[46]
	CaHSP70-2	Upregulated	Slightly lowered expression of CaHSP70sgenes was observed under optimal conditions, but HS increased their expression by many folds, which indicated their profound impacts in combating heat-induced changes and their efficiency in acquiring thermotolerance in pepper.	[37]

.

3.1.2. Role of CaHSP60s in HS Responses

Being a molecular chaperone, HSP60s are involved in assisting protein homeostasis by carrying the protein folding and refolding [56]. Possession of a double-ring-like structure is a key advantage for such chaperonins, which is considered important to attain the proteins’ physiology and their mobilization capabilities [57]. The molecular chaperonins with a molecular weight of 60 kDa are known as HSP60s or are often termed as chaperonin (CPN60). A lot of research has been completed on the HSP60s gene family in bacteria, especially Escherichia coli, where scientists have described its shape and activity, yet little is known about this family in peppers. GroEL in prokaryotes becomes bound by ATP, making a complex with GroES, which has a barrel structure that aids in the correct folding of substrates. Once a protein is properly shaped thanks to ATP and GroES, it moves apart from GroEL and allows the protein to pop out of the cavity, while more ATP is consumed [56]. At the same time, there is not much public knowledge about C. annuum, which is regarded as a serious limitation in this field.

Researchers have identified 16 potential HSP60s genes in pepper using bioinformatics tools [55]. They discovered that silencing CaHSP60-6 increased sensitivity to heat shock, as evidenced by elevated relative electrolyte leakage, lipid peroxidation, and reactive oxygen species (ROS) accumulation in the silenced plants, along with significantly reduced chlorophyll content and antioxidant enzyme activity. These findings suggest that HSP60s may function as a positive regulator in pepper’s defense against heat and other abiotic stresses. Additionally, they observed that the expression of nine representative genes responded to other abiotic stresses (cold, NaCl, and mannitol) and hormonal treatments [ABA, methyl jasmonate (MeJA), and salicylic acid (SA)], with significant findings reported for CaHSP60s [55].

3.1.3. Role of CaHSP70s in HS Responses

HSP70s are the most abundant proteins when the plant goes under HS. These are highly conserved and can be found in all life forms. They hold a central position, as they act as a housekeeping protein, having a molecular weight of 70 kDa, with a characteristic heat shock cognate known as HSC70. The role of HSP70s members is regarded as essential for plants during both non-stress and HS conditions equally [58]. The CaHSP70-15 showed a higher level of expression when the flowers were treated with high temperatures. This increased expression suggested that CaHSP70-15, under normal conditions, may not play a part in developmental regulation but mainly acts as a molecular chaperone that protects the pepper flowers from heat shock.

HSP70s genes are responsible for encoding a highly conserved group of chaperone proteins throughout all life forms, encompassing bacteria, plants, and animals. Their dominant roles in different cellular processes are evident as highly conserved molecular chaperones that are provided by their association with other chaperones, such as sHPS. CaHSP70s may play its role during HSR by governing the hormone signal transduction pathways due to the possession of different stress and hormone signaling-related cis-elements in its promoter regions [37,43,59]. Additionally, the highly conserved sequences of the CaM-binding site are found in some CaHSP70s. It is inferred that CaHSP70s may be involved in calcium signal transduction. In previous research, Ca²⁺ and calmodulin proteins have been reported to be involved in the HS response in Arabidopsis. Nonetheless, knowledge concerning the effect of HS and Ca²⁺ on the regulation of the plant HSP70s family remains very limited.

Plant HSP70s genes were responsible for molecular chaperone activity under HS [60], and overexpression of some HSP70s genes increased tolerance of transgenic plants to HS; however, the cellular mechanisms of thermoprotection by HSP70s are not fully understood [61]. A total of 21 CaHSP70s genes were identified in pepper, as previously reported [37]. Their results revealed that the overexpression of CaHSP70-2 enhanced the thermotolerance of transgenic Arabidopsis plants. In another study, the expression of CaHSP70-1 induced by CaCl₂, H₂O₂, and putrescine (Put) under HS was different between B6 and R9 lines [45]. The different expression patterns may be related to the differences in promoters of CaHSP70-1 from the two lines. These results suggest that CaHSP70-1, as a member of the cytosolic HSP70s subgroup, may be involved in the HS defense response via a signal transduction pathway containing Ca²⁺, H₂O_2, and Put. Another study focused on assessing the mechanism of heat tolerance in chili pepper and evaluated six genotypes for cellular membrane thermostability (CMT) and HSP70s gene expression [62]. Under HS, significantly increased levels of the CaHSP70s gene were detected after 2 h of temperature treatment at 42 °C, which indicated that this gene is quickly and sharply induced by heat shock. The elevated levels of both ABA and H₂O₂ lead to an enhanced migration of ABA from roots, which elevates the synthesis of ABA in the shoots during HS conditions. Inhibition of H₂O₂ accumulation compromises HSP70 expression and reduces the heat tolerance. These results suggest that, under heat stress, ABA triggers the expression of HSP70 in an apoplastic H₂O₂-dependent manner, incriminating the title role of an ABA-dependent H₂O₂-driven mechanism in a systemic response involving root–shoot communication [63].

3.1.4. Role of CaHSP90s in HS Responses

HSP90s are also highly conserved in molecular evolution, responsible for the regulation and maintenance of regular structural conformations of proteins of various kinds. They also play an important role in plant survival via assisting the normal cell growth when they undergo HS. In fungi and animals, HSP90s mediate extensive stress signal transduction, including a role in folding of steroid hormone receptors, protein kinases, and transcription factors, as well as activation of the substrate to initiate stress signal transduction [64].

HSP90s are crucial molecular chaperones that hold a key position in cellular responses to different environmental stresses. They are involved in protein folding, refolding, and degradation, protecting cells from stress-induced damage [65]. Specifically, HSP90s acts as a regulator of HSFs, and HSP90s interacts with HSFs, which are responsible for inducing the expression of heat-inducible genes. Inhibiting HSP90s can induce heat-inducible genes and heat acclimation. HSPs, including HSP90s, protect plants from oxidative stress induced by abiotic and biotic stresses by maintaining the structure and activity of proteins in the antioxidant system. Under HS, HSP90s and other HSPs bind to damaged proteins, helping to refold or degrade them [66].

When cells encounter stressors such as elevated temperatures, HSP90 expression increases. This increased expression is part of the heat shock response, leading to cellular protection. While HSP90 is well known for its role in the heat stress response, it is also involved in cold stress responses. For example, in Cucurbita moschata (Chinese pumpkin), the expression patterns of CmoHSP90s genes vary under heat and cold stresses, indicating their involvement in temperature stress [66]. While several papers discuss HSP90s in general stress responses in plants and other organisms, there is less specific information about C. annuum L. in the provided papers. However, the general principles of HSP90s function would likely apply. For example, HSP90s would be expected to play a role in helping C. annuum L. tolerate both heat and cold stress, contributing to overall stress resistance [65]. During HS, HSP90s would back the plants in maintaining stability and folding of stress-related cellular proteins, preventing aggregation and ensuring proper function. HSP90s likely interacts with heat HSFs in C. annuum L., regulating the expression of other heat shock genes [67].

3.1.5. Role of HSP100s in HS Responses

The caseinolytic proteinase/heat shock protein 100 (Clp/HSP100s) proteins belong to the AAA+ protein group (ATPases associated with various cellular activities) that are involved in the protein dismantling courtesy of the energy provided by the hydrolysis of adenosine triphosphate (ATP) [68]. Compared with other HSPs, which are involved in protein hemostasis in different approaches, HSP100s are specified for the elimination of non-functional proteins. They also help the plants in the assembly of the proteins, which are denatured during the HS, by using the aggregated protein complexes. HSP100’s role in maintaining protein homeostasis in plants has been explicated [69]. There is little known about the expression pattern and molecular phenomenon involved in HSR as provided by the HSP100s in C. annuum. Future studies can focus on finding the expression pattern and molecular changes at the cellular level in HSR in C. annuum.

3.1.6. Functional Hierarchy of HSPs

In response to heat stress, the four primary HSP families in C. annuum function as a cohesive molecular triage system, rescuing, refolding, or degrading heat-sensitive proteins. HSP100/ClpB proteins, found in the stroma and cytosol, untangle protein aggregates using ATP; transcriptomic meta-analysis indicates a 6- to 12-fold increase in expression within 30 min of exposure to 42 °C, with the highest levels in young leaves where photosynthetic enzymes are most at risk [70]. HSP90 operates in two distinct functional groups: cytosolic HSP90.1-3 keeps signaling kinases (such as MAPKs and CaCDPKs) in a state ready for folding, while plastidic HSP90 temporarily interacts with Rubisco activase and the PS II repair complex, accounting for the continued CO₂ assimilation seen in heat-tolerant cultivars [31]. Members of the HSP70 family (CaHSP70-1 to -5) act as a central hub linking stress-granule dynamics and translational control; cross-linking mass spectrometry shows that CaHSP70-3 directly binds eIF4F and PABP, thereby suppressing global translation while selectively stabilizing HSF and HSP mRNAs. Small HSPs (sHSPs, particularly CaHSP17.6 and CaHSP21) form 24-mer “holdase” complexes that prevent irreversible aggregation of thylakoid and mitochondrial proteins; loss-of-function VIGS lines exhibit a 45% increase in lipid peroxidation, confirming their antioxidant-like function [20]. Crucially, these activities are interconnected: HSP70s delivers partially unfolded proteins to HSP90s for ATP-dependent refolding, and severely damaged proteins are transferred to HSP100s for disaggregation or proteasomal degradation. The entire network is regulated by CaHSFs through feedback loops: CaHSFA2 upregulates HSP70s and HSP90s within 15 min, while CaHSFA1d and CaHSFC1 maintain HSP100s and sHSPs expression during extended stress. Consequently, the HSP system in pepper acts as a hierarchical, multi-compartmental chaperone network, with its spatial and temporal coordination determining the extent and duration of thermotolerance [66,67,69].

3.2. Heat Shock Factors (HSFs)

HSFs play a pivotal role during the different developmental stages, as they aid the plants in defending themselves from the varying environmental conditions. The production and quality of C. annuum L., being an economically important vegetable crop, are not exceptions to the adverse effects of different abiotic stresses, which have sternly abridged its productivity. There has been tremendous research in the field of exploring the genomic sequencing of pepper, but the characterization of HSFs has not yet been thoroughly studied. The expression of the sHSP family members can be higher during the HS. It has been well documented that description of possible mechanisms of HSP genes are governed by HSFs and heat stress elements (HSEs). HSFs in plant intracellular include HSFA1 and HSFA2, which can bind to HSEs with their N-terminal DNA binding domain (DBD). The expression of sHSPs results from the complex associations between HSFs and sHSPs, which are crucial for maintaining cellular integrity in pepper. HSEs are located upstream of the HSPs sequence. A previous study showed that HSFs can be classified into three categories detected by CRISPR/Cas genome editing technology: HSFA, B, and C, which are distinguished from each other in their functions [71]. In the HSFs, HSFA is largely regulated by the sHSP cycle (HSFA1 represented as the master regulator in the transcription of sHSPs). HSFA2 not only contributed to the transcription of HSPs but also significantly maintained the expression of sHSP genes in plants [55].

HSFs are transcription factors that play a central role in regulating gene expression under stress conditions, particularly HS. Under normal conditions, HSFs exist as inactive monomers in the cytoplasm [72]. Upon exposure to heat stress, proteins begin to unfold and aggregate. This triggers the activation of HSFs, leading to their trimerization, translocation to the nucleus, and binding to HSEs in the promoter regions of heat shock genes. This binding initiates the transcription of HSPs and other stress-related genes [72,73]. The whole cascade, starting from the leakage of Ca²⁺ and H₂O₂, and a possible description, has been shown in Figure 2.

In [74], the authors have identified 25 CaHSFs in pepper genomics through PCR assays and bioinformatics tools. The HSFs can be categorized into three distinct categories based on the number of amino acids they possess on their respective domains, i.e., HR-A/B domains [75]. Class HSFsA also have the aromatic, hydrophobic, and acidic (AHA) domain at the C-terminus, but class HSFsB and HSFsC do not contain these domains. Thus, HSFsA becomes a transcriptional activator serving to regulate the heat stress response in plants due to the presence of the AHA domain [76,77]. Unlike HSFsA, HSFsB does not contain AHA domains, resulting in their lack of transcriptional activity. A repressor domain (RD) is located at the C-terminus of the HSFB protein, and it is speculated that it functions as a repressor motif, so that HSFB members can act as repressors [53]. HSFC may play an active role in regulating plant heat tolerance, and its positive effect may be related to the induction or upregulation of heat-resistant genes [78].

3.2.1. CaHSFsA

CaHSFA1d from C. annuum L. has been identified as a key regulator of the heat stress response, enhancing plant thermotolerance by modulating the expression of stress and antioxidant-related genes. CaHSFA1d is a member of the heat shock transcription factor family and plays a crucial role in the HSR mechanism. It functions as a master regulator, orchestrating the expression of various HSPs and other stress-responsive genes. The activation of CaHSFA1d under HS leads to the transcriptional induction of HSPs, which are essential for maintaining protein homeostasis and preventing protein aggregation. Additionally, CaHSFA1d regulates the expression of antioxidant-related genes, which help in scavenging ROS generated during HS, thereby reducing oxidative damage. Figure 3 explains the recovery and possible positive outcomes of this molecular understanding for the thermosensitive behavior of C. annuum. Under normal conditions, CaHSFA1d is inactive, often bound to molecular chaperones such as HSP90s. Upon exposure to HS, the increased levels of misfolded proteins dissociate these chaperones from CaHSFA1d, allowing it to form homotrimers and move to the nucleus. In the nucleus, CaHSFA1d binds to HSEs in the promoters of target genes, including HSPs and antioxidant-related genes, thereby inducing their expression.

Recently, the role of CaHSFA1d in HS response has been illustrated [79], and it was revealed that this gene was found to be overexpressed during HS. Furthermore, researchers observed the greater sensitivity of its expression in R9 plants, which is a heat-tolerant pepper line, as compared with a B6 line (thermosensitive). The physiology of CaHSFA1d under heat stress was considered in the pepper plants with silenced CaHSFA1d and the Arabidopsis plants with overexpressed CaHSFA1d. They observed a reduced thermotolerance in pepper with silenced CaHSFA1d, while overexpressing CaHSFA1d in Arabidopsis led to an increased insensitivity to elevated temperatures. Furthermore, an increase in H₂O₂ balance as a result of HS was attained by the plants and increased expression of both the HSFs and HSPs, along with an antioxidant gene AtGSTU5 (glutathione S-transferase class tau 5), which was observed in the transgenic pepper lines.

Previously, researchers have identified 25 CaHSFs subfamilies through PCR assays and advanced bioinformatics tools in the pepper genome [53]. They found that the expression of HSFA2 is significantly induced by HS, ranking just below CaHSFA3 in the thermotolerant line R9 and CaHSFA1d in the thermosensitive line B6, suggesting a potentially dominant role similar to HSFA2 in Arabidopsis and tomato. Notably, CaHSFA2 expression is higher in R9 than in B6 under HS, implying its involvement in the differential thermotolerance of the two lines. However, its expression in R9 declines after 4 h of HS, and in B6, it is slightly downregulated by the end of the treatment, indicating a transient response. This pattern may be influenced by regulatory factors such as the circadian clock, feedback inhibition through interaction with HSP17-2, and repression by HSFB2b. These findings suggest that CaHSFA2 is tightly regulated by both heat stress and internal rhythms, and further investigation is needed to elucidate its full regulatory mechanism.

3.2.2. CaHSFsB

Class B HSFs contain neither a nuclear export signal nor an activation domain, which are found in class A HSFs [80]. HSFB1, HSFB2a, and HSFB2b are HS-inducible genes [81]. CaHSFB2a is a nuclear-localized heat shock factor that positively regulates the response to both Ralstonia solanacearum infection and high temperature–high humidity (HTHH) environments in C. annuum [82]. One study revealed that HSFB2a forms a transcriptional cascade with CaWRKY6 and CaWRKY40, two WRKY transcription factors known for their roles in plant immunity and stress responses. This interaction suggested that HSFB2a played a crucial role in regulating the expression of defense-related genes, thereby enhancing the plant’s resistance to R. solanacearum and tolerance to HTHH. The findings indicate that HSFB2a expression is upregulated under stress conditions, and silencing of HSFB2a using virus-induced gene silencing (VIGS) results in reduced resistance to R. solanacearum and decreased tolerance to HTHH, highlighting its importance in these responses. This study underscores the potential of HSFB2a in improving pepper plant immunity and stress tolerance, which could be leveraged for developing more resilient pepper varieties [82].

The implications of this study are significant for both plant biology and agricultural practices. The findings suggest that HSFB2a could be a key regulator in enhancing pepper plant immunity against R. solanacearum, a pathogen that poses a significant threat to pepper production. This could lead to the development of more resistant pepper varieties through genetic engineering or breeding programs, thereby reducing crop losses and improving food security. Additionally, the role of HSFB2a in HTHH tolerance indicates its potential for improving pepper plant resilience in high-temperature and high-humidity environments, which are common in many pepper-growing regions. Understanding the complex transcriptional network involving HSFB2a, CaWRKY6, and CaWRKY40 could provide valuable insights into the molecular mechanisms underlying plant stress responses and immunity. Future research could focus on elucidating the specific molecular interactions between HSFB2a and the WRKY transcription factors, as well as identifying additional components of the transcriptional cascade. This could lead to the development of new strategies for improving pepper plant resistance and tolerance to multiple stresses, ultimately contributing to more sustainable and productive agricultural systems [82].

3.2.3. CaHSFs

A comprehensive genome-wide analysis of the CaHSF gene family has identified multiple members, each with distinct structural features and expression patterns. The expression profile of the CaHSF gene family under various stress conditions, including HS, revealed that several genes, such as CaHSFA2 and CaHSFC1, are significantly upregulated [74]. This upregulation suggests that these genes play crucial roles in the HSR pathway. The overexpression of CaHSFC1 in transgenic plants has been shown to enhance thermotolerance, highlighting its critical role in HSR.

CaHSFC1 is another important member of the CaHSF gene family that has been implicated in the heat stress response. Similar to CaHSFA2, CaHSFC1 is upregulated under HS conditions, indicating its involvement in the HSR pathway. The specific role of CaHSFC1 includes the regulation of various stress-responsive genes, including those encoding HSPs and antioxidant enzymes. These genes are crucial for maintaining protein homeostasis and reducing oxidative damage under HS. CaHSFC1 likely works in coordination with other HSFs, such as CaHSFA2, to orchestrate a comprehensive heat stress response [83]. This coordinated regulation ensures the induction of multiple plants of the Solanaceae family, enhancing the plant’s overall thermotolerance. The upregulation of CaHSFC1 under heat stress contributes to the plant’s ability to withstand high temperatures by regulating the expression of stress-responsive genes, thereby maintaining cellular integrity and reducing heat-induced damage. Understanding the molecular mechanisms and regulatory networks involving CaHSFC1 and other HSFs provides valuable insights into the HSR pathway and offers potential strategies for developing heat-tolerant crop varieties through genetic engineering. The detailed description of HSFs in pepper has been presented in

Table 2. Heat shock factors and their roles in thermotolerance of C. annuum.

HSF Family	Symbol	Expression	Description	Reference
CaHSFA	CaHSFA1d	Up-/ Downregulated	CaHSFA1d silencing in pepper lines resulted in reduced thermotolerance. CaHSFA1d overexpression led to an increased insensitivity to the elevated temperatures in Arabidopsis.	[85]
	CaHSFA2	Upregulated	CaHSFA2 activates the heat stress response pathway, leading to the induction of heat shock proteins (HSPs) that help maintain protein homeostasis and prevent protein aggregation.	[74,86]
CaHSFB	CaHSFB2a	Upregulated	CaHSFB2a overexpression positively regulates the response to Ralstonia solanacearum infection or high temperature and high humidity, forming a transcriptional cascade with CaWRKY6 and CaWRKY40.	[82]

. Future research should focus on elucidating the specific downstream targets of CaHSFC1 and exploring its interactions with other regulatory proteins to further enhance our understanding of the heat stress response in pepper plants [84].

3.3. Genome-Wide Survey of HSPs/HSFs in C. annuum

Presently, the scientific advancements have enabled our access to a wide range of whole-genome sequences of plants, which helps determine their evolutionary perspectives, roles in plant forms and functions, and diversity of HSFs and HSPs. In C. annuum, a total of 35 putative pepper genes of HSP20s (CaHSP20s) were identified and renamed in [53], based on their molecular weight. Furthermore, 35 HSPs with 38 HSFs were identified to play a substantial role in HS recovery [48]. A total of 35 sHSPs [53], 16 CaHSPs60 [55], and 21 CaHSPs [37] have been reported for C. annuum. Furthermore, there are less data available in the literature about the CaHSP90s and CaHSP100s. Additionally, 25 HSFs have been previously reported [74]. In rice, 25 HSF, 29 sHSPs, 26 HSP70s, 9 HSP90s, and 10 HSP100s family genes were identified [87].

3.4. Comparative Molecular Cascades in Other Solanaceous Plants

Solanaceous plants, of the Solanaceae family, are widely cultivated throughout the globe due to their high nutritional and ornamental value. They have been commonly utilized for health, research, development, and human consumption. The most important economic solanaceous plants include tomato (S. lycopersicum), pepper (C. annuum), potato (S. tuberosum), and eggplant (S. melongena) [88]. The genomic sequencing has been provided for these plants in various studies in response to HS [84,89,90,91].

3.4.1. Molecular Basis of HSR in Potato

Potato (S. tuberosum) is regarded as the top non-grain staple crop, which is preferably grown in temperate climate areas with an optimal temperature range of 20–25 °C [92]. When the temperature exceeds its optimum value for potatoes, the tuberization process stops, and the plants exhibit reduced levels of photoassimilate partitioning in tubers, resulting in low tuber yield, thus affecting the global productivity of the potato [93,94]. In a previous investigation 946], the researchers exposed the potatoes to high temperature (35 °C) for 72 h and observed that 1420 genes were differentially expressed, with 49 different GO types, as revealed by the Gene Ontology analysis. These findings reveal the elevated functionality of the plant’s inherent capabilities in response to HS. They also reported that HSFs and HSPs were not significantly expressed, except StHSP26-CP and StHSP70, which showed a marked increase in expression after 72 h heat treatment. In another study [89], 19 GO enrichment terms and 12 enriched KEGG pathways, along with 1201 differentially expressed genes (DEGs), were reported in potato tubers under HS conditions. The authors of [95] treated the potato tubers with high temperature (37 °C) and observed that there was an increased expression of HSP-related genes during the stress, especially the sHSPs. Overall, these data can bring fruitful outcomes for our better understanding in terms of unravelling the molecular cascades of HSR and developing heat-resilient varieties for better agricultural practices and food security.

3.4.2. Molecular Basis of HSR in Tomato

Tomato (S. lycopersicum) is another economically important crop among the food commodities list, and it also enjoys wide cultivation throughout the globe. Despite its huge annual production, the environmental fluctuations continuously threaten the overall productivity of this crop; especially, losses caused by high temperatures are at the top of the list in abiotic stresses [96]. There have been various approaches to identifying the transcriptomic profiling of heat stress in tomato cultivars. In an investigation [97], the authors have reported a loss in fruit quality and the overall yield of the pepper plant exposed to high temperatures. They have further identified that there were a total of 13 genes that were significantly expressed during the HS period, of which 2 were HSFs, 9 were HSPs, and the remaining 2 were GDSL esterase/lipase (GELPs). They concluded that HSEs’ interactions with the HSEs led to the expression of HSPs, which enabled the plant to acquire thermotolerance. A study has provided the molecular basis of HSR when the tomato lines were exposed to 38 °C for 72 h [98]. The authors have provided the information about the overexpressed proteins, i.e., 27 overexpressed proteins in L0994 and 17 in L6138, with DEGs. A study [99] reported the transcriptomic profiling of tomato, which was given a high temperature treatment (42 °C) for 6 h. The authors identified 4696 genes (2059 upregulated and 2637 downregulated) that were differentially expressed. They also reported that HSPs such as HSP17.6 A, HSP21, and HSP70s exhibited enhanced expression during in vitro experimentation.

3.4.3. Molecular Basis of HSR in Eggplant

Eggplant (Solanum melongena) is no exception from the threats posed by the fluctuating environmental temperatures. Its overall productivity has been significantly reduced during the past few years. It enjoys a global cultivation (1.846 million hectares in 2020) but is favorably grown in most parts of India and China [100,101]. It shows its maximum growth at an optimum temperature range of 22–25 °C. Another investigation has tried to provide the molecular basis for alleviating the impacts caused by HS in eggplants [100]. The authors identified 23 candidate genes that participated in HSR. Furthermore, they have also provided a detailed mechanism of action and expression patterns of these DEGs through (qRTPCR). They conducted yeast experiments, which showed that HSP70, MYB44, AFP2, HSFA8, EGY3, HSFA3, and TIFY6B were involved in the HSR of eggplants. The in vivo experimentation utilized RNA-sequencing approaches to study the transcriptomic profiling of eggplant as a result of heat-induced stress [102]. This study reported that 3067 genes showed a differential expression pattern. A total of 315 genes were found to be overexpressed, while 342 genes showed a downregulation during the HS treatment.

3.5. Molecular Mechanisms of HS Response (HSR)

For organisms to grow and function properly, they must maintain specific internal cellular conditions that allow proteins to acquire their functional conformations and cells to achieve protein homeostasis (proteostasis). Maintaining proteostasis becomes critical when facing abrupt changes in the external conditions, such as an increase in temperature, which can lead to protein misfolding and aggregation, and cellular dysfunction [103]. Thus, organisms must sense, rapidly respond, and adapt to new environmental conditions for survival. The HSR is defined as a transient reprogramming of the gene expression of a conserved response of cells and organisms to elevated temperatures. The induction temperature for the HSR is normally 5–10 °C above the normal optimum growth temperature for that particular species. It has recently been reported that Ca²⁺ plays important roles in the perception, response, and adaptation of plants to HS [104,105]. The alteration of fluidity in the plasma membrane (PM) in plants in response to HS can open cyclic nucleotide-gated calcium channels (CNGCs) controlled by nucleotide cyclases, thereby allowing Ca²⁺ to move into the cytosol from the PM [106]. The Ca²⁺ ions are associated with protein calmodulin 3 (CaM₃) during HS, and the complex of Ca²⁺–CaM₃ interacts with calcium/calmodulin-binding protein kinase 3 (CBK₃) and phosphatase PP₇ to transduce cytosol HSR signals into the nucleus by modulating phosphorylation and dephosphorylation of HSFA1, respectively [105,107]. The features of this response include the induction of HSPs synthesis and the subsequent acquisition of a higher level of thermotolerance [108].

3.6. Understanding the HSR in Pepper: The Role of HSFs

HSFs are pivotal in the HSR of pepper (C. annuum L.) and other plants. Under non-stress conditions, HSFs are kept inactive through association with molecular chaperones such as HSP90s. However, when peppers are exposed to high temperatures, this equilibrium shifts. The accumulation of misfolded proteins due to HS leads to the release of HSFs from their chaperones. Subsequently, HSFs trimerize and translocate to the nucleus, where they bind to HSEs on the DNA, initiating the transcription of stress-responsive genes. This rapid gene expression cascade results in the synthesis of HSPs, which function as molecular chaperones to mitigate the effects of heat stress by preventing protein aggregation and assisting in the refolding of denatured proteins [47,74,79,109]. The whole cascade, involving the sensation of HS signals by the HSFs, their trimerization, and initiation of the HSPs, along with the recovery of misfolded and denatured proteins, is exhibited in Figure 4.

3.6.1. The Interplay of Translation Factors and Stress Granules

The process of protein synthesis is also significantly affected during heat stress. Co-translational protein folding, facilitated by eIF2, eIF4F, and HSP70s/HSC70 complexes under normal conditions, is disrupted [15]. Stress granules form as a cellular response to the overwhelming number of misfolded proteins. These granules sequester translation initiation factors, effectively pausing protein synthesis and reducing the production of additional misfolded proteins that could exacerbate cellular stress [110]. The formation of stress granules is a double-edged sword; while they prevent further protein misfolding, they also represent sites of potential protein aggregation that must be addressed during recovery [111].

3.6.2. Epigenetic Regulation in the HSR of Pepper

The epigenetic landscape plays a subtle but significant role in the HSR of pepper. Epigenetic modifications, such as histone acetylation and DNA methylation, can influence the chromatin state around stress-responsive genes, thereby affecting their expression. For instance, the binding of HSFs to HSEs can recruit histone acetyltransferases (HATs), which acetylate histones and promote a more open chromatin structure conducive to transcription [112]. Conversely, histone deacetylases (HDACs) can remove these acetyl groups, leading to chromatin compaction and gene silencing. These dynamic epigenetic changes are crucial for modulating the expression of stress genes in a controlled manner, ensuring that the plant’s response is both robust and reversible [24].

3.6.3. Recovery Mechanisms and the Return to Homeostasis

In nature, when plants are subjected to HS, their ability to recover is important, as the stronger the ability to recover, the faster the plant can restore its metabolic balance and maintain its normal growth. The regulatory molecular mechanisms and networks of pepper have been reported in [48]. Recovery from HSR involves a series of orchestrated events aimed at clearing protein aggregates and resuming normal cellular functions. HSP70s aids in the formation of stress granules during stress and also plays a critical role in their dissolution once the stress is alleviated. This allows for the resumption of translation and the clearance of protein aggregates. Additionally, the decay of HSP70s mRNA and the sequestration of HSFA1 by HSP70s contribute to the downregulation of stress-responsive genes, facilitating the return to a non-stress state [25].

Constitutive chaperones also assist in protein folding and preserving protein homeostasis. Under physiological conditions, non-stress-regulated genes are transcribed, and their mRNAs undergo canonical cap-dependent translation. Exposure to heat stress induces protein misfolding, which titrates out the HSC70/HSP90s and allows HSF1 to trimerize and translocate to the nucleus, where it binds to the HSEs in the promoter of HSPs and activates transcription. Concomitant with the HSR activation, there is a global transcriptional and translational repression, as shown in Figure 5. The skipping of translational repression in inducible HSP mRNAs, especially HSP70s, is met by a cap-independent mechanism, which increases the number of available chaperones required for coping with the plentiful misfolded proteins and averting their toxic accumulation. When the temperature returns to the optimal levels, the newly synthesized HSPs support the recovery mechanisms of protein homeostasis and revive the functionality by folding the misfolded proteins and disabling the co-chaperone suppressor of G2 allele (SGs) (Figure 5). SGs are responsible for the programmed folding and misfolding of proteins during the HS. The recommencement of regular translational and transcription modifications accords with the decay of HSPs’ mRNAs and leads to the silencing of their transcription [25].

4. Conclusions and Future Implications

The 21st century has witnessed remarkable progress in analyzing gene expression and signaling pathways related to stress responses, as well as understanding the broader landscape of hormone regulation. These advancements, underpinned by genomic information, have provided clarity regarding the overarching control systems regulating responses to drought and high-temperature stress. As the relentless progression of global warming continues, the research focus is shifting toward the genetic and epigenetic understanding of plants’ environmental stress responses under real-world field conditions and intricate ecosystems. The study of heat stress responses in C. annuum L. continues to evolve, with research focused on improving heat tolerance through genetic manipulation of HSP and HSF pathways. Heat stress induces negative physiological changes. Challenges include the integration of these molecular responses with plant growth and development, particularly under variable climatic conditions. Breeding strategies and biotechnological approaches are being explored to enhance the thermotolerance in C. annuum, ensuring stable crop yield in the face of rising global temperatures. The molecular mechanisms underlying HSR in C. annuum L. are orchestrated by a network of HSPs and HSFs that provide essential protection against heat-induced damage. The regulation of these stress-response pathways is critical for plant survival, productivity, and adaptation to global warming scenarios. There is limited research on the molecular mechanisms and the expression pattern of CaHSP90s and CaHSP100s, which is a serious constraint to understanding the genetic and epigenetic basis of HSR completely. Furthermore, the undiscovered families and the members of HSFs needed to be explored to fully understand the process involved in HSR. The future studies can focus on conducting in vivo experimentation and further validations of the results and generally apply these mechanisms to fully explore the research gap. Continued research in this area promises advancements in agricultural practices, contributing to food security in increasingly unpredictable environments.