# Heat stress transcription factors as the central molecular rheostat to optimize plant survival and recovery from heat stress

Ayat Bakery, Stavros Vraggalas, Boushra Shalha, Harsh Chauhan, Moussa Benhamed, Sotirios Fragkostefanakis

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Ayat Bakery, Stavros Vraggalas, Boushra Shalha, Harsh Chauhan, Moussa Benhamed, et al.. Heat stress transcription factors as the central molecular rheostat to optimize plant survival and recovery from heat stress. New Phytologist, 2024, 244 (1), pp.51-64. 10.1111/nph.20017. hal-05051966

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Research review

Heat stress transcription factors as the central molecular rheostat to
optimize plant survival and recovery from heat stress

Author for correspondence:

Sotirios Fragkostefanakis

Email: fragkost@bio.uni-frankfurt.de

Received: 1 April 2024

Accepted: 5 July 2024

Ayat Bakery<fnref n="1" />,²* ID, Stavros Vraggalas<fnref n="1" />* ID, Boushra Shalha<fnref n="1" />* ID,
Harsh Chauhan<fnref n="1" />,³ ID, Moussa Benhamed⁴,⁵,⁶ ID and
Sotirios Fragkostefanakis<fnref n="1" /> ID

New Phytologist (2024) 244: 51–64

doi: 10.1111/nph.20017

**Key words:** acclimation, heat stress response, heat stress transcription factor, recovery, survival, thermotolerance, transcription.

Summary

Heat stress transcription factors (HSFs) are the core regulators of the heat stress (HS) response in plants. HSFs are considered as a molecular rheostat: their activities define the response intensity, incorporating information about the environmental temperature through a network of partner proteins. A prompted activation of HSFs is required for survival, for example the *de novo* synthesis of heat shock proteins. Furthermore, a timely attenuation of the stress response is necessary for the restoration of cellular functions and recovery from stress. In an ever-changing environment, the balance between thermotolerance and developmental processes such as reproductive fitness highlights the importance of a tightly tuned response. In many cases, the response is described as an ON/OFF mode, while in reality, it is very dynamic. This review compiles recent findings to update existing models about the HSF-regulated HS response and address two timely questions: How do plants adjust the intensity of cellular HS response corresponding to the temperature they experience? How does this adjustment contribute to the fine-tuning of the HS and developmental networks? Understanding these processes is crucial not only for enhancing our basic understanding of plant biology but also for developing strategies to improve crop resilience and productivity under stressful conditions.

**I. Introduction**

Organisms experience heat stress (HS) when temperature elevations surpass a threshold that leads to an overall negative effect on the physiological functioning, manifested in reduced photosynthesis, reproductive success and growth, or even death in severe cases (Wahid et al., 2007). In nature, HS commonly occurs during the warmer seasons of the year and in its most severe form as heat waves. As climate scenario projections forecast a higher frequency and severity of heat waves, the agriculture sector is predicted to feel the

negative consequences and the food security to be in peril
(IPCC, 2023).

The temperature threshold that causes HS and the resilience capacity can significantly vary among different species and even strains of a species and are dependent on the environmental niche and the genetic makeup of the organism that has been shaped by evolutionary adaptations (Hatfield & Prueger, 2015). In addition, the physiological stress and the intensity of the response to the stress are dependent on the magnitude and the duration of the thermal extreme, which can also be influenced by the developmental stage and the physiological status of the plant (Bokszczanin et al., 2013). While the genetic component is a major determinant for resilience to HS, acclimation to high temperatures by a previous exposure to mild stress can buffer the physiological response and improve stress resilience (Larkindale & Vierling, 2007).

<footnote n="1">¹Institute of Molecular Biosciences, Plant Cell and Molecular Biology, Goethe University Frankfurt, 60438, Frankfurt am Main, Germany; ²Botany Department, Faculty of Science, Ain Shams University, 11517, Cairo, Egypt; ³Department of Biosciences and Bioengineering, Indian Institute of Technology Roorkee, Roorkee, 247 667, Uttarakhand, India; ⁴Université de Paris Cité, Institute of Plant Sciences Paris-Saclay (IPS2), F-91190, Gif-sur-Yvette, France; ⁵Université Paris-Saclay, CNRS, INRAE, Univ Evry, Institute of Plant Sciences Paris-Saclay (IPS2), Orsay, 91405, France; ⁶Institut Universitaire de France (IUF), Orsay, 91405, France</footnote>

<footnote>*These authors contributed equally to this work.
[Correction added on 05 September 2024, after first online publication: the spelling of Harsh Chauhan has been corrected in the author list.]</footnote>
A common problem that all cells face under HS is proteotoxicity, caused by protein misfolding and denaturation (Mattoo & Goloubinoff, 2014). Therefore, a key characteristic of thermotolerant cells is their ability to synthesize molecular chaperones such as many heat shock proteins (HSPs) as a perquisite for survival. The induction of many HSPs is only a part of the so-called HS response (HSR): a global transcriptome reprogramming that beyond the protection of protein homeostasis aids the scavenging of deleterious chemical species, the protection of housekeeping functions and cellular structures and the conservation of energy resources. HSR includes the upregulation of thousands of genes which code for proteins that have a wide range of molecular functions including primary and secondary metabolism as well as hormone signalling pathways that are employed to ensure the survival and the recovery from HS (Liu & Charng, 2013; Li et al., 2020; Andrási et al., 2021; Olas et al., 2021; Huang et al., 2023).

The core regulators of HSR are the HS transcription factors (HSFs) (Nover et al., 2001; Scharf et al., 2012). HSFs serve as a molecular rheostat, regulating the intensity of the response by integrating environmental temperature cues into the plant and information about the cellular health status via a network of interacting proteins. The swift activation of HSFs is crucial for survival but a prompt attenuation is required to allow for the re-establishment of normal cellular activities, to facilitate recovery from stress and avoid an overproduction of proteins that might have negative effects on growth and development (Scharf et al., 2012).

## II. Plant HSF gene family

The HSF gene family in plants is larger than in other eukaryotes and exhibits a high degree of diversity. The expansion of plant HSF family has resulted from gene duplications and gene losses during evolution and diversification of land plants and might have conferred an evolutionary advantage for colonization in habitats, which require complex adaptation mechanisms (Scharf et al., 2012; Liao et al., 2022).

Heat stress transcription factors have a modular domain structure that includes a DNA binding domain (DBD) in the N-terminal region, followed by a leucine zipper-like coiled-coil oligomerization domain (OD) divided into two hydrophobic heptad repeat regions (HR-A/B) connected via a flexible linker (Nover et al., 2001; Scharf et al., 2012). Besides conserved sequence-specific features, but most obviously based on structural differences in the flexible linker of the OD, plant HSFs are categorized into three classes, namely A, B and C (Scharf et al., 2012). Similar to human and yeast HSFs, class B HSFs have a compact OD linker, while the linker in class A and C members have 21 and 7 aa extensions, respectively. Differences in the OD linker length prevent the formation of hetero-oligomers between members of different classes. HSFA members possess AHA (Acidic, large Hydrophobic and Aromatic aa) motifs, which confer transactivation activity, while class B members harbour the conserved – LFGV – motif that confers co-repressor function (Bharti et al., 2004; Kotak et al., 2004). To date, neither activator nor repressor motifs have been reported in HSFC members. In addition, the C-terminal HSF regions harbour nuclear localization

(NLS) and export signals (NES) required for nuclear import and nucleocytoplasmic transport, respectively (Scharf et al., 2012).

The presence of specific motifs has allowed the further classification of class A, B and C in nine (A1–A9), five (B1–B5) and two (C1–C2) subclasses (Scharf et al., 2012; Berz et al., 2019; Liao et al., 2022; T. Y. Wu et al., 2022). Some subclasses are comprised up to five members, designated by different letters, for example in Arabidopsis HSFA1 subclass a, b, c, and d, based again on amino acid motifs pointing in many cases to common origin and functional similarity (Scharf et al., 2012).

## III. Heat stress sensors and HSF activity elicitors

Plants use an array of interlinked as well as independent mechanisms to perceive changes in environmental temperatures and activate signalling cascades which regulate HSF-dependent gene networks (Guihur et al., 2022; Fig. 1). Under optimum temperatures for growth and development, constitutively expressed HSFs are kept in an inactive state by interaction with HSP70 and HSP90 chaperones (Hahn et al., 2011). The HS-triggered accumulation of unfolded proteins results into the release of HSF by the chaperones due to their higher affinity for unfolded proteins (Hentze et al., 2016). Pharmacological repression or genetic suppression of HSP70 results in the induction of HSF-dependent genes in the absence of a stress (Ikeda et al., 2011; Tiwari et al., 2020), supporting the notion that proteostasis is a primary sensing mechanism to trigger stress activation of the response. The accumulation of reactive oxygen species (ROS) can also induce proteotoxicity and, therefore, lead to the relief of HSFs from chaperone repression. In addition, ROS can trigger intramolecular interactions, which in the case of Arabidopsis HSFA8, lead to the translocation of the protein to the nucleus (Giesguth et al., 2015). Thereby, HSFs can also act as ROS sensors to link redox state to HS response. Mild temperature elevations cause increased membrane fluidity which can be sensed by plasma membrane proteins such as cyclic nucleotide-gated ion channels (CNGC) which trigger the influx of calcium from the periplasm into the cytoplasm (Saidi et al., 2010). Binding of calcium to the CNGC-associated calmodulins, such as AtCaM3, initiates a signalling cascade that leads to the phosphorylation of HSFA1 by calmodulin-binding protein kinases, such as CBK3 (Saidi et al., 2010; Praat et al., 2021). In addition, the histone variant H2A.Z in HSF-regulated genes ensures the limited access of HSFA1 on DNA, and under HS, the eviction of H2A.Z allows for a strong induction of HS-responsive genes already under ambient temperatures (Kumar & Wigge, 2010; Cortijo et al., 2017).

## IV. Regulatory mechanisms of HSF activity

Heat stress transcription factors are in a dynamic equilibrium that shifts between active and inactive states, influenced by the environmental conditions and the fitness of the plant (Fig. 2a). According to the current model, described by Scharf et al. (2012), HSFs undergo transition through a tightly regulated activity cycle in response to HS, starting from the inactive state where they are sequestered by HSPs in the cytosol (Fig. 2b). Upon HS, they are released, undergo oligomerization, translocate to the nucleus, and initiate transcription of heat-responsive genes, including those
<figure><img src="image_4.png" /><figcaption>**Fig. 1** Regulation of heat stress transcription factors (HSFs) by cellular thermosensors acting as stress surveillance. Five mechanisms acting as heat stress surveillance regulate HSF activity. As an example, HSFA1 has been considered for all cases except (2) in which the mechanism applies for Arabidopsis *HSFA2*. (1) Chaperone-mediated sensing of proteostasis is based on the release of an HSF by HSP70 and HSP90 upon the accumulation of unfolded proteins under high temperatures. (2) Heat stress-dependent production of reactive oxygen species (e.g. $H_2O_2$) induce protein misfolding and thereby relief HSFs by the heat shock protein (HSP) repressing function but also induced redox-driven changes in HSF structure that lead to their activation. (3) Calcium influx ($Ca^{2+}$) is triggered by temperature elevations and changes in membrane properties that activate CNGC channels and activation of the calcium-dependent signalling cascade that leads to the phosphorylation of HSFs. (4) Temperature increases can stimulate changes in the structure of some RNAs like that of *HSFA2* and regulate their translation. (5) The presence of H2A.Z variant in the promoters of HS-induced genes restricts the binding of HSFA1 under control conditions, and therefore its eviction is required for the activation of HSR by HSFA1. CaM3, calmodulin; CBK3, CaM-binding protein kinase; CDPK, $Ca^{2+}$-dependent protein kinase; CNGC, cyclic nucleotide-gated ion channel; MAPK, mitogen-activated protein kinase; NOX, NADPH oxidase. Details for each mechanism are provided in the text. HSF is shown with the distinct domains: AD, activation domain; DBD, DNA binding domain; NES, nuclear export signal; NLS, nuclear localization signal; OD, oligomerization domain.</figcaption></figure>

encoding for HSPs and HS-induced HSFs. Persisting stress conditions or a second hotter day can turn HSFs to hyperactivation, further amplifying target gene expression (Fig. 2a). Subsequently, an attenuation phase reduces HSF activity to prevent over-expression of HSPs, leading HSFs back to their basal inactive state. Cells can sustain presynthesized HSFs for a period, which in case of new HS incidents allows them to achieve a more rapid and robust stress response, particularly when the stress conditions become even more severe.

## 1. HSFs under no stress

Under control conditions, HSFs are maintained in an inactive state through binding to HSPs (Hahn *et al.*, 2011). The maintenance of the equilibrium towards the inactive state is

likely favoured by intrinsic steric restrictions that are mediated by temperature and posttranslational modifications (PTMs; Fig. 2c). Mammalian HSF1 possesses a heptad repeat region (HR-C) at the C-terminus of the activation domain, which serves as an intramolecular oligomerization repressor (Gomez-Pastor *et al.*, 2018). A temperature increase induces conformational changes that relief the repressor effect of the HR-C and allow trimerization of HSF1 and its translocation to the nucleus. A similar mechanism has not been reported for plant HSFs; however, it is likely that temperature-dependent conformational changes contribute to their switch from inactive to active state and vice versa. The presence of the Temperature-Dependent Repression (TDR) region in HSFA1 facilitates the maintenance of the inactive state in the absence of stress (Ohama *et al.*, 2016).
<figure><img src="image_5.png" /><figcaption>**Fig. 2** Overview of the activity cycle of major heat stress transcription factors (HSFs). (a) Hypothetical model of changes of heat shock protein (HSP) mRNA and protein, serving as proxy to HSF activity, in response to daily temperature changes that cause heat stress. (b) The transition stages of HSF activity state. (c) Activity cycle of HSFA1 and partner HSFs. As exemplary HSFA1 (A1), HSFA2 (A2) and HSFB (B or B1) members are shown. Under control conditions, HSFA1 genes are positively regulated by other HSFA1 members or other yet to be identified TFs, and negatively controlled by HSFB and PHABULASA (PHB). Binding of HSFA1 by HSP70 and HSP90 favours their monomeric state and prevents their trimerization. Under heat stress, HSFs are released by HSPs, they are phosphorylated and SUMOylated, and HSFA1 oligomers translocate in the nucleus, bind to the promoter of HS-induced genes, including several HSFs. HSFB proteins antagonise HSFA1s for induction, while negative regulators, such as posttranslational modifiers (BIN2, E3 ligase) and interacting proteins such as HSBP can reduce and attenuate the activity of HSFA1. Alternatively, the synthesis of HSFA2 can lead to acclimation and acquired thermotolerance. HSFA2 mRNAs are protected from degradation during HS by sequestration to SGs and PBs by ALBA proteins. Synthesized HSFA2 protein interacts with small HSPs to form HSGs from where is slowly released in case of persisting stress or HS cycles. HSFA2 activity can be repressed by SUMOylation, or enhanced by phosphorylation. Interaction of HSFA2 with HSP90-ROF1 complex enhances the nuclear retention, which is reversed by ROF2. The formation of HSFA1-HSFA2 but also HSFA1-HSFB1-HAC1 complexes with 'superactivator' activity result in the strong upregulation of HS-induced genes and thermotolerance. Green colour indicates positive, and red indicates negative effect. Arrows indicate direction of regulation. ALBA, acetylation lowers binding affinity; BIN2, BRASSINOSTEROID INSENSITIVE2; HAC1, histone acetyltransferase 1; HSBP, heat shock binding protein; HSG, heat stress granule; HSRG, heat stress-regulated gene; PB, processing body; SG, stress granule; TF, unknown transcription factor.</figcaption></figure>

Nuclear translocation of HSFA1d is inhibited by phosphoryla-
tion of a threonine in the NLS by the glycogen synthase kinase 3
(GSK3)-like kinase BRASSINOSTEROID INSENSITIVE2
(BIN2) (Luo *et al.*, 2022). In addition to the repression by
HSP70, the DNA binding of HSFA1 members is restricted in the
presence of class B HSFs acting as competitive repressors but
also by PHABULOSA (PHB) a member of the Class III
HD-ZIP transcription factor family (Li *et al.*, 2023). Furthermore,
proteasome-mediated degradation ensures a limited pool of
constitutively expressed HSFs, as shown for tomato HSFA1a and
HSFB1 (Hahn *et al.*, 2011; Röth *et al.*, 2017; Mesihovic *et al.*
2022).

2. Regulation of the intensity and duration of the active state of HSFs

Exposure of plants to an acute HS results in a prompt synthesis of HSP mRNAs, which occurs within few seconds to minutes (Liu et al., 2021). This increase is transient and peaks within 30-60 min from stress initiation and then gradually diminishes to nearly prestress levels. HSFA1a mediates promoter-enhancer interactions and thereby contributes to chromatin 3D structure in response to HS (Huang et al., 2023). Changes in chromatin structure occur within 1 h of stress in tomato nuclei but diminish after 6 h of stress, similar to the activity of several HSF-dependent genes (Huang
et al., 2023). Therefore, the attenuation of HSFA1a activity might have greater implications for chromatin organization and transcriptional activity. Class B HSFs acting as repressors of HS-induced HSFs modulate the rate of onset, intensity, and the initiation of the attenuation of HSFA-dependent HSR (Ikeda et al., 2011; Fragkostefanakis et al., 2018).

In response to the typical gradual temperature increase during a hot day, the mRNA levels of HSPs peak midday and then decline although the temperature remains high for some time (Li et al., 2020; Fig. 2a). Based on nonplant HSF models, the transient activation of HSFs is due to the accumulation of HSPs and other associated proteins, which desensitize polymerase II from transcription even when the stress persists (Hentze et al., 2016). This leads to a rapid decay in transcription of HS-induced genes, which can protect cells from an overloading with chaperones that potentially can have harmful effects (Fragkostefanakis et al., 2018).

For the attenuation of human HSF1 activity, HSP70 first binds to the region of the transactivation domain that leads to a partial repression of the response, while the dislodging of HSF1 from DNA and its monomerization are mediated by successive cycles of entropic pulling, which lead to the unzipping the triple leucine-zipper (Hentze et al., 2016). The molecular mechanism of regulation of chaperone-based HSF activity control in plants is yet to be described; however, it likely consists of a well-conserved eukaryotic system and an early-evolved mechanism. For example, Heat shock binding protein (HSBP) is a conserved negative regulator of HSFs in eukaryotes which represses the activity of HSFs through direct interaction via the leucine zipper-like OD (Fu et al., 2006; Hsu et al., 2010; Gu et al., 2019; Marko et al., 2019).

### 3. Synergism-based HSF hyperactive state in thermotolerant cells

The activation of HSFA1 members leads to the induction of additional HSFs allowing the formation of hetero-oligomeric HSF complexes with strongly increased transactivation activity (Fig. 2c). They can trigger the upregulation of HSPs under more severe HS conditions, a function that HSFA1 alone cannot fulfil. Such examples are the complexes of tomato HSFA1a with HSFA2 or HSFA7, or among the stress-induced Arabidopsis HSFA2 with HSFA3 and sunflower HSFA2 with HSFA9 (Scharf et al., 1998; Friedrich et al., 2021; Mesihovic et al., 2022; X. Wang et al., 2023). Although HSFA1a and HSFB1 cannot physically interact, they can form ternary co-activator complexes in the presence of histone acetyltransferase 1 (HAC1) as shown in tomato (Bharti et al., 2004). Wheat TaHSFA1 interacts with the HAT member TaHAG1 (histone acetyltransferase of the GNAT family 1), however, whether HAG1 also bridges HSFs from different classes has not been investigated yet (H. Wang et al., 2023). Lily LIHSFC2 can form co-activator complexes with several class A HSFs during HS (Wu et al., 2024). Even more, the formation of complexes of HSFs with other transcription factors, such as BES1 (bri1-EMS-Suppressor 1), belonging similar to BIN2 to brassinosteroid signalling-related regulatory networks can further stimulate HSR and coordinate the crosstalk with plant growth regulation pathways (Albertos et al., 2022).

The preferential formation of HSF complexes under specific temperature ranges or time points of stress response are important for the regulation of HSR. In tomato, under mild HS conditions, HSFA7 is more abundant than HSFA2 and outcompetes it for interaction with HsfA1a (Mesihovic et al., 2022). While both HSFA1a/HSFA7 and HSFA1a/HSFA2 complexes have strong transactivation activity, HsfA1a shows enhanced stability only upon interaction with HSFA2 (Mesihovic et al., 2022). The strong activity in combination with stabilization of HSFA1a makes the HSFA1a/HSFA2 complex as the essential component for the response under severe HS conditions in thermotolerant cells (Charng et al., 2006; Fragkostefanakis et al., 2016).

Plants control the availability of HSFA2 and other HSFs through several mechanisms to ensure the timely formation of 'superactivator' complexes with HSFA1. In Arabidopsis, the HS-induced melting of a hairpin structure in the HSFA2 mRNA under high temperatures allows the translation of HSFA2 protein (Chung et al., 2020). During HS the mRNAs of HSFA2 and other HSFs are protected from degradation by XRN4 (EXORIBONUCLEAR CLEARANCE4) by binding of ALBA (Acetylation lowers binding affinity) proteins that stabilize them in stress granules (SG) and P-bodies (PB) from which they can be released and translated (Tong et al., 2022). In tomato, the newly synthesized HSFA2 interacts with cytosolic class CI and CII small HSPs, leading to its accumulation in cytosolic HS granules (HSGs) which are distinct from SGs and PBs (Mishra et al., 2002; Port et al., 2004). By this, the major pool of HSFA2 is not available for interaction with HSFA1a during the early stages of HSR, but in case of prolonged or repeated cycles of HS. HSFA2 is released from HSGs to form complexes with HSFA1a which can stimulate transcription under more severe stress conditions (Port et al., 2004).

### 4. Fine-tuning HSF activity by PTMs

Stress-induced PTMs contribute to the equilibrium of active/inactive HSFs, thereby ensuring that the activation of the HSR is not only rapid and potent but also precisely moderated to scale back to normal levels when temperature falls below the HS threshold (Fig. 2c). In wheat and tomato, HSFA1a SUMOylation is associated with increased activity, and therefore deSUMOylation of wheat HSFA1a contributes to the attenuation of the response during prolonged stress (Zhang et al., 2018; H. Wang et al., 2023). BIN2-mediated phosphorylation of Arabidopsis HSFA1d supresses nuclear localization and inhibits DNA binding contributing to the attenuation of the response, while brassinosteroid signalling reverses this effect (Luo et al., 2022; Tan et al., 2023).

Interestingly, the same types of PTMs can have opposite effects on different HSFs. SUMOylation of Arabidopsis HSFA2 results in reduced activity (Cohen-Peer et al., 2010), while phosphorylation of HSFA2 by MAP kinase 6 (MPK6) facilitates the nuclear translocation of the factor (Evrard et al., 2013). Similarly, phosphorylation of HSFA4a by MPK3/MPK6 also has a positive effect on the activity of the protein and on stress response (Perez-Salamo et al., 2014). The contrasting effects of PTMs on HSFs acting as master regulators (HSFA1) and the ones acting as secondary responders such as HSFA2 suggest that they play an
important role in the rheostat model, as they can control the very
strong transactivation activity of the HSF hetero-oligomeric
complexes.

5. Alternative splicing as a driver of HSF activity and diversification

Alternative splicing has also been shown to be important modulator
of HSF-dependent HS-responsive networks. It can either redirect
RNA synthesis towards unproductive mRNAs, as shown for
Arabidopsis *HSFA2-II* that is targeted for degradation by the
nonsense mRNA decay (NMD) mechanism, or produce RNAs
that code for protein isoforms with distinct properties and,
therefore, role in HSR (Sugio *et al.*, 2009; Liu *et al.*, 2013).

Some studies have proposed that splice variants with premature termination codons in the DBD-coding region are translated and play an important role in thermotolerance (Fig. 3a). In lily, the splice variant LIHSFA3B-III, which consists only by a truncated DBD, can interact with LIHSFA3A-I and reduce its activity (Wu et al., 2019). In maize, alternative splicing in the same intron of ZmHSF17, an HSFA2 orthologue, is predicted to yield the protein isoform ZmHSF17-II, consisting only a part of the DBD (Zhang et al., 2020). ZmHSF17-II interacts with the DBD region of the full-length ZmHSF17-I resulting in repression of its activity. However, in Arabidopsis, the splice variant HSFA2-III appears under severe HS conditions and codes for a truncated protein with a hydrophobic (Phe/Leu-rich) C-terminal tail that can bind to HSFA2 promoter and induces its expression (Liu et al., 2013).

Some HSFs possess additional introns in the C-terminal coding region (Hu et al., 2020). Alternative splicing in these introns results in protein isoforms with variable C-terminal regions. In tomato, alternative splicing in this region in HSFA2 and HSFA7 leads to the synthesis of the NES-containing isoforms with higher stability than the C-terminal truncated, NES-lacking isoforms, which are rapidly degraded due to their enhanced nuclear retention (Fig. 3b; Hu et al., 2020; Mesihovic et al., 2022). The presence of the C-terminus is also important for the accumulation of HSFA2 in HSGs. The synthesis of NES-lacking isoforms is favoured under milder HS conditions, contributing to the initial response but due to rapid degradation only transiently (Hu et al., 2020; Mesihovic et al., 2022). In wheat, alternative splicing in the second intron of TaHSFA6e results in a protein isoform with an extended C-terminus, which has a stronger transactivation activity (Fig. 3c; Wen et al., 2023). Thereby, alternative splicing of HSFs is influenced by the environment and is an important mechanism for the dynamic regulation of stress response, contributing to the negative and positive regulation of the response.

V. The attenuation of stress response and the anticipation of another hot day

The attenuation of stress response when temperature drops below
the stress threshold is important to ensure that the cellular resources
are diverted towards recovery and restoration of housekeeping
functions. Deactivation of HSFs is central and all mechanisms
mentioned above having a repressive effect on HSF activity, which

are relevant for the timely attenuation of HSF activity (Fig. 2c).
Additional mechanisms, however, have been reported as well. For
example, Arabidopsis HSFA2 interacts with the tetratricopeptide
repeat (TPR)-containing FKBP co-chaperone ROF1 and HSP90
forming a complex that shuttles to the nucleus to induce the
expression of heat-stressed responsive genes, including ROF2
(Aviezer-Hagai et al., 2007; Meiri & Breiman, 2009). During
recovery from stress, ROF2 interacts with the HSP90-ROF1-
HSF2A complex to suppress its transcriptional activity (Meiri &
Breiman, 2009). In addition, PHABULOSA (PHB) represses the
transcription of HSFA1s and in addition physically interacts with
HSFA1s to antagonistically regulate the transcription of HS-
induced genes, including HSFA2 (Li et al., 2023). PHB is a direct
target of the HS-induced and HSFA1-dependent miR165/166 to
ensure that PHB functions only under nonstress conditions (Li
et al., 2023).

In nature, the occurrence of heat waves is not a singular event but
rather a series of consecutive days with extreme temperatures that
can have a cumulative impact on plants. For this, plants ensure their
preparedness for another hot day by maintaining a pool of stress-
induced chaperones, as the first line of defence for an upcoming
stress. The high protein stability of some HSFs as shown for HSFA2
and HSFA7 during recovery from stress allows the more rigorous
protection and stress response when the new stress occurs, thereby
shifting directly from a slow attenuation to hyperactivation (Hu
et al., 2020; Mesihovic et al., 2022).

Plants that have acquired thermotolerance sustain a thermo-
tolerance state for several days after priming, a capacity described as
memory. In Arabidopsis, the chloroplast HSP21 is a memory gene
that can be sustained for several days in primed plants
(Sedaghatmehr et al., 2016). The length of sustained expression
of HSP21 differs between Arabidopsis accessions N13 and Col-0
(Sedaghatmehr et al., 2016) as well as between meristem and leaves
(Olas et al., 2021) HSFA2 positively regulates HSP21 mRNA
accumulation during priming, however, HSP21 protein turnover is
regulated by a metalloprotease FtH6 which is also an HSFA2-
dependent gene (Sedaghatmehr et al., 2022).

This complex loop mechanism implicates HSFA2 in memory
activation as well as resetting. In addition, NEIGHBOR OF
BRCA1 (NBR1) binds to cytoplasmic HSP90-ROF1 and directs
them to autophagic vesicles for turnover, a mechanism that
weakens thermomemory capacity in Arabidopsis (Thirumalaikumar et al., 2020). Interestingly, this mechanism might also reset
thermomemory via the interruption of HSP90-ROF1-HSFA2
complex, which facilitates the transport of HSFA2 in the nucleus
(Meiri & Breiman, 2009; Thirumalaikumar et al., 2020).

Heat stress transcription factor-dependent memory has also been
attributed to modifications in chromatin that control the
expression of the so-called memory genes (Lämke et al., 2016;
Friedrich et al., 2021). Binding of Arabidopsis HSFA2-HSFA3
complexes to the promoters of two types of memory genes
stimulates the histone 3 lysine 4 (H3K4) methylation, a
modification that has been associated with transcriptional memory
(Friedrich et al., 2021). The sustained expression of the memory
gene *HSP21* is dependent on H3K4me3 and the balance between
active H3K4me3 and repressive mark H3K27me3 which is
<figure><img src="image_6.png" /><figcaption>**Fig. 3** Regulation of heat stress transcription factor (HSF) functional diversity by alternative splicing. (a) Alternative splicing in the intron in the DBD-coding region results in protein isoforms having only a truncated DBD with a variable C-terminal extension in lily and maize LIHSFA3B and ZmHSF17, respectively. These isoforms can act directly as repressor of transcription, or interact with full-length protein isoforms and repress their activity. (b) Alternative splicing in the second intron of tomato SIHSFA2 results in two protein isoforms. SIHSFA2-II, which lacks an nuclear export signal (NES), shows a strong nuclear retention and is active *per se*, but is rapidly degraded in the nucleus by 26S proteasome. A major fraction of HSFA2-I that has an NES, is sequestered during heat stress in cytosolic heat stress granules (HSGs) upon interaction with class CI and CII small heat shock proteins (sHSPs). HSFA2-I is slowly released from HSGs during recovery from stress or after repeated HS cycles, and interacts with HSFA1a to form 'superactivator' complexes. Arrows indicate directionality in the process and the thickness the preferred path. (c) Alternative splicing in wheat ZmHSF17 also yield two protein isoforms, with ZmHSF17-II having a C-terminal extension that harbours additional AHA motif and thereby, higher transactivator activity than ZmHSP17-I. Wave lines indicate strength of transcription. HSRG, heat stress-regulated gene.</figcaption></figure>

mediated by the Jumonji C domain-containing protein histone
demethylase 30 (JMJ30) (Yamaguchi *et al.*, 2021). While the
binding of HSFs is transient, the induced modification has a long-
lasting effect which allows the sustained expression of these genes

for several days or their stronger induction if the stress occurs again,
thereby exceeding the limits of ATT (Lämke *et al.*, 2016). HSFA2
recruits CDK8 and MED12, two subunits of the kinase module of
the transcription corepressor complex, Mediator, to HS memory
<figure><img src="image_7.png" /><figcaption>**Fig. 4** Light, thermomorphogenesis and hormone signalling in heat stress transcription factor (HSF) regulation. (a) The blue light receptor CRY1 interacts with HSFA1 and importin 1a (IMP), forming a complex that translocates to the nucleus. HSFA1a/CRY1 complexes regulate genes involved in light-induced thermotolerance. (b) Under warm conditions, COP1 inhibits BIN2-mediated phosphorylation of HSFA1d, which allows its translocation to the nucleus, where it interacts with PIF4, which protects it from the light-activated form of PHYB. This results in expression of thermomorphogenesis-related genes and physiological responses, such as hypocotyl elongation during the day. Additionally, HSFA1 can bind to HSE and PIF4 in G-box cis-elements of HSFA2 and induce its expression, thereby, triggering heat stress response and thermotolerance. (c) Induction of HSF-dependent responses by hormone-related signalling networks. Upper panel: COP1 inhibition of BIN2, reliefs its repressing effect on HSFA1 and the brassinosteroid-related BES1, which they can physically interact and bind to promoter of HSE-containing cis-element genes to induce their expression. Lower panel left: ABA-signalling triggers the AREB1-mediated HSFA6b expression. AREB1 and HSFA6b can interact to upregulate HS-responsive genes. Lower panel right: Ethylene signalling leads to the induction of ERF95 and ERF97 by EIN3. ERF95 and ERF97 bind to HSFA2 promoter and induce its expression to trigger HSR and thermotolerance. Thicker arrows indicate stronger transcription. Arrowheads indicate positive effect on gene expression and bars negative effect.</figcaption></figure>

genes, thereby immediately impacting RNAP-II activity regulation (Crawford *et al.*, 2024). A chromatin-based memory is important as it allows the shutdown of stress response and the reallocation of energy sources to recovery without the need for maintenance of proteins.

# VI. Environmental signals as drivers of HSF activity

## 1. Light and thermomorphogenesis

The transcriptional induction of HSFs depends on the time that HS occurs during the day, as for example Arabidopsis HSFA3 shows a stronger upregulation in the early afternoon than early

morning (*Li et al.*, 2019). HSFB2b acts as a repressor of the morning clock, by binding to PRR7 promoter (Kolmos *et al.*, 2014). HSFB2b is involved in plant growth under warm conditions, and loss-of-function of HSFB2b results in compromised temperature compensation under high temperature for the circadian clock.

Light intensity and quality also influence HSF activity (Fig. 4a). Arabidopsis HSFA1d, HSFA2, and HSFA3 regulate the induction of genes that are early induced in high light conditions, while HaHSFA9 promotes photomorphogenesis and stimulates blue light signalling, suggesting that HSFs can integrate light signalling into the HSR network (Jung & Crisp, 2013; Prieto-Dapena *et al.*, 2017). Plants exhibit an optimized activation of HSR during
the day, which is dependent on the direct interaction of
photoreceptors with HSFs (Fig. 4a). The blue light receptor
CRYPTOCHROME 1 (CRY1) interacts with HSFA1d and
promotes its nuclear translocation through interaction with
importin alpha 1 (OMPa1) (Gao *et al.*, 2023). As mentioned
above, BIN2-mediated phosphorylation of HSFA1d inhibits the
translocation of HSFA1d to the nucleus (Luo *et al.*, 2022; Tan
et al., 2023). Under warm temperatures, the E3 ligase constitutive
photomorphogenic 1 (COP1) suppresses the BIN2-mediated
HSFA1d phosphorylation and allows the translocation of HSFA1d
to the nucleus for the activation of HS-responsive genes (Tan
et al., 2023).

The stimulatory effect of blue light on HSF activity is not
apparent in plants exposed to red/far-red light (Gao et al., 2023).
However, studies have implicated HSFs in the signalling of the
red/far-red light photoreceptor but also thermosensor phytochrome
B (PHYB; Jung et al., 2016; Legris et al., 2016; Tan et al., 2023).
Inactivation of PHYB under elevated temperatures triggers of
phytochrome interacting factor 4 (PIF4)-mediated thermomor-
phogenesis. Interestingly, HSFA1 members interact with PIF4 and
interfere with the interaction of PIF4 with the PHYB under warm
day temperatures, resulting in PIF4 stabilization, and thereby
induction of thermomorphogenesis during the day (Fig. 4b; Tan
et al., 2023). The implication of HSFs in thermomorphogenesis is
further supported by the fact that the loss-of-function hsfa1
quadruple mutant does not display the characteristic temperature-
induced hypocotyl elongation (Li et al., 2023).

Can warm temperatures prime plants against a hot wave? The interplay between thermomorphogenesis (warm) and HSR pathways certainly points in this direction. PIF4 promotes the expression of HSFA2 to enhance Arabidopsis thermotolerance to acute stress, probably due to the induction of *HSFA2* via binding to a G-box *cis*-element in the promoter of HSFA2 (Yang *et al.*, 2022). A knockout *hsfa2* mutant did not display the typical hyponastic response of cotyledons in response to mild temperature increase in the root, supporting a direct role of HSFs in thermomorphogenesis (González-García *et al.*, 2023). Interestingly, ELONGATED HYPOCOTYL5 (HY5), which antagonizes shoot thermomorphogenesis by repressing PIF4 expression (Delker *et al.*, 2014), acts as a negative regulator of HSFA2 under high light and salinity conditions (Yang *et al.*, 2023). The repression is relieved under high light and warm temperatures, when HY5 is destabilized, allowing HSFA2 accumulation. All these results suggest that HSF proteins couple light and temperature responses in order to regulate stress response and thermotolerance during the daytime and therefore can synchronize seasonal adaptations, optimizing plant resilience and growth across varying environmental conditions throughout the year.

2. HSFs as nexus for abiotic and biotic stress responses

Heat stress transcription factors are activated by various stresses and are considered as hubs for multiple stress gene regulatory networks, including those associated with resistance to pathogens. Autophagy is such a central molecular process that is partly mediated by HSFs and serves as an important mechanism for stress resilience.

Tomato HSFA1a regulates autophagy in heat-stressed pollen (Xie *et al.*, 2022), in response to ER stress (Löchli *et al.*, 2022), as well as under water-limited conditions (Wang *et al.*, 2015).

The activation of HSFs under different abiotic and biotic stresses is typically associated with hormone signalling (Fig. 4c). A direct regulation of HSF activity by brassinosteroid signalling pathway via BIN2 and BES1 has been discussed above (Albertos *et al.*, 2022; Luo *et al.*, 2022). In Arabidopsis, abscisic acid (ABA)-induced expression of HSFA6b is mediated by ABA-RESPONSIVE ELEMENT-BINDING PROTEIN1 (AREB1) (Huang *et al.*, 2016). In turn, HSFA6b directly regulates DEHYDRATION-RESPONSIVE ELEMENT-BINDING PROTEIN2A (DREB2A), thereby placing HSFA6b as a positive regulator not only of thermotolerance but also of drought and salinity tolerance (Huang *et al.*, 2016).

In poplar, the auxin indole-3-acetic acid (IAA) repressor IAA17.1 interacts with the HSFA5a, which is highly expressed in roots and induced by salt stress (Song et al., 2024). Overexpression of HSFA5a significantly increased flavonol content, reduced ROS accumulation, enhanced lateral root growth, and salt tolerance in transgenic poplar (Song et al., 2024). In sunflower, HaIAA27 interacts with HaHSFA9 leading to the repression of the latter, suggesting that HaHSFA9 is a target of Aux/IAA protein repression (Carranco et al., 2010).

Ethylene signalling is important for thermotolerance, as it is required to protect against HS-induced oxidative damage (Larkin-dale & Knight, 2002). The transcriptional cascade in response to activation of ethylene signalling is mediated by the ERF family of transcription factors, and it has been demonstrated that ERF95 and ERF97 can directly bind to the promoter of HSFA2 and regulate its expression, thereby linking ethylene signalling to HSR (Fig. 4c; Huang et al., 2021). Arabidopsis HSFA7b is an important regulator for thermomemory in the shoot apical meristem, exerting its function via the regulation of ethylene biosynthesis and signalling genes including ethylene insensitive 3 (EIN3) (John et al., 2023). Interestingly, the root endophyte *Enterobacter* sp. SA187 enhances Arabidopsis and wheat thermotolerance via ethylene-mediated signalling and by inducing thermomemory-related modifications in histones (Shekhawat et al., 2021).

Arabidopsis HSFB1 and HSFB2b act as a repressor of HSR but have a positive role in the development of acquired thermotolerance under HS conditions (Ikeda et al., 2011). HSFB1 acts as an indirect negative regulator of PDF1.2a and PDF1.2b, two defensin genes that are responsive to jasmonic acid (JA) and ethylene to suppress the innate immune response of Arabidopsis to the necrotrophic fungus *Alternaria brassicicola* (Kumar et al., 2009). In Arabidopsis, HSFB1 is induced by SA and regulates the allocation of resources between growth and defence, and its reduced expression in Arabidopsis F1 hybrids might be related to enhanced growth (Pajerowska-Mukhtar et al., 2012; Gonzalez-Bayon et al., 2019).

Silencing of pepper CaHSFB2a compromised the resistance of the plant to *Ralstonia solanacearum*, while overexpression of CaHSFB2a resulted in the induction of stress marker genes and hypersensitive response (HR)-like cell death and H₂O₂ accumulation (Ashraf *et al.*, 2018). In rice, two HSF-dependent JASMONATE ZIM-DOMAIN (JAZ) genes are induced under heat by HTG3/OsHSFA2d (N. Wu *et al.*, 2022), while in
<figure><img src="image_8.png" /><figcaption>**Fig. 5** Involvement of heat stress transcription factors (HSFs) in plant growth and development. (a) HSFA1 and HSFC1b are involved in plant and HSFB4 in root growth, while HSFB2b integrates environmental signals with the circadian clock, including flowering time by regulating the expression of PRR7. (b) HSFA5 is involved in pollen development by controlling in nucleolar functions. The activation of HSFA1 during the early stages of pollen development is important for the induction HEI10 that is associated with meiotic crossovers and the induction of HSFA2, which leads to the upregulated of HSR genes like many heat shock proteins (HSPs) in the absence of stress, seemingly as an acclimation mechanism. HSBP acts as a repressor of HSFA1 function by interfering with the formation of HSF oligomers. HSFB2a is important for ovule development. It is suppressed by a HSFA1-regulated lncRNA (antisense RNA). (c) ABI3 during seed development induces the expression of HSFA9 which alone or in cooperation with HSFA4a upregulate many HSPs, thereby, modulating seed desiccation tolerance and longevity, based on studies mainly conducted on sunflower. In this network, IAA27 acts as a repressor of HSFA9.</figcaption></figure>

Arabidopsis the activation of HSFA1b induces the expression of *AtOPR3* which in turn drives the biosynthesis of JA biosynthesis (Tian *et al.*, 2020). Very recently, Mishra *et al.* (2024) showed that barley HvHSFA2e provides multi-stress tolerance via modulation of hormones and secondary metabolism in overexpression plants.

Altogether, these findings highlight the contribution of HSFs to the development of thermotolerance by intersecting pathogen resistance and hormonal signalling pathways. HSFs, as central hubs to these stresses, can offer the plasticity that the plant requires to prioritize response and protection mechanisms. In support of this, the transcripts of all HSFs were identified at the shoot apical meristem, a hub of plant growth, further supporting the role of HSFs in growth and development (Olas *et al.*, 2021; John *et al.*, 2023).

## VII. HSFs in plant development

Apart from their importance for stress response, HSFs are also involved in plant growth and development. This can be a pro-active mechanism for protection against an upcoming stress or it is associated with the higher demand for chaperones in specific cell types during development (Fig. 5). Evidence that HSFs have a basal activity that is important for housekeeping functions in the absence of stress comes from the stunted growth of Arabidopsis *hsfa1* quadruple mutant (Fig. 5a; Liu *et al.*, 2011). In addition, knockdown of OsHSFC1b in rice also results in reduced growth (Schmidt *et al.*, 2012). Class B HSFs are thought to act as a balance between stress response and growth. A missense mutation in tomato HSFB4a in the bushy root-2 (*brt-2*) mutant is associated

with twisting roots, and slower plant development (Kevei *et al.*, 2022), while a mutation in Arabidopsis *hsfb4*, also known as SCHIZORIZA, results in cell fate segregation defects in roots (Fig. 5a; Kevei *et al.*, 2022 and references therein).

The HSF-driven accumulation of HSPs in meiotic gametophytes is an evolutionary conserved mechanism (Fig. 5b). The accumulation of HSPs in pollen mother cells and meiocytes depends on the HSFA1a-dependent accumulation of HSFA2, which allows the formation of HSFA1a-HSFA2 complexes and thereby HSPs and other HS-induced genes during the early stages of pollen development, as shown in tomato (Fragkostefanakis *et al.*, 2016). It has been assumed that due to the high sensitivity of male gametophyte to high temperatures, the activation of HSFs primes pollen for a possible upcoming stress incident. Whether the accumulation of HSPs or other HS-responsive genes is essential for viable pollen is not yet clear. However, HSFs induce the expression of E3 ligase HEI10, which is involved in meiotic crossovers (Fig. 5b; Kim *et al.*, 2022). The interaction of HSBP with HSFs repressed HEI10 transcription, thereby restricting meiotic HEI10 transcription and crossover number (Kim *et al.*, 2022). An Arabidopsis *hsfa5* mutant, also called *atren1*, exhibits defects in structure and function of the male gametophyte, probably due to nucleolar malfunctions and disturbance in rRNA biogenesis (Reñák *et al.*, 2014). In addition, HSFB2a, which is controlled by an HSFA1-dependent long noncoding RNA, is expressed in the female gametophyte and is important for ovule development (Fig. 5b; Wunderlich *et al.*, 2014).

HSFA9 is highly expressed in seeds and its induction is dependent on ABA-signalling and particularly the ABI3 factor,
**Box 1** Examples of heat stress transcription factor (HSF)-associated natural variation linked to stress tolerance in crops.

**Tomato** (Hu *et al.*, 2020): Wild and domesticated tomato accessions exhibit different splicing efficiency of the second intron of HSFA2 due to intronic single nucleotide polymorphisms (SNPs). During tomato domestication the selection of *HSFA2*<sup>GGG</sup> haplotype was selected against *HSFA2*<sup>AAA</sup>, resulting in enhanced alternative splicing the synthesis of isoform HSFA2-I, which is important for acclimation to HS. Wild tomato species have a more rigorous response to acute stress due to enhanced synthesis of HSFA2-II isoform compared to modern cultivars.

**Soybean** (Bian *et al.*, 2020): Four promoter haplotypes of HSFB2b were identified from wild and cultivated soybeans. Promoter haplotype II found in salt-tolerance wild soybean shows enhanced activity under saline conditions and is associated with increased expression of flavonoid biosynthesis-related genes and, therefore, flavonoid production.

**Rice** (N. Wu *et al.*, 2022): A miniature inverted-repeat transposable element (MITE) in the promoter of *HTG3/OsHSF2Ad* (*heat-tolerance gene on chromosome 3*) is associated with heat-induced expression of OsHSF2Ad and thermotolerance. The variant that lacks the MITE has been selected in rice-growing regions that are prone to heat.

**Grapevine** (Liu *et al.*, 2023): Differences in thermotolerance among Vitis germplasm accessions are associated with variation (Thr315Ile) in the AHA1 motif of HSFA2. *Vitis davidii* VdHSFA2 (tolerant) has higher transactivation activity and regulates more genes than *Vitis vinifera* VvHSFA2 (sensitive).

leading to the accumulation of many HSPs (Fig. 5c; Kotak *et al.*, 2007). In sunflower, HaHSFA9 interacts with HaHSFA4a or HaDREB2 in seeds enhancing the expression of several HSPs, which are important for seed longevity (Almoguera *et al.*, 2009; Tejedor-Cano *et al.*, 2014; Prieto-Dapena *et al.*, 2017). Similarly, in wheat, TaHSFA2d (A6b) showed a seed preferential accumulation in developing seeds and enhanced heat stress tolerance and yield under heat stress conditions (Chauhan *et al.*, 2013).

The dual role of HSFs, balancing between promoting growth under nonstress conditions and facilitating stress resilience, illustrates the importance of the rheostat model. The regulation of HSFs, therefore, is not just about enabling plants to survive adverse conditions but also about ensuring their growth and reproductive success in an ever-changing environment.

## VIII. Conclusion

In 2001, Nover *et al.* in a landmark review on plant HSFs posed the question: 'How many heat stress transcription factors do we need?' Today, there is ample evidence from genetic studies to clearly answer 'many'. Often members of the HSF family have in specific roles not only in HSR but also in developmental processes, disease resistance, and adaptation to other abiotic stresses. Understanding the complex roles and regulator mechanisms that underscore the functions of HSFs within the framework of the rheostat model is important for the development of HSF-based breeding and biotechnological strategies aimed at enhancing crop resilience. Such approaches must carefully consider the balance between

promoting stress tolerance and maintaining or even enhancing growth and reproductive success. The involvement of HSFs in stress resilience and growth and development indicates that they can serve as central hubs in trade-offs. HSFB1 in Arabidopsis is the best example so far showcasing how HSFs can balance trade-offs between growth and immune response (Pajerowska-Mukhtar *et al.*, 2012). Therefore, special consideration must be taken for the manipulation of HSF networks. Furthermore, the richness in the genetic and genomic repositories can also uncover polymorphisms in HSFs that are associated with important agronomic traits. Currently, there are several examples of natural variation in HSFs as a source for crop improvement for abiotic stress tolerance in tomato, ryegrass, rice, grape and soybean (Box 1). Therefore, a holistic perspective is essential for breeding and engineering crops that can thrive under diverse environmental conditions, ensuring food security for future generations.

## Acknowledgements

The authors thank Klaus-Dieter Scharf for helpful comments during the preparation of the manuscript and SF personally recognises his great mentoring and long-standing contribution in the field of heat stress response. SF also acknowledges the constant support from Enrico Schleiff. AB is supported by a DAAD-GERLS scholarship for doctoral studies. This work was supported by the German Research Foundation to SF (DFG, project FR 3776/5). The authors acknowledge EPI-CATCH (CA19125) and RECROP (CA22157) COST Actions for networking opportunities. HC acknowledges Alexander von Humboldt Senior Fellowship for the support. We apologize to all colleagues whose work could not be cited due to space limitations.

## Competing interests

None declared.

## Author contributions

SF conceived and wrote the manuscript with the help from AB, SV, BS, HC and MB. All authors have read and approved the manuscript. AB, SV and BS contributed equally to this work.

## ORCID

Ayat Bakery https://orcid.org/0009-0006-3019-4469
Moussa Benhamed https://orcid.org/0000-0002-2716-748X
Harsh Chauhan https://orcid.org/0000-0002-1396-5464
Sotirios Fragkostefanakis https://orcid.org/0000-0001-5311-7868
Boushra Shalha https://orcid.org/0009-0006-3088-9279
Stavros Vraggalas https://orcid.org/0009-0008-4058-1344

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