MicroRNAs‐mediated heat stress regulations in plants: Mechanisms and targets

Abstract

Heat stress, exacerbated by global warming, can cause significant challenges to agriculture, adversely impacting plant growth, reproduction, and yield. This review examines the crucial role of microRNAs (miRNAs) in mediating plant responses to heat stress across various key crops, including Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), wheat (Triticum aestivum), and other significant species. Under high temperatures, miRNAs regulate gene expression by targeting transcription factors (e.g., SPL, NF‐YA, and Apetala 2 [AP2]), heat shock proteins, and antioxidant enzymes (e.g., copper/zinc superoxide dismutase), thereby modulating pathways involved in hormone signaling, oxidative stress mitigation, and developmental transitions. Advanced high‐throughput sequencing technologies have identified heat‐responsive miRNAs (e.g., miR156, miR398, miR172) and their functional networks, including crosstalk with small interfering RNAs, long noncoding RNAs, and circular RNAs via competing endogenous RNA (ceRNA) mechanisms. These findings highlight miRNAs as promising targets for engineering heat‐resilient crops. However, gaps remain in understanding tissue‐specific miRNA dynamics and their integration with epigenetic and multi‐omics networks. Future research should employ integrative approaches to optimize miRNA‐based strategies for sustainable agriculture in the context of climate change.

Core Ideas

• Heat stress, intensified by global warming, adversely affects plant growth, reproduction, and ultimately crop yields.

• In plants, microRNAs (miRNAs) regulate gene expression by inhibiting the translation of target mRNAs.

• Under heat stress, miRNAs may be either upregulated or downregulated, contributing to enhanced heat tolerance.

• The coordinated interactions among miRNAs, long noncoding RNAs, and circular RNAs modulate gene expressions in response to heat stress.

• Identifying heat stress‐responsive miRNAs offers valuable insights for developing heat‐tolerant crop varieties.

Plain Language Summary

Heat stress is a major threat to plant growth, photosynthesis, and crop productivity, particularly during critical stages like grain filling, often resulting in reduced yields. This review explores the role of miRNAs in enhancing heat tolerance in crops such as rice, wheat, and Arabidopsis by modulating gene expression under high temperatures. Advances in genome sequencing technologies have helped to identify key miRNAs that orchestrate heat stress responses by regulating hormone signaling pathways and boosting antioxidant defenses. These miRNAs also interact with other RNA molecules, including small interfering RNAs, long noncoding RNAs, and circular RNAs, through competing endogenous RNA (ceRNA) networks that further strengthen the plant's resilience to heat stress. Overall, miRNAs emerge as valuable molecular tools for developing heat‐tolerant crop varieties, offering a promising strategy to ensure food security in the face of global warming.

Abbreviations

AFB

auxin signaling F‐box protein

AGO

Argonaute

AP2

Apetala 2

ARF

auxin response factor

ceRNA

competing endogenous RNA

CSD

copper/zinc superoxide dismutase

CSS

copper chaperone for superoxide dismutase

HD‐Zip

homeodomain‐leucine zipper

HSF

heat shock factor

HTT1

heat‐induced TAS1 target1

HTT2

heat‐induced TAS1 target2

LAC

laccase

MYB

myeloblastosis

NF‐YA5

nuclear transcription factor Y subunit alpha 5

PHO2

phosphate2

P P R

pentatricopeptide repeat

ROS

reactive oxygen species

SPL

SQUAMOSA promoter‐binding protein‐like

STTM

short tandem target mimic

TCP

teosinte branched/cycloidea protein

TF

transcription factor

TIR

transport inhibitor response

1. INTRODUCTION

Heat stress occurs when plants endure temperatures above their optimal range (20°C–30°C), disrupting their growth and physiological functions. With global warming increasing the frequencies of extreme heat events, this issue has become a major challenge for plant health and crop productivity. Heat stress causes several changes in plants, starting with dehydration due to increased water loss, which then leads to stunted growth, wilting, and disrupted photosynthesis. High temperatures damage key cellular components such as Photosystem II and ribulose‐1,5‐bisphosphate carboxylase/oxygenase (commonly known as Rubisco), reducing energy production and efficiency (Farooq et al., 2024). Being sessile, plants are constantly subject to fluctuations in abiotic factors, such as temperature changes, in their environment, and therefore need to evolve mechanisms other than evasion to adapt to these changes. Around the world, heat is a major cause of agricultural losses, sometimes in conjunction with drought or other circumstances. According to the Intergovernmental Panel on Climate Change, global temperatures are expected to rise by 0.3°C annually, reaching 1°C above current levels by 2025 and 3°C above current levels by 2100 (Lindsey & Dahlman, 2024). Higher temperatures could alter growing seasons and shift where crops can be grown. While rising temperatures may allow earlier planting in temperate regions like the US Midwest or parts of Europe due to faster soil warming, it can also delay planting in areas like sub‐Saharan Africa, South Asia, and Australia affected by heat‐induced drought, erratic rainfall, or adverse soil conditions. Additionally, accelerated crop maturation may impact yield potential and necessitate adjustments in planting schedules (Marklein et al., 2020). As temperatures continue to rise, the world's major staple crops, such as barley, rice, maize, sorghum, wheat, and soybean, are suffering yield losses globally (Rezaei et al., 2023). With temperatures rising steadily, maintaining high crop yields under heat stress is crucial for achieving significant agricultural goals.

MicroRNAs (miRNAs) are small, noncoding RNAs (ncRNAs), 20–24 nt long, derived from complementary RNA precursors transcribed by RNA polymerase II in subnuclear regions called D‐bodies (Anand et al., 2017). These short regulatory elements bind specifically to target mRNAs to either inhibit their translation or induce their cleavage, thereby blocking protein production as described in Figure 1. In plants, miRNAs play a crucial role in regulating metabolic pathways, development, and responses to biotic and abiotic stresses, including heat stress (R. Kumar, 2014). Their effectiveness mainly relies on how closely their sequences complement with target mRNAs, often encoding transcription factors (TFs). Under high‐temperature stress, key plant processes such as photosynthesis, flowering, and seed formation are disrupted. miRNAs function as molecular switches, turning specific genes on or off to mitigate stress effects. Some miRNAs are upregulated, while others are suppressed, enabling dynamic regulation of stress‐responsive genes (F. Zhang, Yang, et al., 2022). This posttranscriptional control fine‐tunes essential pathways like reactive oxygen species (ROS) detoxification, heat shock protein (HSP) production, hormone signaling, and developmental reprogramming (Y. Zhang, Xu, et al., 2023). Consequently, plants can activate protective mechanisms, conserve energy, maintain homeostasis, and develop acquired thermotolerance. For instance, a recent study developed transgenic petunia lines with altered miR164 expression to target NAC genes under heat stress, using qRT‐PCR (quantative real time PCR), expression profiling, and bioinformatics analyses PPI (protein protein interaction) and gene ontology [GO] enrichment) for functional validation (T. Jiang et al., 2025). These findings highlight the potential of miRNAs as targets for developing climate‐resilient crops.

FIGURE 1.

MicroRNAs (miRNAs) biogenesis and their regulatory roles in plants under heat stress. This figure shows that miRNAs are transcribed in the nucleus as precursor microRNAs (pre‐miRNAs) from miRNA genes via RNA polymerase II under heat stress. These pre‐miRNAs are exported to the cytoplasm through Exportin‐5 and processed by Dicer into mature miRNAs. The mature miRNAs undergo duplex unwinding and are loaded onto the RNA‐induced silencing complex (RISC), where they interact with Argonaute (AGO) proteins to suppress target gene expressions. Specific heat‐responsive miRNAs are either upregulated (e.g., miR156 targeting SQUAMOSA promoter‐binding protein‐like [SPL], miR160 targeting AUXIN RESPONSE FACTOR [ARF], and miR319 targeting TEOSINTE BRANCHED/CYCLOIDEA/PROLIFERATING CELL FACTOR [TCP]) or downregulated (e.g., miR159 targeting MYELOBLASTOSIS [MYB] and miR168 targeting ARGONAUTE 1 [AGO1]).

Under heat stress, plants experience changes in miRNA expressions. Many miRNAs inhibiting genes that enhance stress resistance, such as miR169 and miR159, are downregulated. Conversely, some miRNAs, such as miR156, are upregulated to protect plants by targeting specific TFs to help mitigate the effects of heat (F. Zhang, Yang, et al., 2022). Numerous miRNAs have been reported in dicots to target their genes in response to heat stress. For example, the overexpression of OsmiR397 from rice successfully demonstrated the use of miRNAs for trait enhancement and improving rice production by stimulating panicle branching and enlarging grain size (Kumsa & Kuma, 2023). Similarly, overexpressing miR156 enhanced the tolerance of alfalfa to heat stress (Arshad & Hannoufa, 2022). Research on maize has identified numerous miRNAs with altered expressions under heat stress, emphasizing their roles in regulating pathways such as auxin signaling and stress priming (Ellouzi et al., 2024). Many other miRNAs have been reported in rice, wheat, sunflower, and other crops to be responsive to high temperatures. These miRNAs interact with genes involved in metabolism and development, aiding plants in adapting to higher temperatures. Understanding how plants respond to heat stress is crucial for developing resilient crops and ensuring sustainable agriculture in a warming world. Research has shown that miRNAs regulate gene expressions in a time‐ and location‐specific manner. These small RNA molecules play a crucial role in the growth and development of various plant organs, including stems, leaves, flowers, fruits, and roots, as shown in Figure 2. Furthermore, they are important components of the plant's responses to both biotic and abiotic stresses. This type of gene regulation is also species‐specific (C. Luo, Bashir, et al., 2024; Salami & Moradi, 2024). Previous studies on plant responses to heat stress have mainly focused on TFs (such as NACs, dehydration‐responsive element bindings, heat shock factors [HSFs], TIFYs, and bZIPs) and genes (such as HSPs, LEA, MIP, NADPH, and ROS) that regulate the expressions of heat‐responsive genes (Alshareef et al., 2022; M. Guo et al., 2016; Mizoi et al., 2023). However, both during and after heat stress, plants also require posttranscriptional controls to maintain their cellular equilibrium (Guerra et al., 2015).

FIGURE 2.

Regulatory roles of miRNAs in heat stress adaptation through auxin, gibberellic acid (GA), and abscisic acid (ABA) pathways in plants. This flowchart illustrates the role of miRNAs in auxin, GA, and ABA signaling pathways. The miR160, miR164, and miR393 are upregulated during heat stress in certain plants and are directly involved in the activation of the auxin signaling pathway. The miR160 reduces the levels of AUXIN RESPONSE FACTORS (ARFs; ARF10, ARF16, ARF 17) and directly regulates heat shock proteins (HSPs), helping plants survive by enhancing root architecture under heat stress. Similarly, miR164 targets and reduces NAC transcription factors (TFs; NAC1, NAC22) and directly activates the ARFs for better root growth. The miR393 suppresses TIR1 (transport inhibitor response1), AFBs (auxin signaling F‐box proteins), directly regulates GH3 (GRETCHEN HAGEN 3), ARFs, and manages lateral root development. It modulates auxin signaling to maintain proper root growth and structure during heat stress. In the GA pathway, upregulated miR396 suppresses WRKY TFs and directly regulates GRFs (growth‐regulating factors) to modulate plant cell expansion and its growth under heat stress. Another overexpressed miR319 in the GA pathway represses teosinte branched/cycloidea protein (TCP) TFs (TCP2, TCP3, TCP4, TCP10, TCP24) and controls GA biosynthesis genes (GA20ox, GA3ox). It leads to DELLA protein accumulation and promotes leaf growth and flowering under heat stress. In the ABA pathway, downregulation of miR159 induces the level of MYB (MYELOBLASTOSIS) TFs (MYB33, MYB101, MYB65) and modulates ABA‐responsive genes (ABI3, ABI5) that regulate seed dormancy under heat stress. Similarly, downregulation of miR169 helps to induce the level of nuclear factor Y (NF‐YA5, NF‐YA7), which regulates RD29B and AB15 genes for better root growth. In contrast, miR167 is upregulated in most plants to initiate the ABA pathway and represses AUXIN RESPONSE FACTORS (ARF6, ARF8) under heat stress. It directly regulates RD29B and AB15 to enhance seed germination.

Core Ideas

• Heat stress, intensified by global warming, adversely affects plant growth, reproduction, and ultimately crop yields.

• In plants, microRNAs (miRNAs) regulate gene expression by inhibiting the translation of target mRNAs.

• Under heat stress, miRNAs may be either upregulated or downregulated, contributing to enhanced heat tolerance.

• The coordinated interactions among miRNAs, long noncoding RNAs, and circular RNAs modulate gene expressions in response to heat stress.

• Identifying heat stress‐responsive miRNAs offers valuable insights for developing heat‐tolerant crop varieties.

This review summarizes recent research on notable miRNAs, their interactions, target genes, and regulatory functions in various crops under heat stress, with examples from Arabidopsis, wheat, rice, and many other crops in which miRNAs play an important role in acquiring tolerance to heat stress conditions. WRKY, ARF (auxin response factor), LAC (laccase, a copper‐containing glycoprotein), CSD (copper/zinc superoxide dismutase), CCS (copper chaperone for superoxide dismutase), PPR (pentatricopeptide repeats), nuclear transcription factor Y subunit alpha 5 (NF‐YA5), sulfate transporter (AST), and other genes have been reported to be targeted by specific miRNA families including miR400, miR408, miR397, miR398, miR396, miR395, and miR169 (Noureddine et al., 2022; F. Zhang, Yang, et al., 2022). Understanding these mechanisms is essential for developing crops that can withstand heat stress, thereby contributing to food security in a changing climate.

2. miRNAs THAT MEDIATE HEAT STRESS REGULATION IN ARABIDOPSIS

In Arabidopsis, overexpression of miR156 represses the SQUAMOSA promoter‐binding protein‐like (SPL) genes, SPL2, SPL3, and SPL11, to promote the expression of heat stress‐responsive genes (Yuan et al., 2023). Arabidopsis responds differently to heat and salt stress depending on whether miR156 is overexpressed or underexpressed along with SPL3, suggesting that miR156 may also be involved in physiological processes beyond shoot morphogenesis (Sang et al., 2023; J. Wang, Ye, et al., 2019). Studies have demonstrated that the regulation of the SPL genes by miR156 or miR157 during the vegetative phase of Arabidopsis specifies the qualitative and quantitative changes in leaf shape (J. He et al., 2018). During the vegetative and reproductive phases of plants, including Arabidopsis, sunflower, maize, and tomato, heat‐responsive miRNAs such as miR156, miR159, miR160, miR167, miR396, and miR408 have been reported (Giacomelli et al., 2012; J. He et al., 2019; Keller et al., 2020).

The ability of transgenic Arabidopsis to tolerate heat was greatly improved by overexpressing miR160. Auxin response factor10 (ARF10), ARF16, and ARF17, which regulate the production of HSPs to maintain cellular viability in the presence of heat stress, are three target genes of miR160 (Hao et al., 2022; Zimmerman et al., 2024). In another study, an Arabidopsis mutant in the miR398 gene lost the ability to induce the expressions of heat‐responsive genes, thus making it heat‐sensitive (Guan et al., 2013). In thermotolerant Arabidopsis, miR173 was discovered to specifically target the mRNAs of heat‐induced TAS1 target1 (HTT1) and HTT2 (Halder et al., 2023; J. Jiang et al., 2018; S. Li et al., 2014; J. Zhao, He, et al., 2016). According to microarray studies, HTT1 and HTT2 were found to be greatly induced in Arabidopsis seedlings in response to heat stress (S. Li et al., 2014; Ling et al., 2018). HTT1 and HTT2 overexpression modified the expressions of numerous HSF genes involved in thermotolerance. HTT1 was specifically upregulated at high temperatures in heat‐tolerant plants overexpressing HsfA1a, which directly activated HTT1 and HTT2 by binding to their promoters (Waititu et al., 2020). HSPs Hsp17‐14, Hsp40, and NUCLEAR FACTOR Y, SUBUNIT C interact with HTT1 (S. Li et al., 2014). The findings revealed that HTT1, targeted by TAS1a and activated mainly by HsfA1a, acts as a cofactor for Hsp70‐14 complexes and enhances heat tolerance pathways.

Other specific miRNAs involved in heat tolerance in Arabidopsis thaliana include miR824, miR856, miR857, and miR858. The target gene of miR824 is AGL16 (AGAMOUS‐like 16; At3g57230), a MADS‐box gene. miR858 regulates MYB12 (At2g47460), a member of the R2R3‐MYB family. miR856 targets an unrelated transcript encoding the efflux protein ZAT1 (a zinc transporter). However, the functional roles of the target genes of miR856 and miR857 remain unclear, although miR857 was shown to specifically target a member of the laccase family, LAC7 (At3g09220), with a multicopper oxidase function (F. Zhang, Yang, et al., 2022). By controlling the abundance of TFs and proteins linked to the metabolism of ROS, as well as many mitogen‐activated protein kinase signaling pathways, miRNAs are involved in the response to and the mitigation of the harmful effects related to high‐temperature stress (Ding et al., 2020). miR397 also contributes to heat stress response by controlling l‐ascorbate oxidase expression (Huang et al., 2020). A key plant oxidant, l‐ascorbate, is essential for both enzyme‐ and nonenzyme‐based detoxification procedures that lower excessive ROS activity. It also serves as a cell signaling modulator in a number of cellular processes such as cell division, growth, and cell wall creation (M. Xiao et al., 2021).

In Arabidopsis, the LAC gene, which encodes a copper‐containing glycoprotein crucial for cell wall stability and strengthening cell–cell adhesion during heat stress, is negatively regulated by miR397a. Heat stress suppresses miR397 expression, leading to the activation of LAC (W. Guo et al., 2024).

In Arabidopsis, miR159a and miR159b are downregulated in response to heat stress. These miRNAs specifically target and negatively regulate GAMYB‐related genes such as MYB33, MYB65, and MYB101, which are involved in gibberellic acid signaling pathways crucial for flower development and seed germination (Imran et al., 2022; J. Yu et al., 2024). Other studies showed that the heat‐upregulated miR164a,b targeted the NAC1 (NAC22, NAC100) gene after heat stress treatment (S. Guo et al., 2023). A recent investigation showed that the overexpression of the NAC TF gene, JUNGBRUNNEN1 (JUB1; ANAC042), increased the resistance to heat stress and increased the life span of the plant (Evans et al., 2024).

In Arabidopsis, upregulated miR399b targets the expression of the PHOSPHATE 2 (PHO2) enzyme at the blooming stage under heat stress (Rakhi et al., 2024). Plants respond to phosphate (Pi) deficiency by modulating specific gene expressions. This involves upregulating Pi transporter genes through the activities of miR399, miR827, and the miR399 molecular sponge, IPS1. Simultaneously, the expressions of genes encoding the ubiquitin‐conjugating enzyme E2 (PHOSPHATE2 or PHO2) and Pi‐sensing and transport proteins (SPX‐MFS, a member of the major facilitator superfamily) are downregulated (Wu et al., 2023). These regulatory mechanisms enable plants to adapt to Pi‐deficient conditions efficiently.

Other studies demonstrate that certain miRNAs involved with auxin signaling and plant development are upregulated in Arabidopsis under heat stress, as described in Table 1. They include miR160, miR166a, and miR319a,b. In Arabidopsis, miR160 controls a subset of repressor ARFs that play key roles in auxin hypersensitivity, seed germination, floral organ development, and post‐germination phases (Zimmerman et al., 2024). The expressions of ARF10, ARF16, and ARF17 were shown to be downregulated by miR160 in Arabidopsis (Lin et al., 2018), and miR160 was first identified in Arabidopsis by Hao et al. (2022). miRNAs have been shown to regulate AP2/ERFs (where AP2 is Apetala 2) by silencing their transcripts and inhibiting their translation. miR172 targets the Arabidopsis AP2 transcripts and prevents their translation (Ó’Maoiléidigh et al., 2021). According to Nowak et al. (2022), miR172b was downregulated to induce AP2‐like TFs (such as SMZ, SNZ, TOE1, and TOE2), reducing the effects of heat stress in Arabidopsis. In Arabidopsis, the overexpression of miR172 increased vegetative growth and led to an early‐blooming phenotype (J. Zhao, He, et al., 2016). The miR166a downregulates homeodomain‐leucine zipper (HD‐Zip) genes, including PHV (PHAVOLUTA), REV (REVOLUTA), and HOX9 (homeobox 9), which are involved in heat stress tolerance (HST) (Z. Zhang, Yang, et al., 2024). The HD‐Zip family of TFs belongs to the homeobox protein superfamily. The homeodomain at the N‐terminus of HD‐Zips serves as a DNA‐binding site, while the leucine zipper motif is responsible for dimerization. Other research has demonstrated that miR319a,b, which target the TCP (teosinte branched/cycloidea protein) genes, including TCP2, TCP3, and TCP24, were upregulated in Arabidopsis during heat stress (Fang et al., 2021).

TABLE 1.

MicroRNAs (miRNAs) gene expressions and their targeted genes in Arabidopsis under heat stress.

Types of miRNAs	miRNAs expression	Targeted genes	Targeted gene expressions and functions	References
miR156	Upregulated	SPL2, SPL3, SPL11	Suppresses SPL genes that promote flowering initiation, heat stress memory, and influence leaf morphology and shoot architecture, leading to improved thermotolerance	Yuan et al., 2023
miR398	Upregulated	CSD1	Suppresses the CSD1 gene that encodes antioxidant enzymes that scavenge ROS, reducing oxidative damage and enhancing heat tolerance	Cao et al., 2022
miR173	Downregulated	HTT1, HTT2	Induces HTT genes that regulate heat stress response pathways, improving plant survival under high temperatures	Halder et al., 2023
miR397	Downregulated	l‐ascorbate oxidase	l‐ascorbate oxidase modulates l‐ascorbate levels, helping maintain cellular redox balance under heat stress	Huang et al., 2020
miR397a	Downregulated	Laccases (LACs)	Laccases are involved in lignin biosynthesis; reduced suppression from miR397a promotes lignin production, aiding heat stress adaptation	W. Guo et al., 2024
miR400	Downregulated	PPRs	PPR genes encode stress‐related proteins; induction of these genes alters stress signaling, reducing heat‐induced damage	W.‐B. Xu et al., 2023
miR156a‐f	Upregulated	SPL2, SPL3, SPL11	Suppresses SPL genes that promote flowering and heat stress memory, leading to better adaptation under high temperatures	Yuan et al., 2023
miR159a,b	Downregulated	MYB33, MYB65, MYB101	Induces MYBs, regulating ABA signaling for stress adaptation and influencing reproductive organ development	J. Yu et al., 2024
miR164a,b	Upregulated	NAC1 (NAC22, NAC100)	Downregulates NAC TFs that are essential for modulating lateral root development and auxin signaling	S. Guo et al., 2023
miR166a	Upregulated	HD‐Zip (PHV, REV, HOX9)	Suppresses HD‐Zip genes that control developmental processes such as shoot apical meristem maintenance, leaf polarity, and vascular development	Z. Zhang et al., 2024
miR172b	Downregulated	AP2‐like TFs (SMZ, SNZ, TOE1, TOE2)	Induces AP2 TFs to regulate the embryogenic transition and delay flowering	Nowak et al., 2022
miR319a,b	Upregulated	TCP2, TCP3, TCP24	Downregulates TCP genes that control leaf morphology	Fang et al., 2021
miR399b	Upregulated	PHO2	Suppresses PHO2, reducing the content of phosphate in the shoot under heat stress	Rakhi et al., 2024
miR160	Upregulated	ARF10, ARF16, ARF17	Suppresses ARF genes that promote auxin response, improving root elongation under heat stress	Zimmerman et al., 2024

Abbreviations: ABA, abscisic acid; AP2, Apetala 2; ARF, auxin response factor; CSD, copper/zinc superoxide dismutase; HD‐Zip, homeodomain‐leucine zipper; HTT1, Heat‐Induced TAS1 Target1; HTT2, heat‐induced TAS1 Target2; MYB, myeloblastosis; PHO2, phosphate2; PPR, pentatricopeptide repeat; ROS, reactive oxygen species; SPL, SQUAMOSA promoter‐binding protein‐like; TCP, teosinte branched/cycloidea protein; TF, transcription factor.

3. miRNAs THAT MEDIATE HEAT STRESS REGULATION IN RICE

Rice (Oryza sativa) is a staple food crop, but by 2050, climate change could cause Pakistan to lose up to $19.5 billion due to decreased rice yields. Over 80% of Pakistan's 124 districts, affecting 33 million people, are expected to face severe impacts, with 80% of natural disasters occurring within a decade, leading to an economic loss of $17.1 billion (Rana et al., 2024).

One study demonstrated that when miR169r‐5pr was overexpressed in rice at the flowering stage, HST was improved. In response to high temperatures, the miR169 gene in rice is expressed differentially in the root and shoot (Sailaja et al., 2014).

Similar to Arabidopsis, several miRNAs in rice have been found to be downregulated in response to heat stress. They include miR169d, miR397b, miR159a.1,b, miR168, and miR172. When miR169d was downregulated in response to heat stress, NF‐YA5 was induced (Seo et al., 2024). The regulation of NF‐Y during many plant developmental stages, including root growth, chloroplast biogenesis, flowering time, fatty acid biosynthesis, and embryogenesis, is well‐documented (H. Zhang, Liu, et al., 2023). The overexpression of NF‐Y contributes to downstream gene regulation and helps plants adapt to heat and drought conditions (Sato et al., 2019).

In rice, the heat‐downregulated miR397b targets LACCASE genes (LACs), which encode copper‐containing glycoproteins and are involved in heat tolerance (J.P. Lian et al., 2024).

Additionally, it has been discovered that one family of the heat‐downregulated miRNAs, miR159, targets MYB33/MYB65 (myeloblastosis) genes, a family of genes linked to heat stress (Ramakrishnan et al., 2020; Zuo et al., 2021).

In response to heat stress, miR168 is downregulated, and its target is AGO1 (ARGONAUTE1), which plays a key role in RNA silencing and helps to regulate the expression of stress‐responsive genes such as HSP synthesis, antioxidant production, and other protective mechanisms (Zaheer et al., 2024; J. Zhou et al., 2022). Additionally, it also regulates developmental genes that influence flowering time and root architecture, enhancing rice adaptability to fluctuating environmental conditions (H. Wang et al., 2021). miR172, which targets the gene AP2 (APETALA2), is also downregulated by heat stress (D. Kumar et al., 2024). The AP2/EREBP family of TFs is crucial for several processes including hormone signaling and stress tolerance (C. Liu & Zhang, 2017). OsERF115/AP2EREBP110 from the AP2/EREBP family of rice was identified as a member of group‐Ic and was significantly activated by heat and drought treatment. The OsERF115/AP2EREBP110 proteins were localized to the nucleus in rice protoplasts and blocked the transcriptional activation of the Rab16A promoter by abscisic acid (ABA) (Park et al., 2021). When this protein is overexpressed, it improves thermotolerance in vegetative‐ and seed‐stage plants.

Some other miRNAs are upregulated in response to high‐temperature stress. They include miR156, miR396, miR398, miR319a, miR393b, and miR399a–c. In response to heat stress in rice, miR156 is upregulated, which suppresses the expressions of SPL genes (L. Li, Shi, et al., 2022). In rice and Arabidopsis, anthocyanin levels increase under heat stress while SPL9 is repressed as miR156 expression increases (Y. Wang et al., 2020). The elevated anthocyanin concentration is associated with enhanced tolerance to abiotic stress (Kaur et al., 2023). Similarly, miR396 is also upregulated, which targets OsGRF6 and OsGRF4 under heat stress, promoting rice yield by enhancing panicle branching and grain size (Mo et al., 2023).

MiR398, which targets CSD1 (copper/zinc superoxide dismutase1), is also upregulated in rice in response to heat stress (J. Li, Song, et al., 2022). The cytoplasm and the chloroplast contain the isoforms of CSD1 and CSD2, respectively (Sagasti et al., 2013; Sanyal et al., 2022). Through CCS (copper chaperone for superoxide dismutase), copper is supplied to the CSD1 and CSD2 proteins, enabling their enzymatic activity in rice. CSD1, CSD2, and CCS are downregulated by heat stress, promoting the accumulation of ROS. As a result, miR398 downregulates in response to oxidative stress, which upregulates its target CSD genes to manage oxidative stress more effectively (Guan et al., 2013).

Other miRNAs that are upregulated in rice by heat stress include miR319a, miR393b, and miR399a–c. TCPs, targets of miR319a, are downregulated under heat stress (Gull et al., 2023). TCPs are TFs that regulate the formation of gametophytes, the initiation of flowering, flower development, male and female fertility, and leaf growth (Qian et al., 2024). The miR393b, which targets TIR1 (transport inhibitor response1), an auxin receptor, is upregulated in response to heat stress (J. Jiang et al., 2022). In addition to its role in the auxin signaling pathway, TIR1 regulates the timing of flowering and heat stress response (Goswami et al., 2023). The other family of heat‐upregulated miRNA, miR399a–c, targets PHO2 (PHOSPHATE2) during the flowering stage (Ying et al., 2017). Both PHO1 and PHO2 aid in the long transit of Pi in rice from the root to the shoot (Chiou, 2020). Several miRNAs in rice and their targeted genes are described below in Table 2.

TABLE 2.

Impacts of heat stress on microRNAs (miRNAs) and target gene expressions in rice crop.

Types of miRNAs	miRNAs expression	Targeted genes	Targeted gene expressions and functions	References
miR169r‐5pr	Upregulated	NF‐YA	Aids in mitigating heat stress by indirectly influencing stress signaling pathways	H. Liu et al., 2025
miR169d	Downregulated	NF‐YA5	Induces NF‐YA genes, which help the plant adapt to heat stress by enhancing nutrient uptake and metabolic processes	Seo et al., 2024
miR396	Upregulated	OsGRF6, OsGRF4	Suppresses OsGRF6 and OsGRF4, enhancing grain yield	Mo et al., 2023
miR397b	Downregulated	LACs	Induces LACs that enhance lignin deposition and strengthen cell walls, improving water transport and protecting cells against heat damage	J.‐P. Lian et al., 2024
miR156	Upregulated	SPLs	Suppresses SPL genes that promote flowering initiation, heat stress memory, and influence leaf morphology and shoot architecture, leading to improved heat tolerance	L. Li, Shi, et al., 2022
miR159	Downregulated	MYB33, MYB65	Induces MYB genes that control anther development and flowering time	Zuo et al., 2021
miR168	Downregulated	AGO1	Induces AGO1 that regulates miRNA‐mediated gene silencing, enhancing stress response, heat tolerance, and gene regulation under heat stress	Zaheer et al., 2024
miR172	Downregulated	AP2	Enhances AP2 that controls flowering time and floral development	D. Kumar et al., 2024
miR319a	Upregulated	OsPCF6 OsTCP21	Represses TCP genes that control flowering time and floral and seed development	Gull et al., 2023
miR393b	Upregulated	TIR1	Represses TIR1 to regulate auxin signaling, controlling growth and heat stress adaptation	J. Jiang et al., 2022
miR399a–c	Upregulated	PHO2	Represses PHO2 that is involved in phosphate homeostasis	Goswami et al., 2023
miR398	Upregulated	CSD1	Suppresses CSD1 expression, compromising antioxidant defense and heat tolerance in rice under heat stress	L. Li, Shi, et al., 2022

Abbreviations: AGO1, Argonaute1; AP2, Apetala 2; CSD, copper/zinc superoxide dismutase; LAC, laccase; MYB, myeloblastosis; PNF‐YA5, nuclear transcription factor Y subunit alpha 5; PHO2, phosphate2; SPL, SQUAMOSA promoter‐binding protein‐like; TCP, teosinte branched/cycloidea protein; TIR, transport inhibitor response.

4. miRNAs THAT MEDIATE HEAT STRESS REGULATION IN WHEAT

Wheat (Triticum aestivum) is an essential crop consumed globally. It is also affected by heat stress, and specific miRNAs help the plant adapt to high temperatures. In response to heat stress, wheat exhibits a downregulation of miRNAs such as Tae‐miR818, miR156, miR169, and miR528 (F. Zhang, Yang, et al., 2022). Additionally, the heat‐responsive miRNAs, such as miR528, were found to regulate antioxidant activities and stress‐responsive mitochondrial proteins in wheat (Ravichandran et al., 2019). During the early development of wheat, terminal heat‐tolerant and heat‐sensitive genotypes were used as validation samples for miRNA‐based SSR markers, which have been recognized as key factors in improving terminal heat tolerance in wheat. Two notable diagnostic markers, miR165b and miR159c, exhibited distinct alleles, and through their use, a heat‐tolerant genotype was successfully identified (Sihag et al., 2021).

Furthermore, NF‐YA5 is a target of miR169, which shows dual behavior in wheat, such as miR169d, which is upregulated, but miR169a/c is downregulated in response to abiotic stress (particularly heat stress) in wheat (Gupta et al., 2024; Ragupathy et al., 2016). Heat stress downregulates the expression of miR169, which in turn conserves the expression of NF‐YA and FLOWERING LOCUS C (FLC) (Gol et al., 2017). This causes plants to blossom early or delay in response to heat stress. NF‐YA5 is hypothesized to be present in the guard cells of stomata and is involved in controlling the stomatal aperture (J. Wang, Mao, et al., 2024).

Other studies have shown that miR395a is barely upregulated and induces thermostability to starch biosynthetic enzymes in wheat. They regulate TFs and HSPs, and target APS1/AST (sulfurylase/affinity sulfate transporter) (Kim & Kim, 2021; Shafia Hoor & Nagesh Babu, 2024). miR395 is upregulated in response to high temperatures, increasing thermotolerance and causing glutathione to accumulate (Gahlaut et al., 2018). It was discovered that during heat stress response in wheat, miR398 was upregulated, while its target genes, CSD1, CSD2, and CCS, which control ROS buildup, were downregulated (Tiwari, 2024).

In several crops, including wheat, the miR408 family is significantly linked to abiotic stress, such as excessive heat, where it is upregulated to target the TaTOC1 gene (Gao et al., 2022). A study revealed that the overexpression of miR408, along with the repression of TaTOC1s in transgenic wheat, increased the expression of TaFT1, a crucial gene for flowering time, while the expression of TaCO1 decreased (J.‐P. Zhang, Yu, et al., 2017). The results suggest that miR408 regulates the timing and length of wheat heading by cleaving TaTOC1s mRNA, which enhances the expression of TaFT1 (J.‐P. Zhang, Yu, et al., 2017; X. Y. Zhao, Hong, et al., 2016).

Heat stress causes miR156f that targets SPL genes to be upregulated (Ruan et al., 2024). It is thought that miRNAs have a role in regulating the stress response mechanisms in plants by selectively binding to SPLs, which are known to be crucial for developmental changes in plants, including growth and yield. It has been shown that the miR156/SPL module is crucial for HST, integrating the many stress responses with the plant development process (Jerome Jeyakumar et al., 2020). This knowledge could be immensely helpful to researchers aiming to improve heat tolerance in heat‐sensitive plants such as wheat, with minimal to no yield loss. Heat stress leads to the upregulation of the miR156 family in various cereal crops besides wheat, and its target genes, Ta3711 and Ta7012, are downregulated in wheat (Xin et al., 2010).

In other studies, miR159a,b are downregulated under heat stress, while their upregulated target TaGAMYB contributes to wheat anther development and heat stress responses (Y. Liu, Li, et al., 2022). However, miR160e is upregulated and targets ARFs, which are essential for auxin‐mediated gene regulation. Upregulation of miR160e affects stamen development and the formation of the gynoecium, the female reproductive structure in flowers, highlighting its role in floral organ development through auxin signaling pathways (Ding et al., 2020). On the other hand, the HD‐Zip family of TFs, including Arabidopsis Homeobox 8 (ATHB8), PHABULOSA (PHB), REVOLUTA (REV), PHAVOLUTA (PHV), and CORONA (CAN), are key targets of miR166 (Sessa et al., 2024). Under heat stress, miR166 is upregulated, leading to the suppression of these HD‐Zip TFs, which are essential for plant development, including the regulation of leaf shape, auxiliary meristem development, and floral organ polarity during heat stress (Z. Zhang, Yang, et al., 2024).

In response to heat stress, miR167 is downregulated and induces the expression of ARFs in wheat (Marzi et al., 2024). miR167 targets ARF6 and ARF8 specifically, which regulate auxin signaling and improve the growth and development of the gynoecium and stamen in wheat (X. Chen et al., 2018; Marzi et al., 2024). miR168 is also found to be a crucial miRNA involved in the tolerance to heat stress in wheat. Through its downregulation (Mishra et al., 2023), its target AGO1 participates in plant development and stress response pathways in wheat (Saroha & Sharma, 2020). miR172a,b that target AP2 ‐like TFs is downregulated in response to heat stress (Chatterjee et al., 2023). Young seedlings exhibit higher levels of AP2‐like transcripts, which decrease as plants mature, along with the ability to flower (H. Lian et al., 2021).

In response to heat stress, miR393 is upregulated and targets TIR1/AFB (auxin signaling F‐box protein), which are auxin receptors involved in the auxin signaling pathway and regulate both the timing of flowering and the plant's response to heat stress (Du et al., 2022).

On the other hand, miR399, which targets PHO2, is upregulated and involved in the development of floral reproductive organs and seed germination (D. Luo, Usman, et al., 2024). Several studies about miRNAs in wheat are listed below in Table 3.

TABLE 3.

Impact of heat stress on microRNAs (miRNAs) and their targeted genes expression in wheat.

Types of miRNAs	miRNAs expression	Targeted genes	Targeted gene expressions and functions	References
miR169a/c	Downregulated	NF‐YA5	Induces NF‐YA5, aiding stomatal closure and enhancing wheat survival during heat stress	Gupta et al., 2024
miR395a,b	Upregulated	APS1/AST	Suppresses APS1/AST, regulating sulfur metabolism and improving heat tolerance	Shafia Hoor & Nagesh Babu, 2024
miR398	Upregulated	CSD1, CSD2, CCS	Suppresses CSDs and CCS, which maintain redox homeostasis	Tiwari, 2024
miR408	Upregulated	TaTOC1s	Suppresses TaTOC1s, inducing early flowering	Gao et al., 2022
miR156f	Upregulated	SPLs	Suppresses SPL genes that regulate plant development, including flowering, tillering, and stress responses	Ruan et al., 2024
miR159a,b	Downregulated	GAMYB	Induces GAMYB expression, a gene that regulates flowering, seed development, and abiotic stress responses in wheat, aiding adaptation to heat stress	N. Liu, Xu, et al., 2022
miR160e	Upregulated	ARFs	Suppresses ARF genes that regulate the development of the stamen and accumulation of gynoecium in wheat	Ding et al., 2020
miR166d	Upregulated	HD‐Zip‐III	Suppresses HD‐Zip‐III, which is involved in regulating shoot and root development, enhancing heat stress adaptation by modulating growth and development	Zhang et al., 2024
miR167	Downregulated	ARFs	Induces ARFs, thus regulating auxin signaling and promoting heat stress adaptation	Marzi et al., 2024
miR168	Downregulated	AGO1	Induces AGO1, a key component of RISC, influencing small RNA pathways and affecting plant development and stress responses	Mishra et al., 2023
miR172a,b	Downregulated	AP2	Induces AP2 to delay flowering	Chatterjee et al., 2023
miR393	Upregulated	TIR1, AFB	Suppresses TIR1 and AFB, which encode F‐box proteins that function as auxin receptors	Du et al., 2022
miR399a	Upregulated	PHO2	Suppresses PHO2, resulting in increased phosphate accumulation in plant tissues	D. Luo, Usman, et al., 2024

Abbreviations: AFB, auxin signaling F‐box protein; AGO1, Argonaute1; AP2, Apetala 2; ARFs, auxin response factors; CSD, copper/zinc superoxide dismutase; HD‐Zip, homeodomain‐leucine zipper; MYB, myeloblastosis; NF‐YA5, nuclear transcription factor Y subunit alpha 5; PHO2, phosphate2; RISC, RNA‐induced silencing complex; SPL, SQUAMOSA promoter‐binding protein‐like; TIR, transport inhibitor response.

5. miRNAs THAT MEDIATE HEAT STRESS REGULATION IN OTHER MAJOR CROPS

miRNAs are essential mediators of stress‐responsive pathways that influence hormone signaling, antioxidant response, and protein homeostasis to enhance heat tolerance in crops. In Brassica rapa, a widely cultivated plant, heat stress has a significant impact on miRNA activities. For example, miR399b is downregulated under high temperature, while miR168, miR156g/h, miR167, and miR398 are upregulated. miR398 targets the BrCSD1 gene and plays a critical role in HST (J. Yu et al., 2023). At high temperatures, BrCSD1 expression rises initially. However, it is ultimately suppressed by miR398 after a brief delay, effectively fine‐tuning the heat stress response (Cao et al., 2022; J. Li, Song, et al., 2022; J. Yu et al., 2023). Similarly, miR156g/h is upregulated and targets SPL2, enhancing the expression of flowering genes and contributing to heat stress adaptation by improving the plant's response to high temperatures (Jerome Jeyakumar et al., 2020; X. Yu et al., 2012; F. Zhang, Yang, et al., 2022). Notably, miR156 is also involved in establishing a long‐lasting heat stress memory. Another key miRNA in B. rapa is miR167, which targets ARF6, a gene involved in auxin signal transduction (Ma et al., 2022). miR167 is upregulated under heat stress and activates auxin responses, enabling plants to adapt to heat (P. Luo et al., 2022; X. Yu et al., 2012). Similarly, miR168 is upregulated and targets AGO1 (Rani et al., 2023). AGO1 has a significant function in gene silencing through participation in RNA interference (RNAi) pathways and miRNA biosynthesis. Mature miRNAs must be positioned on AGO1 and form an RNA‐induced silencing complex in order to degrade their target mRNAs (Leitão & Enguita, 2022). Conversely, miR399b, which targets PHO2, is downregulated under high temperatures. This downregulation of the miRNA enhances the uptake of nutrients, such as phosphate and nitrogen, enabling plants to withstand heat‐induced nutritional stress (Ma et al., 2022).

In sunflower (Helianthus annuus), miRNAs also respond dynamically to heat stress. miR396, targeting HaWRKY6, is downregulated under high temperatures, where HaWRKY6 is a TF facilitating responses to abiotic stress (Gahlaut et al., 2018). On the other hand, miR398 in sunflower shows a complex regulatory pattern, as it is both upregulated and downregulated to modulate genes such as CCS and CSD1/2, keeping oxidative stress responses in balance with other cellular functions (Z. Li et al., 2024). This regulation triggers a cascade of responses, including the accumulation of HSFs and HSPs, both of which are vital for mitigating the damaging effects of ROS during heat stress (J. Li, Song, et al., 2022; Ma et al., 2022).

In switchgrass (Panicum virgatum), miR156, miR395, miR164, miR319, and miR393 are upregulated, while miR168, miR528, and miR159 are downregulated in response to elevated temperatures. miR156 delays flowering and increases plant height in switchgrass by inhibiting PvSPL6 under high temperatures. In contrast, PvSPL6 accelerates flowering and reduces internode length and number (Cai et al., 2022). miR395 targets APS/AST and is upregulated under heat stress, transporting sulfate in the leaf to mitigate the effects of increased temperature (Hivrale et al., 2016; Muhammad et al., 2024). Furthermore, miR164, which targets NAC genes (NAM, ATAF, and CUC), is upregulated during heat stress. This upregulation improves root architecture for better nutrient uptake and enhances photosynthesis, ultimately boosting biomass production (Tsai et al., 2023). High temperature induces miR319, which modulates TCP TFs like PvPCF5, enhancing stem diameter, leaf elongation, and plant height, and raising biomass yield (C. Chen et al., 2024; Y. Liu, Yan, et al., 2020). Additionally, the miR393 upregulation targets TIR1, promoting root and flower growth under heat and cold stress (J. Jiang et al., 2022). Conversely, miR528 targets Cu/Zn‐SOD (where SOD stands for superoxide dismutase) genes, enhancing shoot proliferation and regeneration in switchgrass when it is downregulated under heat stress (Han et al., 2024). miR159 targets MYB s (Amini et al., 2023) and is downregulated in organs such as seeds and anthers where MYBs are maintained at relatively high levels, where MYBs play significant roles in seed germination and the development of floral reproductive components (Hivrale et al., 2016). miR168, which targets AGO1, is highly abundant in switchgrass and downregulated under heat stress (X. Liu, Tan, et al., 2020). AGO1 is an integral part of the stress response mechanisms in plants.

In celery (Apium graveolens), miR395 targets APS/AST and is upregulated in response to heat stress (Gahlaut et al., 2018). miR395 regulates the genes encoding APS1, APS3, and APS4 ATP sulfurylases (C. Liu, Ma, et al., 2022). It also affects the efficiency of sulfate transporters 2 and 1 (SULTR2;1 or AST68 [affinity sulfate transporter 68]), and controls the pathway for sulfur uptake (Yuan et al., 2016). Additionally, miR160, which targets ARFs associated with protein folding, is also upregulated, aiding root and flower development (S. Li et al., 2014; Y. Zhang, Zhou, et al., 2022). It was discovered that miR408, which targets phytocyanin genes (plantacyanin, cupredoxin, uclacyanin), is upregulated in celery while downregulated in switchgrass under heat stress, boosting antioxidant defense and copper homeostasis (Gahlaut et al., 2018; Muhammad et al., 2024). This helps reduce oxidative damage, improving HST and stabilizing cellular functions. On the other hand, miR168, which targets AGO1, is downregulated during heat stress and plays a key role in stress response and developmental activities (S. Li et al., 2014; M. Li, Li, et al., 2022). Similarly, in empress tree (Paulownia tomentosa), miR168a,b are also downregulated, with their target AGO1 becoming highly active under high temperatures (D. Xiao et al., 2022). Meanwhile, miR167c,d are downregulated, targeting ARFs, which participate in auxin‐mediated signaling pathways (D. Xiao et al., 2022).

In manioc/cassava (Manihot esculenta), miR156, targeting SPLs, which regulates developmental phase transition, is downregulated by heat stress (J. Liu et al., 2015; Zuo et al., 2021). miR159a is also downregulated, and its targets, HTH (helix‐turn‐helix), function similarly to GA MYB TFs (Ballén et al., 2013; Gahlaut et al., 2018). MYB33 and MYB65 are involved in the development of floral reproductive organs and seed germination (Qian et al., 2024).

In barley (Hordeum vulgare), miR168 primarily targets AGO1B and is upregulated under high temperatures (Kruszka et al., in press). It also regulates other miRNAs at both transcriptional and posttranscriptional levels (Kruszka et al., 2014). miR167 and miR160 are both upregulated in barley and target HvARF8 and HvARF13/17, aiding in auxin‐mediated stress adaptations (P. Luo et al., 2022). HvARFs function in stress adaptations and a number of developmental stages by regulating the auxin‐mediated genes (Hertig et al., 2023). Additionally, miR166a is also upregulated under high temperatures, targeting HD‐Zips (PHV, REV, and HOX9) and influencing plant development (Kruszka et al., [Link], 2014). It also regulates two HOX TFs, HORVU3H026990 and HORVU0H010250 (Kruszka et al., in press).

In sweet potato (Ipomoea batatas), miR160, which targets ARFs, is upregulated in response to both salt and heat stress, promoting root development (Sun et al., 2022). Similarly, miR398 is upregulated in I. batatas and contributes to the responses to heat stress conditions by targeting CSDs, CCS1, and COX5b‐1 (Sun et al., 2022).

In alfalfa (Medicago sativa), overexpression of miR156 targets SPL12a and SPL8a, enhancing HST, with increased antioxidants and proline acting as indicators of stress resilience (Arshad & Hannoufa, 2022). Overexpression of miR319d (sha‐miR319d) from wild tomato (Solanum habrochaites) improves heat stress resistance at 40°C by modulating heat stress responses and ROS signaling. However, it may reduce cold tolerance at 4°C by downregulating GAMYB‐like1, a gene linked to cold stress responses. It illustrates the complex role of miR319d in stress responses in tomato (Solanum lycopersicum) (Shi et al., 2019). In banana (Musa paradisiaca), miR156 enhances HST by targeting SPL genes, with its expression increased by 10% at the microspore release stage when under heat stress (Xia et al., 2023). miR156 plays both positive and negative key regulatory roles in influencing anther development (M. Zhang, Zhang, et al., 2023). Additionally, the overexpression of miR398 in common bean (Phaseolus vulgaris) confers heat tolerance by promoting the cleavage of CSD mRNAs (Y. Zhou et al., 2020).

In cotton (Gossypium herbaceum), heat stress reduces the expression of miR172 and induces the regulation of its targeted gene TOE to modulate floral organ development (J. Chen et al., 2020). Meanwhile, in soybean (Glycine max), miR172 is upregulated and targets AP2 TFs to enhance nodulation and nitrogenase activity (Yan et al., 2013; F. Zhang, Yang, et al., 2022). miRNAs in other major crops are listed below in Table 4.

TABLE 4.

Impacts of heat stress on microRNAs (miRNAs) and targeted gene expressions in other major crops.

Crop species	Types of miRNAs	miRNAs expression	Targeted genes	Targeted gene functions	References
Brassica rapa	miR398	Upregulated	CSD1, CSD2	Suppresses CSD1/2, which mitigates oxidative stress	J. Yu et al., 2023
	miR156g/h	Upregulated	SPL2	Suppresses SPL2, which is involved in flowering regulation	Jerome Jeyakumar et al., 2020
	miR167	Upregulated	ARF6	Suppresses ARF6, which aids in lateral root and flower development	Ma et al., 2022
	miR168	Upregulated	AGO1	Suppresses AGO1, impacting leaf morphology, root development, and flowering time in the plant	Rani et al., 2023
	miR399b	Downregulated	PHO2	Induces PHO2, enhancing nutrient uptake	Ma et al., 2022
Helianthus annuus	miR396	Downregulated	WRKY6	Induces WRKY6 to enhance heat tolerance	Gahlaut et al., 2018
	miR398	Upregulated	CSDs	Suppresses CSDs, reducing oxidative stress	Z. Li et al., 2024
Panicum virgatum	miR159	Downregulated	MYBs	Increases the levels of MYB TFs, thereby influencing the plant's adaptive responses to environmental challenges	Amini et al., 2023
	miR164	Upregulated	NAC (NAM, ATAF, and CUC)	Suppresses NAC genes, promoting lateral root formation and enhancing nutrient uptake under stress	Tsai et al., 2023
	miR156	Upregulated	PvSPL6	Suppresses PvSPL6 to delay flowering under heat stress	Cai et al., 2022
	miR168	Downregulated	AGO1	Induces AGO1, altering the miRNA regulatory network.	X. Liu, Tan, et al., 2020
	miR319	Upregulated	TCPs	Suppresses TCP genes such as PvPCF5, affecting leaf development	C. Chen et al., 2024
	miR528	Downregulated	Cu/Zn‐SOD	Induces Cu/Zn‐SOD, promoting shoot branching and regeneration	Han et al., 2024
	miR393	Upregulated	TIR1	Suppresses TIR1, promoting root and flower growth	J. Jiang et al., 2022
Apium graveolens	miR160	Upregulated	ARF10, ARF16, ARF17	Suppresses ARF10, ARF16, and ARF17, affecting root architecture, seed germination, and growth	Y. Zhang, Zhou, et al., 2022
	miR168	Downregulated	AGO1	Boosts AGO1 expression, regulating stress response mechanisms	S. Li et al., 2014; M. Li, Li, et al., 2022
	miR395	Upregulated	APS, AST	Suppresses APS and AST, affecting sulfur metabolism and glutathione accumulation to enhance stress resistance, thermotolerance, and metabolic efficiency	Gahlaut et al., 2018
	miR400	Upregulated	Phytocyanin genes	Suppresses Phytocyanin genes, stabilizing cellular functions and maintaining metabolic processes	Muhammad et al., 2024
Paulownia tomentosa	miR167c,d	Downregulated	ARFs	Induces ARFs, promoting auxin signaling	D. Xiao et al., 2022
	miR168a,b	Downregulated	AGO1	Induces AGO1 (highly active under high temperatures)	D. Xiao et al., 2022
Manihot esculenta	miR156	Downregulated	SPLs	Induces SPLs, promoting developmental transition	Zuo et al., 2021
	miR159a	Downregulated	MYB‐like HTH	Upregulates MYB‐like TFs, activating genes that aid heat stress management, floral development, and seed germination	Gahlaut et al., 2018
Hordeum vulgare	miR160, miR167	Upregulated	HvARF8 HvARF13/17	Downregulates ARFs, disrupting floral organ formation and reproductive development	P. Luo et al., 2022
	miR166a	Up regulated	HD‐Zips (PHV, REV, HOX9)	Suppresses HD‐Zips involved in vascular patterning and leaf development	Kruszka et al., in press
	miR168	Upregulated	AGO1B	Suppresses AGO1B, aiding heat stress adaptation	Kruszka et al., in press
Ipomoea batatas	miR160	Upregulated	ARFs	Downregulates ARFs, mediating the auxin signaling pathway to enhance root formation	Sun et al., 2022
	miR398	Upregulated	CSDs, CCS, COX5b‐1	Reduces CSDs, CCS1, and COX5b‐1 expression, aiding in ROS accumulation and stress adaptation	Sun et al., 2022
Medicago sativa	miR156	Upregulated	SPL12a, SPL8a	Downregulates SPLs, enhancing the plant's resilience to heat	Arshad & Hannoufa, 2022
Solanum lycopersicum	miR319d	Upregulated	GAMYB‐like1	Enhances antioxidant activity and reduces oxidative stress by downregulating GAMYB‐like1	Shi et al., 2019
Musa paradisiaca	miR156	Upregulated	SPLs	Suppresses the expression of SPL genes that promote flowering and fruiting	Xia et al., 2023
Phaseolus vulgaris	miR398	Upregulated	CSD1, CSD2	Suppresses CSD1 and CSD2, which are involved in oxidative stress responses	Y. Zhou et al., 2020
Gossypium herbaceum	miR172	Downregulated	TOE	Induces TOE expression, enhancing floral organ development	J. Chen et al., 2020
Glycine max	miR172	Upregulated	AP2 TFs	Suppresses AP2 TFs, regulating floral organ, flowering, nodules number, and nitrogenase activity	F. Zhang, Yang, et al., 2022

Abbreviations: AGO1, Argonaute1; AP2, Apetala 2; ARF, auxin response factor; CSD, copper/zinc superoxide dismutase; HD‐Zip, homeodomain‐leucine zipper; MYB, myeloblastosis; PHO2, phosphate2; ROS, reactive oxygen species; SOD, superoxide dismutase; SPL, SQUAMOSA promoter‐binding protein‐like; TCP, teosinte branched/cycloidea protein; TF, transcription factor; TIR, transport inhibitor response.

6. INTEGRATING CROSSTALKS BETWEEN miRNAs AND OTHER RNAs IN PLANTS UNDER HEAT STRESS

Recent advances in high‐throughput sequencing technologies have uncovered the pivotal roles of ncRNAs in regulating plant development and responses to environmental stresses (L. Zhang, Lin, et al., 2023). Under elevated temperatures, a coordinated network of ncRNAs, including miRNAs, small interfering RNAs (siRNAs), long noncoding RNAs (lncRNAs), and circular RNAs (circRNAs), is activated in plants to collectively modulate gene expressions and signaling pathways to promote heat tolerance (J. Zhao, He, et al., 2016). These ncRNAs engage in dynamic crosstalks, forming multilayered and finely tuned regulatory systems that function across transcriptional, posttranscriptional, and epigenetic levels. One prominent mechanism involves the competing endogenous RNA (ceRNA) network, in which lncRNAs and circRNAs serve as molecular sponges, sequestering miRNAs to regulate their downstream target genes involved in hormone signaling, stress tolerance, and developmental transitions. This intricate interplay among ncRNAs is essential for maintaining cellular homeostasis, safeguarding reproductive processes, and enabling adaptive responses to heat stress. Elucidating the complexity of these integrated ncRNA regulatory networks offers valuable insights for engineering climate‐resilient crops through advanced molecular breeding and gene‐editing approaches. The integrated ncRNA network is further dissected below.

6.1. Crosstalk between miRNAs and siRNAs in plants under heat stress

Small RNAs, particularly miRNAs and siRNAs, orchestrate complex regulatory networks that fine‐tune gene expressions in response to environmental stimuli such as heat stress. siRNAs, typically 21–24 nt in length, play a pivotal role in plant heat tolerance by mediating gene silencing through RNAi at both transcriptional and posttranscriptional levels (Abdellatef et al., 2021). Under elevated temperatures, siRNAs not only facilitate the degradation of their target mRNAs but also direct the epigenetic modifications of their target genes, including DNA methylation and histone methylation, thereby contributing to transcriptional gene silencing (Q. Jin, Chachar, et al., 2024; N. Liu & Avramova, 2016). In the RNA‐directed DNA methylation (RdDM) pathway, siRNAs are generated from double‐stranded RNA precursors by Dicer‐like enzymes, particularly DCL3, and subsequently loaded onto Argonaute4 (AGO4) proteins (Ali & Tang, 2025). The AGO4–siRNA complex interacts with nascent transcripts synthesized by RNA Polymerase V, guiding DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2) to catalyze cytosine methylation in the CG, CHG, and CHH sequence contexts (Ali & Tang, 2025; Kumari et al., 2022). This methylation leads to chromatin condensation and transcriptional repression of the target loci, including those of transposable elements (TEs), thus maintaining genomic integrity under heat stress. For instance, 24‐nt siRNAs repress the heat‐activated retrotransposon ONSEN through DNA methylation to mitigate genome instability (Ito et al., 2011). Additionally, siRNAs recruit histone methyltransferases to specific genomic loci, facilitating the deposition of repressive histone marks such as H3K9me2, further reinforcing transcriptional repression (R. Yu et al., 2018). Histone Deacetylase 6 (HDA6) also contributes to chromatin‐level regulations by promoting histone deacetylation and cooperating with RdDM components, such as NRPD1/NRPD2, to silence nearby TEs, such as AtSN1 and Solo LTR in Arabidopsis, thereby stabilizing the expressions of heat‐responsive genes (Popova et al., 2013).

Beyond their independent functions, increasing evidence points to a functional crosstalk between miRNAs and siRNAs in regulating heat stress responses. For example, heat stress induces the co‐expression of miRNAs and phased secondary siRNAs (phasiRNAs) in peanut (Arachis hypogaea), where conserved miRNAs, such as miR1509 targeting serine/threonine‐protein phosphatase 7 (PP7) and ahy‐miR3514 targeting pentatricopeptide repeat (PPR) genes, initiate phasiRNA production from both noncoding and mRNA loci. These phasiRNAs reinforce posttranscriptional gene silencing, creating a regulatory feedback loop that fine‐tunes stress‐responsive pathways (Mittal et al., 2023). This coordinated action exemplifies how miRNAs can directly trigger siRNA biogenesis, integrating distinct RNA silencing layers to enhance heat tolerance. Similarly, in Arabidopsis, the miRNA‐triggered TAS1‐derived siRNA pathway has been implicated in thermoregulation. TAS1a‐derived siRNAs target HEAT‐INDUCED TAS1 TARGET1 (HTT1) and HTT2, modulating the expressions of HSFs and heat shock proteins (Hsps), which are key components in heat tolerance (S. Li et al., 2014). Recent studies in the reproductive tissues of flax (Linum usitatissimum) revealed the dynamic heat stress‐induced reprogramming of small RNA expressions, where key miRNAs such as miR2118 and miR2275 triggered phasiRNA production at the 21‐PHAS and 24‐PHAS loci. However, the majority of the 24‐PHAS‐like loci lacked identifiable miRNA triggers and were significantly downregulated under heat stress, suggesting there may be other unexplored regulatory mechanisms (Pokhrel & Meyers, 2022). In rice (O. sativa), the miR390–OsSGS3a/b–tasiRNA–OsARF3 pathway regulates heat tolerance through modulation of auxin‐responsive genes. OsSGS3a, together with OsSGS3b, is essential for TAS3‐derived tasiRNA biogenesis, which represses OsARF3 TFs acting as negative regulators of heat tolerance. Loss‐of‐function mutations in OsSGS3a (SUPPRESSOR OF GENE SILENCING 3a) reduce tasiRNA accumulation, upregulate OsARF3 expression, and impair floret development under heat stress, underscoring its critical role in reproductive heat tolerance (Gu et al., 2023).

Despite these insights, direct evidence elucidating the coordinated expression patterns and mechanistic interactions between miRNAs and siRNAs under heat stress remains limited. This gap underscores the need for integrative studies employing transcriptomic, degradomic, and epigenomic approaches to decode the synergistic regulation by small RNAs in plant heat tolerance. Such efforts will be instrumental in advancing our understanding of the multilayered RNA regulatory networks and in developing heat‐resilient crop varieties.

6.2. Crosstalks between miRNAs and lncRNAs in plants under heat stress

lncRNAs, typically longer than 200 nt in length, lack protein‐coding potential but serve as pivotal regulators of gene expressions at the transcriptional, posttranscriptional, and epigenetic levels (Statello et al., 2021). Transcribed by various RNA polymerases (Pol I–V), lncRNAs often mirror the structural features of mRNAs, including a 5′‐methylguanosine cap and a 3′‐poly(A) tail. Their origin is diverse, stemming from genomic events such as chromosomal rearrangements, retrotranspositions, and insertions of TEs (L. Zhang, Lin, et al., 2023). Based on their genomic locations relative to the nearest protein‐coding genes, lncRNAs are classified into promoter‐associated, enhancer‐associated, gene body‐associated, and intergenic (lincRNA) types (Chodurska & Kunej, 2025; Marques et al., 2013; Poloni et al., 2024). These molecules are increasingly recognized for their roles in chromatin remodeling, gene regulation, genome stability, and plant adaptation to environmental stresses.

Unlike small RNAs, lncRNAs can act as structural, regulatory, or catalytic molecules that influence diverse biological processes such as translation, metabolism, and intracellular signaling (Mattick et al., 2023; Wierzbicki et al., 2021). Under heat stress conditions, the expressions of specific lncRNAs are significantly modulated, and they contribute to heat tolerance through multiple mechanisms. In the heat‐tolerant wheat (T. aestivum) cultivar TAM107, for instance, TalnRNA5 and TalnRNA27 are notably upregulated in response to high temperatures. The upregulation of TalnRNA5 is associated with increased histone acetylation, suggesting the epigenetic regulation of heat‐responsive genes.

An emerging paradigm in the regulation of gene expression under heat stress is the crosstalk between lncRNAs and miRNAs, often mediated through the ceRNA network. In this context, lncRNAs act as endogenous target mimics (eTMs), sequestering specific miRNAs and thereby influencing the expressions of miRNA target genes. For example, in Chinese cabbage (B. rapa), the heat‐responsive lncRNA TCONS_00048391 functions as an eTM for bra‐miR164a. This interaction regulates the expression of the NAC1 TF (Bra030820), ultimately contributing to enhanced thermotolerance in the heat‐tolerant genotypes GHA and XK (A. Wang et al., 2020). Further evidence for such a regulatory network was provided by a study on Pheonix qiongdaoensis, which identified 25 heat‐responsive lncRNAs (e.g., TalnRNA5, TalnRNA27) and 15 miRNAs (e.g., miR166a, miR319e, miR399f) that interacted with one another as part of a ceRNA network. These regulatory molecules modulate gene expressions related to protein processing, hormone signaling, and key TFs such as NACs and MYBs. Their coordinated action was shown to promote root development and cellular homeostasis under heat stress, as validated through RNA‐seq and qRT‐PCR analyses (J. Xu et al., 2020). In another example, the lincRNA SABC1 was reported to repress the nearby gene encoding the TF NAC3 in cis, which indirectly activates ISOCHORISMATE SYNTHASE 1 (ICS1), a gene involved in salicylic acid biosynthesis. This regulation led to a reduced immune response and improved growth under stress conditions (N. Liu, Xu, et al., 2022). Similarly, in Populus simonii, heat stress triggered the epigenetic reprogramming of DNA methylation and revealed the co‐expression of PsiLNCRNA00268512 and Psi‐MIR396e within the SDMR162 locus. This lncRNA acts as a target mimic, fine‐tuning miR396e expression in a methylation‐dependent manner (Song et al., 2016).

Collectively, these findings emphasize the pivotal roles of miRNA–lncRNA interactions in plant under heat stress. Through the formation of ceRNA networks, lncRNAs can modulate miRNA activities, thereby fine‐tuning gene expressions and ensuring appropriate transcriptional responses to heat stress. This multilayered regulation offers promising avenues for genetic engineering strategies aimed at enhancing crop resilience under climate change scenarios.

6.3. Crosstalks between miRNAs and circRNAs in plants under heat stress

circRNAs are a unique class of endogenous ncRNA characterized by covalently closed loop structures that lack both a 5′‐cap and a 3′‐poly(A) tail. This circular conformation confers high structural stability, protecting them from exonucleolytic degradation and distinguishing them from their linear counterparts. As a result, circRNAs are increasingly recognized as key regulators of gene expressions through mechanisms such as miRNA sequestration, transcriptional regulation, and interactions with RNA‐binding proteins (P. Zhang et al., 2020; D. Zhang, Ma, et al., 2024). In plants, circRNAs have gained attention for their involvement in developmental processes and responses to various abiotic stresses, including heat stress. For example, Tang et al. (2018) identified 149 differentially expressed circRNAs (DEcircRNAs) in maize (Zea mays) in response to environmental stresses such as heat, cold, and drought. Similarly, R. Zhou et al. (2019) reported 73 DEcircRNAs during seed germination in tomato (S. lycopersicum) under high‐temperature stress. GO analysis indicated that the host genes of these DEcircRNAs were mainly enriched in metabolic processes, cellular processes, catalytic activities, and molecular binding, suggesting a regulatory role of circRNAs during the early stages of stress response.

The functional relationship between circRNAs and miRNAs is particularly notable under stress conditions. circRNAs can function as ceRNAs or “miRNA sponges,” binding and sequestering miRNAs to modulate their availability and, consequently, the expressions of miRNA target genes. This circRNA–miRNA–mRNA interaction forms a multilayered regulatory network that fine‐tunes gene expressions under stress. Recent studies have started to explore this crosstalk in the context of heat stress. In cotton (G. herbaceum) restorer lines with differential fertility stability under high temperatures, R. Wang, Zhang, et al. (2024) identified 250 heat‐responsive circRNAs. Among them, the key DEcircRNAs were involved in regulating pollen fertility, and two notable ceRNA modules were thus proposed: circRNA346–miR159a–MYB33 and circRNA484–miR319e–MYB33. These modules suggest a potential regulatory mechanism linking circRNAs and miRNAs to heat tolerance during reproductive development. In another significant study, Y. Wang, Xiong, et al. (2019) conducted strand‐specific RNA sequencing in photo‐thermosensitive genic male sterile rice lines (WXS[S] vs. WXS[F]) across different developmental stages. They identified 9994 circRNAs, including 186 that were differentially expressed. Bioinformatics analyses, including GO, Kyoto Encyclopedia of Genes and Genomes, and ceRNA network prediction, revealed that 15 of these circRNAs might act as miRNA sponges regulating fertility‐ and stress‐related genes during reproductive stages. These findings further support the hypothesis that the circRNA–miRNA crosstalk is critical in modulating plant responses to heat stress, particularly in reproductive tissues.

Despite these advances, the experimental validation of circRNA–miRNA–mRNA networks remains limited, especially under heat stress conditions. Future research should focus on the functional characterization of circRNAs using gene editing technologies such as the CRISPR/Cas system, transcriptomic profiling, and molecular assays to confirm their roles in miRNA sequestration and gene regulation under heat stress.

6.4. Integrating the miRNA–lncRNA–circRNA crosstalk network in plants under heat stress

The regulatory interplay among miRNAs, lncRNAs, and circRNAs establishes a complex and dynamic network that fine‐tunes gene expressions and enhances plant resilience under high‐temperature stress. These ncRNAs can function as ceRNAs, sequestering miRNAs and thereby modulating the expressions of their target genes. For instance, in cucumber (Cucumis sativus), a heat stress‐responsive ceRNA network was identified wherein lncRNAs (TCONS_00031790 and TCONS_00014332) and circRNAs (novel_circ_001543 and novel_circ_000876) competitively bound to miR9748, ultimately influencing the expressions of genes involved in plant hormone signaling pathways associated with heat tolerance (X. He et al., 2020). Similarly, in Populus euphratica, 14 circRNAs and 33 lncRNAs were shown to co‐decoy miR156, promoting the expressions of 12 SPL genes, which play key roles in heteromorphic leaf development and phase transition under heat stress conditions (Qin et al., 2022).

Collectively, these findings underscore the pivotal roles of integrated miRNA–lncRNA–circRNA crosstalks in establishing sophisticated ceRNA regulatory modules, contributing to developmental plasticity and adaptive responses during heat stress in plants. Examples are listed below in Table 5.

TABLE 5.

Regulatory networks of noncoding RNAs (ncRNAs) and microRNAs (miRNAs) in response to heat stress across various crops.

Crops	ncRNA components	miRNAs	Targeted genes	Pathway/functional insight	References
Cucumber (Cucumis sativus)	lncRNAs (TCONS_00031790, TCONS_00014332), circRNAs (novel_circ_001543, 000876)	miR9748	Genes related to indole‐3‐acetic acid (IAA) and 1‐aminocyclopropane‐1‐carboxylic acid (ACC)	ceRNA network sequesters miR9748 to regulate hormone‐mediated heat tolerance	X. He et al., 2020
Populus euphratica	14 circRNAs, 33 lncRNAs	miR156	12 SPL genes	lncRNAs and circRNAs co‐decoy miR156 to promote heteromorphic leaf development under stress	Qin et al., 2022
Chinese cabbage (Brassica rapa)	lncRNA TCONS_00048391	bra‐miR164a	NAC1	lncRNA acts as eTM to sequester miR164a, enhancing heat tolerance	Wang et al., 2019
Phoenix qiongdaoensis	25 lncRNAs, 15 miRNAs (e.g., TalnRNA5, miR166a, miR319e, miR399f)	miR166a, miR319e, miR399f	NAC, MYB transcription factors	ceRNA networks coordinate protein processing, hormone signaling, and root development under heat	J. Xu et al., 2020
Populus simonii	lncRNAs (PsiLNCRNA00268512, miRNA, Psi‐mir396e)	miR396e	Targets within SDMR162 locus	epigenetically modulated lncRNA–miRNA interactions fine‐tune gene regulation under heat	Song et al., 2016
Cotton (Gossypium herbaceum)	circRNAs (circRNA346, circRNA484)	miR159a, miR319e	MYB33	circRNAs act as miRNA sponges to regulate pollen fertility under heat stress	R. Wang, Zhang, et al., 2024
Rice	circRNAs	multiple miRNAs	fertility‐related genes	circRNAs act as miRNA sponges to regulate heat tolerance at reproductive stage	Y. Wang, Xiong, et al., 2019
	miR390, tasiRNA, SGS3	miR390	OsARF3	Thermosensitive SGS3 regulates auxin signaling and ROS detox via miR390–tasiRNA–OsARF3 pathway	Gu et al., 2023
Wheat (Triticum aestivum)	lncRNAs (TalnRNA5, TalnRNA27)	Not specified	heat‐responsive genes	lncRNAs upregulated under heat; associated with histone acetylation to promote gene activation	Xin et al., 2011
Arabidopsis	miRNAs and TAS1‐derived siRNAs	miR173	HTT1, HTT2	miRNA triggers tasiRNA cascade regulating HSFs and HSPs under heat	S. Li et al., 2014
Linum usitatissimum (flax)	miRNAs (miR2118, miR2275), 21‐/24‐PHAS loci	miR2118, miR2275	Reproductive phasiRNAs	Heat‐induced reprogramming of small RNAs; phasiRNAs downregulated, hinting at unexplored regulation	Pokhrel & Meyers, 2022
Peanut (Arachis hypogaea)	phasiRNAs	miR1509, miR3514	PP7, PPR	miRNAs induce phasiRNA cascades, promoting heat tolerance via posttranscriptional silencing	Mittal et al., 2023

Abbreviations: circRNA, circular RNA; eTM, endogenous target mimic; HSF, heat shock factors; HTT1, Heat‐Induced TAS1 Target1; HTT2, heat‐induced TAS1 Target2; lncRNA, long noncoding RNA; MYB, myeloblastosis; phasiRNAs, phased secondary siRNAs; PPR, pentatricopeptide repeat; ROS, reactive oxygen species; siRNA, small interfering RNA; SPL, SQUAMOSA promoter‐binding protein‐like.

The coordinated interactions among ncRNAs, including miRNAs, siRNAs, lncRNAs, and circRNAs, form a multilayered regulatory network essential for plant adaptation to heat stress, as summarized in Figure 3. These ncRNAs modulate gene expressions through transcriptional and posttranscriptional mechanisms, epigenetic modifications, and ceRNA networks. By targeting key TFs and signaling pathways, their crosstalks fine‐tune heat tolerance‐related processes such as pollen development, root growth, and stress hormone regulation. Understanding this dynamic interplay provides a promising framework for developing heat‐resilient crop varieties under changing climate conditions.

FIGURE 3.

Crosstalk of microRNAs (miRNAs) with circular RNAs (circRNAs) and long noncoding RNAs (lncRNAs) in plants under heat stress. This flowchart illustrates the regulatory crosstalk among noncoding RNAs (ncRNAs) in plant heat stress responses. The miR390 triggers tasiRNA production via OsSGS3a/b, which represses OsARF3. Loss of OsSGS3a reduces tasiRNAs, leading to the upregulation of OsARF3 and increased heat tolerance by maintaining reactive oxygen species (ROS) homeostasis. Similarly, miR1509 targeting serine/threonine‐protein phosphatase 7 (PP7) and ahy‐miR3514 targeting pentatricopeptide repeat (PPR) genes initiates phased secondary siRNA (phasiRNA) production, which amplifies transcriptional gene silencing to fine‐tune stress responses. In lncRNAs and miRNAs crosstalk, heat stress demethylases the SDMR162 locus, revealing the co‐expression of PsiLNCRNA00268512 and Psi‐MIR396e. This interaction shows the downregulation of miR396e expression and enhances GRFs (growth‐regulating factors) expression for improved root growth. TalnRNA5 and TalnRNA27 interact with miR166a, miR319e, and miR399f to regulate NAC and MYB transcription factors, promoting root development and cellular homeostasis under heat stress. Similarly, the interaction of TCONS_00048391 with bra‐miR164a regulates the expression of the NAC1 transcription factor (Bra030820), ultimately contributing to enhanced heat tolerance by promoting better lateral root development. The circRNAs regulate pollen fertility through two ceRNA modules: circRNA346–miR159a–MYB33 and circRNA484–miR319e–MYB33, indicating a link between circRNAs, miRNAs, and heat tolerance during reproductive development. The ceRNA network of lncRNAs (TCONS_00031790 and TCONS_00014332) and circRNAs (novel_circ_001543 and novel_circ_000876) competitively binds to miR9748, affecting gene expression in plant hormone signaling pathways linked to heat tolerance. Similarly, 14 circRNAs and 33 lncRNAs co‐decoy miR156, promoting 12 SPL (SQUAMOSA promoter‐binding protein‐like) gene expressions, crucial for leaf development and phase transition during heat stress.

7. miRNAs‐BASED STRATEGIES FOR DEVELOPING FUTURE CROPS AGAINST HEAT STRESS

miRNAs play a central role in regulating gene expression under environmental stresses, including heat stress, by targeting key TFs, signaling molecules, and stress‐responsive genes (de Oliveira et al., 2025). CRISPR/Cas9 technology enables site‐specific modifications such as gene knockouts, insertions, or base editing to enhance the expression of beneficial genes or silence negative regulators of thermotolerance (Yadav et al., 2023). Researchers are now targeting ncRNAs (miRNAs and lncRNAs) using CRISPR/Cas to enhance plant resilience to temperature stress. A study explored their regulatory roles, genome‐editing applications, and potential in developing climate‐smart crops (Labonno et al., 2025). CRISPR/Cas9 can be used to modify miRNA‐binding sites within the 3′ untranslated region (UTR) or coding regions of stress‐related genes, where miRNAs typically bind to suppress gene expression. By introducing targeted mutations at these sites, the binding of miRNAs is disrupted, leading to increased expression of the target genes and improved HST in plants. For instance, CRISPR/Cas9 has been used to edit the miR156 target site in the 3′‐UTR of the TaSPL13 gene in wheat, generating various insertions and deletions that led to a twofold increase in its expression (Y. Liu et al., 2016). With the advancement of genome editing technologies, particularly CRISPR/Cas9, it is now possible to precisely manipulate miRNA genes or their target sites to enhance heat tolerance in plants. For example, researchers utilized dual sgRNA CRISPR constructs to fine‐tune miRNA loci, resulting in graded reductions in mature miRNA levels and correlated improvements in phenotypic traits related to stress in the tetraploid potato (Lukan et al., 2022).

Meanwhile, miRNAs are emerging as valuable molecular markers in plant breeding due to their crucial roles in regulating stress‐responsive genes, including those involved in heat tolerance. Their expression patterns often differ significantly between heat‐tolerant and heat‐sensitive genotypes, making them reliable indicators for screening elite cultivars under heat stress (Mangrauthia et al., 2017). Unlike traditional markers, miRNA‐based markers reflect functional genomic changes, offering a more direct link to stress adaptation mechanisms. For instance, polymorphic miR159c and miR165b‑SSR markers in wheat reliably differentiate heat‑tolerant from susceptible genotypes, facilitating marker‑assisted selection for terminal heat‐tolerance traits (Sihag et al., 2021). Tyagi et al. (2021) developed 13 miRNA‐derived SSR markers from heat‐responsive genes in wheat, where three markers (HT‐169j, HT‐160a, HT‐160b) effectively differentiated heat‐tolerant and susceptible genotypes, aiding germplasm characterization and heat‐tolerance breeding. Likewise, in grapevine, miRNA‐based SSR polymorphisms were analyzed using PCR amplification and Sanger sequencing, identifying 403 SSRs in pri‐/pre‐miRNA regions. The VMIRSSR167c3 marker, based on SSR (simple sequence repeat) length, showed 90% accuracy in distinguishing heat‐tolerant varieties. These results provide a novel molecular marker for the genetic improvement of grape germplasm and will aid future breeding of heat‐resistant cultivars (L. Zhang, Song, et al., 2023). Collectively, these findings underscore the potential of miRNA‐based markers as precise and functional tools for accelerating the development of heat‐resilient crop varieties through molecular breeding.

Recent advances in high‐throughput sequencing technologies, particularly small RNA sequencing (sRNA‐seq), have revolutionized the genome‐wide identification and expression profiling of miRNAs involved in plant responses to heat stress (Sunkar et al., 2012). This approach typically involves isolating total RNA from heat‐stressed and control tissues, constructing sRNA libraries, and subjecting them to deep sequencing. The resulting reads are then processed using robust bioinformatics pipelines such as miRDeep, miRDeep2, psRNATarget, TargetFinder, and alignment tools with reference databases like miRBase to accurately predict and annotate known and novel miRNAs, along with their potential mRNA targets (Palani et al., 2025). For instance, in Ziziphus jujube Mill. (jujube), high‐throughput sequencing of heat‐stressed leaves from heat‐tolerant (Fucumi) and heat‐sensitive (Junzao) cultivars revealed 45 known and 482 novel miRNAs and 13,884 differentially expressed mRNAs. Integrated analysis uncovered 1306 significant miRNA–mRNA interaction pairs, with enrichment in key pathways such as plant hormone signaling, starch metabolism, and spliceosome function, suggesting a critical posttranscriptional regulatory role of miRNAs in genotype‐specific heat stress responses (J. Jin, Yang, et al., 2024). Similarly, in Pinellia ternata, combined transcriptome and miRNAome sequencing under heat stress identified 4960 differentially expressed genes and 1597 heat‐responsive miRNAs. Further integration revealed 41 high‐confidence miRNA–mRNA regulatory pairs, many forming modules linked to MYB‐like proteins and calcium‐responsive transcription coactivators. These findings were further validated via qRT‐PCR, emphasizing the role of miRNAs in modulating heat resistance (Bo et al., 2024).

In another study, 19 novel heat‐responsive miRNAs were identified using the reference genomes of Sorghum bicolor and Z. mays, and their expression was validated via qRT‐PCR in four contrasting wheat cultivars, BT Schomburgk and PBW 343 (heat sensitive), and HD 3086 and Raj 3765 (heat tolerant). Most of these miRNAs, including miRNA2233 cloned from HD 3086, were significantly downregulated under heat stress at the seedling stage, showing a strong negative correlation with targets like HSPs, thus underscoring their potential role in climate‐smart wheat breeding (M. Kumar et al., 2025). In Trichoderma guizhouence NJAU 4742, high‐throughput sequencing and bioinformatics analysis identified heat‐induced Tr‐milRNA1, which downregulated its target gene Trvip36, enhancing lignocellulose secretion under 37°C. The qPCR validation confirmed their inverse expression, and deletion of Trvip36 improved lignocellulose utilization under heat stress (T. Li et al., 2021). Similarly, in Brassica napus, high‐throughput sRNA‐seq of ABA‐treated and control siliques identified 97 novel and 211 known miRNAs, with 23 miRNAs differentially expressed under ABA‐induced conditions; bioinformatics analysis using psRNATarget for miRNA–mRNA interaction prediction and miRDeep2 v2.0.0.8 for novel miRNA identification revealed that key miRNAs such as miR172a, miR395a, and novel13 were significantly downregulated, while novel3 was upregulated, correlating with enhanced expression of fatty acid biosynthesis genes like KAS I and DGAT, as validated by qRT‐PCR, demonstrating the regulatory role of miRNAs in ABA‐mediated unsaturated fatty acid accumulation during seed development (Z. Xu et al., 2024).

Functional validation experiments further strengthen the findings from expression profiling. Gene overexpression, short tandem target mimic (STTM), RNAi, and CRISPR/Cas9 are commonly used to validate the roles of miRNAs under heat stress (Fatima et al., 2025). For instance, a recent study showed that overexpressing miR169r‐5p in rice significantly enhanced spikelet fertility and overall heat tolerance during the flowering stage by downregulating the target gene LOC_Os12g42400. The transgenic lines exhibited an average fertility of approximately 55.8%, compared to about 42.5% in the wild type (Q. Liu et al., 2017). Moreover, another study characterized known and novel ncRNAs, where functional validation of heat‐responsive miRNAs was carried out using RNA‐seq for transcript discovery, qRT‐PCR for expression profiling, RLM‐RACE and PARE for target cleavage confirmation, and luciferase reporter assays for interaction verification, ultimately revealing specific miRNA–mRNA pairs involved in HST mechanisms (Rahman & Sanan‐Mishra, 2025). Similarly, functional validation of miRNAs under heat stress was demonstrated using STTMtransgenic rice lines, where silencing of 35 miRNA families, including miR398, miR172, and miR156, revealed stable improvements in agronomic traits such as panicle length, grain number, and plant height across multiple generations, highlighting their potential in crop improvement under environmental stresses (H. Zhang, Zhang, et al., 2017).

miRNAs have emerged as powerful tools for enhancing crop heat tolerance, both as targets for CRISPR/Cas9‐mediated genome editing and as molecular markers for breeding resilient varieties. By precisely modifying miRNA‐binding sites or leveraging their expression patterns, researchers can optimize stress‐responsive gene networks, paving the way for climate‐smart agriculture. However, while current studies demonstrate promising applications, the full potential of miRNA‐mediated regulation remains underexplored. Such advancements will be critical for developing robust, heat‐tolerant cultivars to address the challenges of global warming. Overall, integrating high‐throughput sequencing with robust bioinformatics pipelines and experimental validation strategies has greatly advanced our understanding of miRNA‐mediated heat stress responses in plants. These technologies have revealed a wide array of heat‐responsive miRNAs and their functional target interactions across various species. Functional studies through genetic engineering approaches like STTM, CRISPR, and overexpression systems further confirm the roles of key miRNAs in conferring heat tolerance. Such insights provide a solid foundation for miRNA‐based crop improvement strategies aimed at enhancing resilience to climate‐induced stresses.

8. CONCLUSION AND FUTURE PERSPECTIVES

miRNAs have emerged as pivotal regulators of plant responses to heat stress, exerting control over key developmental, hormonal, and stress‐responsive pathways through posttranscriptional gene regulation. This review consolidates extensive evidence showing that heat stress significantly alters miRNA expression patterns, triggering both upregulation and downregulation of specific miRNAs, which in turn modulate TFs, HSPs, antioxidant enzymes, and hormone signaling components. Regulatory modules such as miR156‐SPL, miR169‐NF‐YA, miR398‐CSD, and miR172‐AP2 have been repeatedly implicated in mediating thermotolerance across diverse plant species including Arabidopsis, rice, wheat, and maize. Moreover, the integration of miRNA regulation with other ncRNAs (siRNAs, lncRNAs, circRNAs) underscores a highly coordinated multilayered response that reinforces genome stability, protein homeostasis, root architecture, and reproductive development under heat stress.

In parallel with transcriptomic profiling, functional characterization of miRNA–mRNA pairs through techniques such as overexpression, STTM, luciferase reporter assays, RLM‐RACE, and CRISPR/Cas9‐mediated genome editing has substantially enhanced our understanding of miRNA function in thermotolerance. In particular, genome editing tools like CRISPR/Cas9 have been successfully employed to (i) disrupt miRNA loci for loss‐of‐function analyses and (ii) precisely edit miRNA‐binding sites in target transcripts, thereby mitigating miRNA‐mediated repression. This approach enables the fine‐tuning of miRNA‐target interactions to optimize stress responses. Additionally, the development of miRNA‐based molecular markers, including SSRs and SNPs derived from heat‐responsive miRNAs, has proven effective in identifying heat‐tolerant genotypes in breeding populations, thereby bridging molecular research and crop improvement.

Despite these advances, several critical research avenues remain open. One of the foremost challenges is to elucidate the complexity of miRNA regulatory networks under heat stress, including their spatial, temporal, and tissue‐specific dynamics. Future studies should integrate multi‐omics approaches, combining sRNA‐seq, degradome analysis, transcriptomics, proteomics, and epigenomics, with advanced gene‐editing tools such as CRISPR interference, base editors, and multiplex genome editing platforms. These methodologies will be essential to unravel the dynamic feedback circuits, competitive endogenous RNA (ceRNA) interactions, and epigenetic modifications modulated by miRNAs and other ncRNAs under heat stress.

Furthermore, comparative analyses across different plant species are essential to distinguish conserved “core” miRNA modules from lineage‐specific regulatory elements. Such interspecies comparisons, supported by pan‐genome analyses, presence/absence variation of miRNA genes and target sites, and genome‐wide association studies, can identify both universally essential and species‐specific targets for genetic manipulation. Integration of miRNA research with high‐throughput phenotyping andartificial intelligence driven predictive models could facilitate the identification of superior alleles for breeding programs aimed at improving crop resilience to rising global temperatures.

In conclusion, miRNAs offer an exceptional entry point for dissecting and engineering plant heat stress responses. By combining precise functional validation, advanced genome editing, and marker‐assisted selection, it is now possible to develop climate‐resilient crops with enhanced heat tolerance, safeguarding global food security in an era of exceptional environmental challenges.

AUTHOR CONTRIBUTIONS

Muhammad Farooq: Writing—original draft. Hina Tanveer: Data curation. Hafiz Mamoon Rehman: Writing—review and editing. Rabia Areej Cheema: Data curation, visualization. Sehar Nawaz: Data curation. Aneesa Ijaz: Data curation, visualization. Muhammad Arif: Visualization. Hon‐Ming Lam: Conceptualization, funding acquisition, writing—review and editing.

CONFLICT OF INTEREST STATEMENT

The authors declare no conflicts of interest.

Farooq, M. , Tanveer, H. , Rehman, H. M. , Cheema, R. A. , Nawaz, S. , Ijaz, A. , Arif, M. , & Lam, H.‐M. (2025). MicroRNAs‐mediated heat stress regulations in plants: mechanisms and targets. The Plant Genome, 18, e70112. 10.1002/tpg2.70112