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. 2026 Jun 21;68(8):3073–3085. doi: 10.1111/jipb.70324

The multifaceted regulatory network of microRNA396: Orchestrating plant plasticity through coordinating development and various stresses

Hang Yang 1, † , Ying Yan 1, † , Lixia Zhang 1, Junhua Ye 1, Kai Wang 1, Zejun Hu 1, Lingjuan Xie 1, Liming Cao 1, Shujun Wu 1,✉, Zhenying Shi 1,✉
PMCID: PMC13446597  PMID: 42324803

ABSTRACT

MicroRNAs (miRNAs) are a class of small, non‐coding RNAs that regulate gene expression in eukaryotes. Among them, miR396 targets GROWTH‐REGULATING FACTOR (GRF) transcription factors and forms one of the most highly conserved regulatory modules in plants. Recent studies have greatly expanded the functional landscape of the miR396–GRF module, showing that, beyond its canonical role in leaf morphogenesis, it also participates in root meristem regulation, reproductive development, yield formation, tissue regeneration, and responses to diverse abiotic and biotic stresses. In crop plants, this module further controls agronomically important traits. Here, we summarize current knowledge of the evolutionary conservation and diversification of the MIR396 loci, the upstream pathways that control miR396 expression, and the developmental and stress‐related outputs mediated by the miR396–GRF module. We also discuss evidence that miR396 functions as a context‐dependent regulator rather than a simple growth suppressor, and highlight how precise manipulation of the miR396–GRF module may provide new opportunities for crop improvement by optimizing growth, regeneration, and stress resilience.

Keywords: biotic and abiotic stress resistance, crop improvement, developmental plasticity, grain size, GROWTH‐REGULATING FACTOR, microRNA396, tissue regeneration

This review examines the character and function of the microRNA miR396, which helps plants balance growth, reproduction, regeneration, and survival under various stresses, highlighting recent progress and evidence that this pathway shapes crop yield, pest and disease resistance, and tissue regeneration, making it a promising target for precision crop improvement

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INTRODUCTION

miRNAs function as post‐transcriptional regulators of plant growth and development

As sessile organisms, plants must continuously adapt to fluctuating environmental conditions. This requirement has driven the evolution of highly plastic developmental systems governed by multilayered gene regulatory networks (Takeda and Matsuoka, 2008; Moshelion and Altman, 2015; Feng et al., 2022; Dong et al., 2024). Although transcriptional regulation serves as a primary means of genetic network modulation, post‐transcriptional regulation offers an additional rapid and fine‐tuned mechanism that enables adjustment in gene expression in response to transient developmental and environmental cues (Covarrubias and Reyes, 2010; Zhang et al., 2016; Zhang et al., 2025c). Among the major regulators operating at this level, microRNAs (miRNAs) are highly conserved central players that fine‐tune gene expression (Song et al., 2019). These 20–24 nt small RNAs guide ARGONAUTE‐containing RNA‐induced silencing complexes (RISC) to complementary target transcripts, thereby directing transcript cleavage or translational repression (Yu et al., 2026).

Functional studies over the past three decades have established that plant miRNAs participate in nearly all major aspects of plant biology, including phase transition, organ patterning, nutrient homeostasis, and immunity (Song et al., 2019; Silvestri et al., 2024). A subset of miRNA families (and their binding sites in target RNAs) is conserved across land plants, indicating their evolutionary importance. Among these conserved families, MIR396 is particularly notable for coordinating growth regulation with environmental responsiveness (Liebsch and Palatnik, 2020; Lu et al., 2021). Initially characterized as a regulator of cell proliferation through the repression of GRF transcription factors, miR396 is increasingly recognized as a signaling node linking developmental programs to stress‐related pathways.

The expanding functional landscape of miR396

The function of the miR396 family is defined by a highly conserved mature sequence and by its predominant targeting of GROWTH‐REGULATING FACTOR (GRF) genes (Cuperus et al., 2011; Omidbakhshfard et al., 2015). Although very early studies primarily linked miR396 to GRF‐mediated control of cell proliferation in leaf development (Rodriguez et al., 2010; Wang et al., 2011; Das and Nath, 2015), its functional scope is now recognized to be far broader. The miR396 pathway is implicated in the integration of hormonal and developmental signals, including those associated with auxin and gibberellin (GA) pathways (Chen et al., 2020; Lu et al., 2020), and in upstream transcriptional circuits involving regulators such as TEOSINTE BRANCHED1/CYCLOIDEA/PCF4 (TCP4), PLETHORA (PLT), and INDETERMINATE DOMAIN2 (IDD2) proteins (Rodriguez et al., 2015; Lu et al., 2020). In addition to canonical GRF targets, lineage‐specific non‐GRF targets have also been reported, indicating that the regulatory scope and function of miR396 can expand during evolution (Bao et al., 2014). Recent studies further show that miR396 influences agronomically and ecologically relevant traits across multiple species, including grain size and yield in cereals and soybean (Duan et al., 2015; Xie et al., 2024), secondary vascular development in woody plants (Wang et al., 2023b; Liu et al., 2025; Zhang et al., 2025a), nodule senescence in legumes (Yan et al., 2025), and defense responses to herbivores and pathogens in Arabidopsis, rice, and alfalfa (Chandran et al., 2018; Dai et al., 2019; Bhutto et al., 2025). Nevertheless, these functions are highly context‐dependent, and the underlying mechanisms are not equally well resolved across systems. Previous reviews have already provided comprehensive accounts of the canonical functions of the miR396–GRF/GIF module in leaf, root, and floral organ development (Omidbakhshfard et al., 2015; Liebsch and Palatnik 2020; Lazzara et al., 2024). Accordingly, rather than summarizing these well‐established developmental roles in detail again, this review briefly outlines them as a conceptual framework. Focuses are primarily given on recent studies that have expanded the functional scope of miR396, particularly with regard to grain size regulation, reproductive yield traits, regeneration competence, insect resistance, pathogen immunity, and context‐dependent stress adaptation.

EVOLUTIONARY CONSERVATION, EXPANSION, AND DIVERSIFICATION

Ancient origins and evolutionary conservation

miR396 is widely regarded as an ancient plant miRNA family that is conserved across seed plants and likely originated early during vascular plant evolution (Cuperus et al., 2011). Such deep conservation and origin suggest long‐term selective maintenance of the miR396–GRF regulatory relationship and the functional significance of the miR396–GRF module. GRF proteins are plant‐specific transcription factors containing the conserved WRC (Trp, Arg, Cys) and QLQ (Gln, Leu, Gln) domains required for DNA binding and protein interaction, respectively (Omidbakhshfard et al., 2015; Liebsch and Palatnik 2020). The persistence of this regulatory module across distant lineages is consistent with an important role in coordinating proliferative growth with developmental progression (Liebsch and Palatnik 2020). Rather than implying a fixed evolutionary purpose, available evidence supports the view that miR396–GRF co‐evolution has contributed to the robust control of tissue growth across diverse plant contexts.

Genomic expansion and functional diversification

Although the mature miR396 sequence is highly conserved, MIR396 loci have undergone substantial copy‐number expansion in several crop genomes, particularly in polyploid species. Such expansion provides a substrate for sub‐functionalization and, in some cases, for the acquisition of lineage‐specific regulatory roles.

In rice, eight MIR396 loci (OsMIR396a–h) have been identified (Gao et al., 2015; Liebsch and Palatnik 2020). Phylogenetic analysis of precursor sequences suggests that MIR396 members from different species can be broadly grouped into three major clades (Figure 1A). Some rice MIR396 members group with homologs from multiple grasses, whereas others display more lineage‐restricted patterns, consistent with diversification of the MIR396 family in grasses (Yu et al., 2021). Functional divergence among these loci is also supported by differential expression and stress responsiveness. For example, precursor forms of OsmiR396a and OsmiR396b are rapidly induced by brown planthopper (BPH) infestation, suggesting that specific family members have acquired specialized roles in biotic stress responses (Dai et al., 2019).

Figure 1.

Figure 1

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Phylogenetic relationships and tissue‐specific expression patterns of the MIR396 genes in representative species

(A) Phylogenetic analysis of the pre‐miR396 sequences from Arabidopsis thaliana (Ath), Brassica napus (Bna), Chenopodium quinoa (Cqu), Gossypium hirsutum (Ghi), Glycine max (Gma), Hordeum vulgare (Hvu), Oryza sativa (Osa), Solanum lycopersicum (Sly), Solanum tuberosum (Stu), Triticum aestivum (Tae), and Zea mays (Zma). The phylogenetic tree was constructed using the Maximum Likelihood method based on the precursor (pre‐miR396) sequences, with bootstrap support estimated from 1,000 replicates. Major clades are indicated on the right. Group I, Group II, and Group III indicate major clades defined by phylogenetic clustering. (B) Heat maps showing tissue‐specific expression patterns of mature miR396s in Arabidopsis thaliana, Oryza sativa, and Glycine max. Expression values are shown as Log2‐transformed transcript abundance. The pre‐miR396 sequences used for phylogenetic analysis and the mature miR396 expression data used for heatmap construction were retrieved from the PmiREN database (https://www.pmiren.com/). Accession numbers of the PmiREN miRNA loci used in this study are provided in Table S1.

Expansion of the MIR396 family is even more pronounced in hexaploid wheat, where at least 17 loci have been reported (Figure 1A). Comparative analyses indicate that some wheat‐specific MIR396 members may participate in regulatory networks distinct from those of the canonical GRF‐centered branch. For instance, target prediction and enrichment analyses suggest that certain haplotypes, such as TaMIR396n, may be associated with metabolic processes relevant to grain development (Yu et al., 2021). Although these inferences are currently based on computational prediction, they await experimental validation.

Comparable diversification is also observed in sorghum. Expression profiling divides the sorghum MIR396 family into groups with contrasting transcriptional activity across tissues, with SbiMIR396a–c showing limited aerial expression and SbiMIR396d/e accumulating strongly in developing leaves, panicles, and seeds. By actively repressing targets like SbiGRF1/5/8, SbiMIR396d/e function as the primary regulators of floral organogenesis and plant architecture (Wang et al., 2023a). Such partitioning supports the idea that duplicated MIR396 loci can become preferential regulators of particular developmental processes rather than remaining fully redundant.

Copy‐number expansion is closely associated with tissue‐specific expression divergence (Figure 1B). In Arabidopsis, the two MIR396 loci show a relatively simple pattern consistent with their established role in leaf growth control. By contrast, the expanded rice MIR396 family displays more pronounced expression partitioning, and members such as OsMIR396e/f are enriched in reproductive tissues, in line with their contribution to yield‐related traits (Gao et al., 2015; Miao et al., 2020). Similarly, the massively expanded MIR396 family in paleopolyploid soybean displays highly diversified expression, with certain isoforms uniquely accumulating in specialized organs like symbiotic nodules. Together, these observations support the idea that gene duplication has increased the regulatory flexibility of the MIR396 family. However, for many loci, direct functional validation remains limited and should be distinguished from expression‐based inference.

UPSTREAM REGULATION: THE miR319–TCP4–miR396 AND SLR1/IDD2–miR396a CASCADES

To understand how miR396 integrates developmental information, it is essential to examine its upstream regulatory circuits. In Arabidopsis leaves, TCP4 acts as a major promoter of maturation by directly activating MIR396 transcription (Rodriguez et al., 2010; Schommer et al., 2014; Figure 2A). In young primordia, relatively low TCP4 activity is associated with low miR396 abundance and high GRF expression, thereby maintaining proliferative capacity. As leaf development proceeds, TCP4 accumulates and promotes miR396 expression, which in turn clears GRF transcripts and drives the transition from cell proliferation to differentiation. Moreover, in the miRNA network, TCPs are subject to regulation by miR319, thus establishing a regulatory cascade comprising miR319, TCP4, miR396, and GRFs (Schommer et al., 2014). This multi‐step cascade allows for multiple points of signal integration, including hormonal inputs from Jasmonic Acid (JA), which is also regulated by TCPs (Schommer et al., 2008).

Figure 2.

Figure 2

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Regulatory roles of the miR396–GRF module in plant development and yield formation

(A) Schematic model of miR396‐mediated control of leaf morphogenesis in Arabidopsis. In developing leaves, low miR396 levels in the basal proliferative region permit AtGRF accumulation and activation of growth‐promoting transcriptional programs, whereas increased miR396 expression toward the distal region restricts AtGRF activity and promotes cell expansion and differentiation. AtTCP4 acts upstream to induce MIR396 transcription during maturation. The triangle on the left represents the abundance gradient changes of miR396 and AtGRF. (B) Proposed regulatory framework of the miR396–GRF module in the Arabidopsis root meristem. Interactions among AtGRFs, AtGIFs, and AtPLT‐associated pathways contribute to the balance between transit‐amplifying cells (TAC) and the stem cell niche (SCN), highlighting a context‐dependent role of miR396 in meristem maintenance and proliferative control. The light blue cell area in the root tip represents the SCN. (C) Simplified representation of miR396 function in floral organ development. By repressing GRF activity, miR396 influences reproductive organ formation, particularly gynoecium patterning and stamen‐related development. (D) miR396‐mediated regulation of yield‐related traits in rice. Repression of OsGRF4, OsGRF8, and OsGRF6 by OsmiR396 modulates grain size, grain number, and overall yield through multiple downstream branches, including GS3, OsmiR408, and OsGIF1‐associated regulation. Solid arrows indicate positive regulation, promotion; T‐shaped lines indicate repression or inhibition; dashed T‐shaped arrow indicates reduced inhibition. All schematic figures in this review were prepared and assembled using Adobe Illustrator.

Recent work in rice has uncovered how GA regulates cell proliferation via miR396. The DELLA protein SLR1 interacts physically with the transcription factor OsIDD2. The SLR1–OsIDD2 complex binds to the promoter of OsMIR396a and activates its transcription. In the presence of GA, SLR1 is degraded, which leads to a reduction in OsmiR396 levels. The decline in OsmiR396 facilitates an increase in OsGRFs levels, thus promoting stem elongation and cell proliferation (Lu et al., 2020). This finding establishes miR396 as a negative regulator of the GA‐induced cell proliferation.

FUNCTION OF THE miR396–GRF REGULATORY HUB IN PLANT DEVELOPMENT

Leaf morphogenesis: Establishing the mitotic arrest front

In model dicots such as Arabidopsis and tomato, the best‐established developmental role of miR396 is the spatial restriction of GRF expression during leaf growth (Rodriguez et al., 2010; Debernardi et al., 2014; Bresso et al., 2018). Leaf development proceeds along a basipetal gradient: Distal cells exit proliferation earlier, whereas basal cells retain meristematic activity for a longer time. miR396 is a central determinant of this gradient developmental arrangement since it helps define the mitotic arrest front by progressively restricting GRF activity in differentiating regions.

In young leaf primordia, miR396 levels are kept low by upstream repressors or a lack of activators (Figure 2A). This allows for high expression of GRFs (e.g., AtGRF1‐4, AtGRF7‐9 in Arabidopsis; SlGRF1‐5 in tomato) (Rodriguez et al., 2010; Wang et al., 2011; Das and Nath, 2015). These GRF proteins accumulate in the nucleus and interact with the GRF Interacting Factors (GIF) co‐activators. The GRF–GIF complex recruits the SWI/SNF chromatin remodeling complex to the promoters of the cell cycle genes (Vercruyssen et al., 2014; Lu et al., 2021). The SWI/SNF complex actively remodels chromatin, maintaining it in an open, transcriptionally active state that supports rapid cell division and meristematic competence.

As leaves expand, developmental regulators such as TCP4 induce MIR396 expression in a distal‐to‐basal manner (Schommer et al., 2014; Figure 2A). The resulting increase in miR396 reduces GRF transcript abundance, weakens proliferative programs, and promotes the transition to cell expansion and differentiation. Accordingly, constitutive miR396 overexpression leads to smaller and narrower leaves, whereas expression of miR396‐resistant GRFs prolongs cell proliferation and increases leaf size (Rodriguez et al., 2010). Together, these findings establish miR396 as a key temporal and spatial regulator of leaf growth.

Root architecture and the stem cell niche

In roots, the miR396–GRF module participates in the balance between stem‐cell maintenance and transit‐amplifying proliferation. The regulation of the root apical meristem (RAM) involves a complex feedback loop between miR396, GRFs, and the PLT transcription factors (Rodriguez et al., 2015; Baulies et al., 2025; Figure 2B). PLT1 and PLT2 are the main regulators of the root stem cell micro‐environment, which are induced by auxin (Santuari et al., 2016). GRFs repress the expression of PLT genes. In order to prevent the complete silencing of the PLT genes, the PLT proteins themselves induce the expression of MIR396 genes as feedback. Consequently, the induced miR396 exerts a repressive effect on GRFs and thereby alleviates the repression on PLTs. This positive feedback loop has been demonstrated to stabilize the stem cell population. Overexpression of miR396 generally leads to shorter roots by reducing the size of the meristematic zone.

In contrast, studies on Medicago truncatula indicate that reduced miR396 activity can enhance root biomass and mycorrhizal associations (Bazin et al., 2013), suggesting that the developmental consequences of this module extend beyond Arabidopsis.

Reproductive development and yield components

Beyond vegetative growth, the miR396–GRF module also exerts major effects on reproductive development and yield‐related traits. Across multiple species, miR396 generally acts as a brake on growth potential in reproductive organs, whereas partial release of this repression often enlarges seeds, grains, or fruits. However, the magnitude and pleiotropic consequences of this effect differ among species and genetic backgrounds (Duan et al., 2015; Cao et al., 2016; Xie et al., 2024).

Studies indicate that the miR396–GRF module plays a conserved role in floral organogenesis. In Arabidopsis, elevated miR396 activity disrupts gynoecium patterning and compromises both carpel margin meristem‐derived tissues and the archesporial cell lineage, thereby affecting female and male reproductive development (Liang et al., 2014; Lee et al., 2018). In rice, OsmiR396d predominantly regulates spikelet organogenesis by modulating palea–lemma interlocking, sterile lemma development, and floral organ morphology (Liu et al., 2014; Figure 2C).

In crops, especially rice, the same regulatory scheme has been elaborated into multilayered networks that influence agriculturally meaningful traits such as panicle architecture, grain size, grain number, and final yield (Duan et al., 2015; Gao et al., 2015; Cao et al., 2016; Li et al., 2016; Miao et al., 2020; Wang et al., 2024; Xie et al., 2024; Figure 2D).

Direct target de‐repression: In rice, OsGRF4 has been identified as the gene underlying the major grain size quantitative trait loci (QTLs) GS2 and GLW2 (Duan et al., 2015; Li et al., 2016). A naturally occurring 2‐bp mutation in OsGRF4 found in certain high‐yielding Indica varieties disrupts the miR396 binding and thus prevents the miR396‐mediated cleavage, leading to elevated OsGRF4 levels. The elevated OsGRF4 recruits the transcriptional co‐activator OsGIF1, thereby enhancing cell proliferation and expansion in the grain hull and resulting in significantly larger, heavier grains (Duan et al., 2015). In parallel, repression of miR396 can also increase grain number by re‐shaping inflorescence architecture, with OsGRF6 being an important downstream contributor in this branch (Gao et al., 2015). Correspondingly, CRISPR/Cas9‐mediated knockout of MIR396e and MIR396f in rice results in larger grains and longer leaves (Miao et al., 2020). Collectively, these findings indicate that miR396‐targeted OsGRFs influence both grain size and grain number, thereby shaping final yield.

Crosstalk with G‐protein signaling: A newly discovered regulatory link has been found between miR396–GRFs and G‐protein signaling (Zhu et al., 2024). The GS3 gene, which encodes the G‐protein γ subunit, is a negative regulator of grain size. OsGRF4 and OsGRF8 could bind directly to the GS3 promoter and repress its expression. Therefore, the miR396–GRF module promotes grain enlargement by activating cell cycle genes and repressing the growth inhibitor GS3. In MIM396 plants, high GRF levels result in low GS3 levels, which contribute to the large grain phenotype (Zhu et al., 2024).

Regulatory cascade: Additional complexity arises from interactions among miRNA pathways (Yang et al., 2021). In rice, OsGRF8 directly activates MIR408, and reduced miR396 activity can therefore increase miR408 expression through GRF de‐repression. miR408 is an embryo‐specific miRNA that positively regulates grain size. This creates a hierarchical miRNA regulatory cascade in which the miR396–GRF module influences grain filling not only through direct developmental outputs but also through downstream miRNA‐mediated regulation.

In tomato, MIR396a is expressed significantly higher than MIR396b in flowers and fruits, suggesting that it is the dominant isoform regulating reproduction. Suppressing the activity of miR396 using a Short Tandem Target Mimic (STTM396) leads to the release of SlGRFs and an increase in fruit weight of up to 45%, without affecting the ripening or seed‐setting processes (Cao et al., 2016). This supports the idea that the growth‐restrictive function of miR396 is conserved, yet can be exploited in a crop‐specific manner.

The same concept has recently been extended to soybean, where multiplex CRISPR/Cas9 knockout of MIR396 genes increased seed size and hundred‐seed weight (Xie et al., 2024). Intriguingly, distinct mutant combinations exhibited varied ecological adaptations. Triple mutants (e.g., mir396adf) demonstrated superior performance in higher‐latitude regions characterized by yield increases and lodging resistance, while quintuple mutants (e.g., mir396abcdf) exhibited enhanced yield potential in lower latitudes. This emphasizes the necessity for precision in the implementation of this regulatory mechanism within breeding programs.

Taken together, these studies support a coherent view in which miR396 acts as a quantitatively tunable regulator of reproductive growth. While its manipulation offers considerable promise for improving yields, effective deployment will likely require species‐, organ‐ and environment‐specific optimization, rather than constitutive de‐repression across the entire plant.

THE ROLE OF miR396–GRF MODULE IN CELLULAR REPROGRAMMING AND TISSUE REGENERATION

Plant cells retain remarkable developmental plasticity, allowing both regeneration after injury and in vitro reprogramming into new organs or embryos. Recent studies place the miR396–GRF module at the center of these processes, particularly in determining regenerative competence and regeneration speed.

In vivo tissue regeneration: Root tip reconstitution

During root tip regeneration in Arabidopsis, the miR396–GRF module plays a dual role. miR396 contributes to the acquisition of regeneration competence, whereas GRFs promote the pace of meristem reconstruction (Baulies et al., 2025). Elevated GRF activity accelerates meristem recovery, whereas ectopic miR396 expression prevents quiescent center reconstitution, leaving the root in an anatomically open state. Remarkably, these roots still elongate via dispersed stem cell activity (Baulies et al., 2025). This reveals that manipulating the miR396–GRF balance can decouple continuous organ growth from the requirement of a highly organized stem cell niche.

In vitro reprogramming and genetic transformation

In the context of in vitro tissue culture, cellular reprogramming acts as a major bottleneck that is notoriously genotype‐dependent. Somatic embryogenesis (SE) is a process in which somatic cells de‐differentiate and form embryos; this process is tightly linked to stress response and extensively utilized in plant biotechnology. During the induction of SE in Arabidopsis explants, specifically in the cotyledons and shoot apical meristem (SAM), the expression of mature miR396 is significantly upregulated (Szczygieł‐Sommer and Gaj 2019). This surge in miR396 subsequently represses multiple GRFs (such as GRF1, 4, 7, 8, and 9). This dynamic shift modulates the sensitivity of explant tissues to auxin (2, 4‐D) and interacts with stem cell regulators, such as PLT, ultimately acting as a gatekeeper that limits embryogenic transition.

Similarly, in de novo shoot organogenesis, miR396 functions as a negative regulator. A time‐course transcriptomic analysis in tomato revealed that recalcitrant genotypes fail to regenerate because of an inherently high accumulation of miR396a, which continually suppresses SlGRF transcripts (Park et al., 2024). By introducing STTM396a to suppress miR396 activity, researchers successfully released the SlGRFs from repression, boosting shoot regeneration rates from 7% to 27% and enabling the efficient recovery of CRISPR/Cas9‐edited plants in previously intractable varieties (Park et al., 2024).

Leveraging this underlying mechanism, researchers have developed strategies to achieve genotype‐independent transformation. The most prominent approach involves the use of a chimeric GRF‐GIF protein (e.g., GRF4‐GIF1) (Debernardi et al., 2020; Feng et al., 2021). By introducing silent mutations into the miR396 recognition site of the GRF4 gene, the resulting transcript evades miR396‐mediated cleavage. The stabilized expression of this miR396‐resistant GRF4, fused with its co‐activator GIF1, dramatically increases both the regeneration efficiency and the speed of transformation. Originally pioneered in wheat to overcome severe genotype dependencies (Debernardi et al., 2020), this targeted de‐repression strategy has now been successfully translated to diverse crops, including watermelon and medicinal orchids (Feng et al., 2021; Yang et al., 2024b). Furthermore, transient expression of the mutated mTaGRF4‐TaGIF1 complex in wheat has been shown to boost transgene‐free editing frequencies without integrating into the host genome, thus expanding the scope of commercial application (Qiu et al., 2022). Therefore, neutralizing the miR396‐imposed developmental block represents a universal and highly effective toolkit for accelerating plant biotechnology and speed‐breeding pipelines.

MODULATION OF ABIOTIC STRESS RESILIENCE

Plants are constantly challenged by abiotic stresses such as drought, salinity, and extreme temperatures. The miR396–GRF module also acts as a developmental switch, often prioritizing survival over growth during these stress events.

Drought stress

Drought stress has been demonstrated to result in water loss, osmotic imbalance, and reactive oxygen species (ROS) bursts in plant cells (Chang et al., 2024). As a responsive strategy, the miR396–GRF module undergoes species‐specific re‐configuration, reflecting distinct adaptive strategies (Zhou et al., 2010; Xie et al., 2015; Fracasso et al., 2021; Singh et al., 2023; Yang et al., 2024a; Figure 3A).

Figure 3.

Figure 3

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Schematic summary of how the miR396–GRF module contributes to abiotic stress responses in different plant species

(A) Drought‐related regulation of the miR396–GRF pathway differs among species. In Arabidopsis, increased miR396 activity is associated with reduced GRF7 function and altered expression of abscisic acid‐ and osmotic stress‐responsive genes, whereas in tomato, reduced miR396 activity is associated with increased accumulation of GRFs and improved water‐use efficiency. (B) Under salt stress in rice, reduced OsmiR396b accumulation leads to de‐repression of OsGRF6, activation of OsMYB3R, enhanced ROS scavenging, and improved salt tolerance. OsGRF7 promotes OsUGT1/OsUGT5 expression, enhances arbutin accumulation, lowers ROS levels, and improves salt tolerance. (C) In peanut, the AhmiR396–AhGRF3b module contributes to cold tolerance, with reduced AhmiR396 and elevated AhGRF3b being associated with lower ROS accumulation and enhanced cold resistance. (D) In sunflower, reduced HamiR396 results in increased HaWRKY6 expression and enhanced heat sensitivity, indicating that miR396‐mediated repression of HaWRKY6 is required for proper heat‐stress regulation. Solid arrows indicate positive regulation, promotion; T‐shaped lines indicate repression or inhibition; red arrows indicate increased expression, activity, accumulation, or response; green arrows indicate decreased expression, activity, accumulation, or response.

Under drought stress, the expression of GRF7 decreases in Arabidopsis, thereby lifting the repression of the key stress regulator DREB2A (Kim et al., 2012). Overexpression of MIR396 reduces stomatal density and improves drought tolerance in Arabidopsis leaves (Liu et al., 2009). However, evidence from tomato points to a different functional direction. Downregulating SpMIR396a in tomatoes significantly enhanced its water retention capacity and proline accumulation, thereby improving drought resistance (Fracasso et al., 2021). These observations indicate that the drought‐related role of miR396 cannot be generalized as simple upregulation or downregulation for adaptive protection across species (Figure 3A).

In rice, drought‐responsive profiling has revealed reduced miR396 accumulation, a pattern opposite to that observed in some dicot systems (Zhou et al., 2010). This opposite expression pattern reveals that plants fine‐tune the miR396–GRF balance not simply to sacrifice growth, but to optimally manage the trade‐off between growth arrest and stress tolerance under water‐limited conditions.

Salinity stress

Salt stress induces ion toxicity (primarily Na+), osmotic stress, and secondary oxidative damage (Yu et al., 2020). Early functional evidence in rice showed that OsMIR396c is downregulated under salt and alkali stress, and that constitutive overexpression of OsMIR396c reduces stress tolerance in both rice and heterologous Arabidopsis systems, supporting a negative role of miR396 in salinity‐related stress responses (Gao et al., 2010). The miR39–GRF module functions as a regulatory hub for ion homeostasis and oxidative balance in this process (Yuan et al., 2019; Chen et al., 2024; Yuan et al., 2024b; Figure 3B). In the context of salt stress, the expression of OsMIR396b is repressed, resulting in the upregulation of its target gene OsGRF6 (Yuan et al., 2024b). As a transcription activator, OsGRF6 directly binds to and activates the expression of the downstream key transcription factor encoding gene OsMYB3R. OsMYB3R subsequently regulates a series of genes encoding ROS scavenging enzymes, thereby strengthening the cellular antioxidant defense system, reducing hydrogen peroxide accumulation, and protecting cells from oxidative damage. Therefore, plants overexpressing either OsGRF6 or OsMYB3R exhibited significantly improved survival rates under salt stress conditions. In addition, OsGRF7 positively regulates rice salt tolerance through an arbutin‐associated pathway. OsGRF7 promotes the expression of OsUGT1 and OsUGT5, enhances arbutin accumulation, reduces ROS levels, and increases survival under salinity stress (Chen et al., 2024).

Temperature stress: Cold and heat

miR396 also participates in temperature‐stress responses, but the regulatory logic appears to differ across lineages and target modules (Chen et al., 2015; Cao et al., 2024; Farooq et al., 2025). In peanut, the AhmiR396–AhGRF3b module has been implicated in cold tolerance (Zhang et al., 2025b; Figure 3C). Degradome sequencing confirms that AhmiR396 directly cleaves AhGRF3b transcripts. Functional studies reveal that overexpressing AhGRF3b in Arabidopsis significantly enhances cold tolerance, alongside reduced ROS accumulation and increased antioxidant enzyme activity. This suggests that sustaining GRF‐mediated growth processes under cold conditions may facilitate acclimation.

In contrast, during heat stress, miR396 appears to function by targeting distinct regulatory networks to prevent excessive or detrimental responses. In sunflower, miR396 targets the transcription factor HaWRKY6 (Figure 3D). Modulating HaWRKY6 expression via miR396 is crucial for response to the early heat shock, as constitutive expression of a miR396‐resistant HaWRKY6 version led to increased heat sensitivity (Giacomelli et al., 2012). This example is notable because it highlights a non‐GRF target and suggests that lineage‐specific target evolution can diversify the stress‐related functions of miR396.

Taken together, current evidence indicates that miR396 participates in temperature‐stress responses through different target modules in different lineages. However, available data still remain too limited to support a single general model for cold and heat regulation. The main conclusion of this subsection should therefore emphasize functional diversification over broad mechanistic unification.

BIOTIC STRESS AND GROWTH‐DEFENSE TRADE‐OFFS

Allocation of resources between growth and defense is a central challenge in plant biology. The miR396–GRF module has emerged as an important regulator of this balance, influencing defense outputs against herbivores, nematodes, and selected pathogens while simultaneously affecting growth‐related traits (Figure 4).

Figure 4.

Figure 4

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Schematic model of the roles of the miR396–GRF module in biotic stress responses

(A) In rice, BPH infestation induces OsmiR396, which represses OsGRF8 and attenuates flavonoid‐based defense by reducing OsF3H‐associated flavonoid biosynthesis. Suppression of miR396 activity or enhanced OsGRF8 expression increases flavonoid accumulation and improves BPH resistance. (B) In alfalfa, reduced MsmiR396 activity de‐represses MsGRF1, MsGRF4, and MsGRF5, thereby promoting lignin and flavonoid biosynthesis and enhancing resistance to the tobacco cutworm. (C) During cyst nematode parasitism, host miR396 is dynamically regulated in a biphasic manner: early suppression permits GRF accumulation and syncytium initiation, whereas later induction restricts further growth and contributes to syncytium maintenance. (D) In terms of pathogen immunity, reduced miR396 activity leads to the de‐repression of GRF‐dependent defense outputs in Arabidopsis, including the production of ROS, callose deposition, and the expression of defense genes. In rice, repression of OsmiR396 or overexpression of GRFs also contributes to blast resistance. In addition, recent evidence suggests that bacterial blight resistance can also be reinforced through an OsGRF6‐dependent pathway involving OsYUCCA1 and OsWRKY82. Solid arrows indicate positive regulation, promotion; T‐shaped lines indicate repression or inhibition.

Resistance to insects

Piercing‐sucking pests: In rice, BPH infestation induces OsMIR396 expression, thereby weakening defense (Figure 4A). Mechanistically, the OsmiR396–OsGRF8 module controls flavonoid accumulation by regulating flavanone 3‐hydroxylase (OsF3H) gene. Downregulation of miR396 via MIM396 or overexpression of OsGRF8 increases flavonoid content and confers BPH resistance (Dai et al., 2019). Notably, CRISPR/Cas9‐mediated editing of the miR396 binding site in OsGRF8 yields plants with enhanced BPH resistance (Lin et al., 2021).

Chewing Insects: In alfalfa, sequestration of miR396 boosts resistance to the tobacco cutworm (Spodoptera litura) (Figure 4B). This resistance operates independently of the JA pathway. Instead, the de‐repression of GRF transcription factors (e.g., MsGRF1/4/5) constitutively activates the biosynthesis of lignin and specific flavonoids. Enhanced cell wall lignification creates a physical barrier that deters larval feeding (Yan et al., 2023). The observed biphasic miR396 expression upon wounding, initial downregulation for defense activation followed by upregulation for tissue repair, exemplifies the dynamic balance this module governs.

Mediating interaction with nematode

Cyst nematodes (e.g., Heterodera spp.) orchestrate the formation of specialized feeding sites (syncytia) by manipulating the host's miR396–GRF module (Figure 4C). A conserved, biphasic regulatory pattern is observed: During syncytium initiation, nematode infection suppresses miR396, leading to the accumulation of specific GRFs (e.g., AtGRF1/3 in Arabidopsis; GmGRF6/9 in soybean) that drive cell cycle and expansion. Later on, during syncytium maintenance, miR396 is strongly upregulated to cleave GRF mRNAs and arrest further growth. Disrupting this precise temporal regulation through miR396 overexpression, GRF knockdown, or expression of miR396‐insensitive GRF variants severely impairs syncytium development and reduces nematode reproduction. Thus, the temporal hijacking of the miR396–GRF module is essential for successful nematode parasitism (Hewezi et al., 2012; Noon et al., 2019).

The role of miR396 in immunity

Conserved role in pathogen immunity: In Arabidopsis, reducing miR396 activity through target mimicry confers broad resistance to necrotrophic and hemibiotrophic fungi. Pathogen perception triggers miR396 downregulation, de‐repressing GRFs and thus potentiating defenses like ROS burst, callose deposition, and defense gene expression (Soto‐Suárez et al., 2017). Similarly, in rice, overexpression of MIR396 isoforms increases susceptibility to the blast fungus Magnaporthe oryzae, while blocking miR396 or overexpressing OsGRF6/7/8/9 enhances resistance (Chandran et al., 2018; Figure 4D). This negative regulation extends across multiple immune layers, as overexpression of miR396 can even compromise strong, specific resistance mediated by R genes (e.g., Piz‐t) in rice (Bhutto et al., 2025). Beyond blast resistance, recent evidence indicates that the defense outputs of the miR396–GRF module may also extend to bacterial leaf blight resistance through the OsGRF6–OsYUCCA1/OsWRKY82 signaling cascade (Yuan et al., 2024a).

Balancing immunity and agronomic traits: Importantly, not all GRFs contribute equally to the balance between immunity and growth. In rice, OsGRF6 can improve both blast resistance and yield‐associated traits, whereas OsGRF7 enhances resistance with a clearer growth penalty (Chandran et al., 2018). This functional specialization is highly relevant for crop engineering because it suggests that selective targeting of specific GRF family members may be more effective than globally suppressing miR396.

GENOME EDITING OF miR396–GRF MODULE FOR TRAIT IMPROVEMENT

Given its unparalleled control over growth, regeneration, and defense, the miR396–GRF module is increasingly standing out as an attractive target for precision breeding. In view of the functional diversification/specification of the miR396–GRF module illustrated above, the central challenge in implementation is not simply to increase or decrease pathway activity, but to modulate it in ways that minimize pleiotropic penalties while preserving desirable agronomic outputs. Several promising approaches have been conceptualized or validated:

Editing MIR396 genes or promoters: One strategy is to edit MIR396 genes or their promoters to alter the dosage of miR396 in a quantitative manner. Existing examples in several crops, such as rice, wheat, and soybean, and in fruit such as tomato, indicate that reducing MIR396 activity can improve yield‐related traits, but the broader developmental and defense consequences of constitutive de‐repression still need careful evaluation.

Engineering tissue‐specific regulation: A second, conceptually attractive strategy is tissue‐specific manipulation. For instance, driving the expression of MIM396 specifically in seeds could enhance grain filling and size by locally boosting GRF activity, while leaving vegetative architecture and systemic defense responses largely unaffected.

Creating miRNA‐binding site variants in GRF genes: A highly precise strategy involves editing the miR396 binding sites within specific GRF transcripts (e.g., in OsGRF4 or OsGRF8) to generate in‐frame mutations that abolish miRNA binding without disrupting the protein's coding sequence. This results in the accumulation of functional, miRNA‐resistant GRF proteins. This approach has successfully created plants with stable, heritable improvements in traits such as grain size (Duan et al., 2015) and BPH resistance (Lin et al., 2021), demonstrating the potential to selectively enhance both yield and defense without pleiotropic developmental defects.

CONCLUSION

The MIR396 family, together with its targets, has emerged as a major regulatory factor in plant biology. Rather than functioning solely as a growth suppressor, miR396 acts as a context‐dependent integrator of developmental progression, regenerative competence, and environmental responsiveness.

In development, miR396 modulates organ morphology, meristem behavior, and reproductive potential. Expansion of MIR396 loci has further enabled expression divergence and probable functional specialization in many species. Its interaction with the target GRF mRNA enables fine‐tuned rheostatic control across evolution. Furthermore, the miR396–GRF module functions as a critical gatekeeper of cellular reprogramming, releasing this developmental block, which offers a possible toolkit to overcome genotype dependency in tissue culture and genetic transformation.

Under abiotic stress conditions, the effects of miR396 are highly context‐dependent. In some systems, increased miR396 activity is associated with stress‐adaptive transcriptional reprogramming, whereas in others, reduced miR396 improves tolerance or defense.

During biotic interactions, the downregulation of miR396 itself becomes a strategic trigger for defense activation. The consequent de‐repression of GRFs mobilizes diverse defense outputs, from reinforcing cell walls via lignification against insect herbivores to priming broad‐spectrum immune responses against fungal pathogens. However, the breadth of evidence is uneven across pathosystems, and general conclusions should therefore remain appropriately qualified.

Future crop improvement efforts should therefore move beyond constitutive overexpression or knockdown and instead focus on precision engineering of specific MIR396 loci, selected GRF family members, or miR396‐binding sites. Such refined approaches are more likely to produce crops with improved yield, regenerative capacity, and resilience under complex field environments.

CONFLICTS OF INTEREST

The authors declare no conflicts of interest.

AUTHOR CONTRIBUTIONS

S.J. Wu, Y. Yan, and Z.Y. Shi planned and designed the framework. H. Yang undertook most of the writing. Z.Y. Shi and L.M. Cao revised the paper. Z.J. Hu, L.J. Xie, J.H. Ye, L.X. Zhang, and K. Wang participated in the revision of the draft. All authors reviewed the manuscript.

Supporting information

Additional Supporting Information may be found online in the supporting information tab for this article: http://onlinelibrary.wiley.com/doi/10.1111/jipb.70324/suppinfo

Table S1. PmiREN miRNA locus accession numbers used in this study

JIPB-68-3073-s001.docx (18.4KB, docx)

ACKNOWLEDGEMENTS

This work was supported by grants from the Natural Science Foundation of Shanghai (Grant No. 24ZR1462400, 25ZR1401316), Shanghai Agricultural Science and Technology Innovation Program (Grant No. T2024317), and Shanghai Agriculture Applied Technology Development Program (Grant No. X2024‐02‐08‐00‐12‐F00037).

Biographies

graphic file with name JIPB-68-3073-g003.gif

graphic file with name JIPB-68-3073-g002.gif

Yang, H. , Yan, Y. , Zhang, L. , Ye, J. , Wang, K. , Hu, Z. , Xie, L. , Cao, L. , Wu, S. , and Shi, Z. (2026). The multifaceted regulatory network of microRNA396: Orchestrating plant plasticity through coordinating development and various stresses. J. Integr. Plant Biol. 68: 3073–3085.

Edited by: Binglian Zheng, Fudan University, China

Contributor Information

Shujun Wu, Email: wushujun@saas.sh.cn.

Zhenying Shi, Email: shizhenying@saas.sh.cn.

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Supplementary Materials

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Table S1. PmiREN miRNA locus accession numbers used in this study

JIPB-68-3073-s001.docx (18.4KB, docx)

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