The Role of SQUAMOSA-PROMOTER BINDING PROTEIN-like (SPL) Transcription Factors in Plant Growth and Environmental Stress Response: A Comprehensive Review of Recent Advances

Abstract

In plants, SPL is a distinct family of transcription factors. Its protein structure possesses a highly conserved SBP domain comprising two zinc finger structures and nuclear localization regions, and microRNAs (miR156) control the transcriptional expression of the majority of SPL genes. SPLs are key TFs in regulating organ morphogenesis, phase transition/floral induction, and yield-related traits in agronomic and horticultural crops. These biomolecules have been functionally characterized for their role in augmenting plant responses to abiotic and biotic stresses. Present research gaps and viewpoints are addressed herein. Using these extensive data, researchers can more comprehensively understand how SPL genes modulate agronomic features in different ways.

1. Introduction

One of the main challenges facing modern agriculture in satisfying the increasing need for food is creating new elite crop varieties with enhanced agronomic traits, such as ideal plant architecture and flowering time [1,2]. In recent decades, researchers have successfully cloned numerous genes that control key agronomic traits. A subset of these genes encodes transcription factors that are SQUAMOSA-PROMOTER BINDING PROTEIN-like (SPL) [3,4]. The initial finding of SPL genes in Antirrhinum majus (snapdragon) was prompted by the ability of similar proteins, AmSBP1 and AmSBP2, to bind the promoter of the floral meristem identity gene SQUAMOSA (SQUA) [4,5,6]. Functional studies of numerous SPL family members have been completed in a number of economically important crops, in addition to the model plant Arabidopsis [3]. SPLs impact practically every aspect of plant development and growth, suggesting extensive sub- and neo-functionalization during speciation after whole-genome or local duplication events [7]. Another interesting finding is that miR156 (microRNA156s) targets many SPL genes across different plant species. This finding suggests that the miR156/SPL regulatory module has evolved to control different developmental processes in plants [7,8]. The SPL gene family is present in virtually every type of green plant, from algae and mosses to gymnosperms and angiosperms [9]. In this review, we briefly outline the structural features of the SPL gene family. The role of SPL genes in growth and developmental biology is extensively discussed. The regulation of plant responses to abiotic and biotic stresses is presented in detail. Critical discourse, research limitations, and prospective directions are elucidated to facilitate the understanding of SPL TFs by early-stage researchers. It is our expectation that this manuscript will enhance the comprehension of these vital compounds and assist in future breeding initiatives.

2. Structural Composition of SPL TFs

A 76-amino-acid DNA-binding domain, known as the SQUAMOSA promoter binding protein [SBP] domain, is present in all SPL proteins. This domain possesses two zinc binding sites, and a nuclear localization signal (NLS) partially overlaps with the second zinc finger (Figure 1A,B). Upon nuclear import, the SBP domain binds to a consensus binding site that contains a GTAC core motif and flanking sequences that are specific to genes [6]. Outside of the SBP domain, SPL proteins show very little conservation. Although several brief motifs have been found to be preserved throughout SPL subgroups, their exact role remains unclear. A significant number of SPL genes have been found and categorized into various clades (Figure 1A,B) using three plant species, including Arabidopsis, cucumber, and rice, because their coverage covers monocots (rice) and eudicots (Arabidopsis, cucumber), capturing vast phylogenetic diversity. Group I/II possesses extended N-terminal sections, including phosphorylation sites (e.g., MAPK target motifs) that modulate protein stability during stress. Group III/IV contains C-terminal domains abundant in glutamine-rich areas, perhaps enhancing protein–protein interaction. Group VI/VII encompasses additional motifs, such as PLN03210 superfamily domains, associated with chromatin remodeling or RNA-binding activities. In terms of functions, Group I (e.g., AtSPL8, OsSPL14) facilitates the transition from vegetative to reproductive stages and the architecture of the panicle [3,4]. Group II (e.g., AtSPL3, AtSPL4, AtSPL5) regulates flowering timing using miR156-mediated modulation. Group III (e.g., AtSPL9/15, SlSPL-CNR) influences foliar development and fruit maturation. Group IV (e.g., AtSPL13, OsSPL16) is associated with stress adaptation and root development. OsSPL16 (IPA1) enhances drought resistance by regulating root lignin levels. Group VI/VII (for instance, AtSPL7, CnSPL1) is engaged in micronutrient homeostasis (e.g., copper absorption) and the development of reproductive organs (Figure 1B). These genes are extensively discussed below for their role in growth and stress biology.

3. Regulatory Functions of SPL TFs

SPL TFs are essential genes for crop improvement because of their critical roles in controlling plant shape, panicle architecture, and grain formation. Herein, we explain the role of SPL TFs in developmental biology.

3.1. Seed Development

The process of seed development (SD) begins with double fertilization and typically involves three steps in regular seeds: embryogenesis, filling, and desiccation [10]. Given that ROS accumulation is inhibited in miR156 overexpression mutants, the MIR156/SPL9 regulatory module is thought to be involved in controlling ROS accumulation. Members of the MIR156 family displayed upregulated expression throughout SD. For example, in Phaseolus vulgaris L. (common bean), pvu-miR156i exhibits inconsistencies with the reduced levels of its anticipated SPL9 target [11,12]. This finding indicates that the MIR156/SPL9 regulatory module may modulate ROS levels during SD and indirectly aid in preserving genomic integrity [11,12]. By binding to the ABI5 promoter, the Arabidopsis AtSPL9 increases ABA accumulation in seeds and prevents pod germination once seeds have matured [13]. Despite these convincing results, a comprehensive functional study would aid in further elucidating the role of SPL genes in seed development.

3.2. Root Biology

Plants rely on their roots to both anchor themselves to the soil and absorb nutrients and water. The layout and design of a plant’s root system in relation to the soil are referred to as its root system architecture [14,15]. Crown roots, sometimes called adventitious roots, constitute the bulk of a cereal plant’s root system and play a key role in soil anchoring and nutrient and water intake [16]. A mutant strain of rice called lower crown root number1 (lcrn1) was found to have a reduced number of crown roots [16]. lcrn1 is caused by a mutation of a putative transcription factor-coding gene, OsSPL3. The mutant phenotype arises from lcrn1 disruption of the cleavage of OsSPL3 transcripts directed by OsmiR156. The authors of other studies have suggested that OsSPL3 binds to induce the expression of OsMADS50. Overexpression of OsMADS50 in rice produced phenotypes similar to those of lcrn1, thus indicating the importance of the OsmiR156-OsSPL13 module in crown root development [16]. The rate of root regeneration is highly dependent on both age and injury. The process of wound-induced auxin production initiates root regeneration in plants. Plants’ ability to regenerate their roots steadily declines with age [17]. The mechanism by which wounding causes the auxin to burst and age and by which wound signals work together to control the ability of roots to regenerate largely depends on SPL TFs. In Arabidopsis, the increased levels of miR156-targeted SPL2, SPL10, and SPL11 block wound-induced auxin production, suppressing root regeneration with age [17]. Wounding quickly activates a subset of APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) transcription factors, such as ABSCISIC ACID REPRESSOR1 and ERF109, which work as a substitute for wound signals to trigger auxin production. The SPL2/10/11 dampens auxin accumulation at the wound site by directly binding to the promoters of AP2/ERFs and reducing their induction in older plants [17]. In light of the above findings, it seems plausible that miR156-SPL modulates root regeneration via the ERF pathway, and its exact molecular mechanism, if applicable, requires further exploration. Nitrogen (N) is a crucial component for the growth of rice. Rice alters the shape of its roots in response to N-limiting conditions. SPL14 modulates root elongation in conditions of low nitrogen (LN) availability [18]. In one study, it was observed that SPL14 expression was induced by LN supply, and, unlike wild-type plants, its knockdown had a smaller impact on root length under LN conditions. However, when overexpressed under sufficient nitrogen supply, root length was enhanced to the same degree as in WT plants under LN conditions, indicating that SPL14 was induced by the LN conditions and caused changes in root elongation [18].

3.3. Vegetative Growth

SPL TFs have the ability to control the structure, morphology, and number of leaves in plants in a dynamic manner. Transgenic rice plants overexpressing OsSPL14 exhibited shorter leaf growth periods, narrower leaf shapes, and thicker and darker green leaves [19]. In high planting density, the wheat TaSPL8 knockout mutants have erect leaves due to lamina junction loss, compact architecture, and increased spike quantity [20]. The AUXIN RESPONSE FACTOR and CYP90D2 (brassinosteroid biosynthesis gene) promoters are activated by TaSPL8. These findings suggest that TaSPL8 regulates lamina joint formation via auxin signaling and brassinosteroid production [20]. Mutations in the MsSPL8 gene in Medicago sativa resulted in alfalfa seedlings having more leaves and losing their serrations [21]. A double-stranded RNA-binding protein required for appropriate miRNA maturation is encoded by HYL1 (HYPONASTIC LEAVES 1) in Arabidopsis [22]. A characteristic leaf incurvature phenotype is observed in its null mutant, hyl1. BcpLH (Brassica rapa ssp. pekinensis LEAFY HEADS), a near homolog of HYL1, is differentially produced in flat juvenile leaves and extremely incurved adult leaves in Chinese cabbage. BcpLH is shorter and lacks protein–protein interaction domains compared to HYL1. To determine the association between BcpLH and deficiencies in miRNA biogenesis and leaf flatness, antisense-mediated downregulation of BcpLH diminished a specific subset of miRNAs (including miR156) and enhanced the activity of their target genes SPL9, resulting in upward curvature of rosette leaves and early leaf incurvature, combined with the enlargement of leaf heads, precocity, and transition to a round-to-oval shape of leafy heads [22].

OsSPL14 (ideal plant architecture 1), a new “Green Revolution” gene, regulates rice architecture [23]. The ortholog of OsSPL14 in wheat is TaSPL14, and in one study, its knockout significantly reduced plant height [24]. Similarly, in another study, loss of aspSPL14 in garden asparagus generated shorter mutants [25]. In China, tip pruning is a common agricultural technique used to avoid lodging in asparagus cultivation. Given that semi-dwarf plant architecture enhances lodging resistance in garden asparagus, developing semi-dwarf garden asparagus lines would be beneficial in asparagus breeding [25]. Transgenic switchgrass plants overexpressing miR156 possess shorter internodes, impacting their ornamental appeal and inhibiting biomass yield for feed and bioenergy production [26]. In contrast, when PvWOX3a is overexpressed, stems grow longer and wider. When overexpressed in a transgenic switchgrass line that also overexpresses miR156, it induces a significant increase in internode length and diameter and leaf blade breadth in the plants. Remarkably, the miR156OE-27 transgenic line was found to have a lower internode number than double transgenic switchgrass plants. This finding is primarily due to the fact that the top stunted internode grew longer in the double transgenic switchgrass plants, making the stunted nodes visible under the microscope [26]. PlSPL14-overexpressing tobacco led to increased stem diameter, increased xylem thickness, and improved stem strength in Paeonia lactiflora plants; in comparison, PlSPL14-silenced plants exhibited lower stem diameter and weakened stem strength [27]. Reduced expression of Slender rice 1 (SLR1), a negative regulator of the GA signaling pathway, was recorded in the PlSPL14-overexpressed lines [27]. Members of the PHYTOCHROME-INTERACTING FACTOR-LIKE (PIL) family have been recognized as novel repressors of tillering in cereal crops [28]. Overexpression of TaPIL1 decreases wheat tiller quantity, whereas overexpression of the TaPIL1-SUPERMAN repression domain results in enhancement. In addition, TaPIL1 stimulates the transcription of wheat TEOSINTE BRANCHED1 (TaTB1) and interacts with (TaSPL)3/17, which are activators of TaTB1 transcription. Further evidence from this study suggests that PIF/PILs engage in a conserved function in wheat, rice, and Arabidopsis by interacting with SPLs to suppress tillering and branching. It is possible that the conserved feature of TB1’s PIL/SPL regulation of tillering evolved prior to the divergence between monocot and dicot plants [28]. The genes associated with the regulation of cereal crop tillering are crucial candidates for selection in crop breeding aimed at enhancing plant architecture and agronomic performance. In this context, it is of value to examine whether these components can be customized to achieve optimal plant design and ultimately enhance agricultural yields.

3.4. Reproductive Biology

3.4.1. Flower Induction

The floral stage of higher plants marks the start of a new phase of the plant’s life cycle. Genes belonging to the SPL family are crucial regulators of flowering time in plants [29]. A decrease in the expression level of the targeted Gossypium hirsutum (cotton) GhSPLs gene and subsequent attenuation of MADS-box gene transcription may result from the high expression of GhmicroRNA157 in the late stage of flower development. It also slows down the development of flower organs and inhibits the auxin signaling pathway [30]. The regulation of wheat floret development and differentiation was accelerated through the overexpression of TaSPL13-2B [31]. By increasing the expression of SUPPRESSOR OF CONSTANS1 (AtSOC1), FRUITFULL (AtFUL), and APETALA1 (AtAP1), ectopic overexpression of MiSPL3a/b accelerated Arabidopsis flowering by 3–6 days compared to wild-type (WT) plants [32]. Transgenic cucumber plants overexpressing CsSPL13A (CsSPL13A-OE) exhibited early flowering, more male flowers, and longer flower stalks [33]. The flowering integrator gene Flowering Locus T (CsFT) and sugar-mediated flowering gene β-amylase (CsBAM) are elevated by CsSPL13A in cucumbers. CsSPL13A directly interacts with CsFT and CsBAM promoters, indicating that it collaborates with them to regulate cucumber flowering [33].

3.4.2. Phase Transition

The age pathway is regulated by the phytohormone brassinosteroid (BR), which facilitates the transition from the juvenile to adult phase. In Arabidopsis, while BR treatment dramatically increased SPL9, SPL10, and SPL15 expression levels in wild-type plants, they were significantly lower in bri1-301 (BR-insensitive mutant) [34]. The overexpression of pSPL9:rSPL9 exhibited hypersensitivity to BR in hypocotyl elongation and somewhat mitigated the prolonged vegetative phase transition in bri1-301 mutants. Moreover, we demonstrated that BRASSINAZOLE-RESISTANT 1 (BZR1) interacts with SPL9 to collaboratively modulate the expression of downstream genes [34]. The authors of another study highlighted that delays in vegetative phase change are caused by a loss-of-function mutation in the BR biosynthesis gene DWARF5 (DWF5). The defective phenotype is mainly caused by lower levels of SPL9 and miR172 and an elevation in TARGET OF EAT1 (TOE1) [35]. Additional findings from this study indicate that BIN2, a kinase similar to GSK3, directly interacts with SPL9 and TOE1, phosphorylating them to trigger proteolytic disintegration. From the above results, it can be concluded that BRs control the vegetative phase transition in plants by concurrently stabilizing SPL9 and TOE1 [35] (Figure 2A). Additionally, the SPL-mediated phase transition in fruit development has been discussed below.

3.4.3. Tillering

Effective panicle count is directly influenced by tiller number. Besides being the focus of high-yield breeding, this characteristic serves as a signal for ideal plant architecture [36]. Tillering is significantly influenced by the phytohormone auxin. Auxin-deficiency root traits, including shortened lateral roots, decreased lateral root density, and increased root angles, were observed in the high-tillering and semidwarf 1 (htsd1) rice mutant [37]. The mutant htsd1 encodes the auxin influx transporter OsAUX1. The SPL7 binds with the OsAUX1 promoter to activate its expression. The mutant htsd1 showed a lower expression of SPL7 than WT. It can therefore be suggested that the lower expression of SPL7 in the mutant line causes the semi-dwarf and high-tillering phenotype [37]. Based on the results of the aforementioned study, SPL TFs can be categorized as a negative regulator of tiller number. However, this issue can be resolved by deleting a particular cis-regulatory region in the promoter region. Enhancing crop genetics necessitates navigating intricate trade-offs resulting from gene pleiotropy and linkage drag, as illustrated by IPA1 (Ideal Plant Architecture 1), a prototypical pleiotropic gene in rice that augments grains per panicle while diminishing tiller numbers [38]. The removal of a 54-base-pair cis-regulatory region in IPA1 prevents the trade-off between tiller number and grains per panicle, leading to a substantial increase in grain yield per plant compared to the WT line ZH11 (Figure 2B,C). The transcription factor An-1 targets the deleted region “GCGCGTGT” to suppress IPA1 expression in the panicles and roots. An-1 has been shown to be a domestication-related transcription factor that positively regulates awn length and negatively regulates grain number per panicle [38]. It is expected that focusing on gene regulatory regions will aid in elucidating trade-off effects and provide great potential for breeding plants with complementary advantageous characteristics.

3.4.4. Grains and Yield

Nineteen SPL TFs can be found in the rice genome, and some of them have been linked to rice yield [39]. For instance, knocking down OsSPL4 results in improvements in rice panicle primary and secondary branches, grains per panicle, and grain size [40]. OsSPL4 is responsible for regulating the formation of spikelets by facilitating cell division. OsSPL4 is a target of osa-miR156, which can cleave OsSPL4, thus establishing the OsmiR156-OsSPL4 module that modulates rice grain size. The results of an OsSPL4 genetic diversity study suggest that artificial selection introduced the large grain gene in indica rice from Australian variants [40]. The difference between indica and japonica grain morphology is regulated by OsSPL12. In the genomic region of OsSPL12 from 1479 rice types, 33 SNPs were found, 20 in introns and 13 in exons. Five of the thirteen non-synonymous SNPs found in the exons cause changes in amino acids. Of all of the functional domains of OsSPL12, the C-terminal transcription activation domain, which contains one of these SNPs (SNP1066), has the strongest self-activation [41]. OsSPL13 is encoded by GLW7 (GRAIN LENGTH AND WEIGHT ON CHROMOSOME 7) [42]. During genome-wide association studies (GWASs) on 381 japonica rice varieties, it was determined to be a quantitative trait locus (QTL) governing grain weight and length. The GLW7 locus contributes to about 30% of grain length variation and 25% of grain weight variance in this japonica population. The main factor influencing grain size variation in japonica rice is the tandem repeat of the CCATTC sequence from −146 bp to −135 bp in the 5′-UTR of OsSPL13, based on the results of allelic variation analysis using sequencing of the OsSPL13 from 26 small-grain and 21 large-grain varieties [42]. Analysis of gene expression confirmed that OsSPL11, which is involved in regulating grain size, is nuclear-localized and significantly expressed in spikelet hull and juvenile developing grains [43]. Grain length (GL), grain weight (GW), and thousand grain weight (TGW) were all adversely impacted in osspl11 mutant plants; however, GL and TGW were positively impacted in OsSPL11-overexpressing lines [43]. In subsequent investigations, it was verified that OsSPL11 directly stimulates the expression of GW5L to modulate GS [43]. Excluding TaSPL13, which possesses the miR156 recognition element in its 3-untranslated region (3-UTR), nine out of the ten orthologous TaSPL genes in rice possess microRNA recognition elements (MREs) in their last exons [44]. Altering the MRE of TaSPL13 via CRISPR-Cas9 resulted in 12 mutations across the three homoeologous genes. The MRE mutations resulted in an approximate two-fold elevation in mutated TaSPL13. Phenotypic examination revealed that MRE mutations in TaSPL13 led to reduced flowering time, tiller number, and plant height while concurrently increasing grain size and number in wheat. TaSPL13 mutants share phenotypes with Arabidopsis AtSPL3/4/5 and rice OsSPL13/14/16 mutants, indicating potential for wheat yield enhancement [44].

3.4.5. Fruit Development

Fruit development is closely associated with SPL TFs, as reported by in several studies. For instance, SPL13 suppression in tomato increases inflorescences on vegetative and lateral branches, decreases the number of flowers and fruit, shrinks fruit, and decreases yield [45]. SPL13 directly binds to the tomato inflorescence-associated gene SINGLE FLOWER TRUSS (SFT) promoter region to positively regulate its expression [45]. One particular type of trichome, known as a spine, covers the surface of cucumbers. These trichomes are multicellular and unbranched. The esthetic value of cucumber fruits is dictated by the number and density of these fruit spines. In one study, fruit spine development was negatively impacted by CsWOX3, a typical transcriptional repressor. CsWOX3 showed a comparatively high expression level in the female floral organs of cucumber, particularly in the fruit exocarp [46]. CRISPR/Cas9 knockout of CsWOX3 increased fruit spine base width by two- to three-fold; in comparison, overexpression resulted in a decrease of 17% [46]. CsSPL15 can directly bind and activate CsWOX3, inhibiting downstream auxin-related genes, including CsARF18. Increased expression of CsWOX3 and enlarged fruit spines were recorded in CsSPL15-silenced plants [46]. The parthenocarpy of cucumbers is one of the most important agronomic features for determining the amount of fruit that they produce [1,2]. The application of CPPU significantly induced parthenocarpy in a naturally non-parthenocarpic cucumber line [1]. Under CPPU, the expression of CsSPL16-like was increased several-fold and could be considered a marker in the cucumber breeding program [1].

4. Postharvest Biology

Machinery, pathogens, and senescence induce significant losses of vegetables and fruits during harvest, sorting, storage, and transportation. The postharvest attributes of fruits and vegetables are regulated by TFs. SPL TFs have been historically documented to be involved in postharvest biology. For instance, strawberry fruits are subjected to both ABA and nordihydroguaiaretic acid (NDGA, an ABA biosynthesis inhibitor). ABA treatment induces strawberry receptacle ripening by enhancing the expression of miR156c while suppressing its target gene, SPL18 [47]. As a master ripening TF, the tomato SBP-box protein Colorless Non-ripening (SlSPLCNR or SlCNR) was cloned at an early stage and assessed. In a recent study, researchers found that CRIPSR/Cas9-generated slcnr mutants can produce complete red fruits, unlike the original Cnr mutant, which does not ripen [48]. After further investigation into the maturity traits in fruits of slcnr mutants (slcnr-16, slcnr-22, and slcnr-23) that lacked the SlCNR protein entirely, they discovered that these fruits only showed a slight decrease in lycopene content and a three-day delay in the beginning of ripening [49]. Mutating SlCNR did not affect the entire ripening process, with these findings showing that SlCNR is not a master regulator of ripening. SlCNR may have a novel function in the postharvest tomato fruit as a regulator of flavonoids. The results of molecular studies show that SlCNR suppresses SlMYB12 transcription activity and negatively influences flavonoid biosynthesis gene expression. SlSPL-CNR deletion through the utilization of the CRISPR/Cas9 editing technique can result in higher flavonoid contents in tomato fruits [49]. SlSPL-CNR knockout also facilitated the production of cutin in tomato peel [50]. Thirteen genes involved in cutin production, export, and assembly are directly repressed by SlCNR. It can therefore be concluded that SlCNR negatively regulates cutin content and cuticle thickness, impacting fruit firmness and permeability [50]. The exogenous application of 1-methyl cyclopropane (1-MCP) significantly delayed the ripening process of papaya fruit. The expression of CpSPL3, CpSPL6, and CpSPL7 was rapidly triggered following 1-MCP treatment, meaning that they could be considered as key ripening regulators. In banana, overexpression of MaSPL16 accelerated fruit ripening by inducing the transcription of MaNAC029 and ethylene biosynthesis genes (MaACO1) [51]. Based on the above findings, the role of SPL genes is highly complex and maybe species specific. In the case of papaya, the expression of CpSPL6/7 increased following 1-MCP application. On the other hand, the overexpression of MaSPL16 speeded the transition stage of banana fruit. The overexpression of MaSPL16 shortened the time in which banana fruit turn yellow from green.

5. Stress Response

Due to their sessile nature, plants must tolerate abiotic and biotic stresses. Because of these stresses, plant dispersal can be severely restricted, plant growth and development can be altered, and agricultural yields can be reduced [52]. Recent advances in our understanding of the molecular mechanisms that underlie plant responses to stresses have highlighted their multilevel nature. Various processes, such as sensing, transcription, processing of transcripts, and post-translational protein modifications, are involved [52]. Herein, the role of SPL TFs in fine-tuning the plant response to abiotic and biotic stresses is discussed in detail.

5.1. Abiotic Stress

When plants are exposed to abiotic stress, their cellular processes are altered, impacting their physiology in numerous ways. Multiple abiotic stresses, including heat, cold, salt, drought, and waterlogging, pose a threat to economically important plants. The mechanism by which SPL regulates plant tolerance to these stresses is discussed herein.

5.1.1. Cold Stress

Cold is a significant abiotic factor that compromises plant growth and agricultural yield [29]. Rice, a staple meal for half of the world’s population, is vulnerable to low-temperature stress, which results in weak seedling growth, yellowing, stunting, withering, and death [53]. Chilling stress can cause OsSAPK6 to phosphorylate and stabilize IPA1. Subsequently, IPA1 may enhance C-REPEAT BINDING FACTOR 3 (OsCBF3) expression by directly binding to the GTAC motif on the promoter [54]. OsSAPK6, IPA1, and OsCBF3 were all found to be positive regulators of rice chilling tolerance based on genetic data. IPA1 is essential for OsSAPK6’s chilling tolerance function (Figure 3A). Overexpression of OsCBF3 can restore the chilling-sensitive phenotype in IPA1 loss-of-function mutants. The natural gain-of-function allele ipa1-2D can improve seedling freezing tolerance and grain yield [54]. In this study, the freezing tolerance of rice rose with vegetative maturity. The expression of CBF2 was induced by the overexpression of SPL9 (rSPL9), which subsequently improved the chilling tolerance [55]. Upon further investigation, SPL9 was found to directly bind to the CBF2 promoter, activating its expression and increasing freezing tolerance [55]. During fruit development under cold stress, the accumulation of miR156c inhibits the production of MaSPL4 and miR528, which in turn enhances MaPPO activity. This cascade induces the formation of reactive oxygen species and indications of cold injury, notably peel browning [56]. In addition to controlling miR528, MaSPL4 influences genes related to lipid metabolism and antioxidant pathways, highlighting its function as a major regulator in the chilling response that functions independently of the CBF regulon. Thus, the overexpression of MaSPL4 in transgenic banana plants is associated with improved cold tolerance, presumably due to these synchronized molecular modifications [56]. From these results, it can be concluded that, to manage the developmental transition in plants, cold induces miR156, a master age-regulator, which in turn decreases the expression of SPL3 and SPL13. In addition, as plants mature, their cold tolerance capacity increases. As a result, the miR156-SPL pathway mediates the equilibrium between plant development and freezing tolerance.

5.1.2. Drought Stress

Drought is a major abiotic stress affecting the growth and yield of all economically important crops. ABA is a master regulator of drought stress that modulates stomatal movement [57]. Wheat seedlings treated with drought or abscisic acid showed a decrease in the expression of TaSPL6 genes, which were shown to be expressed predominantly in the roots [58]. TaSPL6-A overexpression in wheat increased drought sensitivity. In comparison with WT plants, transgenic lines showed reduced antioxidant enzyme activities, increased water loss from leaves, and greater levels of reactive oxygen species (ROS), malondialdehyde (MDA), and water. In contrast, augmented drought tolerance was achieved in TaSPL6-A- silenced wheat plants [58]. The majority of upland and improved lowland rice varieties carrying the OsSPL10^Hap1 allele showed enhanced drought tolerance [59]. In contrast, lowland and landrace rice varieties primarily possess the OsSPL10^Hap2 allele. Of note, varieties with the OsSPL10^Hap1 allele were found to have low expression levels of OsSPL10 and its downstream genes, OsNAC2, OsAP37, and OsCOX11, and further inhibited ROS and programmed cell death (PCD). Improved drought tolerance in rice was achieved through the induction of rapid stomatal closure and prevention of water loss with the knockdown or deletion of OsSPL10. By modulating OsNAC2 expression, OsSPL10 confers drought tolerance, and OsSPL10^Hap1 may be a helpful haplotype for improving drought tolerance in rice (Figure 3B) [59]. Drought stress causes an increase in microRNA156ab in the apple Malus sieversii (M. sieversii) [60]. Researchers noted increased auxin accumulation, sustained apple plant development, and enhanced plant resilience to drought stress after overexpressing msi-miR156ab. miR156ab-OE transgenic apple lines showed an increase in antioxidant enzyme activity and proline levels, which contributed to better drought tolerance. In Arabidopsis, heterologous expression of MsSPL13 reduced auxin content and suppressed growth under drought stress and normal circumstances [60]. In MsSPL13-OE transgenic Arabidopsis, antioxidant enzyme activity was also inhibited, decreasing drought resistance. In our study, we found that MsSPL13 binds to GTAC motifs in the promoters of auxin-related genes MsYUCCA5, PIN-FORMED7 (MsPIN7), and Gretchen Hagen3-5 (MsGH3-5) to regulate auxin metabolism. It can be concluded that the miR156ab-SPL13 module regulates apple drought tolerance via the auxin metabolism pathway [60]. Based on the results of the above studies, SPL could be considered a negative regulator of drought tolerance in plants.

5.1.3. Flooding Stress

Globally, agricultural and food security are most threatened by floods, second only to droughts, as a result of natural disasters [61]. Between 2008 and 2018, floods caused USD 21 billion in losses, accounting for 19% of all agricultural losses in low- and lower–middle-income countries [61]. Ethylene triggers SUB1A and ABA, which mediate rice and Arabidopsis reactions to flooding stress (FS) [62]. Under FS, miR156-OE alfalfa plants exhibit a rise in ABA-metabolites [63]. Conversely, SPL13-RNAi alfalfa lines also displayed increased FS tolerance with increasing ABA accumulation [63]. In-depth research is still lacking to comprehensively elucidate the role of SPL genes in FS.

5.1.4. Heat Stress

Heat stress presents a serious threat to global crop productivity. Heat stress during flowering appears to represent the greatest harm to crops, as plants are more sensitive to temperature increases during reproductive development than during vegetative growth [64,65,66]. Enhancing crop thermotolerance without compromising yield is therefore vital. SPL1 and SPL12 in Arabidopsis play a redundant role in thermotolerance during the reproductive stage [67]. Heat stress hypersensitivity was observed in spl1-1 spl12-1 inflorescences; in comparison, Arabidopsis and tobacco plants with overexpressed SPL1 or SPL12 showed improved thermotolerance. In the spl1-1 spl12-1 inflorescence, heat stress disrupted 39% of ABA-sensitive genes [67]. Inflorescence thermotolerance was restored in spl1-1, spl12-1, and the ABA biosynthesis mutant aba2-1 through pre-application of ABA and overexpression of SPL1. The molecular network of SPL1 and SPL12 provides fresh insights into plant thermotolerance mechanisms during reproduction [67]. Although no studies have been conducted on major agronomic crops such as rice and wheat thus far, these biomolecules (SPL1 and SPL12) could be considered key biomarkers in enhancing yield under the ever-present threat of heat stress.

5.1.5. Heavy Metal

Soil cadmium (Cd) contamination is a growing threat that has devastating consequences for agricultural output quality and crop yield. Cd toxicity in crops can be addressed through two methods: spraying blocking agents, such as plant hormones, to reduce aboveground accumulation, and genetic engineering to modify metal transport protein expression levels [68]. The OE-NtSPL4a and OENtSPL4aΔ transgenic tobacco were found to have significantly lower Cd levels (85.1% and 46.7%, respectively) compared to WT tobacco [3]. Overexpression of NtSPL4a also impacted mineral nutrient balance in transgenic tobacco. Moreover, Cd toxicity-induced oxidative stress and leaf chlorosis were successfully reduced by overexpressing NtSPL4a/NtSPL4aΔ [3]. The overexpression of OsSPL7 considerably enhanced rice tolerance to Cd [69]. The results of biochemical and molecular analyses suggested that OsSPL7 induces a decrease in OsNramp5 expression, resulting in reduced translocation and an accumulation of Cd in rice roots [69]. The aforementioned studies provide evidence of the key role of SPL genes in regulating plant response to heavy metal stress.

5.2. Biotic Stresses

Plant defense mechanisms are often challenged by different biotic organisms, including bacteria, fungi, viruses, and insects [70]. SPL TFs serve as plant “ammunition” against these challenging conditions. For instance, the miR156-SPL9 module is responsible for controlling plant growth and development, whereas SQUINT (SQN) controls plant maturation by increasing miR156 activity [71]. SQN is involved in the jasmonate (JA) system, which is a key signaling mechanism that controls plants’ response to insect herbivory and infections. Arabidopsis sqn mutants were found to be more susceptible to Botrytis cinerea (B. cinerea) than the wild type. SQN, however, is not a part of the early pattern-triggered immune response [71]. In contrast, sqn loss-of-function mutants treated with B. cinerea exhibited decreased JA accumulation, JA response, and sensitivity to JA, indicating that SQN positively controls the JA pathway. Increased expression of SPL9 was recorded in the sqn mutant following B. cinerea stress. The knockout mutant of spl9 or overexpression of miR156 restored the resistance of Arabidopsis plants to B. cinerea by inducing JA accumulation. The miR156–SPL9 module may mediate the SQN-JA pathway to increase plant resistance to B. cinerea [71] (Figure 4A). Magnaporthe oryzae (M. oryzae), a fungal infection, causes blast disease, which has a major impact on rice output. Global food security is threatened by this disease, which results in annual production losses of 10% to 30% [72]. In contrast with the role of SPL9 against B. cinerea in Arabidopsis, the overexpression of OsSPL10 in rice increased the resistance against M. oryzae by positively regulating the JA signaling pathway. Chitin-induced immunological responses, including ROS burst and callose deposition, are positively regulated by OsSPL10. OsSPL10 physically binds to OsJAmyb, a key TF in JA signaling, positively regulating its protein stability [72]. Suppressing the expression of miR156 triggered its target gene OsSPL14, which led to enhanced resistance to blast disease [73].

Bacterial leaf blight (BLB) in rice is caused by the bacterial pathogen Xanthomonas oryzae pv. oryzae (Xoo), which can cause yield losses of up to 70% [74]. OsSPL7/14/17 proteins bind to OsAOS2 and OsNPR1 promoters, activating transcription and regulating JA accumulation and SA signaling [75]. Overexpression of OsAOS2 or OsNPR1 reduces the susceptibility of the triple mutant osspl7/14/17. Exogenous JA treatment improves resistance in the osspl7/14/17 triple mutant and miR156 overexpressing plants [75]. Research findings indicate that bacterial pathogen-activated miR156/529 inhibits PAMP-triggered immunity (PTI) responses, including pattern recognition receptor Xa3/Xa26-initiated PTI. To aid pathogen infection, bacterial pathogens alter miR156/529-OsSPL7/14/17 modules to inhibit OsAOS2-catalyzed JA accumulation and the OsNPR1-promoted SA signaling pathway. The miR156/529-OsSPL7/14/17-OsAOS2/OsNPR1 regulatory network offers a viable approach to genetically enhance rice disease resistance [75] (Figure 4B).

JA ZIM-domain (JAZ) proteins, such as JAZ3, can interact with the SPL9 protein family. Gradually rising SPL9 levels lead to JAZ3 accumulation and a diminished JA response [76]. Consistent with these findings, the deletion of the OsSPL10 gene enhanced the direct and indirect defenses of rice, making it more resistant to brown planthopper (BPH) [77]. Secondary metabolites involved in defense were shown to accumulate to a greater degree in the spl10 mutant through the terpenoid and phenylpropanoid pathways [77]. Of note, the OsSPL14 knockout lines displayed increased susceptibility to BPH [78]. The vulnerability of spl14 mutants to BPH was attributed to the decreased expression of OsPR4 and OsWRKY45 genes [78]. The functional role of certain SPL genes is tabulated and presented below (

Table 1. Enlisted functions of SPL genes in plant growth and abiotic and biotic stresses.

Gene	Species	Role	References
AtSPL9	Arabidopsis	Inhibition of pod germination.	[13]
OsSPL13	Rice	Mutation caused a low number crown roots.	[16]
AsSPL14	Asparagus	Mutation leads to a semi-dwarf phenotype.	[25]
CsSPL13A	Cucumber	Overexpression of CsSPL13A induced male flowers.	[33]
SPL9	Arabidopsis	Positive regulator of brassinosteroid-mediated phase transition.	[35]
TaSPL13	Wheat	Mutation in the MRE enhanced wheat yield.	[44]
CsSPL15	Cucumber	Silencing leads to an increase in fruit spine density.	[46]
SlSPL-CNR	Tomato	Knocking out the produce tomato with a thick cutin layer.	[50]
SPL9	Rice	Overexpression enhanced the freezing tolerance by activating CBF.	[55]
MsSPL13	Apple	Overexpression significantly reduced apple tolerance to drought.	[60]
SPL13	Alfalfa	RNAi-silencing enhanced flooding tolerance by increasing ABA accumulation.	[63]
SPL1	Arabidopsis	Overexpression boosted the heat tolerance via the ABA pathway.	[67]
NtSPL4a	Tobacco	Reduced cadmium toxicity and uptake.	[3]
SPL9	Arabidopsis	Knock-out mutant exhibited increased resistance to B. cinerea.	[71]

).

6. Conclusions and Future Perspectives

Numerous SPL genes have been found in food crops (rice, maize, and wheat), fodder crops (alfalfa), medicinal plants, and woody plants. The results of recent studies show that SPL TFs are crucial in plant responses to both biotic and abiotic stresses and that they participate in a wide variety of physiological processes, including the formation of tissues and organs, the regulation of hormone metabolism, the dynamics of development, the synthesis of secondary biomass, and more. The dynamic functions of these biological molecules are yet to be fully elucidated. Research gaps and open questions remain, representing key directions for future studies.

(1)

Since SPL genes are largely involved in hormonal metabolism, the functional role of these genes in regulating root development can be further evaluated.

(2)

The comprehensive role of these genes in the fruit development of horticultural crops is poorly understood. For instance, a high expression of SPL16-like in natural parthenocarpic cucumber lines has been observed. Its functional characterization could be vital in producing parthenocarpic fruits.

(3)

The questions of whether the targeted overexpression or silencing of SPL genes can optimize plant architecture to enhance agricultural productivity, and how stress-induced epigenetic modifications or post-translational regulatory mechanisms might be strategically leveraged to develop resilient, high-yielding crop varieties, remain unanswered.

(4)

SPL genes regulate rice responses to fungi, bacteria, and insects by modulating the JA and SA signaling pathways. However, no reports on their regulation in horticultural crops are available. In light of this research gap, the role of SPL genes could be studied in cucumbers under powdery mildew conditions or in tomato plants subjected to B. cinerea.