Plant Aux/IAA Gene Family: Significance in Growth, Development and Stress Responses

Abstract

Auxin plays a crucial role throughout the entire life cycle of plants. The auxin/indole-3-acetic acid (Aux/IAA) gene family serves as a negative regulator of auxin response and is one of the earliest auxin-responsive gene families. It regulates the expression of auxin-responsive genes by specifically binding to auxin response factors. This review summarizes the protein structural characteristics of the Aux/IAA gene family and its typical and atypical transduction mechanisms in auxin signaling. Additionally, it examines the role of Aux/IAA in regulating plant growth and development, as well as its function in modulating plant resistance to abiotic stress through hormonal signaling pathways. Our findings indicate that the Aux/IAA gene family plays a significant role in plant growth and development, as well as in abiotic stress resistance. However, research on the functional roles of the Aux/IAA gene family in crops such as rice, wheat, and maize remains relatively scarce. Furthermore, we identified key questions and proposed new research directions regarding the Aux/IAA gene family, aiming to provide insights for future research on plant hormone signaling and molecular breeding in crop design.

1. Introduction

Auxin is one of the most important plant hormones, playing a crucial regulatory role in growth and development throughout the entire life cycle of plants [1]. It regulates various processes, including tropic growth, tissue differentiation, cell division, elongation, organogenesis, morphogenesis, apical dominance, and flowering time [2,3,4]. Auxin can also interact with other signal transduction pathways to regulate extracellular developmental processes [5,6,7]. The Aux/IAA family, a gene family involved in early auxin response in plants, plays a significant role in the mechanism of auxin action. Aux/IAA proteins and auxin response factors (ARFs) form heterodimers and participate in various physiological processes via the auxin signaling pathway [8]. These proteins are identified as short-lived nuclear proteins. At low auxin levels, Aux/IAA proteins interact with ARFs to inhibit the activation of ARF target genes [9]. At high auxin levels, Aux/IAA proteins are ubiquitinated through interaction with TRANSPORT INHIBITOR RESPONSE 1/auxin signaling F-box (TIR1/AFB) and subsequently degraded via the 26S proteasome pathway, releasing ARF activity [10]. Aux/IAA genes are specific to plants and have been identified in Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), maize (Zea mays), wheat (Triticum aestivum), tomato (Lycopersicon esculentum), and potato (Solanum tuberosum), but are absent in the genomes of bacteria, animals, or fungi [11]. While the biological functions of some Aux/IAA genes in Arabidopsis and rice have been reported, information is limited for crops such as maize, wheat, and potato [12,13]. With the development of multi-omics approaches, significant progress has been made in the research of Aux/IAA genes, although much remains unknown. In this article, we primarily provide an overview of research on Aux/IAA proteins in plants such as Arabidopsis, rice, and maize, aiming to facilitate future studies on plant hormone signal transduction and molecular breeding applications in crop science.

2. Structural Characteristics of Aux/IAA Proteins

Aux/IAA proteins consist of four conserved domains: Domains I, II, III, and IV (Figure 1). Domain I contains a conserved EAR (ERF-associated amphiphilic repression) module that binds to the co-repressor TPL (TOPLESS) [14]. Domain II is highly conserved and serves as one of the core components of auxin signaling transduction, featuring the amino acid core sequence “VGWPPV”. This domain mediates the degradation of Aux/IAA proteins through ubiquitin-mediated proteolytic enzymes, resulting in shorter half-lives [12]. Domain III exhibits a βαα composite fold region, where this binding domain plays a crucial role in mediating protein dimerization and facilitating DNA recognition [15]. Domain IV contains a GDVP motif, likely contributing to electrostatic interactions among proteins [16,17]. Domains III and IV, collectively referred to as Phox and Bem1 (PB1) domains, function as binding regions for ARF proteins, enabling interactions between Aux/IAA and ARF family proteins, thereby repressing the expression of auxin-responsive genes.

3. Auxin Signal Transduction Pathway

Auxin primarily regulates plant growth and development through coordinated signal transduction, anabolism, and polar transport within the plant [18,19]. The auxin transduction pathway is primarily mediated by three core signaling pathways: TIR1/AFB proteins, Aux/IAA transcriptional repressor proteins, and ARF transcription proteins [20,21,22]. In the presence of low or absent auxin concentrations, Aux/IAA proteins function as repressors. The PB1 domain interacts with the CTD domain of ARF, and Aux/IAA proteins recruit the related co-repressor TOPLESS (TPL) to inhibit ARF activity, thereby suppressing the expression of downstream target genes (Figure 2A) [23]. However, at high auxin concentrations, Aux/IAA proteins bind to auxin and TIR1 inhibitors, enhancing the ability of TIR1 to recognize Domain II in Aux/IAA proteins. Auxin binds to the SCF (Skp, Cullin, F-box containing complex) complex to form the SCF^TIR1/AFBs ubiquitin ligase, triggering the ubiquitination and degradation of Aux/IAA via the 26S proteasome, which reduces the binding affinity to ARF proteins (Figure 2B) [24]. This subsequently activates the N-terminal DBD domain of ARF proteins, allowing them to bind to auxin response elements (AuxREs), which activate the expression of auxin-responsive downstream genes [25,26]. Throughout plant development, this signaling pathway regulates auxin-mediated processes through various combinations of Aux/IAA-ARF transcriptional regulatory elements [10].

The complex functions of auxins cannot be fully elucidated by TIR1/AFB-mediated signaling transduction alone. Certain Aux/IAA proteins that lack conserved Domain I or II are incapable of binding to TIR1/AFB proteins, indicating that their mechanisms of action diverge from the classical auxin signaling pathway mediated by TIR1/AFB proteins [27]. This includes the TMK1-IAA32/34 and MPK14-AtIAA33 signaling pathways in Arabidopsis, as well as the SOR1-OsIAA26 signaling pathway in rice [28,29,30]. Reports indicate that high concentrations of auxin can induce cleavage of the carboxy-terminal (C-) region of transmembrane kinase 1 (TMK1), thereby transmitting auxin signals to the nucleus and cytoplasm, which regulates downstream gene expression. Concurrently, the C-terminal region specifically recognizes and phosphorylates AtIAA32/34; the modified AtIAA32/34 inhibits transcriptional activity by binding to ARF, leading to the suppression of cell elongation in the inner cells of the cotyledon apex [29]. Additionally, high concentrations of auxin can promote the activation of mitogen-activated protein kinase 14 (MPK14). The activated MPK14 can bind and phosphorylate the AtIAA33 protein, stabilizing it. At the same time, AtIAA33 competes with AtIAA5 to bind the downstream inhibitor ARF10/16, resulting in the degradation of AtIAA5 protein through the TIR1/AFBs nuclear receptor signaling pathway [30]. An E3 ubiquitin ligase SOR1 (soil-surface rooting 1) was identified in the roots of the rice mutant mhz2, which interacts with the typical protein OsIAA9 and the atypical protein OsIAA26. At low levels of ethylene or auxin, OsIAA9 binds to SOR1, inhibiting its E3 ubiquitin ligase activity and preventing SOR1 from modifying and degrading OsIAA26 protein. Conversely, at higher levels, OsIAA9 is degraded via the canonical auxin signaling pathway, activating SOR1, which subsequently degrades OsIAA26 through ubiquitination, ultimately inhibiting rice root elongation [28]. These studies indicate that auxin exhibits non-canonical signal transduction mechanisms across multiple regulatory levels. However, regarding biological significance, atypical and typical signaling transduction pathways coordinate with one another to control the complex developmental processes of plants [31,32].

4. Research on the Function of Aux/IAA Genes in Arabidopsis

4.1. Regulation of Aux/IAA Genes on the Growth and Development of Arabidopsis

Currently, 29 members of the Aux/IAA gene family have been identified in Arabidopsis [27]. Most members of the Aux/IAA family are associated with root growth and development, as well as other important biological processes (Figure 3). For instance, the AXR5 gene in Arabidopsis encodes the AtIAA1 protein, and the axr5 mutant exhibits auxin resistance. In media containing excess auxin, this mutant inhibits lateral root growth [33]. The overexpression of AtIAA1 has been shown to suppress hypocotyl elongation, leaf expansion, and stem elongation in Arabidopsis. This suggests that AtIAA1 may play a crucial role in regulating cell elongation and cell division in the aerial parts of Arabidopsis, as well as in vascular development [34]. The gene encoding AtIAA3 has a gain-of-function mutant atiaa3/shy2, which affects root hair growth, lateral root formation, and gravitropism, indicating that SHY2/IAA3 regulates multiple auxin responses in roots [35]. AtIAA8 regulates lateral root formation by interacting with the TIR1 auxin receptor and ARF transcription factors in the nucleus [36]. AtIAA12 and AtIAA14 are key regulators of lateral root formation. The AtIAA14/SLR-AtARF7/19 auxin pattern hinders lateral root formation by partially and completely blocking pericycle cell division [37,38,39]. In divided pericycle cells, the BDL/IAA12–MP/ARF5-mediated auxin response ensures the formation of lateral roots regulated by downstream SOLITARY-ROOT (SLR)/IAA14 [40]. Similarly, AtIAA18-AtARF7/19 and AtIAA19-AtARF7 are also essential for regulating lateral root formation [41,42]. Rinaldi et al. found that the mutant atiaa16 exhibits an auxin-deficient phenotype, characterized by reduced main root elongation, shortened root hairs, and a decreased number of lateral roots in seedlings, indicating that AtIAA16 plays a unique role in auxin-regulated root development [43]. AtIAA27 and RNA-directed DNA methylation (RdDM) represent an alternative pathway for transcriptional repression mediated by CLSY1 (CLASSY chromatin remodeling factor). This pathway plays a crucial role in regulating lateral root development, particularly when the canonical auxin pathway is disrupted [44]. The transcription of AtIAA28 is not induced by exogenous auxin, and the atiaa28 mutant exhibits severe defects in lateral root formation, characterized by impaired root hair number and reduced root branching patterns [45]. Additionally, AtIAA28 regulates the specification of lateral root primordia cells and the root branching pattern by modulating the expression of AtGATA23 [46]. In a study involving the Arabidopsis mutant axr2, it was found that the AtIAA7 (AXR2/auxin-resistant 2) gene can trigger photomorphogenic effects in plants and negatively regulate flowering time-related genes [47,48]. Furthermore, it has been observed that axr2-17/iaa7 mutants in Arabidopsis exhibit a lack of response to gravitropism in the stems, defects in hypocotyl gravitropism, and a reduction in hypocotyl length in the absence of light [49]. Rouse and Kubalová et al. [50,51] found that AtIAA17 is involved in several typical phenotypes controlled by auxin signaling, such as hypocotyl elongation, root gravitropism, and the formation of root hairs and adventitious roots. Moreover, AtIAA1, AtIAA3, AtIAA9, and AtIAA17 can be phosphorylated by PHYTOCHROME A (PhyA) in vitro, providing evidence for the involvement of auxin in phototransduction [52].

In addition to the typical Aux/IAA genes previously mentioned, plants overexpressing atypical Aux/IAA genes, such as AtIAA20, AtIAA30, and AtIAA31, exhibited abnormal gravitropic responses in their hypocotyls and roots, accompanied by significant inhibition of root growth. This observation indicates their involvement in the regulation of plant gravitropic responses, as well as in root growth and developmental processes [53]. Compared to wild-type (WT) plants, AtIAA20 and AtIAA30 plants displayed distinct phenotypes, including the formation of ectopic protoxylem cells in the root epidermis, vessel deformation, and the discontinuity or even loss of protoxylem [54].

4.2. The Impact of Aux/IAA Genes on Abiotic Stress in Arabidopsis

In the context of global warming, the increasing frequency of high-temperature droughts and severe soil salinization underscores the importance of plants’ ability to withstand non-biological stress. The Aux/IAA family plays a pivotal role in regulating plant defense responses through hormone signaling pathways [36,55]. Research has demonstrated that the CaNAC46 transcription factor in Arabidopsis enhances the expression of AtIAA4 in response to drought and salt stress [56]. Furthermore, transgenic Arabidopsis overexpressing MeAnn2 under salt stress exhibits improved salt tolerance compared to WT plants. Notably, the expression level of the auxin signaling pathway-related gene AtIAA4 significantly increases, suggesting that MeAnn2 may enhance the salt tolerance of Arabidopsis by modulating the auxin signaling pathway response mediated by AtIAA4 [57]. Under drought and high-salt conditions, AtCPR5-AtIAA12/19 jointly regulate auxin signaling transduction and inhibit lateral root development [58]. In drought conditions, AtIAA5, AtIAA6, and AtIAA19 are directly regulated by DREB2A and DREB2B, which maintain the levels of aliphatic glucosinolates (GLS) to enhance drought tolerance. The absence of AtIAA5/6/19 results in a decrease in GLS levels, consequently reducing the plant’s drought tolerance [59]. Other studies have found that under high chromium (Cr) levels, AtIAA14/SLR1 (SOLITARY-ROOT) inhibits the growth of both main and lateral roots while promoting adventitious root growth [60] (Figure 4).

5. Research on the Functions of Aux/IAA Genes in Rice

5.1. Regulation of Aux/IAA Genes in Rice Growth and Development

Aux/IAA genes play a crucial role in regulating plant development, with 31 identified members in rice [61]. These genes are involved in various growth and developmental processes, including lateral root development, tiller angle formation, and grain development (Figure 5). For instance, OsIAA1 is induced by auxin and is essential for photomorphogenesis and coleoptile elongation in rice [62]. It also negatively influences plant height and leaf angle through auxin and brassinosteroid (BR) hormone signaling [63]. OsIAA3 positively regulates grain size in rice (Oryza sativa) by promoting cell expansion and proliferation of the spikelet hull [64]. Additionally, OsIAA3, OsIAA17, and Gnp4 interact to regulate the activity of the OsARF25 transcription factor (TF), thereby affecting grain length and leaf angle [65,66]. Qiao et al. [67] found that OsARF6 interacts with the auxin signaling negative regulators OsIAA8/20, altering the content and distribution of auxin in rice glume cells, which in turn regulates grain length and weight by affecting the size of glume cells. A recent report shows that overexpression of OsIAA4 in rice leads to reduced sensitivity to exogenous auxin, stunted plant growth, increased tillering angle, and weakened gravitropism compared to WT plants [68]. The overexpression of OsIAA6 enhances drought tolerance; however, the loss-of-function mutant of OsIAA6 exhibits more tiller growth than the WT [69]. Conversely, the overexpression of OsIAA10 increases the number of tillers [70]. Hu et al. [71] found that OsIAA7, OsIAA11, and OsIAA30 regulate the gravitropism and tillering angle of rice stems by either inhibiting or releasing OsARF5. Additionally, the degradation of OsIAA11 is crucial for initiating lateral root formation, as it also regulates lateral root primordia, callus initiation, and the uptake of manganese (Mn) and cadmium (Cd) [72,73,74,75,76]. Chen et al. [77] discovered that transgenic plants overexpressing OsIAA12 or deficient in OsARF17 exhibit a larger leaf angle. OsIAA9, a classical Aux/IAA protein, when expressed as a WT in Arabidopsis, influences lateral root formation and root gravitropism [78]. Yuka et al. found that the gain-of-function mutant Osiaa13 displays a phenotype characterized by reduced lateral roots and defective root gravitropism, demonstrating the involvement of OsIAA13 in auxin signaling and the regulation of genes involved in lateral root primordia in rice [79]. The OsIAA23 gene plays a crucial role in maintaining the post-embryonic development stage of the quiescent center (QC) in semi-dominant mutants by mediating the auxin signaling pathway and is also involved in the development of lateral and adventitious roots [80]. Plants overexpressing OsIAA31 genes exhibit shorter leaves, reduced adventitious roots, and insensitivity to auxin and gravitropism stimuli, indicating its involvement in the growth and development of leaves and adventitious roots [81].

5.2. The Impact of Aux/IAA Genes on Abiotic Stress in Rice

Rice mitigates or resists abiotic stresses such as drought, salt, and low temperature through Aux/IAA regulation (Figure 6). Under high-salt conditions, OsIAA9 and OsIAA20 are significantly upregulated, while OsIAA10 and OsIAA25 are significantly downregulated in response to drought conditions [82]. Furthermore, the abscisic acid (ABA) response gene OsRab21 is downregulated in OsIAA20 RNAi and upregulated in OsIAA20 overexpression lines, indicating that OsIAA20 plays a crucial role in plant responses to drought and salt stress through the ABA pathway transduction mechanism [83]. Jung et al. [69] showed that OsIAA6 is highly induced by drought stress, and the drought resistance of OsIAA6 overexpression lines is significantly enhanced, suggesting that OsIAA6 is involved in the drought stress response. Additionally, transgenic rice overexpressing OsIAA18 exhibits higher chlorophyll content and greater sensitivity to ABA under drought and salt stress compared to WT plants, indicating that OsIAA18 plays a positive role in drought and salt tolerance by regulating stress-induced ABA signaling [84] (Figure 6).

6. Research on the Functions of Aux/IAA Genes in Maize

6.1. Regulation of Aux/IAA Genes in Maize Growth and Development

To date, 40 Aux/IAA genes have been identified in maize, designated as ZmIAA1-ZmIAA40 [85]. Von Behrens et al. [86] identified RUM1 (ROOTLESS WITH UNDETECTABLE MERISTEM 1) in maize, which encodes the Aux/IAA10 protein. This protein plays a crucial role in regulating the formation of lateral roots through a signaling pathway mediated by the maize proteins ZmARF25 and ZmARF34. Furthermore, the analysis of RUM1 highlighted its crucial role in the specific regulation of auxin signaling components and vascular development [87]. Galli et al. [88] reported two genes, BIF1 (BARREN INFLORESCENCE1) and BIF4 (BARREN INFLORESCENCE4), encoding ZmIAA27 and ZmIAA20 involved in the auxin signaling pathway, that regulate maize organ development by mediating auxin transport. Additionally, ZmIAA5 regulates the growth and development of maize roots by interacting with ZmARF5 under the specific binding of ZmTCP15/16/17 [89]. A recent study found that ZmEREB167 directly targets OPAQUE2, ZmNRT1.1, ZmIAA12, ZmIAA19, and ZmbZIP20, inhibiting their expression. It acts as a negative regulator in maize endosperm development and affects starch accumulation and grain size, indicating that ZmIAA12 and ZmIAA19 are involved in maize endosperm development [90]. ZmIAA29 interacts with ZmARF transcription factors to regulate maize flowering time by affecting plant signal transduction and the key genes related to maize flowering [91]. In addition, ZmCLA4 can directly bind to the promoter regions of auxin-related genes ZmARF22 and ZmIAA26, thereby regulating the leaf angle of maize [92] (Figure 7).

6.2. The Impact of Aux/IAA Genes on Abiotic Stress in Maize

Yan et al. [93] demonstrated that under high-salt conditions, the overexpression of ZmbHLH32 enhances salt tolerance in maize, while the knockout mutant of ZmbHLH32 exhibits a reduced capacity for salt tolerance. Experimental results indicate that ZmbHLH32 can directly bind to the promoter region of ZmIAA9 to activate its expression. Furthermore, ZmARF1 interacts with ZmIAA9 and suppresses the expression levels of reactive oxygen species (ROS)-scavenging genes. These findings suggest that the regulatory pathway mediated by the ZmbHLH32-ZmIAA9-ZmARF1 module plays a crucial role in influencing salt tolerance in maize [93]. Zhang et al. [94] found that under drought stress, ZmCCT interacts with ZmFra a1, ZmWIPF2, and ZmIAA8 to regulate the expression of auxin-responsive genes, thereby regulating maize drought tolerance. This indicates that ZmIAA8 is involved in the regulation of maize drought tolerance. Through mutant analysis, ZmIAA2, ZmIAA10, and ZmIAA21 were shown to mitigate aluminum stress and play a critical role in root elongation and lateral root primordium formation [95,96] (Figure 8).

7. Research Progress on the Aux/IAA Family in Other Plants

A total of 34 Aux/IAA genes were identified in wheat, of which 30 exhibit duplications in the A, B, or D subgenomes, resulting in a total of 84 Aux/IAA sequences [13]. In wheat roots, the expression of TaSHY2 and TaIAA7 is downregulated by drought stress and upregulated by cytokinin treatment, thereby inhibiting root growth [97]. Research has demonstrated that mutations in TaIAA21 significantly enhance grain length, width, and weight. Conversely, mutations in TaARF25, which interacts with TaIAA21, result in a reduction in grain size and weight. Experimental evidence indicates that TaARF25 promotes the transcription of ERFs (ethylene response factors); however, mutations in TtERF3 lead to decreased grain size and weight. These findings suggest that TaIAA21 functions as a negative regulator of grain size and weight through the ARF25-ERFs module [98]. Zhao et al. [99] found that TaHST1L mediates the auxin signaling pathway by interacting with TaIAA17, playing a pivotal role in regulating endogenous auxin levels and thus affecting wheat tillering patterns. Su et al. [100] discovered that TaIAA15-1A positively regulates plant drought tolerance by activating the ABA signaling pathway. Zeng et al. [101] found that the TaIAA19 gene may influence wheat gravitropism by regulating the expression of ARF and PIN genes.

In tomato, there are 36 Aux/IAA genes, designated as SlIAA1–SlIAA36 [102]. For instance, SlIAA9 interacts with SlARF6A, SlARF8A, SlARF8B, and SlARF24, thereby elucidating the molecular mechanism by which SlIAA9 regulates leaf development [103]. Additionally, researchers have found that the SlDELLA and SlARF7/SlIAA9 complexes regulate the interaction between gibberellin (GA) and auxin, thus modulating fruit formation [104]. Deng et al. [105] demonstrated that the downregulation of SlIAA15 diminishes apical dominance in tomatoes, affects axillary bud development, promotes lateral root formation, and increases leaf thickness, indicating that SlIAA15 plays a significant role in leaf and lateral root development. Through RNA interference (RNAi) silencing of SlIAA17, Su et al. [106] discovered that SlIAA17 RNAi fruits were larger than wild-type fruits, suggesting that SlIAA17 is involved in regulating fruit size. Bassa et al. [107] reported that SlIAA27 knockout plants exhibited increased auxin sensitivity, altered root development, and reduced chlorophyll content in leaves. The fertility of ovules and pollen significantly decreased following SlIAA27 knockdown, indicating that SlIAA27 is involved in both vegetative and reproductive growth in tomatoes. The latest study revealed that SlKD1 interacts with SlGATA6 through its KNOX2 domain, thereby inhibiting the transcriptional activation ability of SlGATA6 on the downstream target genes SlLAX2 and SlIAA3. This interaction ultimately disrupts auxin gradients and organ abscission through the SlKD1-SlGATA6-SlLAX2/SlIAA3-SlARF2a molecular module [108].

In potato, StIAA9 is highly expressed during tuber initiation and is involved in the formation and development of potato tubers [109]. Furthermore, the inhibition of StIAA2 results in alterations in aboveground morphology and an increase in plant height [110] (

Table 1. Biofunctions of AUX/IAA genes in other plants.

Species	Gene Name	Function	References
Wheat (Triticum aestivum)	TaIAA7	Involved in regulating wheat root system development	[97]
	TaIAA15	Positively regulates wheat drought tolerance	[100]
	TaIAA17	Involved in regulating wheat tillering pattern	[99]
	TaIAA19	Involved in regulating wheat’s gravitropic response process	[101]
	TaIAA21	Regulates wheat grain size and grain weight	[98]
Tomato (Lycopersicon esculentum)	SlIAA3	Involved in regulating organ abscission	[108]
	SlIAA9	Involved in regulating tomato leaf development and fruit formation	[103,104]
	SlIAA15	Involved in regulating tomato plant leaf and lateral root development	[105]
	SlIAA17	Involved in regulating tomato fruit size	[106]
	SlIAA27	Involved in tomato nutrient and reproductive growth	[107]
Potato (Solanum tuberosum)	StIAA2	Involved in regulating potato plant morphology and plant height	[110]
	StIAA9	involved in the formation and development of potato tubers	[109]

).

8. Conclusions and Perspectives

Among the three major gene families involved in early auxin response, the Aux/IAA family was investigated in this study, and their biological functions, phylogenetics, protein structures, expression patterns, and physiological roles in various processes such as plant growth and development were elucidated. Aux/IAA proteins not only participate in the auxin signaling pathway but are also crucial for regulating plant growth and development, as well as responses to abiotic stress, through interactions between auxins and other plant hormones.

The current research primarily focuses on the role of Aux/IAA in various aspects of plant growth and development via the auxin signaling pathway, while there is less attention paid to Aux/IAA’s role in regulating plant stress resistance through interactions with other plant hormones. Previous studies have revealed that interaction modules between ARF-Aux/IAA and other proteins are widespread, yet the mechanisms underlying these regulatory modules remain largely unknown. Furthermore, functional studies on the Aux/IAA family in crops such as wheat, maize, and potato are relatively scarce. Elucidating the biological functions of Aux/IAA in these crops could provide valuable genetic resources for improving crop quality and yield. For instance, genes such as OsIAA3, OsIAA8, ZmIAA12, ZmIAA19, and TaIAA21 serve as regulators of grain size and weight, presenting opportunities for enhancing crop productivity. Therefore, research targeting crop stress tolerance, yield improvement, and the interaction modules of ARF-Aux/IAA is essential.

Studies have demonstrated that Aux/IAA genes originated prior to the divergence of monocots and dicots. However, these proteins were either lost or evolved subsequently, playing a significant role in the development of either group. Furthermore, some non-homologous Aux/IAA proteins in monocots may be crucial in determining species-specific traits and functions, suggesting that monocot Aux/IAA proteins possess unique functions and regulatory mechanisms. Interestingly, even proteins with close homology may exhibit opposite regulatory effects in different plant species. Notably, no sister pairs were identified between dicots and monocots, whereas sister pairs were present within the same species type, exemplified by OsIAAs and ZmIAAs, as well as SlIAAs and AtIAAs [111]. Consequently, the roles of Aux/IAA genes in monocots and dicots warrant further investigation.

In recent years, the application of CRISPR/Cas9 targeted gene knockout technology and single-cell RNA sequencing (scRNA-seq) has significantly increased in plant research, providing new insights into the study of Aux/IAA. Jiang et al. utilized CRISPR/Cas9 to knock out the OsIAA23 gene in rice, generating several Osiaa23 mutants with distinct knockout genotypes, which resulted in varied phenotypes [112]. Jia et al. generated the osiaa19 mutant using CRISPR/Cas9 gene editing. Their findings indicate that the OsIAA19 mutation significantly increased both grain length and weight in rice while showing no significant effects on plant height, tiller number, or flag leaf dimensions (length and width) [113]. By employing targeted knockouts, researchers can address functional redundancy within the Aux/IAA gene family, identify key regulatory elements or signaling components associated with Aux/IAA, and utilize efficient gene editing techniques to accurately target and design these elements. Single-cell RNA sequencing (scRNA-seq) is used to identify transcriptional regulation at the single-cell level, providing a reference gene expression profile for specific cell types or conditions to facilitate the identification of differentially expressed genes (DEGs). When combined with spatial transcriptomics (STs), scRNA-seq enhances our understanding of cellular gene expression within tissues, intercellular interactions, and the visualization of complex cellular relationships. Raquel et al. [114] employed scRNA-seq with auxin-induced synthetic promoters to elucidate the complexity of ARF-regulated transcription. Their findings demonstrated that the transcriptional properties of ARFs are not solely intrinsic; they also depend on the configuration of the cis-elements to which they bind, thereby revealing a dual-layer ARF/cis-element transcriptional code [114]. The application of these innovative technologies offers new insights into the intricate genetic and molecular mechanisms within cells and the interactions between plants and their environment, thereby laying the groundwork for accelerating the analysis of Aux/IAA functions.