Aux/IAA family is ubiquitous in higher plants and encodes typical auxin-induced genes. It has been found in dicotyledons such as Arabidopsis thaliana, Medicago truncatula, peanut, soybean, tobacco, and monocotyledons, including corn and rice. However, it has not been found in bacteria, animals or fungi, so it is likely to be a plant-specific family gene. Aux/IAA gene family plays a key role in the auxin signal transduction pathway and negatively regulates the auxin response gene. In the absence of Auxin, Aux/IAA proteins are supposed to bind with ARFs and prevent the activation of auxin-responsive genes. In the higher levels of auxin, these proteins are ubiquitinated by interacting with TRANSPORT INHIBITOR RESPONSE 1/AUXIN SIGNALING F-BOX (TIR1/AFB) receptors and subsequently degraded via the 26S proteasome, which results in the release of ARFs that regulate the expression of downstream auxin-responsive genes [1]. So far, Aux/IAA family genes have been found in more than 30 plant species, with members ranging from 1 in Marchantia polymorpha and 119 in Brassica napus [2]. Aux/IAA family proteins are nuclear proteins with a short life span, and can be rapidly induced by auxin. Most of them contain four highly conservative and typical domains, all of which have specific functions. Domain I, located at the N-terminal, which is a repressor domain containing an ethylene response factor (ERF) associated with amphiphilic repression (EAR) leucine repeat LxLxL, which may recruit TOPLESS (TPL) co-repressor. Domain II bears a conserved sequence “GWPPV,” relating to protein stability. It interacts with the F-BOX protein SCFTIR1 and thereby regulates the protein turnover by causing rapid protein instability and degradation [3]. Domain III harbored an amphipathic Bαα-fold with function and structure analogous to DNA recognition motif, similarly as observed in the case of MetJ and Arc repression. Domain IV contains an SV40 nuclear localization signal (NLS) “PKKKRKV” and an acidic region. Domains III and IV are mostly involved in the protein-protein interaction. These can form homodimer or heterodimer with protein by electrostatic interaction and form homodimer or heterodimer with other proteins by protein-protein interactions [4]. Aux/IAA family genes are usually located in the promoter region of auxin-responsive genes, which can be combined with other factors to regulate auxin-mediated gene expression. The protein does not bind to auxin-responsive genes directly but regulates the activity of ARFs, which further controls the regulation of the auxin response factor [3, 5]. In the absence of auxin, Aux/IAA proteins directly inhibit transcriptional activity. Auxin response factors interact through domains III and IV, which are comparatively conservative protein families [6]. In the presence of auxin, the SCF component of TIR1 or its related proteins are affected in this way. SCFTIR1 complex enhances the interaction with protein, promoting ubiquitination and proteasome degradation in an auxin-dependent manner [7]. Auxin induces protein degradation and releases and then activates transcription auxin response factors. At the same time, auxin activates transcription, forming a negative feedback mechanism to regulate auxin activity [5, 6].
Most Aux/IAA family members may interact with each other and with other proteins in different combinations, thereby increasing the diversity and complexity of auxin signaling pathways that control many aspects of plant growth and physiology [8, 9]. Some studies have shown that the combination of IAA and IAA may have special functions during embryonic development, lateral root development, phototropism and fruit ripening [10–12]. In plants, not all proteins have the above typical four domains. For example, the proteins in potatoes and tomatoes lack domains I and II [13, 14]. In addition, some proteins do not have domains III and IV like papaya [15]. It is the diversity and changes of the protein domains which may be one of the reasons for the diversity of auxin signal transduction pathway and functions, thereby helping to regulate the full play of its multiple roles in various environmental changes. As Aux/IAA family comprises auxin-responsive genes, therefore, quickly respond to auxin homeostasis changes, which coordinate and regulate many plant developmental processes and help the plants cope with external extreme environmental changes, and this gene family mainly regulates auxin homeostasis within the cells. This gene family is involved in responses to light, apical dominance, leaf morphogenesis, lateral branch formation, floral organ formation, vascular bundle development, germ, and root cell development. Different family members have overlapping functions, and the same member may participate in multiple growth and development processes. In Arabidopsis thaliana, some single-gene deletions have little effect on plant growth and development, but functional gain mutants may lead to pleiotropic phenotypes [2, 16, 17]. A large number of studies have found that once VGWPPV, the conserved sequence of protein domain II mutates, It significantly improves the stability of the protein, making the SCFTIR1 protease complex unable to bind to the protein and undergo ubiquitination modification and degradation, resulting in protein degradation without the regulation of auxin homeostasis [18–21]. This discovery is the basis of auxin functions diversity research. Therefore, most of the studies on auxin function are based on the mutant with abnormal domain II to explore the regulatory role of auxin in plant growth and development. For example, mutilation in IAA6, IAA7, and IAA17 caused abnormal elongation of hypocotyls and leaves and affected leaf shape [22]. Mutation in IAA12 caused loss of radicle growth [23]; mutation in IAA18 led to cotyledon fusion during embryonic development and reduced root development at the seedling stage [24]. The mutation in IAA28 affected the development and formation of lateral roots and the dominance of the shoot tip [25]. The phenotypic differences among different IAA mutants suggest that they may act in different regulatory pathways. Aux/IAA proteins may perform their unique regulatory functions by interacting with their specific ARF partners or working in a network [1, 26, 27]. Three genes, IAA6, IAA9, and IAA17 of the Aux/IAA family, played the related roles of controlling the primitive development of adventitious roots, as verified by using functional deletion mutants of these genes. The proteins of these three genes interacted with ARF6 and ARF8 and inhibited their activity in the development of adventitious roots [28]. The results showed that TIR1 and AFB2 were positive regulators of adventitious root formation. The loss of function of IAA7 in Arabidopsis thaliana caused dwarfism, loss of root,s geotropism, and shortening of the length and number of hypocotyl epidermal cells [29–31]. The mutation in IAA3 of Arabidopsis showed weakened root orientation, decreased lateral roots, shortened hypocotyl, and upwardly curled leaves [32], while a mutation in IAA8 of Arabidopsis negatively affected lateral root formation by interacting with TIR1 auxin receptors and ARF in the nucleus [33]. In Arabidopsis, IAA1, IAA7, and IAA 17 mutants exhibited reduced apical dominance, and root orientation [34–36], and IAA28-1 and SLR/IAA14 mutants showed fewer lateral roots and root hairs [18, 37]. SHY2/ IAA3 mutant seedlings of Arabidopsis have shorter hypocotyls and upwardly curled leaves in the dark [32]. The overexpression of IAA1 in rice increased plant sensitivity to auxin and reduced inhibition of root elongation by interacting with ARF1, but increased the sensitivity to 24-Epibrassinolide and leading to an increase in the number and length of primary roots and an increase in the number of lateral roots [38].
With continuous research of the gene function analysis of the Aux/IAA family, it has been found that Aux/IAA family members are not only involved in the morphological development and growth of plants but also play important roles in resisting abiotic stresses. Studies of some mutants have more precisely found that IAA3 and IAA8 affect primary and lateral root development [33, 39] and that IAA3, IAA5, IAA6, and IAA19 respond to environmental stresses [40, 41]. Aux/IAA regulates the expression of auxin-responsive genes by interacting with ARFs to mediate auxin response. At present, 25 OsARFs and 31 OsIAA genes have been found in rice, but their expression regulation network is very complex, and drought and salt stress can induce multiple OsIAA and OsARF expressions. Under salt stress, OsIAA24 and OsIAA20 were upregulated in rice [42]. The mechanism by which IAA/ARF responds to salt stress by regulating downstream genes is complex [43]. Salt stress can induce the specific binding of the NTM2 gene to the IAA30 promoter, resulting in an increase in the transcription level of IAA30; In the NTM2-1 mutant, IAA30 was expressed normally, and the induction effect of exogenous auxin on IAA30 was significantly reduced, with a significantly lowered inhibitory effect on germination. Based on the above findings, the effects of salt on seed germination are regulated by auxin and NTM2, which negatively regulates auxin levels by regulating the expression feedback of IAA30, an adaptation strategy for plants to ensure that their seeds germinate only under favorable growth conditions under stressful conditions [44]. The low-temperature treatment increased auxin levels in rice seedlings, gene chip and quantitative expression results showed that multiple enzyme-coding genes in the auxin synthesis pathway were upregulated, while multiple members of the GH3 gene family were down-regulated, and the most genes in the auxin signaling pathway were also down-regulated, but there were a few genes such as OsARF13, OsSAUR19 and OsIAA39, and another class of auxin-responsive genes OsIAA20 and OsIAA28 [45]. OsIAA6 may also be involved in ABA-mediated drought-tolerant regulatory mechanisms by regulating the expression of auxin synthesis genes, triggering auxin-mediated drought responses [45]. The OsIAA6 overexpressed plants exhibited significantly increased drought resistance [46]. According to the Spatio-temporal expression analysis of the cassava IAA gene family under stress conditions, we found that most of the cassava IAA gene families have certain expressions after being stressed by salt, high temperature, low temperature, and drought stress, but different genes have specific expressions under different stress conditions, of which IAA9, IAA14, IAA16, IAA17, IAA21, and IAA38 were more than 50 times higher in expression under salt stress. This suggests that different IAA genes may control IAA gene expression in different stress situations. The OsIAA6 gene in rice was highly induced by drought, and after overexpression of the OsIAA6 gene in rice, the drought resistance of plants improved by regulating the auxin biosynthesis pathway. In addition, OsIAA6 was specifically expressed in axillary meristems of the basal stem, which regulates auxin transporter OsPIN1 and the rice tiller inhibitor OsTB1, while mutants knocking out the OsIAA6 gene exhibited abnormal tillers growth [46]. In rice, many members of the Aux/IAA family genes can be induced by hormonal and abiotic stress, among which IAA1, IAA2, IAA9, IAA18, IAA19, IAA22, IAA23, and IAA27 genes can be induced by salt stress and showed upregulation there [38, 43]. The expression of SbIAA2 and SbIAA24 genes in sorghum roots was upregulated under salt stress [47]. Jain (2006) also found upregulation of osIAA9 and OsIAA20 in rice under high-salt conditions [48]. In addition, phylogenetic analysis showed that both the SbIAA24 and OsIAA20 genes were localized in the same branch of the phylogenetic tree, which may suggest that some Aux/IAA genes are also associated with salt stress. Aux1-mediated accumulation of IAA in Arabidopsis roots, involved in aluminum stress inhibition of primary root growth and promotion of lateral root and root hair growth. Shi (2017) found that in Arabidopsis thaliana, the integration of IAA17 and RGA-LIKE3 in the NO-mediated salt stress response is an important component [49]. In Arabidopsis thaliana, DREB promotes transcription of IAA5 and IAA19 genes in response to abiotic stress, and recessive mutations in these IAA genes lead to reduced tolerance to stress [40].
After silencing the SlIAA27 gene in tomatoes, the sensitivity of auxin in plants increased, affecting root development and reducing leaf chlorophyll content [50]. In addition, the expression of SlIAA27 was regulated by fungi, and silencing SlIAA27 has a negative impact on arbuscular mycorrhizal colonization [51]. SlIAA27 plays an important role in the growth and development of tomatoes [50]. Liu et al. (2017) found that the tomato ethylene-responsive factor S1-ERF.B3 was involved in the regulation of ethylene and auxin homeostasis by the downstream regulation of the expression of SlIAA27 [52]. In tomatoes, SlIAA27 regulates the expression of strigolactone biosynthesis genes SI-D27 and SI-MAXI via modulating the expression of transcription factor NSPI [51, 53], which indicates that SI-IAA27 regulates the ethylene and auxin signaling one side and involved in the regulation of strigolactone biosynthesis on the other hand. Recently, the VcIAA27 gene isolated from blueberry showed higher expression in shoots, flowers, and fruits of blueberry, played important roles in the fruit enlargement and ripening, and its overexpression in Arabidopsis caused curled leaves growth, dwarfism, and produced shorter or sterile siliques, indicating negative that VcIAA27 might negatively regulate the auxin signaling pathway [54]. Previous transcriptome analysis of white clover under PEG-induced drought stress showed a strong up-regulation of TrIAA27, suggesting its putative role in mediating drought stress responses in white clover; however, the role of IAA27 has not been studied in mediating drought responses in any species nor it has been isolated and studied in grasses or fodders. Therefore, in the current study, we isolated, cloned, and functionally characterized the white clover gene TrIAA27 of the Aux/IAA family, intending to understand the molecular response mechanism drought stress and thereby to further molecular improvement of white clover against drought stress.