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Article

Evolution Analysis of the Aux/IAA Gene Family in Plants Shows Dual Origins and Variable Nuclear Localization Signals

by
Wentao Wu
1,2,
Yaxue Liu
1,2,
Yuqian Wang
1,2,
Huimin Li
1,2,
Jiaxi Liu
1,2,
Jiaxin Tan
1,2,
Jiadai He
1,2,
Jingwen Bai
1,2 and
Haoli Ma
1,*
1
State Key Laboratory of Crop Stress Biology for Arid Areas, College of Agronomy, Northwest A&F University, Xianyang 712100, China
2
Innovation Experimental College, Northwest A&F University, Xianyang 712100, China
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2017, 18(10), 2107; https://doi.org/10.3390/ijms18102107
Submission received: 28 August 2017 / Revised: 27 September 2017 / Accepted: 1 October 2017 / Published: 8 October 2017
(This article belongs to the Special Issue Auxin)
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Abstract

:
The plant hormone auxin plays pivotal roles in many aspects of plant growth and development. The auxin/indole-3-acetic acid (Aux/IAA) gene family encodes short-lived nuclear proteins acting on auxin perception and signaling, but the evolutionary history of this gene family remains to be elucidated. In this study, the Aux/IAA gene family in 17 plant species covering all major lineages of plants is identified and analyzed by using multiple bioinformatics methods. A total of 434 Aux/IAA genes was found among these plant species, and the gene copy number ranges from three (Physcomitrella patens) to 63 (Glycine max). The phylogenetic analysis shows that the canonical Aux/IAA proteins can be generally divided into five major clades, and the origin of Aux/IAA proteins could be traced back to the common ancestor of land plants and green algae. Many truncated Aux/IAA proteins were found, and some of these truncated Aux/IAA proteins may be generated from the C-terminal truncation of auxin response factor (ARF) proteins. Our results indicate that tandem and segmental duplications play dominant roles for the expansion of the Aux/IAA gene family mainly under purifying selection. The putative nuclear localization signals (NLSs) in Aux/IAA proteins are conservative, and two kinds of new primordial bipartite NLSs in P. patens and Selaginella moellendorffii were discovered. Our findings not only give insights into the origin and expansion of the Aux/IAA gene family, but also provide a basis for understanding their functions during the course of evolution.

1. Introduction

The plant hormone auxin (IAA) regulates various processes in plant growth and development, including apical dominance, gravitropic response, embryogenesis, organogenesis, vascular differentiation, axis and lateral root initiation, and shoot elongation [1,2]. Auxin coordinates these processes via activating genes involved in auxin signal transduction. The early/primary auxin response genes are generally grouped into three major categories: Aux/IAA (auxin/indoleacetic acid), GH3 (Gretchen Hagen 3), and SAUR (small auxin up RNA) [3]. Furthermore, auxin plays a crucial role in making the plant architecture; namely, auxin specifies the different divisions of plants by controlling embryogenesis and postembryonic development [4]. During the course of evolution, changes in auxin perception and signaling can be an essential mechanism to generate varied plants architectures [5,6]. Aux/IAAs are short-lived transcriptional repression factors involved in regulating auxin-responsive transcription in the plant kingdom [7,8]. Typical Aux/IAAs contain four highly conservative domains, designated as domains I, II, III, and IV, although one or some of these domains are lacking in many predicted proteins [8,9]. Domain I, represented by a conservative leucine repeat motif (LxLxLx), is responsible for the repression activity and the recruitment of co-repressor TOPLESS (TPL) [10,11]. Domain II, with the conservative degron-sequence GWPPV, confers instability and rapid degradation of Aux/IAAs by interacting with the F-box protein TIR1 [11,12]. Hence, the introduction of specific point mutations or deletions in the domain II degron-sequence can increase the stability of the proteins [13,14]. The C-terminal domains III and IV are dimerization regions involved in homo-dimerization and hetero-dimerization with other Aux/IAAs or auxin response factors (ARFs) [15]. In addition, Aux/IAAs are localized in the nucleus because they have two nuclear localization signals (NLS) [16,17].
The molecular model of auxin signaling was elucidated in the past two decades, despite this plant hormone having been investigated for nearly a century [18]. Aux/IAAs and ARFs are two kinds of transcriptional regulators. Aux/IAAs generally function as transcriptional repressors of auxin-response genes by controlling the activity of ARFs through protein-protein interactions [19], while ARFs can promote downstream target genes’ expression by binding to AuxRE (auxin response element, TGTCTC) in their promoters through B3-type DNA-binding domains [8,20]. Under a low auxin concentration, ARFs’ transcriptional activity is inhibited by forming an ARF-Aux/IAA hetero-dimer [8,19]. Furthermore, TOPLESS (TPL) is recruited to maintain chromatin in a repressive configuration by recruiting histone deacetylase [21]. However, when the auxin density is relatively high, Aux/IAAs are degenerated through the ubiquitin–proteasome protein (TIR1) pathway triggered by SCF-type E3 ubiquitin ligase complexes. Then, ARFs are released from Aux/IAAs to regulate the expressions of the downstream target genes [19,22,23].
Recently, several Aux/IAAs have been characterized in model plants, indicating that Aux/IAAs participate in diverse cellular and developmental processes. In Arabidopsis thaliana, the IAA1/AXR5 mutant shows a variety of growth defects related to auxin insensitivity phenotypes [24]. Loss-of-function mutation in IAA3/SHY2 has effects on auxin homeostasis and lateral root formation [25]. Inflorescences of a dominant mutant of A. thaliana IAA7/ARX2 have negative phototropism and gravitropism defects [26]. The mutant of AXR3-1/IAA17-1 has enhanced apical dominance and a decreased number of lateral roots, which are associated with an increased auxin response [27]. The gain-of-function mutation of IAA16 inhibits plant growth and causes decreased response to auxin [28]. In monocot rice (Oryza sativa), plants with overexpression of OsIAA4 are characterized by dwarfism, increased tiller angles, reduced gravity response, and less responsiveness to auxin [29]. The loss-of-function mutant of OsIAA6 shows abnormal tiller outgrowth [30]. OsIAA23 is referenced to the postembryonic maintenance of the quiescent center [31]. Several reports highlight that some Aux/IAAs are responsive to abiotic stresses, including cold acclimation, dehydration, and salt stress [32,33].
Aux/IAA gene families have been identified in several plant species, such as 29 members in A. thaliana [34], 31 in O. sativa [35], 35 in Populus trichocarpa [36], 34 in Zea mays [37], 26 in Vitis vinifera [38], 17 in Medicago truncatula [39], 26 in Eucalyptus grandis [40], 63 in G. max [33], and 26 in Solanum tuberosum [41]. Some Aux/IAAs are expressed throughout the plant, but others have tissue-specific and developmental stage-specific expression manners depending on the precise developmental context [42]. Moreover, many homologous Aux/IAAs have similar expression patterns and functions [17,43,44,45,46]. No visible defect is detected in double or triple mutants of the closely-related Aux/IAA genes, suggesting that functional redundancy is widespread in the Aux/IAA gene family [47]. In the auxin signaling pathway, a single Aux/IAA protein can interact with multiple members of the ARFs and vice versa [48]. These genes may achieve more complicated regulatory strategies through changing their interacting partners [49].
To gain a better understanding of auxin signaling in the evolution of plants, the evolutionary history of Aux/IAAs needs to be investigated. In this study, we identify 434 Aux/IAA proteins from 17 plant species covering major lineages of plants and we perform bioinformatics analyses to reveal the evolutionary mechanisms of the Aux/IAA gene family. Our study demonstrates that canonical Aux/IAA genes can generally be divided into five groups, and that they have a variety of domain compositions. Additionally, our results indicate that tandem and segmental duplication events play dominant roles in the expansion of the Aux/IAA gene family. Moreover, it is found that both changes in coding sequence and rearrangements in domain organization contribute to the functional diversity of plant Aux/IAAs.

2. Results and Discussions

2.1. Identification of the Aux/IAA Gene Family in Plants

In order to identify Aux/IAA genes in plantae, we first used 29 A. thaliana and 31 O. sativa Aux/IAAs as the query to perform local BLAST searches against the proteomes of 17 plant species. Then, these candidates were submitted to the NCBI conserved domain database. The presence of AUX_IAA domains was necessary to confirm their identities. As a result, we finally identified 434 Aux/IAA genes (Table S1). The number of Aux/IAA genes ranged from three to 63 across the different plant species. Interestingly, we were unable to detect any Aux/IAA domains in C. reinhardtii. The gene copy number of six species ranged from 20–30, and four species were between 30 and 40 (Table 1). There were three Aux/IAA genes in P. patens; while G. max contained the most Aux/IAA genes (63). It was found that the copy number of Aux/IAA genes was uncorrelated with genome size. For example, G. max and P. abies contained 56,044 and 58,587 genes, respectively; however, the Aux/IAA gene number in G. max (63) was twice that of P. abies (31) (Table 1). There were 29 Aux/IAA genes in A. thaliana, while P. paten only had three Aux/IAA genes, even if the gene loci number of P. paten (32,929 loci) was more than that of A. thaliana (27,416 loci). The regression coefficient analysis was conducted (r = 0.6527; p-value = 0.0022), indicating that the Aux/IAA gene copy number and gene loci number had a weak correlation (Figure S1). The results of the paired t-test with these two variables were found to be remarkably different (p < 0.001). In other words, the genome size was not directly proportional to the number of Aux/IAA genes. Plant species might undergo some forces to prompt diversity in this gene family [50]. In addition, lower plants such as P. patens and S. moellendorffii had smaller Aux/IAA gene families compared to higher plants, typically gymnosperms and angiosperms. This phenomenon indicated that the Aux/IAA gene family experienced a dramatic boost from lower plants to higher plants. The extra genes might have functional redundancy and/or generate new functions, leading to adaption to changing environments [50,51,52,53].

2.2. Classification and Structural Analysis of Aux/IAA Proteins

In order to investigate the motif composition of Aux/IAA proteins, MEME (Multiple Em for Motif Elicitation) analysis was applied to find conservative motifs in Aux/IAA proteins. Consequently, five putative motifs corresponding to Aux/IAAs’ four conservative domains were found: motifs 5, 3, and 2 were related to domains I, II and III, respectively; domain IV contained two independent motifs: motif 1 and motif 4 (Figure 1). Motif 5 had a conservative amino-terminal leucine repeat motif (LxLxLx), which was important for conferring repression, and was also a typical feature of domain I [11]. A conservative sequence “VGWPPV” was found in motif 3, which functioned as a conservative degron sequence and determined the stability of Aux/IAA proteins. Substituting the first proline by serine or the second proline by leucine in this motif could remarkably prolong protein half-life times [11,13]. In domains III and IV, “VKVxM” and “RK” in motif 2 and “GDVPW” in motif 1 were also considerably conservative. These motifs might play crucial roles in composing the β-grasp fold and responsible for protein to protein dimerization [15,54]. A conserved SV40 type NLS “KRxRxxK” sequence in motif 4 might also contribute to dimerization [55,56].
In total, there were 434 Aux/IAA proteins; 253 of them had all five motifs, which accounted for 58.3% of the total number, whereas some proteins only contained motifs 1 and/or 2 (Figure 1). For example, AtIAA29 and AtIAA33 in A. thaliana had both motif 1 and 2. However, in G. max, Glyma.10G000700.1 only had motif 1, and Glyma.13G140500.1 only had motif 2. According to the distribution patterns of conservative motifs, Aux/IAA proteins were generally classified into 11 subgroups. The proteins having all five motifs were regarded as canonical Aux/IAAs and grouped into type I. These canonical proteins had four complete domains, which were confirmed by multiple sequences alignment. In type II, 16 proteins lacked motif 4, which led to incomplete domain IV (Figure 1). Nine proteins were grouped into type III, because they lacked both motif 1 and motif 4 in domain IV. Twenty proteins containing domain I, domain III, and incomplete domain IV were grouped in type IV. Moreover, 30 proteins were grouped into type V for lacking motif 5. In type VI, 23 proteins lacked both motif 4 and motif 5. Nine proteins lacking both domains I and II were grouped into type VII. The second most proteins (35), which only had motifs 1 and 2, were clustered into type VIII. Type IX and type X had half-baked domain IV with single motif 2 and motif 1, respectively. The other Aux/IAAs not belonging to any of the above types were grouped into others. The deficiency in domains might cause various motif compositions. In the past few decades, the functions of a single domain in Aux/IAA proteins were revealed by truncated mutations of Aux/IAAs. Domain I was an active repression domain that was responsible for transfer and dominant over activation domains [11]. Domain II composed a TIR1 auxin receptor binding site. The protein lacking this domain showed an obvious longer half-life time than the canonical Aux/IAA protein. AtIAA20 and AtIAA30 in A. Thaliana both lacked domain II, which were more stable than the canonical types of Aux/IAA proteins. Double mutants expressed a conspicuous phenotype such as the collapse of the root apical meristem [57]. Domain III and domain IV were PB1 domains that facilitated protein-protein interactions with other PB1 domain proteins by electrostatic contacts. Truncations in this region might cause defects in dimerization, which would interrupt auxin-related signaling.

2.3. Phylogenetic Analysis of Aux/IAAs

The evolution analysis of the Aux/IAA family was crucial to understanding auxin signaling in plants. However, it was hard to unveil the origin of this gene family, considering the large gene family size and functional redundancy among gene family members. To solve this question, an unrooted tree was built to reveal the phylogenetic relationship of the Aux/IAA gene family among eudicots, monocots, gymnosperms, and lower eukaryotic plants. The phylogenetic tree containing 434 Aux/IAA sequences from 17 species was weakly supported by the bootstrap test because of the many divergent sequences. In order to improve the quality of phylogenetic analysis, we chose 253 canonical Aux/IAA sequences, accounting for 58.3% of the total proteins, to perform phylogenetic analysis (Figure 2). In addition, we reconstructed the phylogenetic trees of the Aux/IAA family by using the maximum likelihood and three different models. The results were very similar to the topology of the neighbor-joining (NJ) tree with the p-distance model and the Gamma distributed with Invariant sites (G + I) distribution. Hence, the NJ tree was chosen for further investigation. In this phylogenetic tree, the Aux/IAA family could generally be divided into five clades: Clade A (including A. thaliana IAA18, IAA26, IAA28), Clade B (including A. thaliana IAA5, IAA6, IAA15, IAA19), Clade C (including A. thaliana IAA7, IAA14, IAA16, IAA17), Clade D (including A. thaliana IAA1, IAA2, IAA3, IAA4), and Clade E (including A. thaliana IAA8, IAA9, IAA27). Bryophyte Aux/IAA sequences were loosely associated with Clade A, which means that Clade A could probably be traced back to the origin of the plant kingdom. Both P. patens and S. moellendorffii had two canonical Aux/IAA sequences, while higher plants contained more gene copies. This gene family might have undergone a dramatic boost during plant evolution. P. equestris (monocot) and aquatic plant U. gibba (eudicots) sequences were on the edge of other monocots and eudicots in this phylogenetic tree. It was speculated that these two species had a longer evolutionary distance than the other species. The origin of the Aux/IAA gene could be dated back at least to the origin of land plants and the major Aux/IAA and ARF lineages originated before the monocot-eudicot divergence [58]. Our work fully supported this hypothesis: it failed to find the Aux/IAA domain in green alga and some charophytes, which were regarded as the closest aquatic relatives of land plants. This result strongly supported the idea that the Aux/IAA protein family did not exist before the emergence of land plants. The auxin response factor (ARF) was another essential kind of transcription factor in auxin signaling that could combine with Aux/IAA domains III and IV and was also determined to be land plant innovation proteins [4]. It was assumed that the auxin perception and response pathway induced by Aux/IAAs and ARFs first appeared in land plants.
In the phylogenetic tree, significant sequence similarities were observed among the Aux/IAA gene family, which indicated that these genes probably arose from gene duplication events (Figure 2). Additionally, these homologous genes might share the same or overlapping functions. In total, 65 sister pairs were found with strong bootstrap support (>90%), 56 of which were from the same species. G. max contained the largest number of sister pairs (17 pairs). There were six and four sister pairs in Z. mays and A. thaliana, respectively. In addition, we found one sister Aux/IAA gene pair each in P. patens, S. moellendorffii, P. abies, and S. tuberosum, while there were no sister pairs in A. trichopoda, O. sativa, B. distachyon, P. persica, or R. communis in the entire tree. The remaining Aux/IAA gene pairs were from cross species, and six sister pairs existed between O. sativa and B. distachyon Aux/IAA genes. We also observed three kinds of sister pairs from cross species (O. sativa IAA-Z. mays IAA, G. max IAA-P. persica IAA, R. communis IAA-P. trichocarpa IAA) at one time. The non-canonical Aux/IAA genes were excluded from the phylogenetic tree as they have a large sequence diversity that would cause less reliability with low bootstrap values. We assumed that these non-canonical Aux/IAA genes tended to be clustered into additional clades.

2.4. Aux/IAAs and ARFs May Share the Same Origin

The auxin/indole-3-acetic acid (Aux/IAA) family and the auxin response factor (ARF) family are two transcription factor families that regulate the auxin signaling pathway in the nucleus. In the absence of auxin, the Aux/IAAs carboxyl-terminal PB1 domain specifically binds to the corresponding homologous domain in the ARFs, leading to the repression of ARF activity [15,20,54]. In order to reveal the evolutionary relationship between Aux/IAAs and ARFs, we first performed multiple sequence alignments by using full-length amino-acid sequences of several typical Aux/IAAs and ARFs among eudicot (A. thaliana), monocot (O. sativa), gymnosperm (P. abies), and lower eukaryotic plant (P. patens and S. moellendorffii) sequences by ClustalX. It was shown that the structure of the PB1 domain was composed of three α-helices (α1–α3) and five β-sheet strands (β1–β5) (Figure 3). The remarkable sequences conservation and the unique feature compared with typical PB1 domains indicated that ARF and Aux/IAA gene families may derive from an ancient PB1 family [15]. The PB1 domain three-dimensional crystal structures in ARF5 and ARF7 confirmed that the tertiary structures of these domains were ubiquitin-like β-grasp folds [59,60]. However, the three-dimensional structure revealed that these PB1 domains were not typical PB1 domains due to an additional α helix (α3) at the C-terminus region [59,60,61].
Interestingly, although the sequences conservation in domains III/IV of both ARF and Aux/IAA proteins was noticeable, the difference of this region between two gene families was also observed. The most noticeable difference between ARFs and IAA proteins in this domain was that almost all Aux/IAA proteins had the “GDVP” motif linking β3 and α2, while this motif was “GDDP” in almost all ARF proteins (Figure 3). One more acidic residue was present at this motif in ARF proteins than Aux/IAA proteins, which would possibly influence protein to protein electrostatic interactions [15]. We also observed this sequences diversity in moss (P. patens), indicating that the Aux/IAA and ARF gene families’ differentiation event appeared before the divergence of green algae and land plants.
A phylogenetic tree was built based on the full-length amino acid sequences of Aux/IAA and ARF proteins. ARFs were highlighted with green; canonical Aux/IAAs were highlighted with blue; and a novel group of N-terminal truncated Aux/IAAs was highlighted with orange (Figure 4). The N-terminal truncated Aux/IAAs were closer to those of ARF proteins than to Aux/IAAs. Interestingly, those proteins contained the “GDDP” motif rather than the “GDVP” motif in accordance with most Aux/IAA proteins. Several proteins belonging to this group were identified in some plants, such as StIAA5 (PGSC0003DMP400032527) in S. tuberosum [41], Glyma.07G134900.1 and Glyma.13G140500.1 in G. max [33], and MtIAA (Medtr4g060470) in M. truncatula [39]. Therefore, it was speculated that these Aux/IAAs derived from truncated ARF genes that only have the Aux/IAA domain, but lack their typical domains (I and II) (Figure 3). These Aux/IAA genes might not function as repressors. The other types of truncated Aux/IAA genes might derive from sequence mutation events during the process of Aux/IAA gene family expansion. Interestingly, some proteins from lower plants that clustered into the “orange” clade contained relatively long disordered sequences in the N-terminals (634 amino acids in Pp3c9_21330V3.1 from P. patens and 180 amino acids in 438333 from S. moellendorffii). They were possibly byproducts of the origins of Aux/IAAs and ARFs, because these sequences could not be found in higher plants.

2.5. Analysis of Aux/IAA Duplication Patterns during the Course of Evolution

The genome’s and the genetic system’s evolution is mainly driven by gene duplications [62]. Segmental duplication, tandem duplication, and transposition events are three basic gene expansion patterns [63,64]. Among them, segmental duplication and tandem duplication are two main causes of gene family expansion in the plant kingdom [65,66]. Tandem duplications were often caused by unequal crossing-over and characterized as multiple paralogous genes existing in proximal genomic regions [67,68]. In order to investigate the roles of gene duplications in the Aux/IAAs’ expansion, segmental duplication and tandem duplication events were investigated. First, the tandem duplicated gene pairs were searched through their physical location on the chromosome and loci. It was determined that paralogous genes existing in the same chromosome within a 50-kb physical distance were tandem duplicated pairs [65]. Segmentally-duplicated pairs were searched in the Plant Genome Duplication Database [66]. The genomes of P. patens and S. moellendorffii contained three and nine Aux/IAA genes, respectively, and each of them had one segmentally-correlated gene pair. P. abies was generally regarded as having undergone numerous duplications [69,70], but only one tandem duplicated gene pair was founded among 31 Aux/IAA genes in this model gymnosperm (Figure 5). It was found that 64.79% and 38.66% of Aux/IAA genes in eudicots and monocots were segmentally correlated, respectively, while 28.09% and 12.61% of Aux/IAA genes were tandemly correlated, respectively. In more detail, for example in A. thaliana, 22 (75.86%) out of a total of 29 Aux/IAA genes were segmentally correlated, six (20.69%) Aux/IAA genes were tandemly correlated, and three genes (AT1G04240.1, AT3G23030.1, and AT4G14550.1) took part in both segmental and tandem duplications. Among 31 Aux/IAA genes in monocot O. sativa, 19 (61.29%) and seven (22.58%) Aux/IAA genes were from segmental and tandem duplication, respectively. Two Aux/IAA genes (LOC_Os03g43400.1 and LOC_Os12g40890.1) were involved in both segmental and tandem duplications. Interestingly, we found segmental duplicated pairs in lower plants (P. patens and S. moellendorffii), which were encoded by canonical Aux/IAAs in these species (Figure 5). These results proved that the earliest ancestors of land plants contained at least one Aux/IAA gene. In summary, we predicted that both segmental and tandem duplications contributed significantly to Aux/IAA gene family expansion, though segmental duplications played a greater role in such progress. Moreover, monocots and eudicots may undergo different expansion types of the Aux/IAA gene family because they underwent different segmental and tandem duplication patterns.
It was found that the expansion patterns observed in the Aux/IAA gene family coincided with whole-genome duplication events (WGD) during evolution. Two WGD events that occurred in progymnosperms might contribute to today’s Aux/IAA gene family size and diversity [33]. During angiosperm evolution, the fact that these Aux/IAA duplications specifically related to definite single whole-genome duplication events was quite difficult for subsequent extensive genome rearrangements. G. max contained the largest Aux/IAA gene family compared to other plant lineages. It was assumed that WGDs might have contributed to the expansion of Aux/IAA genes in G. max, as its genome had undergone one WGT (whole-genome triplication) and two WGD events (legume WGD and Glycine WGD) [33]. During the course of evolution, plants should also constantly change their genetic constitution to fit the changeable environment. Duplicated genes were believed to be the basis on which plants generated new gene functions and evolutionary novelty [62]. Numerous studies on single, double, even triple mutations suggested an extensive functional redundancy among the Aux/IAA gene family members [44,47,71]. However, Aux/IAA proteins also had distinct functions compared with their homologues [44,71,72]. Their different spatiotemporal expression patterns and specific preference for a particular ARF partner could give us further insights to investigate the functional specificity and similarity of Aux/IAA proteins.

2.6. Estimation of the Molecular Evolutionary Rates of Aux/IAAs

Estimation of the molecular evolution rates of Aux/IAA is an essential basis to understand the evolution process of the Aux/IAA gene family in the plant kingdom. The Ka/Ks values were calculated to estimate the molecular evolutionary rates of the paralogous gene pairs among Aux/IAAs. It was found that most Aux/IAA pairs evolved at a Ka/Ks value lower than one, as expected for the MA_10430843g0010/MA_10430843g0020 gene pair in P. abies and the Potri.006G161200/Potri.006G161400 gene pair in P. trichocarpa, implying that most of these Aux/IAA paralogous gene pairs had evolved under purifying selection. The Ka/Ks values of MA_10430843g0010/MA_10430843g0020 gene pairs and Potri.006G161200/Potri.006G161400 gene pairs were 1.6720 and 1.1686, respectively (Table 2), indicating that these gene pairs experienced positive selection pressure in evolution. The Aux/IAA paralogous gene pairs in dicots were slightly more conservative than those in monocots, as the average Ka/Ks value in dicots’ Aux/IAA pairs was lower than those in monocots, both in segmental duplication (0.2224 and 0.4027 in dicots and monocots, respectively) and tandem duplication (0.1903 and 0.1705 in dicots and monocots, respectively) (Figure 6). Aux/IAA genes might have undergone different selective pressures and patterns between the two seed plant classes. The Ka/Ks values in segmentally and tandemly correlated paralogous gene pairs were compared, and the average Ka/Ks value in segmentally correlated paralogous gene pairs was 0.2362 and in tandemly correlated gene pairs 0.1484 (Figure 6). The Student t-test was performed, and the p-value was 0.0004165, indicating that the average Ka/Ks value in segmentally correlated paralogous gene pairs was significantly greater than tandemly correlated gene pairs. It was assumed that segmental duplication and tandem duplication contributed to Aux/IAA gene family enlargement at different molecular evolutionary rates. Genes or proteins evolve through the interplay between mutation and selection [50]. According to the neutral theory of molecular evolution, synonymous substitution did not affect the encoding of amino acids. Therefore, it was generally considered neutral and did not affect individual fitness. Nonsynonymous substitution could cause changes in the amino acid composition that might alter the protein structure and generate new functions. The selective pressure was estimated by calculating the ratio of the nonsynonymous substitution rate to the synonymous substitution rate (Ka/Ks value). During evolution, some genes reached their optimal state, which tended to abandon mutations that alter the function by purifying selection. However, since plants cannot escape from their environment, positive selection promoted functional changes to better adapt to their changing environment [50]. Therefore, the study of these selective patterns could gain more insight into understanding the genes’ and proteins’ evolutionary patterns. Only one pair of segmentally correlated Aux/IAA genes was found in each of P. patens and S. moellendorffii with a Ka/Ks value lower than 0.5, indicating that Aux/IAA genes duplicated under purifying selection. Two gene pairs (MA_10430843g0010/MA_10430843g0020 and Potri.006G161200/Potri.006G161400) were found to undergo positive selection after being separated by duplication, which might improve the fitness of the organism in a new environment.

2.7. Two Types of Putative NLSs Are Conservative in Aux/IAA Proteins

Aux/IAA proteins are cell nucleus-located transcriptional repressors. IAA-GFP (green fluorescent protein) and IAA-YFP (yellow fluorescent protein) fusion proteins could be exclusively localized to the nucleus [13,16,49]. Two types of putative NLSs were detected in the majority of the Aux/IAA proteins: the bipartite NLS and the SV40-like NLS. A bipartite NLS contained a conservative KR (lysine and arginine) basic doublet located between domains I and II (Part 1) and basic amino acids in domain II (Part 2). A basic residue-rich SV40-like NLS was located in domain IV (Figure S2). These putative NLSs might guide Aux/IAA proteins to the cell nucleus [17].
The first part (Part 1) of the bipartite NLS was relatively conservative in plants, depending on the research. However, in Pteridophyta (S. moellendorffii), the detection of this conservative KR basic doublet failed. In the second bipartite NLS (Part 2), the stretches of K/R residues RxxRK represented this region with substitution within lysine (K) and/or arginine (R). Distinctively, this region in lower plants P. patens and S. moellendorffii was QxxRK (the first R in RxxRK was substituted by Q) and KxxNK (the first R in RxxRK was substituted by K, and the second R in RxxRK was substituted by N), respectively. These amino acid substitutions might alter the protein secondary structures, as well as change their subcellularly-located patterns and functions compared with those in higher plants. To reach a more solid conclusion, we also searched Aux/IAA protein sequences from another two already fully-sequenced species of Bryophyta (Marchantia polymorpha and Sphagnum fallax), and their NLS data are added to Figure 7. Finding the second bipartite NLS (Part 2) in M. polymorpha’s Aux/IAA proteins also failed, while S. fallax’s second bipartite NLS (Part 2) was presented by RxxRK with substitution within lysine (K) or/and arginine (R) in the last lysine (K) site. An SV40-like NLS was composed of KxRxxRK in plants, with substitution within lysine (K) or/and arginine (R), especially in the first lysine (K) site (Figure 7). Many Aux/IAA proteins had the full set of NLSs including the bipartite NLS and SV40-like NLS, while some Aux/IAA proteins lacked part of the NLS or had a degenerated NLS (lysine (K) or arginine (R) substituted by another amino acid). For example, in S. tuberosum, StIAA14 lacked bipartite NLS Part 2, and StIAA6 had degenerated bipartite NLS (Part 2) for the first arginine (R) mutated to cysteine (C). Fifty-two percent of Aux/IAA proteins had bipartite NLS Part 1; 43.4% of proteins lacked this region; and the rest of the Aux/IAA proteins had degenerated bipartite NLS Part 1. Meanwhile, 49.2% of Aux/IAA proteins contained the bipartite NLS Part 2; 15.6% of Aux/IAA proteins lack this region; and the remaining Aux/IAA proteins contained degenerated bipartite NLS Part 2 (Table 3).
The SV40-like NLS was shown to be more conservative than the bipartite NLS, as 71.7% of Aux/IAAs contained intact SV40-like NLS, and 20.9% of proteins had degenerated SV40-like NLS. Recent research indicated that the Aux/IAA proteins contained degenerated NLS in both bipartite NLS and SV40-like NLS, which were specifically targeted to the nucleus [73]. The absence and mutation of the bipartite NLS (Part2) region caused Aux/IAA proteins to be detected not only in the nucleus, but also in cytoplasm [13,73]. It was assumed that both the bipartite NLS and SV40-like NLS were responsible for driving the protein specifically to the nucleus, but more evidence is still needed to prove the function of these NLSs.

3. Materials and Methods

3.1. Bioinformatics Identification of Aux/IAAs

In order to identify candidate Aux/IAA genes in 17 species (Chlamydomonas reinhardtii, Physcomitrella patens, Selaginella moellendorffii, Picea abies, Amborella trichopoda, Phalaenopsis equestris, Zea mays, Oryza sativa, Brachypodium distachyon, Solanum tuberosum, Utricularia gibba, Arabidopsis thaliana, Gossypium raimondii, Prunus persica, Glycine max, Populus trichocarpa, Ricinus communis), multiple searches were conducted. First, the complete proteomes of most species were downloaded from the Phytozome website (Version 11; Available online: www.phytozome.org) [74] and the others were obtained from genome sequencing databases of given species, i.e., Picea abies was obtained from PlantGenIE (plantgenie.org) [75], Phalaenopsis equestris from Orchidbase (ftp://ftp.genomics.org.cn/) [76], and Utricularia gibba from CoGe (http://genomevolution.org/CoGe/) [77] Then, A. thaliana and O. sativa Aux/IAA protein sequences were used as queries to perform local BLAST searches with a −3 expect (E) threshold. Second, the conserved domains of all obtained sequences were examined by the NCBI conserved domain database (Available online: http://www.ncbi.nlm.nih.gov/cdd). The presence of the AUX_IAA domain was used to confirm the identity of Aux/IAA genes (Table S1). ARF genes that also contained the AUX_IAA domain were removed by referring to the presence of the ARF family-specific AUX_RESP domain.

3.2. Motif Prediction and Multiple Sequence Alignment

The MEME web server (Available online: http://memesuite.org/) was used to identify motifs under the following parameters: (1) each motif site distributes zero or one occurrence per sequence; (2) optimum motif widths are between six and 50; (3) the maximum number of motifs is five. To identify the finer domains and motifs, the Aux/IAA protein sequences were aligned by using ClustalX (2.1) with the following parameters: protein weight matrix, Gonnet 250; gap penalty at opening, 10; gap penalty at extension, 0.1.

3.3. Phylogenetic Analyses

Aln files generated by ClustalX (2.1) were converted into MEGA format by using MEGA 7. The neighbor-joining (NJ) tree was built under the p-distance model and G + I distribution. Bootstrap analyses with 1000 replicates were performed for support estimation. Maximum likelihood (ML) trees built under the Jones-Taylor-Thornton (JTT), WAG, and Poisson models were also conducted to prove these results.

3.4. Gene Duplication and Molecular Evolution Analysis

We used the annotation information of the Aux/IAA genes on the Plant Genome Duplication Database to determine their chromosomal locations (Available online: https://phytozome.jgi.doe.gov/pz/portal.html). Tandem duplication gene pairs were identified by comparing their physical locations on chromosomes and their homology (more than 50%). We defined that paralogous genes that exist in the same chromosome within a 50-kb physical distance were tandem duplicated pairs [64]. The segmental duplication regions of the different chromosomes were downloaded from the Plant Genome Duplication Database. In order to obtain the molecular evolutionary rates between these Aux/IAA paralogous gene pairs, we performed pairwise alignment among these gene pairs by using an embedded program ClustalW (codons) in MEGA7 [78]. Then, we used KaKs_Calculator 2.0 to calculate the ratio of the nonsynonymous substitution rate (Ka), the synonymous substitution rate (Ks), and the ω (Ka/Ks) value between paralogous gene pairs with the MYN (Modified YN) model [79].

4. Conclusions

The Aux/IAA proteins constitute a large multi-gene family in various plant species that regulate auxin perception and signaling. A comprehensive analysis of the Aux/IAA proteins in 17 plant species is carried out in the current study. In total 434 Aux/IAA proteins are identified and separated into eleven types according to their motif compositions. The canonical Aux/IAA proteins which have all five motifs can be divided into five clades based on phylogenetic analysis. Our work supports the hypothesis that the Aux/IAA gene family did not exist before the emergence of land plants. And we also find a novel group of truncated Aux/IAA proteins might derive from the C-terminal truncation of ARF proteins. Tandem and segmental duplications contribute to the expansion of the Aux/IAA gene family mainly under purifying selection. In addition, the putative nuclear localization signals (NLSs) in the Aux/IAA proteins are conservative except sequence variation in several lower plants. Typically, two kinds of new primordial bipartite NLSs in P. patens and S. moellendorffii are discovered. Future work will focus on whether the Aux/IAA proteins from the lower plants target into the nucleus and the emergence time of auxin perception and signaling triggered by the Aux/IAA proteins in the course of plant evolution.

Supplementary Materials

Supplementary materials can be found at www.mdpi.com/1422-0067/18/10/2107/s1.

Acknowledgments

This research was supported by the National Natural Science Foundation of China (31500159), the Natural Science Foundation of Shaanxi Province (2016JQ3029) and the Undergraduate Innovation Foundation of Northwest A&F University (No. 1201610712119).

Author Contributions

Haoli Ma and Wentao Wu conceived of and designed the research plans; Wentao Wu, Yaxue Liu, and Yuqian Wang performed most of the experiments and analyzed the data; Huimin Li, Jiaxi Liu, Jiaxin Tan, Jiadai He, and Jingwen Bai provided technical assistance to Wentao Wu, Yaxue Liu, and Yuqian Wang; Wentao Wu wrote the article with contributions from all the authors; Haoli Ma supervised and supported the writing.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. De Smet, I.; Jurgens, G. Patterning the axis in plants—Auxin in control. Curr. Opin. Genet. Dev. 2007, 17, 337–343. [Google Scholar] [CrossRef] [PubMed]
  2. Ljung, K. Auxin metabolism and homeostasis during plant development. Development 2013, 140, 943–950. [Google Scholar] [CrossRef] [PubMed]
  3. Guilfoyle, T.J. Auxin-regulated genes and promoters. In Biochemistry and Molecular Biology of Plant Hormones; Hooykaas, P.J.J., Hall, M.A., Libbenga, K.R., Eds.; Elsevier: Leiden, The Netherlands, 1999; Volume 33, pp. 423–459. [Google Scholar]
  4. Finet, C.; Berne-Dedieu, A.; Scutt, C.P.; Marlétaz, F. Evolution of the ARF gene family in land plants: Old domains, new tricks. Mol. Biol. Evol. 2013, 30, 45–56. [Google Scholar] [CrossRef] [PubMed]
  5. Cooke, T.J.; Poli, D.B.; Sztein, A.E.; Cohen, J.D. Evolutionary patterns in auxin action. Plant Mol. Biol. 2002, 49, 319–338. [Google Scholar] [CrossRef] [PubMed]
  6. Finet, C.; Jaillais, Y. Auxology: When auxin meets plant evo-devo. Dev. Biol. 2012, 369, 19–31. [Google Scholar] [CrossRef] [PubMed]
  7. Abel, S.; Theologis, A. Early genes and auxin action. Plant Physiol. 1996, 111, 9–17. [Google Scholar] [CrossRef] [PubMed]
  8. Hagen, G.; Guilfoyle, T. Auxin-responsive gene expression: Genes, promoters and regulatory factors. Plant Mol. Biol. 2002, 49, 373–385. [Google Scholar] [CrossRef] [PubMed]
  9. Reed, J.W. Roles and activities of Aux/IAA proteins in Arabidopsis. Trends Plant. Sci. 2001, 6, 420–425. [Google Scholar] [CrossRef]
  10. Szemenyei, H.; Hannon, M.; Long, J.A. Topless mediates auxin-dependent transcriptional repression during Arabidopsis embryogenesis. Science 2008, 319, 1384–1386. [Google Scholar] [CrossRef] [PubMed]
  11. Tiwari, S.B.; Hagen, G.; Guilfoyle, T.J. Aux/IAA proteins contain a potent transcriptional repression domain. Plant Cell 2004, 16, 533–543. [Google Scholar] [CrossRef] [PubMed]
  12. Dharmasiri, N.; Dharmasiri, S.; Estelle, M. The f-box protein TIR1 is an auxin receptor. Nature 2005, 435, 441–445. [Google Scholar] [CrossRef] [PubMed]
  13. Ludwig, Y.; Berendzen, K.W.; Xu, C.; Piepho, H.-P.; Hochholdinger, F. Diversity of stability, localization, interaction and control of downstream gene activity in the maize Aux/IAA protein family. PLoS ONE 2014, 9, e107346. [Google Scholar] [CrossRef] [PubMed]
  14. Von Behrens, I.; Komatsu, M.; Zhang, Y.; Berendzen, K.W.; Niu, X.; Sakai, H.; Taramino, G.; Hochholdinger, F. Rootless with undetectable meristem 1 encodes a monocot-specific Aux/IAA protein that controls embryonic seminal and post-embryonic lateral root initiation in maize. Plant J. 2011, 66, 341–353. [Google Scholar] [CrossRef] [PubMed]
  15. Guilfoyle, T.J. The PB1 domain in auxin response factor and Aux/IAA proteins: A versatile protein interaction module in the auxin response. Plant Cell Online 2015, 27, 33–43. [Google Scholar] [CrossRef] [PubMed]
  16. Abel, S.; Oeller, P.W.; Theologis, A. Early auxin-induced genes encode short-lived nuclear proteins. Proc. Natl. Acad. Sci. USA 1994, 91, 326–330. [Google Scholar] [CrossRef] [PubMed]
  17. Wu, J.; Peng, Z.; Liu, S.; He, Y.; Cheng, L.; Kong, F.; Wang, J.; Lu, G. Genome-wide analysis of Aux/IAA gene family in solanaceae species using tomato as a model. Mol. Genet. Genom. 2012, 287, 295–311. [Google Scholar] [CrossRef] [PubMed]
  18. Enders, T.A.; Strader, L.C. Auxin activity: Past, present, and future. Am. J. Bot. 2015, 102, 180–196. [Google Scholar] [CrossRef] [PubMed]
  19. Tiwari, S.B.; Wang, X.J.; Hagen, G.; Guilfoyle, T.J. Aux/IAA proteins are active repressors, and their stability and activity are modulated by auxin. Plant Cell 2001, 13, 2809–2822. [Google Scholar] [CrossRef] [PubMed]
  20. Ulmasov, T.; Hagen, G.; Guilfoyle, T.J. ARF1, a transcription factor that binds to auxin response elements. Science 1997, 276, 1865–1868. [Google Scholar] [CrossRef] [PubMed]
  21. Wang, L.; Kim, J.; Somers, D.E. Transcriptional corepressor topless complexes with pseudoresponse regulator proteins and histone deacetylases to regulate circadian transcription. Proc. Natl. Acad. Sci. USA 2013, 110, 761–766. [Google Scholar] [CrossRef] [PubMed]
  22. Dezfulian, M.H.; Jalili, E.; Roberto, D.K.A.; Moss, B.L.; Khoo, K.; Nemhauser, J.L.; Crosby, W.L. Oligomerization of SCFTIR1 is essential for Aux/IAA degradation and auxin signaling in Arabidopsis. PLoS Genet. 2016, 12, e1006301. [Google Scholar] [CrossRef] [PubMed]
  23. Santner, A.; Calderonvillalobos, L.I.; Estelle, M. Plant hormones are versatile chemical regulators of plant growth. Nat. Chem. Biol. 2009, 5, 301–307. [Google Scholar] [CrossRef] [PubMed]
  24. Yang, X.; Lee, S.; So, J.H.; Dharmasiri, S.; Dharmasiri, N.; Ge, L.; Jensen, C.; Hangarter, R.; Hobbie, L.; Estelle, M. The IAA1 protein is encoded by AXR5 and is a substrate of SCFTIR1. Plant J. 2004, 40, 772–782. [Google Scholar] [CrossRef] [PubMed]
  25. Uberti-Manassero, N.G.; Lucero, L.E.; Viola, I.L.; Vegetti, A.C.; Gonzalez, D.H. The class I protein ATTCP15 modulates plant development through a pathway that overlaps with the one affected by CIN-like TCP proteins. J. Exp. Bot. 2012, 63, 809–823. [Google Scholar] [CrossRef] [PubMed]
  26. Sato, A.; Sasaki, S.; Matsuzaki, J.; Yamamoto, K.T. Light-dependent gravitropism and negative phototropism of inflorescence stems in a dominant Aux/IAA mutant of Arabidopsis thaliana, AXR2. J. Plant Res. 2014, 127, 627–639. [Google Scholar] [CrossRef] [PubMed]
  27. Leyser, H.M.; Pickett, F.B.; Dharmasiri, S.; Estelle, M. Mutations in the AXR3 gene of Arabidopsis result in altered auxin response including ectopic expression from the SAUR-AC1 promoter. Plant J. 1996, 10, 403–413. [Google Scholar] [CrossRef] [PubMed]
  28. Rinaldi, M.A.; Liu, J.; Enders, T.A.; Bartel, B.; Strader, L.C. A gain-of-function mutation in IAA16 confers reduced responses to auxin and abscisic acid and impedes plant growth and fertility. Plant Mol. Biol. 2012, 79, 359–373. [Google Scholar] [CrossRef] [PubMed]
  29. Song, Y.; Xu, Z.-F. Ectopic overexpression of an auxin/indole-3-acetic acid (Aux/IAA) gene OsIAA4 in rice induces morphological changes and reduces responsiveness to auxin. Int. J. Mol. Sci. 2013, 14, 13645–13656. [Google Scholar] [CrossRef] [PubMed]
  30. Jung, H.; Lee, D.-K.; Choi, Y.D.; Kim, J.-K. OsIAA6, a member of the rice Aux/IAA gene family, is involved in drought tolerance and tiller outgrowth. Plant Sci. 2015, 236, 304–312. [Google Scholar] [CrossRef] [PubMed]
  31. Jun, N.; Gaohang, W.; Zhenxing, Z.; Huanhuan, Z.; Yunrong, W. OsIAA23-mediated auxin signaling defines postembryonic maintenance of QC in rice. Plant J. 2011, 68, 433–442. [Google Scholar] [CrossRef] [PubMed]
  32. Ghanashyam, C.; Jain, M. Role of auxin-responsive genes in biotic stress responses. Plant Signal. Behav. 2009, 4, 846–848. [Google Scholar] [CrossRef] [PubMed]
  33. Singh, V.K.; Jain, M. Genome-wide survey and comprehensive expression profiling of Aux/IAA gene family in chickpea and soybean. Front. Plant. Sci. 2015, 6, 918. [Google Scholar] [CrossRef] [PubMed]
  34. Liscum, E.; Reed, J.W. Genetics of Aux/IAA and ARF action in plant growth and development. Plant Mol. Biol. 2002, 49, 387–400. [Google Scholar] [CrossRef] [PubMed]
  35. Jain, M.; Kaur, N.; Garg, R.; Thakur, J.K.; Tyagi, A.K.; Khurana, J.P. Structure and expression analysis of early auxin-responsive Aux/IAA gene family in rice (Oryza sativa). Funct. Integr. Genom. 2006, 6, 47–59. [Google Scholar] [CrossRef] [PubMed]
  36. Kalluri, U.C.; DiFazio, S.P.; Brunner, A.M.; Tuskan, G.A. Genome-wide analysis of Aux/IAA and ARF gene families in Populus trichocarpa. BMC Plant Biol. 2007, 7, 59. [Google Scholar] [CrossRef] [PubMed]
  37. Ludwig, Y.; Zhang, Y.; Hochholdinger, F. The maize (Zea mays L.) auxin/indole-3-acetic acid gene family: Phylogeny, synteny, and unique root-type and tissue-specific expression patterns during development. PLoS ONE 2013, 8, e78859. [Google Scholar] [CrossRef] [PubMed]
  38. Çakir, B.; Olcay, A.C. Genome-wide analysis of Aux/IAA genes in Vitis vinifera: Cloning and expression profiling of a grape Aux/IAA gene in response to phytohormone and abiotic stresses. Acta Physiol. Plant 2013, 35, 365–377. [Google Scholar] [CrossRef]
  39. Shen, C.; Yue, R.; Yang, Y.; Zhang, L.; Sun, T.; Xu, L.; Tie, S.; Wang, H. Genome-wide identification and expression profiling analysis of the Aux/IAA gene family in medicago truncatula during the early phase of sinorhizobium meliloti infection. PLoS ONE 2014, 9, e107495. [Google Scholar] [CrossRef] [PubMed]
  40. Yu, H.; Soler, M.; San Clemente, H.; Mila, I.; Paiva, J.A.P.; Myburg, A.A.; Bouzayen, M.; Grima-Pettenati, J.; Cassan-Wang, H. Comprehensive genome-wide analysis of the Aux/IAA gene family in eucalyptus: Evidence for the role of EgriAA4 in wood formation. Plant Cell Physiol. 2015, 56, 700–714. [Google Scholar] [CrossRef] [PubMed]
  41. Gao, J.; Cao, X.; Shi, S.; Ma, Y.; Wang, K.; Liu, S.; Chen, D.; Chen, Q.; Ma, H. Genome-wide survey of Aux/IAA gene family members in potato (Solanum tuberosum): Identification, expression analysis, and evaluation of their roles in tuber development. Biochem. Biophys. Res. Commun. 2016, 471, 320–327. [Google Scholar] [CrossRef] [PubMed]
  42. Gan, D.; Zhuang, D.; Ding, F.; Yu, Z.; Zhao, Y. Identification and expression analysis of primary auxin-responsive Aux/IAA gene family in cucumber (Cucumis sativus). J. Genet. 2013, 92, 513–521. [Google Scholar] [CrossRef] [PubMed]
  43. Liu, S.; Hu, Q.; Luo, S.; Li, Q.; Yang, X.; Wang, X.; Wang, S. Expression of wild-type PtriAA14.1, a poplar Aux/IAA gene causes morphological changes in Arabidopsis. Front. Plant Sci. 2015, 6, 388. [Google Scholar] [CrossRef] [PubMed]
  44. Muto, H.; Watahiki, M.K.; Nakamoto, D.; Kinjo, M.; Yamamoto, K.T. Specificity and similarity of functions of the Aux/IAA genes in auxin signaling of Arabidopsis revealed by promoter-exchange experiments among MSG2/IAA19, AXR2/IAA7, and SLR/IAA14. Plant Physiol. 2007, 144, 187–196. [Google Scholar] [CrossRef] [PubMed]
  45. Paul, P.; Dhandapani, V.; Rameneni, J.J.; Li, X.; Sivanandhan, G.; Choi, S.R.; Pang, W.; Im, S.; Lim, Y.P. Genome-wide analysis and characterization of Aux/IAA family genes in Brassica rapa. PLoS ONE 2016, 11, e0151522. [Google Scholar] [CrossRef] [PubMed]
  46. Wang, Y.; Deng, D.; Bian, Y.; Lv, Y.; Xie, Q. Genome-wide analysis of primary auxin-responsive Aux/IAA gene family in maize (Zea mays. L.). Mol. Biol. Rep. 2010, 37, 3991–4001. [Google Scholar] [CrossRef] [PubMed]
  47. Overvoorde, P.J.; Okushima, Y.; Alonso, J.M.; Chan, A.; Chang, C.; Ecker, J.R.; Hughes, B.; Liu, A.; Onodera, C.; Quach, H.; et al. Functional genomic analysis of the auxin/indole-3-acetic acid gene family members in Arabidopsis thaliana. Plant Cell 2005, 17, 3282–3300. [Google Scholar] [CrossRef] [PubMed]
  48. Krogan, N.T.; Yin, X.; Ckurshumova, W.; Berleth, T. Distinct subclades of Aux/IAA genes are direct targets of ARF5/MP transcriptional regulation. New Phytol. 2014, 204, 474–483. [Google Scholar] [CrossRef] [PubMed]
  49. Kumar, R.; Agarwal, P.; Pareek, A.; Tyagi, A.K.; Sharma, A.K. Genomic survey, gene expression, and interaction analysis suggest diverse roles of ARF and Aux/IAA proteins in Solanaceae. Plant Mol. Biol. Rep. 2015, 1–21. [Google Scholar] [CrossRef]
  50. Cao, J.; Li, X.; Lv, Y.; Ding, L. Comparative analysis of the phytocyanin gene family in 10 plant species: A focus on Zea mays. Front. Plant Sci. 2015, 6, 515. [Google Scholar] [CrossRef] [PubMed]
  51. Makarova, K.S.; Wolf, Y.I.; Mekhedov, S.L.; Mirkin, B.G.; Koonin, E.V. Ancestral paralogs and pseudoparalogs and their role in the emergence of the eukaryotic cell. Nucleic Acids Res. 2005, 33, 4626–4638. [Google Scholar] [CrossRef] [PubMed]
  52. Pinyopich, A.; Ditta, G.S.; Savidge, B.; Liljegren, S.J.; Baumann, E.; Wisman, E.; Yanofsky, M.F. Assessing the redundancy of MADS-box genes during carpel and ovule development. Nature 2003, 424, 85–88. [Google Scholar] [CrossRef] [PubMed]
  53. Wang, X.Y.; Paterson, A.H. Gene conversion in angiosperm genomes with an emphasis on genes duplicated by polyploidization. Genes 2011, 2, 1–20. [Google Scholar] [CrossRef] [PubMed]
  54. Guilfoyle, T.J.; Hagen, G. Getting a grasp on domain III/IV responsible for auxin response factor—IAA protein interactions. Plant Sci. 2012, 190, 82–88. [Google Scholar] [CrossRef] [PubMed]
  55. Poutrain, P. Molecular characterization of an Aux/IAA of Catharanthus roseus. J. Plant Growth Regul. 2011, 30, 235–241. [Google Scholar] [CrossRef]
  56. Song, Y.; You, J.; Xiong, L. Characterization of OsIAA1 gene, a member of rice Aux/IAA family involved in auxin and brassinosteroid hormone responses and plant morphogenesis. Plant Mol. Biol. 2009, 70, 297–309. [Google Scholar] [CrossRef] [PubMed]
  57. Sato, A.; Yamamoto, K.T. Overexpression of the non-canonical Aux/IAA genes causes auxin-related aberrant phenotypes in Arabidopsis. Physiol. Plant. 2008, 133, 397–405. [Google Scholar] [CrossRef] [PubMed]
  58. Remington, D.L.; Vision, T.J.; Guilfoyle, T.J.; Reed, J.W. Contrasting modes of diversification in the Aux/IAA and ARF gene families. Plant Physiol. 2004, 135, 1738–1752. [Google Scholar] [CrossRef] [PubMed]
  59. Korasick, D.A.; Westfall, C.S.; Lee, S.G.; Nanao, M.H.; Dumas, R.; Hagen, G.; Guilfoyle, T.J.; Jez, J.M.; Strader, L.C. Molecular basis for auxin response factor protein interaction and the control of auxin response repression. Proc. Natl. Acad. Sci. USA 2014, 111, 5427–5432. [Google Scholar] [CrossRef] [PubMed]
  60. Nanao, M.H.; Vinos-Poyo, T.; Brunoud, G.; Thevenon, E.; Mazzoleni, M.; Mast, D.; Laine, S.; Wang, S.; Hagen, G.; Li, H.; et al. Structural basis for oligomerization of auxin transcriptional regulators. Nat. Commun. 2014, 5, 3617. [Google Scholar] [CrossRef] [PubMed]
  61. Sumimoto, H.; Kamakura, S.; Ito, T. Structure and function of the PB1 domain, a protein interaction module conserved in animals, fungi, amoebas, and plants. Sci. STKE 2007, 2007, re6. [Google Scholar] [CrossRef] [PubMed]
  62. Moore, R.C.; Purugganan, M.D. The early stages of duplicate gene evolution. Proc. Natl. Acad. Sci. USA 2003, 100, 15682–15687. [Google Scholar] [CrossRef] [PubMed]
  63. Cao, J.; Li, X. Identification and phylogenetic analysis of late embryogenesis abundant proteins family in tomato (Solanum lycopersicum). Planta 2015, 241, 757–772. [Google Scholar] [CrossRef] [PubMed]
  64. Kong, H.; Landherr, L.L.; Frohlich, M.W.; Leebens-Mack, J.; Ma, H.; DePamphilis, C.W. Patterns of gene duplication in the plant SKP1 gene family in angiosperms: Evidence for multiple mechanisms of rapid gene birth. Plant J. 2007, 50, 873–885. [Google Scholar] [CrossRef] [PubMed]
  65. Cannon, S.B.; Mitra, A.; Baumgarten, A.; Young, N.D.; May, G. The roles of segmental and tandem gene duplication in the evolution of large gene families in Arabidopsis thaliana. BMC Plant Biol. 2004, 4, 10. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  66. Zhu, Y.; Wu, N.; Song, W.; Yin, G.; Qin, Y.; Yan, Y.; Hu, Y. Soybean (Glycine max) expansin gene superfamily origins: Segmental and tandem duplication events followed by divergent selection among subfamilies. BMC Plant Biol. 2014, 14, 93. [Google Scholar] [CrossRef] [PubMed]
  67. Achaz, G.; Coissac, E.; Viari, A.; Netter, P. Analysis of intrachromosomal duplications in yeast Saccharomyces cerevisiae: A possible model for their origin. Mol. Biol. Evol. 2000, 17, 1268–1275. [Google Scholar] [CrossRef] [PubMed]
  68. Hanada, K.; Zou, C.; Lehti-Shiu, M.D.; Shinozaki, K.; Shiu, S.H. Importance of lineage-specific expansion of plant tandem duplicates in the adaptive response to environmental stimuli. Plant Physiol. 2008, 148, 993–1003. [Google Scholar] [CrossRef] [PubMed]
  69. Mohanta, T.K.; Mohanta, N.; Mohanta, Y.K.; Bae, H. Genome-wide identification of calcium dependent protein kinase gene family in plant lineage shows presence of novel D-x-D and D-E-L motifs in EF-hand domain. Front. Plant Sci. 2015, 6, 1146. [Google Scholar] [CrossRef] [PubMed]
  70. Nystedt, B.; Street, N.R.; Wetterbom, A.; Zuccolo, A.; Lin, Y.C.; Scofield, D.G.; Vezzi, F.; Delhomme, N.; Giacomello, S.; Alexeyenko, A.; et al. The norway spruce genome sequence and conifer genome evolution. Nature 2013, 497, 579–584. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  71. Weijers, D.; Benkova, E.; Jager, K.E.; Schlereth, A.; Hamann, T.; Kientz, M.; Wilmoth, J.C.; Reed, J.W.; Jurgens, G. Developmental specificity of auxin response by pairs of ARF and Aux/IAA transcriptional regulators. EMBO J. 2005, 24, 1874–1885. [Google Scholar] [CrossRef] [PubMed]
  72. Knox, K.; Grierson, C.S.; Leyser, O. AXR3 and SHY2 interact to regulate root hair development. Development 2003, 130, 5769–5777. [Google Scholar] [CrossRef] [PubMed]
  73. Audran-Delalande, C.; Bassa, C.; Mila, I.; Regad, F.; Zouine, M.; Bouzayen, M. Genome-wide identification, functional analysis and expression profiling of the Aux/IAA gene family in tomato. Plant Cell Physiol. 2012, 53, 659–672. [Google Scholar] [CrossRef] [PubMed]
  74. Goodstein, D.M.; Shu, S.; Howson, R.; Neupane, R.; Hayes, R.D.; Fazo, J.; Mitros, T.; Dirks, W.; Hellsten, U.; Putnam, N.; et al. Phytozome: A comparative platform for green plant genomics. Nucleic Acids Res. 2012, 40, D1178–D1186. [Google Scholar]
  75. Sundell, D.; Mannapperuma, C.; Netotea, S.; Delhomme, N.; Lin, Y.-C.; Sjödin, A.; Van de Peer, Y.; Jansson, S.; Hvidsten, T.R.; Street, N.R. The plant genome integrative explorer resource: PlantGenIE.Org. New Phytol. 2015, 208, 1149–1156. [Google Scholar]
  76. Cai, J.; Liu, X.; Vanneste, K.; Proost, S.; Tsai, W.-C.; Liu, K.-W.; Chen, L.-J.; He, Y.; Xu, Q.; Bian, C.; et al. The genome sequence of the orchid Phalaenopsis equestris. Nat. Genet. 2014, 47, 65–72. [Google Scholar]
  77. Ibarra-Laclette, E.; Lyons, E.; Hernández-Guzmán, G.; Pérez-Torres, C.A.; Carretero-Paulet, L.; Chang, T.-H.; Lan, T.; Welch, A.J.; Juárez, M.J.A.; Simpson, J.; et al. Architecture and evolution of a minute plant genome. Nature 2013, 498, 94–98. [Google Scholar] [Green Version]
  78. Kumar, S.; Stecher, G.; Tamura, K. MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol. Biol. Evol. 2016, 33, 1870–1874. [Google Scholar] [CrossRef] [PubMed]
  79. Wang, D.; Zhang, Y.; Zhang, Z.; Zhu, J.; Yu, J. Kaks_calculator 2.0: A toolkit incorporating gamma-series methods and sliding window strategies. Genom. Proteom. Bioinform. 2010, 8, 77–80. [Google Scholar] [CrossRef]
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Figure 1. Graphical representation of 11 types of Aux/IAAs and their comparative analysis among the 17 plant species. The chart at the bottom shows the conserve amino acid composition of different motifs on average. The content of conservative amino acids is expressed by the height of characters. The phylogenetic tree at the top is from the Tree of Life Web Project (Available online: http://www.tolweb.org/Green_plants).
Figure 1. Graphical representation of 11 types of Aux/IAAs and their comparative analysis among the 17 plant species. The chart at the bottom shows the conserve amino acid composition of different motifs on average. The content of conservative amino acids is expressed by the height of characters. The phylogenetic tree at the top is from the Tree of Life Web Project (Available online: http://www.tolweb.org/Green_plants).
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Figure 2. Phylogenetic relationship among canonical Aux/IAA genes. The deduced full-length amino acid sequences of canonical Aux/IAA genes were aligned by ClustalX (2.1), and the neighbor-joining (NJ) tree was constructed by Mega7. The bootstrap values with 1000 replicates are placed on the nodes. The Aux/IAAs derived from different categories are shown in different colors.
Figure 2. Phylogenetic relationship among canonical Aux/IAA genes. The deduced full-length amino acid sequences of canonical Aux/IAA genes were aligned by ClustalX (2.1), and the neighbor-joining (NJ) tree was constructed by Mega7. The bootstrap values with 1000 replicates are placed on the nodes. The Aux/IAAs derived from different categories are shown in different colors.
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Figure 3. Evolution relationships between representative Aux/IAAs and ARFs. (A) Multiple sequence alignment of representative Aux/IAAs and ARFs. Conservative domains III and IV are underlined. The height of the bars indicates the number of identical residues at each position. The β-grasp fold in domains III and IV is marked with “β1”, “β2”, “α1”, “β3”, “β4”, “α2”, “β5” and “α3”. The part enclosed by the red line is the conservative motif site “GDDP” in ARFs and “GDVP” in Aux/IAAs. The asterisk (*), colon (:), and dot (.) represent different conservative level from high and low; (B) Putative model for generating Aux/IAAs from truncated ARFs. Some truncated putative Aux/IAA genes were derived from truncated ARF genes, which only compose the Aux/IAA domain (domains III and IV), but lack their typical domains I and II.
Figure 3. Evolution relationships between representative Aux/IAAs and ARFs. (A) Multiple sequence alignment of representative Aux/IAAs and ARFs. Conservative domains III and IV are underlined. The height of the bars indicates the number of identical residues at each position. The β-grasp fold in domains III and IV is marked with “β1”, “β2”, “α1”, “β3”, “β4”, “α2”, “β5” and “α3”. The part enclosed by the red line is the conservative motif site “GDDP” in ARFs and “GDVP” in Aux/IAAs. The asterisk (*), colon (:), and dot (.) represent different conservative level from high and low; (B) Putative model for generating Aux/IAAs from truncated ARFs. Some truncated putative Aux/IAA genes were derived from truncated ARF genes, which only compose the Aux/IAA domain (domains III and IV), but lack their typical domains I and II.
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Figure 4. Phylogeny of Aux/IAAs and ARFs in Physcomitrella patens, Selaginella moellendorffii, and Picea abies. The deduced full-length amino acid sequences of Aux/IAA genes and ARF genes were aligned by ClustalX (2.1), and the neighbor-joining (NJ) tree was constructed by Mega7. The bootstrap values with 1000 replicates are placed on the nodes. The Aux/IAA and ARF clusters are grouped into two clades, but there exists a group of Aux/IAA genes highlighted with orange that clustered closer to the ARF clade.
Figure 4. Phylogeny of Aux/IAAs and ARFs in Physcomitrella patens, Selaginella moellendorffii, and Picea abies. The deduced full-length amino acid sequences of Aux/IAA genes and ARF genes were aligned by ClustalX (2.1), and the neighbor-joining (NJ) tree was constructed by Mega7. The bootstrap values with 1000 replicates are placed on the nodes. The Aux/IAA and ARF clusters are grouped into two clades, but there exists a group of Aux/IAA genes highlighted with orange that clustered closer to the ARF clade.
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Figure 5. Duplication events of Aux/IAAs in the plant kingdom. This phylogenetic tree was decorated with whole-genome duplication events (Available online: http://chibba.pgml.uga.edu/duplication/index/files) and the number of genes derived from segmental duplication, tandem duplication, and both events. The phylogenetic tree of 17 species was from the Tree of Life Web Project (Available online: http://www.tolweb.org/Green_plants). Seg: segmental duplication; Tan: tandem duplication; Seg&Tan: genes belonging both to segmental duplication and tandem duplication; Total: total Aux/IAA gene number in the species.
Figure 5. Duplication events of Aux/IAAs in the plant kingdom. This phylogenetic tree was decorated with whole-genome duplication events (Available online: http://chibba.pgml.uga.edu/duplication/index/files) and the number of genes derived from segmental duplication, tandem duplication, and both events. The phylogenetic tree of 17 species was from the Tree of Life Web Project (Available online: http://www.tolweb.org/Green_plants). Seg: segmental duplication; Tan: tandem duplication; Seg&Tan: genes belonging both to segmental duplication and tandem duplication; Total: total Aux/IAA gene number in the species.
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Figure 6. Molecular evolutionary rate of Aux/IAAs in the plant kingdom. (A) Rates of molecular evolution in segmental duplication of dicot and monocot Aux/IAA genes; (B) Rates of molecular evolution in tandem duplication of dicot and monocot Aux/IAA genes. Two gene pairs are excluded from the plot and exist in the above table; (C) Rates of molecular evolution in segmental duplication and tandem duplication in plants.
Figure 6. Molecular evolutionary rate of Aux/IAAs in the plant kingdom. (A) Rates of molecular evolution in segmental duplication of dicot and monocot Aux/IAA genes; (B) Rates of molecular evolution in tandem duplication of dicot and monocot Aux/IAA genes. Two gene pairs are excluded from the plot and exist in the above table; (C) Rates of molecular evolution in segmental duplication and tandem duplication in plants.
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Figure 7. Presence of conservative amino acids in the nuclear localization signal (NLS) of Aux/IAAs. The content of conservative amino acids is expressed by the height of characters. The amino acids under the symbol * are conservative NLS sites.
Figure 7. Presence of conservative amino acids in the nuclear localization signal (NLS) of Aux/IAAs. The content of conservative amino acids is expressed by the height of characters. The amino acids under the symbol * are conservative NLS sites.
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Table
Table 1. Aux/IAA genes identified from 17 sequenced plant genomes.
Table 1. Aux/IAA genes identified from 17 sequenced plant genomes.
LineageOrganismNo. of Predicted Loci aNo. of Aux/IAA GenesReferences
AlgaeChlamydomonas reinhardtii17,7410This study
MossPhyscomitrella patens32,9263Kumar et al. [49]
LycophytesSelaginella moellendorffii22,2859Kumar et al. [49]
GymnospermPicea abies58,58731This study
AmborellalesAmborella trichopoda26,84616This study
DicotsUtricularia gibba28,49425This study
Solanum tuberosum35,11926Gao et al. [41]
Gossypium raimondii37,50544This study
Arabidopsis thaliana27,41629Liscum and Reed [34]
Populus trichocarpa41,33536Kalluri et al. [36]
Ricinus communis31,22121This study
Prunus persica26,87324This study
Glycine max56,04463Singh and Jain [33]
MonocotsPhalaenopsis equestris31,38416This study
Brachypodium distachyon34,31027This study
Oryza sativa42,18931Jain et al. [35]
Zea mays63,48034Wang et al. [46]
a The data came from the Phytozome Database (Available online: https://phytozome.jgi.doe.gov/pz/portal.html).
Table
Table 2. Molecular evolutionary rate of selective duplicated Aux/IAA gene pairs in P. patens, S. moellendorffii, P. abies, and A. trichopoda.
Table 2. Molecular evolutionary rate of selective duplicated Aux/IAA gene pairs in P. patens, S. moellendorffii, P. abies, and A. trichopoda.
SpeciesParalogous PairsKaKsKa/KsDuplication Types
Physcomitrella patensPhpat.008G051100/Phpat.024G0234000.22421.07150.2092Segmental duplication
Selaginella moellendorffii15405070/154226440.03720.08690.4281Segmental duplication
Picea abiesMA_10430843g0010/MA_10430843g00204.37212.61491.6720Tandem duplication
Amborella trichopodaAmTr_00002.512/AmTr_00002.5140.34303.28960.1043Tandem duplication
Amborella trichopodaAmTr_00061.52/AmTr_00061.540.20003.58480.0558Tandem duplication
Amborella trichopodaAmTr_00061.52/AmTr_00061.550.36653.44830.1063Tandem duplication
Amborella trichopodaAmTr_00061.52/AmTr_00061.570.07210.10400.6933Tandem duplication
Amborella trichopodaAmTr_00061.54/AmTr_00061.550.37131.52160.2440Tandem duplication
Amborella trichopodaAmTr_00061.54/AmTr_00061.570.04290.06970.6163Tandem duplication
Amborella trichopodaAmTr_00061.55/AmTr_00061.570.38902.60670.1492Tandem duplication
Table
Table 3. The amino acid compositions of Aux/IAA NLSs in plants.
Table 3. The amino acid compositions of Aux/IAA NLSs in plants.
SpeciesBipartite NLSSV40-Type NLS
Part 1Part 2
KRDe aLack bRxxRKDe aLack bKRxRxxKDe aLack b
Ricinus communis130812811740
Populus trichocarpa22014221042961
Glycine max41121391685184
Prunus persica133814732040
Gossypium raimondii24118231743572
Arabidopsis thaliana16112121252180
Utricularia gibba15010131201933
Solanum tuberosum160915641942
Brachypodium distachyon12015141122061
Oryza sativa15115161142281
Zea mays19015151722473
Phalaenopsis equestris5386641114
Amborella trichopoda4111538853
Picea abies9616891410156
Selaginella moellendorffii008044440
Physcomitrella patens201021210
a De: degenerated NLS caused by a non-synonymous mutation; b Lack: the typical NLS in the corresponding Aux/IAA protein sequence failed to be detected. “x” means the amino acid is not conservative in the given site.

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Wu, W.; Liu, Y.; Wang, Y.; Li, H.; Liu, J.; Tan, J.; He, J.; Bai, J.; Ma, H. Evolution Analysis of the Aux/IAA Gene Family in Plants Shows Dual Origins and Variable Nuclear Localization Signals. Int. J. Mol. Sci. 2017, 18, 2107. https://doi.org/10.3390/ijms18102107

AMA Style

Wu W, Liu Y, Wang Y, Li H, Liu J, Tan J, He J, Bai J, Ma H. Evolution Analysis of the Aux/IAA Gene Family in Plants Shows Dual Origins and Variable Nuclear Localization Signals. International Journal of Molecular Sciences. 2017; 18(10):2107. https://doi.org/10.3390/ijms18102107

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Wu, Wentao, Yaxue Liu, Yuqian Wang, Huimin Li, Jiaxi Liu, Jiaxin Tan, Jiadai He, Jingwen Bai, and Haoli Ma. 2017. "Evolution Analysis of the Aux/IAA Gene Family in Plants Shows Dual Origins and Variable Nuclear Localization Signals" International Journal of Molecular Sciences 18, no. 10: 2107. https://doi.org/10.3390/ijms18102107

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