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Article

Genome-Wide Identification and Characterization of the Aux/IAA Gene Family in Strawberry Species

by
Xiaotong Jing
1,2,
Quan Zou
1,2 and
Hui Yang
1,2,*
1
Yangtze Delta Region Institute (Quzhou), University of Electronic Science and Technology of China, Quzhou 324003, China
2
Institute of Fundamental and Frontier Sciences, University of Electronic Science and Technology of China, Chengdu 610054, China
*
Author to whom correspondence should be addressed.
Plants 2024, 13(20), 2940; https://doi.org/10.3390/plants13202940
Submission received: 24 August 2024 / Revised: 17 October 2024 / Accepted: 18 October 2024 / Published: 21 October 2024
(This article belongs to the Section Plant Genetics, Genomics and Biotechnology)
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Abstract

:
Auxin is the first plant hormone found to play a dominant role in fruit growth, from fruit set to fruit ripening. Strawberry plants represent a suitable model for studying auxin’s biosynthesis, sensing, and signaling machinery. Aux/IAA genes are a classical rapid auxin-responsive family. However, the Aux/IAA gene family in Fragaria genus is poorly understood. In this study, a total of 287 Aux/IAA genes were identified in the eight strawberry genomes. Their physicochemical properties, domain structure, and cis-regulatory elements revealed the functional multiplicity of the strawberry Aux/IAAs. We used a phylogenetic analysis to classify these genes into 12 classes. In addition, based on synteny analysis, gene duplications, and calculation of the Ka/Ks ratio, we found that segmental duplications promote the evolution of Aux/IAAs in Fragaria species, which is followed by purifying selection. Furthermore, the expression pattern and protein–protein interaction network of these genes in Fragaria vesca revealed various tissue-specific expressions and probable regulatory functions. Taken together, these results provide basic genomic information and a functional analysis of these genes, which will serve to expand our understanding of the direction in which the Aux/IAA gene family is evolving in Fragaria species.

1. Introduction

Fruit development is a complex process involving multiple phytohormones, within which auxin was the first plant hormone to be discovered. Auxins play a critical role in tuning fruit growth, from fruit set to fruit ripening [1]. Auxins mainly exert their effects by coordinating auxin synthesis and metabolism [2,3], polar transport [4], and signal transduction [5] pathways. Through the classical signal transduction pathways, auxin facilitates auxin response factor (ARF) transcriptional activity by eliciting the degradation of the auxin/indole-3-acetic acid (Aux/IAA) transcription repressors [6].
Aux/IAA genes belong to a classical rapid auxin-responsive family. They encode short-lived nuclear proteins, which have half-lives as short as 6–8 min in pea plants (8 min for PSIAA4 and 6 min for PSIAA6) [7], 10–60 min in Arabidopsis (10 min for AtIAA7/AtIAA17 and 60 min for AtIAA28) [8], and 11–120 min in maize (11 min for ZmIAA2 and 120 min for ZmIAA15) [9]. Their half-lives are determined by their domain II. The genome of Arabidopsis thaliana has 29 genes encoding Aux/IAA proteins, with the majority of these genes exhibiting four conserved domains (domains I–IV) [10]. Each domain contributes to a different function. Among them are domain I, which bears a Leu-rich motif (LxLxLx, where L is Leu and x is one of several different amino acids) and is responsible for the repression of proteins [11], and domain II, which contains a conserved GWPPV degron in nearly all Arabidopsis Aux/IAA and directly interacts with the SCFTIR1 complex. This interaction leads to the polyubiquitination and degradation of Aux/IAA proteins [12]; The homodimerization and heterodimerization of ARFs and the Aux/IAAs are induced by domains III and IV by way of the Phox and Bem1p (PB1) domain [13]. Until now, studies about mutations in Aux/IAAs have revealed that this family has distinct functions within a range of developmental processes, including floral organ development, embryo development, lateral root elongation, phototropisms, and so on [14]. The Aux/IAA family has been recognized and studied in various flowering plants, such as the crop plants rice [15] and maize [16]; the vegetable species Solanum [17,18] and Brassica [19,20,21]; the fruits papaya [22] and apple [23]; the tree species Populus trichocarpa [24], Carya cathayensis [25], Acer rubrum [26], Populus simonii [27], and Paulownia fortune [28]; the forage grasses orchardgrass [29] and alfalfa [30]; the medicinal plants Bletilla striata [31], Dendrobium ofcinale [32], Panax ginseng [33], and Artemisia argyi [34]; and bamboo species [35,36].
Strawberry has long been used as an excellent model through which to study the molecular role of the hormone auxin in fruit growth and ripening [37,38]. Achenes, located on the outside of the receptacle, are rich sources of auxin [39]. When part or all of the achenes are removed, the fleshy fruit will fail to develop and ripen or will grow into an abnormal shape [37,40]. Exogenous auxin treatment may serve as a substitute for achenes and can cause horizontal and vertical expansion of fleshy fruit [38,41]. Therefore, the strawberry plant is a suitable means of studying auxin’s biosynthesis, sensing, and signaling machinery. Recent studies have indicated that ARF, Aux/IAA, and DELLA proteins interact with one another and play roles in regulating auxin signaling pathways in strawberry fruit growth [42]. In the Fragaria species, Aux/IAA genes have only been reported in F. × ananassa and F. vesca. For example, FaAux/IAA1 and FaAux/IAA2 transcripts may be involved in the early development of fruit, and their levels are observed to be significantly elevated in the early development stages, after which they decrease sharply in the ripening stage [43]. Additionally, a downward trend has been observed in the expression of FvIAA4 following fertilization, which may negatively impact fruit initiation [44]. The Fragaria genus consists of around 25 species with varying ploidy levels, ranging from diploid (2×) to decaploid (10×) [45]. Among them, F. × ananassa is a young cultivated species, which is an allo-octoploid (8×) and originated from natural crosses between F. virginiana (8×) and F. chiloensis (8×). Diploid strawberry plants, such as F. bucharica, F. chinensis, F. daltoniana, F. hayatai, F. iinumae, F. mandshurica, F. nilgerrensis, F. nipponica, F. nubicola, F. pentaphylla, F. vesca, and F. viridis, etc., are the most abundant of all Fragaria [45]. These wild diploids exhibit varied morphology and physiology. F. vesca was chosen as the genomic reference for Fragaria thanks to its many advantages, including its rapid generation time and ability to easily propagate [46]. It differs from F. vesca, Fragaria genus including F. bucharica, F. chinensis, F. mandshurica, F. nipponica, F. nubicola, F. pentaphylla, and F. viridis, which are self-incompatible [45]. Mature fruits of F. viridis have an apple-like aroma [47], while F. nilgerrensis fruits have peach-like and banana-like aromas [48]. Among these diploid strawberries, F. iinumae, F. nipponica, F. vesca, and F. viridis might contribute to the subgenomes of F. × ananassa [49].
For the purpose of understanding the role of Aux/IAAs in the growth of strawberry fruits, we have comprehensively identified the Aux/IAAs in the octoploid strawberry as well as the extant relatives of each diploid progenitor species. We also examined the sequence structure characteristics of these genes in various species. Additionally, we explored the evolutionary trajectory and expansion mechanisms of Aux/IAAs in Fragaria based on interspecific collinearity. Moreover, the tissue expression patterns and protein–protein interaction (PPI) networks were investigated in the Aux/IAA genes. To sum up, out study will contribute to better annotation of Fragaria spp. genomes, including F. iinumae, F. mandshurica, F. nilgerrensis, F. nipponica, and F. viridis, and provide insights into the fruit development process with the aim of increasing crop production and fruit quality.

2. Results

2.1. Identification and Characterization of Aux/IAAs in Fragaria

To explore the evolutionary histories of the Aux/IAA family in the various ploidy strawberries, we identified the putative Aux/IAA genes from six diploid strawberries and two octoploid strawberries using the BLAST [50] and HMMER [51] search tools. Then, the supposed Aux/IAA genes were further confirmed to contain a conserved AUX_IAA (PF02309) domain in accordance with the Pfam database [52] and NCBI’s Conserved Domain Database [53]. After filtering out the genes found to possess B3 and ARF domains, thereby sustaining the auxin response factor (ARF), 287 Aux/IAAs were identified in the eight strawberry genomes. They were named according to the phylogenetic relationship between strawberries and Arabidopsis. Among these strawberry species, F. × ananassa has the largest Aux/IAA family (82 members), and the fewest (18) members were found in F. iinumae. Table S1 contains detailed information about these genes, such as gene ID, chromosome location, and sequence length. The length of strawberry Aux/IAA coding sequences (CDSs) varied from 309 bp for FaIAA11b to 1221 bp for FveIAA12. In addition, strawberry Aux/IAA protein’s physical and chemical properties were analyzed using the Expasy-ProtParam tool (https://web.expasy.org/protparam/) (accessed on 19 April 2024) in this study (Table S2). The molecular weight of those proteins ranged from 11.76 to 43.92 kD, and the theoretical isoelectric point ranged from 4.81 to 9.54. Furthermore, most Aux/IAA members (87.11%, 250/287) were unstable (instability index > 40), and all members were hydrophilic proteins (GRAVY index < 0).

2.2. Molecular Structure of Aux/IAA Proteins in Fragaria

To explore the conservation of these Aux/IAAs, we investigated the conserved motifs using the MEME Suite (https://meme-suite.org/meme/) (accessed on 30 April 2024), which elucidated the diversity and comparability of the Aux/IAAs. In total, 10 conserved motifs were found in the Aux/IAAs of Fragaria, namely, motifs 1–10 (Figure 1, Figures S1 and S2). Among them, five different conserved motifs, motifs 4, 2, 5, 1, and 3, were mapped to the most Aux/IAA proteins (Figure 1A). Among them, motif 4 has a classical “LxLxLx” motif, using which we identified domain I (Figure 1B), which was missing from the members of IAA14, IAA29, IAA32, IAA33, IAA34, and IAA12 of F. mandshurica (Figure S2D). Motif 2 corresponded to domain III, which was missing from the members of IAA33. All strawberry Aux/IAA proteins contained motif 5, with the exception of FcIAA15c, FiIAA3, and two Aux/IAA16 members, FiIAA16 and FnlIAA16. Motif 5 was included in domain IV. In addition, motif 1 was also included in domain IV, the conserved “GDVP” of which may contribute to electrostatic protein interactions [54]. We found that this function potentially was missing from the IAA15c, IAA29, IAA30, IAA31, IAA32 and IAA34 members, IAA16 of F. iinumae; IAA14 of F. mandshurica; and IAA17 and IAA20 of F. nilgerrensis (Figure S2). Motif 3, with a classical “GWPPV” motif, corresponded to domain II, which plays a role in rapid protein turnover. IAA1, IAA2, IAA32, IAA33, IAA34, and some IAA15 members may lack this function. Furthermore, motif 7 has a conserved “KR” motif located between domain I and domain II, and is highly conserved in the proteins of IAA6, IAA8, IAA9, IAA14, IAA17, IAA27, and IAA29. Interestingly, in this work, we found that motif 8 was observed in the Aux/IAAs, including IAA8, IAA9, and IAA33 members, with the exception of the two octoploid IAA8 members (FaIAA8a and FcIAA8a). The functions of this motif have not yet been reported. Moreover, we also measured the integrity of the Aux/IAAs in Fragaria. This result indicated that diploid Aux/IAA proteins are more complete than octoploid proteins (Table 1).

2.3. Evolutionary Tree Analysis and Classification of the Aux/IAA Gene Family in Fragaria

To probe into the evolutionary relationships among the Aux/IAAs from six diploid strawberries (F. iinumae, F. mandshurica, F. nilgerrensis, F. nipponica, F. vesca, and F. viridis), two octoploid strawberries (F. × ananassa and F. chiloensis), and A. thaliana, a maximum-likelihood tree was produced by MEGA11 [55] and based on the 316 Aux/IAA protein’s amino acid sequences, including 29 members of A. thaliana and 287 members of Fragaria spp. Based on the evolutionary tree, these Aux/IAAs were classified into 12 major clades: IAA1/2/3/4 (Clade A), IAA5/6/19 (Clade B), IAA15 (Clade C), IAA14/17 (Clade D), IAA16 (Clade E), IAA27 (Clade F), IAA8/9 (Clade G), IAA10/11/12/13/32/34 (Clade H), IAA18/28 (Clade I), IAA20/30/31 (Clade J), IAA29 (Clade K), and IAA33 (Clade L) (Figure 2).

2.4. Synteny and Ka/Ks Analysis of Aux/IAAs in Fragaria

A collinearity analysis was carried out using Tbtools-II [56] ao that we might further understand the evolutionary relationships among Fragaria Aux/IAA genes. We explored the distribution of duplicate events in eight strawberry species. Among the 286 genes (with the exception of FviIAA29b), we identified instances of 223 whole-genome duplication (WGD), 43 of dispersed duplication, 11 of tandem duplication, and 9 of proximal duplication events (Table S1). Octoploid strawberries had more WGD events involving Aux/IAA family genes than diploid strawberries. Notably, all Aux/IAAs of F. chiloensis expanded through the WGD, while Aux/IAAs in diploid strawberries expanded mainly through WGD and dispersed duplication (Figure 3A). Additionally, based on the collinearity analysis, 2309 gene pairs, including 8 gene pairs formed through tandem duplication, were found in the strawberry Aux/IAAs. Among them, we identified 5 and 120 gene pairs produced by segment duplications in F. vesca and F. × ananassa, respectively. Additionally, 103 gene pairs experienced segment duplications between the F. vesca and F. × ananassa (Figure 3B). We further calculated the values of Ka and Ks of all gene pairs with the KaKs_Calculator [57] (Figure 3C,D and Figure S3). The findings indicated that all gene pairs exhibited Ka < 1, and 71.50% (1651/2309) of gene pairs exhibited Ks < 1. The Ka/Ks distribution of the gene pairs showed that most had Ka/Ks < 1, implying that the Aux/IAA gene family may largely have undergone purifying selection during the evolutionary process. Moreover, a high value of Ka/Ks (>1) was detected in 45 gene pairs, including the members of IAA3 (5 pairs), IAA6 (6 pairs), IAA18 (5 pairs), IAA29 (7 pairs), IAA32/34 (7 pairs), etc. (Figure S4, Table S3). Among them, three gene pairs—FaIAA29c-FmIAA29b, FaIAA29d-FmIAA29b, and FcIAA29c-FmIAA29b—exhibited Ka/Ks > 2, indicating strong positive selection of these genes.

2.5. Predicting the Promoter Cis-Acting Elements of Aux/IAAs

To detect the feasible regulatory modes of the Aux/IAAs, we analyzed the cis-acting element located within the 2000 bp sequences preceding the translation start sites of genes in the PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/Plantcare/html/) (accessed on 9 May 2024). Then, we mainly analyzed and screened four types of cis-acting elements, which were largely hormone response elements, stress response elements, tissue development-related elements, and light-responsive elements (Figure 4). Five hormone response elements were found: abscisic acid (ABA), auxin, gibberellin acid (GA), methyljasmonate (MeJA), and salicylic acid (SA). Among them, 81.47% (233/286), 47.90% (137/286), 41.26% (118/286), 68.18% (195/286), and 43.71% (125/286) promoters of the Aux/IAA family carry ABA-, auxin-, GA-, MeJA-, and SA-responsive elements in strawberries, respectively. The promoters of the IAA11 subfamily contain many ABA- and auxin-responsive elements. Half of the members of the IAA15 and IAA29 subfamilies contain response elements of ABA and MeJA, respectively. We also discovered six crucial stress-responsive regulatory elements in strawberry Aux/IAA promoter sequences: anaerobic induction (95.45%, 273/286), anoxic-specific (6.99%, 20/286), defense and stress (29.37%, 84/286), drought (54.90%, 157/286), low temperature (32.87%, 94/286), and wound (6.99%, 20/286). The IAA8 and IAA9 subfamilies contain up to six anaerobic induction elements. FcIAA1 not only contains many anaerobic induction elements but also harbors the most low-temperature-responsive elements. Moreover, some tissue development-related elements were observed, such as the endosperm (64 sequences), meristem (105 sequences), palisade mesophyll cells (4 sequences), root (3 sequences), and seed (32 sequences). Among them, the subfamilies of IAA4 and IAA20/30/31 show many meristem expression elements, and the IAA33 subfamily has many endosperm expression elements. Of the IAA11 members, FmIAA11, FcIAA11a, and FaIAA11a, each contain a root-specific regulatory element. In addition, all the strawberry IAA promoter sequences contain a number of light-responsive elements, ranging from 2 (FnpIAA32, FcIAA32b, and FaIAA32c) to 26 (FviIAA11, and FcIAA15f). We found that half of the IAA15 subfamily contain many ABA-, MeJA-, and light-responsive elements, and the other half contain many meristem expression elements. Altogether, an analysis was conducted on the cis-acting elements that will enhance our understanding of the roles of the Aux/IAAs in Fragaria.

2.6. Aux/IAA Genes’ Expression Profiles in Different Tissues of F. vesca

Aux/IAA family members have distinct roles to play in plant growth and development processes. In this work, their expression patterns were studied in the development of the reproductive and vegetative tissues of the strawberry model F. vesca based on the annotation of the F. vesca v4 genome [58]. As shown in Figure 5, FveIAA9 was highly expressed in all tissues and abundantly expressed in the anther 12 and style 1 stages. FveIAA17 showed high expression in the cortex and pith development stages. FveIAA4 and FveIAA14 were significantly expressed in the style 1 stage. Additionally, we found that all Aux/IAA members showed a low degree of expression in pollen. In the seed development process, FveIAA11, FveIAA15b, FveIAA16, FveIAA29b, and FveIAA31 largely accumulated in the ghost and wall development stages; FveIAA8, FveIAA9, and two FveIAA27s (FveIAA27a/b) were present at high levels in the embryo development process. Unlike FveIAA29b, FveIAA29a was weakly expressed in all tissues, as was FveIAA32. In addition, many Aux/IAAs were strongly expressed in vegetative tissues, including leaves, roots, and seedlings.

2.7. Analysis of Aux/IAA Protein–Protein Interaction Network

We identified five PPI networks for the proteins encoded by specific expression genes, including three ghost-and-wall-specific expression genes (FveIAA15b, FveIAA29b, and FveIAA31) and two cortex-and-pith-specific expression genes (FveIAA16 and FveIAA17), which were established based on the known interactions of A. thaliana homologues on the STRING online website (http://cn.string-db.org) (accessed on 7 August 2024) [59,60,61,62] (Figure 6). The majority of interactions occurred between Aux/IAA and ARF proteins. For example, ARF5 and ARF7 were predicted to interact with five Aux/IAA proteins. Interestingly, we found that FveARF5 had an expression pattern that opposed that of the five IAA genes in F. vesca (Figure 6). In addition, it was anticipated that Aux/IAA proteins would engage with other auxin signaling proteins; for instance, five Aux/IAAs (with the exception of IAA29b) also interacted with TIR1. Unlike FveIAA16 and FveIAA17, FveTIR1 was minimally expressed in cortex and pith tissues (Figure 6B,C). IAA17 was also predicted to interact with AFB2 and AUX1, while IAA29 was predicted to interact with YUC8 (Figure 6C,E). Moreover, IAA29 was also predicted to interact with WRKY57, which showed leaf-specific expression in the strawberry (Figure 6E).

3. Discussion

Aux/IAAs act as pivotal factors that regulate the expression of downstream transcription factors in auxin signaling transduction. Following the ongoing advancements in whole-genome sequencing technology, Aux/IAAs of several species have been identified [14,20]. Among these plant species, the number of Aux/IAAs varies significantly and ranges from 1 in Marchantia polymopha [63] to 119 in Brassica napus [21]. Based on different tree construction methods and numbers of sequences, the Aux/IAA family is divided into different clades in different plants. In Populus simonii, 33 PsIAAs were categorized into three subgroups using the neighbor-joining (NJ) approach [27]: all 149 Aux/IAA proteins—including Arabidpsis, rice, and ginseng Aux/IAA family members—were classified into six groups through the maximum-likelihood method using the DCMut + F+ R4 model [33]. To unveil the origins of the Aux/IAAs, Wu et al. [64] used 253 canonical Aux/IAA members from eudicots, grasses, amborellales, gymnosperms, pteridophytes, and bryophytes to analyze evolutionary relationships under the assumption that the auxin response pathway mediated by Aux/IAAs first emerged in land plants. To investigate the evolution of the Aux/IAAs in greater depth, all Aux/IAA genes from 406 species were divided into groups I–VIII. The results indicated that charophytes were clustered into group VI, and basal angiosperms were clustered into all subgroups except group I [65]. In our study, after further division, the Aux/IAA genes of eight strawberries were classified into 12 major clades. This result showed phylogenetic relationships of the Aux/IAA gene family between the different strawberry species, but could not comprehensively represent Aux/IAAs’ evolution in the Fragaria genus in the Rosaceae family, and this topic will require further exploration.
Additionally, a large proportion of Aux/IAAs expanded over the course of their evolution via both segmental and tandem duplications in the plants, among which segmental duplications showed a greater effect [14]. In A. thaliana, 75.86% of Aux/IAAs were found to be duplicated via segmental duplication events [64]. Among 63 soybean Aux/IAAs, 90.5% were from segmental duplications [66]. In this study, we found that diploid and octoploid strawberries of the Aux/IAA family have presumably undergone dissimilar forms of expansion. Some 40~65% of diploid Aux/IAAs originated from segmental duplications, while 95.12% and 100% of Aux/IAAs were derived from segmental duplications in F. × ananassa and F. chiloensis, respectively. F. × ananassa, the cultivated strawberry, is a young domesticated plant. It stems from natural hybrids of F. virginiana and F. chiloensis in Europe [67]. Among other taxa, flowering plants have experienced continuous whole-genome duplication, resulting in genomes made up of several homologous subgenomes [68]. Many scholars have put forward that four subgenomes—F. iinumae, F. nipponica, F. vesca, and F. viridis—are the subgenome progenitors of F. × ananassa [49,69]. Therefore, we identified gene pairs between F. × ananassa and its four subgenomes and found that segmental duplications drove Aux/IAA evolution in Fragaria species, which was followed by purifying selection: their evolution has been conservative (Figure 3C). This result is similar to findings in Panax ginseng [33]. Evolution of protein sequences is influenced by the constraint of purifying selection or the fixation of positive selection at the molecular evolution level [70]. Ka/Ks > 1 is considered the signature of positive selection [71]. In this work, 45 gene pairs were acquired based on intraspecific and interspecific collinearity analyses. Additionally, the gene pairs displayed Ka/Ks values ranging from 1.01 (FveIAA4-FiIAA4) to 3.13 (FaIAA29d-FmIAA29b) (Table S3). In P. ginseng, two gene pairs—PgIAA25-PgIAA75 (homologue AtIAA19) and PgIAA14-PgIAA40 (homologue AtIAA30/31)—underwent positive selection throughout their evolutionary history [33]. Similarly, FaIAA31-FcIAA30a also showed Ka/Ks values of >1 in this study (Table S3). We also found that IAA8, IAA9, IAA14, and IAA27 members expanded at a Ka/Ks value higher than that of strawberries and other plant species. For instance, one gene pair, Potri.006G161200–Potri.006G161400, was found in Populus trichocarpa, which showed high sequence similarity to the Arabidopsis IAA27 gene [64]. Additionally, 13 gene pairs, largely belonging to the IAA8 and IAA9 subfamilies, were detected in turnip plants. Their Ka/Ks values were higher than 1, indicating that they experienced tachytelic evolution in the recent past [20]. Alongside dicots, gene pairs with Ka/Ks values of >1 were also found in gymnosperm, an example of which is MA_10430843g0010-MA_10430843g0020 in Picea abies, which is homologous with AtIAA14 [64].
Four characteristic conserved domains underlie the typical Aux/IAA protein functions [10]. In this study, analyses of conserved structural domains showed that some members are atypical, lacking one or more conserved domains in Fragaria (Table 1). For instance, subfamilies IAA14 and IAA29 lack domain I, which is also missing in the three PoptrIAA29 members (PoptrIAA29.1/2/3) and an Arabidopsis orthologue, AtIAA29 [24,72], indicating that these proteins show an inability to attract the TOPLESS and do not participate in typical auxin signal transduction. In this study, we found that IAA1 and IAA2 subfamily members lack domain II, which determines the stability of Aux/IAAs by recognizing the GWPPV sequence interacting with TIR1/AFB protein [73]. The Arabidopsis orthologues AtIAA20 and AtIAA31 cannot be rapidly biodegraded because they lack domain II [72,74]. Similar results have also been reported in rice and tomato plants [15,75]. Therefore, we speculated that these proteins’ half-lives in defects of domain II are much longer than the standard Aux/IAAs. In addition, IAA32s and IAA34s lack domains I and II and feature a truncated domain IV. Similarly, domains I and II seem to be disappearing from PoptrIAA34 as well as AtIAA34 [24,72]. Some 42 Aux/IAA proteins also exhibit this structure in turnip, which may interact with additional unknown components and participate in other processes such that Aux/IAA’s degradation does not require the mediation of SCFTIR1-dependent proteasomes [20]. Domains III and IV, which are shared with ARF proteins, facilitate homodimerization and heterodimerization with other Aux/IAA members [8,76] and for heterodimerization with ARFs to regulate the transcriptional activation of downstream genes [8,77]. Interestingly, unlike in studies of other plants (such as chinses hickory [25], bamboo [35], and D. ofcinale [32], etc.), all of the Aux/IAA proteins in the Fragaria contain domain III, with the exception of the IAA33 subfamily members, which contain only one conserved domain, domain IV. Some truncated proteins, such as FcIAA15 and FiIAA16, lack domain IV. These atypical Aux/IAAs may conduce to the varying roles of the auxin response processes, a topic that requires further study.
Most aux/iaa mutations in Arabidopsis reveal their functional roles during plant growth and development processes. For example, auxin-resistant 5 (axr5), with a mutation in Aux/IAA1, is resistant to auxin and induces various auxin-related growth defects, including defects in tropism of roots and shoots [78]. iaa2 and iaa6 show similar phenotypes, and accelerate tuberous root development of Tetrastigma hemsleyanum by regulating hormone biosynthesis [79]. Similarly, FveIAA2 shows higher expression in roots. FveIAA2 and FveIAA6 are more pronounced in the wall development process (Figure 5). The wall is a part of the achanes, which cause dotting on the receptacle surface of true strawberry fruit and are essential for the enlargement and ripening of the strawberry receptacle [39,80]. Recent studies indicate that MdAux/IAA2 negatively regulates apple fruit and size of cells, and its expression is enhanced in the absence of auxin, conversely leading to small fruit size in Longfeng apple plants [81]. Two IAA15 members (FveIAA15a and FveIAA15b) were identified in F. vesca. However, their promoter structure and expression patterns differed greatly (Figure 5 and Figure S5). For instance, compared with FveIAA15a, most ABA- and MeJA-responsive elements were located in the FveIAA15b promoter and FveIAA15a was solely expressed in the ghost4 and SAM tissues. The same was true of the two IAA29 members (FveIAA29a and FveIAA29b). IAA29 competitively interacts with WRKY57 to confront the leaf senescence induced by MeJA [82]. In the strawberries, we found that FveWRKY57a exhibited a leaf-specific expression (Figure 6D), and most members of the IAA29 subfamilies contained many MeJA-responsive elements in their promoters (Figure 3). Hence, we inferred that FveIAA29a and FveWRKY57a may be important for the MeJA-induced leaf senescence of strawberry plants. High levels of FveIAA9 were observed in all tissues, but predominantly expressed in flower tissues. This suggested that IAA9 may be connected to the development of flowers. In tomato, SiIAA9 controls multiple processes mediated by auxin signaling, including apical dominance and the development of flower organs and fruit development [83]. In addition, the IAA33 subfamily has only one conserved domain and contains the majority of endosperm development-related cis-elements in strawberries. We suggest that these proteins may participate in embryo and fruit development. Overall, analysis of the structure, evolution, and expression levels of the Aux/IAA family offers an initial insight into the function of these genes in strawberry genus. These results need to be further verified in future investigation.

4. Materials and Methods

4.1. Identification and Characterization of Aux/IAA Genes in Fragaria

The genome information and related annotation files of six diploid strawberries—F. iinumae, F. mandshurica, F. nilgerrensis, F. nipponica, F. vesca (v4.0.a2), and F. viridis—and two octoploid strawberries—F. × ananassa (v1.0.a2) and F. chiloensis—were downloaded from the Genome Database for Rosaceae (https://www.rosaceae.org/) (accessed on 18 January 2024) [84]. The AtIAA protein sequences were obtained from the TAIR (https://www.arabidopsis.org/) (accessed on 18 January 2024). Twenty-nine Aux/IAA genes of A. thaliana were used as query sequences, and BLAST v2.10.0 [50] (E-value 1 × 10−10) searches were performed to obtain orthologous genes in Fragaria genus. The Aux/IAA domain’s hidden Markov model (HMM) (PF02309) was downloaded [52] from the Pfam databas, and HMMER v3.2.1 [51] (E-value 1 × 10−10) was used to search sequences with this model in the Fragaria genome. Finally, the shared sequences obtained by the two search tools served as candidate Aux/IAA gene family members of the strawberries. The Pfam database [52] and the Conserved Domain Database in NCBI [53] were used to identify the conserved domains. All Fragaria Aux/IAA protein’s physical and chemical parameters were calculated with the Expasy-ProtParam tool (https://web.expasy.org/protparam/) (accessed on 19 April 2024).

4.2. Phylogenetic and Duplication Analysis

The protein sequences of Aux/IAA were aligned using MEGA 11 (https://www.megasoftware.net/) (accessed on 29 April 2024) [55]. Evolutionary trees were constructed using the maximum-likelihood method with the JTT + G model (Jones–Taylor–Thornton model with a gamma distribution for among-site rate variation, bootstrap test replicates = 1000 times). Tbtools-II (v2.110) was used to predict segmental duplications and tandem duplications [56]. The synonymous (Ks) and non-synonymous (Ka) substitution rates for gene pairs were calculated by KaKs_Calculator v2.0 [57].

4.3. Analysis of Motif and Cis-Regulatory Elements

The identification of motifs was achieved using MEME Suite [85]. The analysis of cis-regulatory elements was conducted using the 2000 bp upstream of Aux/IAA genes using the PlantCARE website (http://bioinformatics.psb.ugent.be/webtools/Plantcare/html/) (accessed on 9 May 2024), and the subsequent results were illustrated using Tbtools-II [56].

4.4. Analysis of Tissue Expression Patterns in F. vesca

We obtained the expression data in diploid strawberry tissues based on an annotation file (“Expression patterns of all the genes in v4.0.a2 across diverse tissue types”) of the F. vesca v4.0.a2 genome [58]. For anther and carpel tissues, 7, 8, 9, 10, 11, and 12 represent the developmental stages of F. vesca flower. For the other tissues (including style, ovule, embryo, ghost, seed, wall, cortex, and pith), 1, 2, 3, 4, and 5 represent the developmental stages of F. vesca fruit.

4.5. PPI Network Prediction

The PPI network was established based on the STRING website (http://cn.string-db.org) (accessed on 7 August 2024). Then, a BLASTP search of the F. vesca protein database [58] was performed, with the aim of finding the hub proteins of homologous A. thaliana, as predicted using the STRING website.

5. Conclusions

A total of 287 Aux/IAAs were identified in eight strawberry species in this work. Analyses of the sequence structures (including motifs, conserved domains, and promoter) revealed the characterization of Aux/IAAs. In addition, evolutionary trees and collinearity analyses revealed that segmental duplications drove Aux/IAA evolution in Fragaria species, which was followed by purifying selection. Moreover, the tissue expression profiles of Aux/IAAs showed that most genes potentially participated in the development of seeds and fruits in strawberries. In short, this study not only provides a foundation for the functions of Aux/IAAs but also offers proof for the evolution of strawberries.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/plants13202940/s1. Figure S1: Motif analysis of Aux/IAA protein family in F. chiloensis; Figure S2: Motif analysis in Aux/IAA proteins of six diploid strawberry species; Figure S3: The results of Ka, Ks, and Ka/Ks values of gene pairs in six Fragaria species; Figure S4: Ka/Ks > 1 of gene pairs in eight strawberry species; Figure S5: Promoter cis-acting elements predicted in Aux/IAAs from F. vesca; Table S1: Basic information of AuxIAA genes in eight strawberry species; Table S2: Physical and chemical coefficients of Fragaria AuxIAAs; Table S3: Gene pairs exhibiting Ka/Ks > 1 in eight strawberry species.

Author Contributions

Conceptualization, X.J. and H.Y.; methodology, X.J.; software, X.J.; validation, X.J., Q.Z. and H.Y.; formal analysis, X.J.; investigation, X.J. and Q.Z.; resources, Q.Z.; data curation, X.J.; writing—original draft preparation, X.J.; writing—review and editing, X.J., Q.Z. and H.Y.; visualization, X.J.; supervision, H.Y.; project administration, Q.Z. and H.Y.; funding acquisition, Q.Z. and H.Y. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Natural Science Foundation of China (grants 62131004, 62302065, and 62450002), the National Key R&D Program of China (grant 2022ZD0117700), the Zhejiang Provincial Natural Science Foundation of China (grant LD24F020004), and the Municipal Government of Quzhou (grant 2023D036).

Data Availability Statement

The data presented in this study are available in the article and Supplementary Materials.

Acknowledgments

The authors are grateful to Pinyu Zhu for his help and support.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Motif analysis of the Aux/IAAs in F. × ananassa. All schemes follow the same formatting. (A) Conserved motifs of FaIAA proteins; (B) sequence logos of six conserved motifs in F. × ananassa.
Figure 1. Motif analysis of the Aux/IAAs in F. × ananassa. All schemes follow the same formatting. (A) Conserved motifs of FaIAA proteins; (B) sequence logos of six conserved motifs in F. × ananassa.
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Figure 2. Unrooted maximum-likelihood tree analysis of Aux/IAAs. Aux/IAAs were classified into 12 clades (A–L). As shown in the legend at bottom right, bootstrap values are represented by different sizes of bubbles.
Figure 2. Unrooted maximum-likelihood tree analysis of Aux/IAAs. Aux/IAAs were classified into 12 clades (A–L). As shown in the legend at bottom right, bootstrap values are represented by different sizes of bubbles.
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Figure 3. Synteny and KaKs analysis of Aux/IAA genes in strawberries. (A) Gene duplication types of Aux/IAAs. (B) Chromosomal collinearity relationships between F. vesca and F. × ananassa. F. × ananassa and F. vesca chromosomal regions are colored salmon and violet, respectively, and different-colored lines represent orthologous or paralogous gene pairs. (C) The paralogous Aux/IAA gene pairs in F. × ananassa, and orthologous Aux/IAA gene pairs between F. × ananassa and seven Fragaria species (including F. chiloensis, F. iinumae, F. mandshurica, F. nilgerrensis, F. nipponica, F. vesca, and F. viridis) were measured for Ka, Ks, and Ka/Ks. (D) Ka, Ks, and Ka/Ks values of paralogous Aux/IAA gene pairs in F. vesca and orthologous gene pairs between F. vesca and six Fragaria species (F. chiloensis, F. iinumae, F. mandshurica, F. nilgerrensis, F. nipponica, and F. viridis).
Figure 3. Synteny and KaKs analysis of Aux/IAA genes in strawberries. (A) Gene duplication types of Aux/IAAs. (B) Chromosomal collinearity relationships between F. vesca and F. × ananassa. F. × ananassa and F. vesca chromosomal regions are colored salmon and violet, respectively, and different-colored lines represent orthologous or paralogous gene pairs. (C) The paralogous Aux/IAA gene pairs in F. × ananassa, and orthologous Aux/IAA gene pairs between F. × ananassa and seven Fragaria species (including F. chiloensis, F. iinumae, F. mandshurica, F. nilgerrensis, F. nipponica, F. vesca, and F. viridis) were measured for Ka, Ks, and Ka/Ks. (D) Ka, Ks, and Ka/Ks values of paralogous Aux/IAA gene pairs in F. vesca and orthologous gene pairs between F. vesca and six Fragaria species (F. chiloensis, F. iinumae, F. mandshurica, F. nilgerrensis, F. nipponica, and F. viridis).
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Figure 4. Promoter cis-acting elements predicted in the Aux/IAA family of Fragaria. As shown in the upper-left corner of the figure, four types of cis-acting elements were found: hormone response cis-acting elements (ABA, auxin, GA, MeJA, and SA), stress response cis-acting elements (anaerobic induction, anoxic-specific, defense and stress, drought, low temperature, and wound), tissue development-related cis-acting elements (endosperm, meristem, palisade mesophyll cells, root, and seed), and light-responsive cis-acting elements. Bubbles with various sizes and colors indicate the number of different types of cis-acting elements.
Figure 4. Promoter cis-acting elements predicted in the Aux/IAA family of Fragaria. As shown in the upper-left corner of the figure, four types of cis-acting elements were found: hormone response cis-acting elements (ABA, auxin, GA, MeJA, and SA), stress response cis-acting elements (anaerobic induction, anoxic-specific, defense and stress, drought, low temperature, and wound), tissue development-related cis-acting elements (endosperm, meristem, palisade mesophyll cells, root, and seed), and light-responsive cis-acting elements. Bubbles with various sizes and colors indicate the number of different types of cis-acting elements.
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Figure 5. FveIAA gene tree and expression profiles of Aux/IAAs in F. vesca. SAM, FM, and REM represent the shoot apical meristem, flower meristem, and receptacle meristem, respectively.
Figure 5. FveIAA gene tree and expression profiles of Aux/IAAs in F. vesca. SAM, FM, and REM represent the shoot apical meristem, flower meristem, and receptacle meristem, respectively.
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Figure 6. Five PPI networks and tissue expression patterns of Aux/IAAs and related hub genes. (A) PPI networks and tissue-specific expression profile of IAA15 and related hub genes; (B) expression profile of IAA16 and related hub genes from the PPI network; (C) IAA17-related hub genes were selected from the PPI network and their expression levels were analyzed; (D) PPI network and gene expression patterns analysis of IAA29 and related hub genes; (E) PPI network and expression profile of IAA31 and hub genes.
Figure 6. Five PPI networks and tissue expression patterns of Aux/IAAs and related hub genes. (A) PPI networks and tissue-specific expression profile of IAA15 and related hub genes; (B) expression profile of IAA16 and related hub genes from the PPI network; (C) IAA17-related hub genes were selected from the PPI network and their expression levels were analyzed; (D) PPI network and gene expression patterns analysis of IAA29 and related hub genes; (E) PPI network and expression profile of IAA31 and hub genes.
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Table 1. The completeness of Aux/IAAs in Arabidopsis and Fragaria.
Table 1. The completeness of Aux/IAAs in Arabidopsis and Fragaria.
SpeciesProtein No.Truncated ProteinsComplete Proteins
Arabidopsis thaliana2911 (38%)18 (62%)
Fragaria x ananassa8236 (43.9%)46 (56.1%)
Fragaria chiloensis7533 (44%)42 (56%)
Fragaria iinumae187 (38.9%)11 (61.1%)
Fragaria mandshurica229 (40.9%)13 (59.1%)
Fragaria nilgerrensis208 (40%)12 (60%)
Fragaria nipponica279 (33.3%)18 (66.7%)
Fragaria vesca228 (36.4%)14 (63.6%)
Fragaria viridis217 (33.3%)14 (66.7%)
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Jing, X.; Zou, Q.; Yang, H. Genome-Wide Identification and Characterization of the Aux/IAA Gene Family in Strawberry Species. Plants 2024, 13, 2940. https://doi.org/10.3390/plants13202940

AMA Style

Jing X, Zou Q, Yang H. Genome-Wide Identification and Characterization of the Aux/IAA Gene Family in Strawberry Species. Plants. 2024; 13(20):2940. https://doi.org/10.3390/plants13202940

Chicago/Turabian Style

Jing, Xiaotong, Quan Zou, and Hui Yang. 2024. "Genome-Wide Identification and Characterization of the Aux/IAA Gene Family in Strawberry Species" Plants 13, no. 20: 2940. https://doi.org/10.3390/plants13202940

APA Style

Jing, X., Zou, Q., & Yang, H. (2024). Genome-Wide Identification and Characterization of the Aux/IAA Gene Family in Strawberry Species. Plants, 13(20), 2940. https://doi.org/10.3390/plants13202940

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