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Genome-wide identification and expression pattern analysis of the Aux/IAA (auxin/indole-3-acetic acid) gene family in alfalfa (Medicago sativa) and the potential functions under drought stress

BMC Genomics volume 25, Article number: 382 (2024) Cite this article

Abstract

Background

Auxin/induced-3-acetic acid (Aux/IAA) is an important plant hormone that affects plant growth and resistance to abiotic stresses. Drought stress is a vital factor in reducing plant biomass yield and production quality. Alfalfa (Medicago sativa L.) is the most widely planted leguminous forage and one of the most economically valuable crops in the world. Aux/IAA is one of the early responsive gene families of auxin, playing a crucial role in response to drought stress. However, the characteristics of the Aux/IAA gene family in alfalfa and its potential function in response to drought stress are still unknown.

Result

A total of 41 Aux/IAA gene members were identified in alfalfa genome. The physicochemical, peptide structure, secondary and tertiary structure analysis of proteins encoded by these genes revealed functional diversity of the MsIAA gene. A phylogenetic analysis classified the MsIAA genes into I-X classes in two subgroups. And according to the gene domain structure, these genes were classified into typical MsIAA and atypical MsIAA. Gene structure analysis showed that the MsIAA genes contained 1–4 related motifs, and except for the third chromosome without MsIAAs, they were all located on 7 chromosomes. The gene duplication analysis revealed that segmental duplication and tandem duplication greatly affected the amplification of the MsIAA genes. Analysis of the Ka/Ks ratio of duplicated MsAux/IAA genes suggested purification selection pressure was high and functional differences were limited. In addition, identification and classification of promoter cis-elements elucidated that MsIAA genes contained numerous elements associated to phytohormone response and abiotic stress response. The prediction protein–protein interaction network showed that there was a complex interaction between the MsAux/IAA genes. Gene expression profiles were tissue-specific, and MsAux/IAA had a broad response to both common abiotic stress (ABA, salt, drought and cold) and heavy metal stress (Al and Pb). Furthermore, the expression patterns analysis of 41 Aux/IAA genes by the quantitative reverse transcription polymerase chain reaction (qRT-PCR) showed that Aux/IAA genes can act as positive or negative factors to regulate the drought resistance in alfalfa.

Conclusion

This study provides useful information for the alfalfa auxin signaling gene families and candidate evidence for further investigation on the role of Aux/IAA under drought stress. Future studies could further elucidate the functional mechanism of the MsIAA genes response to drought stress.

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Introduction

Indole-3-acetic acid (IAA) is the main natural form of auxin in higher plants and plays an important role in biosynthesis, metabolism, transport, location, signal transduction, and crosstalk with other phytohormones [1, 2]. Auxin signal is the transmission of signal in an organism, particularly through cells. The auxin signaling pathway involves several stages such as signal recognition, expression of downstream auxin related genes, and physiological responses in plants, suggesting it is not singular. Plants can rapidly sense and respond to changes in auxin levels involving several major classes of auxin-responsive genes, including the Aux/IAA and ARF families [3]. Aux/IAA proteins mediate the auxin responses through interaction with ARF transcription factors [4]. Previous studies showed that Aux/IAA family genes were necessary for the regulation of various plant development processes. Detailed explanations of Aux/IAA molecular mechanisms have been obtained in plant model systems, particularly in Arabidopsis thaliana and rice (Oryza sativa) [5]. Once auxin concentration is elevated and the auxin receptor receives an auxin signal, Aux/IAA proteins will degraded by the 26S proteasome, thereby activating ARF transcriptional activity and initiating the downstream auxin response gene-cascade expression [6,7,8,9]. Therefore, precise regulation of plant growth and development processes by Aux/IAA is achieved by finely transforming the spatiotemporal dynamics of auxin levels into gene reprogramming signals. Aux/IAA encoded proteins that are localized to the nucleus and has a short half-life [10], so Aux/IAA proteins are short-lived transcriptional regulatory factor composed of four highly conserved domains, encoded by the early auxin response gene family [11]. Domain I acts as a transcriptional repressor of auxin-regulated genes [12]; Domain II is a TIR1/AFB with a conserved nitrogen removal sequence that can recognize the sequence GWPPV and regulate protein stability of Aux/IAA, interacting with the F-box protein TIR1 to rapidly degrade Aux/IAA protein [13]; Domain III contains the βαα region, and the conserved domain IV represents the acidic region [14], and domains III and IV are capable of homologous or heterodimerization with other Aux/IAA or ARF to regulate the expression of downstream auxin-responsive genes [15].

Aux/IAA is involved in the regulation of a variety of cellular and developmental processes including embryogenesis, axis formation, lateral root initiation, leaf expansion, vascular elongation, tropism, inflorescence development and fruit ripening, apical dominance, and defense response to stress [16,17,18]. Obviously, many typical phenotypes controlled by auxin signaling will be regulated by Aux/IAA [19, 20]. For example, Audran-Delalande et. al [21]. found that inhibition of SlIAA9 expression led to parthenogenesis, while silencing of SlIAA27 or SlIAA17 altered in fruit size, shape and quality [22, 23]. In addition, Aux/IAA can regulate plant growth and development by affecting other hormones. For instance, auxin signaling is involved in fruit ripening regulation through the interaction with ethylene signaling, while the Aux/IAA family genes are implicated in the interaction between the two [24]. There are obvious differences in auxin content and ethylene release during peach (Prunus persica) ripening, accompanied by similar changes in the expression of the key rate-limiting enzyme gene ACS1 and the auxin synthesis gene YUCCA11, suggesting that auxin may be a prerequisite factor for ethylene release during peach ripening [25, 26]. Aux/IAA regulates anther dehiscence, affects pollination and floral organ development by altering the change of jasmonic acid (JA) content [27]. The ethylene response factor ERF3 (ethylene response factor) is upregulated in taiaa21, and ttarf25 is downregulated in tetraploid wheat (Triticum aestivum). Further analysis found that the TaARF25 promotes the transcription of ERF3, and TaERF3 mutation caused reduced grain size and grain weight in tetraploid wheat, indicating that TaIAA21 negatively regulated wheat grain size and grain weight through ARF25-ERFs pattern, thus improving wheat yield [28]. And AtIAA7 inhibited flowering time by regulating gibberellin (GA) related-genes GA20ox1 and GA20ox22 [3]. The AtARF6-AtIAA8 module regulated floral organ development by affecting JA levels [29]. It is well known that the crucial role of auxin in plants goes far beyond these, and it also plays pivotal regulatory parts in resisting biotic and abiotic stresses, as well as the Aux/IAA proteins are a large family of auxin co-receptors and transcriptional repressors that have a central role in auxin signaling [30], so the versatility of the auxin implies the broad functionality of Aux/IAA.

A detailed understanding of abiotic stress tolerance in plants is essential to provide food security in the face of increasingly harsh climatic conditions [31]. The auxin core gene Aux/IAA plays a huge role in plant biotic stress. Among numerous environmental stresses, losses due to drought stress are estimated to be at 30% globally [32], and the yield loss of crops caused by drought exceeds the total loss caused by other non biological factors [33]. Under drought conditions, auxin content is reduced, and transcriptional expression of genes in auxin biosynthesis as well as some auxin responsive genes, including Aux/IAA genes, are also affected by drought treatment [34]. There are many examples that the Aux/IAA genes involve in regulating plant response to drought stress. Under drought and salt stress, proline and chlorophyll content were significantly reduced in rice osiaa20 lines, and the abscisic acid (ABA) response gene OsRab21 was down-regulated in osiaa20 and up-regulated in OsIAA20 over-expression lines, indicating that OsIAA20 played an important role in plant drought and salt stress response through ABA signal transduction pathway [35]. IAA5, IAA6, and IAA19 in the Aux/IAA gene family were maintained glucosinolates (GLS) levels when plants were exposed to drought, while AtIAA5/6/19 deficiency caused decreased GLS levels and decreased drought tolerance, indicating that Aux/IAA genes regulate drought tolerance in A. thaliana by regulation of GLS levels [31]. Alfalfa (Medicago sativa L.) is the most widely planted perennial legume forage, with high biomass and crude protein content, rich in digestible nutrients and mineral elements, and is known as the "king of forage" [36, 37]. It is widely planted in North America, Asia, and other continents, and is also one of the most economically valuable crops in the world. In the United States, it is second only to wheat, maize (Zea mays) and soybeans (Glycine max) are the fourth largest cultivated crops [38, 39]. Drought, as one of the main environmental factors affecting alfalfa productivity, is an important factor limiting its cultivation and promotion [40]. Aux/IAA gene family is also known to play an important regulatory role in protecting plants against drought stress. Therefore, identification of Aux/IAA genes in tetraploid cultivated alfalfa at the genome-wide level and doing related analysis, as well as analysis of expression patterns under drought stress can provide more comprehensive data for finding a new and broader layer of auxin signaling regulatory network. Sequence characteristics, genomic organization, cis-regulatory elements, and evolutionary duplication, as well as the transcriptional expression patterns of Aux/IAA homologous genes under different tissue/developmental stages and abiotic stress conditions were analyzed in the study. Furthermore, we further analyzed the expression levels of 41 MsIAA genes under drought stress. These data help to further elucidate the accurate biological functions of the Aux/IAA genes in alfalfa and provide basic data for drought resistance breeding.

Results

Identification of MsIAA genes family and physicochemical properties analysis of the MsIAA proteins

A total of 41 MsIAA sequences were identified in the alfalfa genome and named MsIAA1—MsIAA41 according to the order of their occurrence in the genome (Table S1). Analyze the physicochemical properties of the encoded proteins of 41 MsIAA genes, including number of amino acids, molecular weight, theoretical isoelectric point, instability index, aliphatic index and grand average of hydropathicity (Table S2). Significant differences exist in the physicochemical properties of the 41 MsIAA proteins, among which, the numbers of amino acids ranged from 59 (MsIAA11) to 1129 (MsIAA14); The molecular weight ranged from 6802.78 Da (MsIAA11) to 126,377.93 Da (MsIAA14). The theoretical isoelectric point were between 4.33 (MsIAA11) and 9.42 (MsIAA2), and the theoretical isoelectric point of 28 MsIAA proteins were less than 7.5, which belonged to acidic proteins and the remaining 13 belonged to alkaline proteins. The instability index of 41 MsIAA family proteins varies between 16.24 (MsIAA29) and 72.96 (MsIAA34), and in which, the instability index of most proteins (31) are more than 40, indicating that they are unstable proteins and the remaining 10 being stable. Except for MsIAA2 with an aliphatic index of 121.26, the aliphatic index of the remaining 40 MsIAA proteins range from 61.23 (MsIAA25) to 95.42 (MsIAA29), all less than 100 and are hydrophilic proteins. With the help of the protein grand average of hydropathicity, 22 MsIAA proteins, whose grand average of hydropathicity is less than -0.5, are hydrophilic proteins, and the other 19 MsIAA proteins are between -0.5 and 0.5, belonging to amphiphilic proteins.

Subcellular localization, signal peptides, leading peptides and transmembrane structural analysis of MsIAA proteins

The subcellular localization prediction of the 41 MsIAA proteins revealed that most of the MsIAA proteins (MsIAA1, 3, 5, 7, 13–23, 25–28, 30, 31, 33–35, 38) were located in the nucleus, while the other 9 proteins (MsIAA4, 6, 8, 10, 12, 29, 36, 40, 41) were located in the chloroplast, 6 proteins (2, 9, 11, 32, 37, 39) were located in the cytoplasm, and only the MsIAA24 was located in the extracellular matrix (Table S3). Addition to, the results of signal peptide analysis showed that all MsIAA proteins have no peptides and signal peptides. However, analysis of transmembrane structure showed that four proteins (MsIAA4, MsIAA6, MsIAA8, and MsIAA29) had typical transmembrane structures, with MsIAA29 having two typical transmembrane structures and the other three having only one segment each. Interestingly, we found that all four genes are localized in the chloroplast, indicating that these four genes can act across the chloroplast membrane both inside and outside (Fig. 1). On the other hand, the 25 MsIAA genes located in the nucleus serve as transcription factors without any signaling peptides, leading peptides, or transmembrane structures, so they do not secrete or transport and only function within the nucleus.

Fig. 1
figure 1

The transmembrane structure analysis of MsAux/IAA proteins. A: MsIAA4; B: MsIAA6; C: MsIAA8; D: MsIAA29

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Analysis of the secondary and tertiary structure of the MsIAA proteins

Analysis of the secondary structure of MsIAA protein, the results found that, except for the extended strand of MsIAA11 protein, alpha helix of MsIAA12, 33, the secondary structures of remaining 37 MsIAA proteins, the largest proportion was random coil, as well as the composition percentage of 34 proteins was shown as random coil > alpha helix > extended strand > beta turn (Table S3). At the same time, we also constructed a tertiary structure of MsIAA protein, which showed significant differences due to different percentages of secondary structure composition. Total of 41 MsIAA proteins each have specific spatial structural types, and no two completely consistent spatial configurations have been observed (Fig. 2). Structure determines the function, so we speculate that its unique spatial configuration may be related to its unique function.

Fig. 2
figure 2

Analysis of tertiary structures of 41 MsAux/IAA proteins

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Phylogenetic analysis of IAA proteins from A. thaliana, G. max, Medicago truncatula and M. sativa

Phylogenetic relationship analysis was conducted on 158 IAA family members from 4 species (25 for M. truncatula, 63 for soybeans, 29 for Arabidopsis, and 41 for alfalfa). The results showed that the IAA family members were clearly divided into two groups: A and B, and further subdivided into 10 classes. Class I-V belongs to group A and consists of 91 members (15 for Arabidopsis, 41 for soybeans, 15 for M. truncatula, and 21 for alfalfa); Class VI-X belongs to group B and consists of 72 members (14 Arabidopsis, 22 soybeans, 10 M. truncatula, and 20 M. sativa). Except for Class IX, which does not include members of M. sativa, the remaining classes all contain IAA members of the four species (Fig. 3).

Fig. 3
figure 3

Phylogenetic analysis of Aux/IAA proteins from Arabidopsis thaliana, Glycine max, Medicago truncatula and Medicago sativa

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Analysis of conserved motif and gene structure of the MsIAA family members

MEME tool was used to identify the conserved motifs of the MsIAA proteins, and we obtained visualizations of the 10 conserved motifs. It was showed that aspartic acid residue (D), glycine residue (G), leucine residue (L), serine residue (S), valine residue (V), methionine residue (M), histidine residue (H), alanine residue (A), phenylalanine residue (F), proline residue (P), arginine residue (R), tryptophan residue (W), lysine residue (K), asparagine residue (N), and tyrosine residue (Y) and cysteine residue (C) were extremely conserved (Fig. 4). Further analysis of the distribution of 10 motifs on 41 MsIAA proteins revealed that motif 1, motif 3, motif 7, and motif 9 were highly conserved motifs in the MsIAA gene family, appeared in most MsIAA gene members, indicating that they were also the four most important motifs in the MsIAA gene family (Fig. 5A). On the other hand, we also found that motif 2 was a mainly highly conserved motif in the MsIAA subfamily Class II, III, IV, and V. In addition, motif 4, motif 5, motif 6, motif 8, and motif 10 were mainly highly conserved in the MsIAA subfamily Class VIII.

Fig. 4
figure 4

Structural analysis of conserved Motif of MsAux/IAA proteins

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Fig. 5
figure 5

Analysis of conserved motifs and domains and gene structure of MsAux/IAA proteins. A: Gene conservative motif analysis; B: Protein conservative domain analysis; C: Gene structure analysis

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Analysis of the conserved domains of the MsIAA protein sequence revealed that all MsIAA contain an auxin Aux_IAA structural domain. Interestingly, 10 MsIAAs (MsIAA1, 13, 14, 19, 21, 22, 23, 27, 31, 38) all contain Aux_IAA, B3, and Auxin_resp domain, and they all belong to Class VIII. Combined with the above analysis, we speculated that motif 4, motif 5, motif 6, motif 8 and motif 10 mainly encoded the B3 and Auxin_resp domains (Fig. 5B). To determine the structural differences of the MsIAA genes in order to reveal their function, regulation and evolution, the MsIAA family numbers were analyzed for gene structure. The results showed that the number of CDS and TR regions in MsIAA genes all varied from 2 to 15, and all MsIAA genes contained CDS sequence, while 32 MsIAAs contained UTR regions and the remaining 9 have no UTR regions. Among the 41 MsIAA genes, MsIAA21 has the most CDS (n = 15), followed by MsIAA1 and MsIAA38 with 14 CDS, while MsIAA9 and MsIAA29 only contain 2 CDS. Through phylogenetic analysis, it was found that the CDS distributions of Class VIII members in Group B of the MsIAA family were the highest (Fig. 5C).

Chromosomal localization and homology analysis of MsIAA gene in alfalfa

To further analyse the collateral homologous gene pair relationship between the MsIAA genes in alfalfa, we identified and mapped the chromosomal positions of all identified MsIAA numbers (Fig. 6). The results found that 41 MsIAA genes were unevenly distributed on seven chromosomes (Chr) in alfalfa, and there was no distribution of MsIAA family members on Chr3. And the MsIAA genes were the most distributed on Chr1 with 12 members, followed by Chr4, Chr7, Chr2, Chr5 and Chr8, with 7, 6, 5, 5 and 5 members, respectively, while only one member on Chr6. We also performed collinearity analysis to clarify the gene duplication events. The results showed that 5 pairs of segmental duplications and 2 groups of tandem duplications (MsIAA27/MsIAA28 and MsIAA6/MsIAA8) were identified in the MsIAA gene (Fig. 7). And 43 pairs of homologous Aux/IAAs were identified between M. sativa and A. thaliana (Fig. 8A), and 50 pairs of homologous Aux/IAAs were identified between M. sativa and M. truncatula (Fig. 8B), while 127 pairs of orthologous Aux/IAAs were identified between M. sativa and soybean (Fig. 8C). Five MsIAAs (MsIAA2, MsIAA5, MsIAA6, MsIAA8 and MsIAA32) had one homologous gene in A. thaliana, and fore MsIAAs (MsIAA4, MsIAA6, MsIAA8 and MsIAA33) had one homologous gene in soybean. To determine whether there was selection pressure acting on the IAA family genes, we further calculated the Ka/Ks values for MsIAA numbers. The results found that the Ka/Ks values of 5 pairs of segmental duplications and 2 groups of tandem duplications were less than 1 respectively, indicating they exert purification of selection effects (x S4).

Fig. 6
figure 6

Distribution and location of MsAux/IAA gene on alfalfa chromosomes. Chr 1 to Chr 8 represent the linkage group of "Zhongmu NO.1" alfalfa. Each black line indicates the location of the Aux/IAA gene. The red line represents a tandem duplicated of the Aux/IAA gene

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Fig. 7
figure 7

Synteny analysis of the MsAux/IAA gene in the genomes of the M. sativa. The gray line represents all the synteny blocks in the alfalfa genome. The green and red lines represent gene pairs and tandem repeat genes in the Aux/IAA gene, respectively. The yellow heat map and the red broken line map represent the gene density and expression level of the MsIAA gene, respectively

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Fig. 8
figure 8

Collinearity analysis between different genomes include Medicago sativa, Arabidopsis thaliana, Medicago truncatula and Glycine max. A: Collinearity analysis between M. sativa and A. thaliana; B: Collinearity analysis between M. sativa and M. truncatula; C: Collinearity analysis between M. sativa and G. max

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Analysis of Cis-regulatory elements of the MsIAA gene family

The cis-regulatory element regulated the expression of genes involved in the stress response by binding to transcription factors, which was located in a specific DNA sequence upstream of the coding sequence of the gene. In this study, ABRE had the most cis-regulatory elements, with 74, followed by CGTCA-motif (n = 43), MBS (n = 32), TGA-element (n = 24), TCA-element (n = 23), TC-rich repeats (n = 17), LTR (n = 12), and AuxRR core (n = 2) (Fig. 9). Interestingly, we found that the 2000 bp upstream sequence of the MsIAA41 gene and MsIAA11 only contained two and four TC-rich repeats cis-regulatory elements, respectively. Except for TC-rich repeats, there are no other cis-regulatory elements at MsIAA41 and MsIAA11. And the MsIAA24 gene only contained a MBS elements.

Fig. 9
figure 9

Analysis of Cis regulatory elements in the upstream promoter of MsAux/IAA gene

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Protein–protein interaction (PPI) network analysis

To effectively identify and visualize potential interactions, we analyzed PPIs for 41 MsIAA proteins that can explain the hub gene of this interaction (Fig. 10, Table S5). It was found that 7 MsIAA were repeatedly annotated, such as MsIAA5/34 (3880.B7FMP7), MsIAA6/8 (3880.A0A072VMX0), MsIAA7/9 (3880.G7I486), MsIAA33/35 (3880.I3SRI4), IAA20/37 (3880.A0A072UBZ5), MsIAA22/30 (3880.G7LIT1), MsIAA27/28 (3880.G7KFN6), respectively. And within the hub gene network, MsIAA38 (3880.A0A072TM91), MsIAA21 (3880.A0A072TTL5) and MsIAA31 (3880.A0A072U1C1) had the highest number of interactions, suggesting that these genes played a crucial role in regulating the function of the IAA gene family. In addition, three of the hub genes, MsIAA12, MsIAA32 and MsIAA41, had yet to be annotated and required further investigation.

Fig. 10
figure 10

The protein–protein interaction (PPI) network of MsAux/IAA genes and related hub genes

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Analysis of the expression patterns of MsIAA gene family numbers under different treatment

Aux/IAAs, as repressors that respond to auxin and regulate its gene expression, play an important role in auxin signal transduction, thereby regulating plant growth and development, such as tissue differentiation and response to abiotic stress. Therefore, this study utilized the alfalfa database to obtain 41 expression patterns of MsIAAs, mainly divided into three types, including specific expression in different tissues (young leaves, natural leaves, sensory leaves, flowers, leaf, root, post elongating stem, node and elongating stem), common abiotic stress (ABA, salt, dry and cold), and heavy metal stress (Al and Pb) (Fig. 11). The tissue-specific expression of MsIAAs were related to its specific function in specific tissues, and the expression patterns of 41 MsIAAs in 8 different tissues were divided into 4 types (Fig. 11A). Under ABA, salt, drought, and cold stress, the expression of MsIAAs were very diverse. Our analysis showed that the expression of MsIAA under different stress conditions was not only related to the type of stress, but also limited by the duration of stress, which is closely related to MsIAAs being short-lived proteins (Fig. 11B). In addition, we also found that under heavy metal stress, except for the type and duration of stress, the expression of MsIAAs were also related to stress concentration, indicating that MsIAA have very complex regulatory mechanisms under abiotic stress (Fig. 11C).

Fig. 11
figure 11

Analysis of the expression pattern of the MsAux/IAA gene family. A. Tissue specific expression pattern analysis; B. Analysis of expression patterns under salt, drought, and cold stresses; C. Analysis of expression patterns under Al and Pb stress

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To reveal the possible function of MsAux/IAA gene family numbers in response to plant drought stress, we analyzed the expression pattern of the 41 MsIAA genes identified in root under the drought stress (Fig. 12). The result found that MsIAA34 showed no or very low expression and was not further analyzed, and MsIAA30 was no expression at 48 h. Among others, 13 MsIAA genes (MsIAA1/4/6/7/9/10/21/23/25/26/32/33/35) were significantly upregulated at 6 h, and the expression level decreased in the following time including 12, 24 and 48 h. Interestingly, MsIAA30 also belonged to this type but lack of 48 h. And 9 MsIAA genes (MsIAA3/5/11/14/17/36/37/39/40) were upregulated under drought stress at 6, 12, 24 and 48 h when compared with CK. On the contrary, 9 MsIAA genes (MsIAA8/15/16/18/19/20/24/31/38) were downregulated under drought stress at 6, 12, 24 and 48 h when compared with the CK. The expression patterns of the remaining eight genes were period-specific, with MsIAA2 upregulated at 12 and 48 h, MsIAA12 and MsIAA22 upregulated at 6 and 12 h, MsIAA13 only downregulated at 12 h, MsIAA27 and MsIAA28 significantly low expression at 24 h, MsIAA29 only significantly high expression at 12 h, as well as MsIAA41 upregulated at 6 and 48 h and downregulated at 12 and 24 h.

Fig. 12
figure 12

The expression pattern analysis of Aux/IAA genes under drought stress treatment for 0, 6, 12, 24 h and 48 h using qRT-PCR. Data are presented as the mean ± standard deviation

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Discussion

Significant progress has been made in regulating the function of IAA genes in auxin signaling in understanding auxin sensing and signaling [30]. However, much remains unknown regarding the processes of Aux/IAA involvement in the auxin signaling pathway. At present, research on Aux/IAA in the auxin signaling pathway mainly focuses on various aspects of plant growth and development, but little attention has been paid to the role of Aux/IAA in the response to auxin mediated environmental interactions [41]. In this study, we performed the genome-wide identification and related analysis of Aux/IAA genes in alfalfa using the whole genome of tetraploid cultivated alfalfa, and analyzed the expression pattern of 41 MsIAA genes under 15% PEG simulation drought by qRT-PCR, which is important to reveal the possible function of Aux/IAA in plants in response to drought stress.

Various protein physicochemical, peptide structure, secondary and tertiary structure analysis reveal functional diversity of the MsIAA proteins

With the increasing popularity of whole genome sequencing technology, the Aux/IAA genes of several species, including most crops, have been identified. The number of Aux/IAA genes varied significantly between different species, such as only one in Marchantia polymorpha [42], 28 Aux/IAA genes in Arabidopsis [43], 25 in M. truncatula [9], 63 in soybean [44], and 119 in Brassica napus [45]. In this study, 41 IAA genes were identified in alfalfa with higher numbers than some leguminous plants including M. truncatula, which may be related to the fact that alfalfa is the main tetraploid cultivated specie, and also indicated that alfalfa Aux/IAA gene was not overextended in the evolution. To further illustrate the function of MsIAA genes, physicochemical properties analysis of encoding protein were performed, and it was found that 41 MsIAA proteins had various physicochemical characteristics. The number of amino acids of most of MsAux/IAA (26) were less than 7, the average value was 6.66; the coefficient of instability of MsAux/IAA (26) was greater than 40, the average value was 46.19, mostly unstable proteins; the average value of aliphatic amino acid index was 74.84, the average value of protein hydrophobicity coefficient was -0.45, most of which were hydrophilic proteins. It can be seen that although there were obvious differences in the specific values, the performance classification of physicochemical properties was relatively single, reflecting the coexistence of functional diversity and unity of Aux/IAA genes, suggesting that different MsIAA genes might function in different microenvironments. Subcellular localization indicated that most of the proteins were localized to the nucleus (25), indicating that the nuclear localization signal in Aux/IAA proteins was conserved [46]. And they did not have any signal peptide, guide peptide, or transmembrane structure, indicating that the Aux/IAA gene in alfalfa did not act as a transcriptional repressor for cell secretion or transport, but only acted in the nucleus. Among them, three genes (MsIAA4/9/8) located on chloroplast had a typical transmembrane domain, and MsIAA29 had two typical transmembrane domains, indicating that these four genes on chloroplast not only functioned in chloroplast, but also secreted and transported, and then played a role in other organelles. These characteristic properties of alfalfa Aux/IAA were similar to the Aux/IAA features in most plants [46], which may be related to the conservatism of Aux/IAA, implying their similar functions.

Gene structure can intuitively determine gene function, therefore, in order to more fully reveal the function of MsIAA, we further analyzed the secondary and tertiary structure of 41 genes. It was found that the secondary structure of the majority of the largest proportion were α-coil and irregular coil. At the same time, we predicted the tertiary structure of all MsIAA genes, the results showed that each gene contained a helix-fold-helix structure, suggesting that the helix-fold-helix structure was the essential spatial structure of the MsIAA gene. The acquisition of physicochemical properties, secondary and tertiary structure can provide clues for further studying the function and interaction of proteins, and it was an indispensable component in the analysis of plant gene families. The wide variability in physicochemical properties among the members of the MsIAA gene family in this study suggested that different MsIAAs proteins may function together and participate in regulating these processes under variable microenvironmental conditions including plant growth and development, as well as different biological and abiotic stresses.

The phylogenetic relationship and domain analysis demonstrates specificity of MsIAA gene function

The elucidation of evolutionary relationships and the prediction of the potential function of genes can be achieved through phylogenetic analysis [47]. Earlier studies showed that the evolutionary relationship of Aux/IAA genes can be divided into five major clades [48]. There were also studies that classified Aux/IAA genes into two major groups [44], A and B similar to Arabidopsis [43] and rice[49], which were further subdivided into 10 subgroups. Analysis of the evolutionary tree constructed in this study made it more reasonable to adopt the second classification method. The evolutionary analysis showed that I-V in class A all contained MsIAA genes, while class IX in group B did not contain MsIAA genes, indicating that the divergence formation of MsIAA transcription repressor family members may be later than the species differentiation in alfalfa.

Wu et. al [48]. had reported that altered protein properties were determined by the absence of domains and relevant scholars have elucidated the function of the domain in Aux/IAA proteins. This study used MEME to identify 10 conserved motifs of MsIAA, the result found that D, G, L, S, V, M, H, A, F, P, R, W, K, N, Y and C residue were extremely conserved, which was similar to the results obtained in most plants [30]. The MsIAA gene domain is the core of the Aux/IAA transcription factor, which can activate the downstream genes by interacting with the promoters of the downstream genes, thereby enabling them to function. Further analysis of the motif distribution revealed that motif1, motif3, motif7, and motif9 were highly conserved in MsIAA proteins, corresponding to domain I-domain IV. These genes were classified into typical Aux/IAA (26), and the remaining 15 members belong to atypical Aux/IAA based on their domain structure, or called atypical members [21], and motif4, 5, 6, 8, and 10 were mainly found in the class VIII. These atypical Aux/IAA genes mainly lack domain I (EAR-motif), suggesting that these members may participate in other biological processes that do not require ARF-mediated auxin regulation [3]. The atypical Aux/IAA proteins played key roles in plant growth and development processes as well as in their adaptation to various environmental conditions, but their specific functions remained unknown [45], which indicated that atypical auxin signaling pathways may be involved in growth and development in M. sativa [50]. These atypical Aux/IAA proteins were more long-lived than typical proteins [51]. It had been shown that this atypical Aux/IAA protein may have special functions in plant auxin response processes and signal transduction [21]. Previous studies had reported that the C-terminus was conserved, and the N termini of Aux/IAA proteins interact with ubiquitin ligase receptors through motif-based interactions [52]. In this study, the C-terminus was composed of motif 1, 2, 3, and 4, and the N-terminus consisted of motif 1, 7 and 9, showing that two domain motif 1 and 3 were conserved in MsIAA, and two domain additional motif 7 and 9 played a major role. In this study, only 21 MsIAAs had four complete domains, implied some variability in the function among the MsIAAs members.

The gene duplication analysis shows that MsAux/IAA genes tend to lose mutations by purifying selection

Gene duplication events determined the diversification and specificity of gene function, which often occurred between members of the same gene family during plant evolution [53, 54]. Previous studies have found that more gene duplication events in plants are segmental tandem duplication, which acts to expand gene families [55]. In this study, only two pairs of tandemly duplicated genes of the MslAA gene family were detected, namely MsIAA27/MsIAA28 and MsIAA6/MsIAA8. And we identified five pairs of segmental duplicated genes, suggesting it played a major role in the amplification of the Aux/IAA gene family in alfalfa. Furthermore, the Ka/Ks ratios were all less than 1, suggesting that MslAAs were under purifying selection, and indicating that M. sativa had undergone local gene duplication and shares an ancient round of gene duplication with other leguminous plants [56], or which indicated that all gene pairs evolved mainly under the influence of purification selection pressure, and the functional differences after fragment replication were limited [44]. These results were consistent with previous study [57], which reported that Aux/IAA genes tend to lose mutations by purifying selection and subsequently adapt to current microenvironmental conditions.

Cis-acting element analysis demonstrates that MsIAA genes regulates the response to abiotic stress in alfalfa

Correlation studies suggested that temporal and spatial changes in gene expression were influenced by promoters, and regulation of gene function was determined by cis-elements within the promoter through interactions with trans-acting factors [58]. Multiple promoter elements associated with abiotic stress and hormones had been identified in the Aux/IAA genes [9]. The analysis of MslAAs promoter found seven major cis-elements that had specific functions, including drought-inducibility (MBS), defense and stress responsiveness (TC-rich repeats), hormone responsive elements (AuxRR-core, ABRE, TGA-element, CGTCA-motif), low-temperature responsiveness (LTR), and it was found that MsIAAs may respond to various stimuli, including auxin, GA, ABA, salicylic acid, JA, drought, salt, heat stress, and low temperature, indicating that these genes were involved in phytohormone signaling and/ or stress response. Many MsIAA genes contained the AuxRE motifs that bind to the downstream ARF in their promoters, which was important for the transcriptional activation in auxin signaling [59]. The presence of these cis-acting elements explained the universality of MsIAAs function as well as the specificity of tissue expression. Some promoter elements were enriched multiple times in the promoter regions of MsIAA genes. Most genes had two or more genetic components, and only three genes each contained one class of cis-elements, MsIAA41 and MsIAA11 gene only contained two and four TC-rich repeats cis-regulatory elements, and MsIAA24 gene only contained a MBS elements. Taken together, multiple cis-regulatory elements exist in the upstream region of the MsAux/IAA genes. These results provide new evidence that support for further studies on the involvement of Aux/IAA genes in plant growth and development processes and in abiotic stress responses.

Many physiological processes including the regulation of signal transduction and gene expression involve protein–protein interactions of plant. Auxin responses were mediated by interaction between Aux/IAA genes and other genes [12]. Thus, studying the interaction of Aux/IAA proteins in M. sativa was significant. In this study, we found that the proteins encoded by MsIAA genes formed a complex regulatory network, among which, three genes (MsIAA38/21/31) were at the core, and three genes (MsIAA12/32/41) acted independently and had no interaction with other MsIAA genes. Moreover, there were seven duplicate genes in alfalfa compared to the M. truncatula, which may depend on the genome size of both species [39, 60].

The expression analysis of MsIAA genes in tissue development, abiotic stress responses, and their potential functions under drought stress

The underlying function of a gene influences its temporal and spatial expression. Several IAA genes showed specific and overlapping expression patterns in different tissues and developmental periods in alfalfa, suggesting that they regulated the initiation and development of different organs [9]. The expression patterns of Aux/IAA genes in turnip (Brassica rapa) and pepper (Piper nigrum) were tissue-dependent [61, 62]. The MsIAA genes also exhibited tissue-specific transcriptional patterns, indicating their important roles in leaf, root, stem or flower development in the study. In addition, we used previous transcriptome data to obtain the expression patterns of 41 MsIAA genes under ABA, salt, mannitol, cold, Al and Pb treatment and showed that different genes respond differently to abiotic stress. The expression patterns generated by the promoter activity and/or the molecular properties of the gene product determine the similarities and differences of the Aux/IAA genes [63]. Overall, the expression of several Aux/IAA genes in different tissues and developmental periods in alfalfa indicated that they participated in the developmental process of specific tissues.

Promoter sequence analysis of MsIAAs revealed the presence of drought-responsive cis-regulatory elements, hence further investigation of its role under drought stress response/signaling was valuable. The quantitative real time polymerase chain reaction (qRT-PCR) is a useful and convenient tool for reflecting the transcript levels of target genes under any condition [64]. More and more evidence suggested that Aux/IAA genes were responsive to drought stress [41]. Therefore, the expression patterns of the 41 AUX/IAA genes using qRT-PCR were analyzed under drought stress. We found that the 41 MsIAA genes were divided into five categories according to their expression patterns, and in the first category, MsIAA34 was not expressed under drought stress. Fourteen MsIAA genes in the second category were upregulated only at 6 h, and these genes responded rapidly to short drought stress, consistent with the characteristics of MsIAA as a short-lived gene [11]. Nine MsIAA genes in the third category were up-regulated, and nine genes in the fourth category were downregulated under drought stress. The expression of eight genes in the fifth category was period-specific under drought stress. Previous studies had found that MtIAA genes showed extensive responses to drought stresses, and many SbIAA genes of Sorghum bicolor had been found down-regulated under drought conditions [9, 65]. Similarly, many Aux/IAA genes in other plant species were induced by drought treatment [34]. Aux/IAA genes were believed to play a negative regulatory role in response to drought stress. For instance, under drought treatment, the expression level of IAA4 in grape (Vitis vinifera) decreased [66]. However, both OsIAA6 and OsIAA20 were upregulated during drought stress and the over-expression lines showed enhanced drought resistance, indicating that OsIAA6 and OsIAA20 were positive regulators of drought stress [35].

It is known that Aux/IAA genes mainly binds to ARF to regulate plant endogenous auxin concentration and then in response to drought stress. Furthermore, Aux/IAA genes can also be integrated with other signal transduction pathways in response to the external environment or intrinsic genetic signals. Previous study has found that IAA5, IAA6 and IAA19 can be directly regulated by DREB transcription factors (DREB2A and DREB2B) and play an important role in drought response [67]. Further studies showed that IAA5, IAA6 and IAA19 regulated plant drought tolerance by regulating glucosinolate levels [31]. Recent study has found that under drought conditions, the interaction between ZmCCT and ZmAux/IAA8 regulates the expression of downstream important drought induced genes [68]. These studies all indicate that Aux/IAA genes can jointly regulate plant drought stress by combining with other pathways to form regulatory modules or expression networks. In this study, nine genes in the third category and nine genes in the fourth category had opposite expression patterns, indicating that Aux/IAA can regulate drought stress in different ways, and the mechanism is also more complex. Thus, information on the Aux/IAA gene family in alfalfa and the functional characteristics of the MsIAA genes under drought stress remains limited. In the future, the functions of MsIAA genes in the drought stress of alfalfa should be further investigated using transgenic or gene-editing techniques.

Conclusion

In this study, we identified 41 MsIAA genes across the genome of M. sativa and analyzed gene structure, protein features, and promoter sequences, and explored their potential roles in regulating drought response. With bioinformatics analysis, we observed that the MsIAAs were highly conserved and can be divided into 10 classes based on phylogenetic analysis. At the same time, the structure of MsIAA protein was also different, indicating that the properties and functions of the protein had changed. Moreover, hormone responsive and abiotic stress-related elements were identified in the promoter of MsIAA genes. Segmental duplication was the main mode of gene number amplification of the Aux/IAA gene family in M. sativa. The expression of Aux/IAA genes showed tissue specificity in root, stem and leaf development of alfalfa, and MsIAA can regulate abiotic stress in plants. Analysis of expression patterns under drought stress revealed that MsIAA had a broad response to drought stress, which can serve as a new perspective on improving the drought resistance of alfalfa. In general, MsIAAs are essential to plant development, auxin response, and abiotic stress responses in M. sativa. Our results are important for further studies on the involvement of auxin under drought stress and contributes additional evidence that will improve future genome annotations for this crop.

Materials and methods

Experimental material and treatment of drought stress

The material used in this experiment was "Zhongmu No.1" alfalfa. Alfalfa seeds with full and single shape were selected for surface sterilization, planted in pots with vermiculite: nutrient soil = 1:1 (20 cm diameter, 20 cm high), and five biological replicates were set. They were grown in a culture room with a 16 h light/8 h dark cycle, relative humidity of 60% and temperature of 22℃. After 15 days, drought treatment (15% PEG-6000 solution) was performed, each seedling to be treated was irrigated (until fully thoroughly), and roots were collected at different stress time points (6, 12 24 and 48 h). Untreated seedling (0 h) was used as a control (CK). All roots were frozen with liquid nitrogen and stored at -80℃.

Identification of the MsIAA gene family and the physicochemical properties analysis

The alfalfa genome data, nucleic acid and protein sequences were downloaded from the online website (https://figshare.com/projects/whole_genome_sequencing_and_ assembly_of_Medicago_sativa/66380) and used for local database construction [39]. The Hidden Markov Model (HMM) profile of Aux/IAA (PF02309) was downloaded from the Pfam database (http://pfam.xfam.org/) and further filtered using HMMER 3.0 software, with an E-value threshold of 1.0 and the remaining parameters by default. Redundancy was removed using the Expasy online database (https://web.expasy.org/decrease_redundancy) [69]. To verify the accuracy of the MsIAA protein sequence after removing redundancy, Aux_IAA domain of the MsIAA protein sequence was further identified on the Pfam website (http://pfam.xfam.org/search#tabview = tab1) and NCBI-CDD Search (https://www.ncbi.nlm.nih.gov/Structure /cdd/wrpsb.cgi), and sequences without the Aux_IAA domain were removed [70]. And the predicted genes are sequentially named MsIAA1 ~ MsIAA41 according to their order of appearance in the genome. Preliminary prediction of physicochemical properties of 41 identified MsIAA protein sequences, including the number of amino acids, molecular weight, theoretical isoelectric point, instability index, aliphatic index, and grand average of hydropathicity were performed by the online network ProtParam (https://web.expasy.org/protparam/) tool. The subcellular localization of all MsIAA genes was predicted by the online tool WoLF PSORT (https://www.genscript.com/wolf psort.html). Signal peptides were analyzed by SignalP 5.0 (http://www.cbs.dtu.dk/services/SignalP-5.0) and transmembrane structures using TMHMM 2.0 (http://www.cbs.dtu.dk/services/TMHMM2.0). And leading peptides prediction were analyzed using Target P-2.0 (http://www.cbs.dtu.dk/services/Target P-2.0). Addition to, secondary structures were predicted using SOPMA (https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat. plpage = npsa_sopma.html). Tertiary structure were predicted by SWISS-MODEL (http: //swissmodel.expasy.org/interactive).

Phylogenetic analysis, gene structure, and motif composition of MsIAA proteins

To analyze the phylogenetic relationship of MsIAA proteins, multiple sequence alignment of MsIAA, AtIAA (Arabidopsis thaliana), MtIAA (Medicago truncatula) and GmIAA (Glycine max) protein sequences was performed using MEGA 7.0 software and automatically trimmed with TrimAL [71]. Phylogenetic tree was constructed by maximum-neighbor-Joining (Neighbor-Joining, NJ) method using MEGA 7.0 software. Referring to the classification of IAA of Arabidopsis and soybean, the MsIAA family genes were classified into Class I—X [44]. The motifs of all 41 MsIAA proteins analyzed by MEME 4.12.0 online tool (http://meme-suite.org/) and their type and order, and the motif features were visualized by TBtools [72]. The parameter settings for MEME is that the maximum number of motifs is 10.

Analysis of chromosome mapping and gene duplication

The CDS sequences and gene sequences corresponding to all MsIAA genes were obtained from the alfalfa genome file, and the intron and exon structures of MsIAA genes were predicted by using the GSDS 2.0 (gene structure display server) (http://gsds.gao-lab.org/) online website. In addition, to explore the distribution characteristics of Aux_IAA genes on chromosomes, MapGene2Chrome V2.0 (http://mg2c.iask.in/mg2c_v2.0/) was used to map the chromosome location based on the GFF3 data from alfalfa genome annotation file. Localization of genes on the chromosome and collinearity analysis of gene duplication within the M. sativa and between different species was completed using TBtools.

Cis-regulatory element analysis

The cis-regulatory elements in the 2000 bp sequence upstream of the transcription start site of the MsIAA gene were identified by the PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/), which are binding sites for transcription factors and regulate the precise initiation and transcription efficiency of gene transcription by binding to transcription factors.

Heatmap analysis of MsIAA gene response to abiotic stress and tissue specificity

Heatmap of the expression profile data of MsIAA genes under abiotic stress and different tissue was obtained by BLASTn alignment of alfalfa database. Heatmap analysis was performed using the OmicShare tools (http://www.omicshare.com/tools), a free online platform for data analysis. And the expression level is expressed as the calculation results of Z-core normalization for the expression data.

Protein–protein interaction (PPI) network analysis

As the genomic data of tetraploid cultivated alfalfa were not utilized as pattern data, the homologous genes of M. truncatula and M. sativa were uploaded to the STRING (v.10.5) database to predict PPI, and the predicted interactions were visualized using Cytoscape software.

RNA extraction and qRT-PCR analysis

Specific PCR primers of 41 MsIAA genes were designed using the NCBI online website Primer-BLAST (http://www.ncbi.nlm.nih.gov/tools/primer-blast) (Table S6). The total RNA was extracted from the 15 samples using an RNA easy Plant Mini Kit according to the manufacturer's instructions (Qiangen, Hilden, Germany). The concentrations of RNA were then detected using an Ultra Micro UV Spectrophotometer (Beijing Dinghaoyuan Biotechnology Co., Ltd., Beijing, China). The total RNA was then reverse-transcribed into cDNA using a Prime Script RT reagent Kit with a gDNA Eraser (Perfect Real Time) (TaKaRa, Japan) and diluted 20 times as template. The qRT-PCR was performed with StepOnePlusâ„¢ Real-Time PCR System (ABI) using SYBR premix Ex Taq (Takara, Japan). Triplicate q-PCR of each sample was performed. The reaction volume was 20 μL and included 10 μL of 2 × SuperReal PreMix Plus, 3 μL of ddH2O, 5 μL of cDNA template, and 2 μL of forward and reverse primer. The total reaction conditions included a pre-denaturation step at 95℃ for 15 min, 40 cycles of PCR amplification (denaturation at 95℃ for 10 s and annealing at 58℃ for 30 s). The relative expression levels of the selected genes were calculated by 2−ΔΔCT method [73], and normalized to the expression levels of the alfalfa Actin gene (AES78237. 1).

Data analysis

Gene expression level data obtained by qRT-PCR were graphed using the GraphPad Prism 8.0.2. software.

Availability of data and materials

All data generated or analyzed in this study are included in this published article and its supplementary material. The draft genome data of autotetraploid cultivated ("Zhongmu No.1") alfalfa was obtained from fgshare (https://fgshare.com/projects/whole_genome_sequencing_and_assembly_of_Medicago_sativa/66380). The Arabidopsis and rice ARF protein sequences were all downloaded from Plant Transcription Factor Database (http://planttfdb.gaolab.org/). Soybean AUX_IAA protein sequence reference Singh et. al. [44], and M. truncatula Aux_IAA protein sequence reference Liu et. al. [9]. Genome-wide transcriptome data of diferent alfalfa tissues were acquired from the Medicago Analysis Portal (https://medicago.legumeinfo.org/). All transcriptome sequencing data analysed during the current study are available in the NCBI SRA repository (https://www.ncbi.nlm.nih.gov/sra/): SRR7091780-SRR7091794 (cold treatment), SRR7160322-SRR7160357 (ABA, mannitol and salt treatments), SRR22519684-SRR22519695 (aluminum treatment), and SRR5279707-SRR5279711 (lead treatment).

References

  1. Emenecker RJ, Strader LC. Auxin-Abscisic Acid Interactions in Plant Growth and Development. Biomolecules. 2020;10(2):281.

  2. Wang Y, Liu H, Li H, Teng R, Zhuang J. Genome-based identification and analysis of the genes involved in auxin biosynthesis and signal transduction during tea plant leaf development. Sci Hortic. 2020;261(61):109030.

    Article  CAS  Google Scholar 

  3. Luo J, Zhou JJ, Zhang JZ. Aux/IAA Gene Family in Plants: Molecular Structure, Regulation, and Function. Int J Mol Sci. 2018;19(1):259.

  4. 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(3):297–309.

    Article  CAS  PubMed  Google Scholar 

  5. Cheng W, Zhang M, Cheng T, Wang J, Zhang Q. Genome-wide identification of Aux/IAA gene family and their expression analysis in Prunus mume. Front Genet. 2022;13:1013822.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Zhang C, Yang Y, Yu Z, Wang J, Huang R, Zhan Q, Li S, Lai J, Zhang S, Yang C. SUMO E3 ligase AtMMS21-dependent SUMOylation of AUXIN/INDOLE-3-ACETIC ACID 17 regulates auxin signaling. Plant Physiol. 2023;191(3):1871–83.

    Article  CAS  PubMed  Google Scholar 

  7. Salehin M, Bagchi R, Estelle M. SCFTIR1/AFB-based auxin perception: mechanism and role in plant growth and development. Plant Cell. 2015;27(1):9–19.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Weijers D, Wagner D. Transcriptional Responses to the Auxin Hormone. Annu Rev Plant Biol. 2016;67:539–74.

    Article  CAS  PubMed  Google Scholar 

  9. Liu R, Guo Z, Lu S. Genome-Wide Identification and Expression Analysis of the Aux/IAA and Auxin Response Factor Gene Family in Medicago truncatula. Int J Mol Sci. 2021;22(19):10494.

  10. Paul P, Dhandapani V, Rameneni JJ, Li X, Sivanandhan G, Choi SR, Pang W, Im S, Lim YP. Genome-Wide Analysis and Characterization of Aux/IAA Family Genes in Brassica rapa. PLoS ONE. 2016;11(4): e0151522.

    Article  PubMed  PubMed Central  Google Scholar 

  11. Song Y, Wang L, Xiong L. Comprehensive expression profiling analysis of OsIAA gene family in developmental processes and in response to phytohormone and stress treatments. Planta. 2009;229(3):577–91.

    Article  CAS  PubMed  Google Scholar 

  12. Szemenyei H, Hannon M, Long JA. TOPLESS mediates auxin-dependent transcriptional repression during Arabidopsis embryogenesis. Science. 2008;319(5868):1384–6.

    Article  CAS  PubMed  Google Scholar 

  13. Tiwari SB, Hagen G, Guilfoyle TJ. Aux/IAA proteins contain a potent transcriptional repression domain. Plant Cell. 2004;16(2):533–43.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Hagen G, Guilfoyle T. Auxin-responsive gene expression: genes, promoters and regulatory factors. Plant Mol Biol. 2002;49(3–4):373–85.

    Article  CAS  PubMed  Google Scholar 

  15. Guilfoyle TJ, Hagen G. Auxin response factors. Curr Opin Plant Biol. 2007;10(5):453–60.

    Article  CAS  PubMed  Google Scholar 

  16. Woodward AW, Bartel B. Auxin: regulation, action, and interaction. Ann Bot. 2005;95(5):707–35.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. De Smet I, Jürgens G. Patterning the axis in plants–auxin in control. Curr Opin Genet Dev. 2007;17(4):337–43.

    Article  PubMed  Google Scholar 

  18. Kazan K, Manners JM. Linking development to defense: auxin in plant-pathogen interactions. Trends Plant Sci. 2009;14(7):373–82.

    Article  CAS  PubMed  Google Scholar 

  19. Wang H, Jones B, Li Z, Frasse P, Delalande C, Regad F, Chaabouni S, Latché A, Pech JC, Bouzayen M. The tomato Aux/IAA transcription factor IAA9 is involved in fruit development and leaf morphogenesis. Plant Cell. 2005;17(10):2676–92.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Koenig D, Bayer E, Kang J, Kuhlemeier C, Sinha N. Auxin patterns Solanum lycopersicum leaf morphogenesis. Development (Cambridge, England). 2009;136(17):2997–3006.

    Article  CAS  PubMed  Google Scholar 

  21. 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(4):659–72.

    Article  CAS  PubMed  Google Scholar 

  22. Bassa C, Mila I, Bouzayen M, Audran-Delalande C. Phenotypes associated with down-regulation of Sl-IAA27 support functional diversity among Aux/IAA family members in tomato. Plant Cell Physiol. 2012;53(9):1583–95.

    Article  CAS  PubMed  Google Scholar 

  23. Su LY, Audran C, Bouzayen M, Roustan JP, Chervin C. The Aux/IAA, Sl-IAA17 regulates quality parameters over tomato fruit development. Plant Signal Behav. 2015;10(11): e1071001.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Liu K, Yuan C, Feng S, Zhong S, Li H, Zhong J, Shen C, Liu J. Genome-wide analysis and characterization of Aux/IAA family genes related to fruit ripening in papaya (Carica papaya L.). BMC genomics. 2017;18(1):351.

    Article  PubMed  PubMed Central  Google Scholar 

  25. Pan L, Zeng W, Niu L, Lu Z, Liu H, Cui G, Zhu Y, Chu J, Li W, Fang W, et al. PpYUC11, a strong candidate gene for the stony hard phenotype in peach (Prunus persica L. Batsch), participates in IAA biosynthesis during fruit ripening. J Exp Bot. 2015;66(22):7031–44.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Zeng WF, Pan L, Liu H, Niu L, Lu ZH, Cui GC, Wang ZQ. Characterization of 1-aminocyclopropane-1-carboxylic acid synthase ( ACS ) genes during nectarine fruit development and ripening. Tree Genet Genomes. 2015;11(2):1–18.

    Article  Google Scholar 

  27. Nagpal P, Ellis CM, Weber H, Ploense SE, Barkawi LS, Guilfoyle TJ, Hagen G, Alonso JM, Cohen JD, Farmer EE, et al. Auxin response factors ARF6 and ARF8 promote jasmonic acid production and flower maturation. Development (Cambridge, England). 2005;132(18):4107–18.

    Article  CAS  PubMed  Google Scholar 

  28. Jia M, Li Y, Wang Z, Tao S, Sun G, Kong X, Wang K, Ye X, Liu S, Geng S, et al. TaIAA21 represses TaARF25-mediated expression of TaERFs required for grain size and weight development in wheat. The Plant journal : for cell and molecular biology. 2021;108(6):1754–67.

    Article  CAS  PubMed  Google Scholar 

  29. Wang J, Yan DW, Yuan TT, Gao X, Lu YT. A gain-of-function mutation in IAA8 alters Arabidopsis floral organ development by change of jasmonic acid level. Plant Mol Biol. 2013;82(1–2):71–83.

    Article  CAS  PubMed  Google Scholar 

  30. Yang E, Yang H, Li C, Zheng M, Song H, Zou X, Chen X, Zhang J. Genome-Wide Identification and Expression Analysis of the Aux/IAA Gene Family of the Drumstick Tree (Moringa oleifera Lam.) Reveals Regulatory Effects on Shoot Regeneration. Int J Mol Sci. 2022;23(24):15729.

  31. Salehin M, Li B, Tang M, Katz E, Song L, Ecker JR, Kliebenstein DJ, Estelle M. Auxin-sensitive Aux/IAA proteins mediate drought tolerance in Arabidopsis by regulating glucosinolate levels. Nat Commun. 2019;10(1):4021.

    Article  PubMed  PubMed Central  Google Scholar 

  32. Diouf L, Pan Z, He SP, Gong WF, Jia YH, Magwanga RO, Romy KRE, Or Rashid H, Kirungu JN, Du X. High-Density Linkage Map Construction and Mapping of Salt-Tolerant QTLs at Seedling Stage in Upland Cotton Using Genotyping by Sequencing (GBS). Int J Mol Sci. 2017;18(12):2622.

  33. Gupta A, Rico-Medina A, Caño-Delgado AI. The physiology of plant responses to drought. Science (New York, NY). 2020;368(6488):266–9.

    Article  CAS  Google Scholar 

  34. Jung H, Lee DK, Choi YD, Kim JK. OsIAA6, a member of the rice Aux/IAA gene family, is involved in drought tolerance and tiller outgrowth. Plant science : an international journal of experimental plant biology. 2015;236:304–12.

    Article  CAS  PubMed  Google Scholar 

  35. Zhang A, Yang X, Lu J, Song F, Sun J, Wang C, Lian J, Zhao L, Zhao B. OsIAA20, an Aux/IAA protein, mediates abiotic stress tolerance in rice through an ABA pathway. Plant science : an international journal of experimental plant biology. 2021;308: 110903.

    Article  CAS  PubMed  Google Scholar 

  36. Dong X, Deng H, Ma W, Zhou Q, Liu Z. Genome-wide identification of the MADS-box transcription factor family in autotetraploid cultivated alfalfa (Medicago sativa L.) and expression analysis under abiotic stress. BMC genomics. 2021;22(1):603.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Liu W, Xiong C, Yan L, Zhang Z, Ma L, Wang Y, Liu Y, Liu Z. Transcriptome Analyses Reveal Candidate Genes Potentially Involved in Al Stress Response in Alfalfa. Front Plant Sci. 2017;8:26.

    PubMed  PubMed Central  Google Scholar 

  38. Liu Z, Chen T, Ma L, Zhao Z, Zhao PX, Nan Z, Wang Y. Global transcriptome sequencing using the Illumina platform and the development of EST-SSR markers in autotetraploid alfalfa. PLoS ONE. 2013;8(12): e83549.

    Article  PubMed  PubMed Central  Google Scholar 

  39. Chen H, Zeng Y, Yang Y, Huang L, Tang B, Zhang H, Hao F, Liu W, Li Y, Liu Y, et al. Allele-aware chromosome-level genome assembly and efficient transgene-free genome editing for the autotetraploid cultivated alfalfa. Nat Commun. 2020;11(1):2494.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Wang W, Shi S, Kang W, He L: Enriched endogenous free Spd and Spm in alfalfa (Medicago sativa L.) under drought stress enhance drought tolerance by inhibiting H(2)O(2) production to increase antioxidant enzyme activity. J Plant Physiol 2023, 291:154139.

  41. Wang M, Feng G, Yang Z, Wu J, Liu B, Xu X, Nie G, Huang L, Zhang X. Genome-Wide Characterization of the Aux/IAA Gene Family in Orchardgrass and a Functional Analysis of DgIAA21 in Responding to Drought Stress. Int J Mol Sci. 2023;24(22):16184.

  42. Bowman JL, Kohchi T, Yamato KT, Jenkins J, Shu S, Ishizaki K, Yamaoka S, Nishihama R, Nakamura Y, Berger F, et al. Insights into Land Plant Evolution Garnered from the Marchantia polymorpha Genome. Cell. 2017;171(2):287-304.e215.

    Article  CAS  PubMed  Google Scholar 

  43. Remington DL, Vision TJ, Guilfoyle TJ, Reed JW. Contrasting modes of diversification in the Aux/IAA and ARF gene families. Plant Physiol. 2004;135(3):1738–52.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Singh VK, Jain M. Genome-wide survey and comprehensive expression profiling of Aux/IAA gene family in chickpea and soybean. Front Plant Sci. 2015;6:918.

    Article  PubMed  PubMed Central  Google Scholar 

  45. Li H, Wang B, Zhang Q, Wang J, King GJ, Liu K. Genome-wide analysis of the auxin/indoleacetic acid (Aux/IAA) gene family in allotetraploid rapeseed (Brassica napus L.). BMC plant biology. 2017;17(1):204.

    Article  PubMed  PubMed Central  Google Scholar 

  46. Zhu W, Zhang M, Li J, Zhao H, Ge W, Zhang K. Identification and Analysis of Aux/IAA Family in Acer rubrum. Evol Bioinformatics Online. 2021;17:1176934321994127.

    Google Scholar 

  47. Begum T, Robinson-Rechavi M. Special Care Is Needed in Applying Phylogenetic Comparative Methods to Gene Trees with Speciation and Duplication Nodes. Mol Biol Evol. 2021;38(4):1614–26.

    Article  CAS  PubMed  Google Scholar 

  48. 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(10):2107.

  49. Jain M, Kaur N, Garg R, Thakur JK, Tyagi AK, Khurana JP. Structure and expression analysis of early auxin-responsive Aux/IAA gene family in rice (Oryza sativa). Funct Integr Genomics. 2006;6(1):47–59.

    Article  CAS  PubMed  Google Scholar 

  50. Mathura SR, Sutton F, Bowrin V. Genome-wide identification, characterization, and expression analysis of the sweet potato (Ipomoea batatas [L.] Lam.) ARF, Aux/IAA, GH3, and SAUR gene families. BMC plant biology. 2023;23(1):622.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Dreher KA, Brown J, Saw RE, Callis J. The Arabidopsis Aux/IAA protein family has diversified in degradation and auxin responsiveness. Plant Cell. 2006;18(3):699–714.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Figueiredo MRA, Strader LC. Intrinsic and extrinsic regulators of Aux/IAA protein degradation dynamics. Trends Biochem Sci. 2022;47(10):865–74.

    Article  CAS  PubMed  Google Scholar 

  53. Li X, Cai K, Pei X, Li Y, Hu Y, Meng F, Song X, Tigabu M, Ding C, Zhao X. Genome-Wide Identification of NAC Transcription Factor Family in Juglans mandshurica and Their Expression Analysis during the Fruit Development and Ripening. Int J Mol Sci. 2021;22(22):12414.

  54. Bowers JE, Chapman BA, Rong J, Paterson AH. Unravelling angiosperm genome evolution by phylogenetic analysis of chromosomal duplication events. Nature. 2003;422(6930):433–8.

    Article  CAS  PubMed  Google Scholar 

  55. Yang Y, Kang L, Wu R, Chen Y, Lu C. Genome-wide identification and characterization of UDP-glucose dehydrogenase family genes in moso bamboo and functional analysis of PeUGDH4 in hemicellulose synthesis. Sci Rep. 2020;10(1):10124.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Young ND, Debellé F, Oldroyd GE, Geurts R, Cannon SB, Udvardi MK, Benedito VA, Mayer KF, Gouzy J, Schoof H, et al. The Medicago genome provides insight into the evolution of rhizobial symbioses. Nature. 2011;480(7378):520–4.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Su Y, He H, Wang P, Ma Z, Mao J, Chen B. Genome-wide characterization and expression analyses of the auxin/indole-3-acetic acid (Aux/IAA) gene family in apple (Malus domestica). Gene. 2021;768: 145302.

    Article  CAS  PubMed  Google Scholar 

  58. Huangfu Y, Pan J, Li Z, Wang Q, Mastouri F, Li Y, Yang S, Liu M, Dai S, Liu W. Genome-wide identification of PTI1 family in Setaria italica and salinity-responsive functional analysis of SiPTI1-5. BMC Plant Biol. 2021;21(1):319.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Tiwari SB, Hagen G, Guilfoyle T. The roles of auxin response factor domains in auxin-responsive transcription. Plant Cell. 2003;15(2):533–43.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Li A, Liu A, Wu S, Qu K, Hu H, Yang J, Shrestha N, Liu J, Ren G. Comparison of structural variants in the whole genome sequences of two Medicago truncatula ecotypes: Jemalong A17 and R108. BMC Plant Biol. 2022;22(1):77.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Xu H, Liu Y, Zhang S, Shui D, Xia Z, Sun J. Genome-wide identification and expression analysis of the AUX/IAA gene family in turnip (Brassica rapa ssp. rapa). BMC plant biology. 2023;23(1):342.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Waseem M, Ahmad F, Habib S, Li Z. Genome-wide identification of the auxin/indole-3-acetic acid (Aux/IAA) gene family in pepper, its characterisation, and comprehensive expression profiling under environmental and phytohormones stress. Sci Rep. 2018;8(1):12008.

    Article  PubMed  PubMed Central  Google Scholar 

  63. Muto H, Watahiki MK, Nakamoto D, Kinjo M, Yamamoto KT. 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(1):187–96.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Si C, Zeng D, da Silva JAT, Qiu S, Duan J, Bai S, He C. Genome-wide identification of Aux/IAA and ARF gene families reveal their potential roles in flower opening of Dendrobium officinale. BMC Genomics. 2023;24(1):199.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Wang S, Bai Y, Shen C, Wu Y, Zhang S, Jiang D, Guilfoyle TJ, Chen M, Qi Y. Auxin-related gene families in abiotic stress response in Sorghum bicolor. Funct Integr Genomics. 2010;10(4):533–46.

    Article  CAS  PubMed  Google Scholar 

  66. Birsen Ç, Ozan K, AhmetCan O. 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(2):365–77.

    Google Scholar 

  67. Shani E, Salehin M, Zhang Y, Sanchez SE, Doherty C, Wang R, Mangado CC, Song L, Tal I, Pisanty O, et al. Plant Stress Tolerance Requires Auxin-Sensitive Aux/IAA Transcriptional Repressors. Current biology : CB. 2017;27(3):437–44.

    Article  CAS  PubMed  Google Scholar 

  68. Zhang Z, Qu J, Lu M, Zhao X, Xu Y, Wang L, Liu Z, Shi Y, Liu C, Li Y, et al. The maize transcription factor CCT regulates drought tolerance by interacting with Fra a 1, E3 ligase WIPF2, and auxin response factor Aux/IAA8. J Exp Bot. 2024;75(1):103–22.

    Article  PubMed  Google Scholar 

  69. Artimo P, Jonnalagedda M, Arnold K, Baratin D, Csardi G, de Castro E, Duvaud S, Flegel V, Fortier A, Gasteiger E et al: ExPASy: SIB bioinformatics resource portal. Nucleic acids research 2012, 40(Web Server issue):W597–603.

  70. El-Gebali S, Mistry J, Bateman A, Eddy SR, Luciani A, Potter SC, Qureshi M, Richardson LJ, Salazar GA, Smart A, et al. The Pfam protein families database in 2019. Nucleic Acids Res. 2019;47(D1):D427-d432.

    Article  CAS  PubMed  Google Scholar 

  71. Capella-Gutiérrez S, Silla-Martínez JM, Gabaldón T. trimAl: a tool for automated alignment trimming in large-scale phylogenetic analyses. Bioinformatics. 2009;25(15):1972–3.

    Article  PubMed  PubMed Central  Google Scholar 

  72. Chen C, Chen H, Zhang Y, Thomas HR, Frank MH, He Y, Xia R. TBtools: An Integrative Toolkit Developed for Interactive Analyses of Big Biological Data. Mol Plant. 2020;13(8):1194–202.

    Article  CAS  PubMed  Google Scholar 

  73. Yang X. Analysis of the copy number of exogenous genes in transgenic cotton using real-time quantitative PCR and the 2-△△CT method. Afr J Biotechnol. 2012:11(23):6226-33.

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Acknowledgements

We are grateful to the National Natural Science Foundation of China (32260349), and the Inner Mongolia Pratacultural Technology Innovation Center Co., Ltd (CCPTZX2023B04).

Funding

This research was supported by the National Natural Science Foundation of China (32260349), and the Inner Mongolia Pratacultural Technology Innovation Center Co., Ltd (CCPTZX2023B04).

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Authors and Affiliations

  1. College of Forestry and Prataculture, Ningxia University, Yinchuan, 750021, China

    Jinqing Zhang, Shuxia Li, Xueqin Gao & BingZhe Fu

  2. Ningxia Grassland and Animal Husbandry Engineering Technology Research Center, Xixia District, Yinchuan, Ningxia Hui Autonomous Region, Yinchuan, 750021, China

    Shuxia Li, Xueqin Gao & BingZhe Fu

  3. Key Laboratory for Model Innovation in Forage Production Efficiency, Ministry of Agriculture and Rural Affairs, Yinchuan, 750021, China

    Shuxia Li, Xueqin Gao & BingZhe Fu

  4. Inner Mongolia Pratacultural Technology Innovation Center Co, Ltd, Hohhot, 010000, China

    Yaling Liu

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Contributions

BZF and JQZ conceived and designed the experiment. JQZ, SXL and YLL performed the experiments. JQZ, XQG and YLL analyzed all the data. JQZ wrote the manuscript. BZF revised the manuscript. All of the authors read and approved the final manuscript.

Corresponding author

Correspondence to BingZhe Fu.

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Ethics approval and consent to participate

Experimental research and studies on plants in this study, including the collection of plant material, are complied with the institutional, national, and international guidelines and legislation. We conducted the experimental research on cultivated alfalfa in accordance with the IUCN Policy Statement on Research Involving Species at Risk of Extinction and the Convention on the Trade in Endangered Species of Wild Fauna and Flora. Alfalfa seeds of "Zhongmu No.1" were gifted by the National Livestock Husbandry Station, Ministry of Agriculture and Rural Afairs of The People’s Republic of China. We have permission to use plant material. "Zhongmu No.1" alfalfa was approved by Beijing Institute of Animal Sciences of Chinese Academy of Agricultural Sciences in November 1997 by the National Grass Variety Approval Committee, registered as a bred variety by Huazhu Geng, Qingchuan Yang, Wenhua Shu, Cong Li and Jiguo Hao. The variety registration number is 177, national approval number is 2003006.

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Zhang, J., Li, S., Gao, X. et al. Genome-wide identification and expression pattern analysis of the Aux/IAA (auxin/indole-3-acetic acid) gene family in alfalfa (Medicago sativa) and the potential functions under drought stress. BMC Genomics 25, 382 (2024). https://doi.org/10.1186/s12864-024-10313-2

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Keywords

BMC Genomics

ISSN: 1471-2164

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