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Article

The TIR1/AFB Family in Solanum melongena: Genome-Wide Identification and Expression Profiling under Stresses and Picloram Treatment

by
Wenchao Du
1,2,*,†,
Umer Karamat
3,†,
Liuqing Cao
1,†,
Yunpeng Li
2,
Haili Li
1,
Haoxin Li
1,
Lai Wei
2,
Dongchen Yang
4,
Meng Xia
1,
Qiang Li
2 and
Xueping Chen
2
1
Biochemistry and Environmental Engineering College, Baoding University, Baoding 071000, China
2
Key Laboratory for Vegetable Germplasm Enhancement and Utilization of Hebei, Collaborative Innovation Center of Vegetable Industry in Hebei, College of Horticulture, Hebei Agricultural University, Baoding 071000, China
3
Guangdong Key Laboratory for New Technology Research of Vegetables, Vegetable Research Institute, Guangdong Academy of Agricultural Sciences, Guangzhou 510642, China
4
College of Plant Protection, Hebei Agricultural University, Baoding 071001, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Agronomy 2024, 14(7), 1413; https://doi.org/10.3390/agronomy14071413
Submission received: 29 May 2024 / Revised: 22 June 2024 / Accepted: 26 June 2024 / Published: 28 June 2024
(This article belongs to the Special Issue Cultivation Physiology, Molecular Biology and Molecular Breeding of Solanaceae)
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Abstract

:
TIR1/AFB proteins are a class of auxin receptors with key roles in plant development and biotic and abiotic stress responses; several have been identified as targets of the auxin-mimicking herbicide picloram. In this study, we identified five putative TIR1/AFB gene family members in the important vegetable crop Solanum melongena (eggplant) and characterized them using bioinformatics tools and gene expression analyses. Phylogenetic analysis of the TIR1/AFBs classified them into three subgroups based on their Arabidopsis and Solanum lycopersicum homologs. AFB6 homologs were present only in S. melongena and S. lycopersicum, whereas AFB2/3 homologs were found only in Arabidopsis. One pair of S. melongena TIR1 homologs were located in syntenic regions in the genome and appeared to have arisen by segmental duplication. Promoter analysis revealed 898 cis-elements in the TIR1/AFB promoters, 125 of which were related to hormones, stress, light, or growth responses, but only SmAFB5 had a cis-acting regulatory element involved in auxin responsiveness (AuxRR-core). RNA sequencing and expression profiling showed that the TIR1/AFB genes were differentially expressed at different growth stages and in response to light, temperature, and drought. Only SmTIR1A expression was significantly induced by picloram treatment and different growth stages. TIR1/AFB expression is regulated by microRNAs (miRNAs) in other plant species, and we identified 6 or 29 miRNAs that potentially targeted the five TIR1/AFB genes on the basis of comparisons with S. lycopersicum and S. tuberosum miRNAs, respectively. Three-dimensional protein structure predictions revealed that all the TIR1/AFB proteins were very similar in structure, differing only in the numbers of alpha helices and in one angle linking an α helix and a β sheet. For measuring the function of TIR1/AFB genes in response to drought, SmAFB5 was selected, and knockdown by virus-induced gene silence (VIGS) 35S::SmAFB5 lines showed resistance to drought compared to controls. These analyses provide insight into the potential functions of TIR1/AFBs during growth and in response to stress; they highlight differences among the SmTIR1/AFBs that may be useful for eggplant breeding.

1. Introduction

Members of the TRANSPORT INHIBITOR RESPONSE 1/AUXIN-SIGNALING F-BOX (TIR1/AFB) protein family are auxin receptors that form co-receptor complexes with Aux/IAA transcriptional repressors [1]. TIR1/AFB proteins serve as positive regulators of downstream auxin-responsive pathways upon the perception of auxin. TIR1/AFB proteins and Auxin/IAA (Aux/IAA) proteins form a co-receptor of auxin or homologs of auxin when few auxins exist [2]. The degradation of Aux/IAA proteins via the 26S proteasome occurs under higher concentrations of auxin or its homologs, and then auxin response factors (ARFs) are released, which are transcriptional regulators of auxin-responsive genes such as Aux/IAA [3,4]. TIR1/AFB proteins of land plants can be divided into three broad groups (TIR1/AFB1/AFB2/AFB3, AFB4/5, and AFB6) on the basis of their N-terminal F-Box domain and leucine-rich-repeat (LRR) domain [5]. AFB6 proteins are found in many land plants (e.g., S. lycopersicum and Pisum sativum) but are absent from the genomes of core Brassica species such as Arabidopsis and Poaceae species such as rice and maize [5,6]. Previous research has shown that the TIR1/AFB1/AFB2/AFB3 group can be divided into two subgroups, TIR1/AFB1 and AFB2/3, which arise through gene duplication prior to angiosperm radiation [5]. However, AFB2/3 proteins have not been reported in Solanaceae species. The loss of specific TIR1/AFB homologs in some species suggests that their functions may not be essential or that they share redundant functions with other TIR1/AFB family members. By contrast, the retention of other TIR1/AFBs suggests that they may have unique or specialized roles.
TIR1/AFB proteins mediate diverse auxin responses [7] and have essential roles in embryogenesis, lateral root initiation [8], seed abortion [9], and leaf and fruit development [10,11]. The expression of some TIR1/AFB genes also changes over the course of plant development. For example, the gene expression of AFB6 in the pea fruit pericarp increased in response to deseeding [6]. Gene expressions of TIR1 and AFB2 changed for pea fruit development [12].
TIR1/AFB proteins also function in response to abiotic or biotic stress [13], including salt stress and fungal disease [14,15]. Research has shown that TIR1/AFB expression can be induced by fungi such as Verticillium dahlia and by small signaling molecules such as hydrogen sulfide and nitric oxide [16,17,18]. TIR1 was upregulated under drought stress, as determined by RNA-Seq in maize or solanaceous crops [19]. TIR1 and AFB2 expression levels were significantly downregulated in spikelets in response to drought stress in rice [20]. An Arabidopsis tir1afb2 double mutant exhibited enhanced tolerance against salt stress compared with wild-type plants [21]. The Arabidopsis tir1-1 mutant displayed defective hypocotyl elongation under elevated temperatures [22]. The repression of TIR1 expression in wheat impaired pollen exine formation in male sterility under cold stress [23]. The afb5 mutant showed resistance to auxin herbicide in Arabidopsis [24] and the Osafb4 mutant harbored resistance to picloram in rice [8].
TIR1/AFB genes have been genetically manipulated or used as breeding targets in Arabidopsis, rice, and other crops [13], but much less is known about the TIR1/AFB genes in Solanaceae plants.
Research has shown that TIR1/AFB genes are targeted and repressed by microRNAs (miRNAs). miRNAs are known to regulate auxin signaling and homeostasis in Arabidopsis and rice by directly cleaving or inhibiting the translation of their target mRNA(s) [25]. For example, miR393 has been identified in many plant species and shown to target TIR1/AFB genes in rice, apple, maize, cotton, and Arabidopsis [26,27,28,29,30]. However, miRNA–TIR1/AFB pairs have not previously been identified in S. melongena (eggplant, also known as aubergine or brinjal).
In this study, we performed a genome-wide analysis of the TIR1/AFB gene family in S. melongena, one of the most economically important vegetable crops in Solanaceae [31]. We identified and characterized five SmTIR1/AFB genes using bioinformatics tools, performed structural modeling of their encoded proteins, and predicted miRNAs likely to regulate their expression. Using VIGS, RNA-sequencing, and RT–PCR data, we revealed the effects of growth stage, light, temperature, and picloram treatment on SmTIR1/AFB expression. These results provide new insights into the evolutionary history and functions of the TIR1/AFB gene family in Solanaceae and support further investigation of these key auxin-response genes as breeding targets in eggplant.

2. Materials and Methods

2.1. Identification and Characterization of the TIR1/AFB Gene Family

We performed BLASTP searches of the Eggplant Genome Database (http://eggplant-hq.cn/Eggplant/blastBefore, accessed on 3 May 2022) [32], using six Arabidopsis TIR1/AFB sequences (At1g12820, At3g26810, At3g26980, At4g03190, At4g24390, At5g49980; https://www.arabidopsis.org, accessed on 2 May 2022) and four S. lycopersicum TIR1/AFB sequences (Sl09g074520, Sl06g008780, Sl04g074980, Sl02g079190; https://solgenomics.net/, accessed on 6 July 2022) as queries, with a permissive e-value cutoff of <0.01. The resulting 12 candidate TIR1/AFB proteins were examined for the presence of an F-Box domain (PF18511) and a Transport inhibitor response 1 protein domain (PF18791) (http://pfam.xfam.org/, accessed on 6 July 2022), and only the five sequences with at least one copy of each domain were retained. These putative S. melongena TIR1/AFB protein sequences were submitted to the ExPASy ProtParam tool (https://web.expasy.org/protparam/, accessed on 6 July 2022) to determine their molecular weight, number of amino acids, isoelectric point, and GRAVY score and submitted to Wolf PSORT (https://wolfpsort.hgc.jp, accessed on 6 July 2022) to predict their subcellular localization [33]. The “Show Genes on Chromosome” feature of TBtools (https://github.com/CJ-Chen/TBtools, accessed on 8 July 2022) was used to visualize the locations of the TIR1/AFB genes on the S. melongena chromosomes.

2.2. Comparative Phylogenetic Analysis and Classification of the S. melongena TIR1/AFB Genes

The predicted protein sequences of the TIR1/AFB genes from Arabidopsis, S. lycopersicum, and S. melongena were aligned using Clustal W, and the resulting alignment was used to construct a neighbor-joining (NJ) phylogenetic tree with 1000 bootstrap replicates using MEGA X software (https://www.megasoftware.net/, accessed on 6 July 2023) [34].

2.3. Identification of Duplicated TIR1/AFB Genes in S. melongena

Duplicated TIR1/AFB genes were identified in the S. melongena genome using the Blast, MCScanX, and Advanced Circos features of TBtools [35]. MCScanX was used to analyze syntenic relationships among the S. melongena genes and the resulting collinearity files were used for the visualization of intrachromosomal synteny with TBtools [36].

2.4. Promoter cis-Elements of the S. melongena TIR1/AFB Genes

The 2000-bp sequences upstream of the start codons of the TIR1/AFB genes were downloaded from the Eggplant Genome database (eggplant-hq.cn) and submitted to the PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 6 July 2023) for cis-element prediction; the results were visualized using TBtools.

2.5. 3D Protein Structure Prediction

The 3D structures of the SmTIR1/AFB proteins were modeled using the web-based PHYRE2 Protein Fold Recognition Server (http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index, accessed on 6 July 2023) [37].

2.6. Gene Structures, Conserved Domains, and Motif Analysis

Conserved protein domains were identified in the SmTIR1/AFB proteins using the NCBI conserved domain search tool (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi, accessed on 6 July 2023) and motif analysis was performed using the MEME Suite (https://meme-suite.org/meme/, accessed on 8 July 2023). The gene structure (exons and introns) was predicted from the coding sequences and genomic sequences of the SmTIR1/AFB genes using the Gene Structure Display Server (GSDS) (http://gsds.gao-lab.org, accessed on 9 July 2023).

2.7. Prediction of miRNAs and Target Genes

miRNAs targeting the SmTIR1/AFB genes were identified by submitting the SmTIR1/AFB coding sequences as target candidates to the psRNATarget database (http://plantgrn.noble.org/psRNATarget, accessed on 12 July 2023) and searching them against known miRNAs from S. lycopersicum and S. tuberosum.

2.8. RNA Sequencing and Data Analysis

2.8.1. Plant Growth and Sample Collection

The homozygous inbred line ‘14–345’ was obtained from the State Key Laboratory for Vegetable Germplasm Enhancement and Utilization of the Hebei Vegetable Germplasm Resource Centre, with good fruit setting and fruit color changing from green (early development stage) to purple (commercial maturity stage) and then to brown (physiological maturity stage) and grown in a growth chamber at 28 °C with a 16 h day/8 h night photoperiod. The uppermost two leaves were collected from three replicate plants at the two-leaf and four-leaf stages for transcriptomic analysis. Three replicate 3-day-old seedlings were also collected at 6 h after treatment with 1 μM picloram or a water control. The sampled tissues were frozen in liquid nitrogen and stored at −75 °C until RNA isolation.

2.8.2. Stress Treatment

Virus-induced gene silencing (VIGS) and tobacco rattle virus (TRV) were used to identify the function of SmAFB5. The VIGS tool (https://vigs.solgenomics.net/, accessed on 6 September 2023) was used to design accurate gene fragments of SmAFB5 for VIGS. Mg-protoporphyrin chelatase (CHlH) encoding genes are widely used in VIGS assays as a marker gene [31]. A 300 bp synthetic sequence corresponding to part of the CHlH and SmAFB5 genes was selected and cloned into the pTRV2 vector according to the high-quality genome of eggplant [25]. The empty vector TRV2 was used as the control. The resultant TRV-mediated VIGS constructs were used to infect two-week-old eggplant seedlings with three true leaves. At least three independent replicates were performed, and each independent replicate contained at least three seedlings. Drought treatment was performed on the 35S::SmAFB5 knockdown plants and controls by ceasing watering, placing them in a greenhouse for 3 days, and then re-watering; the plants were checked and photographed 12 h after recovery. Dark, low-temperature, and drought treatments of seedlings at the cotyledon stage were performed. The light treatment was performed on the cotyledon seedlings after a 24 h light treatment at 25 °C, the low-temperature treatment was performed at 10 °C in the dark, and, for measuring gene expression under the drought treatment, watering was ceased and seedlings were left for 6 days in the dark.

2.8.3. RNA-Seq Library Construction and Sequencing

Total RNA was extracted from replicate samples of leaf tissue according to the instructions of the EASTEP Super Total RNA Kit (Promega, Shanghai, China). Three independent replicates were performed, with each independent replicate including at least three plants, and GAPDH was used as the reference gene. RNA quality was assessed by 1% agarose gel electrophoresis. RNA concentration and integrity were assessed on a Qubit 2.0 Fluorometer (Life Technologies, Waltham, MA, USA) and an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA), respectively. mRNA was isolated from 3 μg of total RNA using poly-T oligo magnetic beads and fragmented using high-temperature and divalent cations in NEB proprietary fragmentation buffer (NEB, Ipswich, MA, USA). RNA-seq libraries were prepared using the NEBNext Ultra RNA Library Prep Kit for Illumina and sequenced on the Illumina HiSeq 4000 platform to obtain 150 bp paired-end reads at Novogene Technology Company (Beijing, China). Raw reads were cleaned and mapped to the eggplant reference genome for the calculation of gene expression, as described previously [38,39].

2.9. Validation of Selected Gene Expression by Real-Time Quantitative Reverse Transcription PCR (qRT–PCR)

Four micrograms of total RNA were reverse transcribed using an EasyScript One-step gDNA Removal and cDNA Synthesis SuperMix kit (TransGen, AE311, Beijing, China), according to the manufacturer’s instructions. GAPDH was used as the housekeeping gene in RNA-Seq library construction and sequencing, gamma tubulin (Smechr0302615) was used as the reference gene for normalization [40], and all primer sequences are provided in Table S3. qRT–PCR was performed with a 20 µL reaction mixture containing ChamQ Universal SYBR qPCR Master Mix and run on a CFX96 TOUCH Real-time qPCR detection system (Bio-Rad, Hercules, CA, USA). There were three biological replicates for each sample type. The PCR program was 3 min at 95 °C, followed by 40 cycles of 95 °C for 10 s and 57 °C for 30 s. The 2−∆∆Ct method was used to analyze relative gene expression [41]. The same RNA samples were used for RNA-Seq and qRT–PCR.

3. Results

3.1. Identification of TIR1/AFB Genes in S. melongena

To identify the TIR1/AFB genes in the S. melongena genome, we performed BLASTP searches against two eggplant genome databases (http://eggplant-hq.cn/Eggplant/home/index and http://eggplant.kazusa.or.jp/index.html, accessed on 18 September 2023), using six Arabidopsis genes and four S. lycopersicum genes as queries. We identified 12 potential TIR1/AFB candidates in S. melongena (Table S1) and retained the five candidates that contained at least one F-Box domain (PF18511) and one Transport inhibitor response 1 protein domain (PF18791). These five S. melongena TIR1/AFBs were predicted to encode proteins of 576 to 621 amino acids; their sequencing IDs, lengths, predicted subcellular locations, instability indices, and grand average of hydropathicity (GRAVY) scores are provided in Table 1. The SmTIR1/AFB genes were located on four of the twelve S. melongena chromosomes: two on E06 and one each on chromosomes E02, E04, and E09 (Figure 1).

3.2. Phylogenetic Analysis and Classification of the S. melongena TIR1/AFB Genes

We constructed an NJ tree using five TIR1/AFB proteins from S. melongena, six from Arabidopsis, and four from S. lycopersicum. The TIR1/AFB sequences formed five groups, three of which contained S. melongena members (Figure 2).
The lower branch level showed a closer phylogenetic relationship between S. melongena and S. lycopersicum. Similar to S. melongena, S. lycopersicum contained three groups. Group I included SlTIR1/AFB1A and SlTIR1/AFB1B, with one each in the AFB4/5 (SlAFB4/5) and AFB6 (SlAFB6) groups. There were three S. melongena sequences in the TIR1 clade (SmTIR1A, SmTIR1B, and SmTIR1C) and one each in the AFB4/5 (SmAFB5) and AFB6 (SmAFB6) clades. The AFB2/3 clade contained only Arabidopsis sequences (AFB2 and AFB3 in Arabidopsis), suggesting the loss of this subgroup in Solanaceae. The function of AFB2/3 genes may be similar to that of TIR1/AFB1 genes, and TIR1/AFB1 homolog(s) in S. melongena may functionally replace Arabidopsis AFB2/3; nonetheless, identifying the specific S. melongena gene(s) that fulfill these functions will be important for breeding.

3.3. TIR1/AFB Gene Structure and Conserved Protein Motifs

All the SmTIR1/AFB genes contained three exons of similar length, but intron lengths differed markedly among individual genes (Figure 3). Motif analysis with MEME tools revealed ten conserved motifs that were present in the same order in all the SmTIR1/AFB proteins. Examination of the motifs revealed that they corresponded to five conserved protein domains: F-box_5, Transport inhibitor response 1, the AMN1 Superfamily, LRR_5, and Protein phosphatase 1 regulatory subunit 42 (PPP1R42). The first three were predicted to be present in all proteins, whereas only SmAFB6 was predicted to contain an LRR_5 domain, and only SmTIR1C was predicted to contain a PPP1R42 domain (Figure 3).

3.4. Gene Duplicates among S. melongena TIR1/AFBs

Tandem, segmental, and whole-genome duplication contribute to plant genomic evolution by giving rise to new gene family members. We used BlastP searches to identify potential segmental duplicates among the TIR1/AFBs; we found one pair of duplicated TIR1 genes (SmTIR1B and SmTIR1C) that appeared to have arisen via segmental duplication or an ancient polyploidization event (Figure 4).

3.5. Promoter cis-Elements in the TIR1/AFB Genes

The 2.0 kb sequences upstream from the translation start sites of the TIR1/AFB genes were submitted to the PlantCARE database for the detection of promoter cis-elements. In total, 898 promoter cis-elements were identified, among which, 125 related to growth, light, stress, and hormone responses were also found, including CAT-box (cis-acting regulatory element related to meristem expression), circadian (cis-acting regulatory element involved in circadian control), MBS (MYB binding site involved in drought inducibility), TC-rich repeat (cis-acting element involved in defense and stress response), elements related to abscisic acid (ABRE), auxin (ARE), methyl jasmonate (MeJA), gibberellin (GAE), salicylic acid (SAE), and others (including TC-rich repeat (cis-acting element involved in defense and stress response), CAT-box (cis-acting regulatory element related to meristem expression), and circadian (cis-acting regulatory element involved in circadian control)) (Figure 5A,B), while most of the other 773 cis-elements were kinds of unnamed or basal cis-elements like TATA-box and CAAT-box. We divided the 125 cis-elements into fourteen important kinds of cis-elements (Figure 5A,B). Among them, eleven important cis-element kinds related to development, stress, and hormones were then focused on to find their specific distributions in the TIR1/AFB gene family members.
Light-responsive elements (LREs) were the most abundant, and stress-responsive elements (SREs) were also found in all SmTIR1/AFB gene promoters (Figure 5C). Nonetheless, differences in the specific cis-elements present in individual promoters hinted at the different functions of the SmTIR1/AFB genes. For example, only the TIR1 homologs contained growth-related cis-elements (REIGs), and SmAFB6 and SmAFB5 contained more elements related to stress response than the other genes. AuxRR, an auxin-responsive cis-element, was present only in SmAFB5 (Figure 5A). Furthermore, 41 cis-elements were found in SmTIR1A, indicating its important functions in response to stress or hormones. These results indicated the specification of SmTIR1/AFB genes in response to hormones, light, and stress, their broad roles during plant development, and the special function of SmTIR1 and SmAFB5 in eggplant.

3.6. 3D Structure of the TIR1/AFB Proteins in S. melongena

The 3D structure of a protein offers insight into how it functions and how it might be controlled or modified; it also provides information on the protein’s biological interactions and helps to predict which molecules will bind to it. In this study, we performed 3D structural predictions of the SmTIR1/AFB proteins using PHYRE2 and found that they shared a conserved 3D structure with similar α helices and β sheets (Figure 6). As expected, the structures of the shorter SmAFB5 and SmAFB6 proteins contained 23 β sheets and 24 α helices, whereas the longer SmTIR1A/B/C proteins contained 23 β sheets and 25 α helices (Table S4). In addition, the five proteins showed subtle differences in a single angle that connected the twelfth β sheet to adjacent α helices and was located in the AMN1 domain, suggesting that this angle may have consequences for protein function (Figure 6 and Figure S1).

3.7. Identification of Potential miRNAs Targeting SmTIR1/AFB Genes

miRNAs help to control plant growth and development through the post-transcriptional regulation of gene expression; they base-pair with complementary sequences in their target mRNAs, silencing them through cleavage or other mechanisms. We searched known miRNAs from S. lycopersicum and S. tuberosum against the SmTIR1/AFB genes using psRNATarget to predict miRNA–SmTIR1/AFB gene pairs in S. melongena. We identified six potential miRNAs targeting three SmTIR1/AFB genes using the S. lycopersicum miRNAs. Among these, four putative miRNAs (miR169, miR482, miR9471a, and miR9471b) targeted SmTIR1C, one putative miRNA (miR1918) targeted SmAFB6, and one putative miRNA (miR159) targeted SmTIR1B.
In total, 29 potential miRNAs targeted five SmTIR1/AFB genes using the S. tuberosum miRNAs (Figure 7). The results showed that miR393 can target most SmTIR1/AFB members except SmAFB5. SmTIR1C and its close homolog SmTIR1B were predicted to be targeted by the largest number of miRNAs (four or nine for SmTIR1C; one or ten for SmTIR1B), followed by SmAFB6 (zero and five) (Table S2 and Figure 7). Fewer miRNAs targeting SmTIR1A (zero or four) than SmTIR1B and SmTIR1C indicated the higher activity and special role of SmTIR1A among them during development or response to stress.

3.8. Transcriptome Analysis of SmTIR1/AFB Gene Expression

We used transcriptome data from the eggplant inbred line 14–345 to examine the expression of SmTIR1/AFB genes in leaves at two stages of plant growth. Only SmTIR1A was upregulated from the four-leaf to the six-leaf stage, whereas the remaining genes were changed little (Figure 8A).
We also used transcriptome data from the seedlings of 14–345 to examine gene expressions of the SmTIR1/AFBs in response to picloram treatment. The results showed that the gene expression of SmTIR1A was also upregulated in response to picloram 6 h after treatment, and the other genes showed no significant difference in expression (Figure 8B). These results may support our speculation that the higher activity of SmTIR1A in response to development and stress may attributed to fewer kinds of miRNA targeting SmTIR1A.
We also used RT-qPCR to analyze SmTIR1/AFB5 expression in seedlings at the cotyledon stage; the results showed that almost all TIR1/AFB genes were responsive to drought stress (BD, watering ceased for 6 days), among which, SmAFB5 was the most significantly upregulated, and gene expression knockdown was then applied to further explore its function in response to drought by VIGS. No genes responded to low-temperature treatment (BL, 10 °C). In the light at 25 °C (LN), SmAFB5 and SmAFB6 were significantly downregulated compared with their expression under dark conditions, SmTIR1C was significantly upregulated, and the expression of the other two genes was unchanged. These results suggest that the SmTIR1/AFB genes may function in response to drought, cold, and/or light, making them useful targets for eggplant breeding (Figure 8C).
The expression patterns of the TIR1/AFB genes measured by quantitative real-time PCR (qRT–PCR) were broadly similar to those obtained from the RNA-seq data (Figure 9). Gamma tubulin (Smechr0302615) was used as the housekeeping gene for normalization.

3.9. Knockdown of SmAFB5 Increases Resistance to Drought

A VIGS experiment was conducted to analyze the function of SmAFB5.
ChlH is widely used in VIGS assays as a marker gene. The leaves of plants transfected with TRV2::SmCHlH changed significantly to a white-yellow color, showing that this system was successful in eliciting VIGS.
Plants transfected with TRV2::SmAFB5 had significantly decreased SmAFB5 expression compared to those transfected with the TRV2 empty vector (TRV2::00), as shown in Figure 10A, suggesting that SmAFB5 was effectively silenced. At 12 h recovery after drought treatment, the control seedlings exhibited wilt leaves, which is a typical symptom of drought injury [35]. The plants subjected to VIGS-mediated SmAFB5 knockdown exhibited resistance to drought and were well recovered (Figure 10B). These results showed that SmAFB5 plays a positive role in regulating drought resistance.

4. Discussion

Auxin regulates cell expansion and cell elongation in plants [42], and TIR1/AFB proteins mediate auxin signaling to affect plant growth [43,44]. As whole-genome sequences have become available for many plants, TIR1/AFB families have been identified and characterized in species such as Arabidopsis and rice using mutants or genome editing [4,8]. TIR1/AFB genes have also been identified in Brassica juncea var. tumida, Populus trichocarpa, and Malus pumila by genome-wide analysis [45,46,47]. Nonetheless, the TIR1/AFB gene family has not previously been investigated in S. melongena.
The number of TIR1/AFB genes varies among plant genomes. Arabidopsis has six TIR1/AFBs, including members of three subgroups: two members each in the TIR1/AFB1, AFB2/3, and AFB4/5 subgroups. Brassica juncea var. tumida contains 18 TIR1/AFBs: ten TIR1/AFB1s, five AFB2/3s, and three AFB4/5s. Like Arabidopsis, it lacks an AFB6 homolog [4,46]. In contrast to Arabidopsis and Brassica juncea, eggplant has five TIR1/AFB genes but lacks an AFB2/3 homolog and contains an AFB6 homolog, similar to tomato (Figure 2).
The evolutionary history of TIR1/AFB in Arabidopsis, tomato, and eggplant was inferred using the neighbor-joining method; the tree was drawn to scale, with branch lengths (next to the branches) in the same units as those of the evolutionary distances. The low numbers on the branch represented the similarity between neighboring sequence pairs. In addition, this result was similar to the observation of the similarity and the low number of branch lengths of the phylogenetic tree of TIR1/AFB family members in Arabidopsis [5].
The S. melongena TIR1/AFBs had similar exon/intron numbers and coding sequences but differed somewhat in motif composition and intron length (Figure 3). Variations in motifs and intron sequences have been identified as potential contributors to the modulation of gene expression [48,49,50]; these differences are suggested to play a role, at least partially, in accounting for the diverse expression levels observed in TIR1/AFB genes during normal growth conditions.
Gene duplication is the major mechanism for the establishment of new genes and gene functions and the generation of evolutionary novelty [51]. Segmental duplications are important for expanding the size of multigene families [52], and studies of the Arabidopsis and soybean genomes have revealed many such large duplications [53]. Previous work suggests that the duplications that gave rise to the main groups of TIR1/AFBs occurred millions of years ago; there have been relatively few recent duplications, and not all TIR1/AFB groups were retained in all flowering plant lineages [5]. In this study, we found one segmental duplication in the TIR1 lineage of S. melongena; it gave rise to SmTIR1B and SmTIR1C, which showed distinct patterns of expression. The functional consequences of this TIR1 duplication will require further investigation. Further experiments, including gene editing, could also be performed to investigate the functional consequences of the difference in the key protein angle revealed by 3D structural modeling (Figure 6).
miRNAs influence plant development and stress responses through post-transcriptional gene regulation [54], and a number of such miRNAs have been identified in Solanaceae species in recent years [55,56]. For instance, miR393 is reported to participate in developmental processes and drought stress responses in tomatoes [57,58]. We identified six putative miRNAs targeting three SmTIR/AFB genes and 29 miRNAs targeting five SmTIR/AFB genes on the basis of miRNAs previously identified in S. lycopersicum and S. tuberosum, respectively (Figure 7). The validation and functional characterization of these miRNA–target gene pairs will be useful for understanding the regulation of auxin responses in eggplant and other Solanaceae.
Different distributions of 125 cis-elements that related to growth, light, stress, and hormone responses of SmTIR1/AFB members were investigated, in which 41 cis-elements were identified in SmTIR1A, and we also found fewer miRNA targeting SmTIR1A in the SmTIR1/AFB family, indicating that SmTIR1A plays a more active role than others in response to stress and plant development. Transcriptome and qRT–PCR analyses showed the increased expression of SmTIR1A under the development stage, picloram, and drought treatments, which certificated the importance of SmTIR1A. However, we only constructed the TRV2::SmAFB5 successfully, and the function of SmTIR1A needs further exploration.
Different patterns of gene expression in the SmTIR1/AFBs (Figure 8) suggest that sub- and/or neofunctionalization occurred [59], and more detailed characterizations of individual gene functions will be needed to support the use of these genes in breeding and gene editing. Previous studies have used genome-wide analysis to investigate transcript levels by, for example, profiling the expression of TIR1/AFBs under salt stress in Arabidopsis [60]. Other studies have also demonstrated the involvement of TIR1/AFB genes in stress tolerance [46,61]. Expressions of the TIR1/AFB family are different in Populus trichocarpa under stress but are not sensitive to auxin [47]. In our study, we confirmed that the expression of SmTIR1/AFBs was highly responsive to light, drought, and temperature stresses; only SmTIR1A was also differentially expressed under auxinic herbicide picloram treatment (Figure 9), suggesting its special role in eggplant. In addition, the increased expression of SmAFB5 under drought treatment and VIGS-induced SmAFB5 knockdown lines showed resistance to drought, indicating its function in drought, but further exploration is needed to uncover this mechanism. In addition, the cis-element analysis showed that AuxRR was only located in SmAFB5, indicating its special function in response to auxin.
Together, these analyses provide insight into the expression and regulation of SmTIR1/AFBs, highlighting differences in their responses to light, temperature, development, and herbicide treatment. In the future, more research will be required to integrate TIR1/AFBs and their regulators into the more extensive network of plant growth regulation.

5. Conclusions

We identified five TIR1/AFB genes in the S. melongena genome, two of which were present on chromosome 6. The SmTIR1/AFBs had similar gene structures and their encoded proteins had similar conserved motifs. Predicted 3D protein structures of the TIR1/AFBs were very similar, differing only in one angle, which may have functional significance. AFB6 was present in tomato and eggplant but not in Arabidopsis, suggesting that its function may be performed by different TIR1/AFB homolog(s) in Arabidopsis. An analysis of promoter cis-elements suggested that TIR1/AFB expression is regulated by hormones, growth stage, and stress conditions, and gene expression analysis confirmed these results. miRNA predictions identified a number of miRNAs that likely regulate the TIR1/AFB genes. These findings support further research on the roles of TIR1/AFB genes in eggplant development and stress response via methods such as overexpression, RNAi, and genome editing.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/agronomy14071413/s1, Figure S1: Alignment of the SmTIR1/AFB proteins, showing the location of residues associated with the angle that differed among the proteins (red box) and was located between an α helix (blue box) and a β sheet (green box); Table S1: 12 genes annotated as TIR1/AFB in the Solanum melongena.; Table S2: Predicted miRNAs and their target genes in Solanum melongena; Table S3: Primers for qRT–PCR analysis; Table S4: Numbers of secondary structures in the SmTIR1/AFB proteins.

Author Contributions

W.D., Q.L. and X.C. conceived the research idea; W.D., U.K. and L.C. collected the materials and wrote the first draft of the manuscript; H.L. (Haili Li), Y.L. and H.L. (Haoxin Li) performed the bioinformatic analyses; L.W., D.Y. and M.X. visualized the data; X.C. and Q.L. interpreted the data. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (grant no. 32172567), the Vegetable Innovation Team Project of Hebei Modern Agricultural Industrial Technology System (grant no. HBCT2018030203), and the Doctoral Fund of Baoding University (grant no. 2023B03).

Data Availability Statement

The raw data supporting the conclusions of this article will be made available by the authors on request.

Acknowledgments

We would like to thank Fengqing Cheng & Shiyao You (Hebei Agricultural University) for their assistances during the preparation of this manuscript.

Conflicts of Interest

The authors declare no competing interests.

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Figure 1. Locations of the five SmTIR1/AFB genes on four of the twelve S. melongena chromosomes.
Figure 1. Locations of the five SmTIR1/AFB genes on four of the twelve S. melongena chromosomes.
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Figure 2. Phylogenetic analysis of TIR1/AFB proteins. Phylogenetic tree of TIR1/AFB proteins from Arabidopsis thaliana, S. lycopersicum, and S. melongena. Phylogenetic analysis of five TIR1/AFB proteins identified in S. melongena. S. melongena TIR1/AFBs were present in three clades and named with reference to their Arabidopsis homologs. Clustal W was used to align the full-length protein sequences and MEGA X was used to construct neighbor-joining phylogenetic trees with 1000 bootstrap replicates.
Figure 2. Phylogenetic analysis of TIR1/AFB proteins. Phylogenetic tree of TIR1/AFB proteins from Arabidopsis thaliana, S. lycopersicum, and S. melongena. Phylogenetic analysis of five TIR1/AFB proteins identified in S. melongena. S. melongena TIR1/AFBs were present in three clades and named with reference to their Arabidopsis homologs. Clustal W was used to align the full-length protein sequences and MEGA X was used to construct neighbor-joining phylogenetic trees with 1000 bootstrap replicates.
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Figure 3. Gene structures (left) and conserved protein motifs and domains (right) of the S. melongena TIR1/AFBs.
Figure 3. Gene structures (left) and conserved protein motifs and domains (right) of the S. melongena TIR1/AFBs.
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Figure 4. Circos plot showing the SmTIR1B and SmTIR1C gene pair (connected in red) that appeared to have arisen via segmental duplication. Gray lines indicate collinear gene blocks.
Figure 4. Circos plot showing the SmTIR1B and SmTIR1C gene pair (connected in red) that appeared to have arisen via segmental duplication. Gray lines indicate collinear gene blocks.
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Figure 5. Identification of cis-acting elements in the TIR1/AFB gene promoters and effects of light and temperature on TIR1/AFB gene expression. (A) cis-acting elements identified in the promoters of the TIR1/AFB genes. ARE, auxin-responsive element. LRE, light-responsive element. ABRE, abscisic acid-responsive element. GAE, gibberellic acid-responsive element. SRE, stress-responsive element. MeJA, methyl jasmonate-related element. SAR, salicylic acid-responsive element. GRE, growth-related element. MYC, MYC element. MYB, MYB binding site involved in light response. AuxRR, an auxin-responsive cis-element present only in SmAFB5, highlighted in a red box. (B) Numbers of different cis-acting elements in the TIR1/AFB promoters. (C) Numbers of cis-acting elements related to hormones (HRE, including ARE, MeJA, SAR, ABAR, and GAE), light (LRE), stress (SRE), and growth (GRE) in the promoters of individual TIR1/AFB genes.
Figure 5. Identification of cis-acting elements in the TIR1/AFB gene promoters and effects of light and temperature on TIR1/AFB gene expression. (A) cis-acting elements identified in the promoters of the TIR1/AFB genes. ARE, auxin-responsive element. LRE, light-responsive element. ABRE, abscisic acid-responsive element. GAE, gibberellic acid-responsive element. SRE, stress-responsive element. MeJA, methyl jasmonate-related element. SAR, salicylic acid-responsive element. GRE, growth-related element. MYC, MYC element. MYB, MYB binding site involved in light response. AuxRR, an auxin-responsive cis-element present only in SmAFB5, highlighted in a red box. (B) Numbers of different cis-acting elements in the TIR1/AFB promoters. (C) Numbers of cis-acting elements related to hormones (HRE, including ARE, MeJA, SAR, ABAR, and GAE), light (LRE), stress (SRE), and growth (GRE) in the promoters of individual TIR1/AFB genes.
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Figure 6. Predicted 3D structures of the S. melongena TIR1/AFB proteins. Coiled shapes represent α helices, flat ribbon shapes represent β sheets, and red circles highlight a single angle that showed subtle differences among the proteins.
Figure 6. Predicted 3D structures of the S. melongena TIR1/AFB proteins. Coiled shapes represent α helices, flat ribbon shapes represent β sheets, and red circles highlight a single angle that showed subtle differences among the proteins.
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Figure 7. Number of miRNAs predicted to target TIR1/AFB genes based on miRNAs identified in S. lycopersicum (A) and S. tuberosum (B).
Figure 7. Number of miRNAs predicted to target TIR1/AFB genes based on miRNAs identified in S. lycopersicum (A) and S. tuberosum (B).
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Figure 8. Expression of the SmTIR1/AFB genes. (A) Expression of the TIR1/AFB genes in leaves at the four-leaf stage (W4) and six-leaf stage (W6). (B) Expression of TIR1/AFB genes in leaves 6 h after treatment with water (CK) or the herbicide picloram (P). 1, 2, and 3 after hyphen represent three independent technical replicates of each treatment, |log2FC| ≥ 1, FDR ≤ 0.01. (C) Expression of TIR1/AFB genes in the dark at normal temperature and irrigation (BN, 25 °C), in the light at normal temperature (LN, 25 °C), in the dark at low temperature (BL, 10 °C), and in the dark with drought stress (BD, watering ceased for 6 days), as measured by RT–qPCR. ** p < 0.01, * p < 0.1 (Student’s t-test, compared with BN).
Figure 8. Expression of the SmTIR1/AFB genes. (A) Expression of the TIR1/AFB genes in leaves at the four-leaf stage (W4) and six-leaf stage (W6). (B) Expression of TIR1/AFB genes in leaves 6 h after treatment with water (CK) or the herbicide picloram (P). 1, 2, and 3 after hyphen represent three independent technical replicates of each treatment, |log2FC| ≥ 1, FDR ≤ 0.01. (C) Expression of TIR1/AFB genes in the dark at normal temperature and irrigation (BN, 25 °C), in the light at normal temperature (LN, 25 °C), in the dark at low temperature (BL, 10 °C), and in the dark with drought stress (BD, watering ceased for 6 days), as measured by RT–qPCR. ** p < 0.01, * p < 0.1 (Student’s t-test, compared with BN).
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Figure 9. Expression of SmTIR1/AFB genes at the two-leaf and four-leaf stages (A) and in response to picloram treatment (B), as measured by RT–qPCR (bars) and RNA-seq (red symbols). For RT–qPCR, gene expression levels were normalized to those of Smechr0302615 (tubulin gamma). In (B), RT–qPCR expression levels in the CK were set to 1. Data are presented as the means ± SE of three biological replicates. * p < 0.1 (Student’s t-test, RNA-seq data).
Figure 9. Expression of SmTIR1/AFB genes at the two-leaf and four-leaf stages (A) and in response to picloram treatment (B), as measured by RT–qPCR (bars) and RNA-seq (red symbols). For RT–qPCR, gene expression levels were normalized to those of Smechr0302615 (tubulin gamma). In (B), RT–qPCR expression levels in the CK were set to 1. Data are presented as the means ± SE of three biological replicates. * p < 0.1 (Student’s t-test, RNA-seq data).
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Figure 10. Effect of silencing SmAFB5 on eggplant susceptibility to drought. (A) Expression of SmAFB5 in leaves 30 days after VIGS. The asterisks indicate statistically significant differences as determined by Student’s t-test (two-tailed). * p < 0.1. (B) Representative seedlings at 12 h recovery after drought treatment. TRV2::00 (TRV2::00 plants treated with drought), TRV2::SmAFB5 (TRV2::SmAFB5 plants treated with drought). Bar = 1 cm.
Figure 10. Effect of silencing SmAFB5 on eggplant susceptibility to drought. (A) Expression of SmAFB5 in leaves 30 days after VIGS. The asterisks indicate statistically significant differences as determined by Student’s t-test (two-tailed). * p < 0.1. (B) Representative seedlings at 12 h recovery after drought treatment. TRV2::00 (TRV2::00 plants treated with drought), TRV2::SmAFB5 (TRV2::SmAFB5 plants treated with drought). Bar = 1 cm.
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Table 1. TIR1/AFB genes identified in S. melongena.
Table 1. TIR1/AFB genes identified in S. melongena.
Gene IDInstability Index Amino AcidsGRAVY ScoreIntrons, ExonsPredicted Subcellular Location
SmAFB687.87587−0.1562, 3Nucleus, cytoplasm
SmAFB538.35621−0.0082, 3Nucleus, cytoplasm
SmTIR1A45.356050.0292, 3Chloroplast, cytoplasm
SmTIR1B52.81576−0.0242, 3Nucleus, cytoplasm
SmTIR1C55.93581−0.062, 3Nucleus
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Du, W.; Karamat, U.; Cao, L.; Li, Y.; Li, H.; Li, H.; Wei, L.; Yang, D.; Xia, M.; Li, Q.; et al. The TIR1/AFB Family in Solanum melongena: Genome-Wide Identification and Expression Profiling under Stresses and Picloram Treatment. Agronomy 2024, 14, 1413. https://doi.org/10.3390/agronomy14071413

AMA Style

Du W, Karamat U, Cao L, Li Y, Li H, Li H, Wei L, Yang D, Xia M, Li Q, et al. The TIR1/AFB Family in Solanum melongena: Genome-Wide Identification and Expression Profiling under Stresses and Picloram Treatment. Agronomy. 2024; 14(7):1413. https://doi.org/10.3390/agronomy14071413

Chicago/Turabian Style

Du, Wenchao, Umer Karamat, Liuqing Cao, Yunpeng Li, Haili Li, Haoxin Li, Lai Wei, Dongchen Yang, Meng Xia, Qiang Li, and et al. 2024. "The TIR1/AFB Family in Solanum melongena: Genome-Wide Identification and Expression Profiling under Stresses and Picloram Treatment" Agronomy 14, no. 7: 1413. https://doi.org/10.3390/agronomy14071413

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