Comparative transcriptome analysis identifies jasmonic pathway genes related to anther dehiscence in Solanum melongena L.

Background: Anther indehiscence is an important form of functional male sterility that can facilitate the production of hybrid seeds. However, the molecular mechanisms of anther indehiscence-based male sterility in eggplant ( Solanum melongena L.) have not been thoroughly explored. Therefore, we performed RNA-seq analysis to investigate the molecular mechanisms of anther dehiscence in eggplant. Results: We used transcriptome sequencing and qRT-PCR assays to compare the anthers of normally developing (F142) and anther indehiscent (S12) eggplant. We identified 2670 differentially expressed genes between lines. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses identified 31 differentially expressed genes related to hormone biosynthesis. We therefore measured the contents of the phytohormones jasmonic acid (JA), IAA, GA3 and ABA in S12 and F142. There were no significant differences in IAA, GA3 or ABA levels between S12 and F142. However, JA levels were significantly lower in S12 than in F142. Five key genes in the JA signaling pathway were differentially expressed in S12 vs. F142. Of these, SmJAZ and SmJAR-like were significantly up-regulated and SmDAD1, SmLOX and SmCOI1 were down-regulated in S12 vs. F142. In DNA-protein interaction studies, SmLOX, SmAOC, SmOPR3, SmCOI1 and SmJAZ1 failed to be enriched on the chromatin of SmDAD1. Protein–protein interaction studies identified a direct interaction between SmDAD1 and SmLOX, but SmDAD1 failed to interact with SmAOC, SmOPR3, SmCOI1 and SmJAZ1. Conclusion: JA is an essential factor affecting anther dehiscence in eggplant. SmDAD1 interacts with SmLOX to alter JA levels, thereby regulating anther dehiscence. Our data represent a valuable resource for further exploring the regulatory mechanisms underlying anther dehiscence in eggplant.


Introduction
Eggplant (Solanum melongena L.), a popular vegetable crop that is thought to have originated in Africa, is widely cultivated in Africa, Asia, Europe, and the Near East [1]. The major characteristics of eggplant exhibit obvious heterosis, and the early use of hybrid vigor in the breeding of eggplant cultivars has been described. The use of reliable male-sterile systems could simplify the process and reduce the labor, cost, and time involved in producing hybrid eggplant seeds [2]. Functional genic male sterility (GMS) was first reported in eggplant in 1954 [3][4][5][6]. These GMS lines show anther indehiscence in which the anthers do not open to release pollen, thereby disabling pollination [7].
Anther dehiscence is a vital process in which mature pollen grains are released from the locules of the anther, thus enabling pollination [8,9]. Although the morphological changes in anthers during dehiscence have been thoroughly described [8,10], the molecular mechanisms controlling anther dehiscence remain relatively unknown.
Jasmonic acid (JA) is a lipid-derived hormone that functions as an important regulator of plant responses to various stresses as well as development [11]. Analysis of plants showed that JA affects wheat development, including germination, growth, flowering time, senescence, and alters tolerance to environmental stresses [12]. Wheat plants with high JA levels are characterized by delayed germination, slower growth, late flowering and senescence, and improved tolerance to short-term freezing [13]. The application of exogenous jasmonate significantly stimulated root hair elongation [1]. JA plays an important role in regulating anther dehiscence [14,15]. JA signal transduction pathways have been investigated in Arabidopsis thaliana [16,17]. JA biosynthesis originated from fatty acids in chloroplasts, and then its metabolic compounds are produced from 12 different pathways in peroxisomes and cytosol, respectively [16].
Gene mutations involved in JA biosynthesis cause failure or delay of anther dehiscence and may lead to male sterility. Several of these genes have been identified, such as anther dehiscence defect 1 (DAD1) [14], AOS [18]; LOX [19]; COI1 [20]; DEHISCENCE 1 (DDE1)/OPR3 [21,22]; and the triple mutation (fad3 fad7 and fad8) [23]. In addition, JAR1 (a JA-amino acid synthetase) has the biological function in regulating the flower opening and closure and anther dehiscence in rice [15]. Some studies have found possible mechanisms that JA is a significant regulator of anther dehiscence.
However, the exact mechanisms of JA activity regulating anther dehiscence remain to be elucidated. It is also unclear how genes in the JA pathway are mutually regulated.
In the current study, we performed transcriptome analysis to identify differentially expressed genes (DEGs) in eggplant S12 (indehiscent anthers) and F142 (normal anther development) in order to uncover differences in the anther indehiscence network. Enrichment analysis of the DEGs and endogenous hormone measurements highlighted the effect of JA signal transduction pathways in anther dehiscence. Finally, we analyzed the relationships between JA pathway genes by yeast twohybrid and yeast one-hybrid analyses. Our results lay the foundation for further uncovering the molecular mechanisms and biological function of anther dehiscence in eggplant.

Morphological Comparison Of F142 And S12
Anther indehiscence is an important form of functional male sterility. Morphological analysis indicated that eggplant accession S12 produced indehiscent anthers, whereas F142 produced normal anthers (Figs. 1A, B, C and D). We used the 2, 3, 5-triphenyl tetrazolium chloride staining method to further investigate pollen viability. The activity of S12 pollen was similar to that of F142 pollen (Fig. 1E, F).
These results indicate that S12 produces anthers with an abnormal appearance but has normal pollen viability.

Transcriptome Assembly
To detect potential molecular differences between eggplant accessions S12 and F142, we performed transcriptome sequencing on anther tissue. The genome-directed stratagemTrinity was used to assemble transcriptome sequences and align single RNA-seq library data with the S. melongena genome (SME_r2.5.1). As a result, the mean of clean reads from three biological repeats per sample was more than 20 million, the mean of mapped reads and uniquely mapped reads were far great than 10 million, and the highest localization ration of each library was roughly 50% in the S. melongena genome ( Table 1 and Table S2). After being assembled the average length of the contig was 1204 bp, while the contig of N50 was 1538 bp. We found that the average GC content in the libraries reached 40-50% (Table S2 and S3). When redundant and short reads were removed, we obtained about 257,800 transcript assembly contigs (TAC) > 100 bp (Table S2). Overall the abundant transcriptome data are enough for further analysis. Analysis Of Biological Processes In S12 We identified 2,670 DEGs in S12 vs. F142 (Table S4 and Figure S2), including 1,928 up-regulated and 742 down-regulated DEGs ( Figure S2). We constructed a heatmap representing the differential expression of the 2670 DEGs ( Figure S5) in S12 vs. F142. We detected dynamic changes in the transcriptomes during anther development in S12 (anther indehiscence). In the heatmap, the original gene expression data were transformed into log2 fold change values (Table S4). Further cluster analysis revealed significant differences in gene expression between F142 and S12.
To characterize the DEGs in detail, we performed Gene Ontology (GO) analysis to uncover their putative functions. We constructed histograms based on the categories of DEGs in anther indehiscent eggplant (S16), including biological processes, molecular functions, and cellular compartments ( Figure S3). Metabolic process was the major group in the biological processes category, including GO terms cellular, single-organism, response to stimulus, and biological regulation. The molecular functions category contained at least 882 DEGs involved in nucleic acid binding transcription factor activity and more than 888 DEGs involved in signal transducer activity, transcription factor activity, and protein binding. Most of the DEGs were present in the top three groups, including the cell, organelle, and membrane ( Figure S4). We performed KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis to categorize all annotated genes. Most of the 2,670 DEGs were categorized into six pathways ( Figure S4). The most highly enriched biological processes in anther indehiscent eggplant were plant hormone signal transduction, protein processing in endoplasmic reticulum, amino sugar and nucleotide sugar metabolism, biosynthesis of amino acids, carbon metabolism and plant-pathogen interactions ( Fig. 2 and S4).

Enrichment Analysis Of Degs In Hormone Signal Transduction Pathways
We performed KEGG pathway analysis to investigate the major regulatory pathways of the DEGs. The  Table 2). Five key genes were identified in the JA signaling pathway, including DAD1, LOX, COI1, JAZ1 and JAR-like, including two that were significantly upregulated and three that were downregulated in S12 vs. F142. One key gene in the CTK signaling pathway and two genes in the ABA signaling pathway were differentially expressed in S12 vs. F142. Two key genes in the GA signaling pathway were significantly upregulated and two were significantly down-regulated in S12 vs. F142. Four key genes in the IAA signaling pathway were differentially expressed in S12 vs. F142. Four key genes in the ETH signaling pathway were also differentially expressed in S12 vs. F142; all four were significantly up-regulated in S12 vs.
F142. Four genes in the BR pathway were significantly upregulated in S12 vs. F142 as well. Finally, nine genes in other hormone signaling pathways were differentially expressed in S12 vs. F142, including two that were significantly down-regulated in the indehiscent accession ( Fig. 4). Endogenous hormone measurements and validation of the expression patterns of several key genes We collected independent anther samples from the plants and performed qRT-PCR analysis to validate the expression levels of several key JA-related genes. In total, we measured the expression levels of seven JA-related unigenes via qRT-PCR. JAZ1 and JAR1 were significantly up-regulated and DAD1, LOX, OPR3, AOC and COI1 were significantly down-regulated in S12. The expression patterns of these genes corresponded well with the FPKM values obtained by RNA-seq (Fig. 6), suggesting that the expression patterns of most unigenes were consistent between the two methods. Finally, we measured JA, IAA, GA 3 and ABA levels in S12 and F142 (Fig. 5). There were no significant difference in IAA, GA 3 or ABA levels in S12 vs. F142. By contrast, JA levels were significantly lower in S12 than in F142. These findings suggest that JA is an important factor for anther dehiscence in eggplant.

Discussion
Transcriptome analysis facilitates the comprehensive investigation of altered gene expression patterns in genetic variants and provides insights into the molecular basis of specific biological processes [27]. Transcriptome sequencing provides a systemic approach for studying gene expression patterns and network interactions underlying various processes in plants. In the current study, to explore the molecular mechanism underlying anther dehiscence in eggplant, we generated a highquality transcriptome dataset from anthers of normally developing (F142) and anther indehiscent (S16) eggplant. The most highly enriched biological processes among the DEGs in indehiscent anthers were plant hormone signal transduction, protein processing in endoplasmic reticulum, amino sugar and nucleotide sugar metabolism, biosynthesis of amino acids, carbon metabolism and plantpathogen interaction.
We identified 31 DEGs (Table 2)  detected in fertile plants [28]. This phenomenon was also observed in tomato mutants [29]. Horner suggested that auxin accumulation causes male sterility in crops, as high IAA levels in pepper induced the production of ETH, which induced male sterility [30]. In addition, Li Yingxian et al. studied K and T types of sterile wheat and determined that during pollen abortion, IAA levels were lower in sterile anthers than in fertile anthers [31]. This phenomenon was also observed in rice [32], citrus [33], and mustard [34].
Moreover, CTK levels were lower in cytoplasmic male sterile barley lines than in their maintainer lines [35]. The excess ABA in the leaves and anthers of cytoplasmic male sterile cabbage lines might be related to the occurrence of microspore abortion [36]. Liu found that in male sterile wheat lines (induced by GENESIS), after induction, ETH levels were significantly higher in sterile lines than in fertile lines during the mononuclear, dinuclear and trinuclear stages of anther development [37]. The rate of infertility increased with increasing induction, and the rate of infertility and the ETH release rate also increased. Finally, wild-type Arabidopsis plants treated with GA and double mutants in the GA signaling repressors RGA and GAI exhibited a loss of fertility [38].
Plant development and responses to environmental signals are coordinated by complex multicomponent signaling networks. JA, a phytohormone derived from fatty acids, is an important component of this regulatory system. JA participates in all stages of plant growth and development. JA also regulates anther dehiscence [15,39]. In the current study, the transcript levels of JA biosynthesis genes were always lower in anther indehiscent plants than in plants with normal anther development.
The JA content was also significantly lower in these plants than in normal eggplant. This observation, which is consistent with the results of transcriptome sequencing, confirms the notion that JA is an essential factor affecting anther dehiscence. This finding validates the results of previous studies. For example, mutations in genes involved in JA biosynthesis typically cause delayed or failed anther dehiscence, including DAD1 [14,40] and OPR3 [21,41].
Based on the current transcriptome data for genes in the JA biosynthesis pathway, we propose that feedback regulation of JA signaling in anther-indehiscent eggplant alters the expression patterns of genes at the mRNA level during anther development [42][43][44]. In this study, we identified 5 JA pathway genes (DAD1, LOX, COI1, JAZ1, and JAR-like) that were differentially expressed in S12 vs. F142 eggplant. DAD1, LOX and COI1 were clearly downregulated in S12, whereas JAZ1 and JAR-like were upregulated in S12 vs. F142. DAD1 is crucial for JA biosynthesis. Arabidopsis DAD1 (At2g44810) encodes the first chloroplastic lipase identified. This enzymes is involved in supplying α-linolenic acid for the JA-biosynthetic pathway [14]. Mutations in DAD1 reduce JA levels in flower buds, causing a delay in their development, failed anther dehiscence during with flower opening, and the lack of pollen grain maturation [14]. In the Arabidopsis coi1, opr3, and dad1 mutants, the anthers fail to crack and the filaments are short: these phenotypes were significantly altered by the external application of JA [21,45].
The JA pathway involves a series of genes-encoded hormone-related factors involved in anther dehiscence [15,25,46]. Previous gene expression analysis demonstrated that overexpressing AtOPR3 selectively affected the expression of various genes of the endogenous jasmonate system, while the expression of other genes remained unaltered. Transgenic wheat plants with high AtOPR3 expression levels exhibited notably altered plant growth and development, including delayed germination, slower growth, and anther indehiscence [47]. These findings indicate that these plant phenotypes are regulated by direct or indirect interactions of these genes.
In the current study, we measured the enrichment of SmLOX, SmAOC, SmOPR3, SmCOI1, and SmJAZ1 on the chromatin of SmDAD1. None of these proteins bound to the promoter of SmDAD1. Maeli (2008) demonstrated that the physical interaction between COI1 and JAZ proteins could be effectively promoted by treatment with biologically active jasmonates (JA-Ile) [28,48]. In addition, AtMYC2 interacts with JAZs [25,49]. Here, we demonstrated that SmDAD1 interacts with SmLOX1 in vitro and in Y2H assays. However, how these proteins regulate anther dehiscence remains unclear and should be addressed in future studies.

Conclusion
The transcriptomes compared results of the normally developing anthers (F142) and indehiscent (S12) anther of eggplant showed that the number of genes differentially expressed in S12 was 2670 and mainly enriched in inositol phosphate metabolism, plant hormone signal transduction, flavonoid biosynthesis, amino sugar and nucleotide sugar metabolism. Gene Ontology (GO) database and Kyoto Encyclopedia of Genes and Genomes (KEGG) database analyses identified 31 genes related to hormone biosynthesis. The contents of JA, IAA, GA 3 and ABA were measured in F142 and S12. It was found that there was no significant difference in IAA, GA 3 and ABA between F142 and S12. On the contrary, the content of JA in S12 was significantly lower than that of F142. Five key genes were identified in the jasmonic acid (JA) signaling pathway, of which SmJAZ and SmJAR-like were significantly upregulated and SmDAD1, SmLOX and SmCOI1 were downregulated. In addition, DNAprotein interaction studies identified failed to enrich of SmLOX, SmAOC, SmOPR3, SmCOI1 and SmJAZ1 on the chromatin of SmDAD1. Protein-protein interaction studies identified a direct interaction between SmDAD1 and SmLOX, but SmDAD1 failed to interact with SmAOC, SmOPR3, SmCOI1 and SmJAZ1. This test provides valuable information for subsequent related research.
Furthermore, the specific regulatory mechanisms of these genes in anther dehiscence need further study.

Materials And Methods Plant Materials and Growth Conditions
The S12 ( Transcriptome analysis using reference genome-based reads mapping Low quality reads, such as only adaptor, unknown nucleotides>5%, or Q20 <20% (percentage of sequences with sequencing error rates <1%), were removed by perl script. The clean reads that were filtered from the raw reads were mapped to eggplant genome (SME_r2.5.1) using Tophat2 [50] software. The aligned records from the aligners in BAM/SAM format were further examined to remove potential duplicate molecules. Gene expression levels were estimated using FPKM values (fragments per kilobase of exon per million fragments mapped) by the Cufflinks software [51].

Identification of differential gene expression
DESeq and Q-value were employed and used to evaluate differential gene expression between F142 and S12. After that, gene abundance differences between those samples were calculated based on the ratio of the 2 FPKM values. The false discovery rate (FDR) control method was used to identify the threshold of the P-value in multiple tests in order to compute the significance of the differences. Here, only gene with an absolute value of log2 ratio ≥2 and FDR significance score <0.01 were used for subsequent analysis.

Sequence Annotation
Genes were compared against various protein database by BLASTX, including the National Center for Biotechnology Information (NCBI) non-redundant protein (Nr) database, Swiss-Prot database with a cut-off E-value of 10-5. Furthermore, genes were searched against the NCBI non-redundant nucleotide sequence (Nt) database using BLASTn by a cut-off E-value of 10-5. Genes were retrieved based on the best BLAST hit (highest score) along with their protein functional annotation.
To annotate the gene with gene ontology (GO) terms, the Nr BLAST results were imported into the Blast2 GO program. GO annotations for the genes were obtained by Blast2 GO. This analysis mapped all of the annotated genes to GO terms in the database and counted the number of genes associated with each term. Perl script was then used to plot GO functional classification for the unigenes with a GO term hit to view the distribution of gene functions. The obtained annotation was enriched and refined using TopGo (R package). The gene sequences were also aligned to the Clusters of Orthologous Group (COG) database to predict and classify functions [52]. KEGG (kyoto encyclopedia of genes and genomes) pathways were assigned to the assembled sequences by perl script.

Hormone extraction and determination
Endogenous hormone extraction and purification are based on the method of Yu mei et al. The high performance liquid chromatograph is Shimadzu LC-60A. Chromatographic conditions: mobile phase was methanol 0.8% glacial acetic acid solution = 55/45, column temperature was 30°C, flow rate was 0.8 mL/min, detection wavelength was 254 nm, injection volume was 10 μL. Each sample was tested 3 times and averaged. The data was statistically analyzed using Graphpad Prism 8 and SPSS 22.0.

Determination of pollen vigor
The pollen viability was determined by TTC method [53]. A small amount of pollen was placed on a clean glass slide, a drop of 0.5% TTC solution was added, stirred, covered with a cover glass, and stained in a 35°C incubator for 20 min. Observe with a microscope and calculate pollen vigor.

Quantitative real-time PCR
The qRT-PCR was performed as previously described [54]. The primers used to test the transcript levels of all genes are shown in Table S4 Meanwhile, the promoters of SmDAD1 was ligated into pGreenII 0800-LUC. All the above recombinants were transformed into agrobacterium (GV3101) then co-infiltrated the leaves of Nicotianabenthamian for measuring the activities of fluorescein enzyme via GLOMAX® multifunctional instrument (Promega, USA).
Yeast two-hybrid assay SmDAD1, SmLOX, SmAOC, SmOPR3, SmCOI1, SmJAZ1 were separately sub-cloned into the activation domain of pGADT7 and pGBKT7 using the BamHI and XhoI sites and then ligated into pGADT7 or pGBKT7 to construct the recombinant plasmids. Using Matchmaker TM Gold Yeast Two-Hybrid System, the recombinants were respectively transformed into Y187 and Y2H (Clontech) for detecting the protein-protein interactions. In addition, toxicity and self-activation detection of the recombinant plasmids had been performed previously.
Pull-down assay

Supplementary Files
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