Transcriptome analysis reveals the molecular mechanisms underlying the enhancement of salt-tolerance in Melia azedarach under salinity stress

Melia azedarach demonstrates strong salt tolerance and thrives in harsh saline soil conditions, but the underlying mechanisms are poorly understood. In this study, we analyzed gene expression under low, medium, and high salinity conditions to gain a deeper understanding of adaptation mechanisms of M. azedarach under salt stress. The GO (gene ontology) analysis unveiled a prominent trend: as salt stress intensified, a greater number of differentially expressed genes (DEGs) became enriched in categories related to metabolic processes, catalytic activities, and membrane components. Through the analysis of the category GO:0009651 (response to salt stress), we identified four key candidate genes (CBL7, SAPK10, EDL3, and AKT1) that play a pivotal role in salt stress responses. Furthermore, the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analysis revealed that DEGs were significantly enriched in the plant hormone signaling pathways and starch and sucrose metabolism under both medium and high salt exposure in comparison to low salt conditions. Notably, genes involved in JAZ and MYC2 in the jasmonic acid (JA) metabolic pathway were markedly upregulated in response to high salt stress. This study offers valuable insights into the molecular mechanisms underlying M. azedarach salt tolerance and identifies potential candidate genes for enhancing salt tolerance in M. azedarach.


Sample collection
In August 2020 (after 40 months of M. azedarach growth), three samples of M. azedarach roots were collected from each plot.The M. azedarach roots were excavated with a shovel and subsequently stored in a − 80 °C freezer for transcriptome analysis.Root samples (R) collected for transcriptome analysis were labelled: HR, MR, and LR for high, medium, and low salinity, respectively.

RNA extraction, library construction, and RNA-Seq
The RNA was extracted from the roots of M. azedarach grown in soils characterized by high, medium, and low salt content using the Trizol reagent kit (Invitrogen, Carlsbad, California, USA) 16 .The quality of the RNA samples was checked using a NanoDrop 2000 Spectrophotometer.A260/280 was in the range of 1.80-2.0.A total of 1 μg of RNA per sample served as the input material for sequencing.Biomarker Technologies, based in Beijing, China, performed the RNA sequencing.Sequencing libraries were constructed using a NEBNext®Ultra™ RNA Library Prep Kit for Illumina ® (NEB, MA, USA), following the manufacturer's prescribed procedures.Index codes were incorporated to distinguish sequences associated with each specific sample.Library quality was assessed using an Agilent Bioanalyzer 2100 system.The sequencing took place on an Illumina Hiseq 2000 platform, generating paired-end reads.
To obtain clean data, reads containing adapters, reads containing poly-N sequences, and low-quality reads were removed from the raw data 17 .Additionally, the Q30 score and the GC content of the clean reads were calculated.All subsequent analyses were conducted using these high-quality clean reads.

Transcriptome assembly and function annotation
Transcriptome assembly was accomplished based on the left.fqand right.fqusing Trinity with min_kmer_cov and all other parameters set to default 18 .Gene function was annotated based on the following databases: NR (NCBI non-redundant protein sequences), Pfam (Protein family), KEGG (Kyoto Encyclopedia of Genes and Genomes), and GO (Gene Ontology).

Differentially expressed genes (DEGs)
Differential expression analysis was performed using the DESeq R package (v.1.10.1).Genes with | log 2 (foldchange) |≥ 1 & an adjusted p-value < 0.05 found by DESeq were assigned as differentially expressed.The GO enrichment analysis of DEGs was implemented by the topGO R packages based on Kolmogorov-Smirnov test.KOBAS software was used to test the statistical enrichment of DEGs in KEGG pathways 19 .

RT-qPCR validation of DEGs
Total RNA was reverse-transcribed into cDNA using a TaKaRa reverse transcription kit (TaKaRa, 6210A).To verify the reliability of RNA-Seq data, 20 DEGs with high expression levels were randomly selected for RT-qPCR validation using ABI Stepone Plus Real-Time PCR System with Tower (ABI, US).Premier 6.0 was utilized for primer design, with the actin gene serving as the internal reference gene.The PCR cycling parameters were initial 95 °C for 2 min, then 40 cycles of 95 °C for 5 s, and 60 °C for 15 s.The experimental data were analyzed by the 2 −ΔΔCt method.

Statistics and data nalysis
An independent sample t-test was performed using IBM SPSS Statistics 20 software (IBM, Armonk, New York, USA), and the significance level of the difference was determined based on p < 0.05.

Transcriptome sequencing quality analysis
From the nine transcriptome libraries, a total of 54.88 Gb of clean reads were obtained, with average sample contributing approximately 6.1 Gb of clean data.It is noteworthy that more than 82% of the sequences within each library were successfully mapped, which underscores the suitability of the assembled transcriptome for conducting differential expression analyses.Furthermore, the sequencing data displayed a high level of quality, with a Q30 base percentage exceeding 93.5% (Table S1).
The saturation analysis of the transcriptome data exhibited a saturable curve.This pattern suggests that the quantity of effective sequencing data obtained was sufficient for the study objectives (Fig. S1).

DEGs analysis of M. azedarach under salt stress
The complete list of DEGs is given in the Supplementary Data (Table S2).The results of PCA indicate that samples from different salinities were distinctly separated from one another, meaning that the transcriptome of M. azedarach roots exhibited substantial variations in response to different salinity levels (Fig. 1a).The heatmap analysis demonstrated that the patterns of up-regulation and down-regulation of DEGs varied significantly under different salinity conditions (Fig. 1b).With increasing salt stress, there was an apparent trend of a growing number of up-regulated DEGs, accompanied by a corresponding decline in the number of down-regulated DEGs (Fig. 1c).The Venn diagram showed that 2916 DEGs were shared in the MR vs. LR and the HR vs. LR comparisons (Fig. 1d).
The GO functional enrichment analysis of DEGs revealed their enrichment in 47 GO annotations.Among these, the biological process (BP) category accounted for 39.62%, the cellular component (CC) category accounted for 38.35%, and the molecular function (MF) category accounted for 22.03% of the enriched terms.Within the BP category, the DEGs were predominantly enriched for terms such as metabolic process (GO:0008152), cellular process (GO:0009987), and single organism process (GO:0044699).In the CC category, the primary enrichments included membrane (GO:0016020), cell (GO:0005623), and cell part (GO:0044464).As the severity of salt stress increased, an increasing number of DEGs was enriched in the GO annotation (Fig. 2).
The enrichment data pertaining to up-regulated DEGs within the 47 GO annotations, as delineated in Table S3, exhibited a consistent pattern.This pattern revealed a notable increase in the number of up-regulated DEGs as salt stress was exacerbated.This trend was particularly prominent in categories associated with metabolic processes, catalytic activities, and membrane.
To identify the metabolic pathways involved in the response to salt stress, we conducted the KEGG pathways enrichment analysis.It revealed a significant enrichment of DEGs in the MR vs. LR in starch and sucrose metabolism, plant hormone signal transduction, amino sugar and nucleotide sugar metabolism, as well as in cysteine and methionine metabolism (Fig. 3a).In the HR vs. LR comparison, there was a notable enrichment in pathways related to plant hormone signal transduction and starch and sucrose metabolism (Fig. 3b).
The signal transduction pathways associated with plant hormones (ko04075) is presented in Fig. 4. At varying severities of salt stress, there was a selective enrichment of M. azedarach genes involved in various phytohormonal signaling pathways, including auxin, cytokinin, gibberellin, abscisic acid, ethylene, brassinosteroid, jasmonic acid, and salicylic acid.Within brassinosteroid metabolism, the genes encoding BRI1, BZR1/2, and CYCD3 were downregulated in both MR vs LR and HR vs LR comparisons.This downregulation would be expected to result in the inhibition of cell elongation and cell division processes.Regarding jasmonic acid metabolism, genes encoding JAZ and MYC2 were upregulated in HR vs LR comparisons (Fig. 4b).
In response to salt stress and based on the GO analysis of BP under the term GO:0009651 (response to salt stress), four key DEGs were identified.The CBL7 and SAPK10 genes were up-regulated in MR vs. LR, the EDL3 gene was up-regulated in HR vs. LR, and the AKT1 gene was up-regulated in both MR vs. LR and HR vs. LR (Table 1).

RT-qPCR
To assess the reliability of the RNA-Seq data, we randomly selected 20 DEGs with high expression levels for validation using RT-qPCR.The RT-qPCR primer sequences can be found in Table S4.
A comparison between the RT-qPCR data for these 20 DEGs and the RNA-Seq results showed that a substantial proportion of the DEGs displayed consistent expression patterns.This congruence between the two datasets (Fig. S2) affirmed the reliability of the RNA-Seq data.The comprehensive curvilinear regression analysis conducted on the 20 DEGs unveiled a notable correlation coefficient of R 2 = 0.792 between the RT-qPCR and FPKM data (Fig. 5).This robust correlation coefficient further confirmed the reliability of the RNA-Seq data and their suitability for this study.

Discussion
The excessive accumulation of Na + in the soil can result in soil compaction, hindered drainage, detrimental impacts on soil health, and ultimately soil degradation 20 .Organic matter and alkaline phosphatase activity play pivotal roles in the global cycles of carbon and phosphorus, respectively 12 .In our previous research, it was found that M. azedarach could significantly decrease Na + content, increase organic matter content, and enhance alkaline phosphatase activity in rhizosphere soil 21 .These findings underscore the positive influence of M. azedarach cultivation in improving soil health and facilitating efficient nutrient cycling within the soil ecosystem.
Similarly, salt stress also exerts a pronounced adverse effect on plant physiology.On the one hand, salt stressinduced osmotic differentials lead to a blockage of water uptake from the soil by the root system, which results in water-loss wilting and even death of the plant 22 .On the other hand, salt stress triggers the accumulation of reactive oxygen species (ROS) in plants, thereby instigating detrimental processes such as lipid peroxidation, membrane degradation, and damage to DNA and proteins 23,24 .Plant membranes, as a key biological barrier, protect cells and organelles from the harmful effects of salt stress 25 , and the stability and function of membrane structural integrity are the basis of normal cell metabolism and overall physiology 26 .Under unfavorable conditions such as salt stress, plants can alter the content and composition of membrane lipids to adapt to the effects of adverse factors 27 .The GO analysis revealed that DEGs were increasingly associated with M. azedarach membranes as the severity of salt stress increased (Fig. 2), which corroborates the above arguments.
The KEGG pathway enrichment analysis undertaken in this investigation revealed that, in comparison to low salinity conditions, DEGs under medium-and high-salinity stress exhibited substantial enrichment in pathways associated with plant hormone signal transduction (Fig. 3a,b).Notably, jasmonic acid (JA) emerged as a pivotal stress-responsive hormone in plants, effectively safeguarding and sustaining the integrity of plant cells under a diverse array of stresses by elevating the expression of antioxidant defense mechanisms 28,29 .Within the JA pathway, MYC2 is a pivotal transcription factor, governing responses to a multitude of biotic and abiotic stresses 30 .When grown in highly saline soil, M. azedarach demonstrated an enhanced capacity to counteract oxidative stress by upregulating genes related to MYC2 (Fig. 4b).
In the GO analysis, within the category denoted as GO:0009651 -response to salt stress, four key DEGs were discerned to be up-regulated as soil salinity increased, namely CBL7, SAPK10, EDL3, and AKT1 (Table 1).Calcium signaling is a key determinant of plant salt tolerance, and CBL7 is a unique set of calcium sensors in plants 31,32 .Meanwhile, SAPK10 and EDL3 genes regulate the ability of plants to adapt to salt stress by modulating abscisic acid-induced antioxidant defense mechanisms 33,34 .The up-regulation of CBL7, SAPK10 and EDL3 genes represents the enhancement of salt tolerance in plants.Similarly, AKT1 plays a crucial role in maintaining K + and Na + homeostasis in plants, and its up-regulation also enhances plant salt tolerance 35 .

Conclusion
The cultivation of M. azedarach may be an effective strategy for improving the properties of saline soil.With increasing soil salinity, M. azedarach had a large number of DEGs within cell membrane components, thus reinforcing the role of the cell membrane as a biological barrier, safeguarding the cytosol and organelles from the detrimental impacts of salt stress.Additionally, M. azedarach upregulated the expression of MYC2 in roots, a pivotal transcription factor within the JA hormone pathway, which could enhance tolerance to oxidative stress.

Figure 1 .Figure 2 .
Figure 1.DEGs of Melia azedarach under salt stress.(a) Principal component analysis based on DEGs.(b) Heatmap analysis of DEGs.(c) Bar graph of up-regulated and down-regulated DEGs.(d) Venn diagram of DEGs.LR roots in low salinity soil, MR roots in medium salinity soil, HR roots in high salinity soil.Biological replicates are indicated by the numbers 1-3.

Figure 3 .
Figure 3. Bubble chart of DEGs enrichment in KEGG pathways.Each circle in the figure represents a KEGG pathway, and the color of the circle represents the q-value.The smaller the value, the more reliable the enrichment significance of DEGs in this pathway.The size of the circle indicates the number of genes enriched in the pathway.The larger the circle, the more genes.LR roots in low salinity soil, MR roots in medium salinity soil, HR roots in high salinity soil.

Figure 4 .
Figure 4. DEGs in plant hormone signaling pathways.(a) MR vs. LR (b) HR vs. LR.The light green background in the figure indicates the genes existing in the KEGG pathway of the species genome; red background indicates up-regulated DEGs involved in the pathway, and green indicates down-regulated DEGs involved in the pathway, blue indicates the pathway involving both up-regulated and down-regulated DEGs.
www.nature.com/scientificreports/This study identified four crucial DEGs associated with the "salt stress response" including CBL7, SAPK10, EDL3, and AKT1.Collectively, these discoveries provide significant insights into the mechanisms through which M. azedarach adapts to salt stress.

Figure 5 .
Figure 5. Linear regression of RT-qPCR and FPKM.LR roots in low salinity soil, MR roots in medium salinity soil, HR roots in high salinity soil.

Table 1 .
Key M. azedarach DEGs for adaptation to salt stress.