Tomato seeds germination in the presence of Bam-FX
Germination of tomato seeds was observed in the presence of BamFX dilutions. Table 1 describes the effect of the BamFX 1:500 dilution (30 min) on the germination of the tomato seeds. The germination rate was 60% in tomato seeds after 48 h and increased to 94% after 72 h. When the seeds soaking time was increased up to 60 min in BamFX 1:500, the germination rate increased up to 68% after 48 h. (Table 1)
When seeds were treated with BamFX1:1000 for 30 min, 70% of the seeds germinated after 48 h, increasing to 96% after 72 h. (Table 1)
Secondary metabolites analysis by using GCMS.
We used GCMS/MS for the analysis of the secondary metabolites from the tomato seeds treated with BamFX and untreated control. The secondary metabolites found in the BamFX treated tomato seedlings - Esters of Fumaric acid, Succinic acid, thiocyanic acid, octadecanoic acid, benzoic acid, hexenoic acid, heptanoic acid, Nicotinic acids, carbamic acid and Diethylmalonic acid.
Fumaric acid, 1-(2-Fluoro-phenyl)-5-oxo-pyrrolidine-3-carboxylic acid (2-chloro-phenyl)-amide, Succinic acid, monoamide, N,N-di(2-ethylhexyl)-, nonyl ester, Thiocyanic acid, [1-(4-amino-1,2,5-oxadiazol-3-yl)-1H-1,2,3-triazol-5-yl]methyl ester, octadecanoic acid, 10-hydroxydecyl ester, Benzoic acid, p-(dimethylsulfamoyl)-, Carbamic acid, N-[10,11-dihydro-5-(2-methylamino-1-oxoethyl)-3-5H-dibenzo[b,f]azepi,Diethylmalonic acid, di(2-chlorophenyl) ester and p-[4,6-Bis[trichloromethyl]-S-triazin-2-yl]benzoic acid ethyl ester were found induced in the BamFX 1:500 treated seeds after 24 h of growth. (Figure 1). Z-3-Methyl-2-hexenoic acid and 6-Acetoxy-4-methyl-hept-4-enoic acid were found decreased in the BamFX1:500 treated seeds than untreated control seeds.
RNA-Seq data analysis
To explore differences in the molecular mechanisms of the defence between BamFX (elicitor treated) and untreated control tomato seedlings, we used Illumina sequencing technology to analyse the transcriptome profiles of the seedlings. A total of 2,35,58,528 raw reads were obtained. Approximately 2,29,54,544 clean reads with >95% Q30 bases (those with a base quality greater than 30) were selected as high-quality reads for further analysis (Table 1). The high-quality reads were mapped to the reference tomato transcript sequences, resulting in the mapping of approximately 96% of the nucleotides. Mapping revealed that transcripts of 18395, 18610, and 18229 genes were detected in the BamFX 1:500 and BamFX 1:1000 treated and untreated control seedlings, respectively.
Functional annotation and classification of DEGs
To identify the DEGs between the control (untreated seedlings) and BamFX-treated seedlings, we employed a general chi-squared test with false discovery rate (FDR) correction and a p-value of 0.05 using DEseq6 software to identify two-fold upregulated and two-fold down-regulated genes. In total 2016 genes, significantly DEGs were detected between the control and the treatment samples, with 1142 upregulated genes and 874 downregulated genes being detected in the BamFX samples (Fig 2).
In search of the possible functions of the Differentially expressed genes, local alignment search by BLAST for non-redundant proteins (NR), nucleotide sequences (NT), Clusters of Orthologous Groups (COG), UniProt, gene ontology (GO), and Kyoto Encyclopaedia of Genes and Genomes (KEGG) databases were performed.
GO enrichment analysis of Differentially expressed genes.
Based on the functions of each DEG, a GO enrichment analysis was performed. All the DEGs were grouped into more than 33 functional groups distributed into three main categories: cellular components, molecular functions, and biological processes (Fig. 3). The GO functions were significantly enriched in the BamFX-treated seedlings.
The ‘organelle’, ‘cell part’, and ‘membrane terms’ from the cellular components category were significantly enriched. The ‘cellular processes’, ‘response to stimulus’, metabolic processes, and ‘biological regulation’ from the biological processes category were significantly enriched. Whereas from the molecular functions category, ‘catalytic activity’ and ‘protein binding’ were significantly enriched.
Also, several DEGs were classified into two functional subclasses involved with transcription regulator activity and transporter activity. Thus, the majority of the identified DEGs were responsible for fundamental processes associated with biological regulation and metabolism (Fig. 3).
KEGG enrichment analysis of DEGs
To group, the biological functions of the DEGs, a KEGG pathway enrichment analysis was performed. All the DEGs were analysed by KEGG pathways. Most of the DEGs were protein processing in the endoplasmic reticulum and plant hormone signal transduction along with the photosynthesis proteins, biosynthesis of amino acids, mitogen-activated protein kinase (MAPK) signalling pathway, and carbon metabolism.
Analysis of DEGs between BamFx-treated and untreated seedlings in the plant hormone signal pathways
In BamFX-treated (1:500 for 30 min) seeds, changes in genes associated with the Jasmonate acid pathway were identified. The significant upregulation of genes in the Jasmonate acid signalling pathways is associated with pathogen infection, plant hormones, and wounding. Solyc12g009220.2 was upregulated in BamFx-treated plantlets.
Most genes associated with the regulation of diverse hormones were differentially expressed between BamFx-treated and untreated seedlings. The transcriptome analysis showed that the expression of genes associated with protein ubiquitination changed significantly. We speculated that these hormone signalling pathways might be involved in differences between BamFx-treated and untreated tomato seedlings.
The transcript levels of most auxin transporter-encoding genes changed significantly in the BamFX-treated seedlings (e.g., Solyc01g007010.3, a RING-type E3 ubiquitin transferase). The gibberellin is important to enhance cell elongation and induce cell division. The gene Solyc07g061720.3 for Gibberellin 2-oxidase was upregulated in the BamFx-treated seedlings. The Phorbol-ester/DAG-type domain-containing protein (Solyc02g068680.1) associated with the intracellular signalling gene was upregulated in the BamFx-treated seeds. Also, we identified six upregulated genes involved in the protein kinase activity signalling pathways in the BamFX-treated seedlings. Solyc01g095770.3 (involved in ion channel activity) was upregulated (Fig 4).
The time-dependent effect of the BamFX (1:500 for 60 min) was found to be regulating many signal transduction pathways. Abscisic acid signalling pathway genes (Solyc09g015380.1) were upregulated in BamFX-treated (1:500 for 60 min) plants. Many signal peptides (Solyc12g049170.2, Solyc12g049150.1, Solyc12g049070.1, Solyc09g075410.3, Solyc12g100110.1, Solyc12g100110.1 Solyc12g100080.1, Solyc07g017570.2) were upregulated in BamFX-treated (1:500 for 60 min) plants (Fig 4).
Protein kinases expression was elevated in the BamFX-treated (1:500 for 60 min) plants. Solyc12g036325.1, Solyc04g079710.3, and Solyc03g119340.3 were upregulated at the lower concentrations of the BamFX (1:1000 for 30 min). Auxin signalling pathway genes (Solyc08g021820.3, Solyc02g082450.3) and an inorganic phosphate transporter (Solyc03g005530.1) were upregulated (Fig 4).
Carboxylic acid pathways
Two important genes were found upregulated in the BamFX-treated (1:500 for 60 min) plants. Aromatic amino acid decarboxylase 1A (Solyc08g068680.3) and Fatty acyl-CoA reductase (Solyc06g074390.3) were upregulated in the BamFX-treated (1:500 for 60 min) plants.
Aromatic amino acid decarboxylase 1A (Solyc08g068680.3) and Cytochrome b561 domain-containing protein (Solyc07g048070.3) were upregulated in BamFX (1:1000 for 30 min) (Fig.4).
Analysis of oxidative stress genes differentially expressed between BamFx-treated and untreated seedlings
The reactive oxygen species are produced during photosynthesis and respiration. The low production of ROS is under strict regulation of the plant cells. Environmental stress can cause an increase in ROS contents. The antioxidant system in plants can remove excess ROS and maintain normal metabolism. In the present study, the significant expression of several candidate genes associated with ROS scavengings, such as Prephenate/arogenate dehydrogenase (Solyc09g011870.2), Fe2OG dioxygenase (Solyc12g006370.2), and L-ascorbate oxidase (Solyc04g054690.3), supported the differential regulation of oxidative stress mechanisms between BamFx-treated and untreated tomato seedlings (Fig. 3 and 4)
When tomato seeds were exposed to BamFx 1:500 for 60 min, peroxidase gene expression was elevated (Solyc01g067870.3, Solyc11g007220.2, Solyc02g014300.2, Solyc02g082090.3, Solyc12g017870.2, Solyc01g009400.3, Solyc01g067860.3, Solyc05g055320.3). This expression profile was not found in seedlings exposed to BamFX 1:500 for 30 min. Other enzymes such as Fe2OG dioxygenase Solyc09g089780.3 were also expressed and upregulated in seedlings treated with BamFx 1:500 for 60 min. (Fig. 3 and 4)
TFs in the BamFx-treated tomato seedlings
Members of the complex family of WRKY TFs are associated with the transcription regulation associated with the plant immune system. In this study, the expression of many WRKY TFs was upregulated very significantly in BamFx-treated seeds . TFs are involved in gene regulation strictly connected with responses to stress; therefore, the genetic manipulation of TFs is highly desirable . In the present analysis, four TFs were differentially expressed in the BamFX-treated seedlings (Fig. 4).
WRKY family members are also directly involved in abiotic stress signalling and tolerance. For example, WRKY23 (Solyc01g079260) responds to auxin regulation, and WRKY70 participates in the defence response to fungus attacks. The present results supported the broad functions of this TF gene family in tomatoes. AP2/ERF (Solyc12g009240.1) and NAC (Solyc07g066330.3) were upregulated in the BamFX-treated (1:500 for 60 min) seedlings (Fig 4).
Defence proteins in BamFX-treated seedlings.
The expression of defence proteins was upregulated in BamFX-treated (1:500 for 30 min) plants. Solyc12g096920.1, Solyc04g007780.3, and Solyc07g009090.3 were up-regulated in the BamFx-treated seeds. In BamFX-treated (1:500 for 60 min) tomato seedlings, the number of genes upregulated was more than with BamFX-treated (1:500 for 30 min) tomato seedlings; and the defence-related gene expression was found upregulated (Solyc07g009040.3, Solyc12g096920.1, Solyc07g009090.3, Solyc07g009030.3, Solyc07g009100.3, Solyc12g009240.1) (Fig 4).