Effect of drought stress on physiological indicators in Axonopus compressus
In the Fig. 1, it was revealed that rate of H2O2 (Fig. 1a) production and MDA concentration (Fig. 1b) increased in drought treated plants. The MDA values and H2O2 production rate increase with the prolonged drought stress treatment, whereas MDA values and the production rate of H2O2 at 36 h after drought induction were significantly increased (p < 0.01). Similarly, the leaf water potential and relative water content decrease significantly under drought, contrarily the electrolyte leakage increased over the drought period in Axonopus compressus. As in case of MDA and H2O2 the values of EL were also increase as the time of drought stress increase. Whilst, the values of leaf water potential and relative water content decrease with the prolonged drought stress.
Fig. 1: Effect of drought on the physiologiacal traits of the Axonopus compressus.
RNA-Seq of Axonopus compressus and de novo assembly
RNA-Seq of the six libraries of cDNA (DS and CK with three repeats of each) resulted in 352.84 million raw reads, of which approximately 347.73 million clean reads de-novo compiled into contigs utilizing Trinity software, tend to range from 54.90 million to 62.25 million reads for each library and approximately 97% of reads with a quality score of Q20 (99% accuracy) (Table 1). The contigs were assembled into 278042 unigenes with an average length of 1066 bp and an N50 length of 1430 bp. All unigenes were over 200 bp long, of which 11.77% (32718 unigenes) were 2,000 bp long.
Functional annotation and classification of unigenes
A total of 135716 (48.81%), 188334 (67.64%), 120455 (43.32%), 189422 (68.13%), 100588 (36.18%), 153958 (55.37%) and 128626 (46.26%) unigenes would have significant levels (E-value <10−5) in KEGG, NR, Swissprot, Trembl, KOG, GO, and Pfam, respectively (Fig 2) (Additional file 1). Out of 278042 unique of high-quality sequences, 191893 (69.03%) unigenes complemented a sequence significantly in at least one of the seven public databases, The five key online databases (GO, KEGG, KOG, NR, and Trembl) were picked out of seven databases to draw a Venn diagram (Fig 3), the unigenes with significant values (Evalue10-5) are also noticed at each junctions of the Venn diagram, in which 94 unigenes corresponds to five databases. Based on the high-score BLASTx matches in the GO proteins database, The BLASTx high-score Predicated matches in the computer database of go proteins were confirmed, and a total of 153958 unigenes had been categorized with Blast2GO (E-value <10-5) and was designated at least once in GO. As shown in Fig 4, the unigenes referred to three main categories of GO and 59 subcategories, namely biological processes (BP), with 28 main sub-classes (405025 unigenes); cellular compartments (CC), with 18 main sub-classes (460094 unigenes); and molecular functions (MF), with 13 main sub-classes (194989 unigenes).
Fig 2. Unigenes matched in seven databases.
Fig 4. Functional classification of GO terms for Axonopus compressus.
Fig 3. Venn diagram of differential BLAST results for Axonopus compressus. The number of unigenes annotated with 5 databasesand the co-annotated gene number are presented
Cellular processes (21.43%) were the biggest subgroups in the category of biological processes, metabolic process (19.70%), biological regulation (9.24%), response to stimulus (9.22%) and regulation of biological process (8.36%). The largest subgroups in the cellular component category were cell (22.53 %) followed by cell part (22.47 %), organelle (17.22%), and membrane (11.72%) respectively. Similarly, the main subgroups in the molecular function category were binding and catalytic activity, that contribute 77.78% and 69.28% respectively, and 115199 unigenes associated to molecular function.
Within the Axonopus compressus unigenes, 112492 (42.64%) had been classified (E-value <10-5) into twenty six KOG clusters (Fig 5). The biggest groups which includes 1) general function prediction only (21955 genes, 19.52%); 2) posttranslational modification, protein turnover, and chaperones (11636 genes, 10.34%); 3) signal transduction mechanisms (11302 genes, 10.05%); 4) Function unknown (5785 genes, 5.14%); and 5) carbohydrate transport and metabolism (5616 genes, 4.99%).
Fig 5. KOG functional classification of the Axonopus compressus transcriptome.
Metabolic pathway analysis of Axonopus compressus by KEGG
A total of 135717 (51.44%) of the 263835 Axonopus compressus unigenes possessed significant correspondence in KO. All these unigenes, such unigenes have been reserved to 103 KEGG pathways and could be splited to 5 categories (Fig 5). The pathways of KEGG includes 4303 unigenes were the members of major group, metabolism (D), 921 associated to genetic information processing (C), 184 related to cellular processes (A), 347 involved to environmental information (B), and 151 related to category (E) of organism systems (Fig. 6) (Additional file 2).
Fig 6. KEGG pathway annotation for Axonopus compressus.
CDS prediction in Axonopus compressus
The BLASTx protein database (NR and SwissProt database) identified 278042 unigenous CDSs, of which 23652 unigenes were larger than 500 bp, 12520 unigenes were larger than 1,000 bp, and 32718 unigenes were larger than 2,000 bp. In addition, the 43,713 unigenes were not linked to the NR and SwissProt database systems. The Estscan (Version; 3.0.3) software were used to interpret their ORF, frequency distribution, length, and related amino acid sequences of the unigene CDSs.
Differentially expressed genes (DEGs) analysis of Axonopus compressus
Amongst the differentially expressed unigenes, the expression of 7444 differs substantially between samples treated with drought-stress (DS) and control (CK) samples. Under drought treatment 4249 numbers were up-regulated and 3195 numbers were down-regulated (p<0.05) (Fig. 7) (Additional file 3).
Fig 7. Volcano plot of differentially expressed unigenes of Axonopus compressus.
Axonopus compressus GO enrichment analysis
By employing the Gene ontology (GO) and the DEG enrichment analysis in Axonopus compressus, 23766 DEGs have been categorized to three GO groups and 4592 numbers associating the DS with CK (number of DEGs annotated in more than one terms), out of which 2766 items found associated to BP, 477 were linked with CC and 1349 were related to MF.
The key 50 DEGs dramatically enriched in three GO categories which are presented in Fig 8. In the top 50 significantly enriched identities (Corrected P-Value<0.05), the 18 found to be linked with BP [carotenoid biosynthetic process GO:0016117, carotenoid metabolic process GO:0016116, cellular amine metabolic process GO:0044106, cellular biogenic amine metabolic process GO:0006576, cellular response to heat GO:0034605, cellular transition metal ion homeostasis GO:0046916, glutamine family amino acid biosynthetic process GO:0009084, Group II intron splicing GO:0000373, heat acclimation GO:0010286, oligosaccharide catabolic process GO:0009313, raffinose catabolic process GO:0034484, raffinose metabolic process GO:0033530, regulation of seed germination GO:0010029, regulation of seedling development GO:1900140 , response to high light intensity GO:0009644, response to hydrogen peroxide GO:0042542, tetraterpenoid biosynthetic process GO:0016109, tetraterpenoid metabolic process GO:0016108], and 6 items were related to CC [chloroplast nucleoid GO:0042644, DNA packaging complex GO:0044815, Nucleoid GO:0009295, Nucleosome GO:0000786, plastid nucleoid GO:0042646, protein-DNA complex GO:0032993], and 14 items were related to MF [4-coumarate-CoA ligase activity GO:0016207, alpha-galactosidase activity GO:0004557, arogenate dehydratase activity GO:0047769, carbon-nitrogen ligase activity, with glutamine as amido-N-donor GO:0016884, ferric iron binding GO:0008199, ferroxidase activity GO:0004322, galactosidase activity GO:0015925, oxidoreductase activity, acting on single donors with incorporation of molecular oxygen GO:0016701, oxidoreductase response, acting on single donors with molecular oxygen incorporation, incorporation of two atoms of oxygen GO:0016702, oxidoreductase response, oxidizing metal ions, oxygen as acceptor GO:0016724, raffinose alpha-galactosidase activity GO:0052692, water channel activity GO:0015250, water transmembrane transporter activity GO:0005372]( Additional file 4, 5).
Fig 8. GO-enriched DEGs in Axonopus compressus.
Axonopus compressus KEGG pathway enrichment analysis
The KEGG pathway enrichment analysis for DEGs revealed that 2569 DEGs participated in 143 various types of pathways in Axonopus compressus. By comparing DS with CK, the number of 2747 DEGs found up-regulated and 2502 DEGs have been identified as down-regulated in deficit water. The twenty key pathways which are found significantly enriched by comparing DS with CK are displayed in Fig 9. The pathways includes Valine, leucine and isoleucine biosynthesis (215 unigenes), Stilbenoid, diarylheptanoid and gingerol biosynthesis (330 unigenes), Starch and sucrose metabolism (1642 unigenes), RNA degradation (1536 unigenes), Protein processing in endoplasmic reticulum (1628 unigenes), Porphyrin and chlorophyll metabolism (559 unigenes), Plant hormone signal transduction (1672 unigenes), Phenylalanine, tyrosine and tryptophan biosynthesis (601 unigenes), Metabolic pathways (1659 unigenes), MAPK signaling pathway – plant (1661 unigenes), Linoleic acid metabolism (323 unigenes), Glyoxylate and dicarboxylate metabolism (1050 unigenes), Glycosphingolipid biosynthesis - globo and isoglobo series (103 unigenes), Galactose metabolism (735 unigenes), Carotenoid biosynthesis (506 unigenes), Carbon fixation in photosynthetic organisms (820 unigenes), Biosynthesis of secondary metabolites - unclassified (223 unigenes), Biosynthesis of secondary metabolites (1661 unigenes), Biosynthesis of amino acids (1655 unigenes), Alanine, aspartate and glutamate metabolism (639 unigenes) (Additional file 6).
Fig 9. KEGG pathway enrichment of DEGs in Axonopus compressus.
Drought stress associated differentially expressed transcription factors (TFs)
The transcription factors play a crucial role in growth and development of plants, and therefore can stimulate and/or suppress transcriptional gene expression to sustain normal physiological function in deficit water stress. In current study, the 352 TFs were observed in Axonopus compressus based on iTAK, including 270 transcription factors and 81 transcription regulatory factors that associated to 32 and 16 families, respectively (Additional file 7, 8). Differential analysis revealed 270 transcription factors were remained differentially expressed by comparing DS with CK, of which 216 transcription factors have been noticed up-regulated and 54 transcription factors were found down-regulated.
qRT-PCR Validation of Transcripts in Axonopus compressus under Drought
To check the validity of RNA-seq results, expression of six transcripts for the three biological replicates was analyzed by qRT-PCR (Figure 10). All the genes were identified as significantly different expression values (DEGs). The finding of qRT-PCR showed that the pattern of expression of these genes was identical to the shown in RNA-seq analysis. To validate the results, the six drought-responsive genes (NAC, MAP Kinase1, MYB2, PIP1, WRKY1, ABI5) were verified using qRT-PCR. The different genes were amplified to compare the gene expression and RNA-seq results, and the findings of this study showed that the expression of drought-responsive genes supporting the results of RNA-seq.
Figure 10: The qRT-PCR expression validation of drought-responsive genes in A. compressus. The bars showed relative expression of (A) NAC (B) MAP Kinase1, (C) MYB1, (D) PIP2, (E) WRKY1, (F) ABI5.