3.4 Transcriptome changes in anthers at different stages of development under drought stress
To study possible molecular adjustments of tomato anthers under drought, Illumina RNA Sequencing (RNA-Seq) was used to investigate the transcriptome of anthers at 3 different development stages: pollen mother cell to meiotic cell stage (PMC-MEI), tetrad to vacuolated microspore stage (TED-VUM) and binucleate to mature pollen stage (BIN-MP) from well-watered (WW) and drought-stressed (DS) plants. After filtering, a total of 924,197,894 clean reads were generated from 18 libraries with high consistency between replicates (Pearson’s r, 0.86-0.99, Table S2). Out of that, 862,078,735 reads, uniquely aligned to specific genomic regions of S. lycopersicum reference genome with an average mapping rate of 98.2 % (Table S3) and were used for differential gene expression (DEG) analysis. In total, 15,431 DEGs were identified (Table S4A). Among them, 3427, 2780, and 815 genes were up-regulated at PMC-MEI, TED-VUM and BIN-MP stages, respectively (Figure 4A, Table S4B), whilst 3450, 3560 and 1399 genes were down-regulated at PMC-MEI, TED-VUM and BIN-MP stages, respectively (Figure 4A, Table S4C), indicating that the number of DEGs decreased with age of anther. Greater number of genes was down-regulated at TED-VUM indicative of its high level of vulnerability to drought stress. Confirmation of RNA-Seq results with RT–qPCR analysis using 18 selected drought-responsive genes revealed that the expression patterns from RT–qPCR analysis were consistent with those from RNA-Seq analysis (Figure S2A).
To have an idea of the possible roles of drought-regulated genes, enrichment analyses of GO terms and KEGG pathways were performed. A total of 225 well-defined functional groups were significantly enriched in drought-responsive genes at different development stages (Tables S5 and S6). All biological processes significantly enriched in up-regulated genes occurred only at one stage such as ‘regulation of gene expression’ (GO: 0010468), ‘transmembrane transport’ (GO: 0055085) and ‘response to water’ (GO: 0009415) at PMC-MEI, TED-VUM and BIN-MP stages, respectively (Table S5A). With regards down-regulated genes, some enriched biological processes were observed at more than one stage, for example, ‘carbohydrate metabolic process’ (GO: 0005975) at TED-VUM and BIN-MP stages; ‘lipid metabolic process’ (GO: 0006629) at PMC-MEI and TED-VUM stage; ‘amide biosynthetic process’ (GO: 0043604) at PMC-MEI and BIN-MP stages and ‘metabolic process’ (GO:0008152) at all three stages. Other biological processes were enriched only at one stage, example ‘photosynthesis’ (GO: 0015979) at TED-VUM stage (Table S 6A), suggesting that carbohydrate biosynthesis function was significantly repressed by drought stress at mid to late stages of anther development. Similarly, KEGG analysis showed only one pathway ‘Ribosome’ was highly significantly enriched in down-regulated DEGs at two stages whereas others were only enriched at TED-VUM stage, such as ‘Photosynthesis’and ‘Biosynthesis of secondary metabolites’ (Figure 4B, Table 7B). Strikingly, only the pathway ‘plant hormone signal transduction’ was highly enriched in up-regulated DEGs at PMC-MEI stage (Figure 4B, Table S7A). Together, our GO and KEGG data suggest drought stress may primarily affect carbohydrate and secondary metabolic processes including hormone pathways in tomato anthers.