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.