Sirtuin 1 affects gene expression during the day
In contrast to the PARPs, alterations to sirtuin expression had a profound effect on the transcriptome. In srt1-4 heterozygous plants, 6490 genes were differentially expressed in the day (sampling at ZT 6) compared to Col-0 (Figure 9a and Supplemental Table 5). This effect was mostly specific to the day, because at night the transcripts of only 49 genes were differently expressed (sampling at ZT 18) (Figure 9a and Supplemental Table 5). Our results suggest that thesirtuin1 might have a very important role in day-time gene expression. To further investigate this hypothesis we compared our transcription profiles with publicly-available light signalling- and sucrose-related transcriptomes (white light, red light, UV-B and sucrose; Supplemental Table 3) and a control transcriptome unrelated to light signalling (heat & salt) downloaded from the EBI (https://www.ebi.ac.uk/gxa/). There were highly significant overlaps between SRT1 -regulated transcript sets and those regulated by light and sucrose, withSRT1 expression affecting the gene expression in the opposite direction to light and sucrose signals (Figure 9b, Supplemental Table 6, 7). A wide range of GO terms were associated with day and SRT1regulation many of which can be associated with light signalling and growth (Supplemental Table 7). These data indicated that SRT1might positively regulate transcripts regulated in the day by light signalling and light induction of sugar production by photosynthesis. By contrast srt2-1 had a relatively small effect on gene expression, with only 235 transcripts at day and 97 at night being differentially expressed (Figure 9a and Supplemental Table 5). The dramatic effect of reduced SRT1 expression on transcript abundance was confirmed by analysis in a srt1-4 hete x srt2-1 which had an overlap of 4143 differentially expressed transcripts with the srt1-4 hete single mutant (Figure 9a).
To confirm the results of the RNAseq data we performed the experiment again three times with new biological material for each experiment and a selected subset of genes to be measured by qRT-PCR to compare to the findings of the RNAseq. At2G36970 (UDP-Glycosyltransferase superfamily protein) was selected because it has a large decrease in abundance in RNAseq in the mutant. AT5G55580 (Mitochondrial transcription termination factor family protein) and AT5G55540(were selected to represent transcripts that had smaller changes in abundance. AT5G62720 (tornado 1), AT4G11830 (phospholipase D gamma 2) and AT2G15880 (Leucine-rich repeat (LRR) family protein) were selected representing large increases in abundance changes in srt1-4 hete in the RNAseq data (Supplemental table 5 and Supplemental Figure 7). The qRT-PCR independently confirmed the RNAseq experiment findings about the effects on gene expression during the day, showing that AT2G36970 , At5G55540 and AT5G55580 had reduced expression and AT5G62720, AT4G11830 and AT2G15880were increased in expression in the srt1-4 mutant during the day (Supplemental Figure 7a and b). Similarly, the qRT-PCR confirmed the findings of the effects on gene expression that we measured at night with RNAseq, demonstrating reduced expression of AT2G36970 ,AT5G55580, At5G55540, AT5G62720 and increased expression at night of AT4G11830 and AT2G15880 in srt1-4 (Supplemental Figure 7c and d). We also examined the abundance of these transcripts by qRT-PCR in two SRT1 artificial microRNA lines, which also knocked down SRT1 expression (Supplemental Figure 8a). The microRNA lines were not quite as effective in knocking down srt1 expression assrt1-4 hete (Supplemental Figure 8a). All the transcripts had similar patterns of abundance between srt1-4 hete and SRTmicroRNA lines in the day, though in line with their reduced effect onsrt1 expression, compared to srt1-4 , the microRNA lines had less of an effect on the abundance of the transcripts (Supplemental Figure 7a). The trend of similar but reduced effects of the microRNA lines compared to srt1-4 was also found at night (Supplemental Figure 7c). For AT2G36970 , AT5G55580, AT5G62720 expression was reduced in srt1-4 and in the microRNA lines, and forAT4G11830 and AT2G15880 expression was increased insrt1-4 and the microRNA lines (Supplemental Figure 7c). The exception being AT5G5540 , for which at night there was little effect of the microRNA lines, but a strong effect of srt1-4 . Thus, in independent experiments we confirmed that srt1-4 and micro RNA lines had similar effects on transcript abundance and the degree of effect was in line with the abundance of the srt1transcript. This demonstrates that the effects on the transcriptome are due to changes in SRT1 expression.
Because we found profound effects of SRT1 expression in the light, and a strong overlap with genes that are regulated by light, either directly by signalling, or potentially indirectly by sugars we also performed qRT-PCR to measure light signalling gene expression in RNA extracted from SRT1 artificial microRNA linesandsrt1-1 , srt1-2 , srt1-3 , srt1-4 hete andsrt2-1 mutants (Supplemental figure 8a). We chose several light signalling related genes (AT2G37678, FHY1 far-red elongated hypocotyl 1, AT5G02200 FHL far-red elongated hypocotyl 1 like, AT2G42870 PAR1 phy rapidly regulated 1, AT1G70290 TPS8 trehalose-6-phosphatase synthase 8 ). There was good correspondence between the effects ofsrt1-4 hete and SRT1 microRNA lines on the expression of these light-regulated signalling transcripts (Supplemental Figure 8b).srt1-4 and micro RNA lines increased the expression of all of the selected light signalling genes confirming that SRT1 affects their abundance (Supplemental figure 8b), as had been found in the RNAseq (Supplemental Figure 8c)..