Loss of SRT1 function causes embryo lethality
To investigate if sirtuin activity contributes to the response of the circadian oscillator to nicotinamide we identified four different alleles of SRT1 (At5G55760) T-DNA insertion mutants (srt1-1 ; SALK_086287, srt1-2 ;SALK_001493,srt1-3 ;SALK_064336 and srt1-4 ;SAIL_552_E02; Figure 4a and Supplemental Table 1). However, qRT-PCR, demonstrated that three of the lines had little effect on the expression of the SRT1 gene compared to Col-0 (srt1-1, srt1-2 and srt1-3; Figure 4b). For this reason, we proceeded only to investigate the T-DNA insertion mutant srt1-4 (SAIL_552_E02) with the insert located 98 bp from the start of exon 5 which resulted in reduced expression of SRT1(Figure 4a, b). We confirmed that the srt2-1 (At5G09230; SALK_149295; Supplemental Table 1) mutant (previously described in Wanget al ., 2010) has an insert located 38 bp from the start of exon 2 and reduced expression of SRT2 (Figure 4a,b). We were unable to isolate homozygous T-DNA lines of srt1-4 because it caused an embryo lethal phenotype which was not present in heterozygotic mutants (Figure 4c). To confirm embryo lethality is due to the absence ofSRT1 , we generated complementation lines with a native promoter and the full length SRT1 gene. All of the complementation lines had normal embryo development, which along with a lack of an embryo phenotype in heterozygotes suggests that it is loss of SIRT1 that affects embryo development, rather than a dominant negative effect of the TDNA insertion (Figure 4c,d). Both SRT1 and SRT2 are highly expressed in siliques and flowers which determine embryo development (Supplemental Figure 4). Because full knock out ofSRT1 was embryo lethal, for the rest of the investigation we used heterozygous srt1-4 plants which were knockdowns, having less than half the expression of SRT1 compared to the Col-0 background (Figure 4b).