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).