Plant material and growth conditions
Surface sterilised Arabidopsis thaliana seeds were sown directly
onto half strength Murashige and Skoog (MS) (Duchefa Biochemie,
Netherlands) 0.8 % bactoagar (BD, USA). Stratification at 4° C in the
dark was for two days, before transfer to Sanyo (UK) growth cabinets (19
– 22° C, 100 µmol m-2 s-2; 12 hours
light/12 hours dark.
T-DNA lines used in this investigation are described in Supplemental
Table 1. Genotyping was carried out according to the instructions athttp://signal.salk.edu/tdnaprimers.2.htmlusing the primers listed in Supplemental Table 2. Left border PCR
products were sequenced to determine the precise locations of the T-DNA
inserts.
For srt1-4 heterozygous (hete ) and srt1-4 hete x
2-1 mutants, we performed genotyping by PCR to identify heterozygous
plants for each experiment. SRT1 artificial miRNAs were designed
using the MicroRNA Designer tool of the WMD3 Web site (Ossowski et
al., 2008) and were introduced by transformation as described below.