Total RNA extraction and northern blot analysis

Total RNAs were extracted from 22 days-old plants using TRI reagent (MRC) following the manufacturer instructions. For 4 days-old seedlings, roots, rosette leaves, inflorescences and green siliques of 7-week-old plants, total RNAs were prepared according to Chang et al. (51). Total tRNAs were enriched from total RNAs as described in (52). This protocol includes a LiCl precipitation step that allows enrichment, in the supernatant, of RNAs of a size smaller than 150 nt (i.e. 5S rRNA, tRNAs and small non-coding RNAs).

For tRNA northern blots, up to 6μg of total tRNAs were separated in 15% polyacrylamide gel, elecrotransferred onto Hybond N+ membrane (GE Healthcare Life Sciences) and hybridized to ³²P radiolabeled oligonucleotides probes in 6X SSC, 0.5% (v/v) SDS at the following temperatures: Y14 P, Y32 P, P12 P, Ala P: 48°C, and P46 P: 44°C. Washing conditions were: 2 times 10 min in 2× SSC and 1 time 30 min in 2× SSC, 0.1% SDS, at hybridization temperature.