Fig. 4

ROMANIAT5-2 controls heat-responsiveness of APUM9 in A. thaliana. a Schematic representation of the ROMANIAT5-2 – APUM9 region in A. thaliana. The yellow block within the 3’ LTR represents a 3P/Gap heat responsive element (HRE). S position of primers for RT of the sense transcripts, A position of primers for RT of the anti-sense transcripts, F and R forward and reverse quantitative PCR primers. META1 is a transposon fragment flanking ROMANIAT5-2 3’ LTR. Silex: the orange block corresponds to the genomic fragment cloned upstream of the 4× “upstream activating sequence” (UAS, violet) and green fluorescent protein (GFP; green). b Schematic representation of the A. lyrata APUM9 locus. Reads per kilobase per million reads (RPKM) for (c) ROMANIAT5 and (d) APUM9 under control, 6 h at 37 °C HS and HS with 48 h recovery at control conditions (HS + R). * P <0.05 in t-test. e RT-PCR analysis of Silex reporter construct response to HS. NS non-stressed control plants, CS and HS control- or heat-stressed plants, respectively, +0 and +5d days of recovery at non-stress conditions, RT+ and RT– samples with and without RT, respectively. 18S rRNA transcript serves as positive control. f GFP signal in control and 24 h heat-stressed (HS2) Silex, detected after 0, 1, 2, or 5 days of recovery. Red – chlorophyll, green – GFP. g Close-up view of plants treated as described in (f). h Strand-specific RT-qPCR of APUM9 and ROMANIAT5-2 in A. thaliana after 6 h HS. i Putative HREs in ROMANIAT5 LTRs in Brassicaceae. j RT-qPCR for ROMANIAT5 in Brassicaceae after 6 and 12 h at 37 °C HS. The values were normalized to UBC28. Error bars indicate standard deviation between three biological replicates and * P <0.05 in Student’s t-test