Plant material and growth conditions
Three independent Tnt1 insertion lines in the HSFA gene Medtr4g126070 of M. truncatula, Mthsfa9-1 (NF10440), 6 bp from ATG), Mthsfa9-2 (NF13157, 4 6 bp from ATG) and Mthsfa9-3 (NF12877,1333 bp from ATG) in the R108 background were obtained after screening by the Noble Foundation. Homozygous lines were screened by PCR with the primers listed in Table S1. For each allele, wild type control plants were simultaneously selected based on the absence of the Tnt1 insertion in the MtHSFA9 gene and are referred to as associated wild type. The homozygous Mthsfa9-1line was backcrossed once with the pollen of the wild type R108. Arabidopsis insertion lines in AtHSFA9 (At5g54070) were obtained from ABRC. Among the three T-DNA insertion lines ordered, homozygous lines were only identified for the Salk_062453 using primers in Table S1.
M. truncatula plants from the different genotypes were grown in batched for 10 plants trays containing a sterile mix of vermiculite at 20°C/19°C, with a 16 h light photoperiod at 200 μmol photons m -²s-². For the high temperature treatment during seed development, plants were transferred to a growth room at 26°C with similar light conditions when 10 flowers had appeared. Flowers were tagged and developing seeds were harvested at different time intervals until pod abscission and after final desiccation. At the abscission stage the pods were dried for 3 days at a relative humidity of 44% generated by saturated solution of K2CO3 at 20°C. The seeds were manually removed from the pods and stored at 20°C in the dark for further after-ripening or kept at -20°C. Arabidopsis plants were grown at 20°C/18°C with a photoperiod of 16 hours. The seeds were harvested after pod abscission and then dried for three days at 44% RH, generated by saturated solution of K2CO3 at 20 °C.