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.