Identification of the seed-specific MtHSFA9 in M. truncatula as the ortholog of HaHSFA9
To identify transcription factors (TF) that underly the regulation of the acquisition of desiccation tolerance (DT) and longevity, we performed a detailed temporal transcriptome study during seed development (Verdier et al. 2013). The construction of a gene co-expression network of genes and seed specific transcription factors that correlated with DT and longevity inferred a number of TFs as putative regulators of late maturation/longevity genes (Verdier et al. 2013). Detailed characterization of this network identified a heat shock transcription factor (Medtr4g126070/HSFA) as a central hub of the module, with the highest betweenness centrality (Fig. 1a, Data S1). In addition, this HSFA was one of the nodes with the closest centrality and number of connected nodes, with only Medtr1g023170, a DREB2D TF, being more connected (Data S1).
A blast of the M. truncatula sequence against Arabidopsis TAIR11 revealed homology with three AtHSFA: At2g26150/HSFA2 (7.55e-75), At3g22830/HSFA6B (1.25e-72) and AT5G54070/AtHSFA9 (7.17e-72). To analyze to which group the M. truncatula HSFA belongs, a phylogenic tree was constructed using homologous HSFA protein sequences retrieved fromM. truncatula, Glycine max and Lotus japonica databases (Fig. 1b). The phylogenetic reconstruction was performed using maximum parsimony and identified Medtr4g126070 as part of a subgroup containing AtHSFA9, HaHSFA9, GmHSFA9.1 and GmHSFA9.2 (Fig. 1b). We therefore referred to the gene as MtHSFA9 . Since the Arabidopsis and sunflower HSFA9 are specifically expressed in seeds, we investigated the expression patterns of MtHSA9 and the other HSFA genes (Fig. 1c). MtHSFA9 is predominantly expressed in seed tissues, with the highest transcript levels at the end of seed maturation (36 days after pollination (DAP)) (Fig. 1c, Fig. S1a). A moderate level of transcript was also detected in shoots submitted to severe drought.