Identification of downstream targets of MtHSFA9
As a first proxy to understand the function of MtHSFA9, putative target genes were identified using ectopic expression in hairy roots ofM. truncatula followed by transcriptome analysis. A total of 420 transcripts were significantly up-regulated, and 253 transcripts downregulated in the35S::MtHSFA9::GFP hairy roots compared to roots transformed with a control plasmid (p<0.01) (Fig. 2a). A total of 11 HSP and sHSP, and three HSF were amongst the highest differentially expressed genes (Data S2). Next we identified two knock out lines in the MtHSFA9 gene, Mthsfa9-1 and Mthsfa9-2 with TNT1 insertions at position 6 and 46 respectively from ATG (Fig. S1b). For each line an associated with type was also selected, corresponding to the line without an insertion at the MtHSFA9 genes at the corresponding position. RNA seq analysis was performed on Mthsfa9 mutants and identified 2323 down-regulated and 2408 up-regulated transcripts in mature mutant seeds compared to wild type seeds (Fig. 2a, Fig. S1c, Data S3). A Venn diagram shows the overlap between the data set from the hairy roots and deregulated genes in the mutant seeds compared to their respective controls (Fig. 2a, Dataset S2). A total of 95 genes were identified as putative targets of MtHSFA9 , with 67 positively regulated and 28 negatively regulated. RT-qPCR validated the reduced transcript level in mature Mthsfa9 seeds forMtHSP70 , MtHSP18.2 and MtHSP17.5 compared to the wild type and associated wild type seeds (Fig 2b-d). Several putative target genes positively regulated by MtHSFA9 encoded members of the class I and class II HSP family (5), three HSP70 and two additional HSF,HSFB2A and HSFA2 . Projection of these putative targets on the co-expression gene network identified 10 genes, out of which eight were directly connected to MtHSFA9 and one connected with MtHSFB1 , another HSF that was deregulated when MtHSFA9 is mutated (Fig. S2a). Other putative targets positively regulated by MtHSFA9 and known to be involved in the regulation of the heat shock response were the co-chaperone regulator peptidyl-prolyl cis-trans isomerase FKBP65/ROF1 (Meiri & Breiman 2009) andBCL-2-associated athanogene6/BAG6 (Nishizawa-Yokoi, Yoshida, Yabuta & Shigeoka 2009). Also, genes encoding for enzymes involved in the synthesis of raffinose family oligosaccharide (RFO seed imbibition 2, a raffinose synthase and galactinol synthase). Analysis of the soluble sugar content in mature seeds of the Mthsfa9 mutants revealed that glucose contents were higher compared to wild type and associated wild type seeds (Fig. 2e), whereas verbascose content was lower (Fig. 2h). No significant difference was detected for sucrose and stachyose, the major soluble sugars in mature seeds (Fig. 2f-g).
Further identification of the biological functions of the downstream pathways modified by MtHSFA9 was investigated by KEGG mapping and Gene Ontology (GO) enrichment (Fig. 2i and Fig. S2b). The main KEGG pathways that were significantly deregulated in the Mthsfa9 seeds were related to biosynthesis of secondary metabolites (flavonoid biosynthesis), carotenoid biosynthesis, porphyrin and chlorophyll metabolism, glutathione metabolism, sphingolipid metabolism, carbon fixation and carbon metabolism (Fig. 2e). Enriched GO categories that were complementary to the KEGG mapped pathways included transport, DNA replication and initiation, sulfur compounds and response to abiotic stimulus (containing all the HSP/HSF) (Fig. S2b).