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).