Figure legends
FIGURE 1. Identification of MtHSFA9 as a seed-expressed gene.
(a) Co-expression network of seed-specific transcription factors
(squares) and transcripts correlating with the acquisition of
desiccation tolerance and longevity. Node size and color represent the
betweenness centrality and edge betweenness (yellow is low, blue is
high). Recalculated from Verdier et al. (2013). (b) Maximum parsimony
analysis of HSFA proteins from legumes (Glycine max ,Medicago truncatula , Lotus japonica), Arabidopsis thalianaand HaHSFA9 from Helianthus annuus . Gene identifiers are provided
in Table S3. Branches corresponding to partitions reproduced in less
than 50% bootstrap replicates (1000) are collapsed. The tree was
obtained using the Subtree-Pruning-Regrafting algorithm using MEGA7
(Kumar et al., 2016). For soybean the two paralogs are shown when
appropriate. Stars indicate the M. truncatula ortholog of
HaHSFA9. (c) Heat map of the HSFA family genes in Medicago
truncatula showing mean-centered transcript levels with high (red) to
low (blue) levels. Data are from the M. truncatula gene
expression atlas
(https://mtgea.noble.org).
FIGURE 2 . Identification of putative targets of MtHSFA9 and
downstream biological processes. (a) Venn diagram between up- or
downregulated genes in hairy roots overexpressing35S::MtHSFA9::GFP::GUS compared to hairy roots transformed with35S::GFP::GUS and in mature Mthsfa9 seeds compared to wild
type seeds. (b-d) Relative transcript levels of MtHSP17.5 (b),MtHSP18.2 (c) and MtHSP70 (d) in wild type seeds (WT),
associated wild type seeds (assocWT) and two Mthsfa9 mutant
alleles. Data represent the average ± S.E.M. of three technical
replicates from 30 seeds. (e-h) Soluble sugar content in mature seeds of
the indicated genotypes, (e) glucose, (f) sucrose, (g) stachyose, (h)
verbascose. Different letters indicate significant differences between
genotypes using ANOVA and post-hoc Student-Newman-Keuls comparisons
(P<0.05). (i) Overrepresentation analysis of KEGG pathways of
differentially expressed genes of mature Mthsfa9 /WT seeds.
FIGURE 3. HSFA9 is not implicated in seed longevity
under dry storage but improves survival during wet storage. (a-b) Loss
of germination of mature seeds from wild type (WT) associated WT and
Mthsfa9 plants during storage at 75% RH and 35°C (a) and at
100% RH and 40°C (b). Data are the mean (± SE) of 3 replicates of 30
seeds. (c-d) Loss of germination of mature seeds from WT (Col-0) andhsfa9 Arabidopsis thaliana plants during storage at 75%
RH and 35°C (c) and 100% RH and 40°C (d). At a given time point of
ageing, data are significantly different when they differ by 15% or
more (χ2 test, P < 0.05). WT, wild type,
assocWT, associated WT.
FIGURE 4 . MtHSFA9 influences the depth of dormancy during late
maturation. (a) Germination curves of freshly harvested seeds of
wildtype, associated WT and Mthsfa9 mutants. Seeds were dried for
3d at 43% RH after pod abscission before imbibition at 20°C in the
dark. Data are the mean of three replicates of 30 seeds. (b) Germination
curves of seeds of mature seeds from wildtype and Mthsfa9 mutants
after stratification for 3d at 4°C. (c) Effect of after-ripening at room
temperature on mean germination time of mature seeds from wildtype and
Mthsfa9 plants grown at 20°C/18°C. Data are the mean of three
replicates of 30 seeds. Stars indicate significant difference between WT
and mutants using ANOVA and post-hoc Student-Newman-Keuls comparisons
(P<0.05). (d) Increase in mean germination time as proxy of
depth of dormancy of wildtype and hsfa9 seeds freshly harvested
at the indicated time during development. 44 DAP corresponds to the
point of pod abscission. Data are the mean of three replicates of 30-50
seeds. WT, wildtype, assocWT, associated WT.
FIGURE 5 . ABA and GA biosynthesis, catabolism and signaling
genes are deregulated by MtHSFA9. (a) Heatmap showing significant
differential transcript levels (log2) of genes related to ABA and GA
biosynthesis, reception and signaling in mature seeds of WT and two
Mthsfa9 alleles. Asterisk indicates a significant difference in
transcript level upon overexpression of MtHSFA9 in hairy roots.
(b-d) Relative transcript levels of MtNCED4 (Medtr5g025270 ) (b),
MtCYP707A (Medtr8g07226) (c) and MtGa2OX2 (Medtr4g096840)
(d) in mature wild type seeds (WT), associated wild type seeds (assocWT)
and two Mthsfa9 mutant alleles. Different letters indicate
significant differences between genotypes using ANOVA and post-hoc
Student-Newman-Keuls comparisons (P<0.05).
FIGURE 6 . ABA content and sensitivity are affected inMthsfa9 mutants. (a) ABA content in mature seeds of freshly
harvested associated WT and Mthsfa9 mutants. (b) Fold reduction
in the germination speed (T50) by imbibition of mature seeds of WT,
assocWT and Mthsfa9 seeds by 10 µM fluridone. (c) ABA sensitivity
of seeds of WT, assocWT and Mthsfa9 mutants as measured by
reduction of germination after incubation in different concentrations of
ABA in the dark. (d) Time to 50% of germination or start of growth for
intact scarified seeds or naked embryos of WT, assocWT and
Mthsfa9 mutants 1 month after harvest. Seed envelopes were
removed 6h after imbibition. Different letters or stars indicate
significant differences between genotypes using ANOVA and post-hoc
Student-Newman-Keuls comparisons (P<0.05).