HSFA9 is not implicated in seed longevity but improves
thermotolerance during wet storage
The main function identified for the sunflower homologue of HSFA9 is a
role in seed deterioration during storage (Prieto-Dapena et al. ,
2006; Tejedor-Cano et al. , 2010). To investigate if MtHSFA9 is
involved in seed longevity, seeds of wild type and Mthsfa9mutants were produced under standard growth conditions, and viability
loss was followed during storage using moderate accelerated storage
conditions (75% RH and 35°C). No difference could be detected in
longevity, both the survival curves and the P50, determined as the
storage time needed for the seed population to lose 50% of their
viability, were similar between wild types and Mthsfa9 mutants
(Fig. 3a). Two additional cultures were grown and storage experiments
were conducted on the harvested seeds, and this confirmed the absence of
a perturbed longevity phenotype of the Mthsfa9 mutants (Fig.
S3a,b).
Next, we hypothesized that MtHSFA9 might have a role in the regulation
of longevity when the mother plants are grown under high temperature
conditions. Indeed in M. truncatula, high temperature (26°C) was
shown to reduce longevity in the A17 genotype (Righetti et al., 2015).
To test this hypothesis, wild type and mutant seeds were produced at
26°C, which shortened seed development compared to 20°C for all
genotypes, with pod abscission occurring at 552-575°days at 26°C
compared to 731-748°days at 20°C. Seed filling was significantly
impacted by the high temperature, in a similar manner for the
Mthsfa9 mutants and wild type (Fig. S4a). Longevity was
significantly higher for all genotypes when seeds were produced at 26°C
compared to 20°C (Fig. S4b and c). However, no significant differences
were observed between wild type and Mthsfa9 seeds. We then
examined if high temperature affected transcript levels of putative
target genes of MtHSFA9 (Fig. S4d-f). Whereas HSP70 transcript
levels were lower in wild type seeds grown at 26°C compared to 20°C,MtHSP17.5 and MtHSP18 .2 transcripts significantly
increased during the high temperature treatment (Fig. S4e-f). Yet,
transcript levels of these three genes were not significantly affected
by heat in the Mthsfa9 mutants compared to the wild type seeds.
Since no longevity phenotype could be observed in the Mthsfa9seeds, we hypothesized that the difference between our study and
previous works (Prieto-Dapena et al. , 2006; Tejedor-Cano et
al. , 2010) might originate from differences in the seed aging
protocols. To verify this, we used the Controlled Deterioration Test
(CDT) protocol by equilibration at 100% RH and 40°C, that we refer to
as ‘wet aging’. Under these conditions, seeds of the Mthsfa9mutants lost their viability much faster than wild type seeds, showing
over three-fold reduction in lifespan compared to wild type seeds (Fig.
3b). We further investigated the effect of RH on seed lots that were
after-ripened for 7 months and submitted to the same storage temperature
(35°C) but different RH (Fig. S5a). Storage of seeds at 100% RH/35°C or
85% RH/35°C also showed a faster deterioration phenotype for the
Mthsfa9 mutants, evident from a reduction in germination speed
and final germination percentage (Fig. S5b,c). Water contents at the
different storage conditions after 56 days of equilibration were 0.14 g
H2O/g DW at 75% RH, 0.18 g H2O/g DW at
85% RH and 0.84 g H2O/g DW at 100% RH regardless of
the genotype (Fig. S5d). This indicated that elevating the water
contents to allow metabolism activates protective or repair processes
governed by HSFA9 that do not occur during dry aging.
To verify whether these findings were only applicable to HSFA9 fromM. truncatula , we isolated an Arabidopsis T-DNA insertion mutant
in which transcript level of HSFA9 (At5g54070) was
strongly reduced (Fig. S6a,b). The transcripts of two putative targets,HSP17.4 and HSP17.6 (Almoguera et al. 2002; Kotaket al. 2007) were significantly decreased in mature mutant seeds,
thereby demonstrating that HSFA9 is a transcriptional activator of these
two HSPs in Arabidopsis seeds (Fig. S6c). Like in M. truncatula ,
mature seeds of the wild type and Athsfa9 mutants lost their
viability at a comparable rate during storage at 75% RH and 35°C (Fig.
3c). In contrast, when seeds were submitted to the CDT protocol,
Athsfa9 mutants lost their viability much faster than wild type
(Fig. 3d). This suggests that HSFA9 or downstream targets begin to
function in the seeds when water content increases through equilibration
between 75% and 85% RH.