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.