INTRODUCTION
During development, seeds acquire a set of physiological characteristics
that are essential to ensure the dispersion of the species and are
therefore crucial for the establishment of seedlings in the field. These
characteristics include dormancy and seed longevity, which play
complementary roles in maintaining the embryo in a protected state in
anticipation of favorable conditions to ensure germination (Finch-Savage
& Bassel 2015; Penfield 2017; Leprince, Pellizzaro, Berriri & Buitink
2017). Both traits vary markedly among plant species with important
consequences on plant phenology, establishment in the field, yield andex situ conservation of the genetic diversity. Dormancy is an
adaptive trait that inhibits freshly matured seeds to germinate under
otherwise favorable conditions or out of the appropriate season. During
storage and/or dispersal, mature seeds progressively lose dormancy over
time, a process known as after-ripening. Longevity is defined as the
ability to survive for extended periods of time during dry storage for
seed crops and in soil seed banks for wild species (reviewed in Longet al. 2015; Sano et al. 2016; Leprince et al.2016). Longevity depends on the ability of a seed to undergo complete
desiccation without loss of viability. Dormancy and longevity are
acquired progressively during seed maturation and are under the control
of the maternal environment and mainly abscisic acid (ABA) (Graeber,
Nakabayashi, Miatton, Leubner-Metzger & Soppe 2012; Zinsmeisteret al. 2016; Penfield & MacGregor 2017).
In legumes, the acquisition of dormancy and longevity occurs during late
seed maturation concomitantly with the upregulation of heat shock
proteins (HSP) and small HSPs (sHSP) (Verdier et al. 2013; Limaet al. 2017). The expression of HSP17.4 during seed
maturation in Arabidopsis parallels the acquisition of dormancy and
desiccation tolerance, and desiccation-intolerant mutants have decreasedHSP17.4 levels (Wehmeyer, Hernandez, Finkelstein & Vierling
1996; Wehmeyer & Vierling 2000). Several functional studies point to a
role of sHSP in seed stress tolerance. In cabbage, the amount of HSP17.6
in dry seeds was positively correlated with germination under water
stress conditions and after storage at 10% moisture and 42°C (Bettey &
Finch-Savage 1998). Arabidopsis and rice seeds overexpressingOsHSP18.2 , a sHSP that accumulates during late maturation,
display an improved tolerance to controlled deterioration (CD) when
stored at 100% RH and 45°C and to osmotic stress during germination
(Kaur et al. 2015). sHSPs are thought to act as ATP‐independent
chaperones that bind stress‐denaturing proteins to prevent their
irreversible aggregation. The ATP‐dependent chaperone machinery,
composed of HSP70, HSP101 and other cochaperones can then proceed to
refold sHSP‐associated proteins (reviewed in (Ohama, Sato, Shinozaki &
Yamaguchi-Shinozaki 2017; Jacob, Hirt & Bendahmane 2017).
Accumulation of HSPs is under the transcriptional control of heat shock
factors (HSF), a large family of transcriptional factors represented by
highly conserved structural features such as a N-terminal DNA binding
domain (DBD) and an oligomerization domain composed of two hydrophobic
heptad repeats (HR-A and HR-B) connected to DBD. Clade A of HSFs are
characterized by the exclusive presence of AHA transactivator motif in
their C-terminal trans-activation domain. To exert their function in
stress tolerance, HSFAs are activated by ABA and function together with
a set of transcription factors such as DREB2A and additional
co-chaperones (Kotak, Vierling, Bäumlein & Von Koskull-Dörlng 2007;
Huang, Niu, Yang & Jinn 2016; Jacob et al. 2017; Bulgakov, Wu &
Jinn 2019). Functional diversification was established among different
HSFA members (Chauhan, Khurana, Agarwal & Khurana 2011; Jacob et
al. 2017; Bulgakov et al. 2019). HSFA9 represents a unique
member of the HSFA family that is specifically expressed in seeds during
development and under the regulatory control of ABA INSENSITIVE 3 (ABI3)
without the need of a heat shock (Kotak et al. 2007).
Seed-specific overexpression of the HSFA9 from Helianthus
annuum in tobacco activated the expression of various HSPs (HSP101,
sHSP-CI, sHSP-CII, and plastid sHSP) and transgenic seeds exhibited
increased resistance to CD (Prieto-Dapena, Castaño, Almoguera & Jordano
2006). In contrast, HaHSFA9 repression in tobacco seeds using an
active repressor version HSFA9-SRDX resulted in the reduction of
seed-specific sHSP proteins and a decreased tolerance against CD but did
not affect desiccation tolerance (Tejedor-Cano et al. 2010),
suggesting a specific role in seed longevity. Additional phenotypes
observed in HaHSFA9 overexpression lines are related to the
protection of the photosynthesis apparatus from dehydration and
oxidative stress in tobacco seedlings (Almoguera et al. 2012) or
tolerance to severe water loss (Prieto-Dapena, Castano, Almoguera &
Jordano 2008).
In Medicago truncatula, the construction of a gene regulatory
network of transcription factors preferentially expressed in seeds
identified a homologue of HSFA9 as the connecting node between
longevity and desiccation tolerance modules (Verdier et al.2013). The objective of this work was to investigate the role of HSFA9
in the regulation of seed longevity and vigor in M. truncatula .
In the dry state, seeds can survive for many years, making it difficult
to assess their longevity within a reasonable time (Hay, Valdez, Lee,
Sta Cruz & Sta. Cruz 2018). To accelerate the deterioration of seeds
during storage in a tractable time scale, CD or accelerating ageing
tests have been proposed by increasing the RH at or above 85% and
temperatures over 40°C during storage, conditions used to assess the
role of HSFA9 in previous studies. However, there are increasing
concerns from biophysics and genetics studies about the reliability of
these tests as a proxy to evaluate seed longevity ex situ in
conditions typically found in gene banks where the low RH and
temperature allowed the seeds to enter into a solid-like state as
opposed to a fluid state (reviewed in (Leprince et al. 2017; Hayet al. 2018; Ballesteros & Walters 2019; Zinsmeister, Leprince
& Buitink 2020). Here we discovered that seed survival was only
affected in hsfa9 mutants from M. truncatula and
Arabidopsis when water contents in the seeds exceeded approx. 0.2 g
H2O g DW, showing that HSFA9 has a role in
thermotolerance during wet storage, when the cytoplasm is in a fluid
state. Our study also reveals an unexpected role for MtHSFA9 in the
regulation of the depth of seed dormancy in M. truncatula .
Further analysis of putative targets indicated a strong deregulation of
genes involved in ABA and gibberellin (GA) metabolism and signaling,
translated in an increase in ABA content and hypersensitivity to ABA in
the Mthsfa9 seeds.