HSFA9 regulates dormancy via modulation of ABA metabolism and signaling
To understand the increased dormancy phenotype of the Mthsfa9seeds, we searched for evidence whether ABA or GA signaling pathways were affected by comparing the transcriptomes of Mthsfa9 seeds and hairy roots ectopically expressing MtHSFA9 . For ABA, five genes were differentially expressed in both analyses. Three genes had higher transcript levels in the Mthsfa9 mutants, and were downregulated when MtHSFA9 was expressed in the hairy roots: anindole-3-acetaldehyde oxidase (AAO , Medtr5g087410) that is involved in ABA biosynthesis, PYL/PYR6 (Medtr5g083270), an ABA receptor, and HVA22 , a marker gene of ABA response (Data S2 and Fig. 5a). In addition, a cytochrome P450 (CYP707A Medtr8g072260), involved in ABA degradation, was also differentially expressed in both studies. We also noticed that one of the most significantly overrepresented GO/KEGG category in the Mthsfa9 mutants was carotenoid metabolism (Fig. 2i, Fig S2b). A closer look at the genes in this pathway revealed that those involved in ABA biosynthesis were upregulated, including a zeaxanthin epoxidase (ZEP) and three 9-cis-epoxycarotenoid dioxygenase (NCED) , whereas two otherCYP707A genes were downregulated (Fig. 5a), suggesting that MtHSFA9 could regulate ABA homeostasis. RT-qPCR confirmed the differential expression of these genes in mature seeds from wild type and Mthsfa9 mutants from a different harvest (Fig. 5b-d). For GA, the comparison of both transcriptomes revealed the biosynthesis gene gibberellin 3-beta- dioxygenase 1 (GA3OX1) being downregulated in the Mthsfa9 seeds and upregulated in the transgenic hairy roots. In addition, several other genes related to GA metabolism were deregulated in the Mthsfa9 seeds, namely two orthologs of gibberellin 2-beta-dioxygenase 2 (GA2OX2) involved in GA inactivation that were upregulated in the mutants, and one gene involved in GA biosynthesis, gibberellin 20 oxidase 2 (GA20OX2) , that was downregulated. A GA receptor, GID1 was also found to have reduced transcript levels in the Mthsfa9 seeds. Another putative link between GA is suggested by the deregulation of the homolog of the protein REVEILLE1 (RVE1 ) both in hairy roots and Mthsfa9 seeds (Data S2). This gene was shown to directly inhibitsGA3OX2 transcription in Arabidopsis and affect dormancy (Yang, Jiang, Liu & Lin 2020). All these results are consistent with the dormancy phenotype in Mthsfa9 seeds.
Considering the higher transcript levels of ABA biosynthesis genes and decreased levels of ABA degradation genes, ABA content was determined in mature seeds. A significant increase in ABA was observed in the mature Mthsfa9 seeds compared to the wild type seeds, with an increase from 317 to 511 and 576 fmol/g DW for Mthsfa9-1 and Mthsfa9-2, respectively (Fig. 6a). To investigate whether the dormancy phenotype of Mthsfa9 seeds was due to further ABA biosynthesis during imbibition, freshly harvested seeds were imbibed in the ABA inhibitor fluridone. For all genotypes, fluridone promoted germination speed. However, compared to their respective mock controls, the impact of the fluridone treatment on the time to 50% germination (T50) was not significantly different between Mthsfa9 and wild type seeds (Fig. 6b). This suggests that dormancy in Mthsfa9seeds could not be explained by de novo ABA synthesis during imbibition but rather originates from differences induced during seed maturation. Further evidence that the dormancy phenotype of Mthsfa9 is related to the maturation program and not activated during imbibition comes from the expression of the three hormone metabolism genes that were deregulated in the Mthsfa9 mutants:MtNCED4 , MtCYP707A and MtGA3OX1 . These three genes showed clear differential expression profiles during imbibition between dormant and non-dormant wild type seeds (Fig. S9a-c). However, imbibition of Mthsfa9 seeds did not show this typical dormancy profile (Fig. S9d-f).
Besides differences in ABA metabolism, several other ABA signaling genes were deregulated in the Mthsfa9 mutants (ABA receptorPYL6 , protein phosphatase 2C, PP2C , BURP domain proteinRD22 , HVA22 protein; Fig. 5a) suggesting thatMthsfa9 seeds could exhibit altered ABA sensitivity during imbibition. This was tested by incubating after-ripened seeds in increasing ABA concentrations (Fig. 6c). Mutant seeds showed hypersensitivity when imbibed in darkness compared to wild type (Fig. 6c).
Physiological dormancy can be imposed by the embryo or the surrounding tissues (testa, endosperm) or combinations of these tissues, and their sum and interaction determine the depth of dormancy (Chahtane, Kim & Lopez-Molina 2016; Penfield 2017). To evaluate whether the dormancy phenotype of the Mthsfa9 mutants came from the embryo or the covering structures, isolated embryos were incubated in water at 25°C and radicle growth was monitored (Fig. 6d). For all genotypes, embryo radicles grew faster in coat-less seeds, as previously observed (Bolingue et al. 2010). However, the time for the radicle to initiate growth was longer for the isolated Mthsfa9 embryos compared to the wild type embryos and the final percentage of seeds with radicles that had grown was smaller in Mthsfa9 compared to wild type (Fig. 6d, Fig. S7e). Thus, the difference in germination/growth in the mutants remains visible without the surrounding layers and appears to reside mostly in the embryo.
The heat shock elements (HSE) that HSFA9 binds to has been shown to bind a modified HSE (Carranco, Almoguera & Jordano 1999). It was suggested that the specific sequence of this heat stress cis-element is crucial for HsfA9 promoter selectivity, and that this selectivity could involve preferential transcriptional activation following DNA binding. We investigated if a similar pattern could be observed for the promotors of the putative MtHSFA9 targets, starting with the two most likely targets: HSP17.5 and MtHSP18.2 (Fig. 2b, c; Kotak et al., 2007). The promotor of HSP17.5 contained one perfect HSE (Table S2, Guo et al. , 2008). HSP18.2 did not contain any perfect HSE, and neither of the two promotors contained the modified HSE detected for HaHSAF9. For the other 22 hormone-related genes that were deregulated in the Mthsfa9mutants (Fig. 5; Table S2), all HSE sites contained at least 2 out of 9 mismatches, but no common sequence could be found that could be tested as a putative binding site for MtHSFA9. However, for six genes (MtPYL6, MtPP2C, AAO, NCED, HVA22 and Ga20Ox ), a similar modified sequence GAAnnTTXnnXAA was identified, and three of these genes also show deregulation in the hairy root ectopically expressingMtHSFA9 (Fig. 5). Since MtNCED4 was one of the most differentially expressed genes (Fig. 5a), a yeast one hybrid assay was carried out to determine if MtHSFA9 could bind to the putative HSE present in the promoter region of this gene (Fig. S8). No interaction could be detected, suggesting that this gene is either not under the direct regulation of MtHSFA9 or that other parts of the promotor are needed for the interaction.
DISCUSSION
HSFA gene family members play a crucial role in plant response to several abiotic stresses by regulating the expression of stress-responsive genes including heat shock proteins (Ohama et al. 2017; Jacob et al. 2017). A peculiar HSF, HSFA9, has been reported in several species with an expression being restricted to seeds, more precisely increasing during the final part of seed development (Almoguera et al. 2002; Kotak et al. 2007; Liet al. 2015). This work tested whether a putative homologue of HSFA9 from M. truncatula, a hub gene involved in seed maturation (Fig. 1) plays a role in seed longevity, defined as the time seeds remain viable after dry storage. We demonstrated that at mild storage conditions, hsfa9 mutants of both M. truncatula and Arabidopsis did not show an altered longevity phenotype, but more humid and hot storage conditions revealed a strong decrease in seed viability (Fig. 3), the later corroborating with earlier studies using heterologous ectopic expression in tobacco (Prieto-Dapena et al. , 2006; Tejedor-Cano et al. , 2010). Further physiological characterization of Mthsfa9 mutant seeds combined with transcriptomic analysis revealed that MtHSFA9 is a regulator of the depth of seed dormancy during seed maturation via the modulation of ABA homeostasis and signalling.
Our data show that HSFA9 does not play a role in seed longevity when mild storage conditions are used. Only when moisture in the seeds was increased by the CD test, seed viability was lost at a faster rate in the hsfa9 mutants. It is well known that the water content and temperature combination in the seeds during storage will determine the rate and type of ageing reactions (Buitink & Leprince 2008; Ballesteros & Walters 2019; Zinsmeister et al. 2020). In our system, seeds were exposed to mild storage conditions (75% RH, 35°C) whereas transgenic tobacco seeds over-expressing HaHSFA9 were exposed to 42°C and 100% RH (Prieto-Dapena et al. 2008). Under these conditions, seeds imbibe and resume metabolism while tolerating the heat stress, considering that the onset of respiration is at 90-92 % RH for a wide range of species (Vertucci & Roos 1990). Consistent with this,OsHSP18.2 gene expression increased 60 fold when rice seeds were stored for 6 days at 45°C, 100% RH (Kaur et al. 2015). These storage conditions are far from ours where the seed water content was still low enough to prevent resumption of metabolism during storage. There are increasing concerns with regard to the similarity between accelerated aging conditions such as the CD test and so called natural/ambient aging conditions (Schwember & Bradford 2010; Nagelet al. 2015; Zinsmeister et al. 2016; Roach, Nagel, Börner, Eberle & Kranner 2018; Hay et al. 2018). Here we demonstrate that at least for the hsfa9 phenotype, results obtained by these methods are not comparable and it is evident that care has to be taken in extrapolating accelerated aging conditions to dry storage. Hay and colleagues (Hay et al. 2018) recommended that seed ageing protocols should be designed based on the potential downstream use of the findings and the biological significance of longevity. Therefore, the role of HSFA9 might be confined to situations in nature, when seeds are buried in the soil and submitted to rehydration-dehydration cycles rather than situations where seeds are stored in the dry state for gene banking purposes. It would be interesting to know if under more moderate soil temperatures (10-25°C), HSFA9 still exerts its protective function on seed viability. Some suggestion that this might be the case comes from the increased tolerance against osmotic stress during imbibition in tobacco seedlings over-expressing HaHSFA9 (Prietro-Dapena et al., 2008) and in Arabidopsis seeds over-expressing a sHSP of rice (Kaur et al. 2015). The most plausible downstream genes regulated by HSFA9 that confer the protection during wet and hot storage are likely to be HSP, since their modulation correlates with seed viability (Prieto-Dapena et al. 2006; Tejedor-Cano et al. 2010; Kaur et al. 2015). Our transcriptome data is consistent with this as MtHSFA9 modulated the expression several (s)HSP (Data S2). Considering the chaperone function of sHSP, the absence of decreased longevity in hsfa9 seeds raises the intriguing possibility that protein aggregation or misfolding might not occur in the dry state where molecular motion is strongly constrained (Buitink 2000, Ballesteros and Walters 2019). Besides sHSP, our transcriptomic data revealed that several components of the chaperone signalling involved in thermotolerance are being deregulated, namely three HSP70, two additional HSF, HSFB2A and HSFA2, as well as ROF1 and BAG6. Interestingly, HSFA2 is a direct target of the master regulator HSFA1 and essential in the induction of the heat shock response (Charng et al. , 2007; Jacob et al. , 2017; Ohamaet al. , 2017). Considering that HSFA2 is induced by ABA (Huang et al. 2016), the increased ABA content in developing Mthsfa9 seeds could have induced HSFA2 and its targets. In Arabidopsis, ROF1 is thought to prolong the thermotolerance by interaction with Hsp90.1 and sustaining the level of HsfA2-regulated small HSPs (Meiri and Breiman, 2009). The ortholog of BAG6, a co-chaperone that also improves basal thermotolerance in Arabidopsis through the regulation of sHSP transcriptional cascade (Echevarría-Zomeño et al. 2016), was co-expressed withMtHSFA9 (r=0.88) in seeds. BAG6 is also a target of HSFA2 (Nishizawa-Yokoi et al. , 2009).
Our results show that freshly harvested seeds of Mthsfa9 mutants germinated much slower than wild type seeds (Fig. 4). Freshly harvested wild type seeds at 40 DAP took 35 d for half of the population to germinate, whereas Mthsfa9 seeds to double the time, 69d. That this was due to a dormancy phenotype was confirmed by the removal of the phenotype by either stratification or after-ripening, leading to germination within a day (Fig. 3). Few studies link HSF or sHSPs to dormancy. A proteome study on wild Lolium rigidum (annual ryegrass) subpopulations selected for low and high levels of primary dormancy revealed that high-dormancy seeds showed higher abundance of sHSP than the low-dormancy seeds, and high-dormancy seeds were more tolerant to high temperatures imposed at imbibition (Goggin, Powles & Steadman 2010). Our data provide direct evidence suggesting that the role of HSFA9 is to regulate the depth of seed dormancy that is acquired during late maturation (Fig. 4d). In Arabidopsis, the depth of dormancy is regulated by DOG1 (Footitt et al. 2020). Interestingly in this species, HSFA9 and its targets were downregulated in thedog1-1 mutant and it was suggested that DOG1 activates this transcriptional cascade independently of ABI3 (Dekkers et al.2016). Another putative cascade in which HSFA9 could be a player in regulating depth of dormancy is via the endosperm expressed transcription factors ICE and ZHOUPI that act during maturation to determine the depth of primary dormancy (MacGregor et al. 2019). During late embryogenesis and in mature seeds, ICE1 inhibits the expression of the transcription factor ABI3 , which itself is a central player in the formation of dormant seeds (Giraudat et al.1992). In Arabidopsis, ABI3 regulates HSFA9 (Kotak et al. 2007), and MtHSFA9 transcripts are also downregulated in developing Mtabi3 mutants (Verdier et al. 2013). Interestingly, ICE has been found to interact with a complex including HSFA1d (Bulgakovet al. 2019). Here, we did not find a difference in dormancy of mature Arabidopsis hsfa9 mutants compared to wild type (Fig. S7). However, Arabidopsis Col-0 accession seeds were not very dormant under our growth conditions, and considering that the differences in dormancy between Mthsfa9 and wild type were highest prior to maturation drying (Fig. 4d), our study might not have been able to reveal potential differences. Alternatively, considering the apparent absence ofDOG1 in M. truncatula genome, MtHSFA9 might play a more preponderant role in dormancy in M. truncatula than in Arabidopsis.
The dormancy phenotype of the Mthsfa9 mutants can be explained by an increase in ABA content in mature seeds together with an increased ABA sensitivity during imbibition (Fig. 6). This phenotype was accompanied by the deregulation of a number of genes involved in ABA homeostasis, as well as the upregulation of a putative ABA receptor (Fig. 5). Whereas it is known that ABA acts upstream of HSFAs and induces its expression (Kotak et al. 2007; Huang et al.2016; Ohama et al. 2017), downstream target genes of HSFAs have so far not been implicated in ABA or GA signalling. The only suggestion that ABA signalling might be affected downstream of HSFAs comes from Huang et al (2016). These authors showed that overexpression ofHSFA6 induced hypersensitivity to ABA, which is opposite of our findings. In addition, the expression of RD22 , a drought-responsive marker gene mediated by the transcription activators MYB/MYC in an ABA-dependent manner (Abe et al. 2003) was unaffected in HSFA6b  mutants but upregulated in the Mthsfa9 mutants (Fig. 5). The ortholog of MYC2 in M. truncatula (Medtr5g030430.1) was also upregulated in the Mthsfa9mutants (Data S3). Amongst the differentially expressed genes both in Mthsfa9 mutants and in hairy roots via ectopic expression ofMtHSFA9 (Fig. 5), we identified several genes that show a typical dormancy-related expression profile during imbibition in dormant versus fully after-ripened M. truncatula seeds, MtNCED4 ,MtCYP707A and MtGa2OX2 (Fig. S9). MtCYP707A(Medtr8g72260) is an ABA 8′-hydroxylase showing highest homology withAtCYP707A1 , a gene that regulates ABA levels and dormancy during maturation (Okamoto et al.2006). Another link between HSFA9 and dormancy could be via the regulation of GA homeostasis. This is supported by the observation that GAox genes were deregulated in mature dry Mthsfa9 seeds. Also, RVE1 was upregulated in the mutants and downregulated in the hairy roots (Data S2). In Arabidopsis, overexpression of RVE1 lead to increased dormancy (Yang et al.2020). This gene directly inhibits GA3OX2 transcription, suppressing GA biosynthesis. We were not able to alter germination speed in Mthsfa9 seeds using paclobutrazol. While this supports a role of MtHSFA9 during maturation rather than imbibition, we cannot exclude that this GA biosynthesis inhibitor does not penetrate the seed coat (Bolingue et al. 2010). While this work suggests that MtHSFA9 acts as a regulator of dormancy depth, future work is needed to decipher whether HSF acts directly on genes regulating hormone metabolism and sensitivity or whether it acts indirectly through the action of specific HSPs under the control of MtHSFA9.