# The BRAHMA-associated SWI/SNF chromatin remodeling complex controls Arabidopsis seed quality and physiology

Magdalena Wrona, Julia Zinsmeister, Michal Krzyszton, Claire Villette, Julie Zumsteg, Pierre Mercier, Martine Neveu, Sebastian Sacharowski, Rafal Archacki, Boris Collet, et al.

► To cite this version:

Magdalena Wrona, Julia Zinsmeister, Michal Krzyszton, Claire Villette, Julie Zumsteg, et al. The BRAHMA-associated SWI/SNF chromatin remodeling complex controls Arabidopsis seed quality and physiology. Plant Physiology, 2024, 197 (1), pp.kiae642. 10.1093/plphys/kiae642. hal-04842520

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1 The BRAHMA-associated SWI/SNF chromatin remodeling complex controls Arabidopsis
2 seed quality and physiology

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5 Magdalena Wrona¹,*, Julia Zinsmeister¹,*, Michal Krzyszton¹, Claire Villette², Julie Zumsteg²,

6 Pierre Mercier², Martine Neveu⁴, Sebastian P. Sacharowski¹, Rafał Archacki³, Boris Collet⁵, Julia

7 Buitink⁴, Hubert Schaller², Szymon Swiezewski¹,# and Ruslan Yatusevich¹,#

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9 ¹ Institute of Biochemistry and Biophysics PAS, Warsaw 02-106, Poland

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11 ² Institut de Biologie Moléculaire des Plantes, Université de Strasbourg, Strasbourg 67084,
12 France

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14 ³ Laboratory of Systems Biology, Faculty of Biology, University of Warsaw, Warsaw 02-096,
15 Poland

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17 ⁴ INRAE, Institut Agro, Université d'Angers, IRHS, Angers 49000, France

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19 ⁵ Université Paris Saclay, INRAE, AgroParisTech, Institute Jean-Pierre Bourgin for Plant
20 Sciences (IJPB), 78000, Versailles, France.

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22 ¶ Present address: INRAE Centre IdF de Versailles-Saclay, Versailles Cedex 78026, France

23 * These authors contributed equally

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26 # Corresponding authors: sswiez@ibb.waw.pl (S.S), yatusev@gmail.com (R.Y.)

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28 Short title: BRM controls seed biology through asDOG1

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33 The authors responsible for distribution of materials integral to the findings presented in this

34 article in accordance with the policy described in the Instructions for Authors
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(https://academic.oup.com/plphys/pages/General-Instructions) are Szymon Swiezewski and

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Ruslan Yatusevich.

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**Abstract**

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The SWI/SNF (SWItch/Sucrose Non-Fermentable) chromatin remodeling complex is involved in various aspects of plant development and stress responses. Here, we investigated the role of BRM (BRAHMA), a core catalytic subunit of the SWI/SNF complex, in *Arabidopsis thaliana* seed biology. *brm-3* seeds exhibited enlarged size, reduced yield, increased longevity, and enhanced secondary dormancy, but did not show changes in primary dormancy or salt tolerance. Some of these phenotypes depended on the expression of *DOG1*, a key regulator of seed dormancy, as they were restored in the *brm-3 dog1-4* double mutant. Transcriptomic and metabolomic analyses revealed that BRM and DOG1 synergistically modulate the expression of numerous genes. Some of the changes observed in the *brm-3* mutant, including increased glutathione levels, depended on a functional *DOG1*. We demonstrated that the BRM-containing chromatin remodeling complex directly controls secondary dormancy through *DOG1* by binding and remodeling its 3' region, where the promoter of the long non-coding RNA *asDOG1* is located. Our results suggest that BRM and DOG1 cooperate to control seed physiological properties and that BRM regulates *DOG1* expression through *asDOG1*. This study reveals chromatin remodeling at the *DOG1* locus as a molecular mechanism controlling the interplay between seed viability and dormancy.
## 55 Introduction

Seeds encapsulate plant embryos in a state of suspended development, poised to resume life cycle upon encountering favorable environmental conditions. Many plant species, including *Arabidopsis thaliana*, produce seeds that can be stored in a dry stage for an extended time. This ability is known as seed longevity. In addition, seeds can also postpone germination despite optimal conditions, in a process known as dormancy that helps to adjust germination capability to changes in the environment. Dormancy established during seed maturation is known as primary dormancy, and is defined as a state in which freshly harvested seeds cannot germinate even under favorable conditions. Primary dormancy can be relieved by different means including after ripening – dry storage, or stratification – exposure to cold in the imbibed state. In contrast, dormancy developed by a non-dormant, imbibed seed in response to an unfavorable germination condition is known as secondary dormancy (Cadman *et al.*, 2006; Finch-Savage & Leubner-Metzger, 2006; Baskin & Baskin, 2014).

DELAY OF GERMINATION1 gene (*DOG1*), the main regulator of seed dormancy, has been identified by population analysis (Bentsink *et al.*, 2006) and further characterized in numerous molecular studies (Carrillo-Barral *et al.*, 2020). Primary dormancy and longevity are both acquired during seed development and are strictly regulated by numerous external and internal factors (Holdsworth *et al.*, 2008; Sano *et al.*, 2016). Interestingly, in Arabidopsis a trade-off between seed longevity and dormancy was described, as deep dormancy was associated with low longevity, suggesting that longevity and dormancy are genetically negatively correlated. On the contrary, *DOG1* has been shown to act as a positive regulator of both dormancy and longevity as *dog1* mutants show low primary and secondary dormancy as well as low longevity (Nguyen *et al.*, 2012; Dekkers *et al.*, 2013; Footitt *et al.*, 2015). As a central regulator of seed biology, *DOG1* expression is extensively regulated (Tognacca & Botto, 2021). Known regulators include two long non-protein coding RNA (lncRNA): one is the *PUPPIES* that activates *DOG1* expression and is transcribed from the *DOG1* promotor (Montez *et al.*, 2023) and a second is *asDOG1* that is transcribed from within the *DOG1* intron 2 in antisense orientation and suppresses *DOG1* expression (Fedak *et al.*, 2016).

Secondary dormancy modulation underlies the dormancy cycling phenomena described for seeds forming soil seed banks (Footitt *et al.*, 2015, 2017). Analysis of histone posttranslational modifications at the *DOG1* gene during dormancy cycling has led to a model where chromatin remodeling at the *DOG1* locus underpins this process (Footitt *et al.*, 2015). Compared to primary
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dormancy, the mechanisms of secondary dormancy establishment in various plant species including *Arabidopsis thaliana* are mostly uncharted. Only few regulators have been identified so far and most of them influence both primary and secondary dormancy (Skubacz & Daszkowska-Golec, 2017; Buijs, 2020). Both types of dormancy are intricately modulated by environmental cues, such as variations in light quality, moisture levels, and transient cold exposure. They are also dependent on internal hormones, namely gibberellic acid (GA) and abscisic acid (ABA, Hauvermale *et al.*, 2015; Sano & Marion-Poll, 2021). ABA plays a pivotal role in initiating and sustaining dormancy, while GA acts as the trigger that breaks dormancy and promotes the germination process (Iwasaki *et al.*, 2022).

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SWI/SNF (SWItch/Sucrose Non-Fermentable) is a highly conserved chromatin-remodeling complex that uses ATP to remodel chromatin. SWI/SNF complexes have been implicated in the control of multiple developmental processes and in orchestrating responses to environmental stimuli in yeast, plants, and animals (Ojolo *et al.*, 2018; Hernández-García *et al.*, 2022; Bieluszewski *et al.*, 2023). In Arabidopsis, several homologous subunits of the SWI/SNF complex have been described and many of these subunits are encoded by gene families, including SNF2-type ATPases: SPLAYED (SYD), BRAHMA (BRM), CHR12/MINU1 and CHR23/MINU2 (Shang & He, 2022; Guo *et al.*, 2022a). These subunits create the basis for plant SWI/SNF complex taxonomy dividing them into BRM-associated (BAS), SYD-associated (SAS) and MINU-associated (MAS) (Guo *et al.*, 2022b; Fu *et al.*, 2023). The BRM-containing complex (BAS) is probably the best-studied chromatin remodeling complex in plants. BRM ATPase contains multiple protein domains, including a bromodomain that binds acetylated histones and is thought to facilitate the complex recruitment to targeted DNA. In addition to BRM, the BAS complex contains other bromodomain-containing proteins - BRDs (BRD1/2/13) (Jarończyk *et al.*, 2021). Studies of Arabidopsis mutants of BAS complex subunits have shown that the BRM containing BAS-SWI/SNF complex is involved in multiple environmental responses and developmental transitions including seed maturation, embryogenesis, cotyledon separation, leaf development, root stem cell maintenance, floral patterning or flowering (Yu *et al.*, 2021; Shang & He, 2022; Guo *et al.*, 2022b; Bieluszewski *et al.*, 2023). Consistent with these findings, Arabidopsis *brm* knockout mutants (*brm-1*) display severe phenotypes including dwarfism, leaf curling, and sterility (Farrona *et al.*, 2004). Conversely, the *brm-3* mutant, which lacks the bromodomain in the BRM protein, is not sterile and exhibits mild phenotypic abnormalities, including seed coat defects (Hurtado *et al.*, 2006; Farrona *et al.*, 2007). Similar analysis of single and multiple mutants of the *BRD1/2/13* genes revealed their redundant role in regulating
vegetative development, flowering, as well as the responses to GA and ABA hormones (Jarończyk et al., 2021; Stachula et al., 2023). Despite the extensive work describing SWI/SNF and BRM's role in seeds development and germination (Han et al., 2012; Ding et al., 2022), the role of the BAS complex in seed biology remains poorly understood.

We and others showed before that apart from promoter regions, chromatin remodelers exhibit extensive binding at the 3'ends of genes (Brzezinka et al., 2016; Archacki et al., 2016; Jégu et al., 2017). This led us to hypothesize that the BAS-containing SWI/SNF complex may regulate antisense transcription to indirectly control the sense gene expression (Archacki et al., 2016). A reporter-effector study in young Arabidopsis seedlings identified DOG1 as one of the genes displaying BRM binding at the 3'end (Archacki et al., 2016). Here, we asked whether the link between BRM and DOG1 may be important in seeds where the role of DOG1 and its regulation through asDOG1 is well established (Fedak et al., 2016). Here we show that BRM is implicated in multiple aspects of seed biology including seed size, seed longevity and seed dormancy. We demonstrate that some of the affected seed properties, including dormancy, are DOG1 dependent and that the BAS complex controls secondary dormancy but not primary seed dormancy through asDOG1 antisense transcription.

## 136 Results

### 137 BRM-mediated gene expression and metabolite composition in mature Arabidopsis seeds

To investigate the role of BRM in Arabidopsis seed biology in the context of DOG1, we created *brm-3dog1-4* double mutant and compared it with the wild type and single mutants in downstream analyses. *BRM* knockout allele *brm-1* is sterile, we therefore used the T-DNA insertion line *brm-3* (Tang *et al.*, 2008) and the double mutant *brm-3dog1-4*. 3'RNA-seq data analysis identified 77, 211 and 911 transcripts with decreased transcript levels in *brm-3*, *dog1-4* and *brm-3dog1-4*, respectively, and 167, 385 and 1391 transcripts with increased expression (absolute fold change > 1, FDR < 0.05) (Fig. 1A). As expected, comparison of differentially expressed genes in *brm-3* and *dog1-4* single mutants showed strong overlap with genes misregulated in double *brm-3dog1-4* mutant (Fig. 1B, Supplementary Figure S1C). Moreover, genes misregulated in *brm-3* and *dog1-4* also showed a substantial overlap (Fig. 1B). Interestingly, *dog1-4*, *brm-3* and double *brm-3dog1-4* mutants showed a substantial number of genes misregulated in the same directions (up and/or down), suggesting that DOG1 and BRM act synergistically in controlling gene expression in seeds (Fig. 1B). Self-clustering of expression profiles among genes misregulated in *brm-3* identified nine groups of genes (Fig.1C). Three
groups showed opposite changes in *brm-3* and *dog1-4* and at least partial suppression of the *brm* mutation-caused defects in the double *brm-3dog1-4* mutant (Fig.1C). Based on this, we considered the BRM effect on those genes as *DOG1*-gene dependent (Fig. 1C, D).

Transcriptomic analysis showed that both BRM and DOG1 are important players in seed biology and gene expression regulation. Our data revealed that in seeds, BRM and DOG1 control expression of a large number of genes synergistically. There is, however, a substantial subset (55 out of 244) of genes that are regulated by BRM in DOG1 dependent manner (Fig. 1C, 1D).

Gene Ontology (GO) analysis identified 20 and 30 significantly enriched GO terms among genes that were down- and up-regulated in the *brm-3* mutant, respectively. Among those, many GO terms represented molecular functions involved in response to oxidative stress, with some related to glutathione metabolism, binding and transferase activity, which are essential for the control of reactive oxygen species (ROS) (Supplementary Table 1). Single *dog1-4* mutant and *brm-3dog1-4* double mutant showed similar GO term profiles. Those included response to multiple factors, like stimulus or ABA for down-regulated genes (Supplementary Fig. S2A), as well as GO terms related to translation – for up-regulated genes (Supplementary Table 1).

Next, we analyzed the metabolic profiles of *brm-3*, *dog1-4* and *brm-3dog1-4* double mutant mature seeds using a non-targeted comparative metabolomics approach based on high-resolution mass spectrometry. Molecular features detected in each single mutant and in the double mutant *brm-3dog1-4* were compared to Col-0 wild type (WT). This identified a total of 410, 112 and 418 differentially abundant molecular features (metabolites) in *brm-3*/WT, *dog1-4*/WT and *brm-3dog1-4*/WT comparisons, respectively (Wilcoxon rank sum test, fold change≥2; p-value<0.05, Supplementary Fig. S1). Among them, 223 were more abundant and 187 were less abundant in *brm-3* in comparison to Col-0 WT. In *dog1-4* mutant, 30 were more and 82 less abundant when compared to WT. Finally, in the double mutant, 254 metabolites were more, and 164 were less abundant when compared to Col-0 WT, indicating a significant metabolic remodeling in *brm-3* and *brm-3dog1-4* mutants. Assigned masses allowed us to identify a putative molecular formula for 170 out of 410 differentially expressed metabolites for *brm-3*/WT, 37 out of 112 for *dog1-4*/WT and 143 out of 418 compounds for double mutant (Supplementary Table 1). To visualize the metabolic changes between mutants and the Col-0 WT, we used a chemical enrichment analysis named ChemRICH (Chemical Similarity Enrichment Analysis for Metabolites) according to Barupal and Fiehn, 2017, which provides differentially enriched clusters of metabolites families. Such clusters were identified for each mutant separately (Fig. 1E and
Supplementary Table 1). For *brm-3*, we detected 26 differentially abundant metabolic families (p-value<0.05), with a decrease in compounds such as phosphatidylcholine, phosphatidylethanolamine, aldehydes, flavonoids and glucosinolates and an enrichment of indoles, polyunsaturated alkamides, phenols, unsaturated fatty acids and amides (Fig. 1E). Interestingly, a 1.6 FC (p-value=0.0012) enrichment in glutathione was found in *brm-3* mutant compared to Col-0 WT (Fig. 1F, Supplementary Table 1). Similarly, in the seeds of the *dog1-4* mutant metabolomic analysis revealed decrease in flavonoids and peptides levels and an enrichment in phenols and glucosides (Fig. 1E). Interestingly, in *dog1-4* mutant we found a -2.08 FC decrease (p-value=0.0059) in glutathione enrichment compared to Col-0 (Fig. 1E, 1F). In the double mutant *brm-3dog1-4*, we found a broad variety of metabolic families up or downregulated compared to Col-0 WT. We noticed an enrichment in phospholipids ethers, phenols, saturated and unsaturated lysophosphatidylcholine, lactones, saturated fatty acids and indoles; and lower levels of phosphatidylcholine, phosphatydylethanolamine, glucosinolates, flavonoids and glucosides (Supplementary Table 1). Interestingly, as observed for the single mutants, the double mutant glutathione was downregulated compared to the wild type (Fig. 1E, 1F, Supplementary Table 1), with a FC of -1.85 (p-value=0.00058). We conclude that similarly to RNA-seq analysis, metabolomic analysis shows that *dog1* enhances metabolic changes in the *brm-3* background as many changes are only visible or more pronounced in the double *brm-3dog1-4* mutant. In addition to untargeted analysis of metabolites, a targeted analysis of soluble sugars and hormones was performed in the mature seeds of the mutants. This analysis showed that ABA levels were slightly reduced in *brm-3* and *dog1-4* mutant seeds while double mutant seeds showed intermediate levels of ABA, with no significant difference to either Col-0 or single mutants (Supplementary Fig. S3A). The small effect on ABA suggests that phenotypic defects observed in *brm-3* and double mutant are probably not driven through ABA. Similarly, analysis of GA levels in dry seeds showed no significant difference for *brm-3* seeds and higher but not significant levels for *dog1-4* and double mutant seeds (Supplementary Fig. S3B). Given the published role of sugars in seed maturation, we also analyzed levels of sucrose, raffinose and stachyose (Li *et al.*, 2017; Salvi *et al.*, 2022). Seeds of *brm-3* and *brm-3dog1-4* contained significantly higher levels of raffinose and lower levels of sucrose, resulting in increased RFO/sucrose ratio (Supplementary Fig. S4A, 4B, 4C). Likewise, stachyose levels were also lower in seeds of the *brm-3* single and *brm-3dog1-4* double mutant (Supplementary Fig. S4D). In contrast, seeds of *dog1-4* mutant did not show changes in sugar levels. These results suggest that BRM is implicated in sugar level control in seeds but independently of DOG1 (Supplementary Fig. S4).
In summary, our metabolomic analysis revealed that *brm-3* and *dog1-4* share some common
differentially enriched compounds compared to the wild type, while much more diverse families
of metabolites are differentially enriched in the double mutant, confirming the synergistic action
of BRM and DOG1 in seeds biology (Supplementary Fig. S1, Supplementary Table 1).
Interestingly, our data suggest the glutathione level is controlled by BRM in a DOG1 dependent
manner, as it was increased in *brm-3* mutant while in *dog1-4* and *brm-3dog1-4* double mutant
glutathione was less abundant compared to Col-0 WT seeds (Fig. 1F, 1E, Supplementary Fig.
S5).

226 **BRM regulates seed quality and physiology**

227 To assess the BRM and DOG1 effect on seed quality, we first checked the seed size in single and
228 double mutants (Fig. 2A, B and Supplementary Fig. S6A). The *brm-3* mutant seeds were 20%
229 bigger compared to the wild type and the *dog1-4* mutant, with an average size of 0.27, 0.21 and
230 0.23 mm² for *brm-3*, *dog1-4* and the wild type, respectively (Fig. 2B, Supplementary Fig. S6A).
231 The *brm-3* mutant seeds were also 25% heavier (p-value < 0.00095) compared to the wild type.
232 In addition, even though *brm-3dog1-4* seeds were not significantly larger than Col-0 WT seeds,
233 double mutant seeds were significantly heavier than Col-0 seeds (Supplementary Fig. S6D). In
234 addition, germination rate of *brm3* and *brm3dog1-4* double mutants in the presence of ABA was
235 similar to Col-0 WT seeds (Supplementary Fig. S6C). As previously reported for *brm-3* (Farrona
236 et al., 2007), *brm-3* and *brm-3dog1-4* mutants had significantly reduced seed yield while *dog1-4*
237 mutant did not show a difference compared to the wild type (Fig. 2C). Our data suggest that seed
238 quality measured by seed size, weight but not yield is affected by BRM in DOG1 dependent
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240 Glutathione is one of the main antioxidants in seeds and its level decreases during seed ageing
241 (Ranganathan & Groot, 2023). Our metabolomic analysis showed changes in glutathione levels
242 prompting us to analyze the role of BRM in longevity. Seeds of *brm-3* mutant showed increased
243 longevity compared to the wild type (Supplementary Fig. S6B), as seen in the analysis of the
244 time required for 50% loss of viability (P<sub>50</sub>, Fig. 2D). As published by Dekkers et al. 2016, we
245 observed that the P<sub>50</sub> of the *dog1-4* mutant was lower compared to the wild type, demonstrating a
246 role of DOG1 in enhancing longevity. Interestingly, the P<sub>50</sub> of the double mutant *brm-3dog1-4*
247 was only slightly higher than for the single *dog1-4* mutant. In addition, we performed a
248 germination analysis of 4-year-old naturally aged seeds of the tested mutants. This analysis
249 revealed a germination phenotype similar to that observed in artificially aged seeds: the *brm3*
mutant showed significantly higher germination rates compared to Col-0 WT seeds, but this effect was suppressed in the *brm3dog1-4* double mutant (Supplementary Fig. S6E). This suggests that BRM role in seed longevity is partially DOG1 dependent.

As we found BRM affects seed longevity, we were further interested in its effect on seed's vigor. We analyzed salt sensitivity during germination of after-ripened seeds (Fig. 2E). After-ripened seeds of *brm-3* showed no difference in germination in presence of 100mM NaCl compared to Col-0. In contrast to what has been observed for freshly harvested stratified seeds, the *dog1-4* mutant showed reduced germination when compared to Col-0 WT (Montez *et al.*, 2023). The *brm-3dog1-4* double mutant behaved like the *dog1-4* single mutant suggesting that BRM is not involved in salt-mediated germination delay (Fig. 2E). Given the role of DOG1 in dormancy, we performed primary and secondary dormancy tests on the mutants. Primary dormancy was tested by germination of freshly harvested seeds and secondary dormancy was analyzed on seeds pre-treated with high temperature in the darkness (Footitt *et al.*, 2015; Krzyszton *et al.*, 2022). In agreement with published results (Krzyszton *et al.*, 2022), we observed that the *DOG1* gene is required for primary as well as secondary dormancy, as the *dog1-4* mutant showed nearly 100% germination of both freshly harvested seeds and seeds induced into secondary dormancy. In contrast, the *brm-3* mutant displayed a stronger secondary but unaffected primary dormancy when compared to Col-0 WT seeds (Fig 2F, G), suggesting a specific function of BRM in secondary dormancy regulation. Likewise for *brm-3*, stronger secondary seed dormancy was also observed in *brm-5*, *3xbrd* and *swp73a*, other SWI/SNF subunit mutants (Supplementary Fig. S7, S8A).

In summary, we showed that BRM is an important regulator of seed biology required for many aspects of seed development and environmental sensing. Our data reveals a genetic requirement of the functional *DOG1* gene for BRM-mediated control of seed longevity and secondary dormancy. The lack of primary seed dormancy defects and stronger secondary seed dormancy observed in *brm-3* and *brm-5* mutants is surprising, as primary and secondary dormancy levels are usually correlated in Arabidopsis mutants (Buijs, 2020; Sajeev *et al.*, 2024).

## BRM binds to the DOG1 3' region and regulates asDOG1 antisense expression

Our genetic analyses suggested that BRM-mediated regulation of secondary dormancy requires the *DOG1* gene. Previously, it has been shown that the *DOG1* gene 3' end is bound by BRM and BRD in seedlings (Yu *et al.*, 2020, Yu *et al.*, 2021, Archacki *et al.*, 2016, Supplementary Fig. 9)
S9A, S9B). To confirm BRM binding at the *DOG1* locus in seeds, we performed ChIP-qPCR experiment using a transgenic line expressing BRM-GFP under a native promoter in the background of *brm-1* null mutant (Jarończyk *et al.*, 2021). We observed BRM binding mostly within exon 2 and exon 3 of the *DOG1* gene, matching the location of previously described by us antisense lncRNA *DOG1* promoter (Fig. 3A). This suggests that BRM could control the *DOG1* gene expression in seeds via as*DOG1* (Fedak *et al.*, 2016).

In non-dormant dry seeds, BRM was bound at exon 2 and exon 3 regions (as*DOG1* promoter), similar to genome-wide ChIP data from Arabidopsis seedlings (Fig. 3B, Supplementary Fig. S9). During SD induction, BRM was predominantly bound at exon 3 (Fig. 3B). This suggests that secondary dormancy induction may lead to changes in the way BRM controls *DOG1* sense and antisense expression. This is in agreement with our phenotypic analysis (Fig. 2G, Supplementary Fig. S7) that demonstrated that *brm-3* seeds show an enhanced propensity to enter secondary dormancy while seeds collected from *brm-3dog1-4* double mutant are unable to enter dormancy.

Next, we performed RT-qPCR analysis of *DOG1* gene expression in the Col-0 WT and selected mutants during secondary dormancy induction. Upon imbibition at 4 hours, we observed a strong *DOG1* mRNA reduction followed by a gradual rebuild of *DOG1* mRNA levels during secondary dormancy induction (Fig. 3C). This is in agreement with previously published by us and others' results (Buijs G. 2020; Krzyszton et al. 2022; Sajeev et al. 2024). Interestingly, we observed a much stronger increase in *DOG1* mRNA levels in *brm-3* mutant (Fig 3C). To test if the observed BRM role in *DOG1* expression regulation during secondary dormancy induction requires activity of the whole BAS complex, we used a single *swp73a* and a triple *BRD1/2/13* (*3xbrd*) mutant that both are components of BAS SWI/SNF complex (Guo *et al.*, 2022; Fu *et al.*, 2023). We observed hyper activation of *DOG1* expression in *3xbrd* and *swp73a* mutant seeds (Fig. 3C, Supplementary Fig. S8B) that was very similar to the one observed in *brm-3*. We also note a stronger dormancy phenotype during secondary dormancy induction for *3xbrd* and *swp73a* mutants (Supplementary Fig. S7, S8A). The observed upregulation of *DOG1* expression in the *brm-3*, *swp73a* and *3xbrd* mutants suggests a previously unrecognized role for BRM and the SWI/SNF BAS-complex in the suppression of *DOG1* expression during secondary dormancy establishment. Gene expression analysis showed increase in the mRNA levels of the *BRM* and *SWP73A* genes but not for *BRD1*, suggesting that the main SWI/SNF subunits are co-induced with *DOG1* gene during secondary dormancy establishment. However, BRM expression was not affected in *dog1-4* mutant compared to Col-0 WT seeds during induction (Supplementary Fig.
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S10A). Thus, our genetic and RT-qPCR analyses suggested that BRM function upstream of the DOG1 gene.

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In parallel to sense transcript expression during secondary dormancy induction, we analyzed *asDOG1* transcript levels. Similarly, to sense transcript expression, we observed a gradual accumulation of antisense transcript during dormancy induction (Fig. 3D). All *brm-3*, *swp73a* and *3xbrd* mutants showed a clear reduction in the levels of *asDOG1* expression when compared to the Col-0 seeds at later stages of dormancy induction (Fig. 3D, Supplementary Fig. S9C). Together with BRM binding at *DOG1* 3'end this suggests that BRM directly regulates *asDOG1* transcription. To test this possibility, we used the *p<sub>AS</sub>DOG1* promoter-driven IRES-LUC reporter line and crossed it with the *brm-3* mutant. RT-qPCR using LUC primers showed significant downregulation of *LUC* transcript in *brm-3*, suggesting that BRM directly regulates *asDOG1* promoter activity (Fig. 3F).

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## BRM regulation of DOG1 gene expression requires asDOG1

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Observed by us direct regulation of *DOG1* antisense expression by BRM and the lack of BRM binding to canonical *DOG1* promoter suggested a model where BRM regulates *DOG1* sense expression through *DOG1* antisense. To test this model, we first asked if BRM can regulate *DOG1* expression in the absence of *asDOG1*. We used a previously published (Fedak *et al.*, 2016) transgenic truncated *DOG1* gene (*p*<sub>DOG1sh</sub>*DOG1::LUC) with an antisense promoter deleted and crossed it to a *brm-3* mutant (Fig. 3E). Importantly, no significant differences were observed between Col-0 and *brm-3* suggesting that BRM is unable to regulate the *DOG1* gene expression when the *DOG1* 3' region is removed. Surprisingly, *p*<sub>DOG1sh</sub>*DOG1*::LUC did not show the induction of expression that we observed for endogenous sense mRNA.

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Therefore, the inability of BRM to regulate *DOG1* gene with antisense deleted (*p*<sub>sense</sub>*DOG1-LUC*), together with the fact that in *brm-3* mutant *p*<sub>AS</sub>*DOG1* activity is suppressed, suggest that BRM acts through antisense. The fact that *p*<sub>sense</sub>*DOG1-LUC* transgene did not recapitulate endogenous *DOG1* sense expression inductions suggest that some of the elements required for *DOG1* induction upon secondary dormancy induction are located in regions deleted in the construct.

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To test this possibility, we took advantage of a set of TATA mutations shown by us previously to greatly reduce *asDOG1* transcript expression (Yatusevich *et al.*, 2017). We engineered these mutations in an antisense promotor of the *DOG1* genomic reporter construct creating *LUC*-
346 *DOG1-deltaTATA* transgenic lines. RT-qPCR analysis using primers that amplify only 347 transgene-originating *DOG1* mRNA showed that removal of antisense transcription indeed 348 resulted in strong upregulation of *DOG1* sense transcript at early stages of dormancy induction 349 (Fig. 3G, Supplementary Fig. S11A, S11B). Together the transgenes analysis shows that BRM 350 requires *asDOG1* to control *DOG1* expression during secondary dormancy induction and that 351 *asDOG1* acts as negative regulator of *DOG1* expression during secondary dormancy 352 establishment. Similar to *brm-3* and *brm-5* mutants, transgenic *LUC-DOG1* lines carrying 353 dTATA mutations showed stronger secondary seed dormancy compared to WT *LUC-DOG1* 354 lines, confirming *asDOG1* acts as a negative regulator of *DOG1* expression during secondary 355 dormancy (Supplementary Fig. S11C).

356 BRM is part of the SWI/SNF complex and our data show that both BRM and another SWI/SNF 357 complex subunits - BRDs are implicated in seed secondary dormancy control through *asDOG1* 358 antisense transcript promoter regulation. SWI/SNF is a chromatin remodeling complex that 359 utilizes ATP to remodel chromatin at target loci (Mashtalir *et al.*, 2018). To test if BRM- 360 mediated regulation of *DOG1* sense expression through *asDOG1* is accompanied by DNA 361 accessibility changes, we performed FAIRE (Formaldehyde-Assisted Isolation of Regulatory 362 Elements) during secondary dormancy induction (Omidbakhshfard *et al.*, 2014). On the third day 363 of secondary dormancy induction, *brm-3* showed a marked increase in DNA accessibility using 364 FAIRE at the end of exon 2 compared to dry seeds (Fig. 4A, 4B). This increase was even more 365 pronounced in the last intron and exon 3 of the *DOG1* region on the 5th day of induction, 366 centering around the *DOG1* antisense promoter region (Supplementary Fig. S8D). This region 367 colocalized with the BRM binding site identified in ChIP experiments (Fig. 3A, B; Fig. 4A, B; 368 Supplementary Fig. S8C, S8D). In agreement with BRM involvement in *DOG1* regulation during 369 secondary dormancy, the FAIRE experiment in dry seeds failed to detect a localized increase at 370 *DOG1* 3' end in *brm-3* compared to Col-0 WT dry seeds (Fig. 4A). Thus, this suggests that 371 during secondary dormancy induction, BRM is directly bound at *DOG1* 3' end and locally 372 remodels chromatin presumably to regulate *asDOG1* expression. This is consistent with the 373 *asDOG1* requirement for BRM's ability to suppress *DOG1* gene expression during secondary 374 dormancy induction.

375 **BRM affects DOG1 splicing and polyadenylation**

376 To gain a deeper understanding of *DOG1* regulation by BRM, we analyzed the misregulation of 377 *DOG1* splicing and polyadenylation in *bmr-3* seeds during the induction of secondary dormancy.
Our RT-qPCR analysis examined early time points (2- and 4-days post-induction) for splicing and up to 14 days post-induction for polyadenylation. We detected increased levels of the short proximally polyadenylated *DOG1* mRNA (*shDOG1*), but not the long form (*IgDOG1*), during secondary dormancy induction in *brm-3* (Fig. 4C, 4D). We also found a significant increase in *β* (beta) and *γ* (gamma) *DOG1* mRNA splicing forms in *brm3* mutants compared to Col-0 seeds (Fig. 4E-H). Increased *β*, *γ* and *shDOG1* were not only observed in *brm-3* but also in *3xbrd* mutant, suggesting that they are a result of BRM activity linked to SWI/SNF complex. The increase in *β*, *γ* and *shDOG1* mRNA is consistent with the observed stronger *brm-3* mutant secondary seed dormancy phenotype as *shDOG1* has been reported to be the predominant *DOG1* isoform that can complement the *DOG1* mutant phenotype and *β* and *γ* mRNA isoforms lead to production of the same protein as encoded by *shDOG1*. Interestingly, in the *ntr1* mutant, known as a spliceosome disassembly factor (Dolata *J et al.*, 2015), we observed a significant reduction in *aDOG1* mRNA splicing forms, while other *β*, *γ* and *δ* splicing forms showed similar kinetics to one observed in Col-0 WT (Supplementary Fig. S15). The differential effect of *brm-3* and *ntr1* on *DOG1* splicing suggest that BRM does not control *DOG1* splicing through NTR1.

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## 394 DISCUSSION

### 395 BRM containing SWI/SNF complex controls seed physiological quality

We show that *brm-3* mutant exhibit multiple seed-related phenotypes, including enlarged seed size, reduced seed yield, increased seed longevity and enhanced secondary dormancy induction but no change in primary dormancy or germination in the presence of salt (Fig. 2, Supplementary Fig. S14). Given the central role of the *DOG1* gene in seed biology as well as BRM binding to *DOG1* locus, we tested the interplay of *BRM* and *DOG1* genes in seeds. We found that some of the *brm-3* mutant phenotypes are genetically dependent on *DOG1* gene as a double mutant of *brm-3dog1-4* shows a reversal of the longevity and secondary dormancy phenotypes (Fig. 2). Transcriptomic analysis in dry seeds showed that around 20% of genes misregulated in *brm-3* are *DOG1* gene-dependent as *dog1-4* mutation can partially or fully suppress the *brm* mutation effect on their expression in the *brm-3dog1-4* double mutant (Fig. 1D). In addition, we observed a pronounced misregulation of gene expression in the double *brm-3dog1-4* double mutant (Fig. 1A) which suggests a synergistic function in the case of the majority of affected genes.

### 408 Longevity and metabolite accumulation
GO terms analysis among differentially regulated genes suggested changes in genes involved in the biosynthesis of metabolites known to be important in seed biology (Supplementary Table 1) that were mostly consistent with changes observed in seed metabolome analysis (Supplementary Fig. S1A, 1B). RNA-seq data showed that genes related to glutathione metabolism were enriched among genes downregulated in *brm-3dog1-4* double and *dog1-4* single mutant and upregulated in single *brm-3* mutant (Supplementary Fig. S2, S5). This is consistent with our metabolomic analysis that showed an increase in *brm-3* and lower levels of glutathione in *dog1-4* and *brm-3dog1-4* mutants (Fig. 1F, Supplementary Fig. S5). Given the published link between glutathione level, dormancy and longevity in Arabidopsis (Cairns *et al.*, 2006; Nguyen *et al.*, 2015; Koramutla *et al.*, 2021), we note that the observed changes in glutathione level are probably responsible for the observed *DOG1* gene-dependent partial increase in longevity of *brm-3* mutant. This observation is corroborated by the suggested previously positive role of *DOG1* in longevity based on analysis of natural variation and *DOG1* NIL line analysis (Nguyen *et al.*, 2012).

Changes in soluble sugar contents have been suggested to be involved in germination and longevity regulation (He *et al.*, 2016). In legume species, a correlation between lower seed storability and a lower ratio between RFO and sucrose has been reported (Pereira Lima *et al.*, 2017). While in Arabidopsis, this correlation remains unclear, as an increase in the RFO/sucrose ratio was not found to be correlated with seed vigor (Bentsink *et al.*, 2000; Li *et al.*, 2017). We found a significant decrease in RFO/sucrose ratio in both single *dog1-4*, *brm-3* and double mutants (Supplementary Fig. S4B). However, there is no significant difference in this ratio between *dog1-4* and *brm-3* mutants, despite the difference in seed longevity between them. Thus, our data suggest that in Arabidopsis, there might be no direct link between RFO and seed longevity. However, it is interesting to further investigate galactinol contents as it has been linked to seed biology and has not been measured by us (De Souza Vidigal *et al.*, 2016).

In addition, in dry *brm-3* mutant seeds, we observed significant alterations in various classes of tryptophan-derived metabolites, including auxin, camalexin, and indole-glucosinolates. The deregulation of genes involved in the tryptophan-derived metabolite pathways (such as *MYB34*, *MYB51*, *MYB122*, *NITs*, *ARFs*, *CYP79B3* etc.) in the *brm* mutant may contribute to the observed auxin-related phenotypes and reduced seed yield of *brm-3* (Supplementary Fig. S2B). While camalexin and indole-glucosinolates are recognized as plant defensive secondary compounds against pathogens and herbivores (Stotz *et al.*, 2011; Nguyen *et al.*, 2020), their specific biological roles in seeds dormancy remain to be fully elucidated.
442 The role of BRM in glutathione accumulation in seed is DOG1-dependent

443 Glutathione is an important player in redox signaling and is involved in protection against
444 excessive oxidation in multiple plant tissues (Mhamdi *et al.*, 2010). Accumulation of oxidative
445 damage during dry seed storage is probably the most important factor behind deterioration of
446 seed quality and eventually loss of viability determining seed longevity (Kumar *et al.*, 2015). Our
447 transcriptomic analysis showed that genes related to glutathione metabolism were misregulated in
448 *brm-3* mutant in the opposite direction to changes observed in *dog1-4* mutant, including *GPX1*
449 and *GPX6* that are responsible for glutathione biosynthesis. We also observed multiple other
450 misregulated genes in different pathways related to glutathione synthesis, degradation and
451 recycling. Genes coding for GLUTATHIONE S-TRANSFERASEs: GSTU9, GSTU10, GSTU11,
452 GSTU12 and GSTU19 were significantly up-regulated (FDR < 0.05, FC > 2) in mature seeds of
453 *brm-3* mutant compared to Col-0 WT seeds (Fig. 1F, Supplementary Fig. S2, S5). No consistent
454 change of the transcript levels of these genes were observed in *dog1-4* seeds.

455 In agreement with the observed deregulation of glutathione-related transcripts, we observed a
456 higher level of glutathione in the *brm-3* mutant, and depletion in *dog1-4* and double mutant
457 compared to Col-0. Those results are concordant with the reduced longevity in both *dog1-4* and
458 double mutant, and the increased longevity of the *brm-3* mutant.

459 ABA and GA hormonal signaling in *brm-3* mutant seeds during SD

460 Our analysis of hormone levels in dry seeds revealed relatively minor differences in the single
461 mutants *brm-3* and *dog1-4* regarding GA and auxin content (Supplementary Fig. S3B, S3C).
462 Interestingly, we observed a significantly elevated auxin level in the *brm-3 dog1-4* double
463 mutant (Supplementary Fig. S2C), correlating with increased expression of auxin pathway genes
464 (Supplementary Fig. S7B). A slight reduction in ABA levels was detected in *dog1-4* and slightly
465 in the double mutant (Supplementary Fig. S3A). Members of the SWITCH2 (SWI2)/SNF2
466 chromatin-remodeling complexes play a role in seed germination under ABA treatment.
467 BRAHMA (BRM) directly suppresses the expression of ABI5 and, consequently, the *brm-3*
468 mutant exhibits increased ABA sensitivity during seed germination (Han *et al.*, 2012). The role
469 of ABA and its signaling pathway in seed biology has been extensively studied, including its role
470 in secondary seed dormancy establishment (Auge *et al.*, 2015; Ibarra *et al.*, 2016).

471 In agreement with published results, we have previously reported that quadruple *nced2469* mutant failed to enter into secondary dormancy (Lefebvre *et al.*, 2006; Krzyszton *et al.*, 2022).
472 Here, we show that *nced2469* shows no defect in *DOG1* expression during secondary dormancy
474 induction when compared to Col-0 seeds (Supplementary Fig.S10B). Also, analysis of the
475 expression of genes related to ABA biosynthesis and catabolism (NCED4/5 and CYP707A2)
476 showed only minor fluctuations during secondary dormancy induction in Col-0 seeds and no
477 major differences when compared to brm-3 or dog1-4 mutants (Supplementary Fig. S12A,
478 S13A). In contrast, we observed a strong induction of RGL1, RGL2 and GAI genes - known
479 negative regulators of the GA pathway, during secondary dormancy induction. Interestingly
480 RGL1 and RGL2 but not GAI showed strong upregulation in brm-3 mutant when compared to
481 WT seeds during dormancy induction (Supplementary Fig. S12B). In contrast, in dog1-4 mutant
482 only RGL1 and GAI genes were induced (Supplementary Fig. S13B). While we did not analyze
483 the levels of gibberellins during secondary dormancy induction, this may suggest a role of GA
484 catabolism genes rather than ABA in enhanced secondary dormancy induction in brm-3 (Ibarra
485 et al., 2016; Chang et al., 2018) and required functional DOG1.

486 **BRM controls secondary seed dormancy through DOG1 antisense**

487 In agreement with the genetic interplay between *BRM* and *DOG1* genes, we detected direct BRM
488 binding to exon 2 and exon 3 of the *DOG1* gene in dry seeds. Interestingly, during secondary
489 dormancy induction, BRM binding increased towards the 3' end of the *DOG1* locus (Fig. 3A, B).
490 Together with the observed deregulation of the *DOG1* gene expression and changes in chromatin
491 accessibility, this suggests that BRM controls secondary dormancy directly through *DOG1*.
492 Primary and secondary dormancy are intrinsically linked and multiple factors including DOG1,
493 AFP2, ABI5 and ABI3 have been shown to affect both primary and secondary dormancy (Han *et
494 al.*, 2012; Ibarra *et al.*, 2016; Chang *et al.*, 2018). Here, we show that BRM is specifically
495 implicated in secondary but not primary dormancy control. To the best of our knowledge, this is
496 the first example of where a factor is required only for secondary but not primary seed dormancy.

497 DOG1 is a known positive regulator of dormancy (Bentsink et al., 2006). Here we show that
498 during secondary dormancy induction, sense DOG1 transcript is induced (Fig. 3C). This is in
499 agreement with published by us and others requirement of functional DOG1 gene for secondary
500 dormancy establishment (Ibarra et al., 2016; Krzyszton et al., 2022; Sajeev et al., 2024). Our data
501 show that in brm-3 seeds DOG1 transcript is upregulated while asDOG1 is downregulated during
502 secondary dormancy induction, when compared to Col-0 (Fig. 3C, 3D). We also observe BRM
503 binding to DOG1 3'end region, and that BRM regulates asDOG1 but not DOG1 sense promoter
504 activity during secondary dormancy establishment (Fig. 3). Supporting a direct role of BRM in
505 control of asDOG1 we observed increased DNA accessibility at asDOG1 promoter region of the
506 *DOG1* locus in *brm-3* mutant during secondary dormancy establishment (Fig. 4A). Interestingly,
507 BRM binds to the 3' end region of selected differentially expressed genes (DEGs) involved in
508 ABA and GA pathways (such as *RGL3*, *NCED4*, *CYP707A1* etc.) (Supplementary Table 2).

509 Previous research has shown that *asDOG1* is a negative regulator of *DOG1* expression, as its
510 deletion results in high *DOG1* expression (Fedak *et al.*, 2016, Yatusevich *et al.*, 2017). BRM
511 appears to positively control antisense, thus also negatively regulating *DOG1* gene expression
512 (Fig. 4, 5). The mechanism of *DOG1* silencing by its antisense, aka *1GOD*, is not yet fully
513 understood. We hypothesize that during secondary dormancy establishment, BRM indirectly
514 limits *DOG1* induction by enhancing *asDOG1* expression (Fig. 3D). In agreement, we show that
515 mutation of TATA boxes located in the antisense promoter region resulted in much stronger
516 *DOG1* upregulation compared to not mutated *DOG1* transgene and, in agreement, enhanced
517 secondary dormancy phenotype in seeds. Our model suggests that observed by us in Col-0
518 upregulation of *asDOG1* during secondary dormancy induction serves to limit *DOG1* induction
519 attenuating dormancy strength. In contrast to BRM function in secondary dormancy, both RNA-
520 seq and RT-qPCR showed no major differences in *DOG1* sense and antisense transcripts levels
521 between Col-0 and *brm-3* mutant in dry seeds. This agrees with the observed lack of primary
522 dormancy defects in *brm-3* as well as in *3xbrd* and *swp73a* mutants (Fig. 2F, Supplementary Fig.
523 S14).

524 Notably, the introduction of triple *3xbrd* mutation into the *brm-1* knockout mutant background
525 did not enhance the *brm-1* phenotype, confirming the conclusion that BRD subunits operate
526 within the same complex as BRM (Stachula *et al.*, 2023). Also, here we show that *3xbrd* and
527 *swp73a* display similar *DOG1* expression changes to *brm-3* mutant consistent with BRM
528 operating as part of BAS SWI/SNF complex in controlling *DOG1* expression (Fig. 3,
529 Supplementary Fig. S9).

530 Thus, we propose a model where BRM-containing the SWI/SNF complex binds to the *DOG1*
531 3'end region and in response to secondary dormancy-inducing conditions remodels nucleosomes
532 which activate *asDOG1* antisense promoter (Fig 5). This leads to *asDOG1* transcript expression
533 that limits the activation of *DOG1* and subsequently to a strong dormancy establishment.
534 Surprisingly, analysis of *p sense* DOG1-LUC transgene that lacks a function *DOG1* antisense
535 transcript in seeds showed that *DOG1* 5'region is insufficient to support *DOG1* expression
536 upregulation in response to secondary dormancy induction. This suggests that in addition to
537 *DOG1* antisense that limits the full activation of *DOG1* expression, the *DOG1* 3'end region
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contains unknown positive regulators responsible for secondary dormancy-mediated *DOG1*
expression induction.

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Analysis of *DOG1* splicing and polyadenylation during secondary seed dormancy induction in Col-0 WT, *brm3* showed increased levels of *β* and *γ* alternatively spliced mRNA as well as increased levels of *shDOG1* resulting from selection of proximal termination site (Fig. 4F, 4G). Whereas in the *ntr1* mutant, we observed predominantly changes in *α* and not significantly in *β* *DOG1* mRNA splicing forms compared to Col-0 WT seeds (Supplementary Fig. S15). Given that our work implicated NTR1 in splicing control through Pol II speed on *DOG1* we speculate that defects observed in *brm-3* are unlikely to results from NTR1 dependent splicing defects or direct Pol II speed regulation. One possibility is that changes in *DOG1* splicing and termination site selection observed in *brm-3* and *3xbrd* result from defects in antisense expression in these mutants. The proximity of *DOG1* alternative splice sites - proximal termination site and antisense promoter make deletional confirmation of this hypothesis difficult, if possible. We, however, showed that BRM regulates the *DOG1* antisense promoter in seeds in the absence of sense promoter driving *DOG1* alternative splicing or proximal termination site selection.

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In summary, our work explores the functions of the BRM-containing SWI/SNF complex in seed biology. We observe that BRM is required for multiple aspects of seed physiology as underpinned by metabolomic and transcriptomic analysis. We show that BRM controls some of the seed-related phenotypes including longevity and secondary dormancy regulation through the *DOG1* gene. Our analysis demonstrates that in response to environmental signals triggering secondary dormancy induction BRM containing SWI/SNF complex controls *DOG1* expression through *DOG1* antisense.

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561

**Materials and Methods**

562

**Plant Material and Growth Conditions**

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*Arabidopsis thaliana* plants were grown in pots with mixed coconut and normal soil in a greenhouse with a long-day photoperiod (16-h light/8-h dark) at 22°C/18°C. After harvest, seeds were stored in paper bags at room temperature. Ecotype Col-0 plants were used as a wild type (WT). The *brm-3* (SALK_088462) and *dog1-4* (SM_3_20886) mutants were obtained from the Nottingham Arabidopsis Stock Centre (NASC) and are described in (Farrona et al., 2007; Fedak et al., 2016). Transgenic reporter lines: genomic *LUC-DOG1dTATA*, *asDOG1dTATA-LUC*,
senseDOG1-LUC, asDOG1-LUC, and genomicLUC-DOG1 were generated and characterized previously (Fedak et al., 2016; Yatusevich et al., 2017). The *brm-5*, *swp73a* (*SM_3_30546*), *3xbrd*, *ntr-1*, *4xnced* and *brm-1/BRM-GFP* lines were described previously (Tang et al., 2008; Dolata *et al.*, 2015; Sacharowski *et al.*, 2015; Jarończyk *et al.*, 2021; Krzyszton et al., 2022). The double mutants: *brm-3dog1-4*; *brm-3*p*sense*DOG1-LUC; *brm-3p*ASDOG1-LUC; *brm-3genomicLUC-DOG1dTATA*; *brm-3p*ASDOG1dTATA-LUC were generated by crossings and homozygous plants were identified using *brm-3* T-DNA insertion primers described in Jarończyk *et al.*, 2021.

For salt stress, sterilized after-ripened seeds were sown on agar plates supplemented with 100mM NaCl. After sowing, plates were taped, wrapped with silver foil, and kept for stratification for 3 days at 4°C. Then plates were unwrapped from the silver foil and transferred to the growth chamber under long-day conditions (16h light/8h dark) to check the germination rate (Montez *et al.*, 2023).

## 581 Seed Longevity Measurement

To perform artificial ageing, mature post-harvested *Arabidopsis* seeds were stored in the darkness at 35°C in hermetically closed containers with saturated NaCl (75% of relative humidity). 50 seeds per each biological replica were imbibed on blue paper (Anchor) plates after different storage times (0, 7, 14, 21, 28, 35, 42, 49, 56 days) and placed in a phytotron at 22°C/long day photoperiod. The final germination percentage was counted after 10 days. P50 was determined as the time after which seeds lost 50% of their germination capacity (Zinsmeister *et al.*, 2020).

## 588 Seed primary dormancy assay

Freshly harvested seeds were sown on plates with water-soaked blue paper (Anchor) and sealed with tape. Plates with the seeds were put in the growth chamber under long-day conditions (16 hours light/8 hours dark) at 22°C/18°C. The germination rate was scored every day until 100% germination was observed. Control plates were initially stratified for 3 days at 4°C to break seed dormancy and to ensure that the seeds were not dead.

## 594 Seed secondary dormancy assay

Seeds stored for at least 3 months that showed full loss of primary dormancy were used for secondary dormancy induction. Seeds were sown on water-soaked blue paper plates, sealed and kept in the dark. Plates were incubated at 30°C for 4 hours, 1, 3, 5, 7, 10 or 14 days. After high incubation, the plates were transferred to the growth chamber at 22°C under long-day conditions. 19
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Seed germination was assayed after 4 and 7 days. The control plates were placed in a phytotron immediately after sowing the seeds, without a dormancy induction.

601

RNA extraction, cDNA synthesis and RT-qPCR analysis

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RNA extraction from seeds was performed using the phenol-chloroform protocol. The frozen seeds were ground to a powder using an electric drill and then mixed with 600 µl of RNA extraction buffer (100 mM Tris pH 8.5, 5 mM EDTA pH 8.0, 100 mM NaCl, 0.5% SDS, 1% β-mercaptoethanol). Afterwards, samples were centrifuged for 5 min at 14,000 g at 4°C. The supernatant was transferred to new tubes and 250 µl of chloroform was added and samples were shaken at room temperature (RT) for 15 min. Then 250 µl of phenol was added and samples were shaken for a further 15 min and centrifuged for 10 min at 14,000 g at 4°C. Then, 550 µl of the aqueous layer was transferred to new tubes and mixed with 550 µl of phenol-chloroform-isoamyl alcohol 25:24:1. Samples were shaken for 10 min at RT and centrifuged for 10 min at 14,000 g at 4°C. Then, 500 µl of supernatant after transferring to new tubes was mixed with 50 µl of 3 M sodium acetate and 400 µl of pure ice-cold isopropanol and incubated for 15 min at RT. After the incubation, samples were centrifuged for 30 min at 14,000 g at 4°C. Finally, the RNA pellet was washed in 1 ml of 80% ethanol, dried and resuspended in Milli-Q water. DNase treatment of RNA samples was performed using a TURBO DNA-free™ Kit (ThermoFisher Scientific), according to the manufacturer's protocol. DNase treatment effectiveness was checked by PCR with pp2A primers. Reverse transcription of RNA was performed using a RevertAid First Strand cDNA Synthesis (ThermoFisher Scientific) or RevM First Strand cDNA Synthesis (KleverLab) kits according to the manufacturer's protocol. Two types of cDNA synthesis were performed: using 1,000 ng of RNA and oligo(dT) primers for DOG1 analysis and gene-specific synthesis for asDOG1 analysis using 2,500 ng of RNA and primers with overhangs as described (Fedak et al., 2016). qPCR was performed with SYBR Green mix and specific primers for PCR amplification and with using LightCycler 480 real-time system (Roche). The sequences of all primers are published previously (Cyrek et al., 2016; Fedak et al., 2016) and provided in the Supplementary Table 2. RT-qPCR results were normalized to the expression level of the UBC21 (AT5G25760) gene.

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RNA-sequencing and data analysis

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3'RNA-seq and data analysis were performed as described previously using 500 ng of total RNA as starting material (Krzyszton *et al.*, 2022).
630 Chromatin Immunoprecipitation (ChIP)

631 Chromatin was isolated from dry, non-dormant mature seeds and after 1, 3 and 5 days of
632 secondary dormancy induction for WT and brm-1/BRM-GFP lines (Jarończyk et al., 2021). ChIP
633 was performed as described previously (Kowalczyk, 2017) with some modifications. The 60mg
634 of frozen seeds were ground to a powder using a pestle and mortar and suspended in 10ml of MC
635 buffer (0.1M sucrose; 10mM sodium phosphate pH 7; 50mM NaCl). Then 37% formaldehyde
636 was added to the final concentration of 1% and samples were mixed softly on a rotating wheel for
637 10 min at 4°C. After mixing, 625µl of 2M glycine was added and samples were rotated for
638 another 10 min. Then the samples were filtered through a double layer of Miracloth Quick
639 Filtration Material, and centrifuged for 10 min at 1500xg at 4°C. After centrifugation, samples
640 were resuspended in 5ml of Honda buffer (0.44M sucrose; 1.25% Ficoll; 2.5% Dextran T40;
641 20mM HEPES-KOH pH 7.4; 0.5M EDTA; 0.5% Triton X-100; 10mM β-Mercaptoethanol and
642 freshly added 0.0005M PMSF and 1×Complete EDTA-free protease inhibitor) and spun for
643 10min at 1800xg at 4°C. The nuclear pellet was resuspended in 500µl ChIP Lysis/sonication
644 buffer (50mM HEPES-KOH pH 7.4; 150mM NaCl; 1mM EDTA; 1% Triton X-100; 0.8% SDS;
645 10mM β-Mercaptoethanol and freshly added 0.0005M PMSF and 1×Complete EDTA-free
646 protease inhibitor) and sonicated twice 30sec-ON/30s-OFF for 25min using Bioruptor Sonication
647 System (Diagenode). 1/10 of each sample was saved as input control and 20µl for sonication
648 control. Sonication efficiency was verified by running de-crosslinked samples on 1% agarose gel.
649 GFP-Trap® Agarose beads were prepared according to the manufacturer's protocol (Chromotek).
650 The lysates of sonicated samples were added to equilibrated beads and placed on a rotating wheel
651 in a cold room for 2-4 hours. After incubation, beads were centrifuged for 2500 x g for 5min at
652 4°C and washed twice for 5min with 1ml of low salt wash buffer (150mM NaCl; 1% Triton X-
653 100; 2mM EDTA; 20mM Tris pH 8.0; 0.1% SDS). Then beads were washed for 5min with 1ml
654 of high salt wash buffer (500mM NaCl; 1% Triton X-100; 2mM EDTA; 20mM Tris pH 8.0;
655 0.1% SDS) and centrifuged for 2500 x g for 5min at 4°C. Then 500 µl of
656 phenol:chloroform:isoamyl alcohol mixture (25:24:1; pH 8.0) were added and the samples were
657 shaken for 10 min at 22°C. After centrifugation for 10 min at 14,000 g the upper aqueous layers
658 were collected and 0.1 volume of 3M sodium acetate (pH 5.2), 1 µl of glycogen (ThermoFisher
659 Scientific), and 1 ml 96% ethanol were added. The mixed samples were held at -80°C for >1
660 hour and then centrifuged for 30 min at 14,000 g at room temperature. The pellets were washed
661 with cold 70% ethanol, air-dried and suspended in water. For the quantification of DNA
662 fragments, samples were tested by qPCR. The sequences of all primers are given in the
663 Supplementary Table 2 or published by (Dolata et al., 2015).
664 Formaldehyde-assisted isolation of regulatory DNA elements (FAIRE)

665 The FAIRE method was performed according to the published protocol (Omidbakhshfard et al., 2014) with minor modifications. For nuclei isolation, 100 mg of the dry and secondary dormancy-induced seeds of *Col-0* and *brm-3* were used. Chromatin was sheared by sonication and the sonication efficiency was checked by electrophoresis in agarose gel as previously described for the ChIP protocol. To separate NDR (nucleosome-depleted regions), the sheared chromatin DNA was extracted by PCI (phenol:chloroform:isopropanol) method. The enrichment was assessed by qPCR using primers indicated in Supplementary Table 2. Calculations were performed using the ΔΔCt method with normalization of a crosslinked sample (FAIRE) to non-crosslinked sample (UN-FAIRE) as described in Omidbakhshfard et al., 2014 and then to *PP2A* – AT1G13320 (Supplementary Table 2) as an internal control.

675 METABOLOME ANALYSIS

676 Sample preparation

677 Mature post-harvested seeds (50 mg) in biological triplicates were ground with metal beads for 2x 90s on a Tissue Lyser (Qiagen) at 30 Hz in 1.5 ml of cold (-20°C) methanol spiked with an internal standard of deuterium labelled abscisic acid (²H₆ ABA, 0.2µgg/ml). Samples were shaken for 10 minutes at RT, and centrifuged for 5 minutes at 13,000 rpm, RT. The supernatant was transferred to a glass vial and the extract was dried with a SpeedVac concentrator (Savant SPD121P, ThermoFisher Scientific) at RT. The pellets were extracted twice with 1.5 ml methanol, shaken, centrifuged and collected in the same glass vial to be evaporated. After this, dry samples were solubilized in 100 µl of methanol.

685 Non-targeted metabolites analysis

686 Extracts were analyzed by liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS) using an UltiMate 3000 UHPLC system (ThermoFisher Scientific) coupled to the ImpactII (Bruker) high-resolution Quadrupole Time-of-Flight (QTOF) mass spectrometry according to Villette et al. (2018) and Graindorge et al. (2022). Chromatographic separation was performed on an Acquity UPLC ® HSS T3 C18 column (2.1 × 100 mm, 1.8 µm, Waters) coupled to an Acquity UPLC HSS T3 C18 pre-column (2.1 × 5 mm, 1.8 µm, Waters) using a gradient of solvents A (H₂O, 0.1% formic acid) and B (methanol, 0.1% formic acid). Chromatography was carried out at 35°C, at a flux of 0.3 ml min⁻¹, starting with 5% B for 2 min, reaching 100% B at 10 min, holding 100% B for 3 min and coming back to 5% B in 2 min (run time 15 min).
Samples were kept at 4 °C, 5 µL were injected in full loop mode with a washing step after sample injection with 150µL of wash solution (H2O/MeOH, 90/10, v/v). The spectrometer was equipped with an electrospray ionization (ESI) source and operated in positive and negative ion modes on a mass range from 20 to 1000 Da with a spectra rate of 8 Hz in Auto MS/MS fragmentation mode. The end plate offset was set at 500 V, the capillary voltage set at 2.5 kV, the nebulizer at 29 psi, the dry gas at 81 min⁻¹, and the dry temperature of 200°C. The transfer time was set at 20–70 µs (positive mode) and 40.8–143 µs (negative mode) and the MS/MS collision energy was at 80–120% with a timing of 50–50% for both parameters. The MS/MS cycle time was set to 3 s, the absolute threshold to 816 cts, and active exclusion was used with an exclusion threshold at 3 spectra, release after 1 min, and the precursor ion was reconsidered if the ratio current intensity/previous intensity was higher than 5. A calibration segment was included at the beginning of the runs allowing the injection of a calibration solution from 0.05 to 0.25 min. The calibration solution used was a fresh mix of 50 mL isopropanol/water (50/50, v/v), 500 µL NaOH 1M, 75 µL acetic acid, and 25 µL formic acid. The spectrometer was calibrated on the [M+H]⁺/[M−H]⁻-form of reference ions (57 masses from m/z 22.9892 to m/z 990.9196 in positive mode; 49 masses from m/z 44.9971 to m/z 996.8221 in negative mode) in high-precision calibration (HPC) mode with a standard deviation below 1 ppm before the injections for each polarity mode, and re-calibration of each raw data was performed after injection using the calibration segment. Molecular features were processed with MetaboScape version 4.0 (Bruker, Bremen, Germany). Molecular features were considered and grouped into buckets containing one or several adducts and isotopes from the detected ions with their retention time and MS/MS information when available. The parameters used for bucketing are a minimum intensity threshold of 10,000 (positive mode) or 1,000 (negative mode), a minimum peak length of 3 spectra, a signal-to-noise ratio (S/N) of 3, and a correlation coefficient threshold set at 0.8. The [M+H]⁺, [M+Na]⁺, [M+K]⁺, and [M+NH4]⁺ ions (positive mode); [M-H]⁻ and [M+Cl]⁻ ion (negative mode) were authorized as possible primary and seed ions. Replicate samples were grouped and only the features found in 80% of the samples of one group were extracted from the raw data. The obtained lists of features from positive and negative ion modes were merged. The parameters used for metabolite annotation were as follows. The maximum allowed variation on the mass (Δm/z) was set to 3 ppm, and the maximum mSigma value (assessing the good fitting of isotopic patterns) was set to 30. The merged list of features was annotated using SmartFormula to generate a raw formula based on the exact mass of the primary ions and the isotopic pattern. Analyte lists were derived from KNApSAcK (http://www.knapsackfamily.com/KNApSAcK_Family/), PlantCyc (https://plantcyc.org/), 23
729 FooDB (http://foodb.ca), LipidMaps (https://www.lipidmaps.org/), and SwissLipids
730 (https://www.swisslipids.org/) to obtain a level 3 annotation according to Schymanski
731 classification (tentative candidates based on exact mass and isotopic profile) (Schymanski et al.,
732 2014). Spectral libraries (Bruker MetaboBASE Personal Library 3.0,
733 MoNA_LCMSMS_spectra, MSDIAL_LipidBDs-VS34) were searched to obtain level 2
734 annotations (probable structure based on library spectrum match (MS2 data) according to
735 Schymanski et al (2014). PubChem IDs, SMILES and InChiKeys were obtained from PubChem
736 (https://pubchem.ncbi.nlm.nih.gov/idexchange/idexchange.cgi) for chemical enrichment studies
737 using ChemRICH tool (Barupal & Fiehn, 2017).

738 Sugar determination

739 Soluble sugar contents were assessed by HPLC (Dionex) according to Rosnoblet et al. 2007.
740 Analysis was performed on four replicates of 15 mg of mature Arabidopsis seeds. In brief, Seed
741 were ground in a mortar in the presence of 1 ml 80% methanol containing melizitose as the
742 internal sugar standard. After heating at 76°C for 15 min, the liquid was evaporated under
743 vacuum. The residue was dissolved in 1 ml distilled water and centrifuged for 1 min at 13 000 g.
744 Sugars were analyzed by HPLC on a Carbopac PA-1 column (Dionex Corp.) (Rosnoblet et al.,
745 2007).

746 Targeted hormone analysis

747 Auxin, ABA and GA were analyzed by ultra-high-performance liquid chromatography (UHPLC)
748 on an UltiMate 3000 UHPLC system (ThermoFisher Scientific)coupled to EvoQ Elite (Bruker)
749 mass spectrometer equipped with an electrospray ionization (ESI) source in MS/MS mode as
750 described in (Zumsteg et al., 2023).

751 Statistical analyses

752 Statistical tests were done using a Student-Newman-Keuls test or a two-tailed t-test, implemented
753 in Microsoft Office Excel.

754

755 Accession Numbers

756 Sequence data from this article can be found in the GenBank/EMBL data libraries under
757 accession numbers listed in Supplementary Tables 1 and 2.

758
759 Supplementary Data

760 Supplementary Figure S1. Untargeted metabolite analysis of mature seeds of *brm3*, *dog1-4* and
761 *brm3 dog1-4*.

762 Supplementary Figure S2. Heatmap showing significant and differential transcript levels of genes
763 of selected pathways.

764 Supplementary Figure S3. HPLC quantification of hormones in dry mature seeds of mutants
765 compared to Col-0 WT.

766 Supplementary Figure S4. UHPLC quantification of total soluble sugars in dry mature seeds of
767 mutants compared to Col-0 WT.

768 Supplementary Figure S5. Glutathione biosynthesis pathway regulation by BRM is *DOG1* gene-
769 dependent.

770 Supplementary Figure S5. Seed physiology is affected in brm3 mutants compared to wild type.

771 Supplementary Figure S6. Secondary seed dormancy phenotype of the selected mutants.

772 Supplementary Figure S7. Inactivation of SWP73A - BAS SWI/SNF specific subunit - results in
773 stronger dormancy, higher *DOG1* expression and decreased expression of *asDOG1*.

774 Supplementary Figure S8. ChIP-qPCR on BRM binding to *DOG1* locus.

775 Supplementary Figure S10. RT-qPCR expression analysis of the *DOG1* mRNA level.

776 Supplementary Figure S11. RT-qPCR analysis of selected ABA and GA marker genes during
777 secondary dormancy induction.

778 Supplementary Figure S12. RT-qPCR analysis of selected ABA and GA marker genes in *dog1-4*
779 mutant.

780 Supplementary Figure S13. RT-qPCR expression analysis of the native *DOG1* vs *LUC::DOG1*
781 transgene with *dTATA* mutations in antisense region of independent transgenic lines.

782 Supplementary Figure S14. Primary seed dormancy phenotype of *3xbrd*, *brm-3*, *dog1-4* and
783 double *brm-3dog1-4* mutants in comparison to Col-0 WT seeds.

784 Supplementary Figure S15. *DOG1* gene scheme and splicing analysis.

785 Supplementary Table S1. GO terms and ChemRICH metabolite analyses.
786 Supplementary Table S2. List of all primers used in this study.

787 **Funding**

788 This work was funded by a National Science Centre, Poland OPUS16 grant (UMO-
789 2018/31/B/NZ3/03363), SONATA BIS UMO-2018/30/E/NZ1/00354. SPS was supported by a
790 NCN Preludium grant UMO-2015/17/N/NZ2/01919. JZ funded by European Union's Horizon
791 H2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement
792 No 101038075 and from EMBO scientific exchange grant No 9107.

793

794 **Acknowledgments**

795 The authors thank Dimitri Heintz (metabolomics platform) and Jérôme Mutterer (Imaging
796 platform) from IBMP institute, for their help and support. The authors declare that they have no
797 conflict of interest.

798 **Author Contributions**

799 RY and SS conceived and designed the research project. RY, MW, JZ, MK, SPS, RA,CV, JZ,
800 BC, PM and MN performed experiments. RY, MK, JZ, CV, JZ, HS, JB and SS analyzed data.
801 RY and SS wrote the paper with contribution of all authors.

802 **Data availability**

803 The 3'RNA-seq data generated for this study have been deposited at the Gene Expression
804 Omnibus (GEO) under the accession code GSE251921.

805

806

807 **Figure Legends**

808 **Figure 1.** *Seeds transcriptome and metabolome of *brm* and *dog1* single and double mutants.*

809 A. Identification of differentially expressed genes in mature, dry *brm-3*, *dog1-4* and *brm-3dog1-4* seeds compared to Col-0 Wild Type (3'RNA-seq, differential analysis was performed using 810 DESeq2 – genes with FDR < 0.05 and absolute fold change > 1 were considered as differentially 811 expressed). B. Analysis of overlap between genes whose expression was affected in analyzed 812 mutants (extended graph version shown in Supplementary Figure S1C). C. Self-clustering of 813 expression profiles for genes differentially expressed in *brm-3*, identifies the *DOG1*-dependent 814 genes among ones affected in *brm* mutant seeds. Number of DEGs is indicated on panels. D. 815 Heatmap of genes' expression for genes misregulated in *brm-3* mutant across mutants used. 816
817 Genes marked as *DOG1*-gene dependent show suppression of the *brm* effect in the double *brm-3dog1-4* mutant. Second column colors correspond to specific color of the cluster. **E.** Chemical enrichment analysis of *brm* and *dog* single and double mutants. Colored circles represent clusters of metabolites from given chemical families (red, increased cluster; blue, decreased cluster; purple, increased and decreased metabolites in a cluster). The number of metabolites as indicated as circle size. **F.** The graph represents the fold change of glutathione levels compared to Col-0 in the seeds of mutants indicated values were ranked into groups as indicated by the respective letter using a Student-Newman-Keuls test, n=?.

825
826 Figure 2. The *brm-3* mutant seeds showed multiple morphological and physiological defects.
827 A. Scanning electron visualisation of seed from Col-0 WT, *brm-3*, *dog1-4* and *brm-3dog1-4* mutants. Bar corresponds to 200 nm. B. Seeds size analysed using Boxed robot, C. Seed yield analysed based on total number of seeds produced by mature plants. D. Seed longevity analysed using artificial aging. E. Germination in presence and absence of 100mM NaCl. F. Primary seed dormancy analysed with freshly harvested seeds. G. Secondary dormancy analysis for seed of *brm-3*, *dog1-4* and *brm-3dog1-4* mutants. Asterisks indicate significant differences compared to Col-0 dry seeds. Statistical analysis applies to all figure panels; t-test, *, P<0.05, **, P<0.01 and ***, P<0.0001; n=4, one biological replicate is a mixture of independent 5 plants; error bars represent standard deviation.

836

837 Figure 3. **BRM directly regulates DOG1 antisense transcription to control seed secondary dormancy.**
838 A. BRM ChIP-qPCR in dry seeds and B. seeds subjected to 3 days of secondary dormancy induction. Col-0 and BRM-GFP *brm-1* seeds were analysed using GFP antibodies. x-axis shows beginning of amplicon relative to TSS, TSS=0. Percent of input normalized to PP2A gene region. C. RT-qPCR analysis of *DOG1* sense and D. antisense transcripts in Col-0, *brm-3* and *3xbrd* mutants during secondary dormancy induction; E. RT-qPCR analysis of reporter lines activity during secondary dormancy induction for *p*<sub>sense</sub>*DOG1-LUC* and *F. p*<sub>AS</sub>*DOG1-LUC* lines in Col-0 and *brm-3* background. G. RT-qPCR for endogenous and *LUC-DOG1 deltaTATA* line activity during secondary dormancy induction shows that inactivation of *asDOG1* results in stronger induction of *DOG1* during secondary dormancy induction. H. BRM, SWP73A and BRD1 genes expression analysis during SD induction in Arabidopsis seeds. RT-qPCR analysis in C-H is normalized using *UBC21* gene. Statistical analysis applies to all figure panels, t-test, *, P<0.05, **, P<0.01 and ***, P<0.0001; n=4, one biological replicate is a mixture of independent 5 plants; error bars represent standard deviation.

839

840

841

842

843

844

845

846

847

848

849

850
851

Figure 4. The *brm3* mutant shows enhanced chromatin accessibility at *DOG1* 3'end during secondary dormancy induction. A. FAIRE in Col-0 and *brm-3* seeds on the 3rd day and B. 5th day of secondary dormancy induction. Chromatin accessibility at *DOG1* shown as % recovery to non-crosslinked samples (UNFAIRE) and relative to PP2A. The x-axis shows beginning of amplicon relative to TSS, TSS=0. C-F. RT-qPCR analysis of α-, β-, γ-, δ-DOG1 mRNA splicing forms during secondary seed dormancy induction. Transcript level of short (G) and long (H) polyadenylated DOG1 mRNA forms The x-axis shows time/days of secondary dormancy induction. Statistical analysis applies to all figure panels; t-test, *, P<0.05, **, P<0.01 and ***, P<0.0001; n=4, one biological replicate is a mixture of independent 5 plants; error bars represent standard deviation.

862

Figure 5. Model of the BRAHMA-associated SWI/SNF complex control of Arabidopsis seeds quality and physiology. The BRAHMA-associated SWI/SNF complex controls seed yield, seed size and plant hormonal crosstalk to a large extent independently of DOG1. The BRAHMA controls longevity and secondary dormancy by controlling DOG1 expression through DOG1 antisense (in red color; dark grey arrows). The BRAHMA also either directly or through DOG1 antisense negatively controls DOG1 gene expression (grey arrow) by affecting its alternative splicing and alternative polyadenylation.

870
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<figure><img src="image_2.png" /><figcaption>Figure 1</figcaption></figure>

Figure 1. Seeds transcriptome and metabolome of *brm* and *dog1* single and double mutants.

A. Identification of differentially expressed genes in mature, dry *brm-3*, *dog1-4* and *brm-3dog1-4* seeds compared to Col-0 Wild Type (3'RNA-seq, differential analysis was performed DESeq2 – genes with FDR < 0.05 and absolute fold change > 1 were considered as differentially expressed).

B. Analysis of overlap between genes whose expression was affected in *brm* analyzed mutants (extended graph version shown in Supplementary Figure S1, the x-axis is set). C. Self-clustering of expression profiles for genes differentially expressed in *brm-3*, identifies the *DOG1*-dependent genes among ones affected in *brm* mutant seeds. Number of DEGs is indicated on panels. The y-axis is scaled expression. D. Heatmap of genes' expression for genes misregulated in *brm-3* mutant across mutants used. Genes marked as *DOG1*-gene dependent show suppression of the *brm* effect in the double *brm-3dog1-4* mutant. Second column colors correspond to specific color of the cluster. E. Chemical enrichment analysis of *brm* and *dog* single and double mutants. Colored circles represent clusters of metabolites from given chemical families (red, increased cluster; blue, decreased cluster; purple, increased and decreased metabolites in a cluster). The number of metabolites as indicated as circle size. F. The graph represents the fold change of glutathione levels compared to Col-0 in the seeds of mutants indicated values were ranked into groups as indicated by the respective letter using a Student-Newman-Keuls test, n=4.
<figure><img src="image_3.png" /><figcaption>Figure 3</figcaption></figure>

Figure 3. BRM directly regulates *DOG1* antisense transcription to control seed secondary *dormancy*. A. BRM ChIP-qPCR in dry seeds and B. seeds subjected to 3 days of secondary dormancy induction. Col-0 and BRM-GFP *brm-1* seeds were analyzed using GFP antibodies. x-axis shows beginning of amplicon relative to TSS, TSS=0. Percent of input normalized to PP2A gene region. C. RT-qPCR analysis of *DOG1* sense and D. antisense transcripts in Col-0, *brm-3* and *3xbrd* mutants during secondary dormancy induction; E. RT-qPCR analysis of reporter lines activity during secondary dormancy induction for *psense DOG1-LUC* and *F. pAS DOG1-LUC* lines in Col-0 and *brm-3* background. G. RT-qPCR for endogenous and *LUC-DOG1 deltaTATA* line activity during secondary dormancy induction shows that inactivation of *asDOG1* results in stronger induction of *DOG1* during secondary dormancy induction. H. BRM, SWP73A and BRD1 genes expression analysis during SD induction in Arabidopsis seeds. RT-qPCR analysis in C-H is normalized using *UBC21* gene and the x-axis shows time/days of secondary dormancy induction (h-hours; d-days). Statistical analysis applies to all figure panels, t-test, *, P<0.05, **, P<0.01 and ***, P<0.0001; n=4, one biological replicate is a mixture of independent 5 plants; error bars represent standard deviation (±SD).
<figure><img src="image_4.png" /><figcaption>Figure 4</figcaption></figure>

Figure 4. The *brm3* mutant shows enhanced chromatin accessibility at *DOG1* 3'end during secondary dormancy induction. A. FAIRE in Col-0 and *brm-3* seeds on the 3rd day and B. 5th day of secondary dormancy induction. Chromatin accessibility at *DOG1* shown as % recovery to non-crosslinked samples (UNFAIRE) and relative to PP2A. The x-axis shows beginning of amplicon relative to TSS, TSS=0. C-F. RT-qPCR analysis of *α*-, *β*-, *γ*-, *δ*-DOG1 mRNA splicing forms during secondary seed dormancy induction. Transcript 1 level of short ( G ) and long ( H ) polyadenylated DOG1 mRNA forms The x-axis shows time/days of secondary dormancy induction (h-hours; d-days). Statistical analysis applies to all figure panels; t-test, *, P<0.05, **, P<0.01 and ***, P<0.0001; n=4, one biological replicate is a mixture of independent 5 plants; error bars represent standard deviation (±SD).
<figure><img src="image_5.png" /><figcaption>Figure 5</figcaption></figure>

Figure 5. Model of the BRAHMA-associated SWI/SNF complex control of Arabidopsis seeds quality and physiology. The BRAHMA-associated SWI/SNF complex controls seed yield, seed size and plant hormonal crosstalk to a large extent independently of DOG1. The BRAHMA controls longevity and secondary dormancy by controlling *DOG1* expression through *DOG1* antisense (in red color; dark grey arrows). The BRAHMA also either directly or through *DOG1* antisense negatively controls *DOG1* gene expression (grey arrow) by affecting its alternative splicing and alternative polyadenylation.
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