Global epigenetic analysis revealed dynamic fluctuations in levels of DNA methylation and histone modifications in the calli of Fagopyrum with different capacity for morphogenesis

Buckwheat characterises with high susceptibility to in vitro tissue culture conditions, which have been researched extensively to study a plethora of processes. F. tataricum morphogenic callus (MC) is characterised by its capacity for morphogenesis for up to ten years of culture, displaying an extraordinary level of genome stability, and comprises of proembryogenic cell complexes (PECC),which are the structures resembling somatic embryos arrested on the pre-globular stage. The non-morphogenic callus (NC) that appears on the surface of MC after approximately two years of culture due to endoreduplication cycles, is characterised by aneuploidy, rapid growth rate and high level of oxidative stress. F. esculentum embryogenic callus (EC) has different morphological and histological features, remains stable for up to three years of culture, has a dense, globular structure, and is capable of forming embryoids from the masses of embryogenic cells, but does not produce a non-embryogenic clone. In this work, immunocytochemical analyses revealed dynamic epigenetic changes in Fagopyrum calli. We demonstrated that; decreased level of H3K4me2 seems to be associated with pluripotency acquisition in F. esculentum EC and F. tataricum MC; DNA hypomethylation appears to be connected with the acquisition of the embryogenic potential and PECC reinitiation in F. tataricum MC. Moreover, we observed that H4K16ac and H4K5ac exhibited the highest variability during the course of passage in NC. Elevated levels of these modifications on day zero and day six for H4K16ac and H4K5ac, respectively, seem to be connected with endoreplication peaks, the processes which are characteristic of this callus. Epigenetic changes accompany the dedifferentiation and re-differentiation processes in long-term callus cultures of Fagopyrum with different capacity for morphogenesis.


Introduction
The buckwheat genus comprises 23 species, and the two most cultivated are Fagopyrum esculentum Moench (common buckwheat) and Fagopyrum tataricum Gaertn.(Tartary buckwheat) (Tomasiak et al. 2022).Buckwheat is highly susceptible to in vitro conditions which have been researched extensively to study the induction of callus, plantlet regeneration from a variety of explants, organogenesis, somatic embryogenesis (SE) and synthesis of secondary metabolites (Fei et al. 2019;Rumyantseva et al. 2005;Rumyantseva et al. 2003;Srejovic and Neskovic 1981;Takahata and Jumonji 1985;Yamane 1985).F. tataricum in vitro tissue culture of the morphogenic callus (MC) characterised by low chromosome variability and the capacity for morphogenesis for up to ten years of culture displaying an extraordinary level of stability (Betekhtin et al. 2017;Rumyantseva et al. 2003).On the contrary, the non-morphogenic callus (NC) of F. tataricum appears on the surface of MC after approximately two years of culture due to endoreduplication cycles (Betekhtin et al. 2019(Betekhtin et al. , 2017)).NC is characterised by aneuploidy, rapid growth rate and high content of hydrogen peroxide, an indication that NC is in a constant state of oxidative stress (Betekhtin et al. 2017;Kamalova et al. 2009).F. esculentum embryogenic callus (EC) displays different morphological and histological features, remains stable for up to three years of culture, has a dense, globular structure, and is capable of forming embryoids (Rumyantseva et al. 2005) but does not produce a non-embryogenic clone.
Sessile organisms, such as plants, are subjected to the ever-changing environment and have developed a high level of phenotypic plasticity, which to a vast degree, is controlled by the post-translational modifications (PTMs) (Bennett et al. 2021;Ghosh et al. 2021;Miguel and Marum 2011).Under certain circumstances, differentiated plant cells have the ability to return to a previous developmental state (dedifferentiate) and regain pluri-or totipotency, resulting in organogenesis and SE, processes largely involving PTMs (Birnbaum and Sanchez Alvarado 2008;Simsek Geyik et al. 2022;Xu and Huang 2014).The process of dedifferentiation and re-entry into the cell cycle was found to be associated with the global reorganisation of chromatin and changes in gene expression (Avivi et al. 2004;Williams et al. 2003;Zhao et al. 2001).In vitro tissue culture is a good system for studying these processes since they can be easily induced, influenced and modified under controlled conditions.According to the available evidence, it is believed that in vitro conditions destabilise the genetic and epigenetic programs of the plant tissue and can result in alteration of chromosome and DNA sequence, transposon activation or repression, generation of somaclonal variants and recalcitrance (Ckurshumova and Berleth 2015;Neelakandan and Wang 2012).
DNA methylation, one of the most researched modifications, is associated with a variety of molecular mechanisms, including inactivation of the chromatin, gene regulation and plant cell differentiation, among others (Ghosh et al. 2021;Kaeppler et al. 2000;Park et al. 2008;Springer and Schmitz 2017).Highly dynamic mechanisms of global and local DNA methylation changes were found to occur during cell dedifferentiation and re-differentiation processes in callus formation (Horstman et al. 2017;Li et al. 2017b).Recent studies conducted in peach (Prunus persica) demonstrated the beneficial influence of DNA hypomethylation during the transition from leaf to callus (Zheng et al. 2022), and subsequently that genes involved in DNA and histone methylation are up regulated in five-year old callus when compared to newly formed callus (Gao et al. 2023) showing dynamic changes in these epigenetic marks during the callus culture.In Glycine max and Zea mays on the other hand, losses in DNA methylation were observed after many years of consecutive culture which led to the conclusion that tissue culture results in diminishing ability to maintain DNA methylation (Han et al. 2018;Ji et al. 2019).Studies on Eleuterococcus senticosus embryogenic and non-embryogenic calli demonstrated significantly lower global DNA methylation rates in embryogenic calli (Chakrabarty et al. 2003).Similar results were obtained for Agave furcroydes, where hypermethylation on a high but stable level was present throughout the culture in non-embryogenic callus (Monja-Mio et al. 2018).DNA methylation has also been found to be engaged in SE (Chakrabarty et al. 2003;Fraga et al. 2012;Nic-Can et al. 2013;Park et al. 2008).Results obtained for Daucus carota showed that DNA hypomethylation suppressed the formation of embryogenic cells (Yamamoto et al. 2005).DNA methylation contributes to a plethora of processes; however, its role in the cell differentiation/dedifferentiation switch and SE and related processes seem to be species-dependent.
Callus formation and its developmental processes were found to involve global changes in global epigenetic make-up (Bednarek and Orłowska 2019;Miguel and Marum 2011;Us-Camas et al. 2014).Marks involved in open chromatin state such as acetylation on histone H3 and H4, as well as methylation H3K4me3 and H3K36me3 have a tendency to be enriched in the callus cells when compared to somatic cells, while marks responsible for heterochromatin such as H3K9me2/3 and H3K27me2/3 are decreased in dedifferentiated cells (Hemenway and Gehring 2023;Lee and Seo 2018).Histone methylation is an indispensable player in the epigenetic regulation of the gene expression orchestrated by environmental and developmental cues in plants (Cheng et al. 2020;Hu and Du 2022;Yu et al. 2009).Recent research demonstrated that reduced levels of H3K4me2 allow the activation of genes essential for the acquisition of regenerative competency during the callus culture of Arabidopsis (Ishihara et al. 2019).Another study on Arabidopsis reported the reduced accumulation of H3K4me3 during early stages of callus formation (Lee et al. 2019).Similarly, in Coffea canephora global levels of H3K4me were decreased at the beginning of SE induction (Nic-Can et al. 2013).Another modification associated with active transcription H3K36me3 was found to be accompanying the transition processes from differentiated somatic cells to dedifferentiated pluripotent stem cells during the formation of callus in Arabidopsis, leading to the conclusion that this PTM promotes early stages of cellular dedifferentiation by creating an open chromatin structure and maintaining transcriptionally competent state (Lee et al. 2017;Ma et al. 2022;Zhang et al. 2015).Histone acetylation is associated with relaxed euchromatin and transcriptional activation (Kouzarides 2007).It has been reported that reduced levels of histone acetylation due to the compromised function of histone acetyltransferases and deacetylases had a negative effect on morphogenic responses such as callus formation (Kim et al. 2018;Lee et al. 2016;Rymen et al. 2019;Zhang et al. 2020).Indirect evidence of the correlation between dynamic epigenetic changes during SE induction was also demonstrated in Hevea brasiliensis, Peaonia ostii and Arabidopsis, where patterns of expression of the histone acetyltransferases (HAT) and histone deacetylases (HDAC) genes fluctuated during the SE induction (Ci et al. 2022;Li et al. 2017a;Wickramasuriya and Dunwell 2015).In Hevea brasiliensis, reduction in histone deacetylases was observed in the early stages of callus differentiation (Li et al. 2020) Study on Arabidopsis demonstrated that histone acetylation is essential for in vitro acquisition of pluripotency by promoting transcriptional activation of several root-meristem gene loci (Kim et al. 2018).There is limited evidence available in literature decoding the exact function of the histone acetylation, however, the research implicates that acetylation is an essential factor for the appropriate regulation of transcription (Birnbaum and Roudier 2017;Us-Camas et al. 2014).
This study was conducted in order to decipher how the epigenetic modifications influence cell fate transition in long-term cultivated Fagopyrum callus lines with different capacity for morphogenesis.Depicting levels of DNA and histone methylation as well as histone acetylation during the transition from non-embryogenic to embryogenic state will help to comprehend what role the PTMs play in the acquisition of embryogenic potential, thereby expanding the limited evidence available on this topic.

Plant material
The seeds of F. tataricum, sample k-17 were acquired from the collection of the N.I.Vavilov Institute of Plant Genetic Resources, Saint Petersburg, Russia (plants were grown in field conditions during the period from May to September).For F. esculentum commercially available seeds of the Panda cultivar (the Malopolska Plant Breeding, Poland) were used.EC of F. esculentum and F. tataricum MC were induced from immature zygotic embryos.NC of F. tataricum appears on the surface of MC after approximately two years of culture (Betekhtin et al. 2017).For the present study, EC's age was approximately one year old and MC's eight years old, and NC's five years old.Calli were cultivated in the dark in an incubator at 25 °C ± 1 on an RX medium, according to Betekhtin et al. (2019).It composed of Gamborg B5 including vitamins (Duchefa, Netherlands) (Gamborg et al. 1968), 2 g L −1 N-Z amine A (Sigma-Aldrich, USA), 2 mg L −1 2,4-dichlorophenoxyacetic acid (2,4-D, Sigma-Aldrich), 0.2 mg L −1 kinetin (KIN, Sigma-Aldrich, USA), 0.5 mg L −1 3-indoleacetic acid (IAA, Sigma-Aldrich, USA), 0.5 mg L −1 1-naphthaleneacetic acid (NAA, Sigma-Aldrich, USA), 25 g L −1 sucrose (Chempur, Poland) and 7 g L −1 phyto agar (Duchefa, Netherlands).EC of F. esculentum, MC and NC of F. tataricum were subcultured every four and two weeks, respectively.

Histological and immunostaining procedures
Proembryogenic cell complexes (PECC) from F. tataricum MC were carefully selected in sterile conditions under the stereoscopic Nikon microscope (Japan) and transferred onto a fresh medium for each time point, i.e. day zero, two, six and eleven of the passage.For immunocytochemical analysis NC of F. tataricum and EC of F. esculentum pieces of calli were put onto fresh medium for each time point.The same day, material from one petri dish of F. tataricum MC and NC and F. esculentum EC were fixed in 4% paraformaldehyde (Sigma-Aldrich, USA) in 1 × phosphate buffered saline (PBS), pH 7.3 and placed in the vacuum desiccator for three hours with subsequent incubation in 4 °C overnight.The next day, the fixative was carefully replaced with 1 × PBS and followed by dehydration in a graded ethanol series diluted in 1xPBS solution twice for 15 min in each concentration (10%, 30%, 50%, 70% and 90%) and 99,8% twice for 30 min each.The fixation process was repeated on day two, six and eleven.
The embedding procedure was performed according to Wolny et al. (2014).The de-embedding process involved placing the slides in 99,8% ethanol three times for 10 min, followed by rehydration in ethanol/ 1xPBS solutions: 90%, 70%, 50%, 30% v/v and 1xPBS for 10 min each.The immunostaining and histological processes then commenced.For the histology, slides were stained with 0.05% aqueous solution of Toluidine blue (TBO, Sigma-Aldrich, USA) for 5 min and mounted with distilled water.Observations and photographs were performed with an Olympus BX43F microscope equipped with a Olympus XC50 digital camera.
The immunostaining method used in this experiment was previously described by Braszewska-Zalewska et al. (2010);Braszewska-Zalewska et al. (2012);Braszewska-Zalewska et al. (2013).De-embedded slides were incubated with 5% bovine serum albumin (BSA, Sigma-Aldrich, USA) in 1xPBS for 1 h in the humid chamber at room temperature and subsequently, the primary antibody in 1% BSA in 1xPBS (1:100) was applied and incubated at 4 o C overnight.For 5mC primary antibody incubation was preceded with 2N HCl (Sigma-Aldrich, USA) digestion for 45 min.Next, the slides were rinsed by three washes in 1xPBS and secondary antibody in 1% BSA in 1xPBS (1:100) was applied.Samples were incubated at 37 o C in the humid chamber in the dark for one hour.After the incubation, three washes in 1xPBS were performed and nuclei were counterstained with 4',6-diamidyno-2-fenyloindol (DAPI, 2.5 g/ml in Vectashield antifade buffer, Vector Laboratories, USA).The negative control was performed for each of the used modification by conducting the immunostaining procedure omitting the addition of the primary antibody.The antibodies used in the immunocytochemical analysis are listed in Table 1.

Fluorescence intensity measurements and statistical analysis
Images were obtained using Olympus FV1000 confocal system (Olympus, Poland) equipped with an Olympus IX81 inverted microscope.Fluorescence of DAPI (excitation 405 nm, emission 425-475 nm) and Alexa488 (excitation 488 nm, emission 500-600 nm) was acquired from 60 × Plan Apo oil-immersion objective lens (NA 1.35), a 50 mW 405 nm diode laser and a 100 mW multi-line argon ion laser (Melles Griot BV, the Netherlands).Axial series of two-dimensional fluorescence images of the optical sections through the nuclei (z-stacks) was collected with the use of two separate photomultipliers (R6357, Hamamatsu, Japan) set to work in the integration mode at 4 µs pixel dwell time and 12-bit signal digitization (4096 intensity levels).Alexa488 and DAPI fluorescence intensity levels were measured in the ImageJ version 1.53 s software (Wayne Rasband, National Institutes of Health, USA).Images were converted to eight-bits and segmented with the threshold value parameter.
The fluorescence intensity of Alexa488 and DAPI was calculated as mean values from the Integrated Density (IntDen) parameter per one nucleus, which depicted the sum of all of the pixels within the region of interest.Results are presented in relative units.500 nuclei were analysed for each callus type, four time points, for seven histone modifications, DNA methylation and two controls (unmodified histone H3 and H4).Numerical data is included in Supplementary Materials Fig. S1.Fluorescence data analysis and plotting was done using R, a software environment for statistical computing and graphics in R Studio 2022.12.0 Build 353 (Script included in Supplementary Materials Fig. S3), an integrated development environment for R (Team RC 2022;Team 2022b).An ANOVA's test with the R Stats package (Team 2022a) followed by Tukey's HSD test at the significance level p ≤ 0.05.Standard errors were also calculated via the stats package.The package agricolae (Statistical Procedures for Agricultural Research) was used to find significant differences between means (Mendiburu 2021).Subsequently, data was plotted with packages dedicated to data visualization; ggplot2 (Wickham 2022) and ggpubr (Kassambara 2022).Letters on the graphs indicate statistically significant differences between samples.

Results and discussion
In general, callus tissue is of remarkable properties, it contains cells of increased developmental potency and can be formed through a variety of initial pathways which include the same gene regulatory network responsible for stress, hormonal and developmental cues (Feher 2019; Hemenway and Gehring 2023).These processes have also been linked with the chromatin accessibility indicating they are closely intertwined (Bednarek and Orłowska 2019;Birnbaum and Roudier 2017).F. tataricum MC characterises with a stable regeneration potential, the ability to undergo SE and organogenesis while maintaining low chromosome number (2n = 16) throughout approximately ten years of culture (Betekhtin et al. 2017;Kamalova et al. 2009;Rumyantseva et al. 1989).MC's PECCs have a distinctive structure typical for embryogenic cultures, of the somatic embryos arrested at the pre-globular stage due to the presence of auxin in the culture media as demonstrated previously by Rumyantseva et al. (2003), Souter and Lindsey (2000).PECCs are known to be able to regenerate through organogenesis as well as SE, which makes it a perfect system for studying its response to hormonal cues, whereas an abnormal line, NC appears on the surface of MC due to endoreduplication cycles and is subdued to constant oxidative stress (Betekhtin et al. 2017), making it suitable for research on epigenetic mechanisms involved in stress response pathways.Since they originate from the same species, but are so extraordinary different in structure and responses to tissue culture conditions, comparative analyses can be performed to decipher the role of epigenetic mechanisms behind their development.Since, the closely related species F. esculentum produces an EC typical for that type of callus, able to regenerate through the means of SE, and is not as genetically stable as F. tataricum MC callus, it was significant to research the levels of the same epigenetic modifications to shed some light on similarities in mechanisms regulating responses to stress, hormonal and developmental signals.

Morphology and histology of Fagopyrum esculentum EC, Fagopyrum tataricum MC, NC
Calli were examined on days zero, two, six and eleven of the passage (Fig. 1).Images of the morphology were acquired with the Keyence VHX-970F digital microscope (Japan) equipped with an ultra-small high-performance zoom lens VH-Z20R/Z20T and wide-area illumination adapter OP-87298.It provided real-time depth composition of images with the 3D scanning technique.The microscope produced images of extraordinary clarity and texture, due to a high resolution dynamic range.In this experiment, day zero is the beginning of a new passage; a time point at which callus is transferred onto fresh RX medium.Most F. esculentum callus induction was obtained from hypocotyls, (Adachi et al. 1989;Hao et al. 1998;Jin et al. 2002;Kwon et al. 2013;Yamane 1985), however this could result in low embryogenic potential and elevated chromosome number.Callus induced from immature zygotic embryos characterised by the presence of meristemoids, was able to produce high amounts of somatic embryos suggesting that the genotype is an important factor for SE (Berbec and Doroszewska 1999;Nesković et al. 1987).EC of F. esculentum has a dense, globular structure, milky glittering surface and is comprised of embryogenic masses (Fig. 1 a-d).Upon transfer to the fresh medium, the secretion is triggered which is marked with red arrows (Fig. 1 a,b).EC of F. esculentum (Fig. 2a) contains embryogenic cells (Fig. 2a, rectangle, a1 arrowheads, a2 arrow, a3 arrow), which are poorly vacuolised, have dense cytoplasm and centrally located nucleus (Fig. 2, a3, arrow).They are surrounded by highly vacuolated parenchymatous cells (Fig. 2a, a1, a2 asterisks).
Nucleus has a near-cell wall position.Phenolic containing (PC) cells that accumulate phenolics in their vacuoles are present (Fig. 2a, arrow).EC of F. esculentum also contains cells exhibiting epidermal phenotype (Fig. 2a, a2 open head arrows) which can form embryoids (Rumyantseva et al. 2005).Upon transfer on fresh medium, in MC of F. tataricum PECC, which has a dense structure and light colour (Fig. 1e) is triggered to disintegrate, release secretion (Fig. 1 e, red arrows) giving rise to 'soft' callus cells (SCCs).This is evident on day two and day six (Fig. 1 f, g) where the number of SCCs rises and they become prominent brownish, with loose structure and more secretion (Fig. 1f, red arrows).SCCs may act as support tissue, providing nutrients such as proteins and sugars as well as other conditioning factors, which are secreted into the medium during the disintegration of the PECCs.SCCs do not divide but are metabolically active which postpones death through senescence (Betekhtin et al. 2017).New PECC blowout (white asterisks) supported by SCCs (black asterisks) is noticeable on day 11 (Fig. 1h).Previous study conducted on F. tataricum revealed two cycles of PECCs reinitiation during the course of the passage (Betekhtin et al. 2019(Betekhtin et al. , 2017;;Rumyantseva et al. 2004).First one occurs approximately on the eleventh day, and the second one around day twenty-second, both supported by the spikes of the mitotic activity and release of the extracellular polymers into the culture medium (Rumyantseva et al. 2004).In this study one cycle was studied, since the events of the PECCs disintegration and reinitiation undergo the exact same processes after day eleven.PECC's surface (Fig. 2b) is covered by the PC cells (Fig. 2b F. tataricum NC (Fig. 1i-l) has unorganised, frail structure, is characterised by very fast growth rate and is devoid of the ability for morphogenesis (Betekhtin et al. 2017).F. tataricum NC (Fig. 2c) comprises exclusively of highly vacuolated parenchymatous cells with irregularly shaped wall-adjacent nucleus (Fig. 2c, arrows) that frequently contains multiple nucleoli (Betekhtin et al. 2017;Rumyantseva et al. 2003) (Fig. 2c, inset, open arrows) PCs are components of the antioxidant defence system in tissue cultures and take part in the regulation of morphogenesis processes (Debeaujon  . 2003;Sakar and Naik 2000).It has been previously demonstrated that MC accumulates much more phenolic compounds than the NC (Akulov et al. 2018).Moreover, NC cells poorly synthesize chlorophyll as a result of anomalies in the plastid development and lack the capacity to perform photosynthesis.Transformation of MC to the NC seems to be a result of the mutational or epigenetic changes in the DNA and subsequent silencing of genes involved in the PC biosynthesis (Akulov et al. 2018).

Epigenetic modifications in Fagopyrum esculentum EC
The analysis of the level of epigenetic modifications in F. esculentum EC were preceded with the measurement of the unmodified histones H3 and H4 as controls where no significant differences were displayed across the examined days of the passage (Fig. S2 a,b).Levels of DNA methylation across the examined days of the passage displayed significant differences (Fig. 3a).The content of 5mC decreased from day zero to day two which subsequently increased on day six and eleven.A similar result was obtained for Beta vulgaris, where hypomethylation was observed during callus formation (Zakrzewski et al. 2017).Similarly, in Oryza sativa callus, DNA hypermethylation was associated with dedifferentiated state (Stroud et al. 2013).In Triticosecale global DNA demethylation took place during tissue culture regeneration (Machczyńska et al. 2014).H3K36me3 levels (Fig. 3b) fluctuated from high on days zero and six to low on days two and eleven, whereas H3K4me (Fig. 3c, d) PTMs showed the lowest levels on day six and eleven.The deposition of H3K4me3 and H3K36me3 results in an open chromatin and transcriptional activation.H3K4me3 is typically enriched at the transcription start site and is associated with its initiation whereas deposition of H3K36me3 is more widespread and associated with transcriptional elongation (Roudier et al. 2011).H3K4me and H3K36me3 are linked to a variety of developmental processes and responses to environmental cues (Roudier et al. 2011).In Arabidopsis, a mechanism responsible for gene priming involving LYSINE-SPECIFIC DEMETHYLASE 1-LIKE 3 (LDL3) eliminates H3K4me2 during the callus formation and pluripotency acquisition (Ishihara et al. 2019).H3K4me3 increase was observed in Oryza sativa callus and was found to be mainly involved in DNA replication (Zhao et al. 2020) hence suggesting that dynamic cell differentiation in F. esculentum EC takes place during the first days of the passage.H3K18ac was on an overall high level and decreased with the passage progression (Fig. 4a), a pattern displayed also by the levels of the acetylation of H4K12 (Fig. 4b) correlating with the results obtained for histone H3K4me2 methylation.H3K18ac is also mainly found in regions adjacent to the transcription start site and correlated with transcription enhancers.Likewise is H4K12ac which is located on active coding genome regions where it creates binding sites for regulatory factors and promotes transcription (Ayyappan et al. 2015;Inacio et al. 2018).In Brassica napus high levels of H3Ac and H4Ac were detected in the vacuolated microspore, which is a totipotent cell capable of reprogramming and re-entry into the cell cycle upon induction (Rodriguez-Sanz et al. 2014).H4K16ac (Fig. 4c) levels were the lowest on day zero and kept increasing with the passage progression.H4K5ac (Fig. 4d) presented a reverse pattern, decreasing across the passage, with the exception of an increase on day eleven.Both modifications are associated with active transcription suggesting that on day eleven gene transcription might occur in higher frequency and might be related with the acquisition of the embryogenic potential (Moronczyk et al. 2022).
It has been previously reported that an increase in histone H4 acetylation was associated with the progression through the S-phase of the cell cycle (Desvoyes et al. 2014).In Vicia faba, Hordeum vulgare and Arabidopsis the intensity of histone H4 acetylation at lysine 5 and 12 was correlated with the cell cycle and evident 'deposition-related' acetylation at eu-and heterochromatin during DNA replication and subsequent deacetylation at heterochromatin towards mitosis.On the contrary, significant acetylation of H4K16ac within the chromocenters of endopolyploidy nuclei suggested delayed deacetylation after the endoreduplication (Jasencakova et al. 2001(Jasencakova et al. , 2000(Jasencakova et al. , 2003)).

Epigenetic modifications in F. tataricum NC and MC
Unmodified histone H3 and H4 levels in F. tataricum NC and MC were measured and showed no substantial differences across the examined days of the passage (Fig. S2 c-f).
Immunocytochemical analyses revealed dynamic epigenetic changes in F. tataricum calli with different morphogenic potential.Differences in calli structure on the cellular level development might be a contributor to such immense changes in epigenetic modifications.NC is characterised by aneuploidy (Betekhtin et al. 2017;Kamalova et al. 2009).
Increased cell ploidy has been related to an extended depth of the nuclear lobes as well as the nucleus area.The occurrence of high-level polyploid cells seemingly justifies the presence of lobed nuclei in the NC cells (Betekhtin et al. 2017).NC exhibited relatively stable but high level of DNA methylation (Fig. 3i), and histone H3K18, H4K12 acetylation (Fig. 4i, j) probably due to the rapid growth rate and high content of hydrogen peroxide, malonic dialdehyde, low catalyse activity and high superoxide dismutase activity indicating that NC is in a constant state of oxidative stress (Kamalova et al. 2009).Additionally, NC has a high level of DNA damage across the passage, which is a normal physiological state for the NC and its cells appear to be adapted to it (Betekhtin et al. 2017).This can be correlated with the high levels of DNA methylation (Fig. 3i) and significantly reduced and unstable levels of the acetylation of H4K16 and H4K5 (Fig. 4k, l) during the course of the passage.
The most noticeable and prominent pattern of the epigenetic modifications across the course of the passage in F. tataricum MC was DNA methylation (Fig. 3e).On day zero and eleven the levels of 5mC were significantly decreased when compared to day two and six.This might be due to the fact that PECCs on day zero have the highest morphogenic capacity, and upon disintegration and the loss of the capacity the 5mC content rises.Subsequent reinitiating of PECCs on day eleven results in regaining the morphogenic capacity and a decrease in the DNA methylation level.The levels of 5mC in NC (Fig. 3i), on the other hand were on a much higher level when compared to MC, however except for day six stayed on a stable level across the course of the passage.Betekhtin et al. (2019) examined the expression of DNA methyltransferases in F. tataricum calli and demonstrated significantly higher expression of MET1 and MET2 in the NC, while demethylases (DME1, DME2, DME3) expression was on the higher level in MC.Similar results were obtained for Siberian ginseng where significantly lower global DNA methylation rates in embryogenic calli were present in contrast to non-embryogenic calli (Chakrabarty et al. 2003).Additionally, in Agave furcroydes, hypermethylation on high but stable level was present throughout the culture in non-embryogenic callus and in the embryogenic clone the hypomethylation was observed in the beginning and towards the end of the passage (Monja-Mio et al. 2018), which correlates with the results obtained for MC of F. tataricum.H3K36me3 (Fig. 3f, j) was on the stable level with no vast fluctuations throughout the days of the passage in MC and in NC.H3K4me marks (Fig. 3g, h) were present in decreased levels on days zero and two in MC, similarly to Coffea canephora global levels of H3K4me which were decreased at the beginning of SE induction (Nic-Can et al. 2013).As mentioned earlier, elimination of H3K4me2 was observed in Arabidopsis during the pluripotency acquisition (Ishihara et al. 2019), therefore the differentiation/dedifferentiation switch might be initiated during the first days of the culture in MC.In NC, the level of H3K4me2 (Fig. 3k) was on the stable high level throughout the culture, whereas in H3K4me3 (Fig. 3l) it was decreased on days zero and two, comparable to MC. H3K18ac levels in MC and NC (Fig. 4e, i) were on an overall high level.However, an interesting pattern was prevalent in NC, where the lowest level presented on day zero and eleven, elevating on day two and six.H4K12ac is the mark associated with euchromatin and active transcription and in MC and NC (Fig. 4f, j) the pattern is opposing, namely in MC the H4K12ac level was the highest on day zero and eleven, whereas in NC those days levels were the lowest.This might be due to active gene expression correlated with PECCs reinitiating in the MC.An increase in the acetylation of histone H4 and acetylation of H3K18 has been associated with the progression through the S-phase of the cell cycle (Jasencakova et al. 2001(Jasencakova et al. , 2003)).Therefore, the elevated levels of H4K12ac (Fig. 4j) in NC on days two and six as well as H4K5ac (Fig. 4l) on day zero and H4K16ac (Fig. 4k) on day six might be a result of the endoreplication (Betekhtin et al. 2017).The levels of H4K16ac in MC (Fig. 4g) presented a pattern of lower levels on day zero and eleven and elevated levels on day two and six, which seem to correlate with intense cell division and an increase in the mitotic activity (Rumyantseva et al. 2003).H4K5ac in MC (Fig. 4h) presented a gradual increase from day zero to day eleven, while in NC the significant reduction of this modification was observed on day zero two and eleven.Elevated level of this modification on day six may suggest that upon this day the transcription is activated and rapid transcription takes place.Betekhtin et al. (2019) examined the expression of genes connected with ethylene biosynthesis, a factor with a significant role in the senescence processes, 1-AMI-NOCYCLOPROPANE-1-CARBOXYLIC SYNTHASE (ACS2 and ACS6), 1-AMINOCYCLOPROPANE-1-CARBOXYLIC ACID OXIDASE(ACO1) and ETHYLENE RESPONSE FAC-TOR (ERF), in MC and NC.Their research showed that, ACS2 and ACS6 expression was higher in the NC, which led to the conclusion that such vast differences in the expression of these genes can result in the ethylene overproduction and the induction of fast senescence processes in the NC.Additionally, an increased expression of the ERF1 in NC in comparison to MC seem to be related to the high levels of oxidative stress present throughout the culture.It was demonstrated, that ERF1 is upregulated in the response to abiotic stress triggers such as salt and cold treatments or water deficit (Lestari et al. 2018;Makhloufi et al. 2014).

Conclusions
This study investigated the influence of the epigenetic modifications on the cell fate transition in long-term cultivated in vitro Fagopyrum callus lines with different capacity for morphogenesis.It was exhibited that; decreased level of H3K4me2 seems to be connected with pluripotency acquisition in F. esculentum EC and F. tataricum MC; DNA hypomethylation appears to be correlated with the acquisition of the embryogenic potential and PECC reinitiation in F. tataricum MC; out of all examined PTMs, H4K16ac and H4K5ac revealed the highest variability during the course of passage in NC.Elevated levels of these modifications on day zero and day six for H4K16ac and H4K5ac respectively, seem to be correlated with endoreplication peaks, the processes which are distinctive for this callus.To sum up, while F. esculentum EC displays the characteristics typical to irregular, disorganised callus tissue, F. tataricum MC behaves in a completely different way.The cyclical development, disintegration and reinitiation of PECCs allowed to examine epigenetic changes and associate them with these processes.Comprehending how the chromatin reprogramming underlies the changes in the cell fate can provide a way for future manipulation towards the crop improvement, since once established during the callus formation, those modifications can be inherited through the epigenetic allele's transmission and become strategies for the crop manipulation in the future (Hemenway and Gehring 2023;Lee and Seo 2018).In the future study, annotation of the genes involved in the process of dedifferentiation/re-differentiation switch, their expression and their epigenetic status in connection of the PTMs should be considered.
, rectangle, b1 arrows).Below the PC cell layer, lay poorly vacuolised meristematic cells (Fig. 2b1, double arrows) with dense cytoplasm.Round nuclei and centrally located nucleolus-the source of embryogenically determined cells, from which embryoids and new PECCs can form.Parenchymatous, storage cells (Fig. 2b1, open arrows) containing large starch grains in their plastids and wall-adjacent large nucleolus are located below and constitute the largest part of the PECC.

Fig. 1
Fig. 1 Morphology of F. esculentum EC a-d; embryogenic complex of F. tataricum MC e-h; red arrows indicate the secretion, black asterisks indicate soft callus, white asterisks newly formed PECCs; NC of F. tataricum (i-l) on day zero, two, six and eleven; Scale bars: 0,5 mm

Fig. 3
Fig. 3 The levels of fluorescence intensity in DNA methylation and histone H3 methylation; in F. esculentum EC a-d and F. tataricum MC e-h and NC (i-l) across the passage.Letters indicate statistically significant differences between samples, P ≤ 0.05, n = 500

Fig. 4
Fig. 4 The levels of fluorescence intensity in histone H3 and H4 acetylation in F. esculentum EC a-d F. tataricum MC e-h and NC (i-l) callus; letters indicate statistically significant differences between samples, P ≤ 0.05, n = 500