Quantitative changes in DNA methylation induced by monochromatic light in barley regenerants obtained by androgenesis

Changes in DNA methylation are one of the best known mechanisms of epigenetic regulation of gene expression, which in the process of induced androgenesis is associated with reprogramming of haploid microspores development towards the formation of embryos, as a result of exposure of anthers in ears and then anthers culture in vitro to stress factors. The aim of the study was to test the hypothesis of whether the use of monochromatic light during induced androgenesis might be associated with epigenetic phenomena. The experiments were carried out on DH plants of spring barley (Hordeum vulgare L.) obtained by androgenesis modifi ed by monochromatic light: blue, green and red. A quantitative evaluation of the eff ect of light on the degree of DNA methylation was performed using RP-HPLC for the comparison of regenerants obtained under standard, control conditions (darkness) with those obtained with light usage. The diff erences in the amount of methylated cytidine in comparison to the control were: 0.40%, 0.16% and -0.55%, for blue, red and green light, respectively. The level of global genomic DNA methylation from control plants was in the range 21.32-21.52%. Methylation changes in response to monochromatic light used during callus formation in anthers culture, determined by RP-HPLC, are signifi cant although small.


Introduction
DNA methylation is a modifi cation of deoxyribonucleic acid, which occurs during the S phase of the cell cycle, under the infl uence of DNA methyltransferases, specifi c against nitrogenous bases: cytosine or adenine, which are part of the nucleotides deoxycytidine (dC) and deoxyadenosine (dA). It involves the attachment of methyl groups (-CH 3 ) mainly to the 5 th carbon atom in the pyrimidine ring and less frequently on the methylation of NH 2 + groups located either at the 4 th carbon atom of cytosine or at the 6 th carbon atom of adenine (purine base). Methylation occurring in CpG dinucleotides and CpXpG trinucleotides, where X is A, T, C is defi ned as symmetrical methylation, while in CpXpX sequences, as unsymmetrical.
The methyl group donor is 5'-adenosine methionine and the reaction products are:5-methyl-2'-deoxycytidine (5mdC), the main product of DNA methylation as well as N 4 -methyl-deoxycytidine and N 6 -methyl-deoxyadenosine. The amount of 5mdC depends on both the enzymatically catalysed methylation as well as demethylation, which may be passive or active. Passive demethylation occurs during replication and is not accompanied by melyltransferase-1 (DNMT1), thus resulting in a lack of conservative methylation (Guz et al., 2010). In contrast, active demethylation is catalyzed by bifunctional DNA glycosylases from the D family during the repair of replication errors (Li et al. 2018). It is also associated with modifi cations of histones and, most likely, non-coding RNA (Parrilla-Doblas et al., 2019, Zhang et al. , 2012. In mammals, the share of 5mdC in total dC content is 3-4%, which is 0.75-1% in relation to all nucleosides (Guz et al., 2010). It is estimated that 70-80% of CpG dinucleotides in the entire mammal genome are methylated (Law and Jacobsen, 2010). On the other hand, the amount of methylated cytidine in the plant genome is 20-30% (Finnegan et al., 1998). In the Arabidopsis thaliana genome CpG, CpXpG and CpXpX are methylated approximately at 24%, 6.7% and 1.7%, respectively (Law and Jacobsen, 2010). The degree of DNA methylation changes with tissue age, wherein the methylation non-related to CpG islands is characteristic for diff erentiating cells (Peredo et al., 2006).
The androgenesis in vitro of haploid microspores allows for reprogramming their development from the gametophyte, leading to the formation of functional pollen grains, to sporophyte, leading to embryo formation. This process is accompanied by changes in genomic DNA methylation. Although in mammals most aspects of epigenetic regulation of both embryo as well as cancer cell development are quite well understood (Guz et al., 2010), the mechanisms of plant DNA methylation patterns reset remains predominantly unexplored. Changes in patterns of genomic DNA methylation in plant tissues may appear as a response to environmental stresses, associated with the redox signalling system (Bednarek and Orłowska, 2020). It has been shown that these changes are not only specifi c to individual species, but also diff er within genotypes (Karan et al., 2012). Among others, plant regeneration by tissue culture in vitro also induces variation in the level of DNA methylation, depending on plant regeneration system, genotype of donor plants, explant, nutrient medium, as well as time duration of the culture. Among factors likely affecting changes in DNA methylation is also light. It was found that the total amount of methylated cytidine increased in DNA of barley DH regenerants obtained via androgenesis or somatic embryogenesis in comparison with the donor plant, whereas in triticale it decreased (Machczyńska et al., 2014;Orłowska et al., 2016). However, if one or more subsequent generative propagation cycles are carried out, the amount of 5mdC stabilizes in successive plants thus obtained. Quantitative changes of methylated DNA are accompanied by changes in methylation patterns (Bednarek i Orłowska, 2020;Machczyńska et al., 2014;Niedziela, 2018;Orłowska et al., 2016).
Quantitative determination of genomic DNA methylation can be carried out by RP-HPLC (Reversed Phase-High Performance Liquid Chromatography). This method was used for analyses of genomic DNA of cereal plants in response to abiotic stress (Niedziela, 2018), as well as in studies of barley and triticale in vitro regeneration (Orłowska et al., 2016;Machczyńska et al., 2014). The purpose of the present work was to evaluate whether modifi cations of the androgenesis process in anther cultures in vitro, consisting of the use of monochromatic light at the callus induction stage, aff ects changes in the level of methylation of genomic DNA in regenerants.

Materials and Methods
DNA from leaves of spring barley regenerants (Hordeum vulgare L.), genotype 2dh/8, were used for the experiment. Regenerants were obtained in anther culture in vitro, proceeded according to a modifi ed protocol by using diff erent monochromatic lighting at the stage of callus formation on induction medium (usually run in darkness). Regenerant plants were grown in dedicated trays with wells for single plants, in a phytotron at 18/14° C and in a 16/8 h day/night photoperiod. Leaves for DNA isolation were taken from plants in the tillering stage. The ploidy of regenerants genome was determined using a CyFlow Ploidy Analyzer fl ow cytometer (Sysmex Polska Sp. Z o.o.). This stage of the experiment and its detailed results are the subject of a separate publication (Siedlarz et al., 2020). Here, the subject of research was the DNA of regenerants obtained as a result of androgenesis in vitro modifi ed at the time of callus induction by monochromatic LED light: blue 454.63 nm, green 525.95 nm and red 630.84 nm. The control group consisted DNA of regenerants obtained under standard conditions, i.e. in the dark during callus induction (Orłowska et al., 2016, Bednarek and Orłowska, 2020, Siedlarz et al., 2020. DNA was isolated from pairs of regenerates Quantitative changes in DNA methylation induced by monochromatic light in barley... obtained from the anther of the same ear: 10 in control conditions and 10 in light modifi ed conditions. A total of 60 DNA samples were isolated from leaves of plants in the tillering phase using the DNasy Mini Prepkit kit (Qiagen GmbH, Hilden, Germany), according to the manufacturer's methodology. DNA concentration and purity were determined using a UV-Vis NanoDrop 2000c/2000 spectrophotometer (Thermo Scientifi c, USA). The quality of the samples was verifi ed electrophoretically in 1.4% agarose gel.

Results and Discussion
The experiment was conducted to check whether modifi cations of the in vitro androgenesis by the usage of monochromatic light at the callus induction stage aff ects changes in the level of genomic DNA methylation of regenerants. DNA which was isolated from 60 plants, more specifi cally 30 pairs of plants, obtained under control and light-modifi ed conditions, were subjected to quantitative RP-HPLC analysis. Since the donor plants were characterized by a constant level of DNA methylation, as they came from generative reproduction (Orłowska et al., 2016), we assume that the modifi cations of methylation observed in this experiment were induced by light.
There is no information available on how many generative cycles are needed to stabilize/eliminate (if possible at all) the eff ects of tissue cultures on DNA methylation. Nevertheless, it has been shown that in both the barley and triticale genome the level of methylation stabilizes after one/two cycles (Machczyńska et al., 2014;Orłowska et al., 2016). Thus, to stabilize DNA methylation changes induced in regenerants genome DNA during in vitro tissue culture, the use of regenerants should be considered as donor plants. The total range of methylation changes in the barley genome determined in the present experiment was small and ranged from 21.12 to 21.87%. Also, Orłowska et al.  (Fiuk et al., 2010). In the present experiment, the total amount of 5dmC in the DNA of control plants averaged 21.4% and did not diff erentiate the tested regenerants obtained under unmodifi ed light conditions (Table 1). However, in the group of regenerants obtained using light at the callus induction stage, diff erences were observed: for blue light conditions the amount of 5dmC was the smallest, signifi cantly diff erent from the value registered for parallel control plants, while for green light it was the largest and also diff erent from the control group (Table 1). The results obtained for the group exposed to red light were ambiguous. They did not diff er signifi cantly from the value obtained for the group of plants under blue light. However, they did not diff er from the control plants either. The diff erence in the amount of methylated cytidine between control plants and plants regenerated using light was on average: + 0.40%, + 0.16% and -0.55% for blue, red and green light (Fig.  1). In regenerants obtained in conditions modifi ed by red light, diff erences from control ranged from -0.86% to + 0.92%. Those results were ambiguous and require further investigation. The changes in the global level of cytidine methylation in cereal plants described in the literature, induced by generative reproduction, mainly concerned comparisons between donor plants and regenerants under standard conditions for particular laboratories, or between the applied plant regeneration systems. It is interesting that within the Hordeum vulgare L. species, in vitro cultivation of wild forms caused changes in cytosine methylation in both level and patterns of methylation in comparison to the donor plant, in which higher level of DNA methylation were found (Li et al., 2007). An inverse relationship was observed for the barley cultivar Scarlett, since the lowest average value of global methylation was observed in donor plants, while the average value of DNA methylation and their off spring was 20% and 20.13%, respectively (Orłowska et al., 2016). Higher levels of methylation were observed in triticale DNA for plants obtained in cultures of spontaneously released microspores, compared to regenerants from the culture of immature zygotic embryos. Diff erences between donor plants and regenerants have been shown. The average methylation of the genome of a donor plant was 25.4%, whereas regenerants 24.1%. The fi rst generative progeny was characterized by 23.6% DNA methylation, the second 23.8%, and the third generative progeny-23.9%. (Machczyńska et al., 2014). Changes in the level of genomic DNA methylation are specifi c for each species, and also depend on the in vitro culture conditions. Exposure to UV-B light of an annual plant, big sagebrush seedlings obtained from apical meristem shoots induced a signifi cant reduction in the total level of DNA methylation (Pandey and Pandey-Rai, 2015). The presented results also indicate that the type of monochromatic light used during callus induction in anthers in vitro culture causes changes in the level of methylation of genomic DNA, in a manner depending on the wavelength of light used, i.e. the color of the monochromatic light.