Molecular Responses of Arabidopsis MET1 Cytosine Methyltransferase Mutants To Salinity

In this study, we investigated the morphological and molecular responses of Arabidopsis met1-7 and met1-3 null mutants under salinity stress. In this context, global DNA methylation changes of mutants exposed to salt stress were compared and expressions of DRM2, Pol IV and Pol V genes known to be involved in DNA methylation in plants were analyzed. We found that met1-7 and met1-3 mutants have a higher rate of hypomethylation than Col-0 under all conditions. According to the results of gene expression analysis, the increase in expression of DRM2, Pol IV and Pol V genes involved in CNN methylation in mutants than Col-0 plant suggests that hypomethylation is directly related to CG regions, but in met1 mutants, the lack of CG methylation is tried to be compensated by RdDM. In addition, we analyzed the expression of the TERT gene as a stress response indicator in order to examine the effect of salt stress on the telomerase enzyme in met1-7 and met1-3 mutants. Contrary to expected, we found that there was an increase in the expression of TERT gene in the salt stress applied plants. Within the scope of all the data, it is thought that in met1 mutants RdDM pathway is activated in order to deal with the lack of DNA methylation in CG islands. As a conclusion, it is believed that the morphological and molecular data obtained on the effects of NaCl application to met1 mutants will help us to understand the epigenetic basis of stress mechanisms.


Introduction
Plants have a major impact on ecosystem balance. They have important roles such as being food sources for animals and humans. However, it has become di cult to provide the required amount of food due to the increasing population and the decrease in agricultural areas as a result of salinity and drought stress. The rst step to nd solutions to these problems is to understand the relevant pathways very well in model organisms such as Arabidopsis thaliana.
After salt exposure molecular mechanisms are activated in plants in order to tolerate stress conditions. Epigenetic modi cations, one of these molecular mechanisms, affect the stability of the genome by changing the covalent bonds between DNA and protein without changing the DNA structure. Studies have shown that epigenetic modi cations can mediate stress tolerance memory during repeated exposure to certain environmental stresses. Some genetic changes that can occur in plants' reproductive cells throughout their lifetimes can then be passed on to subsequent generations. For this reason, plant systems are a rich resource for the study of epigenetic inheritance ( DNA methylation, which is one of the most common epigenetic modi cations in A. thaliana, accounts for about 30% of its genome (Haag and Pikaard 2011;Sahu et al. 2013). Cytosine methylation in plants occurs in three types: CG, CNG as symmetric and CNN as asymmetric (where N represents A, T, or C) (Kong et al. 2018). There are 3 enzymes mainly involved in the methylation of cytosine in plants. The rst enzyme is methyltransferase 1 (MET1), which is responsible for methylation of CG sequences and a homologue of the mammalian DNMT1.
The second enzyme responsible for methylation of CNG and a small amount of CNN sequence is chromomethylase 3 (CMT3). This plant-speci c enzyme is largely deposited in TE's and induces ectopic methylation of protein-coding genes. It is also thought to depend on direct physical binding to H3K9me2 to target chromatin and methylation. The third enzyme, domains rearranged methylase (DRM), which is a homologue of DNMT3 in mammals, is mainly responsible for maintaining asymmetric CNN methylation with siRNAs and plays a minor role in the maintenance of CNG methylation. Also it is required for de novo methylation of cytosines in all sequence contexts (Ashapkin et al. 2002 and this hypomethylation is not only related to the MET1 gene. Besides this, DRM2-related de novo methylation is also affected. Considering these studies, molecular mechanisms must be understood well in order to make plants resistant to the stress conditions they encounter and maintain this resistance for generations. Consequently, it was decided to examine the effect of salt on met1-7 and met1-3 mutants to understand the role of the MET1 gene under salt stress.
In this study, to understand the relationship between the hypomethylation caused by NaCl stress and the MET1 gene, A. thaliana Col-0 plants and null mutants of met1-7 and met1-3 were exposed to salt stress. To this extent, methylation level of the genome and the expressions of DRM2, Pol IV and Pol V genes, which are known to be involved in the RdDM mechanism were analyzed. Also the expression of the TERT gene was analyzed in order to examine the effects of salt stress on the telomeres of met1 mutants.

Plant material and growth conditions
A. thaliana Col-0 (Wild type), met1-7 and met1-3 seeds were kindly provided from Dr. Ralf Stracke The Col-0, met1-7, and met1-3 seeds were surface sterilized and placed on Murashige and Skoog basal medium (MS) (Murashige and Skoog 1962) which was supplemented with sucrose (3%, w/v) and pH was adjusted to 5.8. Agar (0.9%, w/v) was added for solidifying and the medium was sterilized by autoclaving at 121°C under 1 atm pressure for 15 minutes. Col-0, met1-7 and met1-3 seeds were germinated under uorescent light in a plant growth chamber [ Bioinformatic analysis on MET1 enzyme Self-fertilization and seeding studies performed in our laboratory gave positive results in met1-7, but were inconclusive in met1-3 similar with Saze et al. (2003). The fact that the two met1 mutants morphologically different signi cantly from each other and after observing only met1-7 mutant seed yields, the regions where the T-DNA insert entered were examined in more detail to investigate the answer that may have caused this difference.
The bacterial Ti-plasmid (T-DNA) inserts disrupt the conserved regions by entering the beginning of the 2nd exon in the met1-7 mutants and at the beginning of the 7th exon in the met1-3 mutants (TAIR 2021a, 2021b). The locations of the T-DNA inserts in the mutants are shown in Fig. 1.
The amino acid sequences of the A. thaliana MET1 enzyme (NP_199727.1) were obtained from the NCBI site (https://www.ncbi.nlm.nih.gov/protein/ ) and the catalytic sites of the enzyme were obtained using the

RNA isolation and qPCR analysis
For qPCR analysis, total RNA was isolated with Hibrizol (Hibrigen, Turkey) from A. thaliana Col-0, met1-7, and met1-3 plants exposed to NaCl stress. Then, the RNAs were checked for their integrity, purity and quantity. Their purities and quantities were checked by NanoDrop 2000 (Thermo Scienti c) and their integrities were analyzed by agarose gel electrophoresis (%1 TAE). cDNA was synthesized using the High-Capacity cDNA Reverse Transcription Kit (Thermo Scienti c, 4368814) according to the manufacturer's recommended protocol. qPCR ampli cation was performed with a Roche LightCycler Nano instrument by using 2X SYBR Green Master mix (Hibrigen, 0220-UB-775). The following PCR protocol was applied: 50˚C/30 s, followed by 40 cycles of 94˚C/30 s, 59°C/61°C/65°C for 30 s (59°C for TERT, 61°C for Actin, Pol IV, Pol V and 65°C for DRM2 respectively) and 72°C for 30 s. qPCR reactions were carried out with 2 technical replicates for each 3 biological replicates. The fold changes were calculated using 2 −ΔΔCt values, and relative expressions were shown as log2 fold changes (Livak and Schmittgen 2001). The primer sequences are given in Table 1. TERT gene (AT5G16850) primer pair was designed using the Primer3 program (http://primer3.ut.ee/).

Morphological effects of stress application
The morphologies of the met1-7, met1-3 null mutants and the control plants after 7 days exposure to NaCl stress are given in Fig. 2. As a result of the morphological analysis, leaf number for different NaCl concentrations in the control group Col-0 and met1-7, met1-3 null mutants did not change signi cantly between the groups. But the number of leaves was signi cantly reduced within all plant groups except in the met1-3 mutants between the 100 and 150 mM NaCl concentrations.
It was observed that the leaf surface areas decreased signi cantly between control and 150 mM NaCl concentrations in the Col-0 plants. Also, it was observed that the leaf surface areas of met1-3 mutants were affected more than the met1-7 mutants after 150 mM NaCl application.
And nally, it was observed that the root length decreased signi cantly except between the control group and the 100 mM NaCl application in both mutants. Compared to Col-0, met1-3 was more sensitive to salt stress than met1-7 plant and root length was signi cantly shortened in all groups. The root length was signi cantly shortened between the control group of met1-7 and the Col-0, also between each NaCl concentrations applied to met1-3 and Col-0. Lastly, the root length was decreased between both mutants for each concentration. The graphs of the morphological analysis are given in Fig. 3, and in addition to the graphical data, average values and standard errors are also given in Table 2.
Table2 Determination of the morphological effects of different NaCl concentrations in Col-0, met1-7 and met1-3 plants. Each value represents the average of repetitions and the standard error (± SH)

Global DNA methylation
As a result of the methylation analysis, mutants were found to be less methylated than Col-0 in each NaCl concentration. In control conditions, met1-3 mutants had less methylation compared to met1-7, but there was no signi cant change in met1-3 between applied NaCl concentrations, while hypomethylation occurred between control and 100 mM in met1-7. In addition, it was observed that between 100 and 150 mM NaCl treatments methylation increment has occurred. These ndings could indicate that the treatment with 150 mM NaCl could have activated additional mechanisms in the met1-7 mutant other than met1-3. However, when considering the entirely, mutants were found to be less methylated than Col-0 at each NaCl concentration. The results of methylation analysis are given in Fig. 5, and the percentages of methylation levels and standard errors are given in Table 3.
Table3 Determination of the global DNA methylation percentages and standard errors of Col-0, met1-7 and met1-3 plants after different concentrations of NaCl treatment Expression of DRM2, Pol IV, Pol V and TERT genes As a result of the analysis, expression of the DRM2 gene decreased by 0.4 fold in A. thaliana Col-0 plant after 100 mM NaCl application compared to control, and increased by 1.4 and 1.2 fold in met1-7 and met1-3, respectively. And there was no change in Col-0 after 150 mM NaCl application, it was increased by 1.65 and 1.45 fold in met1-7 and met1-3, respectively. In the expression change of Pol IV gene, it was determined that 100 mM NaCl application caused an increase in met1-7 and met1-3 groups 1.37 and 2 fold respectively, while 150 mM NaCl application caused an increase of 1.7, 2.73 and 1.95 fold at Col-0, met1-7 and met1-3 plants, respectively. The expression of Pol V gene after 100 mM NaCl application caused an increase of 1.3, 2.1 and 1.55 fold in Col-0, met1-7 and met1-3 groups respectively. After 150 mM NaCl application, the expression of Pol V gene in Col-0 plant was decreased by 0.7 fold, while in mutants it was increased by 2.85 and 1.8 fold, respectively. Lastly, the expression of TERT gene in Col-0 after 100 mM NaCl application decreased by 0.6 fold, while in met1-7 and met1-3 mutants it was increased by 1.7 and 1.5 fold, respectively. After 150 mM NaCl application, the expression of TERT gene in Col-0 plant was decreased by 0.7 fold, while in met1-7 and met1-3 mutants it was increased by 1.6 and 1.2 fold, respectively. As a result of the two-way ANOVA with post-hoc Tukey test, there was a statistically signi cant increment of TERT gene expression between Col-0 and met1 mutants at 100 mM NaCl application. Statistical signi cance was not determined in the expression analysis among other genes. The graphical representation of the gene expression analysis is given in Fig. 6 and relative fold changes are given in Table 4. have found that the met1-3 mutant is more sensitive to salt stress than the met1-7 mutant, morphologically. In many studies, this morphological sensitivity observed in epigenetic mutants compared to Col-0 plants supports the importance of epigenetic mechanisms in plant development.
In addition to the analysis of mutants' responses to salt stress, seed productivity was also analyzed. In order to obtain homozygous mutant seeds, seeds were tried to be obtained by self-fertilization method. Seeds could be obtained from the met1-7 mutant, while they could not be obtained from the met1-3 mutant. This has led us to think that in the met1-3 plant, unlike met1-7, the insertion of the T-DNA insert at the beginning of the 7th exon may cause some defects in the alternative arrangements of RNA transcripts, and this may disrupt the functioning of some pathways involved in gametophyte formation. In previous studies, it was stated that the seeds of the met1-3 mutant were smaller than normal and the plants in the rst generation were sterile (FitzGerald et al. 2008). However, the cause of infertility is not speci ed. We attribute the reason for this problem, which we encountered with this study, that the T-DNA insert entering the met1-3 mutant exactly coincides with the active region of the MET1 protein. Bioinformatic analysis showed that the T-DNA insertion in met1-3 mutant most probably disrupts the active site where S-adenosylmethionine (SAMe) binds to the methyltransferase1 protein. It is thought that the disrupted region in the met1-7 mutant may not cause any impairment in its function, although it disrupts the 3D structure of the protein. It is thought that MET1 protein, which is known to play an active role in embryogenesis, causes infertility due to its inability to function properly. As with many cellular signaling pathways, molecular responses to salt stress are expected to be in uenced by negative regulation as well as positive regulation of the gene expression (Zhu 2000). In parallel with our data, Gao et al. (2020) stated that gene expression of DMR2 decreased in Ginkgo biloba at 150 mM NaCl stress, but increased in chickpea roots. These results suggest that the inactivation of the MET1 gene has an effect on hypomethylation, and in this case DRM2-related de novo methylation plays a more effective role as a compensator in met1-7 and met1-3 mutants. Also, expressions of Pol IV and Pol V genes were examined in order to evaluate the NaCl stress application in terms of RdDM in the absence of functional MET1 enzyme.

Stress responses in plants cause changes in methylation in
Similar to our ndings, Naydenov et al. (2015) observed that Pol IV and Pol V expression also increased under heat stress and stated that the expression of these genes could change not only through RdDM but also by acting in other regulatory processes.
DNA methylation has an important role in regulating promoter activity. Many studies have shown that promoter hypomethylation occurs mostly in the CG regions and is also dependent on the methyltransferases MET1 and DRM2. Accordingly, it was concluded that the TERT gene could be controlled by the MET1 system. Defects in DNA methylation are known to lead to developmental abnormalities. Zangi et al. (2020) suggested that mutations occurring in methylation systems may affect TERT gene expression, leading to developmental anomalies. In their study, they have shown that expression levels of the TERT gene increased nearly 14 fold in homozygous met1 mutant plants compared to wild type. At the same time, TERT gene expression increased nearly 2 fold in heterozygous met1 mutants compared to wild type. They have reached the conclusion that the TERT gene is regulated by methyltransferases and may be involved in developmental abnormalities caused by mutation in the MET1 methyltransferase system. Zangi et al. (2020) also suggested that the mutation involved in the MET1 methyltransferase systems decreased the methylation of CG islands in the promoter of the TERT gene and consequently increased the expression of the TERT gene. It is known that overexpression of telomerase leads to telomere elongation. Therefore, it is thought that overexpression of telomerase in plants with the met1 mutation may be associated with limited growth and developmental abnormalities in Arabidopsis. It is possible that the effects and de ciencies of the met1 mutation can be compensated by other methylation systems, thereby improving the phenotype (Zangi et al. 2020). Despite this, Ogrocká et al. (2012) reported that DNA methylation in the putative TERT promoter region is not a dominant factor in the regulation of TERT transcription. They analysed the telomerase activity in met1-3 mutant seedlings, they have found no signi cant change in TERT transcription. Whereas, repeated analysis with met1-3 mutants revealed signi cantly lower telomerase activity in young leaves, the amount of TERT transcript in young leaves of Col-0 samples and 7 day seedlings of met1-3 mutants was similar. Our result is different from Zangi et al. (2020) but similar to Ogrocká et al. (2012). The expression of TERT gene is similar in Col-0 and met1 mutants not treated with NaCl. However, there is an increase in TERT expression compared to Col-0 in NaCl treated mutants. According to these ndings, it was concluded that to cope up with NaCl stress, TERT expression was upregulated due to the increasing RdDM in the mutants.
In conclusion, hypomethylation occurred at the genome level in mutants and they were left behind morphologically compared to Col-0 plants. It is consistent with the previous reports showing that the MET1 gene negatively affects the morphological characteristics of plants under NaCl stress. The reason for the increased expression of DRM2, Pol IV, Pol V, and TERT genes after NaCl application to met1 mutants is thought to support the RdDM pathway to cope with DNA methylation de ciency in CG islands.
In order to make plants resistant to stress, molecular stress mechanisms must be understood. It is thought that the data obtained from this study will contribute to the basic knowledge to understand the effects of salt stress on epigenetic mechanisms.

Declarations
Author contributions YVY and NTK designed the experiments. YVY and BA performed the experiments. All authors analyzed data, wrote and approved the manuscript. All authors contributed to the nal version of the manuscript.

Funding
This study was supported by the Research Fund of the Istanbul University (Project ID: 36576).
Financial interests Figure 1 The locations of the T-DNA inserts in the met1-7 and met1-3 mutants (TAIR, 2021a and 2021b). A. thaliana plants exposed to 0, 100 and 150 mM NaCl during seven days. Row A belongs to Col-0 plants exposed to different concentrations of NaCl. Row B belongs to met1-7 plants exposed to different concentrations of NaCl. Row C belongs to met1-3 plants exposed to different concentrations of NaCl Morphological analysis of A. thaliana Col-0, met1-7 and met1-3 plants. A represents the differences between leaf numbers of plants exposed to 0, 100 and 150 mM NaCl. B represents the differences between leaf surface areas of plants exposed to 0, 100 and 150 mM NaCl C represents the differences between root lengths of plants in each group individually exposed to 0, 100 and 150 mM NaCl D represents the differences in root lengths of plants exposed to 0, 100 and 150 mM NaCl between Col-0 and mutant groups E represents the differences in root lengths of plants exposed to 0, 100 and 150 mM NaCl between met1-7 and met1-3 mutant groups