Salt stress that plants encounter in nature is usually caused by NaCl (Ngara and Ndimba 2014). High NaCl concentrations in soils cause ionic stress by disrupting the ionic balance of Na+, Cl−, K+ and Ca2+ together with osmotic stress. Excessive amounts of Na+ ions entering the stem cells are carried to other leaves, tissues and organs and tried to be tolerated. This situation causes morphological changes in plants (Yang and Guo 2018; Liu et al. 2019). Considering all this information, in this study leaf number, leaf surface area and root length of epigenetic mutant plants treated with 100 and 150 mM NaCl during 7 days were examined. Similar to our morphological results, Arıkan et al. (2018) reported that the application of 100 and 150 mM NaCl to Arabidopsis Col-0 plants restricted root and shoot growth. Also, Baek et al. (2011) stated that the met1-3 mutant was hypersensitive to NaCl and they observed a salt-sensitive phenotype in root growth, and this situation could be attributed to the loss of methylation in the putative small RNA target region in the AtHKT1 promoter. In a previous study, it was stated that after 75 and 150 mM NaCl application, the met1-3 mutants were sensitive to salt stress, although not at the same degree as the ddm1 mutants (Yao et al. 2012). Moreover, Huang et al. (2013) stated that the epigenetic mutants (ros1 and rdm16ros1) of Arabidopsis plants were adversely affected morphologically with 75, 100 and 125 mM NaCl application at seedling stage. Similar to all this research, we 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 first generation were sterile (FitzGerald et al. 2008). However, the cause of infertility is not specified. 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.
Stress responses in plants cause changes in methylation in the coding region of genes involved and regulate gene expression (Sudan et al. 2018). For this reason, detecting epigenetic changes in the plant genome shows great importance in the understanding of stress responses of plants. DNA methylation is mostly observed on cytosine bases. The main enzymes responsible for cytosine methylation in plants are MET1, CMT3 and DRM2. The MET1 enzyme, which has a great role in these regulations, is responsible for 80-90% of the cytosine-level methylation in plants (Zangi et al. 2020). In this study, we used the null mutants met1-7 and met1-3, in which the functional MET1 enzyme of A. thaliana plant is not synthesized. The methylation changes of these met1 mutants were investigated and the 5-mC content at the genome level was determined. Likewise, Arıkan et al. (2018), the application of 100 and 150 mM NaCl during 7 days to A. thaliana Col-0 plant caused hypomethylation at the genome level. Boyko et al. (2010) reported in their study that unlike Arıkan et al. (2018), they encountered hypermethylation in the progeny of Arabidopsis plants exposed to salt stress. Moreover, Zhong et al. (2009) indicated that salt stress caused hypermethylation in some regions of the genome even though hypomethylation was found overall in the genome as a result of application of 100 and 150 mM NaCl during 5 days to bread wheat (“Triticum aestivum L.”).
As with many cellular signaling pathways, molecular responses to salt stress are expected to be influenced 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. Similar to our findings, 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 deficiencies 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 significant change in TERT transcription. Whereas, repeated analysis with met1-3 mutants revealed significantly 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 findings, 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 deficiency 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.