The promoters of OsGLP genes exhibited differentially methylated sites under drought and salt stress in rice cultivars

DNA methylation at cytosine residues governs the regulation of stress responsive genes in plants to generate response against various environmental abiotic stress conditions. Here, we analyzed the epigenetic consequences of drought and salinity on cytosine methylation dynamics of promoter regions of stress responsive Germin-like protein (GLP) genes in rice (Oryza sativa). The bisul�te sequencing technique was employed to identify differential methylation status at cytosine residues in selected promoter region of three OsGLP genes (OsGLP4-1, OsGLP8-10 and OsGLP8-12) in leaves and roots of two elite Indica rice cultivars (tolerant KS282 and sensitive Super Basmati) under drought and salt stress. Our results identi�ed cultivar, tissue and stress-dependent differentially methylated cytosine residues, however, the extent of methylation was found to be different depending upon CGN, CHG and CHH sequence contexts. Among all three OsGLP genes, promoter region of OsGLP8-12 was observed with most methylated and differentially methylated sites depending upon a particular variety, tissue or stress condition whereas no methylated sites were observed in promoter region of OsGLP4-1 in all samples. Moreover, in promoter regions of OsGLP8-10 and OsGLP8-12, several differentially methylated sites in response to stress treatments were identi�ed either near or within cis-regulatory elements (CREs) involved in gene regulation or abiotic stress response indicating the association between promoter methylation and regulation of OsGLP genes under drought and salinity. This study provides insights to argue that altered methylation status of speci�c cytosine residues in the promoter regions of OsGLP genes can be a key regulatory mechanism associated with their regulation under abiotic stresses in contrasting rice cultivars.


Introduction
Rice (Oryza sativa), a vital commercial crop, is reported to be severely affected by drought and salt stress (Panda, Mishra, and Behera 2021; Reddy et al. 2017).However, there exists variation in tolerance for these stresses in different cultivars (A.Kumar and Dash 2019).Despite the higher levels of similarities within genomes the variability in response to stress conditions may be attributed to the molecular mechanisms involved in regulation of stress responsive genes (Garg et al. 2015).Among these mechanisms, epigenetic regulation being quite dynamic process, has been associated to provide e cient control to enhance plant tolerance towards a particular stress factor (Singroha 2019).
DNA methylation, methylation of cytosine nucleotides to 5-methylcytosine, is one of the most important epigenetic marks in genomes of eukaryotes that plays signi cant role in governing chromatin structure, replication and gene regulation (Sapna et al. 2020).In plants, cytosine methylation occurs at three different types of sequence contexts: CGN, CHG and CHH (where N = A, C, T or G and H = A, C, T).The variation in methylation status of cytosine residues has been documented to be associated with suppression or activation of gene expression, however, this regulation is dependent upon a particular tissue type, sequence context of cytosine residues and the genomic region (Skorupa et al. 2021;Yaish et al. 2018).In this aspect, changes in DNA methylation status within or near CREs in the promoter region have also been suggested to regulate gene expression by directly interfering the interaction with transcription factors (Fei et al. 2017).Additionally, the role of DNA methylation in regulation of gene expression in plants under various abiotic stress conditions has also been reported (Garg et al. 2015;Rajkumar et al. 2020;Probst and Mittelsten Scheid 2015).
Earlier, studies have indicated the effect of drought and salinity in modulating cytosine methylation in various plant species.For example, in rice, 12.1% variation in methylation status was observed in different rice varieties, tissues and growth stages in response to drought stress (Singroha 2019).Another study highlighted the reduced level of methylated cytosine residues in salt treated roots of soybean plant as compared to control samples (Chen et al. 2019).In terms of gene expression, salt-tolerant rice variety Pokkali showed elevated expression of DNA demethylases whereas salt-sensitive IR29 exhibited activation of methyltransferases and demethylases in response to salt stress (Ferreira et al. 2015).
Interestingly, changes in methylation status have been reported to play crucial role in differential regulation of stress responsive genes in several plant species including rice (H.Wang et al. 2016).For instance, salt stress has been reported to decrease the level of methylated cytosine residues in the promoter region of OsMYB91 in rice, which may explain its elevated expression under salt stress leading to stress tolerance (Zhu et al. 2015).
Among various stress responsive genes in plants, GLP genes play a positive role in providing protection against abiotic stress conditions including drought and salt stress.In recent years, many reports have revealed the stress and tissue speci c varied expression of OsGLP genes in response to both stress conditions (Banerjee, Gantait, and Maiti 2017; L. Li et al. 2016;Anum et al. 2022).The rice genome consists of 43 OsGLP genes distributed among different chromosomes (1, 2, 3, 4, 5, 8, 9, 11 and 12) with highest number on chromosome number 3, 8 and 12 in the form of clusters (Das et al. 2019).Several OsGLP genes particularly from chromosome 3, 8 and 4 have been indicated to show varied expression in response to drought and salt stress in different rice cultivars.In this aspect, OsGLP4-1 and some OsGLP genes from chromosome number 3 and 8 were observed with modulated expression upon exposure to drought and salt stress in different rice cultivars (L.Li et al. 2016;Anum et al. 2022).Interestingly, detailed analysis of transcriptomic data on OsGLP genes has also revealed that OsGLPs genes from same chromosome may exhibit variation in their regulation depending upon a particular cultivar, tissue or stress treatment.For instance, OsGLP8-10 showed down-regulation in roots of both tolerant KS282 and sensitive Super Basmati rice variety only under drought stress whereas OsGLP8-12 was observed with upregulation under both drought and salt stress only in roots of Super Basmati (Anum et al. 2022).Despite of the high sequence similarity in the gene regions of OsGLP genes, differential expression of OsGLP genes from same chromosome indicates the possible role of variation in promoter region (Mahmood et al. 2010) and epigenetic effect such as DNA methylation in regulation of their expression.However, currently no report is available regarding the DNA methylation analysis in promoter region of OsGLP genes in contrasting rice cultivars under drought and salt stress.
In this study, we explored the role cytosine methylation pattern in the promoter region of 3 OsGLP genes (OsGLP4-1, OsGLP8-10 and OsGLP8-12) from chromosome number 4 and 8 in leaves and roots of two important rice cultivars: tolerant KS282 and sensitive Super Basmati under control, drought and salinity.
To achieve this, we rst analyzed the impact of drought and salt stress on DNA methylation in promoter regions of selected OsGLP genes as well as compared the differential methylation status of these promoter regions in two contrasting rice cultivars.Second, we investigated the tissue and stress speci c potential role of differentially methylated sites in regulation of OsGLP genes by identifying CREs involved in gene regulation and abiotic stress response in selected promoter regions.We found several differentially methylated sites within and near identi ed CREs suggesting the association between promoter methylation and regulation of OsGLP genes under both stresses.This is the rst study reporting the insightful information about the DNA methylation status of GLPs.The ndings can pave the way for understanding the regulatory mechanisms of epigenetic marks in contrasting rice cultivars and can be utilized for enhancing the associated stress resistant traits in rice.

Plant material
Two rice cultivars with contrasting response speci cally to salt stress (Ijaz et al. 2019) were utilized as plant material, including tolerant KS282 and sensitive Super Basmati.Seeds from both varieties were de husked and surface sterilized (Anum et al. 2022).

Growth Conditions And Stress Treatments
The seeds were grown on Murashige and Skoog (MS) media in test tubes under 16 h photoperiod in a growth room (temperature 25°C ± 1) for two weeks.Two weeks old seedlings were randomly grouped for control, salinity and drought conditions (3 replicates/group with 15 plant/replicate).Drought stress was given by placing seedlings on dried lter paper for 24 h and seedling were treated with salinity stress by keeping them in 200 mM NaCl solution for 24 h (L.Li et al. 2016).The leaves and roots for stress treated seedlings of both rice varieties were harvested, snap frozen and kept at -80°C for extraction of genomic DNA.

Isolation And Bisul te Conversion Of Genomic Dna
Genomic DNA was isolated from control and treated leaves and roots of KS282 and Super Basmati using DNeasy Plant Mini Kit (Qiagen, Germany) as per manufacturer's instructions.DNA concentration and quality was determined by using NanoDrop™ 1000 Spectrophotometer (Thermo Scientifc) and agrose gel electrophoresis.The extracted DNA was stored at -20°C for downstream processes.Un-methylated cytosine bases in isolated genomic DNA from control and treated samples were modi ed to uracil by sodium bisul te conversion using EpiTect Bisul te Kit (Qiagen, Germany) following manufacturer's instructions.

Primer Designing And Optimization
To perform methylation analysis in promoter region of three OsGLP genes (OsGLP4-1, OsGLP8-10 and OsGLP 8-12), bisul te sequencing was performed with bisul te converted DNA from control and treated samples of leaves and roots of both varieties.For this purpose, bisul te sequencing primers were designed for the selected promoter region for each OsGLP gene using MethPrimer software (L.C. Li and Dahiya 2002; Manning et al. 2006) (Online Resource 1).To obtain the best results for bisul te sequencing, primers were selected with a size of 24 to 26 nucleotides and product length of less than 500 bp (303 bp for OsGLP4-1, 358 bp for OsGLP8-10 and 259 bp for OsGLP 8-12).Designed primers were optimized with melting temperatures ranging between 52 to 58°C.

Pcr Ampli cation And Puri cation For Bisul te Sequencing
Bisul te converted DNA was utilized as template for PCR ampli cation of targeted regions with bisul te sequencing primers using HotStar Taq Polymerase (Qiagen, Germany) on a GeneAmp PCR System 9700 (Applied Biosystems).The PCR reaction mixture (for one reaction) was prepared as follows: 2.5 µl 10x PCR buffer, 0.5 µl dNTP mix, 1 µl of each speci c bisul te primer, 0.125 µl Hotstar Taq polymerase, 2 µl bisul te converted DNA and H 2 O to make nal volume of 25 µl.Following temperature pro le was used for ampli cation: initial activation step (15 min 95°C), 35 cycles (1 min 94°C; 1 min at optimal annealing temperature for each speci c primer; 1 min 72°C) and nal extension step (10 min 72°C).Puri cation of PCR products was performed by GeneJET PCR Puri cation Kit (Thermo Scienti c) according to manufacturer's instructions and well-puri ed PCR products from all control and treated samples with each speci c primer were sequenced commercially.

Data analysis
For investigation of methylation, analysis of bisul te sequencing data was performed by determining cytosine methylation status for all three sequence contexts: symmetrical CGN and CHG as well as nonsymmetrical CHH by using CyMATE software (Karan et al. 2012;Hetzl et al. 2007).Further, comparative analysis of all bisul te sequences was accomplished by aligning the sequences for converted samples with reference sequence (for each OsGLP gene targeted promoter region) by ClustalX and BioEdit (S.Kumar, Singh, and Awana 2018).PLACE and PlantCARE were utilized for identi cation and analysis of CREs in promoter regions of OsGLP genes (Fei et al. 2017).

Methylation analysis of selected promoter regions of OsGLP genes under control condition
To analyze the cytosine methylation status in the promoter regions of OsGLP genes in control leaves and roots samples of KS282 and Super Basmati, bisul te sequencing was performed in selected promoter regions of 3 OsGLP genes (OsGLP4-1, OsGLP8-10 and OsGLP8-12).Promoter region was selected within 1kb upstream region of transcription start site for OsGLP4-1 (from − 153 to -455 bp), OsGLP8-10 (from − 436 to -794 bp) and OsGLP8-12 (from − 528 to -787 bp).As a result of methylation analysis in control samples using CyMATE software, among three OsGLP genes, highest number of methylated cytosine residues was identi ed in OsGLP8-12 whereas no methylated cytosine residues were identi ed in promoter regions of OsGLP4-1 and OsGLP8-10 in both rice varieties (Fig. 1a).However, the level of methylation was found to be different depending upon CGN, CHG and CHH sequence context as well as rice varieties.Overall, the level of methylation was found to be higher in CGN context than CHG and CHH contexts, In this aspect, in both leaves and roots, promoter region of OsGLP8-12 exhibited 90.90% methylated CGN positions and 9.09% CHG positions in KS282 whereas 78.6% methylated CGN positions and 21.4% CHG positions were observed in Super Basmati (Fig. 1b).Interestingly, no methylated residues were observed at CHH position in any analyzed promoter of the OsGLP genes from control sample of both varieties.

Drought induced differential methylation patterns in promoter regions of OsGLP in leaves and roots of rice varieties
To investigate the location, class and methylation status of cytosine residue in the selected promoter region of OsGLP genes in response to drought stress, comparative methylation analysis of bisul te sequences was performed in promoter regions of 3 OsGLP genes (OsGLP4-1, OsGLP8-10 and OsGLP8-12) in leaves and roots of KS282 and Super Basmati under control and drought conditions by utilizing cytosine methylation map generated by CyMATE and multiple sequence alignment by CLUSTALX/BioEdit.For promoter region of OsGLP4-1, overall analysis indicated no differentially methylated sites in any of the bisul te converted sequences from control and drought treated leaves and roots of KS282 and Super Basmati (Fig. 2, Online Resource 2).However, the analyzed promoter segment of OsGLP 8-10 was observed with differentially methylated sites in roots of KS282 and Super Basmati under drought stress with KS282 exhibiting higher methylation rate (22.22%) at CGN positions as compared to Super Basmati (11.11%) (Fig. 3, Online Resource 3).Among 3 OsGLP genes, bisul te sequence of OsGLP8-12 promoter that was observed with most methylated sites in both KS282 and Super Basmati exhibited differentially methylated sites with respect to a particular variety, tissue and stress type.For example, in control samples, 23.40% methylated cytosine residues in KS282 and 29.17% in Super Basmati were observed with differential methylation status at CHG position whereas single CGN site was found with differential methylation status (de-methylation) in roots of Super Basmati under drought stress (Fig. 4, Online Resource 4).

Salt induced differential methylation patterns in promoter regions of OsGLP in leaves and roots of rice varieties
To understand the impact of salt stress on methylation pattern in promoter regions of OsGLP genes, bisul te sequences of promoter regions of 3 OsGLP genes (OsGLP4-1, OsGLP8-10 and OsGLP8-12) were analyzed in control and salt treated leaves and roots of KS282 and Super Basmati by utilizing cytosine methylation map generated by CyMATE and multiple sequence alignment by CLUSTALX/BioEdit.Similar to drought stress, the comparative methylation map and multiple sequence alignment of control and salt treated samples from both rice varieties showed no differentially methylated positions in the bisul te converted selected promoter region of OsGLP4-1 (Fig. 5, Online Resource 5).However, in contrast to drought stress, salt stress induced no change in methylation status of cytosine residues in the selected promoter segment of OsGLP8-10 and all of the possible methylation sites were found to be unmethylated in leaves and roots samples of both rice varieties under control and salt stress conditions (Fig. 6, Online Resource 6).The comparative analysis of bisul te converted sequences of OsGLP8-12 promoter from control and salt treated samples revealed the methylation patterns similar to drought treated samples with single but same salt stress induced de-methylated CGN site in roots of Super Basmati.Interestingly, in addition to differentially methylated sites, OsGLP8-12 promoter sequence from KS282 and Super Basmati showed difference in their nucleotide sequence at two different sites where cytosine was replaced with adenine at one mutated site and guanine was replaced with adenine at the other site in KS282 while master sequence and sequences from Super Basmati shared same sequence (Fig. 7, Online Resource 7).

Stress induced cytosine methylation changes around CREs in promoter regions of OsGLP genes
To better understand the importance of modulated methylation patterns at speci c cytosine residues on the functionality of selected genes with respect to a particular stress treatment, the location and methylation status, analysis of CREs was performed in the promoter regions of OsGLP genes.Selected promoter sequences for OsGLP4-1, OsGLP8-10 and OsGLP8-12 were analyzed in detail by PLACE and PlantCare, and signi cant CREs for abiotic stress and transcription regulation were identi ed.For instance, the analyzed sequence of OsGLP4-1 consisted of various CREs mainly including MYB, MYC, Wbox, CTCC-motif, TATA-Box and CAAT-box.However, all the possible sites of methylation were found to be un-methylated within or around these CREs (Fig. 8).Furthermore, several important CREs (MYB, W-box, GT1-motif, ABRE/ACGT, CTCC-motif, TATA-Box and CAAT-box) were also determined in promoter region of OsGLP8-10 and one of the two differentially methylated sites (identi ed in bisul te sequence analysis) was found in close proximity with W-box CTCC, MYB and GT1 motif while the other site was observed near GT1 motif and CAAT-box (Fig. 9).Characterization of CREs in the promoter region of OsGLP8-12 also indicated the presence of MYB, W-box, LTR, CTCC-motif, CAAT-box, TATA-box and ABRE/ACGT-motif and several methylated and differentially methylated sites were observed near or within these CREs.Investigation of differentially methylated sites with respect to CREs showed that one of the differentially methylated sites at CHG position was present nearly after CTCC-motif, MYB and W-box motifs indicating that these motifs were followed by un-methylated cytosine in KS282 while methylated cytosine in Super Basmati in all samples.In addition to this, single site that was differentially methylated in response to salt and drought stress (de-methylated in roots of Super Basmati under salt and drought stress) was identi ed within ABRE/ACGT-motif.Interestingly, one of the two mutated sites observed in OsGLP8-12 promoter sequence in KS282 was determined near this differentially methylated CRE motif (Fig. 10).

Discussion
Evidences are growing in favor of DNA methylation at cytosine residues in uencing the regulation of stress responsive genes to generate plant response against various environmental abiotic stress conditions including drought and salinity.However, the role of modulated methylation status of cytosine residues in regulating the expression of stress responsive GLP genes under drought and salt conditions has not been documented.In current study, epigenetic consequences of salt and drought stress on cytosine methylation dynamics of promoter regions of OsGLP genes have been assessed in two contrasting rice varieties.The leaves and roots speci c methylation pattern in promoter regions of 3 OsGLP genes (OsGLP4-1, OsGLP8-10 and OsGLP8-12) was analyzed in tolerant KS282 and sensitive Super Basmati under both stress treatments.Among 3 OsGLP genes, comparison of cytosine DNA methylation sites (CGN, CHG and CHH) in control leaves and roots samples indicated higher methylation density in the promoter region of OsGLP8-12 in both rice varieties.Importantly, the bisul te sequence of OsGLP8-12 exhibited site-dependent differential methylation level at three sequence contexts (CGN > CHG > CHH) in KS282 and Super Basmati.Similar to this study, higher methylation levels in CGN context have also been reported in earlier studies performed on different genes in rice and other plants (Garg et al. 2015;Hossain et al. 2017;Cokus et al. 2008).One of the major reasons for high level of methylation at CGN and CHG sites may be due to the maintenance of methylation status in these sequence contexts which leads to the methylation of newly replicated DNA strands (Rajkumar et al. 2020).Additionally, although same methylation pattern was observed in KS282 and Super Basmati, however, the level of methylation at each sequence context was found to be dependent upon a particular rice variety indicating the differential role of DNA methylation in two different rice cultivars.
Drought stress has been reported to cause major variations in expression of abiotic stress responsive genes including OsGLP genes.In our recent work OsGLP4-1, OsGLP8-10 and OsGLP8-12 have been shown to exhibit modulated expression under drought stress in leaves and roots of KS282 and Super Basmati (Anum et al. 2022).Interestingly, in current study, analysis of promoters of these OsGLP genes for altered DNA methylation pattern against drought stress indicated few differentially methylated sites in the analyzed regions depending upon a particular tissue and rice variety.OsGLP8-10 that was least expressed in drought treated roots of KS282 and Super Basmati was observed with methylated sites in its promoter region in roots under drought stress with varied level of methylation in both cultivars.Furthermore, OsGLP8-12 that was previously reported to be up-regulated only in roots of Super Basmati under drought stress (Anum et al. 2022) was observed with un-methylated CGN site in its promoter region in drought treated roots.A study on whole genome methylome showed that promoter methylation seemed to be associated with tissue speci c gene expression in plants (Surdonja et al. 2017).Moreover, in accordance with our study, drought stress has been documented to induce cultivar and site speci c alteration in methylation status of DNA in two contrasting rice varieties with an increased differential methylation in promoters of abiotic stress responsive genes including drought stress (Sapna et al. 2020; W. S. Wang et al. 2011;Zheng et al. 2013).In view of previous reports as well as current study it can be suggested that differentially methylated sites in the promoter regions of OsGLP genes may play a role in modulating their cultivar and tissue speci c expression in response to drought stress.
All three OsGLP genes analyzed in this study for their DNA methylation analysis have also previously been identi ed as salt stress responsive in the course of expression analysis (Anum et al. 2022).Analyzing methylation variation revealed minor but cultivar, tissue and site speci c changes in the methylation status in the selected promoter regions of some OsGLP genes.OsGLP8-12 that was reported with elevated expression during transcriptomic analysis only in salt stress treated roots of Super Basmati was detected with de-methylation at CGN sequence context in the same sample in present study indicating the importance of differential methylation at CGN site in salt stress response.Similar to this, a previous report on differential methylation in seedlings of contrasting rice cultivars has also indicated variety and site speci c variation in methylation status with an effect on gene expression suggesting the role of cytosine methylation in salt stress response.However, in contrast to our analysis, major differences in methylation status were reported in CHH context in association with altered expression of stress responsive genes in rice seedling under salt stress (Rajkumar et al. 2020).This may suggest the signi cance of site speci c differential methylation in generating tissue speci c abiotic stress response.In addition to this, several other studies have also revealed the association of salt stress induced removal of cytosine methylation in promoter region of stress related genes with their increased expression in various plant species including rice indicating its role in regulating gene expression under stress conditions (Zhu et al. 2015; Zhang et al. 2020).
Furthermore, the role of CREs present in the promoter regions of stress responsive genes in regulating gene expression by interacting with transcription factors under abiotic stress conditions is well recognized (Bjornson, Dandekar, and Dehesh 2016).In this study, in silico analysis of selected promoter regions of OsGLP genes revealed the presence of CREs involved in gene regulation and abiotic stress response including salt and drought stress.Also, the results identi ed cultivar, tissue and stressdependent differentially methylated cytosine sites within or in close proximity of several identi ed CREs mainly including MYB, W-box and ABRE/ACGT in the promoter regions of OsGLP8-10 and OsGLP8-12.Previously, expression and promoter methylation study of TaGAPC1 gene in wheat also reported its differential expression and differential methylation of cytosine residues on CREs in two wheat cultivars with contrasting drought tolerance (Fei et al. 2017).Although, it has been documented that the binding of a transcription factor can be blocked by the DNA methylation at speci c sites in the promoter sequence, however, there is also a possibility that methylation in the promoter sequence may not affect the binding of transcription factor (Kulakovskiy and Makeev 2009; Skorupa et al. 2021).Overall, differential methylation patterns in the selected promoter regions suggest that altered methylation status of speci c sites within or near CREs can be a crucial regulatory mechanism in modulating the expression of OsGLP genes under drought and salt stress in two contrasting rice varieties.

Conclusion
Present study revealed the differential methylation patterns in promoter regions of OsGLP genes in two contrasting rice cultivars under drought and salinity.Among all three OsGLP genes selected for methylation analysis, promoter region of OsGLP8-12 exhibited most methylated and differentially methylated residues depending upon a particular variety, tissue or stress condition whereas no methylated residues were observed in promoter region of OsGLP4-1 in all samples.Also, several differentially methylated sites in response to stress treatments in selected promoter regions of OsGLP8-10 and OsGLP8-12 were identi ed either near or within stress related cis-regulatory elements indicating the potential role of site speci c alterations in cytosine methylation in regulating response of OsGLP genes to these stress conditions.Overall results suggested that the stress, tissue and cultivars speci c methylation changes in promoter regions may be a key alternative regulatory mechanism in modifying the expression of OsGLP genes for generating drought and salt stress response in rice cultivars.

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Figure 1 Analysis
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