Comprehensive Analyses of Nitric Oxide-Induced Plant Stem Cell-Related Genes in Arabidopsis thaliana

Plant stem cells are pluripotent cells that have diverse applications in regenerative biology and medicine. However, their roles in plant growth and disease resistance are often overlooked. Using high-throughput RNA-seq data, we identified approximately 20 stem cell-related differentially expressed genes (DEGs) that were responsive to the nitric oxide (NO) donor S-nitrosocysteine (CySNO) after six hours of infiltration. Among these DEGs, the highest number of positive correlations (R ≥ 0.8) was observed for CLAVATA3/EMBRYO SURROUNDING REGION-RELATED (CLE) 12. Gene ontology (GO) terms for molecular function showed DEGs associated with signal transduction and receptor activity. A promoter study of these DEGs showed the presence of cis-acting elements that are involved in growth as well as the regulation of abiotic and biotic stress. Phylogenetic analysis of the Arabidopsis stem cell-related genes and their common orthologs in rice, soybean, poplar, and tomato suggested that most soybean stem cell-related genes were grouped with the Arabidopsis CLE type of stem cell genes, while the rice stem cell-related genes were grouped with the Arabidopsis receptor-like proteins. The functional genomic-based characterization of the role of stem cell DEGs showed that under control conditions, the clv1 mutant showed a similar phenotype to that of the wild-type (WT) plants; however, under CySNO-mediated nitrosative stress, clv1 showed increased shoot and root length compared to WT. Furthermore, the inoculation of clv1 with virulent Pst DC3000 showed a resistant phenotype with fewer pathogens growing at early time points. The qRT-PCR validation and correlation with the RNA-seq data showed a Pearson correlation coefficient of >0.8, indicating the significantly high reliability of the RNA-seq analysis.


Introduction
Stem cells are pluripotent cells that have gained the attention of the scientific community in the recent past because of their tremendous applications in the biomedical field, specifically in cancer and organ regeneration. However, it was out of our notice that stem cells are an integral part of our life as they are the ultimate precursors of most of the life-saving commodities such as food, fuel, and the oxygen we breathe [1]. Therefore, plant stem cells are the most important cells affecting human life directly or indirectly. Despite concrete differences in cellular biophysics and the properties between animal and plant cells, the basic concept and mechanism of action of their stem cells is curiously the such as germination [17], flowering time [18], flower development [19], apical dominance [19,20], and auxin-mediated root growth and development [21]. Similarly, the effects of NO on internode elongation and hypocotyl emergence have also been studied. In a study using NO donors, it was observed that NO inhibited hypocotyl growth and enhanced de-etiolation and chlorophyll in Arabidopsis and potato [17]. Reports also suggested the role of NO in the induction of adventitious roots development in cucumber [22], whereas the NO scavenger, 2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), inhibited its formation. Previous studies also suggested the role of NO in maize root elongation [23]. In a recent study, we also discovered the role of the NO-responsive RAPID ALKANIZATION FACTOR (RALF) genes in root growth and development. These genes showed a negative role in growth and development as the ralfl1 and ralfl22 mutants produced longer roots compared to the wild type [24].
The role of NO in plant defense is well explored. Sensing avirulent microbial pathogen activates the production of reactive oxygen intermediates (ROIs) and reactive nitrogen species (RNS), which play important roles in the plant defense system. An avirulent pathogen infection can start a network of inducible defense mechanisms in both local and systemic plant tissues [25]. Concluding all this, NO plays diverse roles in plant growth, development, and defense responses; however, the exact mechanism of growth regulation is yet to be understood. Therefore, the increasing evidence of the production and deleterious effects of RNS along with ROIs is gaining increasing importance day by day, and need to be explored further. In a recent study, using the RNA-seq mediated transcriptomic analysis, we identified global changes in gene expression in response to one of the common NO donors, S-nitrosocysteine (CySNO), and found that NO regulates diverse biological processes [26]. In the current study using the high-throughput RNA-sequencing data, we identified approximately 20 stem cell and stem cell-related genes that showed differential expression to one mM of CySNO. By using both in silico and in vivo approaches, we suggested that the NO-responsive stem cell-related genes are mostly involved in plant growth; however, the promoter analysis suggests their putative roles in plant defense as well. This study might help scientists divert their attention toward creating strategies to use pluripotent stem cells in plant defense.

Transcriptome-Wide Identification and Characterization of Plant Stem Cell Genes
A number of DEGs that showed significant differential expression in response to one mM of CySNO were identified. A detailed list of those DEGs has been reported earlier [26]. Here, we manually searched for the presence of stem cell or stem cell-related genes in the list and found approximately 20 stem cell genes that showed significant (p ≤ 0.05) differential expression to CySNO. The list was carefully checked for any duplicate values. A heatmap with a dendrogram representing hierarchical clustering was generated to visualize the differences in expression values between the treated and control samples using the fragments per kilobase of transcript per million mapped reads (FPKM) values of the control and CySNO-treated samples using R version 3.3.1.R (https://www.r-project.org/). A principal component analysis (PCA) plot was also generated for the same samples to find any dispersion in the data using R.

Correlation and Gene Ontology Analyses
All of the NO-induced stem cell genes were then processed using the Phytomine tool of the Phytozome database (https://phytozome.jgi.doe.gov/pz/portal.html) in order to find the correlations among these genes. We identified both positive and negative correlations, which were visualized with red and blue colors, respectively. To further understand the regulatory role of the NO-induced plant stem cells, an analysis of the associated gene ontology (GO) terms for the biological processes, molecular functions, and cellular components was performed using the web interface of PANTHER version 11.1 (http://pantherdb.org), as described earlier [27]. Briefly, the locus IDs of the 20 differentially expressed NO-induced stem cell-related genes were searched using the appropriate search field, while Arabidopsis thaliana was selected as the organism of choice. The "statistical overrepresentation test" was selected as the method of analysis with default settings. The analyzed data for all the GO terms; that is, GO biological process, molecular function, and cellular location, were observed, and the GO terms with an enrichment p-value < 0.05 were downloaded for each annotation dataset and compiled using Microsoft Excel for quantitative presentation.

Promoter Analysis
Promoters play a key role in the regulation of genes, as they have specific sites for binding transcription factors that have the mechanistic control of gene transcription. Therefore, we sought to predict the regulatory roles of the NO-induced stem cell genes. For this, the promoter sequences of the CySNO-induced stem cell-related genes 1.5 kb upstream of their transcription initiation site were retrieved from The Arabidopsis Information Resource (TAIR) center (https://www.arabidopsis. org/). All of the sequences were analyzed for regulatory elements using the Plant Cis-acting Regulatory Elements (PlantCARE) web interface (http://bioinformatics.psb.ugent.be/webtools/ plantcare/html/) [28]. The identified regulatory elements were manually screened, and only some of those involved in abiotic and biotic stresses were mapped using the Regulatory Sequence Analysis Tool [29].

Phylogenetic Analysis
To determine the evolutionary relationship among the stem cell-related genes from different species, the Arabidopsis stem cell protein sequences were queried against the protein sequences of rice (Oryza sativa), soybean (Glycine max), tomato (Solanum lycopersicum L.), and poplar (Poplar trichocarpa) in the Phytozome database, and only the proteins that were common in all the species were selected. We found approximately 28 orthologs in rice, 21 in soybean, four in tomato, and 15 in poplar. The protein sequences of these proteins were retrieved from Phytozome and aligned using the ClustalW tool; the resulting alignment was then used to generate a phylogenetic tree using the maximum likelihood method based on the Jones Taylor-Thornton (JTT) matrix-based model [30]. All of the positions having less than 95% site coverage were eliminated; that is, fewer than 5% alignment gaps, missing data, and ambiguous bases were allowed at any position. All of the evolutionary analyses were conducted using MEGA7 [31].

Plant Material and Growth Conditions
To further validate the roles of the NO-induced stem cell-related genes in plant growth and defense, we selected the CLAVATA genes for functional genomics study using the reverse genetics approach because of their defining roles in maintaining OC. Among the CLAVATA family genes, CLV1 and CLV3 are considered to be the most important ones, as they are involved in a feedback loop to maintain OC; therefore, we selected CLV1 and CLV3 for downstream analysis. The Arabidopsis thaliana seeds, including the wild-type seeds in Landsberg erecta (Ler-0) and the Columbia 0 background (Col-0) and mutant lines clv1 (CS37, At1G75820) and clv3 (CS8066, At2G27250), were obtained from the Nottingham Arabidopsis Stock Centre (http://arabidopsis.info/BasicForm), and those with the enhanced disease susceptibility 1-2 (eds1-2) gene were kindly provided by Prof. Gary J. Loake, Edinburgh University, United Kingdom. The atgsnor1-3(At5g43940) [32] and cat2 (At4g35090) [33] were used for comparison as sensitive controls for nitrosative and oxidative stresses, respectively. The CLAVATA mutants and eds1-2 were in the Ler genetic background, while the remaining mutants were in the Col-0 background. All of the seeds were sterilized in 50% bleach solution for five minutes, incubated with 1% (v/v) TritonX-100, and rinsed with sterile distilled water three times. Then, the seeds were stratified at 4 • C overnight prior to sowing either on 1 2 Murashige and Skoog (MS) medium or in soil under long-day growth conditions (16 hours of light and eight hours of darkness at 23 ± 2 • C).

Redox Stress Assay
Plants were exposed to oxidative and nitrosative stresses, as described previously [24]. In brief, the plants were grown on 1 2 MS medium supplemented with 0.75 mM of S-nitrosoglutathione (GSNO) and 0.75 mM of S-nitrosocysteine (CysNO) for nitrosative stress and with two mM of H 2 O 2 and one mM of methyl viologen for oxidative stress conditions. The control plants were grown only on 1 2 MS medium. The data on seed germination, cotyledon development frequency (CDF), and root and shoot lengths were recorded at appropriate intervals until 14 days of sowing (unless stated otherwise). The CDF was used for green developed seedlings, and was calculated as described earlier [34].

Pathogen Inoculation and Pathogenicity Assessment
To evaluate the response of CLV genes in basal defense, the wild-type and mutant lines were subjected to virulent Pseudomonas syringe pv. tomato (Pst) strain DC3000 inoculation. The pathogen was maintained and inoculated according to Yun et al. [35]. In short, the pathogen was grown on Luria-Bertani (LB) agar medium supplemented with 100 mg/mL of rifampicin as the antibiotic for selection and incubated at 30 • C overnight. Then, single colonies were then used to inoculate the LB broth that had rifampicin, and it was incubated at 30 • C with shaking overnight. The overnight-grown liquid culture was then centrifuged at 8000 rpm for three minutes, and the bacterial pellet was re-suspended in 10 mM of MgCl 2 solution. The concentration of the cells was adjusted to 5 × 10 5 colony-forming units (CFUs), and the cell suspension was infiltrated into the abaxial side of the leaves. The control inoculant contained 10 mM of MgCl 2 only. The inoculated leaf samples were collected at zero hours to three days post-inoculation for bacterial colony counts and subsequent gene expression analysis.
Plants were assessed phenotypically for any symptom development due to pathogen growth over time and photographed using a digital single-lens reflex (DSLR) Canon camera. For pathogenicity assessment, the leaf discs from the inoculated leaves, which were one cm in diameter, were homogenized in one mL of 10 mM of MgCl 2 solution, and then subjected to serial dilution. The diluted homogenate was spread on the LB medium containing rifampicin and incubated under optimal growth conditions. The log colony-forming unit (CFU) was calculated using the dilution factor and the number of colonies grown.

q-RT PCR and Gene Expression
Leaf samples from the plants inoculated with Pst DC3000 were used to extract RNA using the Trizol ® (Invitrogen, USA) method according to the description by Imran et al. [36]. The RNA was used to synthesize the first strand of cDNA using the reverse transcriptase (RT) kit (BIOFACT, Korea) according to the manufacturer's recommendations. The cDNA that was synthesized was used as a template for gene expression analysis. A 20-µL reaction mixture containing 2X Real-Time PCR Master Mix [(including SYBR ® Green I) BIOFACT, Korea] with 10 nM of each primer was processed in a two-step PCR reaction using the Eco™ real-time PCR (Illumina, USA). A "no-template control" containing nuclease-free water instead of the cDNA template was used as the negative control. The thermal cycling conditions included initial denaturation at 95 • C for 15 minutes, followed by 40 cycles of 95 • C for 10 seconds and 60 • C for 30 seconds. Actin was used as the internal reference gene.
For validating the RNA-seq mediated transcriptional changes in stem cell-related genes, the wild type Col-0 plants were infiltrated with one mM of CySNO, and the samples were collected at zero and six hours post-infiltration. For validation, we selected a few stem cell-related genes that showed at least a fourfold change in gene expression when treated with one mM of CySNO after six hours of infiltration in RNA-seq analysis [26]. RNA extraction and qRT-PCR was performed as described in the above paragraph. The fold change was calculated and correlated with the RNA-seq data using the Pearson correlation coefficient in Microsoft Excel (version 2016 https://www.office.com/). The primers that were used in this study are listed in Supplementary Table S1.

Transcriptome-Wide Identification and Characterization of Stem Cell-Related Genes in Response to Nitric Oxide
To identify stem cell-related genes, we manually searched the list of all the differentially expressed genes (DEGs) in the CySNO-induced transcriptome [26]. We found approximately 20 different stem cell-related genes that showed differential response to one mM of CySNO ( Figure 1A; Supplementary  Table S2). A heatmap representing expression patterns and a dendrogram showing hierarchical clustering among the control and CySNO-treated samples ( Figure 1A) were generated. A PCA plot showing the dispersion in data is also shown in Figure 1B. All of these genes showed at least a two-fold change (Supplementary Table S2). These genes included the Breast Cancer-Associated Ring 1 (BARD1) that particularly binds with H3K4me3 of the target gene (such as WUS). BARD1 is also required for organizing the shoot apical meristem and development of a quiescent center (QC) in the root apical meristem (RAM) [37,38]. Another important DEG was Arabidopsis Response Regulator 7 (ARR7), which helps in the phosphorylation of the aspartic acid residue that induces the protein to activate the transcription of target genes. Similarly, we found MERISTEM DISORGANIZATION 1 (MDO1), which was an uncharacterized protein that is required for the maintenance and organization of meristems by reducing DNA damage [39]. The CLAVATA genes were another important gene class in stem cell DEGs (Supplementary Table S2). The CLV1 protein, which is involved in signal regulation during meristem maintenance and regulates the balance between meristem cell proliferation and differentiation [40], and the CLV2 protein, which is a receptor-like protein involved in the perception of CLV3 and mediates organ and meristem development in Arabidopsis [41], showed response to the nitric oxide (NO) donor CySNO (Supplementary Table S2). We also found the CLE family members that are mostly involved in regulating root meristem maintenance. Among others were SCARECROW, which is a transcription factor that is required for the asymmetrical cell division of the cortex [42]; WUSCHEL-related homebox 13 (WOX13); APETALA 2 (AP2), which is required for early floral meristem identification and subsequently the transition of inflorescence meristem into floral meristem [43,44]; CORYNE (CRN), which is supposed to be involved in the CLV3 peptide in addition to the modulation of root, shoot, and flower meristem organization [45]; and FANTASTIC FOUR 3 (FAF3), which that can repress WUS and influence shoot meristem size [46] (Supplementary Table S2).
Interestingly, approximately 75% of the CySNO-induced stem cell-related genes were down-regulated. We further sought to determine a correlation among these genes, and selected the ones with high correlation coefficient factors (R ≥0.8). We found that the highest number of positive correlations were for AT1G68795 (CLE12), which had positive correlations (R >0.8 unless stated otherwise) with 15 other genes, and negative correlations with three genes ( Figure 1C). This was followed by AT5G65700 (BAM1), which had 14 positive correlations with other genes ( Figure 1C). Similarly, the lowest positive correlations were found for AT5G43820 (ZLL), which encodes Argonaute10 and showed only one positive correlation. Furthermore, the highest number of negative correlations was found for AT3G54220, which regulates the radial organization of the root, and for AT5G64770 (RGF9), which encodes a root meristem growth factor; these each had negative correlations with 11 genes ( Figure 1C).
We further analyzed all 20 NO-responsive stem cell-related genes for the GO terms of biological processes, molecular function, and cellular components. In the GO terms for biological processes, we only found an association with one GO term, which was response to endogenous stimulus ( Figure 1D). Similarly, in the GO terms for molecular functions, the highest category in terms of fold enrichment (p-value = 0.0000655) was signal transducer activity. Another category studied was the GO terms for cellular components, in which the majority of genes were associated with plasma membrane, while the highest fold enrichment was found for cell junction ( Figure 1D).

Promoter Anlaysis for Identification of Cis-Regulatory Elements
Cis-regulatory elements are non-coding DNA sequences in the promoter of a gene that serve as potential binding sites of other regulatory proteins such as transcription factors (TFs) [47]. The presence of particular cis-regulatory elements in the promoter can predict the function of a gene, as these elements are reported to play a key role in the mechanistic control of the transcription of these genes [27,48]. Therefore, we were prompted to determine the cis-regulatory elements in the promoters of the CySNO-induced stem cell-related genes. For this, we analyzed the promoter region

Promoter Anlaysis for Identification of Cis-Regulatory Elements
Cis-regulatory elements are non-coding DNA sequences in the promoter of a gene that serve as potential binding sites of other regulatory proteins such as transcription factors (TFs) [47]. The presence of particular cis-regulatory elements in the promoter can predict the function of a gene, as these elements are reported to play a key role in the mechanistic control of the transcription of these genes [27,48]. Therefore, we were prompted to determine the cis-regulatory elements in the promoters of the CySNO-induced stem cell-related genes. For this, we analyzed the promoter region one Kb upstream of the transcription initiation site. About 14 cis-regulatory elements that were involved in growth and abiotic or biotic stress conditions were found. These elements included the CAT-box in 100% sequences; ARE in 90% sequences; CGTCA-motif in 65% sequences; STRE in 45% sequences; ERE in 40% sequences; GARE, W-box, and MBS in 35% sequences, A-box, ABRE, and AT-rich motif in 10% sequences, and GATA, EIRE, and OCS in 5% sequences ( Figure 2). one Kb upstream of the transcription initiation site. About 14 cis-regulatory elements that were involved in growth and abiotic or biotic stress conditions were found. These elements included the CAT-box in 100% sequences; ARE in 90% sequences; CGTCA-motif in 65% sequences; STRE in 45% sequences; ERE in 40% sequences; GARE, W-box, and MBS in 35% sequences, A-box, ABRE, and ATrich motif in 10% sequences, and GATA, EIRE, and OCS in 5% sequences ( Figure 2).

Figure 2.
Promoter analysis of the CySNO-induced stem cell-related genes. Promoter sequences one Kb upstream of the transcription initiation site ATG were retrieved from The Arabidopsis Information Resource (TAIR; https://www.arabidopsis.org/) and analyzed for the identification of cis-regulatory elements. Only those cis-acting elements that were involved in biotic and/or abiotic stress conditions were mapped here.

Phylogenetic Analsysis of Arabidopsis Stem Cells and their Orthologs in other Species
We were further interested in checking whether the selected stem cell-related genes were evolutionary conserved. Therefore, we retrieved the protein sequences of the stem cell-related genes from Arabidopsis, rice, soybean, tomato, and poplar, and analyzed them through MEGA 7.0 [31]. We found that most of the soybean and populus stem cell-related proteins were grouped together with the Arabidopsis CLE type of stem cells proteins; however, some of the soybean stem cell proteins were also grouped with the Arabidopsis receptor-like kinases such as BAM1 (At5g65700), CLV1 (At1g75820), and CLV2 (At1g65380) (Figure 3). Furthermore, the majority of rice stem cell-related proteins were grouped together with the Arabidopsis receptor-like kinase type of stem cell proteins ( Figure 3). Tomato had the least number of stem cell-related proteins homologous to the Arabidopsis stem cellrelated proteins, and they were grouped together with the Arabidopsis receptor-like kinase type of stem cell proteins (Figure 3).

Phylogenetic Analsysis of Arabidopsis Stem Cells and their Orthologs in Other Species
We were further interested in checking whether the selected stem cell-related genes were evolutionary conserved. Therefore, we retrieved the protein sequences of the stem cell-related genes from Arabidopsis, rice, soybean, tomato, and poplar, and analyzed them through MEGA 7.0 [31]. We found that most of the soybean and populus stem cell-related proteins were grouped together with the Arabidopsis CLE type of stem cells proteins; however, some of the soybean stem cell proteins were also grouped with the Arabidopsis receptor-like kinases such as BAM1 (At5g65700), CLV1 (At1g75820), and CLV2 (At1g65380) (Figure 3). Furthermore, the majority of rice stem cell-related proteins were grouped together with the Arabidopsis receptor-like kinase type of stem cell proteins ( Figure 3). Tomato had the least number of stem cell-related proteins homologous to the Arabidopsis stem cell-related proteins, and they were grouped together with the Arabidopsis receptor-like kinase type of stem cell proteins (Figure 3).
Genes 2019, 10, x FOR PEER REVIEW 9 of 19 Figure 3. Molecular phylogenetic analysis of stem cell-related proteins. Analysis was performed using the stem cell-related protein sequences from Arabidopsis, rice, soybean, tomato, and poplar. The evolutionary history was inferred by using the maximum likelihood method based on the JTT (Jones Taylor-Thornton) matrix-based model [1]. The tree with the highest log likelihood (-34197.0000) is shown. The initial tree(s) for the heuristic search were obtained automatically by applying the neighbor-join (NJ) and BioNJ algorithms to a matrix of pairwise distances estimated using the JTT model, and then selecting the topology with a superior log likelihood value. The analysis involved 97 amino acid sequences. All of the positions with less than 95% site coverage were eliminated; that is, fewer than 5% alignment gaps, missing data, and ambiguous bases were allowed at any position, leaving only three positions in the final dataset. The evolutionary analyses were conducted in MEGA7 [2].

Interactome of CySNO-Induced Stem Cell-Related Genes
Gene interaction is the key to understanding the functional relationships among genes or their corresponding gene products [49]. Therefore, we sought to determine the interaction of the CySNOinduced stem-cell related genes. Both experimentally confirmed and inferred interactions were studied using the Search Tool for the Retrieval of Interacting Genes/Proteins [STRING (https://stringdb.org/)]. We found interesting interactions, specifically for the clavata protiens. The Clavata 1 (CLV1) proteins were found to interact with CLV2, BAM2, BAM1, and CRN. These interactions were predicted from the curated as well as experimentally proven databases ( Figure 4A). Both BAM1 and Figure 3. Molecular phylogenetic analysis of stem cell-related proteins. Analysis was performed using the stem cell-related protein sequences from Arabidopsis, rice, soybean, tomato, and poplar. The evolutionary history was inferred by using the maximum likelihood method based on the JTT (Jones Taylor-Thornton) matrix-based model [1]. The tree with the highest log likelihood (-34197.0000) is shown. The initial tree(s) for the heuristic search were obtained automatically by applying the neighbor-join (NJ) and BioNJ algorithms to a matrix of pairwise distances estimated using the JTT model, and then selecting the topology with a superior log likelihood value. The analysis involved 97 amino acid sequences. All of the positions with less than 95% site coverage were eliminated; that is, fewer than 5% alignment gaps, missing data, and ambiguous bases were allowed at any position, leaving only three positions in the final dataset. The evolutionary analyses were conducted in MEGA7 [2].

Interactome of CySNO-Induced Stem Cell-Related Genes
Gene interaction is the key to understanding the functional relationships among genes or their corresponding gene products [49]. Therefore, we sought to determine the interaction of the CySNO-induced stem-cell related genes. Both experimentally confirmed and inferred interactions were studied using the Search Tool for the Retrieval of Interacting Genes/Proteins [STRING (https://string-db.org/)]. We found interesting interactions, specifically for the clavata protiens. The Clavata 1 (CLV1) proteins were found to interact with CLV2, BAM2, BAM1, and CRN. These interactions were predicted from the curated as well as experimentally proven databases ( Figure 4A). Both BAM1 and BAM2 seem to be involved in a feedback loop regulating ERL2 that further regulates inflorescence and organ shape organization, including stomatal patterning ( Figure 4A). The CLAVATA stem cell-related genes mostly work in coordination with hormonal pathways involved in growth and development, such as auxin and cytokinin. Previous reports suggested that the induction of CLV1 and CLV3 blocks WUS, which induces CLV3 for meristem differentiation [9]. Furthermore, WUS positively regulates SHOOTMERISTEMLESS, which promotes cytokinin (CK) synthesis. High auxin levels can inhibit ARABIDOPSIS RESPONSE REGULATOR (ARR), resulting in the high levels of CK ( Figure 4B). Thus, the complex coordination of genes is required to maintain meristem integrity and stability.
Genes 2019, 10, x FOR PEER REVIEW 10 of 19 BAM2 seem to be involved in a feedback loop regulating ERL2 that further regulates inflorescence and organ shape organization, including stomatal patterning ( Figure 4A). The CLAVATA stem cellrelated genes mostly work in coordination with hormonal pathways involved in growth and development, such as auxin and cytokinin. Previous reports suggested that the induction of CLV1 and CLV3 blocks WUS, which induces CLV3 for meristem differentiation [9]. Furthermore, WUS positively regulates SHOOTMERISTEMLESS, which promotes cytokinin (CK) synthesis. High auxin levels can inhibit ARABIDOPSIS RESPONSE REGULATOR (ARR), resulting in the high levels of CK ( Figure 4B). Thus, the complex coordination of genes is required to maintain meristem integrity and stability.

CLV1 and CLV3 Differentially Regulate Nitrosative and Oxidative Stresses
The effect of redox-active molecules, such as NO and H2O2, on stem cells has not been investigated so far. Therefore, we were tempted to test the phenotypic performance of the Arabidopsis clv1 and clv3 mutant plants grown on the media supplemented with either CySNO and GSNO (for nitrosative stress) or H2O2 and MV (for oxidative stress), and obtained interesting results. First, the response of clv1 and clv3 to nitrosative as well as oxidative stress was significantly different from each other, as clv3 was more resistant to the effects of CySNO and H2O2, but highly susceptible to GSNO and MV compared to clv1, which showed a completely opposite response, as indicated by the CDF measurements recorded after seven days of stress ( Figure 5A,B). More interestingly, the resistant response of clv3 and susceptible response of clv1 was significantly higher than the known response of the atgsnor1-3 plants. The same pattern of CDF was observed after 14 days of stress ( Figure 5A,B). Although the germination percentage of clv1 was similar to that of clv3 on the CySNO media, a significantly higher number of clv1 seeds germinated on the GSNO media, and less germinated on the media supplemented with H2O2 and MV compared to the clv3 seeds after four days of sowing ( Figure 5E,F). Although the root lengths of clv1 and clv3 plants were inherently shorter than those of

CLV1 and CLV3 Differentially Regulate Nitrosative and Oxidative Stresses
The effect of redox-active molecules, such as NO and H 2 O 2 , on stem cells has not been investigated so far. Therefore, we were tempted to test the phenotypic performance of the Arabidopsis clv1 and clv3 mutant plants grown on the media supplemented with either CySNO and GSNO (for nitrosative stress) or H 2 O 2 and MV (for oxidative stress), and obtained interesting results. First, the response of clv1 and clv3 to nitrosative as well as oxidative stress was significantly different from each other, as clv3 was more resistant to the effects of CySNO and H 2 O 2 , but highly susceptible to GSNO and MV compared to clv1, which showed a completely opposite response, as indicated by the CDF measurements recorded after seven days of stress ( Figure 5A,B). More interestingly, the resistant response of clv3 and susceptible response of clv1 was significantly higher than the known response of the atgsnor1-3 plants. The same pattern of CDF was observed after 14 days of stress ( Figure 5A,B). Although the germination percentage of clv1 was similar to that of clv3 on the CySNO media, a significantly higher number of clv1 seeds germinated on the GSNO media, and less germinated on the media supplemented with H 2 O 2 and MV compared to the clv3 seeds after four days of sowing ( Figure 5E,F). Although the root lengths of clv1 and clv3 plants were inherently shorter than those of the WT Ler plants grown on control media, the root lengths of both the mutants were significantly reduced on both nitrosative and oxidative stress media ( Figure 5G,H). On the other hand, the shoot lengths of the clv1 and clv3 plants were significantly higher than those of the WT Ler plants grown on the stress media. Especially, the clv1 plants produced significantly longer shoots on the media supplemented with CySNO, GSNO, H 2 O 2 , and MV ( Figure 5I,J). Another interesting result was the completely different response of the WT Ler and Col-0 ecotypes to all stress media, wherein the Ler plants were found to be generally more resistant than the Col-0 plants ( Figure 5). Furthermore, the different responses of the clv1 and clv3 plants to different nitrosative and oxidative stress donors suggest a significant redundancy in the function of clv1 and clv3, as well as the different involvement of these two genes in the perception of different types of stresses. the WT Ler plants grown on control media, the root lengths of both the mutants were significantly reduced on both nitrosative and oxidative stress media ( Figure 5G,H). On the other hand, the shoot lengths of the clv1 and clv3 plants were significantly higher than those of the WT Ler plants grown on the stress media. Especially, the clv1 plants produced significantly longer shoots on the media supplemented with CySNO, GSNO, H2O2, and MV ( Figure 5I,J). Another interesting result was the completely different response of the WT Ler and Col-0 ecotypes to all stress media, wherein the Ler plants were found to be generally more resistant than the Col-0 plants ( Figure 5). Furthermore, the different responses of the clv1 and clv3 plants to different nitrosative and oxidative stress donors suggest a significant redundancy in the function of clv1 and clv3, as well as the different involvement of these two genes in the perception of different types of stresses.

CLV1 and CLV3 Positively Regulates Basal Defense at Early Time Points
The characteristic phenotypic aberrations were observed for both these lines (atclv1 and atclv3) under normal conditions as previously described by Clark et al. [50] and Koornneef et al. [51]. As these knock-out (KO) lines were in the Landsberg (Ler) background, the WT and eds1-2 Ler plants were used as comparative controls. The results of the pathogenicity test showed that although both KO lines showed disease symptoms, they were significantly resistant to the Pst DC3000 infection compared to the WT and eds1-2 Ler plants, as indicated by the symptoms and colony counts after one and two days of inoculation. However, the bacterial CFU counts in both clv mutants after three days were slightly higher than those in the WT plants ( Figure 6A,B). Interestingly, the qRT-PCR results showed that the expression of PR1 and PR2 did not correlate with the colony counts, as the expression of both these genes in the clv1 and clv3 plants was significantly lower that those in the WT plants after one, two, and three days of the PstDC3000 inoculation ( Figure 6C,D). This suggests that the resistance of the clv mutants to PstDC3000 is either upstream or independent of PR1 and PR2 gene expression. media supplemented with either CySNO or S-nitrosoglutathione (GSNO) for nitrosative stress conditions and with H2O2 and Methyl Viologen (MV) for oxidative stress conditions. (A) Cotyledon development frequency (CDF) of the indicated genotypes one week after sowing under nitrosative stress and (B) oxidative stress conditions. (C) CDF after two weeks of sowing under oxidative and (D) nitrosative stress conditions. (E) Percentage germination of the indicated genotypes under nitrosative and (F) oxidative stress conditions. (G) Root lengths of the indicated genotypes after two weeks under nitrosative and (H) oxidative stress conditions. (I) Shoot length under nitrosative and (J) oxidative stress conditions. All data points are the mean of at least three replicates, and experiments were repeated twice with almost similar results. The error bar represents the standard error (±SE). The Y-axis in panel 'h' was interrupted to clarify lower values.

CLV1 and CLV3 Positively Regulates Basal Defense at Early Time Points
The characteristic phenotypic aberrations were observed for both these lines (atclv1 and atclv3) under normal conditions as previously described by Clark et al. [50] and Koornneef et al. [51]. As these knock-out (KO) lines were in the Landsberg (Ler) background, the WT and eds1-2 Ler plants were used as comparative controls. The results of the pathogenicity test showed that although both KO lines showed disease symptoms, they were significantly resistant to the Pst DC3000 infection compared to the WT and eds1-2 Ler plants, as indicated by the symptoms and colony counts after one and two days of inoculation. However, the bacterial CFU counts in both clv mutants after three days were slightly higher than those in the WT plants ( Figure 6A,B). Interestingly, the qRT-PCR results showed that the expression of PR1 and PR2 did not correlate with the colony counts, as the expression of both these genes in the clv1 and clv3 plants was significantly lower that those in the WT plants after one, two, and three days of the PstDC3000 inoculation ( Figure 6C,D). This suggests that the resistance of the clv mutants to PstDC3000 is either upstream or independent of PR1 and PR2 gene expression.  All data points are the means of the three replicates, and error bars represents ± standard error (SE) (n = 3). The background in panel 'a' was modified for more clarity, while the Y-axis in C was interrupted to visualize smaller values.

qRT-PCR Validation of RNA-Seq Data
We further validated the transcriptional changes in the stem cell-related genes by selecting the representative genes and analyzing them through qRT-PCR. These genes were selected based on the fold change in their expression levels in response to treatment with one mM of CySNO. As the majority of the genes (75%) were down-regulated; therefore, we selected more down-regulated genes for validation analysis. We observed that out of the seven studied genes, six showed the same pattern as the RNA-seq results, except for the CLV2 gene that was down-regulated in the RNA-seq results and up-regulated in the qRT-PCR analysis. We compared the datasets from both RNA-seq and qRT-PCR analyses by measuring the Pearson correlation coefficient and a found higher correlation (R = 0.865), indicating higher similarity among the two datasets (Figure 7). replicates, and error bars represents ± standard error (SE) (n = 3). The background in panel 'a' was modified for more clarity, while the Y-axis in C was interrupted to visualize smaller values.

qRT-PCR Validation of RNA-Seq Data
We further validated the transcriptional changes in the stem cell-related genes by selecting the representative genes and analyzing them through qRT-PCR. These genes were selected based on the fold change in their expression levels in response to treatment with one mM of CySNO. As the majority of the genes (75%) were down-regulated; therefore, we selected more down-regulated genes for validation analysis. We observed that out of the seven studied genes, six showed the same pattern as the RNA-seq results, except for the CLV2 gene that was down-regulated in the RNA-seq results and up-regulated in the qRT-PCR analysis. We compared the datasets from both RNA-seq and qRT-PCR analyses by measuring the Pearson correlation coefficient and a found higher correlation (R = 0.865), indicating higher similarity among the two datasets (Figure 7).

Figure 7.
Validation of the RNA-seq analysis using qRT-PCR. Among the CySNO-induced stem cell-related genes, six were selected based on the fold change (Fold change >4) in their expression levels and analyzed through qRT-PCR. The transcript accumulation of these genes was compared with the RNA-seq data, and the Pearson correlation coefficient was calculated using Microsoft Excel. R represents the Pearson correlation coefficient. All of the data points are the means of the three replicates, and the error bars represent ± SE.

Discussion
Nitric oxide, a small gaseous molecule and newly emerged redox-active, is an important signaling component during both biotic and abiotic stress conditions in both animals and plants [16,52]. Therefore, it attracted the attention of plant scientists in the last couple of decades mainly because of its role in plants. Since then, it has been found to be a potent molecule affecting various physiological processes, such as seed germination, stomatal regulation, disease resistance, and plant

Discussion
Nitric oxide, a small gaseous molecule and newly emerged redox-active, is an important signaling component during both biotic and abiotic stress conditions in both animals and plants [16,52]. Therefore, it attracted the attention of plant scientists in the last couple of decades mainly because of its role in plants. Since then, it has been found to be a potent molecule affecting various physiological processes, such as seed germination, stomatal regulation, disease resistance, and plant responses to abiotic environmental conditions [15,16,53,54]. Its mechanism of action was unclear until it was found to transfer its bioactivity through S-nitrosylation, which is a post-translational modification in which NO covalently combines with the cysteine thiols of other proteins making S-nitrosothiols (SNOs) [55]. Thus, it can regulate physiological processes either by directly controlling gene transcription or by changing the redox status of cells by producing RNS. Studying global changes in gene expression in response to NO will make us understand the optimum genetic makeup during stress conditions through using various techniques such as microarray [56], amplified fragment length polymorphism [57], qRT-PCR [58], and RNA-seq [26,59].
Stem cell study is another trending topic in life sciences owing to their possible role in the medical field. Higher plants develop organs throughout their life; therefore, they must retain a pool of undifferentiated cells by regulating the proliferation of meristematic cells. In the Arabidopsis CLAVATA gene family, most genes are responsible for maintaining STM and OC [8,9]. Mostly, stem cells are associated with plant growth and development, and their role in plant disease resistance is poorly understood. Therefore, in the current study, we focused on stem cell or stem cell-related genes that showed differential expression in response to the NO donor S-nitroso-L-cysteine [26]. As NO is mostly produced in response to stress in plants [60], it would be interesting to determine the role of stem cell-related genes in plant growth and defense response.
Among the 20 CySNO-induced stem cell-related genes, 75% were down-regulated, suggesting that most of the CySNO-responsive stem cell-related genes were either involved in the NO production pathways or in the negative feedback loop of genes that are positively regulated by NO. For example, AtWOX13 (AT4G35550) that encodes a WUSCHEL-related homeobox gene family member is up-regulated, whereas both CLV1 and CLV2 were down-regulated in response to CySNO ( Figure 1A; Supplementary Table S2). Reports suggested that WUSCHEL (WUS) is required for stem cell identity, whereas the CLV genes regulate the pathway that limits the size of the shoot meristem. Furthermore, CLV1 and CLV3 suppress WUS expression to maintain the size of the stem cell population in the shoot meristem. The knock-out (KO) lines clv1 and clv3 showed irregular growth patterns; thus, CLV and WUS work in a negative feedback loop to maintain the OC [61]. Similarly, another gene BARD1 (AT1G04020) was down-regulated in response to CySNO ( Figure 1A; Supplementary Table S2). Interestingly, BARD1 is also implicated in the regulation of WUS (up-regulated to CySNO) expression. Han, Li, and Zhu [37] reported that BARD1 regulates SAM organization and OC maintenance by limiting WUS expression to the OC. They confirmed through functional genomics that the bard1-3 mutants resulted in severe SAM defects, while the double mutants wus-1 and bard1-3 resulted in the prematurely terminated SAM structures, suggesting that BARD1 functions through the regulation of WUS [37].
A correlation among the CySNO-induced stem cell-related genes suggested that the highest positive correlation (>0.8) was found for CLE12, which had positive correlations with 15 genes, followed by BAM1 ( Figure 1C). The CLE gene family comprises 32 members of Arabidopsis, and members of this family are involved in vital growth processes, such as root growth, rosette growth, root dwarfing, SAM arrest, asymmetric leaf development, etc. [10]. Most of the CLE gene family members are involved in the regulation of root growth with differential effects [10]. The GO terms for molecular function revealed that the major GO terms to which the stem cell-related genes were associated were related to signaling, receptor activity, and transmembrane receptor protein ( Figure 1D). This might be because a strong signaling capacity is required for the stem cell-related genes to maintain SAM. This can be confirmed by the role of CLV in maintaining SAM through the suppression of WUS, as supported by the reports suggesting that the CLV3 peptide is perceived by at least four different receptor-like proteins to repress the WUS activity [9,40]. The analysis of the promoter regions for cis-regulatory elements has suggested the presence of cis elements involved in abiotic and biotic stress tolerance. These include ABRE (TACGTG), which is involved in osmotic stress response and drought stress regulation [62]; TATA-box, a basic promoter element for enhanced transcription [63]; EIRE (TTCGACC), which is involved in maximal elicitor-mediated activation [64]; and MBS, the promoter element that harbors the transcription factor (TF) binding site for the MYB TF [65]. In addition, the W-box (TTGACT/C), which is the binding site for the WRKY TF, is considered to be involved in the regulation of multiple regulatory pathways involved in both abiotic and biotic stresses [27]. Previous studies also suggested the presence of the TF binding sites in NO-responsive genes. Through a bioinformatics approach, Palmieri et al. [66] have analyzed approximately 28447 Arabidopsis genes, and found that several TF binding sites occurred at least 15% more often in the promoters of NO-induced genes.
A phylogenetic analysis revealed that the majority of the soybean and poplar stem cell-related proteins were grouped together with the Arabidopsis CLE type of stem cell proteins (Figure 3). Similarly, a majority of the soybean stem cell-related genes were grouped together with the Arabidopsis receptor-like kinases type of stem cell proteins, such as BAM1 (At5g65700), CLV1 (At1g75820), and RLP12 (At1g71400) (Figure 3), indicating that the stem cell-related proteins of Arabidopsis and soybean share more common features than those of the other species. Furthermore, it was suggested that the types of stem cells might vary depending upon the types of species and their life forms.
An interactome study of the CySNO-induced stem cell-related genes suggested both experimentally proven and computationally inferred protein interactions. Among them, CLV1, CLV2, and CRN are important stem cell proteins that are involved in the maintenance of stem cells. Both CLV2 and CRN are involved in the perception of CLV3 ( Figure 4A). An extensive in vivo analysis has proven that CLV1 and CLV3 interact to repress WUS expression in order to maintain stem cell organization [8,9,40]. CLAVATA also works in coordination with phytohormones that are involved in growth and development. Reports suggested that WUS positively regulates STM, which promotes cytokinin synthesis. Similarly, high auxin levels can block the expression of ARR, resulting in high cytokinin levels ( Figure 4B).
Further investigation into the physiological roles of CLV1 and CLV3 indicated that under control conditions, both clv1 and clv3 responded differently. However, under the CySNO-induced nitrosative stress condition, both clv1 and clv3 plants showed increased root and shoot length compared to the wild-type plants ( Figure 5G,I). This indicates that both CLV1 and CLV3 negatively regulate shoot and root growth under CySNO stress conditions. CLV1 (At1g75820) was down-regulated in response to CySNO (Supplementary Table S2), which implies that CySNO negatively regulates the CLV1 expression; therefore, an opposite situation would be expected from the clv1 KO mutant. Furthermore, CLV1 in combination with CLV3 inhibits the WUS expression; therefore, clv1 would be unable to repress WUS, which induces STM to promote the production of cytokinin [40,67], which is an important growth-inducing hormone.
Another interesting aspect of both clv1 and clv2 mutants was to study their role in the basal defense system. Interestingly, clv1 and clv3 showed resistant phenotypes, and showed less pathogen growth, specifically at two days post-inoculation ( Figure 6B); however, the PR1 and PR2 gene expression study did not reflect the resistant phenotype. Our results suggested decreased PR1 and PR2 transcript accumulation in response to virulent pathogens, suggesting that the resistance toward PstDC3000 might be upstream or independent of the SA pathway. However, further investigation is required in order to explore the exact mechanism. We also validated the RNA-seq-mediated transcriptional changes in the CySNO-induced stem cell-related genes and correlated these with qRT-PCR. The Pearson correlation coefficient of 0.86527 indicates the high reliability of the RNA-seq data.
This study will add to our understanding of the role of NO in stem cell-mediated growth and defense response toward virulent pathogens.

Conclusions
This study concludes that nitric oxide-induced stem cell related genes are conserved across the plant kingdom, as they have orthologs in other species such as rice, soybean, tomato, and poplar plants.
The promoter study suggested their putative role in plant defense; however, in vivo analysis using the inoculation of mutant lines clv1 and clv3 with virulent PstDC3000 showed a resistant phenotype, but reduced PR gene expression. Furthermore, stem cell-related genes differentially regulated nitrosative and oxidative stress. Together all of the NO-induced stem cell-related genes may directly regulate plant growth, but indirectly regulate the plant defense system.