In this study, rice seeds (Oryza sativa cv. Rajendra Kasturi) were obtained from Bihar Agricultural University, Sabour, Bhagalpur. Plants were grown up to the four-leaf stage and used for salicylic acid (SA) and wounding treatments under greenhouse conditions. We have taken three biological replicates for both mock and treatment conditions. Each replicate consisted of four individual plants. For 2, 4-epi-brassinolide (EBR) and heat stress (HS) treatments, surface-sterilized seeds (Prasad et al., 2016) were grown on Murashige and Skoog (MS) medium (HiMedia) in magenta boxes (Tarson, India) for 15 days at 25°C under a 14/10 h light/dark cycle in a growth chamber. Rajendra Kasturi is a short-grained, high-yielding aromatic cultivar but is very prone to several abiotic and biotic stresses, reducing its productivity (Kumar et al., 2020).

Twenty-one-day-old rice plants were treated with 3 mM sodium salicylate. To prepare the working solution, a 1 M stock solution of sodium salicylate was initially prepared by dissolving the required amount of sodium salicylate in absolute ethanol. The volume was then adjusted to the desired final volume with distilled water. To prepare the 2, 4-epi-brassinolide (EBR) stock solution, a 100 mM/mL EBR solution was dissolved in absolute ethanol, and a 100 µM/mL working solution was prepared from this stock. For the experiment, a final concentration of 1 µM EBR was used, and for the mock treatment, 0.02% ethanol (the solvent used for EBR) was added to the MS media in test tubes. For the preparation of MS media, MS salts and vitamins (HiMedia) were added at a concentration of 4.42 g/L. Fe-EDTA (200X) was prepared by dissolving 557 mg FeSO₄•7H₂O and 745 mg Na₂EDTA, and 5 mL/L of this solution was added to the MS media. Additionally, 7.5 g/L of Clarigel (HiMedia) was included, and the pH of the media was adjusted to 5.8.

X. oryzae pv. oryzae (NCBI GenBank: MH986180) was used for the experiment, following the leaf clipping method as previously described (Kumari et al., 2020, 2022). The 21 day old rice leaves were clipped with scissors dipped in a bacterial suspension (1 × 10^8-9 cfu/mL) in saline (0.9%) with 0.05% Triton-X-100, and mock treatments used sterile water with 0.05% Triton-X-100 (Kumari et al., 2020, 2022).

For HS treatment, 15-day-old rice seedlings grown on MS media were exposed to 42°C for 1 hour and 4 hours, then returned to the growth chamber set to 20°C. Untreated seedlings served as controls.

To comprehensively investigate the presence of PR1 proteins in B. distachyon, H. vulgare, B. rapa, Z. mays, and O. sativa, we conducted BLASTP and HMMER (HMMER 3.1b2; http://hmmer.org/; accessed on 29 April 2020) searches as described previously (Prakash et al., 2017). All the twelve OsPR1 protein sequences were individually subjected for similarity searches against B. distachyon, H. vulgare, B. rapa, and Z. mays using BLASTP searches with these sequences as queries (https://www.ncbi.nlm.nih.gov/) (Agarwala et al., 2018) (accessed on 12 May 2020). The top ten hits from each plant species were selected and combined. Through manual curation, redundant, incomplete, and closely similar entries in terms of amino acid sequences were eliminated. Additionally, we conducted multiple sequence alignments (MSA) of all the newly identified putative PR proteins along with OsPR1s to verify the presence of a conserved 10-amino-acid-long sequence [W(10x)C(8x)C(x)H(12x)GC(4x)C(9x)C(1x)Y(10x)P]. The conservation of the 10 amino acids is a typical characteristic of PR1 proteins (Mitsuhara et al., 2008). Finally, eighty one putative PR1 sequences were used for phylogenetic tree construction. using the neighbor-joining (NJ) method with bootstrapping (1000 replicates) in the MEGA 11 Program (http://www.megasoftware.net; Analysis version 1.01; accessed on 20 August 2020) (Tamura et al., 2021).

All PR1 proteins were subjected for domain and subcellular localization studies. The domain prediction was performed using the Pfam tool (Pfam 37.0; http://pfam.xfam.org/search/sequence; accessed on 10 September 2021) (Mistry et al., 2021) and subcellular localization was predicted using the Balanced Subcellular Localization Predictor tool (BaCelLo) (http://gpcr.biocomp.unibo.it/bacello/info.htm; accessed on 10 September 2021) (Pierleoni et al., 2006). Chromosomal positions of PR1 genes were studied using Phytozome v12.1 database (https://phytozome.jgi.doe.gov/pz/portal.html; Analysis version v12.1; accessed on 20 August 2020). Chromosome maps of O. sativa PR1 gene were constructed using Chromosome Map Tools Oryza base (http://viewer.shigen.info/oryzavw/maptool/MapTool.do; accessed on 27 August 2022).

The Rice Annotation Project Database (RAP-DB), (http://rapdb.dna.affrc.go.jp/tools/dump; accessed on 21 January 2021) (Sakai et al., 2013) was utilized to obtain 1000 base pair upstream sequences of 23 OsPR1 genes. These retrieved sequences were subsequently analyzed using PlantPan3, (http://plantpan3.itps.ncku.edu.tw/; accessed on 21 January 2021) (Chow et al., 2016), for the identification of transcription factor binding sites (TFBS), and PlantCARE, (http://bioinformatics.psb.ugent.be/webtools/plantcare/html; accessed on 21 January 2021) (Lescot, 2002), for the analysis of cis-regulatory DNA elements in plants.

In addition, we retrieved 1000 base pair upstream sequences for newly identified PR1 genes in various plant species using Phytozome 12 (https://phytozome.jgi.doe.gov/pz/portal.html; Analysis version v12.1; accessed on 30 January 2021) (Goodstein et al., 2012). TFBSs and cis-regulatory DNA elements within these sequences were analyzed using the same approach as described above. A detailed flow chart for the identification of cis-regulatory sequences in rice and other plants is depicted in Supplementary Figure 1 .

The relative expression of 11 OsPR1 genes, including the reference gene OsPR1a, in rice seedlings treated with salicylic acid (SA), epibrassinolide (EBR), mechanical wounding, and heat stress (HS) was measured by qRT-PCR analysis. Total RNA was extracted from frozen plant tissues using Promega’s SV Total RNA Isolation System following the manufacturer’s protocol (Promega, Madison, WI). For cDNA synthesis, 1µg of total RNA was reverse transcribed using random hexamer primers according to manufacturer’s protocol (Promega, Madison, WI). The resulting cDNA was diluted in nucleases free water (1:5) and used for qRT-PCR (Kumari et al., 2022; Chaudhary et al., 2024). The qRT-PCR was carried out in a Light Cycler system (Applied Biosystems) using SYBR Green dye. Each reaction mixture (10 µL) contained 5 µL of SYBR Green dye (2×) (Promega, Madison, WI), 0.5 µL of forward/reverse gene-specific primers (10 µM), and 1 µL of diluted cDNA.

Three independent biological replicates as well as two technical repeats were conducted for each qRT-PCR analysis. The qRT-PCR conditions included an initial denaturation at 95°C for 2 minutes, followed by 40 cycles of amplification consisting of denaturation, annealing, and extension at 95°C, 53°C, and 72°C for 20 s, 30 s, and 30 s, respectively. The specificity of amplification was confirmed by melting curve analysis. ACTIN was used to normalize the expression data of OsPR1 genes. The fold-change in expression levels of genes was estimated using the 2^-ΔΔCt method as described previously (Livak and Schmittgen, 2001).

Gene expression data was statistically analyzed using the computer software SPSS. Significance of differences were analyzed by one-way analysis of variance (ANOVA).

The BLASTP analysis yielded 82 PR1 proteins using OsPR1 as the query sequence, and the HMMER search results identified 145 PR1 proteins. To ensure accuracy, we manually excluded PR1 proteins that were common between the BLASTP and HMMER searches. The initial extensive list of 227 PR1 proteins was refined to 81 proteins based on the presence of a conserved 10-amino-acid sequence [W(10x)C(8x)C(x)H(12x)GC(4x)C(9x)C(1x)Y(10x)P]. Among the 81 PR1 proteins analyzed, 23 were identified in Oryza sativa, while putative orthologs were found in Brachypodium distachyon, Hordeum vulgare, Brassica rapa, and Zea mays, totaling 58 proteins across these species. In this study, 23 OsPR1 genes were selected for phylogenetic analysis, with a subset of 11 OsPR1 genes chosen specifically for expression analysis experiments.

Each gene sequence was designated according to its chromosomal position and gene order ( Tables 1 , 2 ). For example, OsPR1-21 (LOC_OS02G54530.1) is located on chromosome 2, while OsPR1-61 (LOC_OS06G24290.1) is situated on chromosome 6. Notably, chromosome 7 houses a cluster of eight PR1 genes, namely OsPR1-71, -72, -73, -74, -75, -76, -77, and -78. This cluster is organized in a consecutive arrangement from the 5’ end to the 3’ end, all aligned in the sense orientation, as described by Mitsuhara et al. (2008).

The neighbor-joining (N-J) method of MEGA 11 Program (Tamura et al., 2021) was used to create a phylogenetic tree to compare OsPR1 and its potential homologs in both monocot and dicot plants. The analysis resulted in the formation of three main clusters: Cluster I, II, and III ( Figure 1 ). Further, these clusters were divided into groups (a-f) based on clade division.

Cluster I contains the largest number (50) of PR1 proteins, including Group C proteins such as OsPR1a and OsPR1-74. Previous reports have used OsPR1a as a versatile marker for both biotic and abiotic studies in rice (Yan et al., 2022). Within Cluster I, OsPR1-011 (OsPR1b) is positioned closer to OsPR1-75. Notably, OsPR1-011 demonstrates upregulation after salicylic acid (SA) treatment but downregulation following wounding (Mitsuhara et al., 2008). Similarly, our study observed the same trend for OsPR1-75. In Cluster I, the Group a, b, and c proteins are clustered together, containing ZmPR1-12 and OsPR1-101, ZmPR1-81 and OsPR1-012, and ZmPR1-73 and OsPR1-73 within the same clade BdPR1-14 (Bradi1g57590) and OsPR1-101. Notably, both of these genes were observed to be upregulated following SA treatment (Mitsuhara et al., 2008; Kakei et al., 2015). Cluster II represents the second largest group of PR1 proteins ( Figure 1 ). In Groups B and C of Cluster II, HvPR1-61 and BrPR1-82 along with OsPR1-022 and OsPR1-22, are grouped within the same clade ( Figure 1 ).

All the identified PR1 proteins contain a conserved motif belonging to the cysteine-rich secretory protein (SCP or CAP) domain, which ranges from 104 to 145 amino acids ( Tables 1 , 2 ). Subcellular localization predictions indicated that the majority of PR1 proteins are secretory in nature ( Tables 1 , 2 ). However, some proteins, such as OsPR1-76, HvPR1-52, HvPR1-53, and HvPR1-73, were predicted to localize in the nucleus. ZmPR1-52 was predicted to localize in the chloroplast.

Our findings indicate that BdPR1, HvPR1, BrPR1, ZmPR1, and OsPR1 genes are situated on distinct chromosomes ( Figure 2 ). Notably, chromosome 7 exhibited the highest concentration of PR1 genes, comprising 25.5% of the total. Chromosomes 1 and 3 collectively harbored 16% of the PR1 genes, while the remaining genes were distributed across other chromosomes ( Tables 1 , 2 ; Figure 2 ).

Plant hormones, salicylic acid (SA) and brassinosteroids (BR), have been identified as key regulators of the plant defense system (Kumari et al., 2022). While the role of SA in regulating PR1 genes during plant defense is well-established (Takatsuji et al., 2010), the precise impact of BR on PR1 gene expression is largely unknown. In the present investigation, a total of 11 OsPR1 genes, whose significant roles had not been previously studied, were meticulously selected for comprehensive expression analysis, along with the OsPR1a marker gene included for reference.

Upon subjecting the rice seedlings to SA treatment, eight genes OsPR1-21(~21-fold induction), OsPR1-22 (~37 fold induction), OsPR1-61 (~6-fold induction), OsPR1-71(~1.5-fold induction), OsPR1-72 (~45-fold induction), OsPR1-74 (~9.5-fold induction), OsPR1-75 (~5.6-fold induction), and OsPR1-77 (~3.1-fold induction)) showed upregulation, while OsPR1-76 displayed downregulation. Notably, OsPR1-73 and OsPR1-78 were not amplified in uniform manner in treated or mock samples ( Table 3 , Figure 3A ).

Following EBR-treated, seven OsPR1 genes OsPR1-21, OsPR1-22, OsPR1-61, OsPR1-71, OsPR1-74, OsPR1-77, and OsPR1-78 were found to be upregulated whereas three genes OsPR1-72, OsPR1-75, and OsPR1-76 were downregulated. The maximum induction was observed in OsPR1-78 (~12- fold induction), followed by OsPR1-61 (~10-fold induction) and OsPR1-21 (~5-fold induction) ( Figure 3C ).

Upon 4h of HS treatment, OsPR1-76 showed the highest upregulation (~28-fold induction) while OsPR1-77 showed the lowest upregulation (~14 -fold induction). Both OsPR1-74 and OsPR1-61 showed upregulated to the same level (~10-fold induction) upon 4h of HS. OsPR1-72 and OsPR1-75 genes were downregulated ~2– ~3-fold after 4 h of HS, whereas OsPR1-22, OsPR1-61, and OsPR1-77 were downregulated (~1.5-fold) after 1 h of HS treatment. OsPR1-74 showed the highest upregulation (~15-fold induction) after 1 h of HS-treatment, followed by OsPR1-72 (~10-fold induction), while the remaining upregulated genes showed upregulation in range of ~ 1-3-fold. Following exposure to HS, OsPR1-73 and OsPR1-78 were unamplified ( Figure 3B ).

The leaves of 21 day old rice seedlings were subjected to precise and effective cross-cross wounding employing sterile scissors as described previously (Mitsuhara et al., 2008). Our result revealed that the maximum expression was observed in OsPR1-74 gene (~17-fold induction), followed by the OsPR1-22 gene (~4-fold induction). Three genes-OsPR1-75 (~4-fold), OsPR1-71 (~2.7-fold), and OsPR1-72 (~4.3-fold) were found to be downregulated ( Figure 3A ). The responses of all 11 OsPR1 genes to different treatments are summarized in Table 3 .

Genes that were highly upregulated at 24 hours post-infection with X. oryzae pv. oryzae included OsPR1-74 (~12-fold), followed by OsPR1-72 and OsPR1-22 (~10-fold), OsPR1-76 (~7.86-fold), OsPR1-75 (~4.64-fold), and OsPR1-61 (~2-fold). In contrast, OsPR1-73 and OsPR1-78 did not show consistent amplification across all biological replicates ( Figure 3D ).

The promoter sequences located up to 1 kilobase (kb) upstream from the translation start site of each PR gene in Oryza sativa (rice) were meticulously analyzed using PlantCARE and PlantPAN 3 programs. The primary goal was to identify potential plant cis-acting regulatory elements (PCAREs) and transcription factor binding sites (TFBSs) associated with defense responses. A diverse set of important defense-responsive TFBSs, such as bHLH, bZIP, C2H2, EIN3, GATA, LEA, MYB, Myb/SANT, NAC, NAM, and WRKY, were successfully identified in both the positive and negative strands of the promoter sequences of OsPR1 genes ( Figures 4 , 5 ). The analysis further focused on determining the enrichment and distribution of all TFBSs within proximal (<500 bp) and distal (>500 bp) regions of all OsPR1 genes. Remarkably, it was observed that the majority of these TFBSs were significantly enriched in the proximal upstream regions of most OsPR1 genes. This finding highlights the potential significance of these regulatory elements in modulating the expression of OsPR1 genes during defense responses in rice ( Figures 4 , 5 ). In our experiment, the highest number of WRKY TFBSs was found in OsPR1-73 (93); however, this gene was not expressed under any of the treatments. In contrast, a significant enrichment of WRKY TFBSs was observed in OsPR1-72 (88), which was upregulated 45-fold and 10-fold following SA and heat stress treatments, respectively ( Supplementary Table 2 ). This suggests that WRKY TFBSs play a major role in PR1-mediated biotic and abiotic responses. OsPR1-76 has the highest number of AP2 (175) and bZIP (61) TFBSs ( Supplementary Table 2 ) and shows maximum expression during 4 hours of heat stress (28-fold), as well as 1 hour of heat stress and wounding (3-fold). The presence of a large number of AP2 and bZIP TFBSs may play a significant role in regulating both biotic and abiotic stress responses.

The study conducted on Oryza sativa (rice) identified a total of 57 cis-acting regulatory elements (CAREs) in its promoter regions. These cis-elements varied in length from 4 to 10 base pairs (bp), with the majority being 6 to 7 bp long. The cis-elements were further categorized into different functional groups, revealing that a significant proportion (63%) were associated with stress responsiveness, with light-responsive elements constituting 34% of the stress-responsive group. The occurrence frequency of different cis-elements was nearly identical in both the forward and reverse strands of the 1-kilobase promoter region. Interestingly, the distribution analysis revealed that most cis-elements were clustered between 16 to 493 bp on the forward strand (+) and 546 to 842 bp on the reverse strand (−) ( Figure 5 ).

Furthermore, specific cis-elements such as TATA, CAAT box (not shown in Supplementary Table S3 ), Box 4, and G-box were found to be more abundant in the OsPR1 genes compared to other cis-elements. These findings provide valuable insights into the regulatory mechanisms governing the expression of OsPR1 genes and highlight their responsiveness to various stress conditions in rice. The distribution of CAREs was unequal throughout the upstream region of the genes, with nearly equal numbers found in the proximal (<500 bp) and distal (>500 bp) regions. Gene expression is correlated with the presence of PCAREs and the number of transcription factor binding sites (TFBSs). Following SA, EBR, wounding, and heat stress treatment (HST), the OsPR1-71 and OsPR1-74 genes were upregulated and enriched with the greatest number of PCAREs. OsPR1-74 was found to be enriched with defense-responsive TFBSs such as WRKY (32), AP2 (74), and developmentally related TFBSs such as TCP (186) and α-amylase (10) ( Supplementary Table 2 ). OsPR1-71 and OsPR1-74 were enriched with W-box (TTGACC), SA-responsive element TCA (CCATCTTTTT), and EBR-binding element MBS (CAACTG), suggesting SA-mediated EBR expression of the genes ( Supplementary Table 3 ). In addition, the OsPR1-71, OsPR1-74, OsPR1-76, and OsPR1-21 genes, which were upregulated after 1 h and 4 h of heat stress treatment (HST), contain the highest number of hormone-responsive PCAREs. These genes were enriched with the maximum number of methyl jasmonate-responsive TGACG-motif and CGTCA-motif, suggesting their role in basal thermotolerance. Previous research in Arabidopsis has shown that jasmonic acid (JA) can mitigate the effects of high light and high temperature conditions, which is crucial for plants to thrive in various agroclimatic environments. Furthermore, OsPR1-72 and OsPR1-76 contain the GC-motif (CCCCCG), which is associated with anoxic-specific inducibility ( Supplementary Table 3 ). This finding suggests that the OsPR1 genes may be involved in submergence regulation.

The promoter sequences of BdPR1, HvPR1, BrPR1, and ZmPR1 were studied to retrieve important stress-responsive TFBSs and CAREs. Comparatively, within the promoter sequences of all PR1 genes, stress-responsive TFBSs exhibit a higher prevalence compared to those associated with developmental processes. In BdPR1, HvPR1, and BrPR1, the number of total CAREs varies from 52 to 53, with 61% of them being stress-responsive. In contrast, ZmPR1 has a total of 49 CAREs, with 70% being stress-responsive ( Supplementary Table 4 ). The numbers of developmental-related CAREs in BrPR1 and ZmPR1 are compared to those in BdPR1 and HvPR1 ( Supplementary Table 4 , Figure 6 ). All the PR1 genes were enriched with TATA-box, CAAT-box, G-box, MYC, ARE, Myb, abscisic acid (CGTACGTGCA), and methyl-jasmonate (TGACG, CGTCA) responsive PCAREs ( Supplementary Table 4 ). The maximum number of TFBSs was found in BdPR1-11 [bHLH (252), bZIP (140), WRKY (109), NAC;NAM (102), BES1 (44), Myb-related (31), AP2;ERF (93)), followed by BdPR1-13 (bZIP (239), EIN3 (11)] ( Figure 7 ). In ZmPR1, the maximum enrichment of defense-responsive TFBSs was found in ZmPR1-51 and ZmPR1-31, though the total enrichment was lower compared to others. In contrast, the enrichment of TFBSs in members of HvPR1 and BrPR1 was the highest ( Figure 7 ).

The specific binding of transcription factors (TFs) to their respective transcription factor binding sites (TFBSs) is a crucial mechanism for mediating the transcriptional regulation of target genes. This process allows TFs to exert precise control over gene expression by interacting with specific DNA sequences in the regulatory regions of their target genes (Narlikar and Ovcharenko, 2009; Jayaram et al., 2016). The evidence of high throughput experimental methods accelerate the identification of TFBSs (Jayaram et al., 2016). However, in silico identification of TFBSs was still a method of choice to study various defense makers of plants (Boeva, 2016; Kumari et al., 2018). PR1 genes were acting as defense markers through activating a series of elements including transcription factors, MAPK and ROS. PR1 is a family of proteins whose expression regulates different functions during stress. The in silico analysis of the promoter regions of PR1 genes revealed the presence of multiple cis-regulatory elements, providing strong evidence for their involvement in multiple stress responses.

WRKY (W box) has an extensive role in biotic and abiotic stress along with a wide range of development process (Choi et al., 2015; Rinerson et al., 2015). Different PR1 genes of this study enriched with conserved consensus sequence TTGAC(C/T/A/G) ( Supplementary Table 3 ). WRKY proteins play a crucial and diverse role in plant responses to various stresses and are involved in essential processes related to plant development, embryogenesis, dormancy, as well as responses to abiotic stresses like drought, salt, and heat in rice (Kalde et al., 2003; Jiang and Deyholos, 2009; Chen et al., 2012; Rushton et al., 2012). In addition, OsWRKY03 regulates a cascade of Jasmonic acid (JA) and salicylic acid signaling pathways to protect plants against bacterial and fungal infections (Liu et al., 2005). WRKY have been shown to exert a positive regulatory effect on the heat tolerance of plants. For example, in pepper plants, CaWRKY40 plays a vital role in the response to high-temperature stresses, contributing to enhanced heat tolerance. Similarly, in rice, OsWRKY11 is involved in the plant’s resistance to heat stress, further emphasizing the significance of WRKY in facilitating heat stress adaptation in different plant species (Wu et al., 2009; Cai et al., 2015). In Arabidopsis, it was observed that PR1 genes expression levels were notably higher in plants overexpressing WRKY39 compared to wild-type plants following heat treatment. This finding led researchers to speculate that WRKY39 likely acts as an upstream regulator of PR1, influencing its transcription and subsequently contributing to the plant’s response to heat stress (Li et al., 2010).

The basic region/leucine zipper motif (bZIP) plays a crucial role in regulating diverse stress and developmental responses in plants. These bZIP proteins exert their regulatory functions by binding to specific transcription factor binding sites (TFBSs) on DNA. Notably, plant bZIP proteins have a preference for recognizing the ACGT core in DNA sequences, particularly in regions known as the A-box, C-box, and G-box (Jakoby et al., 2002), CCAAT-box, TGA-element, NON, ABRE (Holdsworth et al., 1995; Jakoby et al., 2002; Liao et al., 2008) AS-1 (Després et al., 2000), TATCCAT/C-motif (Kong et al., 2018). G-box element imparts response to abscisic acid ABRE (ABA-responsive element), methyl-jasmonate, Anaerobiosis, defense responses ethylene induction as well as in seed specific expression found in most of studied PR1 genes (Kong et al., 2018). The TATCCAT/C-motif (Kong et al., 2018) and TGACGTCA, commonly referred to as the G-box, is abundantly present in the promoter regions of many stress-inducible genes, including the PR1 promoter. This interplay between SA, the TGA subfamily, and NPR1 represents a crucial mechanism underlying the activation of promoters involved in the plant’s defense against pathogens (Zander et al., 2014). In a previous report were found that AS-1 cis element is an oxidative stress-responsive element and activated by SA by binding TGA cis element (Zander et al., 2014). bZIP proteins have impart their major role in activating a number of defense genes (Alves et al., 2013).

AP2 is one of the biggest plant TF super families which play a significant role in regulating plant growth and stress responses (Najafi et al., 2018). The AP2 (APETALA2)/ERF family is categorized into three distinct subfamilies based on the number of AP2/ERF domains they possess. These subfamilies are known as AP2, ethylene responsive factor (ERF), and RAV. Each subfamily is distinguished by its unique arrangement of AP2/ERF domains (Nakano et al., 2014). The ERF subfamily plays a crucial role in regulating a wide array of genes associated with both biotic and abiotic stresses in rice. These stresses include challenging environmental conditions like drought, high salinity, and cold temperatures (Pandey et al., 2014). The AP2 subfamily is known to be involved in regulations of developmental process such as flower development, ovule development, seed set and regulating organ-specific growth while RAV subfamily genes response to expression induced by ethylene, brassinosteroids, and biotic and abiotic stresses (Xu et al., 2013). ERF subfamily members bind to the conserved nucleotide sequences AGCCGCC (Xu et al., 2013) and TACCGACAT (GCC-box) (Pandey et al., 2014) in the upstream regions of genes while the RAV binds to CAACA and CACCTG motif (Song et al., 2013). AP2/ERF transcription factors have been identified as regulators that can trigger the synthesis of ethylene (ET), salicylic acid (SA), and jasmonic acid (JA). These signaling molecules play a significant role in enhancing the expression of pathogenesis-related (PR) genes during instances of pathogen infestation (Singh, 2002; Brown et al., 2003). Moreover, the presence of AP2/ERF transcription factor binding sites (TFBSs) in defense-responsive genes has been found to strengthen resistance against specific biotic and abiotic stresses (Berrocal-Lobo et al., 2002). The ERF family of TFs was shown to regulate abiotic and biotic stresses (Pandey et al., 2014) as well as involved in ethylene mediated PR genes expression which confer tolerance against cold and dehydration stresses (Liu et al., 2007). In the AP2/EREBP family, a subset called DREBs is classified under the EREBP subfamily and plays a pivotal role in plants’ response to various abiotic stresses. Notably, specific DREB members, such as rice OsDREB2B and maize ZmDREB2A, are known to be induced and expressed in response to high-temperature stress. This induction of DREBs assists plants in coping with the challenges posed by elevated temperature conditions (Qin et al., 2007; Matsukura et al., 2010). CG-1;CAMTAs is comprised of core sequence CGTG (Silva, 1994) which associated with ABRE cis- element. In our study of PR1 genes we found the same consensus sequence and another cis-element chs-CMA1a (TTACTTAA) were also present. Calmodulin binding transcription activators (CAMTAs) are proteins that exhibit responsiveness to a diverse range of external signals, including cold, wounding, and drought. Additionally, they are influenced by hormonal signals like ethylene and ABA (Reddy et al., 2000; Yang and Poovaiah, 2003). Our research findings indicate that Calmodulin signaling responsive genes are predominantly linked to ABRE (CGTG) cis-elements.

This may be possible that PR1 signaling response is linked to ABA hormone signaling. bHLH can recognize the MYC (CATTTG), G-box, E-box, WRE3 (CACCT) (Miyamoto et al., 2012). bHLH transcription factors (TFs) play a crucial role in various aspects of plant biology, including SA and JA mediated stress responses, light-induced hormone signaling, wound and drought stress management, shoot branching, as well as fruit and flower development, root development, and other pathways (Carretero-Paulet et al., 2010). Moreover, bHLH TFs are also involved in inducing ABA-dependent signaling to cope with cold stress, specifically in rice (Chinnusamy et al., 2003). MYB transcription factors (TFs) are universally present in eukaryotes and are associated with specific TFBS sequences, such as TAACTG, CAACTG, AACGG, and C/TAACNA/G, as reported by Xu et al. (2008) and Qin et al. (2012). These MYBs play essential roles in various biological processes, including plant development, metabolism, and stress responses. In rice, MYB TFs have been demonstrated to be crucial in managing abiotic stresses like dehydration, salt, and cold stresses (Abe et al., 1997). They are also involved in the ABA and SA-signaling pathways, which confer responses to both biotic and abiotic stresses (Ambawat et al., 2013). One specific member of the MYB family, OsMYB55, has been shown to enhance the tolerance of rice plants to high temperatures. This is achieved by increasing the expression of downstream genes like OsGS1;2, GAT1, and GAD3, which are involved in amino acid metabolism (El-Kereamy et al., 2015).

TCP proteins possess a distinctive TCP domain, and their binding sites exhibit variation, including sequences like GGNCCCAC, GTGGNCCC, and TGGGCC, as identified by Giraud et al. (2011). These TCP genes play a crucial role in influencing the development of axillary structures, petals, and stamens in plants (Cubas et al., 1999). The SBP protein has been shown to interact with the GTAC core sequence, as demonstrated by Wang et al. (2015). In plants, the SBP protein is actively involved in various developmental processes, including flower and fruit development, architecture formation, responses to copper and fungal toxins, as well as regulation of GA (gibberellic acid) levels (Wang et al., 2009). B3 cis -elements are combining with core sequences of CATGC (RY-element) verified in all PR1 genes. The B3 family of proteins plays significant roles in regulating flowering time, organ growth, and organ polarity in plants. These B3 members are distributed in various plant tissues, including pistils, stamens, germinated seeds, and roots (Swaminathan et al., 2008). Together with these TFBSs, many more found in PR1 genes, which regulate the multiple stress along with developmental process.