Abstract Background Drought stress has become a pervasive environmental challenge, significantly impacting all stages of plant growth and development under changing climatic conditions worldwide. In coconut, drought stress critically impairs reproductive development, notably reducing the quality of pollen and gametes during fertilization. Therefore, the seedlings of the aromatic coconut variety were subjected to drought stress for varying durations: control (no stress), 7 days, 14 days, and 21 days to find the potential molecular mechanisms and genes related to coconut drought tolerance through transcriptomic analysis. Our study may provide a theoretical basis for investigations into drought stress tolerance that will be useful for further coconut improvement. Results We assessed antioxidant enzyme activity and conducted comparative transcriptome analyses of aromatic coconut under different drought conditions (7, 14, and 21 days). Our findings revealed significant rises in superoxide dismutase (SOD), peroxidase (POD) activities and proline (Pro) content across all drought periods compared to control plants, suggesting that these enzymes play a crucial role in the adaptive response of coconuts to drought stress. RNA-seq data identified 280, 729, and 6,698 differentially expressed genes (DEGs) at 7, 14, and 21 days, respectively. Principal Component Analysis (PCA) revealed that coconut samples were scattered and separated across different treatment points, suggesting the presence of differentially expressed genes (DEGs), particularly in the 21 day drought treatment (GH21d). KEGG pathway analysis indicated that DEGs were significantly enriched in pathways related to plant-pathogen interaction, plant hormone signaling, and mitogen-activated protein kinase (MAPK) signaling. Functional annotation of these DEGs revealed key candidate genes involved in several hormone signaling pathways, including abscisic acid (ABA), jasmonates (JA), auxin (AUX), brassinosteroids (BR), ethylene (ET), and gibberellin (GA), along with MAPK pathway which may regulate plant adaptation to drought stress through processes such as plant growth, cell division, stomatal closure, root growth, and stomatal development. This study provides valuable insights into the genetic and molecular basis of drought tolerance in coconuts, paving the way for the improvement of drought-tolerant coconut varieties. Conclusions Under drought stress, the expression of genes related to plant growth, stomatal closure, cell division, stress response, adaptation, and stomatal development appears to play a critical role in drought tolerance in coconut. Our results revealed that multiple genes may contribute to the drought tolerance mechanism in coconut through various hormone signaling pathways, including ABA, JA, auxin, BR, GA, and ethylene. These findings offer new insights into the key molecular mechanisms governing drought tolerance in aromatic coconut. Furthermore, the candidate genes and pathways identified in this study could be valuable for developing strategies to enhance drought tolerance in coconut plants. Clinical trial number Not Applicable. Supplementary Information The online version contains supplementary material available at 10.1186/s12870-025-06554-2. Keywords: Drought stress, Antioxidant enzymes, Transcriptome, Plant hormone signal transduction, MAPK signaling pathway, Differentially expressed genes (DEGs) and coconut Background The coconut (Cocos nucifera L.) is a versatile and nutritious food widely used in cooking, baking, cosmetics, and medicinal purposes [[36]1, [37]2]. Additionally, it plays a substantial role in ecological balance, and the agrarian economy, as well as providing livelihoods for millions of people in tropical and subtropical regions worldwide [[38]3]. Furthermore, coconut trees can grow in sandy and saline soils, making them highly adaptable to coastal environments and valuable in a changing climate [[39]4, [40]5]. Recently, Coconut plantations are becoming an attractive industry for farmers due to minimal care and investment requirements. Thus, global coconut production has increased progressively over the past two decades, from an estimated 51 million metric tons in 2000 to 61.5 million metric tons in 2022 [[41]6]. Coconut trees have a high-water requirement, and optimum soil moisture is essential for plant growth and development, any fluctuation from optimal water availability results in stress conditions, adversely affecting nutrient uptake, grain quality, and yield [[42]5, [43]7]. Accordingly, there are challenges in adapting coconut plants to changing climate conditions. Heat and water stress, increasingly common in tropical regions, are anticipated to have detrimental effects on the reproductive processes of coconut trees, which ultimately affect production [[44]8]. Drought stress is the most widespread environmental factor, affecting almost all stages of plant growth directly or indirectly and being a major cause of about 50% yield loss [[45]9]. Global climate change is anticipated to increase drought frequency and severity in the future [[46]10], potentially causing serious problems for plant growth dynamics and ecosystems on more than 50% of arable land by 2050 [[47]11, [48]12]. However, the effect of drought stress depends on the plant species, growth stages, age, drought intensity, and duration, all of which influence plant responses to drought [[49]13, [50]14] but are considerably manageable through nutrient and water management [[51]15]. Under stress conditions, plants produce excess reactive oxygen species (ROS), that can damage living tissues and result in cell death. Moreover, ROS functions as signal transduction molecules, conveying signals to the nucleus through the mitogen-activated protein kinase (MAPK) pathway, thereby playing an important role in diverse adaptation mechanisms [[52]16]. Thus, the production and scavenging of ROS are crucial for balancing plant defense systems, which are maintained by ROS-detoxifying enzymes generated from the overexpression of stress-responsive genes. Proline plays a crucial role in stress signal transduction and also functions as an antioxidant. Consequently, antioxidant synthesis increases in plant cells through the expression of linked genes, particularly superoxide dismutase (SOD: EC 1.15.1.1), which plays a significant role in the antioxidant defense system and aids in plant adaptation to various abiotic stress [[53]17]. In plants, drought stress leads to stomatal closure, reducing enzymatic activities, water movement, and CO[2] uptake, thus critically affecting photosynthetic activity and hampering the reproductive process [[54]18]. Visible morphological effects of drought include seed germination failure, altered leaf relative water content and water use efficiency, reduced leaf number, leaf size, stem extension, and root growth [[55]19, [56]20]. Water stress combined with high temperatures may alter several physiological processes throughout the growing period, causing significant changes at the molecular, cellular, and organismal levels [[57]21]. Plants mainly use three survival strategies under drought stress: drought escape, avoidance, and tolerance through specific metabolic changes and gene expressions [[58]18, [59]22]. These mechanisms include encouraging primary root development, establishing deeper root systems for water uptake, regulating stomatal movement or leaf surface to minimize water loss, and accelerating flowering to complete the life cycle [[60]23–[61]25]. In contrast, plant hormones play a critical role in regulating plant adaptation to drought stress through changes in biosynthesis and signaling pathways, including abscisic acid (ABA), jasmonates (JA), auxin (AUX), brassinosteroids (BR), ethylene (ET), and gibberellin (GA) [[62]26]. Climatic factors such as temperature, precipitation, and humidity could greatly impact the growth, development, and geographical distribution of coconut trees [[63]5]. Drought stress significantly influences coconut tree growth and productivity by limiting water availability and nutrient uptake, however, its efficiency depends on the season and genotype [[64]27]. Reduced fruit sets in drought-prone areas can be attributed to lower-quality pollen and/or gametes produced under drought stress during fertilization [[65]28]. In response to drought stress, coconut trees activate a range of adaptive mechanisms, including morphological changes, expression of drought-resistance genes, synthesis of stress-related hormones, and production of osmotic regulatory substances, all of which work together to alleviate the adverse effects of water stress. Understanding these drought resistance mechanisms and improving drought-resistant varieties are crucial for maximizing yield under water deficit conditions, and ensuring food security in changing climatic conditions [[66]27]. In the 1990s, molecular markers were introduced into coconut breeding programs, aiding in the assessment of genetic variation and facilitating the identification of desirable traits and genes for further improvement. The advent of high-throughput sequencing has propelled research progress in genomics and transcriptomics. Recently, RNA-seq-based transcriptome analysis has become one of the most efficient and cost-effective strategies to investigate novel genes and interactive mechanisms in plants under abiotic stress conditions [[67]23]. RNA-seq technologies have been employed to analyze the coconut transcriptome for genetic improvement, identifying drought-responsive genes, complex metabolic pathways, and biochemical processes [[68]29]. Plants utilize a set of complex physiological and molecular regulatory mechanisms to survive under stress, such as drought [[69]4]. As climate change raises temperatures and alters rainfall patterns, it is crucial to understand the impacts of drought on coconuts and its underlying mechanism. Despite technological advancements, the molecular mechanisms by which plants exhibit potential under drought stress remain ambiguous. To our knowledge, no specific genes or metabolic pathways have been reported for the development of drought-tolerant coconut varieties. Therefore, efforts need to focus on understanding the potential molecular mechanisms and genes related to coconut drought tolerance to maximize fruit yield. Thus, this study aimed to identify the key regulators of coconut transcriptomes under drought stress conditions. Our study may provide a theoretical basis for further investigations into drought stress tolerance in palm crop species. Methods Plant materials and drought treatments For this experiment, six months old seedlings of aromatic coconut varieties were collected from the germplasm of the Coconut Research Institute in Wenchang, Hainan, P.R. China. The seedlings were thoroughly watered for 3 days and then left to dry naturally. A total of 24 healthy seedlings were selected and divided into two main treatment groups: control and drought treatment, with 12 seedlings in each group. Each treatment group was further subdivided based on stress duration: 0 days (no stress), 7 days, 14 days, and 21 days. Each treatment group included three biological replications, with each replication consisting of three seedlings. The experimental unit was a plastic pot with a hole in the base to drain excess water. The potted plants were placed in a growth chamber to avoid outdoor drought stress, ensuring favorable growth conditions with a controlled temperature of 25 °C and a light cycle of 16 h of light followed by 8 h of darkness. They were left for seven days to acclimate before the drought stress treatments commenced. However, water was added daily to maintain the moisture level to ensure the proper growth of control plants. For the drought stress treatment, no watering was done from the beginning of the treatment, lasting for a total of 21 days. Consequently, the soil moisture content in both the control and drought-treated groups was measured and recorded at each sampling point. Soil moisture levels and the phenotypes of coconut seedlings were monitored to confirm drought stress (Fig. [70]1A-B). Leaf samples were collected from the inner side of the second leaves on the 7th, 14th, and 21st days and designated as GH7d, GH14d, and GH21d, respectively. Plants with normal watering during the corresponding period were used as the control (CK7d, CK14d, and CK21d). The collected leaf samples were rapidly transferred in liquid nitrogen and then kept in a refrigerator at -80 °C for further RNA extraction and RNA-seq analysis. Fig. 1. [71]Fig. 1 [72]Open in a new tab Coconut seedlings under different drought stress. A. Soil moisture content (%) change; B. Phenotype change; Data are the mean values of three biological replicates Estimation of antioxidant enzyme activities and proline content Three fresh leaf samples were collected from each treatment group at 0 days (control), 7 days, 14 days, and 21 days and immediately transferred in liquid nitrogen. Frozen coconut leaves were ground into a fine powder using liquid nitrogen (0.5 g) and then homogenized in 0.1 M Tris-HCl buffer (pH 7.5), containing 50 mg of polyvinylpyrrolidone (PVP) on ice. The homogenate was centrifuged at 12,000 g for 15 min at 4 °C, and the resulting supernatant was used to assess superoxide dismutase (SOD) activity. SOD activity was determined based on the protocol developed [[73]30]. One unit of SOD activity was defined as the amount of enzyme required to inhibit 50% of nitro blue tetrazolium (NBT) photoreduction, as observed at 560 nm. Peroxidase (POD: EC 1.11.1.7) activity was measured at 436 nm following the method described in [[74]31]. Additionally, 0.1 g of fresh leaf sample was ground, and the extracted solution was analyzed using assay kits (Suzhou Grace Biotechnology Co., Ltd., Jiangsu, China). Finally, proline (Pro) content was measured spectrophotometrically (UV2600, SHIMADZU, Japan) according to the manufacturer’s instructions. RNA extraction, sequencing, and transcriptome analysis Tissue samples were collected from the fresh leaves of coconut seedlings for each treatment group at 0 days (control), 7 days, 14 days, and 21 days. For each sample, three functional leaves from seedlings subjected to the same treatment were excised and pooled to form a single biological sample. This process was repeated three times to generate biological replicates for each time point. Total RNA was extracted using a modified cetyltrimethylammonium bromide (CTAB) method. RNA integrity and purity were observed visually using 1.5% agarose gel electrophoresis. Additionally, RNA concentration and integrity were measured and quantified using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and an Agilent 2100 Bioanalyzer system (Agilent Technologies, Palo Alto, CA, USA), respectively. Sequencing of the samples was performed using the BGISEQ-MGI2000 platform at BGI Genomics (Wuhan 430073, China). All the raw data have been deposited to CNGB Nucleotide Sequence Archive with the accession number CNP0006465 which are available in the [75]https://db.cngb.org/. Raw data in FASTQ format were filtered to remove unwanted adapters, sequences with more than 5% unknown bases, and low-quality reads (quality score ≤ 10 in more than 20% of the sequence) [[76]32]. The filtered reads were aligned to the sequence from the reference genome using the HISAT (v2.1.0) package [[77]33]. Gene expression levels were evaluated using the RSEM (v1.2.8) program [[78]34]. Unigenes were described by selecting the lengthy transcript, and functional annotation was managed through comparisons with the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) databases. Read count data were analyzed using DESeq2 software to identify differentially expressed genes (DEGs) within the treatment groups, following fold change values (|log2(FC)| ≥1.5) and adjusted p-values (Padj < 0.05) [[79]35]. The KEGG Pathway enrichment analysis of the differentially expressed genes (DEGs) was conducted using the KEGG Orthology Based Annotation System (KOBAS) software [[80]36], based on the KEGG database. The protein-protein interaction was analyzed using associated protein sequences with a high confidence (0.700) interaction score by the STRING database (V 12.00) [81]https://string-db.org/ [[82]37]. Quantitative real-time polymerase chain reaction (qRT-PCR) verification Nine candidate drought-responsive DEGs were randomly selected to validate the reliability of the transcriptome data through qRT-PCR assay. Gene-specific primers were designed using Primer Premier 5 software, ensuring a melting temperature range of 55–60 °C, GC content of 50–60%, and an amplicon size of 80–200 base pairs (bp). Primer quality was assessed using PCR Primer Stats software. The primers and sequences used in qPCR analysis are provided in supplementary file 1. The qRT-PCR procedure followed the technique described [[83]38], with CnACT used as a reference gene. Five biological replicates per sample were included to ensure the reliability of statistical analysis. The cycle threshold (CT) values from each sample were normalized against the reference gene primer, and relative changes in gene expression were calculated using the 2^–ΔΔCT method [[84]39]. Statistical analysis In this experiment, all data are presented as the mean ± standard deviation (SD) of the three replications. Analysis of variance (ANOVA) was completed using SPSS software (version 20.0), with results considered statistically significant at P < 0.05. Charts and heat maps were created using GraphPad Prism (version 8.0; GraphPad Software, San Diego, CA, USA) and Microsoft Excel 2016. Volcano and bubble plots were produced using the online platform HiPlot ([85]https://hiplot.com.cn/cloud-tool/drawing-tool). Results Antioxidant activities and proline levels in aromatic coconut leaves response to drought stress Drought inhibits systematic cellular metabolism, resulting in the production of ROS. However, the production of antioxidative enzymes such as SOD and POD plays a decisive role in sustaining ROS homeostasis in the cells, indicating their contribution to plant adaptation under water stress [[86]17, [87]40]. We evaluated the activities of two major antioxidant enzymes, SOD and POD, in coconut leaves and observed significant differences among the drought periods. A gradual increase in SOD and POD activities was detected in response to drought but stabilized at different time points. SOD activity increased significantly up to 14 days of drought stress, then remained steady at 21 days (Fig. [88]2A). Conversely, POD activity showed a significant increase at 7 days of drought stress, a slight increase at 14 days, and a decreasing tendency at 21 days (Fig. [89]2B). Additionally, we observed a significant increase in proline (Pro) levels in coconut seedlings up to 14 days, followed by a slight decrease at 21 days under water stress conditions (Fig. [90]2C). The highest activities of both SOD, POD and PRO were recorded at 14 days of drought treatment, while the lowest activities were observed in the control group. Notably, a significant difference was observed in SOD, POD activities and PRO compared to control treatments in all drought points except SOD activities in control plants (Fig. [91]2). The increased activities of SOD, POD and PRO during the drought periods demonstrate their critical role in balancing cell metabolism in drought stress and could be a potential trait for selecting stress tolerance coconut plants. Fig. 2. [92]Fig. 2 [93]Open in a new tab Relative expression of antioxidant enzymes under different drought time points: (A) Superoxide dismutase (SOD), (B) Peroxidase dismutase (POD) and (C) Proline content (PRO) at 0 day, 7 day, 14 day, and 21 day, respectively Differential transcriptome profile of coconut under drought stress To explore the molecular mechanisms of aromatic coconut under drought, RNA-seq was performed with samples harvested at 0 days (control), 7 days, 14 days, and 21 days of water stress. Each library contained 41.18–43.86 million clean reads, where each of them displayed more than 6 GB in length. The data quality was high, with more than 85% of reads having a Q30 score and a clean reads ratio exceeding 90% when compared to the reference genome, indicating that the data is robust and consistent for further analysis (supplementary file 2). Differentially Expressed Genes (DEGs) were calculated in different coconut samples with a log2 fold-change (logFC) of ≥ 1.5 in their expression across the time points, specifically CK7d-vs-GH7d, CK14d-vs-GH14d, and CK21d-vs-GH21d. Interestingly, the total number of DEGs steadily increased with the duration of drought. The highest numbers of DEGs, 6698 were observed in CK21d-vs-GH21d indicating a significant response to prolonged (21 days) drought durations (Fig. [94]3A-B). Furthermore, the highest numbers of upregulated and downregulated DEGs were also exhibited in CK21d-vs-GH21d treatment, suggesting that coconut plants may inhibit or activate specific gene expressions to cope with drought stress. When the DEGs of these different treatment groups are compared by a Venn diagram, we identified the DEGs that were both specific to drought point and shared across the treatment (Fig. [95]3C). For functional predictions, DEGs with logFC ≥ 1.5 and FDR < 0.05 were analyzed to detect the differential regulatory genes common among the different drought treatments, helping to identify the tolerance mechanisms in coconut. Results revealed twenty-nine differentially expressed genes that were common across the different drought points (Fig. [96]3C). Fig. 3. [97]Fig. 3 [98]Open in a new tab DEGs identified in aromatic coconut under drought stress; (A) Number of upregulated and downregulated differential genes at 7, 14, and 21 days, drought points, respectively (B) Volcano plot of differentially expressed genes at CK21d-vs-GH21d. The x-axis denotes the fold change in gene expression. The y-axis denotes the significance level of the difference (C) Venn diagram showing the different numbers of mutual and specific DEGs at different drought treatments (D) Principal component analysis between different drought treatment groups Additionally, Principal Component Analysis (PCA) was executed to observe the differences between drought treatments. Significant variations were recorded in the expression levels within the treatment groups. In the scatter plot, the X-axis (PC1) demonstrated 91.57% variation, while the Y-axis (PC2) showed 5.00% variation in the data, with PC1 being the primary distinguishing factor (Fig. [99]3D). The data showed that all the drought treatment groups clustered separately, with the 21-day treatment group (GH21d) showing distinct differences. Gene ontology classification and enrichment analysis of DEGs at different drought stress Gene Ontology (GO) analysis was conducted to further characterize the putative biological, cellular and molecular functions of the differentially expressed genes (DEGs). The number of DEGs varied across different treatments and a total of 38 enriched GO items were recorded, including 19 molecular function (MF), 17 biological process (BP), and two cellular components (CC) categories at extended drought. The number of GO terms decreased with shorter stress durations, with the lowest numbers observed in the CK7d-vs-GH7d and CK14d-vs-GH14d treatment combinations, respectively (supplementary file 3). Among the GO terms, the top five were considered for responding to drought at three distinct time points of coconut leaves (Fig. [100]4). In the MF category, the most abundant genes were categorized under binding, catalytic activity, transporter activity, transcription regulator activity, ATP-dependent activity, and antioxidant activity across all water treatment groups. In the BP category, the major terms included cellular process, metabolic process, biological regulation, response to stimulus, and localization. However, the CC category was limited to two main items: cellular anatomical entity and protein-containing complex across all treatments (Fig. [101]4A-C). The annotated genes, based on GO terms, indicated that drought responsive genes might be linked to metabolic processes, response to stimulus, regulation of biological processes, transporter activity, transcription regulator activity, ATP-dependent activity, and cellular anatomical entity under the BP, MF, and CC categories, respectively. This suggests that these genes play significant roles in the coconut’s response to drought stress by participating in various essential biological, cellular, and molecular functions. Fig. 4. [102]Fig. 4 [103]Open in a new tab Top five GO terms of aromatic coconut varieties; (A) CK7d-vs-GH7d (B) CK14d-vs-GH14d, and (C) CK21d-vs-GH21d, under three drought periods at 7, 14, and 21 days, respectively Based on the analysis of DEGs enriched in the uppermost 20 GO terms with the lowest Qvalue < 0.05, these were chosen from all the treatment combinations at various drought points (Fig. [104]5). The DEGs related to the CC category, including cytoplasm, chloroplast and plastid, displayed main functional GO terms in CK21d-vs-GH21d treatment combination (Fig. [105]5A). The data revealed that the most represented CC subcategories originated in the maximum drought period. Additionally, MF GO terms were significantly enriched with a large number of DEGs in the catalytic activity in CK14d-vs-GH14d, drought combination (Fig. [106]5B). Previous report showed that soil moisture is crucial for nutrient uptake from the soil, which is related to catalytic activity [[107]41]. This suggests that the enriched catalytic activity GO terms might be involved in drought tolerance. Fig. 5. [108]Fig. 5 [109]Open in a new tab GO enrichment analysis of aromatic coconut under drought stress; (A) cellular component, (B) molecular function, and (C) biological process following the lowest Qvalue < 0.05 of the top 20 selected GO terms of six treatment groups Moreover, the DEGs associated with the BP category were most useful considering drought stress response, where “response to abiotic stimulus” directly contributed to drought stress response [[110]42]. The major BP functional classes, such as small molecule metabolic process, oxoacid metabolic process, organic acid metabolic process, and carboxylic acid metabolic process, showed enrichment in drought group, CK21d-vs-GH21d. Additionally, the important subcategory “response to abiotic stimulus” was enriched in CK21d-vs-GH21d treatment (Fig. [111]5C). This suggests that the significant differences in DEG enrichment varying with drought frequencies could originate from the differences in enriched genes number in each of those shared GO terms. Therefore, the functional predictions and GO analysis highlighted the involvement of various biological processes, cellular components, and molecular functions in the drought stress response of coconut. The findings suggest that genes related to small molecule metabolic processes, response to abiotic stimulus, and catalytic activity play significant roles in the coconut’s adaptation to drought conditions. Functional annotation and DEGs enrichment analysis using KEGG pathways of aromatic coconut under drought stress The enrichment analysis using KEGG pathways was performed to compare gene functions in aromatic coconut leaves under varied drought stress, aiming to identify the molecular processes related to changes in gene expression. The KEGG pathways were classified into five main clusters: organismal systems, cellular processes, environmental information processing, genetic information processing, and metabolism (Fig. [112]6). These major classes were further divided into 17 sub-classes based on Qvalue < 0.05. Notably, the total number of (DEGs), both upregulated and down-regulated, increased with the duration of drought stress. Therefore, the highest abundance of up-regulated and down regulated genes was observed in CK21d-vs-GH21d (21 days), compared to other treatment groups CK14d-vs-GH14d and CK7d-vs-GH7d (supplementary file 4). Fig. 6. [113]Fig. 6 [114]Open in a new tab KEGG pathway classes in aromatic coconut under drought stress from both up and down-regulated DEGs in CK7d-vs-GH7d, CK14d-vs-GH14d, and CK21d-vs-GH21d Considering the number of drought responsive genes and enriched pathways, “metabolism” emerged as the largest class with the highest number of pathways and genes. Within this category, “global and overview maps” displayed the highest number of both up-and down regulated DEGs. Additionally, higher numbers of upregulated DEGs were recorded in other dominant categories such as “carbohydrate metabolism,” “amino acid metabolism,” and “lipid metabolism” across all drought treatment groups. Conversely, within the metabolism category, pathways such as “metabolic pathways,” “biosynthesis of secondary metabolites,” “phenylpropanoid biosynthesis,” “amino sugar and nucleotide sugar metabolism,” “starch and sucrose metabolism” and “photosynthesis,” exhibited a notable count of downregulated DEGs across all drought stress time points (Fig. [115]6; supplementary file 4). In the second category, “genetic information processing,” most subclasses showed upregulation in DEGs expression. Additionally, the “Environmental Information Processing” category included core subclasses such as “plant hormone signal transduction,” “MAPK signaling pathway plant,” “phosphatidylinositol signaling system” related to signal transduction, and “ABC transporters” linked to membrane transport in aromatic coconut. Remarkably, all the subclasses exhibited a higher number of downregulated DEGs across all treatment points (Fig. [116]6). However, “plant hormone signal transduction” and “MAPK signaling pathway-plant” are crucial subcategories in this group and hold significant importance within the plant kingdom. In the “Cellular Processes” category, downregulated DEGs dominated during lower drought stress treatment (CK7d-vs-GH7d) but upregulated DEGs were prevalent during longer drought durations (14 and 21 days), except in the “Endocytosis” subclass. Finally, in the “Environmental Adaptation” category, the “plant pathogen interaction” subgroup unveiled a prevalence of downregulated DEGs throughout the drought stress periods (Fig. [117]6; supplementary file 4). This result suggests that plant pathogen interaction within organismal systems in KEGG pathways may increase susceptibility to pests and diseases under drought stress in aromatic coconut. In plants, the DEGs involved in drought tolerance and adaptation under water stress are crucial for understanding how plants respond to such stress. To elucidate the function of these DEGs, KEGG enrichment analysis was performed on coconut leaves cultured under various drought treatments: 7 days, 14 days, and 21 days, with a control. The findings revealed a total of 100 pathways consisting of 9,839 DEGs (supplementary file 4). The top twenty enriched pathways were selected for a bubble plot analysis (Fig. [118]7). Among these, the highest number of DEGs were found in the plant pathogen interaction, MAPK signaling pathway plant, and plant hormone signal transduction pathways. These pathways were consistently enriched and showed a gradual increase in DEGs with the duration of drought treatments, CK7d-vs-GH7d, CK14d-vs-GH14d, and CK21d-vs-GH21d, respectively (Fig. [119]7A-C). However, at the 14 day drought treatment (CK14d-vs-GH14d), there was a slight alteration in gene expression in the phenylpropanoid biosynthesis and flavonoid biosynthesis pathways. Previous reports demonstrated that the DEGs related to “plant hormone signal transduction” and MAPK signaling pathway had significant roles in expression under stress conditions [[120]43, [121]44]. Fig. 7. [122]Fig. 7 [123]Open in a new tab Bubble plot of DEGs in different KEGG pathways under drought treatments CK7d-vs-GH7d, CK14d-vs-GH14d, and CK21d-vs-GH21d, respectively (A-C). Each bubble represents a pathway, with its size indicating the number of enriched genes. The color of the bubbles reflects the level of significance for the identical pathway Analysis of DEGs involved in the plant hormone signaling under drought stress in coconut In plants, drought tolerance is a multifaceted trait involving the activation of multiple genes across various regulatory systems. Phytohormones play a vital role in activating associated genes under drought stress, enabling plants to survive in adverse conditions [[124]26]. The DEGs allied with the “plant hormone signal transduction” pathway displayed significant changes in expression in response to drought stress, suggesting their potential role in plant adaptation to water-stressed environments [[125]43]. In this study, we considered genes involved in plant hormone signaling pathways in aromatic coconut under drought conditions. Based on annotation data, we identified 201 DEGs associated with cell enlargement, stem growth, stomatal closure, cell division, cell elongation, and stress response within the plant hormone signaling pathway. These DEGs are involved in various cellular processes through different hormone signaling pathways, such as abscisic acid (ABA), jasmonates (JA), auxin, brassinosteroids (BR), and gibberellin (GA) (supplementary file 5). The brassinosteroid signaling pathway recorded the highest number of DEGs, with eighteen genes showing upregulation and 49 exhibiting downregulation. In the ABA-induced signaling pathway, 22 DEGs were upregulated, while ten DEGs were downregulated. In contrast, five DEGs showed up-regulation, and 20 genes were downregulated in the JA signaling pathway under drought stress. In the auxin signaling pathway, an equal number of DEGs (each 22) showed upregulation and down regulation. Furthermore, the GA signaling pathway displayed six upregulated and eighteen downregulated DEGs (supplementary file 5). Thus, the highest number of downregulated DEGs were associated with BR, JA, and GA hormonal signaling pathways, while the ABA signaling pathway had the highest number of up regulated DEGs (Fig. [126]8; supplementary file 5). These results indicate that multiple hormonals signaling related genes participate in the coconut’s response to drought stress. Fig. 8. [127]Fig. 8 [128]Open in a new tab Expression pattern of DEGs associated with ABA, JA, Auxin, BR, and GA hormone in “Plant hormone signal transduction” pathway under drought treatments (A) CK7d-vs-GH7d; (B) CK14d-vs-GH14d; (C) CK21d-vs-GH21d, respectively. The color indicates the level of expression of the corresponding treatment Based on the expression levels (logFC value), the representative protein governing DEGs involved in each hormone signaling pathway are presented in the heatmap (Fig. [129]8). Notably, higher expression levels and the maximum number of expressed genes were observed in the CK21d-vs-GH21d treatments at 21 days of drought stress. In contrast, the lowest number of expressed genes was recorded in the CK7d-vs-GH7d and CK14d-vs-GH14d treatments at 7 and 14 days, respectively (Fig. [130]8). Our observations suggest that the expression of DEGs is specific to drought frequency, with multiple genes are involved in the plant hormone signal transduction pathway in coconut under drought stress. Analysis of DEGs involved in the MAPK signaling pathway under drought stress in coconut In plants, the drought tolerance mechanism is a complex trait involving multiple genes within regulatory networks. One crucial signaling system in this process is the Mitogen-Activated Protein Kinase (MAPK) pathway, which plays a significant role in controlling various cellular responses under stress conditions such as drought, salinity stress, and oxidative stress. The MAPK signaling pathway is well-recognized for its contribution to plant tolerance and adaptation to stressful environmental conditions [[131]44]. Thus, we investigated genes involved in MAPK signaling pathways and their regulatory mechanisms in the response to drought stress in aromatic coconut. Based on annotation data and previous reports, we identified 77 drought responsive DEGs associated with defense response, root growth, stress adaptation, and stomatal development. Among these: Thirty genes (22 upregulated and eight downregulated) were associated with ABA signaling. Eight genes (two upregulated and six downregulated) were involved in the JA pathway. Twenty-five genes (eight upregulated and 17 downregulated) were related to ethylene signaling. Thirteen genes (three upregulated and 10 downregulated) were involved in stomatal development under the MAPK signaling pathway during drought stress (supplementary file 6). Based on their expression levels (logFC value) and their linkage with specific pathways, the protein-governing DEGs involved in different hormone signaling pathways and stomatal development are displayed in the heatmap (Fig. [132]9). Notably, higher expression levels and a maximum number of expressed genes were observed in the CK21d-vs-GH21d treatments at 21 days of drought stress. In contrast, the lowest number of expressed genes was recorded in the CK7d-vs-GH7d and CK14d-vs-GH14d treatments at 7 and 14 days, respectively. These findings propose that the expression of identified genes is specific to the frequency of drought stress, and various genes are involved in the MAPK signaling pathway in response to drought stress in aromatic coconut. Fig. 9. [133]Fig. 9 [134]Open in a new tab Expression pattern of DEGs associated with ABA, JA, Ethylene hormone, and Stomatal development in MAPK signaling cascade under drought treatments (A) CK7d-vs- GH7d; (B) CK14d-vs-GH14d; (C) CK21d-vs-GH21d, respectively. The color indicates the level of expression of the corresponding treatment Protein-protein interaction within DEGs regulating drought tolerance in coconut Drought is a complex issue in plants, recognized by multiple sensors, with signals conveyed through distinct parallel signaling networks. These networks trigger responses such as stomatal closure, root growth, and synthesis of stress-related proteins and their governing genes [[135]19]. The protein-protein interaction network analysis was conducted to identify DEGs involved in the drought tolerance mechanism in coconut. This analysis was based on the protein functions associated with defense response, root growth, stress adaptation, stomatal development (MAPK), cell enlargement, plant growth, stomatal closure, cell division, and stress response (plant hormone response) among the DEGs. In this study, 22 proteins were found to be involved in interaction networks among 42 proteins, with the remaining genes possibly functioning independently in their related pathway under drought conditions (Fig. [136]10). Among these, we identified core proteins including AUX1, PIF3, BAK1, NPR1, EIN3, MYC2, CTR1, and ETR1. These proteins are related to various functions such as defense response (5), root growth (3), stress adaptation (2), and stomatal development (1), which are likely to regulate drought tolerance in coconut. Interestingly, no proteins related to ABA signaling were involved in these interaction networks (Fig. [137]10), suggesting that ABA-related genes might work independently to regulate stomatal closure during stress adaptation in coconut. These results reveal that these proteins may be part of a regulatory network that contributes either through an independent pathway or in collaboration with related pathways in response to drought stress in coconut. Fig. 10. Fig. 10 [138]Open in a new tab The protein-protein interaction networks between core genes and genes related to cell development and plant hormone response in coconut transcriptome. The functional interaction was analyzed at STRING database (V 12.00) Validation of the candidate genes for drought tolerance through qPCR To confirm the consistency of the transcriptome data, nine DEGs associated with drought tolerance were randomly designated for qRT-PCR analysis (Fig. [139]11). All the genes were selected based on their expression levels and their association with different hormone signaling pathways such as plant hormone signal transduction, and MAPK signaling pathway under varied drought stress. The qRT-PCR gene expression results showed almost similar trends in differential expression patterns to those observed in the RNA-seq data with some exceptions (Fig. [140]11). Our result demonstrates that transcriptomic data are reliable and valuable for the further improvement of drought tolerant coconut varieties. Fig. 11. [141]Fig. 11 [142]Open in a new tab Expression levels comparison of selected genes in aromatic coconut under different drought stress conditions. Data represent the mean ± SD of five independent biological replicates Discussion Coconut cultivation, ranging from tropical to subtropical regions, is believed to play a crucial role in providing nutrition and maintaining ecological balance [[143]2, [144]3], thereby supporting coastal livelihoods. Drought significantly impacts the growth and productivity of coconut trees by limiting water and nutrient uptake [[145]5] and producing lower-quality pollen and/or gametes during fertilization [[146]28]. Plants generally employ three survival strategies under drought conditions: escape, avoidance, and tolerance which involve in accelerating the reproductive stage, maintaining cell water potential, and altering gene expression [[147]22]. Adaptations to drought stress include stomatal closure, root regulation, balancing cell water pressure, enzyme synthesis, signal transduction, and gene expression [[148]19, [149]45]. Therefore, identifying genes associated with drought tolerance to facilitate the genetic engineering of coconut is essential for improving their ability to adapt to water scarce areas. RNA-seq is an influential tool for perusing differential gene expression in aromatic coconut, helping to understand the molecular mechanisms and predict candidate genes involved in drought stress. Additionally, the activity of antioxidant enzymes in plants is often used as a biomarker for assessing stress responses [[150]4]. Therefore, we measured the activity of SOD and POD to determine their role in supporting drought tolerance in aromatic coconut. However, details for discussion are provided below with the sub-headings. Antioxidant activities and proline levels in aromatic coconut leaves under drought stress In response to drought stress, plants initiate various drought responsive mechanisms, including morphological and physiological changes, biochemical processes, gene expression, hormone production, and the synthesis of secondary molecules to cope with the adverse environment. Under drought conditions, plants produce ROS from photosynthetic and respiratory electron leakage in chloroplasts. ROS, such as oxygen radicals, act as messengers in cell signaling but can cause cellular damage and decrease plant productivity when they exceed equilibrium thresholds [[151]16]. The generation of ROS is a fundamental process in higher plants, serving as an indicator molecule that activates protective responses through transduction pathways under stress environments [[152]46]. Balancing ROS production and scavenging is essential for plant defense systems, maintained by both enzymatic and non-enzymatic antioxidant systems under stress. Plants have evolved mechanisms to cope with oxidative stress, maintaining cellular homeostasis through regulated gene expression and continued activities of various antioxidant enzymes [[153]40]. Plants possess a natural antioxidative defense system, where SOD is a key component. POD and catalase (CAT: EC 1.11.1.6) also play crucial roles by neutralizing potential oxidants and initiating protective mechanisms to balance cell metabolism. SOD acts as the first line of defense in plants under drought stress, dismutation superoxide (O2-) into hydrogen peroxide (H[2]O[2]), which is then decomposed by POD to mitigate the negative effects caused by ROS in plant cells [[154]46, [155]47]. Furthermore, SOD activity is considered an important biomarker for selecting highly tolerant plant cultivars, as it is positively correlated with antioxidant capacity [[156]4, [157]40]. Additionally, proline tends to accumulate in response to various stresses [[158]38], helping to protect plants from the detrimental effects of drought stress and acting as a detoxifying agent for ROS. The results of the present study revealed that the activities of enzymatic antioxidants, such as POD, SOD and PRO levels, were enhanced in coconut leaves in response to various drought stress (Fig. [159]2). In all drought treatments, a significant variation was recorded in SOD, POD activities and PRO levels compared to control treatments. An increasing trend was observed in SOD, POD activities and PRO levels as drought periods extended, though the saturation points differed. SOD activity and PRO levels increased significantly during the first two weeks of drought stress, stabilizing at 21 days. In contrast, POD activity significantly increased in the first week of drought stress but decreased after two weeks of stress. Previous studies have reported similar trends in the activities of antioxidant enzymes and proline content in coconut under stress [[160]38, [161]48]. These data suggest that both biochemical traits may have regulatory elements that function within biological and genetic networks under drought stress conditions. However, SOD activity is controlled by hormones such as abscisic acid and other signaling molecules through gene expression [[162]17]. Furthermore, ROS work as secondary messengers, conveying signals to the nucleus through the MAPK pathway, helping plants to survive in stressful conditions, including drought [[163]44]. In this study, the activities of SOD and POD were analyzed as key antioxidant enzymes involved in regulating ROS, either directly or indirectly, to mitigate free radicals in plant systems. Our findings suggest that these antioxidant enzymes play a crucial role in enhancing drought tolerance in coconut seedlings. Still, this study has some limitations. While assessing ROS generation would provide deeper insights into oxidative stress regulation, we were unable to include direct ROS activity measurements due to technical constraints. Future studies incorporating a more comprehensive analysis of ROS levels and additional antioxidant enzymes would further strengthen our understanding of the molecular mechanisms underlying drought stress responses in coconut. Analysis of RNA-Seq data and transcriptome profiling of coconut leaves under drought stress Drought tolerance in plants is a complex mechanism controlled by a set of gene actions. In this study, we used RNA-seq to explore the expression differences in the coconut transcriptome under varied drought durations of 0, 7, 14, and 21 days, providing insights into the functional study of genes as previously reported [[164]38]. Compared to the reference genome, all the data displayed more than 85% clean reads with a Q30 score exceeding 90%, indicating their high integrity. We observed differentially expressed genes (DEGs) according to the expression duration, providing clues for identifying drought specific responses in coconut and associating transcripts with biological functions. Gene Ontology (GO) terms provided information on the biological roles of transcripts in response to drought stress, aiding in the identification of functional genes and their tolerance mechanisms. Major biological process (BP) groups, such as cellular processes, metabolic processes, biological regulation, response to stimulus, and localization, were exposed across the three drought treatment combinations (Fig. [165]4). Additionally, the BP functional subclass “response to abiotic stimulus” was found over three treatment groups CK7d-vs-GH7d, CK14d-vs-GH14d, and CK21d-vs-GH21d at 7, 14, and 21 days of drought stress (Fig. [166]4A-C). DEGs related to the BP category are involved in the drought stress response, with “response to abiotic stimulus” reported as major contributors [[167]42]. Consequently, functional genes and pathways have been classified into different regulatory groups based on their expression differences. This suggests that DEGs are divided into various functional groups under a complex regulatory network that changes with drought period. Based on the GO enrichment analysis, GO items such as cytoplasm, chloroplast, and plastid were recorded in the CK21d-vs-GH21d drought point (Fig. [168]5A). Additionally, molecular function (MF) GO terms were significantly enriched with a large number of DEGs related to catalytic activity in the CK14d-vs-GH14d drought combinations (Fig. [169]5B). Catalytic activity is crucial for nutrient uptake from the soil under drought conditions, as reported [[170]41]. Moreover, major biological process (BP) functional classes, such as small molecule metabolic process, oxoacid metabolic process, organic acid metabolic process, and carboxylic acid metabolic process, were found in the CK21d-vs-GH21d drought groups. The response to the abiotic stimulus was also enriched in the CK21d-vs-GH21d treatment (Fig. [171]5C). These data suggest that DEG enrichment varies with drought frequencies and might be associated with the maximum drought period in coconut. The DEGs involved in plant drought tolerance and adaptation are crucial for understanding the responses to stress. To elucidate the molecular mechanisms of these DEGs, KEGG enrichment analysis was performed on coconut leaves subjected to various drought combinations. Considering the number of drought-responsive genes and enriched pathways, “metabolism” emerged as the main class with the highest number of genes. Additionally, higher numbers of upregulated DEGs were recorded in other dominant categories such as “carbohydrate metabolism,” “amino acid metabolism,” and “lipid metabolism” across all drought treatment groups. Conversely, within the metabolism category, pathways such as “metabolic pathways,” “biosynthesis of secondary metabolites,” “phenylpropanoid biosynthesis,” “amino sugar and nucleotide sugar metabolism,” “starch and sucrose metabolism” and “photosynthesis,” exhibited a notable count of downregulated DEGs across all drought stress time points. Metabolites and biomolecules are predominantly associated with different pathways related to flavonoids, carbohydrate, amino acid, lipid, and nucleotide metabolism, and regulating plant secondary metabolites under environmental stress [[172]38, [173]49]. Finally, major drought stress signal transduction pathways and regulatory genes were identified through subsequent analysis of KEGG and GO enrichment. The results revealed that more signaling pathways were identified in plant-pathogen interaction, MAPK signaling pathway-plant, and plant hormone signal transduction pathways in response to drought stress. Similarly, previous report demonstrated that response to stimuli and signal transduction are essential processes in plants in abiotic stress response specially drought [[174]42, [175]50]. Plants have various adaptation mechanisms to cope with environmental stresses through molecular networks, signaling pathways, and the expression of stress- responsive genes [[176]22, [177]23]. Stomatal regulation is a key mechanism for minimizing water loss through reduced transpiration under drought conditions, although it results in decreased CO2 intake due to stomatal closure [[178]19, [179]51]. Conversely, roots play a crucial role by increasing root length and density to enhance adaptation to drought stress [[180]20]. Signal transduction regulates various pathways involved in stress responses, supported by hormones [[181]52]. In contrast, plant hormones play a critical role in regulating plant adaptation to drought stress through changes in biosynthesis and signaling pathways, including abscisic acid (ABA), jasmonates (JA), auxin (AUX), brassinosteroids (BR), ethylene (ET), and gibberellin (GA) [[182]26]. In this study, we conducted an in-depth analysis of gene expression related to signal transduction pathways, including plant hormone signaling and the MAPK cascade, which are crucial for responding to stimuli and signal transduction under stress conditions [[183]42, [184]44]. However, proteins and receptor kinases function as sensors on the cell membrane, receiving external signals and conveying them to target genes to initiate stress responses. Drought tolerance mechanism in coconut through plant hormone signal transduction pathway Cell signaling via cell-surface embedded receptors is crucial for the survival and development of plants. In plants, this signaling is primarily mediated through Receptor-Like Kinases (RLKs), which are considered one of the major types of receptors. These RLKs play a pivotal role in perceiving external stimuli, such as environmental stresses [[185]53]. In this study a large number of DEGs are involved in phytohormonal signaling transduction under drought stress, however, these are discussed below in detail. ABA-induced signaling in coconut leaf transcriptome under drought stress Abscisic acid (ABA) plays an important role in plant development, controlling various signal transduction cascades to adjust to drought stress [[186]24, [187]26]. ABA activates different physiological processes, including the biosynthesis of biomolecules, stomatal closure, alteration of root architecture, and transcriptional and post-transcriptional gene expression [[188]54]. ABA is particularly important for drought adaptation by regulating stomatal closure in plants. Under drought stress, PYR/PYL acts as an ABA receptor in the ABA signaling networks and shows a positive correlation with the initiation of ABA receptor genes [[189]55]. In contrast, the protein phosphatase 2 C (PP2C) interacts with sucrose non-fermenting 1-related protein kinase2 (SnRK2), which directs the molecular mechanisms facilitating ABA-induced stomatal closure in plants [[190]26]. The expression of these associated genes may inhibit the stomata guard cells, reducing gaseous exchange and activating regulatory elements in plants [[191]56]. In this study, we identified several genes involved in the ABA signaling pathway under drought stress through the expression of drought-responsive genes (Fig. [192]12). Under drought stress, the genes COCNU_16G003900, COCNU_16G006150, and COCNU_10G002700, which govern the ABA receptor proteins (PYR/PYL), showed downregulated expression. Additionally, genes including COCNU_06G002610, COCNU_07G010020, and COCNU_10G007630, which regulate PP2C expression, as well as COCNU_03G007040 and COCNU_07G004780, related to SnRK2 expression, exhibited upregulation (Fig. [193]12). This upregulation promotes the regulation of the ABA response binding element ABF regulating gene (COCNU_13G004260), which controls stomatal closure by reducing water evaporation and improving the stress tolerance of coconut. Previous studies have indicated that the expression of ABF is induced by drought stress in the ABA signaling pathway, which positively regulates the drought tolerance of plants [[194]57]. These results suggest that the expression of ABA signal transduction-related genes is crucial for showing drought tolerance in coconut. Fig. 12. [195]Fig. 12 [196]Open in a new tab Expression patterns of different genes in the “plant hormone signal transduction” cascade. The color scale specifies the FPKM values for these genes. Red colors denote relatively upregulated DEGs, while green colors represent relatively downregulated DEGs Jasmonic acid signaling in coconut leaf transcriptome under drought stress Jasmonic acid (JA) and its precursors play vital roles in plant growth and development, including reproductive parts production, promoting leaf senescence, root formation, and regulating defense mechanisms against abiotic stresses such as drought [[197]58]. JA contributes to stress adaptation strategies by promoting stomatal closure and deep root growth, helping plants adapt to water stress conditions. Additionally, the exogenous application of JA has been shown to increase drought tolerance in plants [[198]26]. On the other hand, JA does not work independently but participates in various signaling systems with other phytohormones to cope with stress [[199]59]. In plants, multiple physiological, biochemical, genetic, and molecular mechanisms induced by JA are involved in surviving and adjusting to stress environments, either alone or in combination with other phytohormones [[200]60]. Previous reports have revealed that JA regulates plant tolerance to drought stress through the signaling pathway involving the CORONATINE INSENSITIVE 1 (COI1) receptor, JASMONATE ZIM-DOMAIN PROTEIN (JAZ) repressors, and MYC2, a JA-responsive transcription factor [[201]61]. Therefore, the JAZ-MYC component plays a crucial role in the JA signaling pathway through the expression of JA-responsive genes [[202]58]. The present study demonstrated that drought tolerance in coconut seedlings was associated with the downregulation of the JAR1 gene (COCNU_14G001780), which controlled the downregulation of JAZ genes (such as COCNU_03G001450, COCNU_03G004790, COCNU_06G020370, COCNU_13G001890) and MYC2 governing genes (such as COCNU_03G015250, COCNU_08G000560, COCNU_10G005790) (Fig. [203]12). These results suggest that JA-responsive genes, including JAR1, COI1, JAZ, and MYC2, might be important for improved drought tolerance in coconut. Recently, in the JA signaling pathway, JAZ and MYC2 were found to be induced by drought stress and potentially play a significant role by inhibiting the transcription of downstream genes that regulate stomatal closure and root formation in plants [[204]58, [205]62]. Auxin-regulated signaling in coconut leaf transcriptome under drought stress Auxin regulates various aspects of plant growth and development, including seed germination, stem elongation, flowering, and fruit enlargement [[206]63]. It also helps in root branching, pollen fertility, and coordinates plant responses to drought tolerance mechanisms [[207]23], showing tissue-specific responses to exogenous application [[208]64]. In the auxin signaling pathway, auxin-responsive genes Aux/IAA act as co-receptors and transcriptional repressors, contributing to drought tolerance in Arabidopsis [[209]65]. Additionally, auxin-responsive genes SAUR and GH3 play substantial roles in plant drought tolerance [[210]66]. Auxin response factor (ARF) binds to the promoter regions of genes, triggering or suppressing transcription to improve stress tolerance. The TIR1/AFB-Aux/IAA-ARF complex has a major function in the drought tolerance of the plant auxin signal transduction pathway [[211]67]. In our study, we identified various genes tightly linked to the drought tolerance auxin signaling pathway in coconut seedlings, including AUX1 (COCNU_04G000380), TIR1 (COCNU_06G010260), IAA (COCNU_01G021130, COCNU_06G004290), ARF (BGI_novel_G000157, COCNU_06G018800), GH3 (COCNU_01G007120, COCNU_05G002640), and SAUR (COCNU_07G006310, COCNU_07G014290), which showed both upregulation and downregulation. Interestingly, all auxin signaling related proteins were identified in this study. Among them, AUX1 and TIR1-related genes exhibited downregulation under drought stress (Fig. [212]12). These findings suggest that the Aux/IAA-ARF complex might control drought tolerance in coconut seedlings, either alone or in combination with other related genes, by enhancing root initiation and branching, as previously described [[213]50]. Brassinosteroids-regulated signaling in coconut leaf transcriptome under drought stress Brassinosteroids (BRs) are considered steroidal phytohormones participated in different biological and cellular processes in plants, including stem and leaf expansion, flower development, fruit maturation, root initiation, chlorophyll production, and modulation of gene expression [[214]68]. Besides, they also help in stomata formation in plants, enhancing plant resistance to biotic and abiotic stresses [[215]25]. The exogenous application of BR can improve photosynthesis capacity, which shows a positive correlation with stress tolerance [[216]68]. BR binds to the cell surface receptor kinase BRASSINOSTEROID INSENSITIVE1 (BRI1) and BRI1-associated receptor kinase (BAK1), triggering a signal transduction cascade that includes the BSU1 protein and BRASSINOSTEROID-INSENSITIVE 2 (BIN2) proteins, which may control the target genes [[217]69]. In contrast, ZmBSK1 gene has shown increased tolerance to protect maize plants by inhibiting the antioxidant defense system under drought stress [[218]70]. Thus, BIN2 activates the expression of downstream genes in BR signaling which provided the novel functions of CqBIN2 gene in regulating drought tolerance in plants [[219]50, [220]71]. Furthermore, CYCD is a key regulatory factor promoting transformation in controlling cell division [[221]72]. The TCH4 gene contributes to cell wall maintenance, which plays a crucial role in plant secondary growth and resistance to abiotic and biotic stress [[222]73]. Our experimental analysis showed both up and downregulation of genes in BRI (BGI_novel_G000763, BGI_novel_G000764, COCNU_08G004860) and BAK1 (COCNU_06G003410, COCNU_15G001430), with TCH4 (COCNU_02G018860, COCNU_11G013000) showing up-regulation. Additionally, the genes BKI1 (COCNU_06G003980), BIN2 (COCNU_03G013950), BZR1_2 (COCNU_06G003640), and CYCD3 (COCNU_01G010750) exhibited downregulation, but the BSK gene (BGI_novel_G001529, COCNU_06G015620) showed up-regulation under drought stress treatments (Fig. [223]12). The results revealed that both the up and down expression of BRI and BAK1 might lead to the upregulation of BSK. Finally, the signals inhibit the downregulated expression of BIN2, TCH4, and CYCD3 genes, resulting in drought stress responses in coconut. Recently, similar results found in Mesona chinensis Benth grass under drought stress [[224]43]. Gibberellin-regulated signaling in coconut leaf transcriptome under drought stress Gibberellin (GA) is integral to the plant cell division and elongation system throughout the entire life cycle and enhances drought tolerance by suppressing plant growth [[225]74]. Additionally, GA deactivation inhibits guard cells, which contributes to stomatal movement during initial drought stress and controls the transpiration rate in plants [[226]75]. The exogenous application of GA has been shown to improve drought tolerance by enhancing the physiological and biochemical activities of canola plants [[227]76]. In contrast, transgenic plants with lower GA levels exhibit smaller leaves with increased stomatal density, collectively reducing the transpiration rate [[228]77]. In the GA signaling pathway, GIBBERELLIN-INSENSITIVE DWARF1 (GID1) acts as a GA receptor and triggers DELLA proteins, initiating the GA-GID1-DELLA cascade, which affects leaf growth and stem elongation [[229]78]. Phytochrome-interacting factors (PIFs), recognized as transcription factors (TFs), play vital roles in the adaptation of tobacco plants by inhibiting growth and development under drought stress [[230]79]. In this study, the GID1 genes (COCNU_01G016750, COCNU_scaffold012153G000190) and PIF3/4 genes (COCNU_01G022490, COCNU_14G009040) displayed both up- and downregulation among the differentially expressed genes (DEGs). Interestingly, the DELLA governing genes (COCNU_03G008100, COCNU_13G005160) showed down-regulation in coconut seedlings under drought stress (Fig. [231]12). These results indicate that the GID1–DELLA complex might trigger the DELLA proteins to bind with PIF3/4 genes, leading to the activation of GA in response to drought stress in coconut. Previous findings confirm the collaborative function of GID1-DELLA-PIF3 genes in enhancing drought resistance in plants [[232]77, [233]79]. Drought tolerance mechanism in coconut leaf transcriptome through MAPK signaling pathway Based on the transcriptomic data, we further analyzed the KEGG pathways and identified key drought responsive genes associated with abscisic acid (ABA), jasmonic acid (JA), ethylene signaling, and stomatal development pathways within the MAPK cascade. Previous studies have highlighted the roles of these three hormones; ABA, JA, and ethylene in drought tolerance mechanisms [[234]26]. Under drought stress, we observed the core components governing genes in the ABA signaling pathway [[235]80]: PYL-associated genes: COCNU_10G002700, COCNU_16G003900, and COCNU_16G006150; PP2C-related genes: COCNU_06G002610, COCNU_07G010020, COCNU_10G007630, and COCNU_16G008310; SnRK2-linked genes: COCNU_03G007040 and COCNU_07G004780; MAPKKK18-connected gene: COCNU_02G004810; MKK3-correlated gene: COCNU_05G010800 and MPK1/2-accompanied gene: COCNU_14G005810 (Fig. [236]13). Notably, the genes encoding proteins PYL, PP2C, and SnRK2 are also expressed within the ABA pathway, playing critical roles in the hormone signaling system in plants. We propose that PYL functions as a receptor, and the interaction between protein phosphatase 2 C (PP2C) and SnRK2 aids in the regulation of MAPKKK18. This interaction ultimately inhibits the expression of MKK3 and MPK1/2 genes under drought stress. Fig. 13. [237]Fig. 13 [238]Open in a new tab Expression patterns of different genes in the ABA and JA-regulated signaling pathways of the MAPK cascade. The color scale specifies the FPKM values for these genes. Red colors denote relatively upregulated DEGs, while green colors represent relatively downregulated DEGs Previous studies have shown that in the MAPK cascade, the MAP3K17/18-MKK3-MPK1/2 signaling pathway is triggered by the PYR/PYL/RCAR-SnRK2-PP2C module under the ABA signaling network [[239]81]. Our findings suggest that the functions of these genes within the ABA module are critical for processes such as stomatal closure and root growth, which are essential for drought tolerance in coconut [[240]24, [241]82]. These insights could provide theoretical knowledge on drought tolerance mechanisms and aid in the development of drought-resistant coconut varieties. In the jasmonic acid (JA) signaling pathway, we identified several key genes involved in drought stress response in aromatic coconut. These include: MYC2-related genes: COCNU_02G001280, COCNU_03G015250, COCNU_07G003710, COCNU_08G000560, and COCNU_10G005790; and MKK3-allied gene: COCNU_05G010800 (Fig. [242]13). Previous research has demonstrated that the MKK3-MPK6-MYC2 signaling cascade is crucial for seedling development in Arabidopsis thaliana [[243]83]. Moreover, MYC2 is known to play a significant role in regulating stomatal closure and root formation in plants under drought stress conditions [[244]58, [245]84]. Our findings suggest that the MKK3 gene may support the downregulation of MYC2 genes, which could operate independently or in conjunction with other transcription factors. This regulation may be part of a mechanism that enables aromatic coconut to adapt to drought stress environments by modulating stomatal closure and root growth. This insight into the JA signaling pathway further enhances our understanding of the molecular responses in coconuts under drought conditions and provides potential targets for developing drought tolerant coconut varieties. Ethylene, synthesized in plants, acts as a crucial signaling molecule engaged in drought tolerance by regulating the expression of CHIB genes within the MAPK cascade [[246]85]. Key components of ethylene signaling include the endoplasmic reticulum (ER) and a series of proteins such as constitutive triple response 1 (CTR1), ethylene-insensitive 3 (EIN3), EIN3-like (EIL), ethylene response factor (ERF), and ethylene response sensor (ERS). In this pathway, ETR/ERS and EIN3 function as receptors that transmit signals to CTR1, which in turn inhibits genes associated with EIN3 transcription factors [[247]86]. In this study, we identified a number of genes associated with drought tolerance in coconut seedlings through ethylene signaling, including ETR/ERS (BGI_novel_G001528, COCNU_07G010070), CTR1 (COCNU_07G007110, COCNU_09G008920), MKK9 (COCNU_03G009700), EIN3 (BGI_novel_G000063, BGI_novel_G001241), ERF1 (COCNU_07G010400, COCNU_scaffold001401G000010), CHIB (COCNU_01G022440, COCNU_02G014140), copA/RANI (BGI_novel_G000854, BGI_novel_G001899) and XRN4 (COCNU_02G012310) (Fig. [248]13). We hypothesize that the upregulation of CTR1 genes might activate or repress EIN3, which directly binds to the target gene ERF1 and represses CHIB gene expression under drought stress in coconut. ERF genes are known to control both transcriptional and post-translational mechanisms, contributing to plant growth, development, hormone responses, and associated with anthocyanin accumulation [[249]87, [250]88]. Our findings suggest that the down-regulation of the ERF1 gene inhibits the expression of CHIB genes, which could enhance the defense response in coconut seedlings under drought conditions. This complex interaction within the ethylene signaling pathway indicates a finely tuned regulatory mechanism that allows coconut seedlings to adapt to drought stress by modulating key genes and pathways involved in ethylene signaling. Additionally, stomata are essential for proper photosynthesis as they act as channels for the exchange of oxygen, carbon dioxide, and water between the plant’s outer and inner tissues [[251]89]. Various signals, including those from the EPIDERMAL PATTERNING FACTORS (EPFs) family, plant hormones, and environmental factors, play vital roles in regulating stomatal density and the different steps of stomatal development. A well-known MAPK cascade, consisting of YODA, MKK4/5, and MPK3/6, mediates upstream signals to regulate stomatal distribution in plants. The ERECTA (ER) and ER-LIKE (ERL) receptors receive extracellular EPF signals, which regulate stomatal patterning and leaf shape, and SPCH promotes gene expression involved in these processes [[252]90]. Under water stress conditions, EPF1 has been shown to reduce stomatal density, thereby improving drought tolerance in barley [[253]91]. It has also been reported that de novo genes derived from noncoding regions are linked to photosynthesis and plastid modification, which regulate plant growth and development [[254]92]. In this study, we identified several genes involved in stomatal development that contribute to drought tolerance in coconut seedlings, including EPF1/2 (COCNU_06G009520), ER/ERLs (BGI_novel_G000764, COCNU_08G004860), MKK4/5 (COCNU_01G004520, COCNU_01G008740), MPK3 (COCNU_04G014540), and SPCH (COCNU_07G008420). Among these, the upregulation of EPF1/2 and the downregulation of MKK4/5, MPK3, and SPCH-related genes might reduce stomatal density under drought stress (Fig. [255]13). These results suggest that genes related to stomatal development are crucial for drought tolerance in coconut by regulating stomatal density [[256]93]. In this study, we identified several genes involved in stomatal development that contribute to drought tolerance in coconut seedlings, including EPF1/2 (COCNU_06G009520), ER/ERLs (BGI_novel_G000764, COCNU_08G004860), MKK4/5 (COCNU_01G004520, COCNU_01G008740), MPK3 (COCNU_04G014540), and SPCH (COCNU_07G008420). Among these, the upregulation of EPF1/2 and the downregulation of MKK4/5, MPK3, and SPCH-related genes suggest a potential reduction in stomatal density under drought stress (Fig. [257]13). These findings indicate that genes regulating stomatal development play a critical role in drought tolerance by modulating stomatal density to minimize water loss. However, this study has some limitations. While we acknowledge the importance of chlorophyll content estimation as an indicator of photosynthetic efficiency under drought stress, we were unable to conduct this experiment due to technical and logistical constraints. Future research integrating chlorophyll content measurements alongside stomatal density analysis would provide a more comprehensive understanding of the physiological responses to drought stress in coconut seedlings. Furthermore, no DEGs related to proline were identified in our transcriptomic analysis under drought stress. This absence may be due to the transcriptional activation of proline biosynthesis genes not being detectable within the 21-day drought stress period and/or proline accumulation not being a dominant stress-response strategy in coconut plants. Further studies based on functional genomics and metabolomics are required to dissect the role of coconut leaf transcriptome under drought-stress conditions. Conclusions The antioxidant activities and proline content exhibited significant effects on coconut leaves under drought stress, indicating their potential as reliable biomarkers for selecting stress-tolerant coconut plants. Comparative transcriptome analysis of aromatic coconut leaves in response to 7, 14, and 21 days of drought stress revealed key genes and regulatory pathways associated with drought tolerance in coconut. KEGG enrichment analysis highlighted the involvement of DEGs in plant hormone signal transduction and the MAPK signaling pathway cascade. Under drought stress, the expression of genes related to plant growth, stomatal closure, cell division, stress response, stress adaptation, and stomatal development may play a crucial role in drought tolerance in coconut. While significant progress has been made in identifying drought tolerance mechanisms in aromatic coconut, gaps remain in understanding the molecular mechanisms governing stomatal development and ROS activity in stress adaptation. Our findings suggest that multiple genes contribute to coconut drought tolerance through various hormone signaling pathways, including ABA, JA, auxin, BR, GA, and ethylene. Additionally, the identified candidate genes and pathways could be instrumental in developing strategies to enhance drought tolerance in coconut plants. Future multi-omics studies integrating proteomics and metabolomics are necessary to further elucidate the molecular mechanisms underlying drought stress tolerance in coconut seedlings. Electronic supplementary material Below is the link to the electronic supplementary material. [258]12870_2025_6554_MOESM1_ESM.xlsx^ (10.3KB, xlsx) Supplementary Material 1: List of primers used in qRT-PCR assay. [259]12870_2025_6554_MOESM2_ESM.xlsx^ (9.3KB, xlsx) Supplementary Material 2: RNA-seq data summary of coconut seedlings under drought stress. [260]12870_2025_6554_MOESM3_ESM.xlsx^ (12.9KB, xlsx) Supplementary Material 3: GO analysis of coconut seedlings under different drought combinations. [261]12870_2025_6554_MOESM4_ESM.xlsx^ (23KB, xlsx) Supplementary Material 4: Up-regulation and down-regulation of differentially expressed genes. [262]12870_2025_6554_MOESM5_ESM.xlsx^ (22.3KB, xlsx) Supplementary Material 5: DEGs involved in hormone signaling pathways under drought stress. [263]12870_2025_6554_MOESM6_ESM.xlsx^ (17.1KB, xlsx) Supplementary Material 6: DEGs involved in MAPK signaling pathway under drought stress. Acknowledgements