Introduction

Date palm (Phoenix dactylifera L.), a member of Arecaceaefamily, is a dioecious, monocot, perennial woody fruit palm, harbouring a remarkably complex genomic landscape. It is a resilient icon of Arabian Peninsula, boasts a large and contiguous genome, exceeding 760 Mbp1,2,3,4. With its origins potentially tracing back to Iraq, this iconic plant holds significant historical importance in the Arabian Peninsula. Its cultivation spans both the New World (the American continent) and the Old World (Africa, Asia, and Europe), with cultivation zones stretching from the East Indus Valley to the West Atlantic Ocean5. Global date production currently stands at ca. 9.91Ā million tonnes, cultivated across 1.28Ā million hectares6. Within this global landscape, Saudi Arabia emerges as a hotspot of date palm diversity, boasting over 400 cultivated cultivars and ranking second in global date palm production, with 1.61Ā million tonnes harvested from an area exceeding 156 thousand hectares6. Date palm stands as an emblem in the arid regions in Saudi Arabia, embodying cultural significance and economic importance. As a quintessential desert crop, its resilience to drought is of paramount interest to researchers. Three cultivars, Khalas, Reziz, and Sheshi, stand out among 400 cultivars for their popularity driven by consumer preference in Saudi Arabia7, while Khalas is widely cultivated in the Eastern province of Saudi Arabia. These cultivars have garnered popularity for their distinctive attributes, contributing to their prominence in the regionā€™s agricultural landscape.

Saudi Arabia is nestled with vast Arabian Peninsula drenched with arid land agriculture. Numerous abiotic factors, including prolonged drought, scorching temperatures, and saline soils, significantly impact the yield and lifespan of date palm cultivars8. This dryland crop thrives under harsh environmental conditions and has the ability to withstand prolonged drought periods, high temperatures, and saline soils. However, climate change casts an ominous shadow over this resilient species. These abiotic stressors not only directly affect date palm health and yield but also exacerbate the spread of pests and diseases9. Estimates predict that abiotic stresses, especially drought alone, could cause a staggering 50% reduction in average crop yield by mid-century10. This phenomenon disrupts plant growth, photosynthetic activity, nutrient and water relations, and assimilate partitioning, ultimately causing significant yield reductions11. To face this escalating challenge, developing and screening drought-tolerant date palm cultivars has become a high priority. However, unlike other crops, date palm breeding programs have not adopted sophisticated approaches, relying instead on random selection based on yield and quality12. Therefore, a high level of heterogeneity within date palm germplasm regarding drought tolerance is expected.

When plants confront drought conditions, alterations occur in their photosynthetic machinery, leading to changes in photosynthate source/sink relationships and overall plant development13,14. Water uptake by roots fuels both photosynthetic conversion and biomass production, with transpiration returning most absorbed water to the environment. Stomata play a crucial role in balancing these processes, facilitating CO2acquisition for photosynthesis alongside water loss through stomatal conductance. During drought stress, plants primarily employ stomatal closure to limit transpiration, minimizing water loss15. Drought triggers over-accumulation of reactive oxygen species (ROS), leading to reduced photosynthetic activity by reduction in chlorophyll synthesis and enhancing chlorophyll degradation16,17. This intricate interplay of physiological processes underscores the multifaceted strategies employed by plants to navigate and adapt to water scarcity. As drought persists, the relative water content (RWC) of leaves diminishes, leading to reduced stomatal conductance, photosynthetic rates, and transpiration. Studies conducted on date palm cultivars Sheshi18and Khalas19revealed that water stress triggered a decline in chlorophyll levels, photosynthetic rates, stomatal conductance, and transpiration, while concurrently boosting water use efficiency (WUE). Under controlled drought conditions, these cultivars showcased an augmented WUE alongside decreased gas exchange characteristics20.

Drought tolerant plants possess an orchestra of genetic traits that enable them to endure prolonged periods of water scarcity with remarkable resilience. From morphological adaptations, such as deeper roots and smaller leaves, to intricate physiological, biochemical, and molecular adjustments, plants undergo a dynamic transformation under drought stress. Such multifaceted responses aim to maintain cellular function and ultimately recover from stress cessation21. At molecular level, two groups of such genes are recognized: direct defenders and regulatory maestros. The defenders, such as chaperones, osmotin, mRNA binding proteins, late embryogenesis abundant proteins, osmolyte biosynthesis, sugar and proline transporter, and water channel proteins, directly protect from drought22. While, the regulatory maestros, including phosphatases, transcription factors, signaling molecules, and protein kinases, orchestrate a diverse array of downstream genes22,23. Collectively, these intricate networks fine-tune plant responses to the drought challenge.

Recent strides have been made in comprehending the intricate cascade of gene expression during osmotic stress, particularly within the abscisic acid (ABA)-dependent and ABA-independent signaling pathways. ABA stands as a crucial stress-signaling hormone in plants. In the ABA-dependent pathway, intensified ABA production works to alleviate drought stress. It triggers subsequent cascade pathways, including activation of ion channels for stomatal closure, elevation of cytosolic calcium levels, and the generation of ROS. This complex pathway relies on a delicate interplay between ABA-responsive elements (ABREs) and their binding partners, called ABRE-binding transcription factors (ABFs). Simultaneously, an ABA-independent pathway operates, featuring dehydration-responsive elements/C-repeats (DRE/CRTs) and their cognate DRE-binding proteins, notably DREB224,25. This coordinated symphony of gene expression goes beyond direct defense mechanisms. Upregulation of genes involved in ATP synthesis, photosynthesis, late embryogenesis, secondary metabolites, photosystem, carbohydrate metabolism, monooxygenases, and DREB/CBF collectively works to alleviate the adverse effects of drought stress26. However, the drought response is not solely governed by ABA.

Plants have sheer biological and genetic diversity, and they deploy diverse transcriptome and proteomic strategies to counteract the effects of drought stress. Importantly, these strategies and their underlying mechanisms can vary even between closely related species. Hence, exploring and understanding the drought-responsive genes in different cultivars of the same plant species is crucial to decipher their unique stress adaptation mechanisms. Unfortunately, despite global cultivation and socio-economic importance of date palm, it remains one of the less studied and characterized plants in terms of its genome. To date, only a limited number of studies have explored its stress-responsive genes at the molecular level27,28,29,30,31. This dearth of information extends to the genome itself, as minimal characterization and annotation of the date palm genome present a significant hurdle in understanding its drought tolerance mechanisms.

Transcriptome analysis, particularly employing suppression subtraction hybridization (SSH), offers a powerful tool to decipher the intricate molecular responses of a plant to different stresses. Compared to microarrays, SSH is advantageous for uncovering novel genes involved in stress responses32. Additionally, cDNA Subtraction provides a unique method for amplifying differentially expressed sequences, overcoming limitations of traditional methods33. Leveraging this technique, we delved into the physiological and molecular responses of Khalas, Reziz, and Sheshi to drought stress under ambient conditions. The outcome of the study revealed a notable impact of drought on leaf physiological characteristics. Interestingly, both similarities and differences were observed in drought-responsive gene regulation across these cultivars. The implications of these findings are discussed in further detail, shedding light on the nuanced mechanisms underlying plant responses to drought stress.

Results

Measurement of environmental vaiables

The experiment was conducted over a 90-day period, spanning from April to June 2021, a time marked by increasingly harsh environmental conditions that exacerbated drought stress. During this period, the recorded temperatures exhibited a discernible upward trajectory. The average of maximum tempearture increased from 39.87 oC in April to 46.99 oC in May and further rose to 47.41 oC in June. Notably, the highest recorded temperature of 51.13Ā Ā°C occurred in the first week of June (Fig.Ā 1A), further stressing the plants during this critical stage.

Fig. 1
figure 1

Different environmental variables, including temperature, solar radiations, wind speed, evapotranspiration (ETc), and relative humidity, measured throughout the course of experiment time.

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Solar radiations, wind speed, and evapotranspiration (ETc) revealed analogous trends over the experimental duration. The most pronounced fluctuations were noted during the second week of April, marked by peak solar radiations at 0.43 kW.māˆ’2, maximal wind speed 9Ā km.hāˆ’1, and the highest ETc recorded at 11.45%. Subsequently, from May until mid-June, a sustained pattern prevailed, followed by a resurgence of variability in mid-June leading to a substantial decline by the end of June (Fig.Ā 1B, C and D).

Relative humidity demonstrated a highly variable pattern throughout the experimental timeline, featuring multiple surges surpassing the 60% threshold. Nonetheless, the average relative humidity remained 29.06% in April, 22.36% in May, and 20.10% in June (Fig.Ā 1E).

Effect of drought on phenotype and physiological attributes

Phenotypic observation of date palm cultivars under drought stress revealed varying responses, with Khalas demonstrating superior resilience compared to Reziz and Sheshi (Fig.Ā 2A). Leaves of the Sheshi were more supple and chlorotic, indicating reduced chlorophyll contents and compromised physiology. Reziz leaves showed similar symptoms but to a lesser degree, while Khalas leaves remained relatively green and firm, indicating better chlorophyll retention and physiological integrity. While, all control (irrigated) plants displayed vibrant green leaves and softer texture, suggesting higher chlorophyll contents and intact physiology.

Compared to control plants, all investigated physiological parameters in drought-stressed date palm plants showed significant changes after 90 days. These changes included reduced RWC (Fig.Ā 3A), photosynthetic rate (Pn; Fig.Ā 3B), stomatal conductance (gs; Fig.Ā 3C), and transpiration rate (E; Fig.Ā 3D) at pā€‰ā‰¤ā€‰0.05. Conversely, intercellular CO2 (Ci; Fig.Ā 3E) was significanly upregulated (pā€‰ā‰¤ā€‰0.05), while water use efficiency (Fig.Ā 3F) was at par. Among the cultivars under drought stress, Khalas and Reziz outperformed Sheshi in all investigated physiological parameters. This suggests that Khalas and Reziz possess greater tolerance to drought stress compared to Sheshi.

Fig. 2
figure 2

Phenotypic response (A) and chlorophyll content (B) of date palm cultivars under control (irrigated) and drought (stressed) conditions. Photographs and data represents measurement taken after 90 days of drought induction.

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Fig. 3
figure 3

Effects of drought stress on the different physiological attributes of date palm cultivars, including (A) relative water content (RWC), (B) photosynthetic rate (Pn), (C) stomatal conductance (gs), (D) transpiration rate (E), (E) intercellular CO2 concentraion (Ci), and (F) water use efficiency (WUE). Data represents measurements taken after 90 days of drought induction. Statistical differences (pā€‰ā‰¤ā€‰0.05) are indicated by different letters above the bars in the bar graph. Y-bars on each bar (where larger that than the data point) represent the the standard deviation within replicate.

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Sequencing of the drought-responsive ESTs

In this study, we leverage EST analysis to unravel the secrets of drought tolerance in three date palm cultivars under natural field conditions. To investigate the drought-responsive ESTs, a separate cDNA library of each date palm cultivar was constructed using middle leaf tissue from drought-exposed plants. Through sequencing and quality control, a collection of 1026 high-quality ESTs were generated: 300 from Khalas, 343 from Reziz, and 383 from Sheshi. This valuable dataset provides a window into the unique genetic programs employed by each cultivar to overcome water scarcity challenges. All these ESTs were submitted to the DNA Database of Japan (DDBJ) under Mass submission system with ID# DDBJ: NSUB001506 (). This comprehensive EST library, enriched for drought-responsive genes, serves as a vital foundation to uncover the specific genes and pathways employed by each cultivar to combat drought stress.

Functional annotation of the ESTs

The functional annotation of isolated ESTs from individual date palm cultivar was performed using OmicsBox (ver 3.1.11) to infer their cellular components (CC), molecular functions (MF) and biological process (BP), and. Comprehensive functional annotation of drought-responsive ESTs identified distinct transcriptional patterns across three date palm cultivars. This suggested that each cultivar deployed a unqiue repertoire of genes differentially expressed across diverse biochemical pathways, suggesting independent adaptation and coping mechanisms. In Khalas, 194/300 (~ā€‰65%) of ESTs were annotated, with 104 (~ā€‰35%) linked to enzyme commission (EC) codes and 66 (~ā€‰21%) associated with KEGG pathways. Notably, Reziz and Sheshi exhibited lower annotation rates, with 104/343 (~ā€‰30%) and 109/383 (~ā€‰29%) ESTs annotated, respectively. Within these smaller annotated sets, Reziz contributed 41 EC and 19 KEGG-linked ESTs, while Sheshi contributed 48 EC and 37 KEGG-linked ESTs.

The CC analysis revealed distinct organelle-specific expression patterns in the three date palm cultivars (Fig.Ā 4). Khalas displayed a marked preference for genes related to the plastids (28 ESTs), followed by nucleus (23 ESTs), membrane and integral membrane components (15 ESTs), cytosol (14 ESTs), ribosomes (12 ESTs), chloroplast, thylakoid, and stroma (8 ESTs), and endoplasmic reticulum (6 ESTs). Likewise, Reziz showed enrichment for ESTs associated with nucleus (14 ESTs), followed by membrane and integral membrane components (12 ESTs), chloroplast, thylakoid, and stroma (8 ESTs), and photosystem I and II (6 ESTs). In contrast, Sheshi exhibited a unique expression profile characterized by high levels of ESTs encoding chloroplast thylakoid (46 ESTs), photosystem II (38 ESTs), photosystem I (34 ESTs), chloroplast (22 ESTs), protein containing complex (10 ESTs), and ribonucleoprotein complex (4 ESTs). These findings suggest distinct cellular roles and functional specializations among Khalas, Reziz, and Sheshi.

Fig. 4
figure 4

Inferred gene ontology (GO) analysis of ESTs in Khalas, Reziz, and Sheshi. GO terms were organized contingent on p-valuesā€‰ā‰¤ā€‰10. Each bar (x-axis) represents a specific GO term, categorized into three main branches: cellular components (green), molecular functions (pink), and biological processes (cyan). The height of each bar (y-axis) corresponds to the relative abundance of ESTs associated with that specific GO term.

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The MF analysis of drought-responsive ESTs identified substantial differences in transcriptional activity across three date palm cultivars (Fig.Ā 4). Notably, Khalas and Sheshi showed a more robust response to drought stress than Reziz, as evidenced by the upregulation of a greater number of ESTs. Khalas exhibited the highest abundance of ESTs (44 ESTs) pertaining to transferae activty, followed by oxidoreductase activity (27 ESTs), Hydrolase activity (27 ESTs), catalytic activity (24 ESTs), ATP-dependent activity (22 ESTs), metal ion binding activity (14 ESTs), and Ubiquitin binding (14 ESTs). The remaining MF categories included chaperon activity, small molecule sensor activity, molecular transducer, and nutrient reservoir activtiy. In contrast, Reziz expressed fewer drought-responsive ESTs, with the highest number (10 ESTs) associated with transferase activity, followed by structural molecule activity (8 ESTs), ATP and GTP binding (8 ESTs), hydrolase activity (6 ESTs), transcription regulation (6 ESTs), GTPase activity (6 ESTs), metal ion binding (6 ESTs), and protein binding (5 ESTs). Interestingly, Sheshi exhibited a distinct expression profile characterized by the highest EST abundance related to catalytic activity (47 ESTs), chlorophyll binding (39 ESTs), oxidoreductase activity (27 ESTs), meta ion binding (19 ESTs), ATP binding (18 ESTs), hydrolase activity (15 ESTs), and lyase activity (12 ESTs). Additional enriched MFs in Sheshi included electron transfer, ATP hydrolysis, electron transport, iron-sulfur cluster binding, and nucleic acid binding.

The BP analysis unveiled that in Khalas, 33 drought-responsive ESTs were predominantly involved in membrane organization, 30 in transcriptional regulation, 18 in carbohydrate metabolism, 18 in photosynthesis, 18 in amino acid metabolism across different levels, 16 in lipid metabolism, and 15 in DNA repair (Fig.Ā 4). Reziz exhibited a lower diversity, with 14 ESTs primarily associated with cellular signalling, followed by 7 and 6 ESTs related to phosphorylation and biological regulation, respectively. Notably, a larger group of 28 ESTs in Reziz were categorized as ā€œOther,ā€ encompassing diverse biological processes. In contrast, Sheshi displayed a broader spectrum of drought responses, with 38 ESTs associated with photosynthesis and light harvesting, followed by those involved in metabolic precursor generation (18), light stimuli response (17), amide metabolism (13), electron transport chain (12), cellular biosynthesis (11), and additional photosynthetic functions (11). Similar to Reziz, Sheshi also harbored a large pool of 70 ESTs in the ā€œOtherā€ category (Fig.Ā 4).

Gene set enrichment analysis

Traditional enrichment analysis relies on predefined gene expression thresholds, limiting its ability to detect subtle gene changes. In contrast, Gene Set Enrichment Analysis (GSEA) examines gene sets collectively, improving sensitivity to detect coordinated gene expression patterns. GSEA often provides complementary insights compared to traditional methods34. GSEA ranks genes by Signal2Noise and assesses whether pre-defined gene sets are overrepresented at the top or bottom of this list, indicating enrichment in one biological state over another. The GSEA of three date palms is mentioned in Figure S1.

The GSEA revealed distinct patterns of ESTs enrichment among three date palm cultivars. Khalas demonstrated overrepresentation of ESTs associated with cellular component organization, cellular and metabolic processes, catalytic activity, molecular functions, and metal ion binding. Reziz exhibited enrichment in ESTs related to molecular functions, cellular and intrecellular anatomoical entities, lyase activity, biological regulation, metabolic processes, signaling, and nuclear functions. In contrast, Sheshi displayed a predominance of ESTs involved in cellular component, membane bounding organelles, organelle lumen, photosynthetic membrane, photosystem, and cytosolic ribosomal components. These findings suggest cultivar-specific adaptations to environmental challenges and differential utilization of cellular resources.

KEGG pathways and enzyme code distribution analysis

The KEGG pathway analysis35 identified that three date palm cultivars deployed not only some common pathways but also distinct and indigenous pathways to ameliorate drought stress (Fig.Ā 5). Khalas exhibited the most extensive transcriptional reprogramming, involving ESTs mapped to 66 distinct pathways, compared to 55 and 36 pathways in Reziz and Sheshi, respectively (Table S1). Khalas exhibited a significantly stronger bias towards nitrogen metabolism compared to Sheshi throughout its stress response and expressed the highest number of ESTs (18) related to nitrogen metabolism (encoding 7 enzymes) (Fig.Ā 5A). This was followed by 7 ESTs (encoding 3 enzymes) associated with purine metabolism, 7 ESTs (encoding 3 enzymes) with thiamine metabolism. Notably, five ESTs each were associated with galactose metabolism, glycolysis/gluconeogenesis, and pyruvate metabolism (encoding two enzymes each). Khalas also showed upregulation of ESTs related to carbon fixation in photosynthetic organisms (5 ESTs, one enzyme), glycerolipid metabolism (5 ESTs, 2 enzymes), and several other pathways (4 ESTs, one enzyme each), including alkaloid biosynthesis, pentose and glucuronate interconversion, methane metabolism, folate biosynthesis, flavonoid biosynthesis, and fructose and mannose metabolism. Notably, 1 EST (encoding 1 enzyme) was also transcribed to counter bacterial infection.

Fig. 5
figure 5

KEGG pathways identified in three date palm cultivars: Khalas (A), Reziz (B), and Sheshi (C). The central portion (pie graph) displays the pathway names and relative abundance, while the outer section features a histogram depicting the number of ESTs (purple) and the number of genes encoding these enzymes (blue).The Venn diagram illustrates the number and percentage of common and unique pathways among the three date palm cultivars (D).

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In Reziz, 8 ESTs (encoding 3 enyzmes) were associates with carbon fixation in photosynthetic organisms, this constituted the highest number of ESTs linked to a specific pathway. Six ESTs (3 enzymes) were involved in purine metabolism, followed by 5 ESTs (2 enzymes) for glycolysis/gluconeogenesis, 4 ESTs (two enzymes) for thiamine metabolism, and 4 ESTs (2 enzymes) for the pentose phosphate pathway. Starch and sucrose metabolism involved 3 ESTs (2 enzymes), while secondary metabolite biosynthesis utilized 2 ESTs (one enzyme). Notably, Reziz plants under drought stress also transcribed one EST related to a bacterial (E. coli) infection pathway (Fig.Ā 5B).

In Sheshi, the KEGG pathway analysis unveiled a dominance of gene involved in carbon fixation in photosynthetic organisms, with 7 ESTs encoding 3 enzymes. This was followed by 6 ESTs each for photosynthesis (3 enzymes), purine metabolism (3 enzymes), glyoxalate and decarboxylate metabolism (2 enzymes), and thiamine metabolism (2 enzymes). Additionally, 4 ESTs (2 enzymes) were associated with nitrogen metabolism, while 3 ESTs (one enzyme) involved in phenylalanine, tyrosine, and tryptophan biosynthesis. Two ESTs each were identified for plant-pathogen interaction (2 enzymes), pyruvate metabolism (one enzyme), and glycine, serine, and threonine metabolism (one enzyme) (Fig.Ā 5C).

Using Venn analysis, we explored EST-associated pathway expression patterns in three date palm cultivars (Fig.Ā 5D). Venn analysis unveiled that a set of 9 common pathways (8.2% of total mapped pathways), including carbon fixation in photosynthesis, purine metabolism, oocyte maturation, terpenoid biosynthesis, thiamine metabolism, response to bacterial infection, cell-proliferation (related to cancer), and PI3K-Akt-signalling pathways, were expressed in three date palm cultivars, representing shared metabolic and cellular processes. Notably, 10 common pathways were upregulated in both Khalas and Reziz, while a larger set of 18 pathways (16.4%) were shared between Khalas and Sheshi. Reziz and Sheshi, on the other hand, exhibited minimal overlap, with only one common upregulated pathway. Beyond these shared pathways, each cultivar also displayed a distinct profile of unique upregulated pathways. Khalas and Reziz had a substantial number of unique pathways, with 29 (26.4%) and 35 (31.8%), respectively. In contrast, Sheshi had a much smaller set of unique pathways, with only 8 (7.3%) (Table S2).

Enzyme commission (EC) numbers constitute a hierarchical system for categorizing enzymes based on their specific catalyzed reactions. EC distribution analysis (Fig.Ā 6) revealed that transferases dominated the repertoire of identified ESTs, with 55 representatives. Khalas contributed the majority with 42 ESTs, followed by Reziz (10 ESTs) and Sheshi (3 ESTs). Oxidoreductases were close behind with a total of 52 ESTs, with Khalas again leading (27 ESTs), contrasting with Reziz (0 ESTs) and Sheshi (25 ESTs). Notably, Khalas also harbored a relatively higher proportion of ESTs pertaining to hydrolases (23 ESTs) compared to Sheshi (12 ESTs) and Reziz (9 ESTs), despite these constituting a smaller overall group (44 ESTs total). Lyases related ESTs were also transcribed across all three date palm cultivars (Khalas: 23 ESTs, Reziz: 2 ESTs, Sheshi: 11 ESTs). The remaining classes, ligases, translocases, and isomerases, were present in lower numbers. Translocases were represented by 9 ESTs (Khalas: 5 ESTs, Reziz: 0 ESTs, Sheshi: 4 ESTs), ligases by 7 ESTs (Khalas: 4 ESTs, Reziz: 1 ESTs, Sheshi: 2 ESTs), and isomerases by 4 ESTs (Khalas: 3 ESTs, Reziz: 1 ESTs, Sheshi: 0 ESTs).

Fig. 6
figure 6

Distribution of KEGG Orthology (KO) categories among drought-responsive ESTs in three date palm cultivars (Khalas, Reziz, and Sheshi). The 3D bar graph illustrates a specific enzyme class, visualized by a unique color and labeled with the corresponding KO identifier. The height of each bar corresponds to the number of ESTs belonging to that enzyme class.

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Discussion

Inspired from our earlier work31that discerned and appraised drought-responsive ESTs in three date palm cultivars under controlled glasshouse conditions, the current study ventured into the complex realm of ambient conditions. Controlled environments offer meticulous control over parameters such as water availability, temperature, heat, and light intensity, leading to defined plant responses to stresses. Nonetheless, ambient environment is far more complex, multifaceted, and unpredictable than the controlled environment and plants face a multitude of interacting stressors. Furthermore, drought in the ambient environment is often episodic, with periods of water stress interspersed with rainfall events and fluctuating humidity levels. Consequently, we anticipated distinct physiological, molecular, and gene expression responses compared to the controlled glasshouse setting. Our findings confirmed this notion, revealing striking differences in the response of the three cultivars to natural drought compared to the controlled conditions explored previously. Notably, leaves of all cultivars exhibited a surprisingly different phenotype, differing from control plants observed in the previous study31. Leaf texture became less supple and more chlorotic, indicating reduced chlorophyll contents (Fig.Ā 2). Conversely, control plants maintained a significantly different phenotype, characterized by vibrant green leaves and a softer texture, suggesting higher chlorophyll abundance. Drought stress impedes chlorophyll biosynthesis at multiple stages, from the initial formation of 5-aminolevulinic acid to the final incorporation of chlorophyll into light-harvesting complexes36. This leads to significant reductions in chlorophyll content across various plant species. Concomitantly, drought-induced oxidative stress damages chloroplasts, exacerbating chlorophyll loss37.

Unlike most plants where young leaves and roots are most affected by stress38,39, but date palms exhibit a unique response to drought. Drought primarily impacts mature leaves in this species (M. Munir personal communication). This is likely due to the palmā€™s unique structure ā€“ a single shoot with a crown of leaves (monopodial) and no apparent branching. Additionally, the middle region of leaves, containing mesophyll cells responsible for photosynthesis and with high metabolic activity, also houses a high concentration of actively transcribed genes. Therefore, for this study, we focused on middle leaves of date palm to quantify and assess the drought stress at the transcriptomic level.

Under natural drought conditions, most investigated physiological attributes, including RWC, Pn, gs, and E, were down-regulated to varying degrees in all three date palm cultivars. However after 90 days of drought stress, Khalas and Reziz exhibited greater resilience, maintaining higher levels of these attributes compared to Sheshi. These findings align with previous reports in both glasshouse and natural drought settings31,40,41,42,43, suggesting inherent advantages in Khalas and Rezizā€™s drought-combating mechanisms and potentially superior drought-resistant physiology44,45. Under drought, photosynthesis hampered by both stomatal and non-stomatal limitations. We may speculate stomatal limitation played a major role in restricting photosynthesis in date palm under drought stress. As water stress intensified, stomatal conductance declined (Fig.Ā 3C), leading to reduced CO2uptake alongside water loss through transpiration. This stomatal closure, a well-documented early response to water stress41,46, allows for some carbon assimilation at the expense of water conservation. However, under prolonged stress, sustained stomatal closure can increase susceptibility to photodamage, heat stress, and photosynthesis47,48, ultimately impacting Ci (Fig.Ā 3E) and Pn (Fig.Ā 3B). Khalas and Reziz maintained better photosynthetic rate than Sheshi, which may be accredited to their higher chlorphyll contents and RWC.

WUE serves as a crucial metric for crop performance and it often increases in various plant species under stress43,49,50 due to stomatal closure, which restricts leaf conductance, thereby reducing both photosynthesis and transpiration. Our study observed a similar increase in WUE under drought stress conditions (Fig.Ā 3F). Notably, all three date palm cultivars demonstrated greater resilience in regulating RWC, WUE, Pn, gs, Ci, and Ecompared to those under controlled drought. This may accredied to gradual onset of drought, dynamic and sibtle environmental conditions31. Moreover, as plants initiate adaptive mechanisms to cope water stress, the increase in respiration under lower RWC is anticipated to correspond with an increase in metabolism51. Rising temperatures along with high wind speed intensify evapotranspiration, pulling crucial moisture from the plant and further exacerbating the impact of drought52,53,54. Additionally, high temperatures have the potential to impede nutrient uptake and disrupt hormonal balance, resulting in inhibited growth and diminished fruit production55. Ultimately, the complex interplay of these factors determines the severity of water stress and the date palmā€™s capacity to adapt and survive.

The observed disparities in date palms physiological responses to drought stress between field and controlled environments were possibly arouse from the inherent complexity of field conditions. Field environments expose plants to a combination of stresses (drought, heat, salinity, biotic factors) that can interact and influence physiological responses differently than the single stress of drought imposed in controlled settings56,57. This study has witnessed a highly dynamic environmental variables (temperature, humidity, wind, and light) throughout the experimental period (Fig.Ā 1), influencing plant water use efficiency and stress perception, which are less likely to occur in controlled environments. Additionally, field soils exhibit significant heterogeneity in texture, structure, organic matter, and nutrient availability, impacting water infiltration, retention, and root development compared to the uniform conditions in controlled studies58. Plant interactions and soil microbial communities further contribute to the observed differences. Competition for resources, such as water and nutrients, among plants in field settings can significantly alter physiological responses compared to plants in controlled conditions59.

While physiological cues initiate the first line of defense against drought, plants ultimately orchestrate stress response at the molecular level through intricate regulatory networks. Several studies have quantified different repertoire of stress-responsive ESTs in different date palm cultivars28,29,31. Strikingly, our data under ambient conditions revealed upregulation of ESTs implicated in diverse metabolic and cellular processes, encompassing photosynthesis, carbohydrate and amino acid metabolism, reproductive functions, terpenoid biosynthesis, defense against bacterial infection, cell cycle regulation, protein and metal ion binding, structural molecule synthesis, and transport activities (Figs.Ā 3 and 4). These findings diverge from earlier reports on date palm ESTs under controlled drought conditions31, salinity stress60, and even natural conditions61. Two factors likely contribute to this disparity. First, our use of a subtractive hybridization approach, comparing stressed and control plants, might have missed ESTs with relatively low expression levels. Second, the specific molecular mechanisms of stress response may differ substantially between controlled and natural environments. This divergence highlights the remarkable plasticity of date palmā€™s molecular toolkit in response to diverse abiotic stresses. Our findings highlight the value of conducting research under natural conditions, rather than solely relying on controlled environments, to gain a more comprehensive understanding of plant stress responses.

The comparative CC analysis of three date palm cultivars under field drought conditions revealed interesting insights into their diverse adaptation strategies and compared to, our prior work conducted under controlled glasshouse drought conditions31. Khalas under natural drought deployed ESTs related to plastids, nucleus, endosomes, membranes, ribosomes, and thylakoid, suggesting prioritized efforts towards maintaining photosynthesis and cellular membrane integrity through gene regulation. This stands in contrast to its response in glasshouse drought conditions, where ESTs mainly targeted membrane integral components, thylakoid membranes, chloroplasts, photosystems I and II, and the chloroplast envelope were upregulated31. Reziz exhibited a distinct strategy, focusing on a broader response encompassing various cellular components under natural drought. The enriched ESTs involved the nucleus, chloroplast stroma, cytoskeleton, and Golgi membranes, suggesting a more nuanced adaptation than Khalas, suggesting a more nuanced response compared to Khalas and highlights potentially unique adaptation mechanisms. While under controlled drought conditions, chloroplast-related ESTs were the most abundant, followed by those linked to membrane components, thylakoid membranes, chloroplast stroma, and ribosomes31. Sheshi prioritized maintaining photosynthetic machinery under both ambient and controlled drought conditions, as indicated by the enrichment of ESTs related to thylakoid membranes, photosystems I and II, and chloroplasts. However, controlled drought elicited a greater emphasis on membrane integrity, as evident by the higher abundance of ESTs associated with membrane integral components31. Across all cultivars, CC analysis revealed specific gene expression patterns associated with various organelles and structures. During the onset of drought, genes encoding components of the thylakoid membranes and photosystems I and II in plastids, particularly chloroplasts, are regulated to conserve water by remodeling thylakoid architecture and enhancing non-photochemical quenching to protect these delicate membranes from photodamage energy under drought stress62,63,64. Chloroplast stroma witnesses a surge in the activity of ribosomal proteins, encoded by nuclear genes, to ramp up protein synthesis for drought response65. Endosomal trafficking, facilitated by cytoskeletal elements and Golgi membranes, ensures efficient delivery of essential molecules within the cell66,67. Membrane transporters meticulously regulate ion fluxes across membranes, maintaining cellular osmotic balance68. Communication becomes paramount, with signaling pathways orchestrated by genes encoding protein kinases and phosphatases, relaying drought signals from the cell surface to the nucleus, triggering appropriate responses69,70,71. This dynamic interplay between gene expression and organelle function underscores the remarkable adaptability of date palms to drought stress. We may speculate that the differential gene expression observed in date palms under natural and controlled drought conditions is influenced by a complex interplay of factors. Gradual onset and varying intensity in natural drought can induce long-term survival mechanisms, such as genes related to antioxidant production, root development, and hormonal responses72,73,74. Conversely, controlled droughtā€™s abrupt imposition often elicits acute stress responses like stomatal closure and osmotic adjustment. Natural drought frequently coincides with other stressors, such as heat, salinity, and nutrient deficiency, broadening the gene expression profile compared to the more isolated water stress in controlled environments73. Soil conditions, including structure and microbial activity, can further modulate root-associated gene expression under natural drought74. Additionally, the capacity for acclimation, which is often afforded by the gradual nature of natural drought, can influence gene expression patterns75. Plant species, genotype, and developmental stage also contribute to the diversity of drought responses, underscoring the complexity of gene regulation in this context.

The MF analysis revealed distinct EST expression patterns among the three date palm cultivars under natural drought conditions, differing substantially from controlled drought conditions. Khalas prioritized metabolic adaptation and stress response under natural drought, with high expression of ESTs associated with transferase, oxidoreductase, hydrolase activities, ATP-dependent functions, and metal ion binding (Fig.Ā 4). This suggests a focus on metabolic adaptation and stress response under natural drought conditions. Conversely, in our previous findings, it focused on photosynthetic maintenance and cellular protection by expressing ESTs related to ribulose-bisphosphate carboxylase activity, monooxygenase activity, chlorophyll binding, and metal ion binding31. Reziz displayed similar trends, favoring transferase activity in field drought alongside structural molecule activity, ATP/GTP binding, and hydrolase activity. While in our prior findings it ameliorated controlled drought conditions with an emphasis on ATP binding, ribosomal constituents, and photosynthetic genes31. Sheshi exhibited a strong upregulation of ESTs for cellular maintenance and stress response (catalytic activity, oxidoreductases, metal ion binding) alongside chlorophyll binding and ATP binding in natural drought. Under controlled conditions, chlorophyll binding remained dominant, followed by metal ion binding, electron transport, and ribosomal proteins31. All three date palm cultivars orchestrated unique molecular tools to show resilience to drought and each tool played a distinct role to ameliorate the drought stress. Transferases, versatile catalysts, modify essential molecules like hormones, proteins, and antioxidants, detoxify the drought generated ROS, protecting proteins and cellular membranes. During drought, they fine-tune ABA signaling pathways for stomatal closure, synthesize osmoprotectants like proline to maintain cell turgor76,77. Oxidoreductases, vital for electron transfer, fuel ATP-dependent pathways crucial for energy production and stress response. They regulate chlorophyll binding for efficient light capture and scavenge ROS through antioxidant cycles78,79. Hydrolases, adept at breaking down complex molecules, degrade cellular components during senescence to mobilize resources for vital processes under drought stress80,81, and construct/remodel cell wall structures and regulate cell wall extensibility in stress response82to regulate water loss through cell walls by enhancing water use efficiency83. ATP/GTP binding proteins act as molecular switches, regulating diverse cellular processes in response to drought. They orchestrate ATP-dependent pathways for protein synthesis, signal transduction, and ion transport, ensuring plant survival84,85. Metal ion binding proteins, metallothioneins, are biological chelators that bind and sequester toxic metals that accumulate during drought stress, protecting cellular machinery30,86,87,88. Additionally, chlorophyll binding proteins are critical in light-harvesting, photoprotection, and sustaining photosynthetic efficiency and are central to drought resilience in plants89. Light-harvesting proteins, especially chlorophyll-binding a/bproteins, are the most abundant proteins in plants with LHCB1-to-LHCB6 known types90. Down-regulating or impairing any member of LHCB resulted in compromised ROS homeostasis, reduction in drought tolerance, and declined responsiveness of stomatal movement to ABA91. Our findings reveal a fascinating divergence in the molecular strategies employed by date palm cultivars to navigate drought stress. In the ambient environment, they prioritized metabolic adaptation, utilizing enzymes like transferases, oxidoreductases, and hydrolases for detoxification, resource mobilization, and cell wall remodeling. While in the controlled drought, they elicited responses focused on maintaining core cellular functions like photosynthesis and protein synthesis.

The BP analysis of three date palm cultivars under natural drought conditions revealed distinct patterns. Khalas diverted his biological resources to membrane organization, transcription regulation, carbohydrate metabolism, photosynthesis, lipid metabolism, and DNA repair. While, under controlled drought conditions, it focused on oxidation-reduction process, photorespiration, protein-chromophore linkage, translation, pentose phosphate pathway, and photosynthesis31. Reziz tried to ameliorate the natural drought by boosting cellular signaling, phosphorylation, biological regulation, transcription, carbohydrate metabolism, and cytokinin-acting signaling, while it coped to controlled drought by focusing on oxidation-reduction process, translation, photosynthesis, photorespiration, and ATP synthesis31. On the other hand, Sheshi neutralized the natural drought by boosting photosynthesis, generation of metabolites, increasing response to light stimuli, amide metabolism, electron transport chain, and photosynthesis. While it coped to controlled drought by diverting resources to protein-chromophore linkage, photosynthesis, electron transport chain, translation, oxidation-reduction process, and PSI and II31. All the upregulated genes under natural drought conditions play a vital role in coping drought stress. Membrane organization, the gatekeeper of cellular traffic, takes center stage, with genes encoding aquaporins orchestrating the water flow and ion flux across cell membranes, optimizing water uptake and minimizing loss83. Transcription regulation, the conductor of gene expression, tunes up stress-responsive genes, while downregulating non-essential ones, like those in photosynthesis, to conserve energy92,93. Carbohydrate metabolism genes take center stage, churning out osmoprotectants like proline to maintain cell turgor under osmotic stress. Additionally, stressed leaves exhibited upregulation in genes encoding vacuolar and cytoplasmic enzymes involved in glucose, fructan, and fructose biosynthesis pathways94. Yet, photosynthesis, the engine of energy production, can falter under drought due to stomatal closure and reduced light capture. To compensate, lipid metabolism shifts towards synthesizing protective lipids for membranes by upregulating the genes for fatty acid desaturation increasing membrane fluidity and resilience to dehydration and safeguarding photosynthetic machinery95,96,97. DNA repair mechanisms stand guard, patching the inevitable DNA damage caused by drought-induces oxidative stress98. Drought stress itself triggers a cascade of regulatory changes in plants, not just through the activation of cellular signaling pathways, but also by their own fine-tuning through transcriptional and post-translational modifications like phosphorylation. This precise protein phosphorylation acts as a molecular switch, enabling plants to activate or deactivate specific signaling pathways to cope with the stress99,100.

KEGG pathway unveiled that Khalas showed resilience to drought by upregulating the nitrogen metabolism through expressing 18 ESTs (encoding 7 enzymes), while prior studies have shown reduction in nitrogen metabolism, due to downregulation of nitrogen reductase gene in wheat101, rice102, and barley103. Nitrogen is essential for plant growth, productivity, and stress tolerance. Drought hampers nitrogen uptake and assimilation by reducing transpiration and affecting nitrate and ammonium transport104. Nitrogen metabolism is crucial for drought tolerance in plants. Key enzymes like glutamine synthetase (GS) and glutamate synthase (GOGAT) are inhibited by drought, leading to reduced growth and ammonium toxicity105. While nitrogenā€™s role in stress alleviation is known, nonetheless it impact on mitigating drought by stomatal conductance and antioxidant enzymatic activity106. Reziz responded to drought by upregulating the highest number of 8 ESTs (encoding 3 enzymes) related to carbon-fixation in photosynthetic organisms. Optimized carbon fixation, driven by the Calvin-Benson cycle, enhances drought tolerance in photosynthetic organisms. Plants with efficient enzymes and CO2concentrating mechanisms, such as C4 photosynthesis, maximize water use efficiency107. Sheshi upregulated the highest 7 ESTs (encoding 3 enzymes) related to antenna protein involved in photosynthesis. Antenna proteins adjust light capture during drought, optimizing energy utilization and minimizing photodamage108. These dynamic adaptations safeguard photosynthetic machinery, preserving plant resilience109. In water-stressed samples, reduced amount of Chl, LHCs and reaction center proteins result in reduced light absorption, and decreased amount of oxygen evolving complex proteins result in reduced utilization of the absorbed energy. Consequently, reduction of PSII activity was almost similar (nearly 50%) under limiting as well as under saturating light intensities. In the same vein, the downsizing of the light-harvesting Chl-binding proteins of PSI i.e., LHCI (Lhca1 and Lhca4) and PSI core complex protein subunits III (PsaF), V (PsaK) and VI (PsaH) in water-stressed seedlings, resulted in diminished light absorption as well as light utilization by PSI, as was evident from the reduction of PSI reaction by 30% at limiting and higher light intensities. In mature plants, only minimal amounts of Chl and proteins are synthesized, mostly to replace those photo destroyed in PSI and PSII and LHCs, due to light. Therefore, it appears that mature plants do not downsize the components of the photosynthetic apparatus in response to drought. Several studies have demonstrated that abiotic stresses, including water-stress, cause ROS production due to an over-reduction of photosynthetic electron transport chain25,29,64,65. Abiotic stresses also impair reaction centers of well-developed seedlings or mature plants, leading to reduced transfer of absorbed light energy from light harvesting complexes to photodamaged reaction centers and consequently generation of higher amounts of singlet oxygen via photosensitization reactions of Chlorophyll. Across all three date palm cultivars, the KEGG pathways results demonstrated that ESTs were related to 157 pathways (66 in Khalas, 55 in Reziz, and 36 in Sheshi), sharing 9 common pathways (Fig.Ā 5). Purine metabolism is upregulated in plants during drought110,111,112, it provides precursors essential signaling molecules and nucleic acids needed for stress response, its intermediate, allantoin, acts as osmoprotectant, and its degradation liberates nitrogen for biosynthesis113. Stress-responsive genes induce alternative pathways like Crassulacean acid metabolism, fixing CO2at night, conserving water during growth114. Progesterone-mediated oocyte maturation in some woody plants becomes dormant during drought, strategically delaying reproduction until favorable conditions return115. Terpenoid backbone biosynthesis, the foundation for diverse secondary metabolites with defensive and signaling functions, can be upregulated under drought to provide protection against oxidative stress and herbivores116,117.

Comparative analysis of Enzyme Commission (EC) number distribution revealed distinct patterns across three date palm cultivars under natural drought. While all cultivars expressed ESTs belonging to various enzyme classes (Fig.Ā 6). Both Khalas and Reziz exhibited the highest number of ESTs related to transferases, suggesting a heightened focus on metabolic reprogramming and detoxification under ambient conditions. In contrast, Sheshi prioritized oxidoreductases, potentially emphasizing energy production and ROS management. This contrasts with our previous findings under controlled drought, where Khalas leaned towards lyases and Reziz and Sheshi favored oxidoreductases31. This shift in enzyme expression compared to controlled drought highlights the flexibility of these cultivars in tailoring their responses to drought stress.

Despite mapping a substantial portion of our EST collection (~ā€‰40%) to existing repositories, a significant number remained uncharacterized due to the limited availability of gene annotation data. This challenge, also encountered in our previous work, highlights the presence of potentially novel genes with crucial roles in date palm drought tolerance. These unmapped ESTs represent a fascinating trove of potentially novel genes with crucial roles in drought tolerance. Exploring these unmapped ESTs in future studies could unlock valuable insights and pave the way for developing date palm cultivars resistant to drought and other abiotic stresses.

Conclusively, Khalas, the indigenous cultivar, exhibited the greatest resilience, followed by Reziz and Sheshi. All cultivars displayed physiological decline under drought, but Khalas and Reziz maintained higher levels of water content, photosynthesis, and gas exchange traits compared to Sheshi. Analysis of gene function revealed distinct strategies among the cultivars. Khalas prioritized maintaining photosynthesis and cellular integrity, while Reziz adopted a broader response encompassing various cellular components. Sheshi focused on protecting its photosynthetic machinery under both natural and controlled drought. Furthermore, the study identified cultivar-specific differences in metabolic pathways. Khalas upregulated nitrogen metabolism, potentially enhancing its response compared to other plants. Reziz focused on carbon fixation, potentially improving water use efficiency. Sheshi upregulated light-capturing proteins, likely optimizing light utilization under drought stress. Overall, this study provides valuable insights into the diverse drought response strategies of date palm cultivars. The findings highlight the importance of using natural environments for studying plant stress responses and suggest the presence of potentially novel drought tolerance genes in date palms. These findings can inform future efforts to develop drought-resistant date palm varieties.

Materials and methods

Plant material, experiment time, and experimental site

A field experiment was conducted between April to June of 2021 at the Research and Training Station, King Faisal University (KFU), Saudi Arabia (Latitude 25Ā° 27ā€™ 19.0908ā€ N, Longitude 49Ā° 70ā€™ 94.446ā€ E). The research adhered to the guidelines set forth by the Date Palm Research Center of Excellence, KFU, Saudi Arabia. Uniform five-year-old offshoots of commercial date palm cultivars Khalas, Reziz, and Sheshi were purchased from SAPAD Tissue Culture Date Palm Co., Dammam, Saudi Arabia, by M. Munir. These plants were produced through meristem tissue culture technique in the SAPAD laboratory and the date palm cultivars used in this study were taxonomically identified by M. Munir (Plant Physiologist). These cultivars were not formally deposited in a public herbarium due to their commercial status. The plants were directly planted into soil and acclimatized with adequate watering before exposure to drought conditions. The experiment employed a completely randomized block design with three replicates per treatment and three control plants for each cultivar. Control plants were irrigated on alternate days at 100% field capacity, while no water was applied to induce drought stress during the experimental period. The control plants of each date palm cultivar were labeled as ā€˜irrigatedā€™, whereas the plants exposed to natural drought were labeled as ā€˜stressedā€™.

Determination of relative water content

$$\boldsymbol R\boldsymbol W\boldsymbol C\boldsymbol=\boldsymbol\;\frac{\mathbf{Leaf}\boldsymbol\;\mathbf{fresh}\boldsymbol\;\mathbf{weight}\boldsymbol-\mathbf{Leaf}\boldsymbol\;\mathbf{dry}\boldsymbol\;\mathbf{weight}}{\mathbf{Leaf}\boldsymbol\;\mathbf{turgid}\boldsymbol\;\mathbf{weight}\boldsymbol-\mathbf{Leaf}\boldsymbol\;\mathbf{dry}\boldsymbol\;\mathbf{weight}}\boldsymbol\times\mathbf{100}$$

After 90 days of drought stress (April-June, 2021), the assessment of RWC was conducted, as described earlier31,118. To measure the fresh weight of the drought-stressed leaves, three fully expanded leaves of each cultivar from the second whorl were excised and weighed. To measure the fresh weight of the drought-stressed leaves, three fully expanded leaves (fronds) of each cultivar from the second whorl were excised and weighed. Subsequently, leaves were immersed in distilled water for 16Ā h to yield turbid weight. Leaves were then oven-dried at 70Ā Ā°C for 72Ā h to yield dry weight. RWC was calculated from the following equation:

Estimation of physiological and environmental variables

Different physiological attributes, including leaf chlorophyll content, photosynthesis (Pn), stomatal conductance (gs), transpiration (E), intercellular CO2 concentration (Ci), and water use efficiency (WUE) were estimated, as described earlier31. Physiological attributes were measured at the beginning (day 0) and after 90 days of drought stress. Ten leaves were used for each attribute to ensure reliable data. Leaf chlorophyll content was determined using a chlorophyll meter (SPAD 502, Konicaā€“Minolta, Japan), while the remaining physiological attributes were estimated using the Li-6400XT photosynthesis system (LiCor Inc., Lincoln, NE, USA)31,119. Photosynthesis and photosynthetic attributes were measured using an Infra-Red Gas Analyzer (IRGA). A 6Ā cm2Ā leaf section was placed within the IRGA chamber, maintained at 25Ā Ā°C. The CO2levels of the reference and sample were maintained at 400 Āµmol m2Ā sāˆ’1 with an airflow rate of 500 Āµmol sāˆ’1.

To assess the physiological and molecular responses observed in our date palm study under drought stress, we continuously monitored their real-time exposure to ambient environment throughout the experimental period. The ambient environmental data encompassing temperature (minimum, maximum, and average), average solar radiation, average wind speed, average evapotranspiration (ETc), and relative humidity (minimum, maximum, and average) were obtained from a weather station installed 200Ā m away from the research field.

Leaf sample collection and total RNA isolation

Two grams (gm) of fully expanded leaflets from the second whorl were collected at fortnight intervals throughout the experiment, starting from day 0 and continuing for 90 days. The collected samples were pooled, snap-frozen in liquid nitrogen, and stored in the main laboratory-1056 of Department of Biochemistry, Date palm Research Center of Excellence, KFU, at ā€’80Ā Ā°C until used. The voucher number (CL-DPRC-KRS-21) of the specimen was generated through Laboratory Information Management System (LIMS). The total RNA isolation procedure from these collected samples has been previously demonstrated31. The quality and quantity of the isolated RNAs were assessed electrophoretically and spectrophotometrically (2100 Bioanalyzer, Agilent, USA). To maintain RNA integrity, all plasticware and utensils used during extraction were pretreated with diethyl pyrocarbonate (DEPC, 0.8%) solution.

cDNA synthesis and preparation of drought-responsive libraries

Purified total RNA was used to yield mRNA using the Poly A purist MAG kit (Thermo Fisher Scientific, Massachusetts, USA). Following isolation, cDNA synthesis, including both first and second strands, was performed with the SMARTer cDNA synthesis kit (Takara Bio Inc., California, USA).

To identify genes differentially expressed during drought stress, cDNA libraries enriched for drought-responsive transcripts were constructed for each date palm cultivar using the ClontechĀ® PCR-Selectā„¢ cDNA Subtraction Kit (Takara Bio Inc., California, USA). This method involved hybridizing cDNA from drought-stressed plants (tester) with cDNA from control plants (driver). Unhybridized cDNA, representing differentially expressed transcripts, was then subjected to second-strand synthesis, PCR amplification, and cloning into TOPO-TA vectors (Thermo Fisher Scientific). These vectors were subsequently transformed into Escherichia coli (strain DH5Ī±) and plasmids isolated using the GeneJET Plasmid Miniprep kit (Thermo Fisher Scientific).

ESTs sequencing and assembly

The resultant recombinant plasmid clones were sequenced in their entirety (Macrogen Inc, South Korea). Vector sequences were subsequently removed from the raw reads using MegAlign software (DNAStar, Lasergene, Madison, WI, USA) to obtain clean EST sequences.

Annotation and enrichment analysis of the ESTs

Functional annotation and mapping of drought-responsive date palm ESTs employed a multi-tiered approach, comprising of InterProScan120, gene mapping, and gene ontology (GO), in OmicsBox (v3.1.2)121. Initial annotation assigned putative functions by aligning ESTs against protein databases from various plant species and detailed procedure has already been described in prior study31. Initially, ESTs were annotated based on BLASTx results against the nr database, employing a stringent E-value threshold of ā‰¤ā€‰0.05, resulting in a limited number of ESTs for GO analysis. Subsequently, analyses were repeated with a more permissive E-value of 10 to maximize GO annotation coverage. Unmatched ESTs were further interrogated against the Pfam database within OmicsBox for protein family identification. For ESTs showing multiple BLAST hits, the highest scoring hit was used in downstream analyses.

The next step involves performing GSEA122 to identify statistically significant pathways enriched with the ESTs identified in the previous analysis. GSEA ranks pathways based on their enrichment score, calculated from highly expressed ESTs. In GSEA, highly expressed ESTs were scored through the enrichment score (ES) value and the permutation test were performed and the significant p-value was calculated. Finally, the standardized ES value (NES value) was corrected by various tests to obtain the FDR value. ESTs with NESā€‰>ā€‰1 and FDR.qvalā€‰<ā€‰0.25 are deemed credible.

Pathway and enzyme code analyses of the ESTs

To comprehensively understand the ESTsā€™ roles within the intricate biological mechanisms at play, KEGG pathway analysis35 was performed in the OmicsBox. This approach, particularly valuable for non-model organisms like date palms, allows for seamless integration of gene annotation and expression data onto enriched metabolic and signaling pathways. Likewise, EC distribution of the ESTs was inferred. All annotated ESTs were subjected to the analysis using default settings, with results visualized in both tabular and graphical formats for intuitive interpretation.

Statistical analysis and ESTs submission

Statistical analyses were conducted using Genstat software (14th Edition, VSNi, Hemel Hempstead, England). For significant differences identified by ANOVA (pā€‰ā‰¤ā€‰0.05), Duncanā€™s Multiple Range Test (DMRT) was applied to assess the significant difference between treatment means. Finally, all the ESTs used in the study were submitted to DNA database of Japan (DDJB) and can be accessed online https://ddbj.nig.ac.jp/search/entry/bioproject/PRJDB17451 [Khalas], https://ddbj.nig.ac.jp/search/entry/bioproject/PRJDB17452 [Reziz], and https://ddbj.nig.ac.jp/search/entry/bioproject/PRJDB17453 [Sheshi].

Ethical statement

All procedures and protocols adhered to the ethical standards set forth by the indigenous institutional committee.

Conclusions

Conclusively, this study delved into multifaceted drought resilience of three date palm cultivars (Khalas, Reziz, and Sheshi) to natural drought under ambient environment, integrating both physiological and transcriptomic analyses. While drought imposed significant stress on all cultivars, affecting key physiological parameters, Khalas exhibited demonstrably higher resilience compared to Reziz and Sheshi, by maintaining robust water content, photosynthesis, and gas exchange, while uniquely prioritizing nitrogen metabolism to bolster its stress response. Transcriptomic analysis unveiled nine common pathways employed by all cultivars to cope with drought stress, alongside several unique pathways specific to each cultivar, highlighting their inherent potential for adaptation to natural stress. Interestingly, Khalas exhibited the most extensive molecular response, followed by Reziz and Sheshi. The GSEA revealed cultivar-specific enrichment of ESTs, highlighting distinct adaptations to environmental challenges. Khalas prioritized cellular organization, metabolic processes, and metal ion binding, while Reziz focused on molecular functions, cellular anatomy, and regulatory processes. Sheshi emphasized cellular components, membrane-bound organelles, and photosynthetic machinery. Importantly, the observed responses under natural drought conditions differed substantially from those observed in our previous controlled glasshouse studies. This underscores the crucial role of field-based research in capturing the true complexities of plant responses to natural stressors. Consistent with previous work, a significant number of identified genes lacked homology to known Gene Ontology (GO) terms, suggesting that these date palm cultivars have unique genome architect. This opens exciting avenues for future research into the specific functions of these genes and their potential role in drought tolerance.