1 INTRODUCTION

The scientific community has faced challenges in reaching a consensus on the precise definitions of drought and beyond broad interpretations, such as "a deviation from typical water availability" (Slette et al., 2019). Although the terms used to describe drought can differ and be inconsistent, researchers strive to define tree species' and ecosystems' responses and adaptations during the post-drought period (Vilonen et al., 2022). A recent study defined drought as an insidious natural hazard that proceeds gradually and subtly, causing harmful effects (Wlostowski et al., 2022). The World Health Organization (WHO) defines drought as a prolonged period of dryness within the natural climate cycle, which can occur in any location worldwide (WHO, 2023). In an early chapter named Encyclopedia of World Climatology, drought was described as an insidious natural hazard that arises from a lack of precipitation compared to what is expected or deemed "normal." When this occurs over a season or an extended period, the amount of water available is inadequate to fulfil the demands of human activities (Wilhite, 2005). From the perspective of a plant biologist, drought in plants results from a water deficit in the soil, which prevents a specific plant or canopy from receiving the necessary amount of water to sustain its water requirements at a given time. This can significantly alter the plant's water status (Tardieu et al., 2018). A plant physiologist can fundamentally define drought as a condition that dehydrates cells and tissues. Any abnormal water loss by the plant disturbs its water status by creating a water deficiency (Hura et al., 2022). However, a slight water deficiency is typical and does not impair the plant's functioning (Henckel, 1964).

Drought conditions could endanger forest ecosystems, already subjected to significant stress, leading to drastic changes in forest tree mortality and distribution (Flory et al., 2022). Therefore, it is essential to understand how tree species respond to drought to gain a deeper understanding of forests' vulnerability to the effects of climate change (Gazol et al., 2018). Interestingly, it was reported that, among trees within forests, the largest trees are affected at twice the rate of smaller trees due to climate change, and abiotic stress gradients of water, temperature, and competition regulate the intensity of the height-mortality relationship, threatening critical ecological, economic, and social benefits (Pascual et al., 2022). Forest ecosystems are "THE" crucial factors that sustain the health of Earth planet, and the woody plant species play a critical role in maintaining these ecosystems. They absorb carbon dioxide from the atmosphere, produce oxygen, and provide habitat for countless species of animals and plants (Uprety et al., 2023). Forests provide many benefits for society and can help alleviate human-caused climate change. Nevertheless, climate change-associated effects may threaten the carbon sinks of forests in the 21st century and biodiversity conservation, altering the ranges of tree species and forest community assemblages, impacting the carbon cycle and leading to forest vulnerability to stresses (Anderegg et al., 2020; Forzieri et al., 2021; Pascual et al., 2022). With anthropogenic forcing anticipated to increase warming further, as well as the frequency and duration of heat waves and soil drought, tree mortality is also expected to increase.

However, climate change is causing a rise in the frequency and severity of droughts that ipso facto constitute one of the principal threats to the health of forest ecosystems, including forest dieback, decreased productivity, and increased susceptibility to pests and diseases (Hammond et al., 2022). For example, Ma et al. 2023 elucidated how prolonged drought induces hydraulic failure in trees, disrupting water transport mechanisms and leading to reduced growth rates and increased mortality. Moreover, studies by Liu et al. (2024) and Adams et al. (2017) highlight the vulnerability of specific tree species to drought-induced mortality, with particular sensitivity observed in coniferous forests and tropical rainforests. Furthermore, investigations by Raharivololoniaina et al. (2023) underscore the complex interplay between drought, insect outbreaks, and tree mortality, emphasizing the cascading ecological consequences of prolonged water scarcity. Drought can significantly impact forest trees' growth, development, and survival. Indeed, water availability is crucial for trees because it affects their photosynthesis and respiration rates (van Kampen et al., 2022). These processes sustain plant growth and development through complex molecular regulation. Over the past decade, there have been significant advancements in Next Generation Sequencing and biochemical techniques, which have enabled the scientific community to gain valuable insights into the molecular basis of drought stress tolerance of woody plant species (Estravis-Barcala et al., 2020). The irony is that tree species that adapt to drought conditions can drastically impact other living organisms. The survival and existence of several species, such as insects, depend on how well trees can cope. Indeed, insects and other terrestrial arthropods are vital to forest ecosystems, but drought conditions due to climate change are causing significant reductions in their diversity (Frank, 2021; Gely et al., 2020). Thus, understanding how tree species face and adapt to drought benefits other living species.

Contemporary advances in high-throughput DNA sequencing have greatly improved our knowledge of how forest trees respond to water deficiency. These technical developments have allowed a more exhaustive exploration of the molecular and genetic mechanisms underlying drought tolerance in woody plants. This research is critical given the increasing prevalence of drought due to climate change and its impact on forest productivity. The present review aims to provide new updates and a comprehensive understanding of the responses of woody plants under drought conditions. It will delve into the visible changes in trees under drought stress, including shoot and root growth alterations. Furthermore, the article will explore recent developments in tree drought tolerance, including genetic and molecular mechanisms, such as changes in gene expression and epigenetic modifications.

2 FROM THE TRIASSIC TO THE ANTHROPOCENE: LET'S DIG INTO THE HISTORY OF FOREST TREES AND DROUGHT

“Trees have never been the ideal subject to study” (Aitken & Bemmels, 2016). Forests worldwide are facing severe challenges, including deforestation due to human activities like logging and land conversion and environmental stressors such as drought, exacerbated by climate change(Figure. 1) (Armstrong McKay et al., 2022). Like every other environmental phenomenon, drought has a history, and every plant species comes from an ancestor. Before delving into the current physiological and genetic knowledge regarding forest tree adaptation and response to drought, this section explores the broader context of forest adaptation and evolution. It highlights how ancient forest species have historically navigated drastic climatic changes, including periods of drought, during past geological eras.

Indeed, as many research articles have addressed drought's impact on current forest trees, it is essential to recognize that the genetic and physiological strategies that trees employ today are rooted in their evolutionary history (Skelton et al., 2021; Piovesan et al., 2021; Grant et al., 2017). The connection between past and present can emphasize the importance of integrating fields such as paleoecology with contemporary physiological and genetic research to fully grasp the complexities of tree survival under drought conditions. However, the significant lack of studies and understanding of ancient forest tree species' responses to drought remains extremely challenging. Still, investigating or providing an overview of the history of drought and trees, particularly based on the current findings in paleoecology, can offer a fascinating window into how today's trees might navigate and adapt to the challenges of the harsh modern environment.

Forests have been on Earth for millions of years, evolving and adapting to various climatic and geological changes (Rivers et al., 2023). Ancient forests were vast and diverse, playing a crucial role in the ecological balance. The evolutionary history of these forests has shaped the genetic and physiological adaptations that modern tree species employ to cope with environmental stressors like drought. For example, ancient forest species (AFS) that have persisted through multiple geological epochs and faced drastic drought events have developed specific ecological traits that enable them to survive in environments with fluctuating water availability. These traits, such as deep root systems and drought-tolerant leaf structures, are not just a result of current climatic conditions but are deeply rooted in the evolutionary pressures faced by their ancestors (Bergès & Dupouey, 2021; Brodribb et al., 2020). The slow colonization of current forests by AFS indicates their specialized growth traits that evolved under stable environmental conditions (Hermy & Verheyen, 2007). These traits, such as specific root structures or growth patterns optimized for nutrient-poor or undisturbed soils, may confer low dispersal capacity and reduced recruitment success in altered soils (Bergès & Dupouey, 2021). The evolutionary pressure on AFS to develop growth traits suited to ancient ecosystems has, in turn, limited their ability to rapidly adapt to the dynamic conditions of modern forests, where natural processes like succession or human-driven reforestation have altered the landscape (Kimengsi et al., 2023).

Although there is evidence of the significant influence of mankind in the increase of drought conditions and forest species diversity and distribution in the current epoch (Anthropocene), along with other phenomena that affected tree species development, the history of drought and deforestation happened on Earth before the apparition of mankind. Research on the consequences of drought on forest composition and diversity is supported by evidence from historical shifts in vegetation patterns during periods of significant climatic stress, such as in the early Triassic period after the Permian-Triassic extinction event (252 to 201 million years ago) (Jiao et al., 2023; Vajda et al., 2020). During this period, drought conditions led to new, low-diversity, drought-tolerant plant associations dominated by conifers and the seed fern Lepidopteris. The occurrence of false rings in early Triassic wood, along with sedimentary features indicative of seasonal drought (like desiccation structures, ferruginous concretions, and microspherulitic siderite), suggests that this period experienced significant drought conditions (Santos et al., 2023; Vallejos Leiz et al., 2022). This change is attributed to immigration from warmer and better-drained regions with drought-tolerant species. The plants adapted to drier settings likely existed in small populations with low preservation potential in the late Permian period (260 to 251 million years ago) (Bashforth et al., 2021; Richey et al., 2021). These species likely evolved in response to the dry, arid conditions of the late Permian period, demonstrating an early example of how severe environmental stressors can drive the evolution of plant drought tolerance. In fact, the physiological responses observed in modern tree species, such as osmotic adjustments, reflect these ancient adaptations. Indeed, Zhu et al. (2016) acknowledged that SNF1/AMPK-related kinases proliferated and diversified during evolution to mediate the signalling of various abiotic stresses. This suggests that the SNF1/AMPK-related kinases, a family of protein kinases, have evolved and expanded their functions over time to play a crucial role in helping plants respond to different environmental stresses, such as drought. Under stress conditions, SNF1/AMPK-related kinases can modulate plant growth pathways (Zhang, Zhao et al., 2020). The genetic mechanisms underlying these responses, including the regulation of stress-responsive genes, have been fine-tuned over millions of years through natural selection. This evolutionary perspective provides a deeper understanding of how trees today continue to adapt to the increasingly unpredictable climate of the Anthropocene. More empirical evidence is needed to support these statements.

In the current geological epoch (Anthropocene), one specific challenge mentioned by Hammond et al. (2022) is the occurrence of "hotter droughts." These are periods of reduced rainfall combined with higher temperatures, which are becoming more common due to climate change. In this sense, rising temperatures could affect plant water status by enhancing the vapour pressure deficit and reducing stomatal aperture, eventually reducing secondary metabolism, carbon storage, and plant resistance. These conditions are particularly stressful for forests and can lead to widespread forest die-off events, where large areas of forest lose their trees (Veuillen et al., 2023), making the future of several ecosystems uncertain, particularly on how forests will respond to increasing temperatures, changes in precipitation patterns, and the frequency and severity of extreme weather events.

As the scientific community debates the right path of action to tackle the current climate crisis and to stop the increase of drought, the tree species, veterans of the Permian extinction, stand tall, almost chuckling at humanity's late completion. They had witnessed the Permian extinction (conifers, ginkgophytes, or cyadophytes) and had mastered the art of survival long before Homo sapiens stepped on the scene. Thus, understanding how drought evolves with global climate changes and forest tree adaptation and development is necessary for mankind. Substantial research, tools, and molecular databases have been made to monitor the progress of drought (Curtis, 2023), high-throughput genomics (Falk et al., 2019), or advocate for the lack of studies about the molecular response of forest trees to abiotic stress compared to annual crops (Cisse et al., 2023). Indeed, drought evolution and climate changes are increasingly shaping the environmental landscape, with significant implications for water scarcity, agricultural productivity, and ecosystem health. Since its inception, tools such as the U.S. Drought Monitor have been crucial, documenting these trends and providing vital data to inform policy, management strategies, and public awareness. As shown in Figure. 2, the U.S. Drought Monitor represents a significantly advanced tool that plays a critical role in drought assessment and management in the United States, proposing a classic and regularly updated picture of drought conditions and their severity across different areas. Moreover, based on modern physiological tools, researchers have found that the adaptation strategies of several forest trees such as Olea europaea (Ennajeh et al., 2008), Pinus palustris (Samuelson et al., 2019), and some Acacia species (Mendham & White, 2019) on managing efficiently limited water resources and surviving prolonged drought periods are remarkably sophisticated. Although compared to the small forest species that possess a Vegetative Desiccation Tolerance, which can “dry without dying” (Gao et al., 2023), forest tree species cannot stay utterly desiccated. Still, they can withstand dry conditions to a significant extent.

3 THE PHYSIOLOGY OF WOODY SPECIES UNDER DROUGHT STRESS ENCOMPASSES VARIOUS CONCEPTS AND SIGNIFICANT CHARACTERISTICS

Like all organisms, trees undergo eco-physiological changes in response to drought conditions, including alterations in metabolic processes, gene expression, and cellular functions. The nature and magnitude of these responses depend on the stressor's intensity and frequency, as well as the developmental stage of the plant (Rosso et al., 2023). Forest trees, in their natural environments, face periodic drought conditions. They have evolved various physiological and morphological adaptations to survive these challenging periods. Estravis-Barcala et al. (2020) highlighted the different adaptive strategies trees have developed to cope with and survive drought, categorized into mechanisms that either avoid or tolerate drought, as illustrated in Figure. 3.

Indeed, trees use various strategies to avoid drought by reducing water loss or enhancing water uptake (Dubbert et al., 2023; Shao et al., 2023). These physiological adjustments are closely tied to growth responses. Indeed, trees must reduce growth rates to conserve resources under stress, thus linking growth reduction directly to drought avoidance strategies. For instance, tropical forest species such as Clitoriafair childiana, Ceiba pentandra, or Hura crepitans grew deeper or more extensive root systems to access water from deeper soil layers, which allowed them to tap into moisture reserves (Kühnhammer et al., 2023) unavailable to species with more superficial roots. Trees such as Eucalyptus reduced their number and surface of leaves, resulting in smaller leaves or fewer leaves that can change their orientation to reduce direct sun exposure (Fernández-de-Uña et al., 2023; Jiang et al., 2021). Some tree species might lose their leaves to adapt to water scarcity; the North American ocotillo (Fouquieria splendens) is an example of a drought-deciduous plant. It loses its leaves during dry periods and re-grows them when water is available. When the soil dries out, the ocotillo leaves undergo senescence and are shed, reducing water loss through transpiration (Whitford & Duval, 2020). Mediterranean woody shrubs might adapt to drought through phenotypic plasticity by developing leaves with a lower Specific Leaf Area (SLA), meaning thicker, smaller leaves are less prone to water loss. Observing changes in SLA can indicate how plants adapt to drought conditions (Carrascosa et al., 2023). These morphological changes, such as reduced leaf area, are directly linked to reduced growth rates, as the plants conserve water and reduce the risk of hydraulic failure during drought periods.

Moreover, growth traits such as biomass accumulation, root-to-shoot ratio, and overall plant vigour are tightly linked to the plant's ability to maintain cellular homeostasis and function during drought stress (Teixeira, 2022). Characteristics such as turgor loss points are critical leaf traits. It's the point at which the cells in a leaf lose their turgidity, leading to wilting. This point varies among plant species and is an essential indicator of drought tolerance (Rosso et al., 2023). The accumulation of compatible solutes like sugars and amino acids plays a crucial role in maintaining cell homeostasis, essential for sustaining cell expansion and growth under drought conditions. These solutes play a role in osmotic adjustment, which can prevent growth inhibition due to water deficit (Takahashi et al., 2020; Zulfiqar et al., 2020). These osmolytes have been regulated in Citrus trees during drought. Several studies have acknowledged how drought regulates osmolytes or compatible solutes in plants under drought to maintain cell homeostasis. Indeed, Sousa et al. (2022) investigated the metabolic responses of Valencian sweet orange trees grafted on different rootstocks to drought stress, revealing distinct metabolic responses in both leaves and roots of three Citrus scion/rootstock combinations under drought stress and subsequent rehydration. They demonstrated that drought stress induced significant changes in metabolite profiles, including alterations in sugars, organic acids, and amino acids. Importantly, these responses varied among the different scion/rootstock combinations, indicating genotype-specific adaptations to water scarcity. Upon rehydration, partial recovery of metabolite levels occurred, suggesting the reversibility of drought-induced metabolic changes. The mechanisms underlying their drought tolerance involve several factors: firstly, efficient water uptake and root architecture contribute to improved water use efficiency. Secondly, osmotic adjustment mediated by the accumulation of compatible solutes such as sugars and amino acids helps maintain cell turgor under water-deficit conditions. Thirdly, antioxidant defence systems counteract oxidative stress caused by drought, ensuring cellular integrity and function. Such metabolic adjustments enable trees to survive drought conditions and directly influence growth patterns by altering resource allocation and energy balance (Sousa et al., 2022; Schweiger et al., 2023). The osmotic adjustment is a vital physiological response that forest species employ to withstand drought stress by preserving cell turgor at lower water potentials. This adaptation allows them to continue their cellular activities under drought conditions. In Acer saccharum (Sugar Maple), the accumulation of soluble sugars in its leaves enables it to maintain cell turgidity and continue photosynthesis during drought (Walters et al., 2023). Proline, an amino acid that plants often accumulate in response to osmotic stress, served as an osmoprotectant and stabilizing proteins and membranes in Dalbergia odorifera, as Cisse et al. (2021) suggested. Quercus tree species exposed to drought increase several specific metabolites, such as sucrose, fructose, and glucose, showing an osmotic potential and osmotic adjustment, illustrating their high ability to tolerate drought (Aranda et al., 2021). Other plants like Fagus sylvatica showed accumulation of organic acids in their leaves under drought stress, resulting in organic matter accumulation (Brunn et al., 2023), which maintains cell turgidity and osmotic stability function under drought (Venekamp et al., 1989). Populus deltoides has been found to increase glycerol under drought conditions, suggesting that glycerol helps maintain water status. P. deltoides has been found to increase glycerol under drought conditions, suggesting that glycerol helps maintain water status and provides drought tolerance (Tschaplinski et al., 2019). Besides the osmolytes, substances such as alkanes have a prominent role in trees' physiological adaptation to drought. Tree species can produce leaves with waxy coatings that reduce water loss by creating a barrier to slow water evaporation from the leaf surface. Negin et al. (2023) showed a strong correlation between the accumulations of alkanes and reduced cuticular water loss in tree tobacco (Nicotiana glauca). Indeed, Alkanes are crucial for the plant's ability to recover after drought. They seal the cuticle and prevent water loss when the stomata are closed (Bueno et al., 2020; Negin et al., 2023).

Moreover, drought stress is a significant factor associated with photosynthetic pigment biosynthesis limitation in many trees. Woody tree fragrant rosewood (Dalbergia odorifera) significantly reduced its photosynthetic pigments content and net photosynthetic rate to avoid drought damages that can profoundly affect their survival rate (Cisse et al., 2021; Cisse et al., 2022). This reduction in photosynthetic capacity is often associated with decreased growth rates, as energy and resources are diverted toward survival rather than biomass accumulation. Indeed, several forest species undergo photosynthetic and osmotic adjustments to avoid the adverse effects of drought. Hu, Zhang et al. (2023) showed that the yellow horn (Xanthoceras sorbifolium) adopted the same photosynthetic pathway to minimize water loss while still capturing carbon dioxide for photosynthesis. However, the yellow horn also reduced photosynthesis efficiency, as in several CAM (Crassulacean acid metabolism) plants, which negatively affect plant growth and development (Herrera, 2000; Hu, Zhang et al., 2023). The plant stomata is one of the prominent structures in plant leaves that play a massive role in regulating gas exchange and water loss during photosynthesis (Verma et al., 2020). Stomata are microscopic pores located primarily on the leaves of plants. Each stomatal pore is flanked by specialized guard cells, which control its opening and closing (Clark et al., 2022). Many tree species close their stomata to reduce water loss through transpiration in response to drought. While conserving water, stomata closure also limits photosynthesis and growth, further illustrating the trade-off between growth and survival under drought conditions.

Furthermore, organic compounds such as phytohormones are integral to the plant's ability to adapt to changing environmental conditions and optimize growth and development throughout its life cycle (Parwez et al., 2022). Phytohormonal changes play a crucial role in the response of forest trees to drought stress, affecting both their growth and survival. Under drought conditions, the levels of several key phytohormones are altered to help the plant cope with water scarcity (Pandey et al., 2017). Abscisic acid (ABA) is one of the primary hormones that increase during drought, leading to stomatal closure to reduce water loss through transpiration, as shown in Populus euphratica (Yang et al., 2020). This hormonal response is critical for maintaining water status but can also limit carbon dioxide intake, reducing photosynthesis and growth. For example, in Cinnamomum camphora, ABA accumulation under drought has been shown to correlate with reduced growth rates due to its impact on photosynthesis and stomatal conductance (Duan et al., 2020). In Populus, the content of free auxin in the developing xylem decreases during stress, including drought, while the levels of auxin conjugates (inactive forms of auxin) increase. The decrease in free auxin and the increase in auxin conjugates indicate that the plant is deliberately reducing the activity of auxin (Popko et al., 2010). This down-regulation of auxin signalling is important because it helps the plant adapt to stressful conditions by limiting growth and focusing on survival. In poplar, this may affect how the tree develops wood (xylem) and other tissues during stress, possibly slowing growth to prevent damage or excessive water loss. In addition to ABA and auxins, drought stress often triggers changes in the levels of ethylene, cytokinin, jasmonic acid, salicylic acid, and gibberellins (Ullah et al., 2018). Ethylene is generally associated with stress responses and can promote leaf senescence, reducing photosynthetic capacity and growth (Chen et al., 2023). These phytohormonal adjustments form part of complex signalling pathways that allow trees to prioritize survival overgrowth under drought conditions. However, more investigations are needed to understand how these plant hormones are regulated, especially in forest trees under drought.

The study of the adaptation of tree species to water deficiency at morphological and physiological levels is an area of active research, and a significant amount of data is available. However, as climate change alters environmental conditions, ongoing data collection and analysis are critical to updating our current understanding. These physiological changes, driven by complex genetic mechanisms, highlight the profound influence of molecular responses on tree adaptation to drought. This understanding leads us to the next chapter, where we will delve deeper into the transcriptomic landscape and examine how gene expression modulates these critical adaptive processes.

4 INSIGHTS INTO THE MOLECULAR RESPONSES (ACCLIMATION) OF TREE SPECIES TO DROUGHT STRESS: THE OMNIPRESENCE OF THE NEXT GENERATION SEQUENCING

Conducting experiments with woody species presents challenges compared to annual crops, primarily due to the large size of trees, their extended life cycles, and the complexity of their natural environments. Significant genetic studies of forest trees have produced valuable information crucial for understanding these species' economic and evolutionary aspects in the last decades (Estravis-Barcala et al., 2020; Hamanishi & Campbell, 2011; Hammond et al., 2022). Indeed, genetic studies revealed crucial information about tree growth rates, disease resistance, adaptation to different environmental conditions, including drought, and other traits that could benefit forestry and conservation efforts. For example, the connection between genetic markers and growth response to drought is evident in identifying heritability traits like plant height and wood density, which are associated with drought resistance. This connection highlights how genetic traits influence growth patterns under drought conditions (Rowland et al., 2023). Emerging genomic selection tools, which utilize DNA-based data to forecast genotype performance under specific environmental conditions such as drought, offer a solution to the constraints of conventional breeding approaches. This method can significantly reduce the time required to evaluate and select desirable traits in tree species, as it doesn't rely on long-term progeny trials (Ismael et al., 2022). Using genetic markers associated with the characteristics of interest (like drought tolerance) can make a more accurate tree selection. In a recent research study, Soro et al. (2023) show that young white spruce clones have varied responses to drought. The significant heritability levels observed imply that selecting and breeding trees based on their drought response at a young age could be an effective strategy. Interestingly, the heritability of apical growth was raised under drought conditions, suggesting that genetic control of growth traits is heightened under stress, making these traits more prominent and selectable in breeding programs aimed at improving drought tolerance. Moreover, Next Generation Sequencing (NGS), a spectrum of modern sequencing technologies, has revolutionized genomic research in studies focused on tree species and all living species under different conditions (Tsumura, 2023). The high-throughput DNA sequencing developed by Illumina, Inc. Kim et al. (2021) has been essential in advancing genomic research, contributing to a better understanding of studying gene expression under drought stress. Such research provides insights into how specific genetic factors regulate growth processes under drought, linking molecular responses directly to growth outcomes.

Tree species such as Populus, commonly known as poplar, which plays a significant role in forest ecosystems, a crucial model plant species in tree research due to its rapid growth and the availability of a fully sequenced genome (Populus trichocarpa) (Velioğlu et al., 2023). In forestry, Populus serves as an essential model for understanding the responses of trees to different environmental conditions, including drought (Zeng et al., 2023). Indeed, Populus belongs to the family Salicaceae and is widely used in forest tree biology, especially in studying tree physiology, genomics, and environmental interactions. With the new sequencing technologies, researchers have provided significant results about the molecular (gene expression) mechanisms underlying the response of poplars to drought, as shown in Table 1 (Table 1 provides more examples about Populus to show that more studies were focused on Populus). The application of transcriptomic analysis constitutes a powerful tool for understanding how the Populus species regulate their physiological traits in response to drought. Under drought stress, Populus alba activates BAM1a, BAM1c, AMYb, and AMYc for starch degradation across organs (Fox et al., 2023), necessary for several soluble sugar accumulations that counteract the osmotic stress. Indeed, the increase in sucrose synthesis in the roots will likely provide energy for root functions even under drought stress in P. alba, while sucrose degradation is reduced. The limited allocation of sucrose from aboveground organs to the roots (as suggested by the downregulation of invertases) implies that P. alba is restricting growth in favour of maintaining essential functions. It has been shown that AMY genes encoding amylases in cassava (MeAMY1, MeAMY2, MeAMY5, and MeBAM3) that regulate the enzymes amylases that break down starch into sugars are crucial for initiating starch degradation under drought stress. The sugars released are then available for glycolysis and respiration, directly supporting growth. BAM genes in Vitis vinifera (VvBAM1 and VvBAM3) encoding the enzyme Beta-amylase can activate the release of maltose from starch, which can be further metabolized to support energy-demanding processes such as growth under stress.

Molecular research studies have also been conducted on several other forest species exposed to drought, as shown in Table 1. Indeed, by comparing transcripts of species like Fagus sylvatica and pedunculate oak (Quercus robur), researchers have harnessed ecological traits for drought resistance, with deep root systems and controlled leaf shedding being key focus areas (Polle et al., 2019). Indeed, it has been found that 12 genes are involved in drought response in two major European oak species, Q. robur (pedunculate oak) and Q. pubescens (pubescent oak), based on comparative transcriptomics (Kotrade et al., 2019). These genes were highlighted for their differential expression and functional significance in drought response pathways and networks. Indeed, PP2C27 (Protein phosphatase 2C 27), RD22 (Response to desiccation 22), and STP13 (Sugar transport protein 13) are integral to the abscisic acid (ABA) signalling pathway, crucial for drought stress modulation. Meanwhile, genes like BCA2 (Beta carbonic anhydrase 2) and GPT2 (Glucose-6-phosphate/phosphate translocator 2) are related to photosynthesis, while BOR2 (Boron transporter 2) is involved in nutrient transport. Genes such as RD22 (Responsive to Dehydration 22) have been shown to play a crucial role in water deficit response in Pinus pinaster and are directly linked to growth regulation under drought by modulating cellular processes that indirectly impact tree growth. (de María et al., 2020). The expression of RD22 (a marker of drought stress) largely depends on the hormone abscisic acid (ABA), which accumulates in plants during drought stress. Although the exact function of RD22 is not fully understood, it has been suggested to be involved in the phenotypic and physiological adjustment in Phyllostachys edulis through PheDi19-8 (Drought-induced 19) and PheCDPK22 (Calcium-Dependent Protein Kinase) (Wu et al., 2020). Further, previous research has acknowledged that heat Shock Factor (HSF) genes (PheHsfA4a-1, PheHsfA4a-2, PheHsfA4d-1, and PheHsfA4d-2) in moso bamboo (Phyllostachys edulis) are the essential genes involved in drought response and their impact on the species (Xie et al., 2019). Haas et al. (Haas et al., 2021) showed molecular regulatory networks in Norway spruce (Picea abies) that manage resource allocation during drought stress and recovery through genes such as MYB96 or MYB15. MYB96 helps the plant survive drought by focusing growth on the primary root, as demonstrated in Arabidopsis (Seo et al., 2009), where it integrates ABA and auxin signals to arrest lateral root growth., while MYB15 ensures that the plant does not remain in a perpetual state of drought stress response once the immediate threat has passed, it has a negative regulatory function toward DREB1/CBF genes (Dehydration-Responsive Element/C-repeat) (Agarwal et al., 2006), which suggest its crucial role in tree recovery under drought.

Moreover, a recent article has reported for the first time a comprehensive transcriptomic profile related to drought for Abies pinsapo, which successfully identified specific genes and genetic variants associated with drought resilience. The study continues beyond investigating the drought response but also at post-drought recovery, comparing resilient and sensitive phenotypes (Cobo-Simón et al., 2023). Based on NGS, the study revealed that stomatal closing and inhibition of plant growth-related genes during immediate drought were activated, suggesting an isohydric dynamic. Extended drought revealed the prevalence of transcription factors and genes related to cellular damage and homeostasis protection in A. pinsapo. It highlights that resilient individuals activated photosynthesis-related genes and inhibited aerial growth-related genes, indicating a strategic shift in biomass allocation to improve water uptake and carbon balance. Indeed, NGS is a robust platform that has contributed to elucidating molecular mechanisms underpinning the responses of many non-model forest trees under drought stress. Indeed, studies have shown through NGS the global transcriptome variation under drought of Acer Saccharum (Mulozi et al., 2023), Carya illinoinensis (Zhu et al., 2023), Cyclocarya paliurus (Li, Wan et al., 2023), Pterocarya stenoptera (Zhang, Wang, Li et al., 2023), Quercus mongolica (Li, Jiang et al., 2023), Artemisia californica (Atamian & Funk, 2023), Quercus brantii (Safari et al., 2023), Atraphaxis bracteate (He et al., 2024), Cryptomeria japonica (Nose et al., 2023) and Acer truncatum (Li, Guo et al., 2023). This research provided massive vital genes and transcription factors involved in different plant metabolisms crucial for drought tolerance. This is necessary to improve plant resistance to drought by genetically modifying key genes involved in the plant's stress response pathways, particularly those associated with abscisic acid (Polle et al., 2019). Plants can draft or activate a comprehensive set of defence mechanisms through these genetic modifications, which means various responses and adaptations are triggered to counteract the adverse effects of drought conditions.

5 GENOME-WIDE ANALYSIS AND EPIGENETIC REGULATION OF FOREST TREES TOWARD DROUGHT CONDITIONS

The genome-wide analysis (GWA) is an emerging molecular tool in molecular biology that can provide a static picture of plant genome through Genome-Wide Association Studies (GWAS) or Whole Genome Sequencing (WGS) (Brachi et al., 2011; Chen et al., 2019; Wang et al., 2020), different from the transcriptomic analysis that focuses on the transcriptome, which uses several techniques such as RNA sequencing (RNA-Seq), quantitative PCR and microarrays (Guo et al., 2021). The application of genome-wide analysis in comprehending and responding to the challenge of drought in forest trees can help identifies the genetic basis of traits contributing to drought tolerance (Figure. 4). The identification of genetic traits is crucial in plant stress studies. It enables researchers to understand how these traits influence tree growth under drought conditions, linking genetic findings directly to growth outcomes. By understanding these traits, conservation efforts can be more targeted and effective, ensuring that the most resilient genotypes are preserved. This approach is critical to conserving adaptive genetic variation, guiding selective breeding programs, and directing genetic modifications to prepare forest ecosystems for the challenges posed by future drought conditions under the influence of climate change (De La Torre et al., 2022; Hamanishi & Campbell, 2011; Jin et al., 2020). However, the functions of many genes still need to be better understood. This knowledge gap persists because identifying a gene's sequence does not necessarily reveal its role in the cell or organism. Many genes encode proteins whose functions cannot be inferred directly from their sequence alone. Nevertheless, the functions of several genes involved in drought resistance have been reported previously through genome-wide analysis. For instance, P. trichocarpa fructose-bisphosphate aldolase genes (PtrFBAs), such as PtrFBA60, have a prominent regulatory function in plant drought tolerance via the ABA pathway (Feng et al., 2023). FBAs genes localized in different cellular compartments, such as plastid and cytoplasm (Carrera et al., 2021), were suggested to delay growth under drought conditions through stress-induced changes in gene expression, energy reallocation, impaired root function, and metabolic adjustments in Arabidopsis (Lu et al., 2012). The expression of AtFBA genes in Arabidopsis (AtFBA1, AtFBA2, AtFBA5, and AtFBA7) have undetectable expression in Arabidopsis roots under drought, which might be involved in impairing root function. The study also highlighted that sugars like sucrose, glucose, and fructose induce some AtFBA genes. Since sugar signalling is linked to growth regulation, the induction of specific AtFBA genes in response to sugars and their stress-related modulation suggests that FBAs could be involved in a complex regulatory network that balances growth and stress responses. Additionally, many members of the R2R3-MYB gene family were identified in the entire P. trichocarpa genome and several R2R3-MYB genes have been found to play a role in poplar drought tolerance (Zhang, Wang, Chen et al., 2023). Furthermore, R2R3-MYB transcription factors have also been shown to be involved in regulating Populus euphratica growth under drought conditions (Sun et al., 2023). The role of MYBs in plants under drought was highlighted in a previous study (Roy et al., 2016). The study presented the MYBs as prominent in plants against water scarcity; they regulate a variety of pathways and gene networks, including ABA-dependent (AtMYB2, AtMYB74, AtMYB102, AtMYB60, AtMYB96) and ABA-independent (BcMYB1 and LcMYB1) stress signalling, and miRNA-mediated gene regulation. Indeed, in response to ABA, miR159 is induced, leading to the cleavage of MYB33 and MYB101 transcripts. This regulation modulates the plant's sensitivity to ABA. MYB Transcription Factors such as AtMYB96 have been shown in the crosstalk of ABA-auxin signalling. AtMYB96 influences lateral root meristem activation, crucial for root architecture adaptation under drought conditions. This adaptation helps in efficient water uptake, contributing to enhanced drought tolerance. Furthermore, the exhaustive genome-wide analysis showed that four Phyllostachys edulis dehydration response element binding genes (PeDREB) were induced by drought. Precisely, PeDREB28 over-expression indicated that PeDREB28 is potentially involved in regulating redox homeostasis under drought (Hu, Liang et al., 2023). DREB transcription factors are essential regulators of drought stress response in Moso bamboo. They activate stress-responsive genes by binding to specific DNA motifs, which can interact with ABA hormonal pathways and are part of a broader network of transcription factors that enhance the plant's resilience to drought. The study demonstrated that PeDREB28 interacts with the ABA signalling pathway and can modulate the expression of DlaPYL3, a gene involved in the ABA receptor family, suggesting that DREB factors might help fine-tune the plant's response to drought by modulating ABA-related gene networks (Hu, Zhang et al. 2023). Furthermore, based on GWAS, many genes have been suggested to play a prominent role in drought tolerance. Among them Liriodendron chinense carotenoid cleavage oxygenase gene (LcNCED3b) (Xue et al., 2023), P. trichocarpa targeting protein for Xklp2 (PtTPX2) (Qi et al., 2023), Phyllostachys edulis somatic embryogenesis receptor-like Kinase (PeSERK3) (Zhang, Huang et al., 2023), and Liriodendron chinense NAC genes (LcNAC6/18/41/65) (Liu et al., 2023) were suggested to be involved in drought tolerance. The expression of Phoebe bournei genes related to GATA-binding transcription factors PbGATA16, PbGATA22, and PbGATA5 appeared crucial in dealing with drought (Yin et al., 2023). More GWAS in forest trees under drought is urgent because there remains a significant gap in our understanding of many genes. Numerous genetic variants have yet to be identified or fully characterized, leaving their roles in drought tolerance largely unknown.

As organisms with extended lifespans, trees face diverse environmental challenges throughout their existence, water deficiency being a significant example, and epigenetic mechanisms can provide a rapid response system for trees to acclimate to such stressors. Indeed, epigenetic mechanisms, such as DNA methylation and histone modification, play a crucial role in the adaptation of trees to environmental stressors, particularly drought (Batalova & Krutovsky, 2023; Bräutigam et al., 2013; Estravis-Barcala et al., 2020). These mechanisms propose a layer of control over gene expression that can swiftly and dynamically respond to changing environmental conditions, which is essential for the survival and adaptation of tree species. Epigenetic research in forestry also contributes to our understanding of community ecology and evolutionary biology in the context of actual global climate change (García-García et al., 2022; Kurpisz & Pawłowski, 2022). Indeed, there is an emerging field called ‘ecological epigenetics’, which can revolutionize our understanding of forest organism-environment interactions (Bräutigam et al., 2013; Herrel et al., 2020). This field promises to improve our understanding of biological diversity and resilience, particularly in the context of worsening drought conditions around the globe. In fact, ecological epigenetics can provide new insights into how forest trees interact and adapt to different levels of water deficiency through a deep investigation of the connections between epigenetic and phenotypic variation and the role of epigenetic changes in evolutionary processes (Amaral et al., 2020; Godwin & Farrona, 2020; Tomczyk et al., 2022). Significant research has been conducted in tree species under drought involving DNA methylation in regulating gene expression (Klupczyńska & Ratajczak, 2021). A comparative study showed changes in epigenetic markers, precisely 5-methylcytosine levels in the seeds of two maple species during water deficiency (Plitta-Michalak et al., 2018). Indeed, methylation of cytosine bases in DNA (forming m⁵C) is a crucial mechanism of epigenetic gene regulation. Phenotypic plasticity in response to drought, reported by Lafon-Placette et al. (2018) involves correlated variations in the shoot apical meristem (SAM), DNA methylation, and expression on genes involved in phytohormone pathways in Populus euramericana. The SAM of poplar responds to drought and re-watering by rapid and complex gene expression and DNA methylation changes. It reports in Populus the evidence of global DNA demethylation in gene bodies and significant methylation of transposable elements. So, the plant maintains transposon silencing under drought. The study also highlighted that specific phytohormone pathways, such as jasmonic acid, salicylic acid, ethylene, and brassinosteroids, are activated during drought and recovery in the SAM. Among them, DNA hypo-methylation and down-regulation of the hormone-responsive genes may involve classical epigenetic mechanisms, such as gene silencing by the Polycomb Repressive Complexes (PRCs), which repress gene expression through the H3K27me3 marks (Blackledge et al., 2021). Cooperation between DNA methylation and Polycomb complexes could be a significant mechanism of plant-coordinated development and stress response through regulating hormone biosynthesis and the involved compound pathways. During recovery from drought, it was marked in the Populus SAM that global DNA hypermethylation, particularly on gene bodies, correlated to the downregulation of the demethylase gene DEMETER. Hypo-methylation of transposable elements implicates a reduced activity in the RdDM pathway. The conclusion indicates the existence of massive chromatin remodelling during plant recovery (Lafon-Placette et al., 2018). A 2017 epigenetic study about Pinus halepensis (Fox et al., 2018) showed an epigenetic activity of several genes that might be involved in drought responses, including those controlling the methylation of the transcriptional repressor H3K9me and the transcriptional activator H3K4me. The epigenetic regulation of those expressed genes might be partially related to transposable element activation. A recent article identified 3,243 differentially methylated and expressed genes in Morus alba exposed to drought, with significant enrichment in key functional categories, suggesting the prominent role of methylation in stress response pathways. The study also revealed an 8.64% increase in overall DNA methylation levels in drought-stressed mulberry plants, predominantly in mCG methylation sites (Li et al., 2020). Moreover, research about the impact of DNA methylation variation in Populus tomentosa tolerance to drought has revealed that non-CG methylation affects gene expression and resilience to drought and that methylation patterns can be stable despite environmental changes (Zhou et al., 2023).

6 CONCLUSION AND PERSPECTIVES

Drought negatively affects forest ecosystems, an increasingly relevant concern due to climate change. The disparity in molecular-level research between woody and annual crop species is notable, emphasizing the need for more focused genomic studies on forest trees. The complexity of the poplar genome compared to Arabidopsis underscores this necessity. High-throughput DNA sequencing has advanced our understanding of trees' drought responses, but further research is imperative. Future research should prioritize the complete genome sequencing of a broader range of tree species; this will provide deeper insights into their unique genetic adaptations to drought stress. Additionally, epigenetic studies in forest trees under drought conditions are crucial. These studies will reveal how environmental stressors influence gene expression without altering the DNA sequence, offering a new dimension to our understanding of trees' adaptive mechanisms. This integrated genomic and epigenetic approach will be pivotal in developing conservation strategies and enhancing the resilience of forest ecosystems against the escalating threat of climate change. Future research on incorporating genomic and epigenetic data into ecological and climatic models to predict how forest ecosystems will respond to future climate scenarios can significantly advance our understanding of how forest trees respond to drought stress and develop effective strategies to mitigate the impacts of climate change on forest ecosystems. Moreover, research on the function of non-coding RNAs in regulating gene expression related to drought response is a future hot spot in the field. Notably, studies focusing on identifying and characterizing microRNAs and long non-coding RNAs relate to drought responses. Hence, it is crucial to assess the susceptibility of forests to drought stress and delve into the underlying mechanisms to formulate effective strategies for adaptation and mitigation.

AUTHOR CONTRIBUTIONS

Writing-review, investigation and editing, E.-H.M.C, L. S. P., G.K.B and F.Y. All authors have read and agreed to the published version of the manuscript.

FUNDING

This research was supported by the ARS (United States) Project # 8042-21600-001-000D.

INFORMED CONSENT STATEMENT

Not applicable.

CONFLICTS OF INTEREST

The authors declare no conflict of interest.