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. 2025 May 14;45(13):6–20. doi: 10.1093/treephys/tpaf060

Elucidating the drought-responsive changes in poplar cuticular waxes: a GWAS analysis of genes involved in fatty acid biosynthesis

Melike Karaca-Bulut 1, Eliana Gonzales-Vigil 2, Wellington Muchero 3, Shawn D Mansfield 4,5,✉
Editor: Chung-Jui Tsai
PMCID: PMC12666377  PMID: 40367349

Abstract

Drought and episodic drought events are major impending impacts of climate change, limiting the productivity of plants and especially trees due to their inherent high transpiration rates. One common mechanism used by plants to cope with drought stress is to change the composition of their leaf cuticular waxes. Cuticular waxes are essential for controlling non-stomatal water loss and are typically composed of a homologous series of very-long-chain fatty acid-derived compounds, as well as flavonoids, tocopherols, triterpenoids, and phytosterols. In this study, we compared the cuticular waxes of 339 natural accessions of Populus trichocarpa (Torr. & Gray) (black cottonwood) grown under control and drought conditions in a common garden. A Genome-Wide Association Study (GWAS) was then used to identify candidate genes associated with cuticular wax biosynthesis and/or its regulation. Although no major differences were observed in total wax load when subject to drought conditions, the amounts of the individual wax constituents were indeed responsive to drought. Specifically, changes in alkenes, alcohols, esters and aldehydes were evident, and suggest that they contribute to the drought response/tolerance in poplar. GWAS uncovered several genes linked to fatty acid biosynthesis, including CER1, CER3, CER4, FATB, FAB1, FAR3, FAR4, KCS and a homologue of SOH1, as well as other candidate genes that may be involved in coordinating the drought responses in poplar trees. Our findings provide new evidence that genotype-specific shifts in wax composition, rather than total wax accumulation, contribute to drought adaptation in poplar. Additionally, we show that genetic variation in key wax biosynthetic genes drives cuticular wax plasticity in P. trichocarpa under drought, identifying putative molecular targets for improving stress resilience in trees. This study expands our understanding of the adaptive mechanisms of the cuticle and their potential for enhancing drought tolerance in poplar species.

Keywords: alkenes, drought stress, leaf waxes, Populus trichocarpa

Introduction

Poplars (Populus spp.) are among the most productive tree species in the northern hemisphere (Dickmann et al. 2001) and are known to accrue significant above-ground biomass in a relatively short period of time. This growth characteristic is primarily due to the rapid and continuous development of leaves, as well as their considerable leaf area index and photosynthetic rates (Larcheveque et al. 2011). Poplar trees are ecologically important as key riparian species, but also economically important (Muchero et al. 2015) and can be employed for carbon sequestration and phytoremediation purposes (Larcheveque et al. 2011, Bilek et al. 2020).

Poplar trees are dioecious and display a wide geographic range in the northern hemisphere. It has been suggested that historical climate played a major role in shaping the existing significant genetic variation in poplars (Chhetri et al. 2019). Populus trichocarpa (Torr. & Gray) is adapted to relatively moist regions of the coastal side of the Rocky Mountains with a broad latitudinal range, spanning from north of California to south of Alaska, and inland to the Rocky Mountains of Alberta, Idaho and Montana (DeBell 1990). These trees are also recognized as being among the fastest-growing forest trees in diverse climates (Gornall and Guy 2007).

The broad genetic and phenotypic variation of P. trichocarpa across diverse geographical regions is evident in its variable flowering period, spanning from March to June, and its close correlation with growth, physiological traits, and climatic factors (Soolanayakanahally et al. 2009, McKown et al. 2014). Since they are wind-pollinated, poplars display enhanced gene flow and heterozygosity, thereby facilitating rapid adaptation (McKown et al. 2014). Drought stress (including episodic drought) is becoming more prevalent with a rapidly changing climate, and can significantly limit the productivity of trees due to their inherently high transpiration rates; however, since poplars are especially vulnerable to water stress, which can impact their natural distribution, they have developed a number of adaptations to drought stress (Larcheveque et al. 2011). Drought affects the entire tree, with roots being the first to detect water shortage, and as water depletion continues, shoot growth is limited while root growth is sustained, increasing the root-to-shoot ratio (Bogeat-Triboulot et al. 2007, Polle et al. 2019). Stomatal closure, regulated by abscisic acid (ABA), controls xylem hydraulics and water transport, impacting CO2 intake, transpiration, and photosynthesis during short-term drought (Chaves et al. 2003). However, prolonged drought can lead to xylem cavitation and tree mortality (Chaves et al. 2003). Abscisic acid levels rise in response to drought, but the magnitude of the changes in gene expression varies across poplar species (Brunner et al. 2015). The cuticle provides another mechanism to prevent water loss. Cuticular wax chemistry, influencing leaf water permeability, plays a crucial role in the plant's overall response to environmental stress. In turn, alterations in cuticular wax can impact drought tolerance, affecting stomatal density and leaf morphology (Bianchi 1995, Chaves et al. 2003, Gonzales-Vigil et al. 2017).

The cuticle, a hydrophobic layer that covers the aerial portions of epidermal cells of all land plants, is largely made of cutin and cuticular waxes (Waters 2003, Jetter et al. 2006, Riederer 2006, Yeats and Rose 2013). The cuticular wax, which is made up of a series of very-long-chain fatty acid (VLCFA)-derived metabolites, wax esters and other compounds such as flavonoids, tocopherols, triterpenoids and phytosterols, is crucial for regulating non-stomatal water loss that occurs through the leaf surface and aids in maintaining optimal plant water status (Jetter et al. 2006, Samuels et al. 2008). Cuticular wax biosynthesis in plants includes two primary routes: the alcohol-forming and alkane-forming pathways (Samuels et al. 2008). In the alcohol-forming pathway, very-long-chain (VLC) acyl-CoAs are reduced by fatty acid reductase 3 (FAR3/CER4) into primary alcohols, which can either be exported to the surface or esterified into wax esters by wax ester synthase/acyl-CoA:diacylglycerol acyltransferase 1 (WSD1) (Rowland et al. 2006, Li et al. 2008). In the alkane-forming pathway, VLC-acyl-CoAs are converted into aldehydes and then into alkanes by an eceriferum (CER1–CER3) enzyme complex (Chen et al. 2003, Bourdenx et al. 2011). Secondary alcohols and ketones are produced through further oxidation by mid-chain alkane hydroxylase (MAH1) (Greer et al. 2007). The composition of cuticular wax varies depending on tissue type, age and species, as well as exposure to biotic or abiotic stresses such as pathogens, water deprivation, or temperature (Cameron et al. 2002, Kunst and Samuels 2003, Jeffree 2006, Jetter et al. 2006, Samuels et al. 2008). For example, when leaves mature, the relative abundance of alkanes in cuticular waxes of some genotypes of P. trichocarpa remains the same, while the relative abundance of alkene has been shown to increase (Gonzales-Vigil et al. 2017). However, the abundance of alkenes can then also decrease in more mature leaves (Chen et al. 2023). Similarly, some individuals can inherently produce alkenes, while others do not appear to synthesize these compounds at all (Gonzales-Vigil et al. 2017).

Poplar trees, like other land plants, exhibit varying responses to drought. Plants exposed to salt and drought stress often accumulate reactive oxygen species, which in turn negatively impact membranes, proteins, and nucleic acids (Cheng et al. 2019). It has also been shown that plants that grow in dry climates usually have thicker cuticles than those that thrive in more temperate climates, and that total wax load increases rapidly a few days post-exposure to a dry environment (Shepherd and Wynne 2006). In Populus euphratica, which has heteromorphic leaves with broad-ovate and lanceolate leaf forms, the cuticular wax plays a crucial role in adaptability and survival in water-limited conditions (Song et al. 2021).

Given that P. trichocarpa grows in a variety of environments, comparing the wax profile of accessions that originate from the wide geographic range, but are grown in the same environment, may aid in understanding the role of the individual wax components in adaptation. Moreover, it is important to test the response of diverse germplasm to drought conditions, as they are expected to become more prevalent in the future (Hamanishi and Campbell 2011). Incorporating the complex chemical properties of cuticular waxes with GWAS not only can aid in our comprehension of their functional significance, but also holds promise to reveal unique insights into the underlying genetic mechanisms facilitating adaptive responses and resilience in changing environments.

This study surveyed the variation in wax load and composition of 339 natural accessions of P. trichocarpa when grown in a common garden under drought and well-watered conditions. The wax composition was then used to identify single nucleotide polymorphisms (SNPs) associated with the variation in chemical composition of the leaf waxes. These findings provide valuable insights into the genetic basis of wax traits in response to environmental stress and lay the foundation to understand the broader implications of these genetic variations on the adaptability of P. trichocarpa to challenging environments.

Materials and methods

Plant material

Leaf samples from 834 P. trichocarpa genotypes were collected from a common garden at the GreenWood Resources Inc.'s research field site in Boardman, Oregon, USA (Lat: 45°49′58.4″N, Long: 119°33′37.7″W), in May 2019 (Figure 1). Of these, 339 genotypes were present in two plots: one receiving full irrigation (well-watered control or non-drought plot) and the other receiving only 60% irrigation (drought plot), with irrigation time varying seasonally. Although irrigation commenced at the same time of day, in the summer, the non-drought plot was irrigated for 10 h/day, while the drought plot received 6 h/day. In winter, irrigation was 2 h/day for the non-drought plot and 1.2 h/day for the drought plot. Adjustments were made throughout the experiment to maintain a 60% reduction in watering. The trees were established at the Boardman site during the spring of 2016. Dormant cuttings were collected from the Corvallis, OR field site, which was established in 2009, as previously described (Evans et al. 2014). Cuttings were rooted under greenhouse conditions at the University of Arizona (Flagstaff, Arizona) before planting. Irrigation manipulation was conducted from 2017 to 2019 during the growing seasons. Weather data during this period is shown in Figure S1 available as Supplementary Data at Tree Physiology Online. A plastochron index (PI) was assigned to each leaf sampled, with the first fully unfolded leaf (~5 cm) serving as the reference for the sampling of the first source leaf (PI = 5). Samples were collected over two consecutive days, and the first source leaf from each genotype in both plots was collected and stored at −20 °C before wax extraction.

Figure 1.

Figure 1

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Control and drought-treated plots in Boardman, Oregon, USA. The poplar genotypes were planted at the same time, using a sample spacing (distance) in both drought and non-drought plots. The canopy density in the drought site is clearly lower than that in the non-drought plot.

Wax analyses

Wax extraction was performed according to Gonzales-Vigil et al. (2017), where three 6-mm diameter leaf discs were collected from each source leaf (PI-5) avoiding the midvein. Following total wax extraction, the samples were analyzed on an Agilent 8860 gas chromatography-flame ionization detector (GC-FID) equipped with a 30 m × 320 μm × 1 μm HP-1 column. One microliter of the sample was injected onto the column at an initial oven temperature of 50 °C for 2 min, then increased at 40 °C/min to 200 °C and held for 1 min, and finally increased at 3 °C/min to 320 °C and held for 15 min. Helium was employed as carrier gas at a flow rate of 1 mL/min, while the temperature of the injector and detector were set to 300 and 320 °C, respectively. The amount of each wax component in each sample was quantified by determining the peak area of each compound and then normalizing the area against the area of the internal standard (10 mg/mL of n-tetracosane) and the tissue surface area employed for each sample. The surface area was calculated by multiplying six times the area of one side of a leaf disc, with the equation 6Πr2, where r = 3.175 mm.

Compound identification was achieved by GC mass spectrometry (GC–MS) using an Agilent 8890 GC system equipped with a 30 m × 250 μm × 0.25 μm DB1-MS column by comparison against a the National Institute of Standards and Technology (NIST) GC–MS library (2017), and then validated with standards, when available. The 7010B Triple Quadrupole MS detected the analytes with the following parameters: EI 70 eV, m/z 40–500 and 1 scan/s. One microliter of the sample was injected onto the column at an initial oven temperature of 50 °C held for 2 min, then increased at 40 °C/min to 200 °C and held for 1 min, finally raised at 3 °C/min to 320 °C and held for 10 min. Helium was used as a carrier gas at a flow rate of 1.2 mL/min. Inlet mode was set to pulsed splitless at 300 °C and 9.78 p.s.i. pressure. The total flow of the inlet was 104.2 mL/min with a septum purge flow of 3 mL/min in switched mode. The temperature of the mass selective detector (MSD) transfer line was 280 °C.

Statistical analysis

Statistical analysis was completed using MetaboAnalyst (RRID:SCR_015539) 5.0 software (Zhiqiang et al. 2021) and R software version 4.0.2 (2021). Paired t-test were used to compare the means of the leaf wax loads between treatment and control groups (n = 339) for each wax component at a significance level of P < 0.05. To reduce the number of variables contributing to the total variation, principal component analysis (PCA) was performed using MetaboAnalyst 5.0.

Genome-Wide Association Study

A Genome-Wide Association Study (GWAS) was conducted using each wax component as a unique phenotype. In short, whole-genome resequencing, SNP/InDel calling and SnpEff analysis of the P. trichocarpa GWAS mapping population were as previously described (Evans et al. 2014, Zhang et al. 2018). A total of 9,751,445 SNP/InDels with minor allele frequencies ≥0.05 were used for the phenotype to genotype correlations using the Genome-wide Efficient Mixed Model Association (GEMMA) software toolkit (Zhou and Stephens 2012) with kinship as a covariate to control for relatedness. A threshold was set where the false discovery rate (FDR) was <0.05. Deviation of P-values from expectation was evaluated using quantile-quantile (QQ) plots with lambda (λ) as the test statistic.

Results

This study examined the natural variation in the wax composition of unrelated genotypes of P. trichocarpa when grown under well-watered (control or non-drought) and reduced water (drought) conditions in a common garden field site for three years. Leaf surface lipids extracted from 339 paired genetic clonal accessions grown at the two plots were analyzed by GC-FID to investigate the effects of drought on the general wax chemistry. This was followed by a GWAS of the individual wax components (treated as individual phenotypes) to uncover genotype-wax correlations as affected by the drought conditions. Among the identified compounds, the wax components included odd-chain alkanes (C25–C31) and n-hexacosane (C26); odd-chain alkenes (C25–C29) and n-hexacosene (C26); primary alcohols (C22–C30) and esters with C44, C46 and C48 chain lengths. In addition, lignoceric acid (C24 fatty acid), octacosanal (C28 aldehyde), benzoic acid octadecyl ester and glycerol monostearate were detected.

Although total wax amount remains unchanged, individual wax compounds show variation

To test the effect of the watering regimes on cuticular waxes, the means of the total wax loads of samples grown in the drought and control treatments were compared. No significant differences were observed between the treatments when analyzed collectively, with leaves collected from the drought plot having on average 6.80 ± 2.01 μg/cm2 of total wax, while those grown on the control plot had 6.75 ± 2.04 μg/cm2 total wax (Figure 2A). This implies that there is no general response across all accessions to the drought treatment. However, an inspection of individual genotypes clearly indicates that some individuals display notable increases in total wax content, while others show decreases (see Figure S2 and Table S1 available as Supplementary Data at Tree Physiology Online).

Figure 2.

Figure 2

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The wax distribution of (A) total wax, (B) alkanes, (C) alkenes, (D) alcohols, (E) esters, and (F) individual wax compounds collected from poplar leaves of genotypes grown in both drought and control conditions at a common garden in Boardman, Oregon. Wax amounts are presented as averages and error bars show the standard error of the mean. Asterisks indicate a significant difference between drought and non-drought following a comparison of means by a paired t-test, where *P < 0.001,  **P < 0.0001. Abbreviations: FA, C24 fatty acid; AL, C28 aldehyde; BA, benzoic acid octadecyl ester; GM, glycerol monostearate.

Next, individual constituents were categorized by compound class depending on their functional groups. Paired t-tests were used to compare the means of total alkanes, alkenes, alcohols, wax esters and other identified compounds, as well as the total unknown compound abundances across treatments. Total alkenes, alcohols and esters were found to be significantly different between treatments (P-value < 0.001), while no significant changes were observed for total alkanes, other identified compounds and unknown waxes (Figure 2B–E).

Alkanes were the dominant class in the cuticular wax of poplar trees grown in the common garden. Although the overall abundance of alkanes remained unchanged (Figure 2B), C25, C26 and C27 alkanes increased significantly under drought conditions (Figure 2F). In contrast, the alkenes exhibited a 39% increase (Figure 2C) under drought conditions, with the C25, C27 and C29 alkenes largely contributing to the increase (Figure 2F). Conversely, both the alcohols (Figure 2D) and wax esters (Figure 2E) decreased significantly (18 and 15%, respectively) with drought treatment. Specifically, C24 and C30 alcohols and all esters with 44, 46 and 48 carbons decreased significantly in response to the drought treatment (Figure 2F). Among the other identified compounds, octacosanal (C28 aldehyde) increased by 61%, while benzoic acid octadecyl ester decreased by 27% under drought (Figure 2F).

Interestingly, the presence of wax esters was strongly affected by the growth conditions, which displayed an apparent bimodal distribution under drought conditions (Figure 2E). A detailed examination of individual genotypes highlighted 41 accessions that showed no detectable accumulation of esters in the drought-treated plot, whereas at least one wax ester was detected in the control plot. Additionally, two genotypes had undetectable esters when grown at either location, while in four genotypes, esters were detected in the drought but not in the control plot (see Figure S3 and Table S2 available as Supplementary Data at Tree Physiology Online). Differences in the accumulation of alkenes were also detected, with some genotypes (127/339) not producing detectable alkenes at either location, others only producing alkenes in the drought plot (22), and a third group accumulating alkenes only in the control plot (17) (Table S3 available as Supplementary Data at Tree Physiology Online). To investigate if the genotypes failing to accumulate alkenes or esters come from the same location, the site of origin of the 339 genotypes was examined. Alkenes and esters production in P. trichocarpa did not display a geographical relationship to their place of origin in the native range (Figure 3). Specifically, the same 21 genotypes exhibited no detectable alkenes and esters under drought conditions, while only 4 genotypes showed this lack of both alkenes and esters in the control plot (see Tables S2 and S3 available as Supplementary Data at Tree Physiology Online).

Figure 3.

Figure 3

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Geographical distribution of poplar genotypes based on (A) alkene and (B) ester production across drought and control conditions. Genotypes are marked as follows: green dots indicate production of alkene or ester at both sites; purple dots represent production in drought but deficiency in control conditions; orange dots show deficiency in drought but production in control conditions; black dots denote deficiency in both conditions. Interactive map of poplar sampling sites created using Leaflet in RStudio (Cheng et al. 2024).

These observations indicate that alkene accumulation is highly variable and a responsive trait compared with other compounds (see Tables S3 available as Supplementary Data at Tree Physiology Online). The alkene-minus (deficient) (AM) (n = 127) genotypes were therefore selected for further analysis of their wax composition and compared with alkene-plus (producing) genotypes (AP) (n = 212), including the genotypes producing alkenes on at least one site (Figure 4 and see Tables S3 available as Supplementary Data at Tree Physiology Online). When the AP and AM genotypes were compared within sites, statistically significant differences in total wax (Figure 4A) and alkane amounts (Figure 4B) were observed in clones grown on both the control and drought sites. In contrast, a statistically significant difference in alcohol load between AM and AP genotypes was found only in plants grown on the control site (Figure 4D), with AP genotypes exhibiting higher alcohol levels. On average, AP plants displayed a 17% higher total wax load than AM genotypes when grown on the control site, and a 23% higher load when grown on the drought site. Similarly, the alkane load was 15% higher among the AP genotypes when grown on the control plot and 17% higher when grown on the drought plot, compared with AM genotypes (Figure 4B).

Figure 4.

Figure 4

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The wax distribution of (A) total wax, (B) alkane, (C) alkene, (D) alcohol, and (E) ester wax compounds on both AP and AM poplar leaves from genotypes grown in both drought and control conditions at a common garden in Boardman, Oregon. Wax amounts are presented as averages. Asterisks indicate a significant difference between drought and control following a comparison of means by a t-test, where *P < 0.05, **P < 0.001 and ***P < 0.0001.

When the AP and AM genotypes were compared within themselves across the two treatments—drought and control—the AP genotypes showed a 32% increase in alkenes (Figure 4C), a 13% decrease in alcohols (Figure 4D), and a 10% decrease in esters (Figure 4E) when grown under drought conditions. Similarly, AM genotypes showed an 11% decrease in alcohols (Figure 4D) and a 19% decrease in esters (Figure 4E) under drought conditions. However, these variations were quite similar across both genotype groups. The decrease in alcohols and esters observed in both the AP and AM genotypes under drought conditions indicates a common response to drought, rather than a distinct difference between these groups. Consequently, the overall drought-induced changes in chemical composition appear to be similar for both AP and AM genotypes, with little variation between them in response to drought versus control conditions. The comprehensive chemical characterization of cuticular waxes in these accessions revealed subtle, yet consistent differences in how various compound classes, such as alkanes, alcohols and esters, respond to drought treatment. While the overall trend of decreased alcohol and ester levels under drought conditions was observed across both AP and AM genotypes, a few accessions exhibited distinct chemical profiles, with specific compound classes differing notably from the general population, suggesting that these accessions may possess unique mechanisms for drought adaptation (see Figure S2 available as Supplementary Data at Tree Physiology Online).

GWAS reveals key wax-related genes associated with drought and non-drought traits

To identify genetic variants associated with individual leaf cuticular wax traits, a GWAS was completed using 9,751,445 SNP/InDels and the individual leaf cuticular wax traits of 678 P. trichocarpa genotypes (339 paired individuals grown on two sites). In total, 22,269 SNPs from the drought treatment and 10,075 SNPs from the control group passed the FDR P-value cutoff and were determined to be significantly associated with the accumulation of at least one of the cuticular wax constituents. From these, we manually filtered the significant SNPs for those located in or nearby fatty acid biosynthetic pathway genes and were shown to be significantly associated with the phenotypes, particularly the content of C24 fatty acid, C28 aldehyde, C31 alkane, and C48 ester (Table 1). To further explore these associations, Gene Ontology (GO) analysis was also performed to identify trends in the genes linked to wax composition (Table 2).

Table 1.

Candidate genes linked to fatty acid biosynthesis, based on GWAS results.

Treatment Phenotype Chr Pos P-value Gene ID SNP effect Best Arabidopsis hit Annotated function
Drought C24 Fatty Acid Chr06 19,118,963 5.51432E-07 Potri.006G177500 Intergenic AT5G57800.1 Fatty acid hydroxylase superfamily (CER3)
Drought C28 Aldehyde Chr03 9,808,076 1.0557E-13 Potri.003G069801 Upstream gene variant AT1G74960.3 Fatty acid biosynthesis 1
Drought C28 Aldehyde Chr09 11,495,657 1.0557E-13 Potri.009G145100 Upstream gene variant AT3G44540.1 Fatty acid reductase 4
Drought C28 Aldehyde Chr14 11,909,624 1.0557E-13 Potri.014G152900 Upstream gene variant AT1G02205.2 Fatty acid hydroxylase superfamily (CER1)
Drought C28 Aldehyde Chr14 11,909,633 1.0557E-13 Potri.014G152900 Upstream gene variant AT1G02205.2 Fatty acid hydroxylase superfamily (CER1)
Drought C28 Aldehyde Chr14 11,909,959 1.0557E-13 Potri.014G152900 Upstream gene variant AT1G02205.2 Fatty acid hydroxylase superfamily (CER1)
Drought C28 Aldehyde Chr14 11,909,973 1.0557E-13 Potri.014G152900 Upstream gene variant AT1G02205.2 Fatty acid hydroxylase superfamily (CER1)
Drought C28 Aldehyde Chr14 11,909,978 1.0557E-13 Potri.014G152900 Upstream gene variant AT1G02205.2 Fatty acid hydroxylase superfamily (CER1)
Drought C28 Aldehyde Chr14 11,909,981 1.0557E-13 Potri.014G152900 Upstream gene variant AT1G02205.2 Fatty acid hydroxylase superfamily (CER1)
Drought C28 Aldehyde Chr18 9,431,077 1.0557E-13 Potri.018G072700 Downstream gene variant AT1G08510.1 Fatty acyl-ACP thioesterases B
Drought C28 Aldehyde Chr18 9,435,017 1.0557E-13 Potri.018G072700 Intergenic AT1G08510.1 Fatty acyl-ACP thioesterases B
Drought C28 Aldehyde Chr18 9,436,749 1.0557E-13 Potri.018G072700 Intergenic AT1G08510.1 Fatty acyl-ACP thioesterases B
Drought C28 Aldehyde Chr18 9,437,351 1.0557E-13 Potri.018G072700 Intergenic AT1G08510.1 Fatty acyl-ACP thioesterases B
Drought C28 Aldehyde Chr18 9,438,227 1.0557E-13 Potri.018G072700 Intergenic AT1G08510.1 Fatty acyl-ACP thioesterases B
Drought C28 Aldehyde Chr18 9,438,829 1.0557E-13 Potri.018G072700 Intergenic AT1G08510.1 Fatty acyl-ACP thioesterases B
Drought C28 Aldehyde Chr18 9,441,159 1.0557E-13 Potri.018G072700 Intergenic AT1G08510.1 Fatty acyl-ACP thioesterases B
Drought C28 Aldehyde Chr18 9,442,497 1.0557E-13 Potri.018G072700 Intergenic AT1G08510.1 Fatty acyl-ACP thioesterases B
Drought C28 Aldehyde Chr18 9,442,498 1.0557E-13 Potri.018G072700 Intergenic AT1G08510.1 Fatty acyl-ACP thioesterases B
Drought C28 Aldehyde Chr18 9,442,524 1.0557E-13 Potri.018G072700 Intergenic AT1G08510.1 Fatty acyl-ACP thioesterases B
Drought C28 Aldehyde Chr18 9,446,018 1.0557E-13 Potri.018G072700 Intergenic AT1G08510.1 Fatty acyl-ACP thioesterases B
Drought C28 Aldehyde Chr18 9,452,021 1.0557E-13 Potri.018G072700 Intergenic AT1G08510.1 Fatty acyl-ACP thioesterases B
Drought C28 Aldehyde Chr18 9,467,081 1.0557E-13 Potri.018G072700 Intergenic AT1G08510.1 Fatty acyl-ACP thioesterases B
Drought C28 Aldehyde Chr18 9,473,705 1.0557E-13 Potri.018G072700 5′ UTR variant AT1G08510.1 Fatty acyl-ACP thioesterases B
Drought C28 Aldehyde Chr19 10,940,196 1.0557E-13 Potri.019G075200 5′ UTR variant AT3G44540.1 Fatty acid reductase 4
Drought C31 Alkane Chr18 12,747,519 9.70179E-07 Potri.018G099400 Upstream gene variant AT5G57800.1 Fatty acid hydroxylase superfamily (CER3)
Drought C31 Alkane Chr18 12,747,568 5.86157E-07 Potri.018G099400 Upstream gene variant AT5G57800.1 Fatty acid hydroxylase superfamily (CER3)
Drought C48 Ester Chr15 15,132,242 1.0557E-13 Potri.015G146600 In gene AT4G23420.1 NAD(P)-binding Rossmann-fold superfamily protein
Non-drought C48 Ester Chr06 19,086,010 5.38686E-07 Potri.006G177500 Upstream gene variant AT5G57800.1 Fatty acid hydroxylase superfamily (CER3)
Non-drought C48 Ester Chr06 19,087,619 1.70785E-07 Potri.006G177500 Intergenic AT5G57800.1 Fatty acid hydroxylase superfamily (CER3)
Non-drought C48 Ester Chr06 19,092,274 8.75408E-07 Potri.006G177500 Intergenic AT5G57800.1 Fatty acid hydroxylase superfamily (CER3)
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Table 2.

Gene Ontology (GO) terms resulting from the GWAS analysis of both drought and non-drought genotypes are presented in the table. The table emphasizes GO terms with the highest count of SNPs. Additionally, it focuses on those associated with annotations related to fatty acid biosynthesis.

Treatment Gene Ontology The number of SNPs Keyword FA pathway related
Drought GO:0006468 1371 Protein phosphorylation No
GO:0005515 1242 Protein binding No
GO:0008152 778 Metabolic process No
GO:0006355 762 Regulation of DNA-templated transcription No
GO:0003677 517 DNA binding No
GO:0016020 453 Membrane No
GO:0005634 428 Nucleus No
GO:0005506 282 Iron ion binding Yes
GO:0003824 121 Catalytic activity Yes
GO:0006633 37 Fatty acid biosynthetic process Yes
GO:0016790 14 Thiolester hydrolase activity Yes
GO:0080019 3 Alcohol-forming very long-chain fatty acyl-CoA reductase activity Yes
Non-drought GO:0005515 724 Protein binding No
GO:0006468 403 Protein phosphorylation No
GO:0016491 396 Oxidoreductase activity Yes
GO:0006355 334 Regulation of DNA-templated transcription No
GO:0016020 296 Membrane No
GO:0016788 240 Hydrolase activity, acting on ester bonds No
GO:0004659 215 Prenyltransferase activity No
GO:0005506 174 Iron ion binding Yes
GO:0006633 64 Fatty acid biosynthetic process Yes
GO:0050660 25 Flavin adenine dinucleotide binding Yes
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The candidate genes include ECERIFERUM1 (CER1) and ECERIFERUM 3 (CER3) (fatty acid hydroxylase superfamily), which are annotated with oxidoreductase activity (GO:0016491), fatty acid biosynthetic process (GO:0006633) and iron ion binding (GO:0005506); FATB (fatty acyl-ACP thioesterases B), which is associated with thiolester hydrolase activity (GO:0016790) and fatty acid biosynthetic process (GO:0006633); FAB1 (fatty acid biosynthesis 1), which is associated with metal ion binding (GO:0046872), Mg-ATP binding (GO:0005524) and the phosphatidylinositol metabolic process (GO:0046488); FAR4 (fatty acid reductase 4), which is linked to coenzyme binding (GO:0050662) and alcohol-forming very long-chain fatty acyl-CoA reductase activity (GO:0080019) and a SOH1 homologue (NAD(P)-binding Rossmann-fold superfamily protein), which is annotated with oxidoreductase activity (GO:0016491). In addition to the GO terms linked to fatty acid biosynthesis mentioned above, other relevant GO terms were identified and associated with the candidate genes (Table 2).

Additionally, a second independent GWAS was performed for AP and AM genotypes only, across both sites to investigate potential genetic differences driving their divergent wax compositions (Table 3). The analysis revealed that both drought and control sites share two candidate genes directly related to fatty acid synthesis: very long-chain 3-oxoacyl-CoA synthase/very-long-chain beta-ketoacyl-CoA synthase (PtKCS1) and long-chain-fatty-acyl-CoA reductase/acyl-CoA reductase (CER4 or FAR3).

Table 3.

Candidate genes linked to fatty acid biosynthesis, based on GWAS results from comparing genotypes producing alkenes and genotypes deficient in alkenes within each plot.

Plot Chr Pos P-value Gene ID Neighborgene Annotated function
Drought Chr09 11,470,337 9.23867E-06 Potri.009G144900 NA Long-chain-fatty-acyl-CoA reductase/Acyl-CoA reductase
Drought Chr09 11,471,498 1.18729E-05 Potri.009G144900 NA Long-chain-fatty-acyl-CoA reductase/Acyl-CoA reductase
Drought Chr10 10,463,158 3.40818E-07 NA Potri.010G079500 Very-long-chain 3-oxoacyl-CoA synthase/Very-long-chain beta-ketoacyl-CoA synthase
Drought Chr10 10,460,928 6.73786E-05 Potri.010G079500 NA Very-long-chain 3-oxoacyl-CoA synthase/Very-long-chain beta-ketoacyl-CoA synthase
Non-drought Chr09 11,471,498 4.77966E-06 Potri.009G144900 NA Long-chain-fatty-acyl-CoA reductase/Acyl-CoA reductase
Non-drought Chr09 11,470,337 7.71865E-05 Potri.009G144900 NA Long-chain-fatty-acyl-CoA reductase/Acyl-CoA reductase
Non-drought Chr09 11,467,711 8.48326E-05 Potri.009G144900 NA Long-chain-fatty-acyl-CoA reductase/Acyl-CoA reductase
Non-drought Chr10 10,463,158 1.12555E-10 NA Potri.010G079500 Very-long-chain 3-oxoacyl-CoA synthase/Very-long-chain beta-ketoacyl-CoA synthase
Non-drought Chr10 10,460,928 7.92536E-07 Potri.010G079500 NA Very-long-chain 3-oxoacyl-CoA synthase/Very-long-chain beta-ketoacyl-CoA synthase
Non-drought Chr10 10,639,519 6.50206E-06 NA Potri.010G080400 Very-long-chain 3-oxoacyl-CoA synthase/Very-long-chain beta-ketoacyl-CoA synthase
Non-drought Chr10 10,572,370 6.50206E-06 NA Potri.010G080200 Very-long-chain 3-oxoacyl-CoA synthase/Very-long-chain beta-ketoacyl-CoA synthase
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Discussion

Plants have evolved an array of strategies to defend against drought, including stomatal closure, increased root growth, and reductions in leaf size or number to limit transpiration (Fischer and Polle 2010, Arve et al. 2011, Ryan 2011). For example, P. euphratica improves its drought tolerance by modifying chloroplast structures to optimize photosynthesis as well as altering its osmotic adjustment to improve water acquisition. These adaptations, along with other physiological mechanisms such as high light tolerance and temperature resilience, allow P. euphratica to withstand extreme conditions (Song et al. 2021).

In this study, aerial images of the test plots clearly show a reduction in canopy density and overall green cover, largely due to fewer or smaller leaves (Figure 1). Clearly, the 60% reduction in watering affected overall growth. Despite the suggested role of cuticular wax abundance in reducing non-stomatal water loss (Xu et al. 2016, Song et al. 2021), our results indicate that total wax load was similar at both sites when all genotypes were averaged. However, a closer inspection of genotypes revealed more complex responses. For instance, some genotypes (e.g., 18, 19, 1010, CHWH 27–5, etc.) exhibited an increase in wax load under drought conditions, while others (e.g., 56, 877, 1003, CNYH 28–3, etc.) showed a decrease (see Figure S2 and Table S1 available as Supplementary Data at Tree Physiology Online). It is also possible that genotypes exposed to drought are employing additional strategies beyond changes in total wax to cope with water deficiency. Unlike studies in Arabidopsis (Kosma et al. 2009), rose (Jenks et al. 2001), wheat (Uddin and Marshall 1988), cotton (Bondada et al. 1996), tobacco (Cameron et al. 2006), peanut (Samdur et al. 2003), and pea (Sánchez et al. 2001) that have all demonstrated significant increases in leaf wax accumulation during drought, our results suggest that P. trichocarpa may require more severe drought conditions to trigger similar bulk increases, if P. trichocarpa indeed responds this way.

Changes in wax composition in P. trichocarpa: increased alkene and decreased alcohols and esters in response to drought

Despite the overall wax load remaining constant, we observed a notable shift in composition, characterized by an increase in alkene abundance and a significant decrease in both alcohols and esters. This observation is consistent with findings in drought-treated alfalfa, where changes in composition led to no significant change in the total wax load (Ni et al. 2012).

Alkanes are the predominant compounds found in the wax of poplar leaves, in both control and drought conditions, and no difference in total alkane production was apparent during drought treatment. There were, however, significant increases in C25, C26 and C27 alkanes in the drought-treated trees, while changes in the dominant C29 and C31 were not apparent. Although total wax production did not differ statistically, the changes in alkane components have previously been suggested to be crucial for selecting drought-resistant plants (Ni et al. 2012). For example, in alfalfa, drought induced a 36–78% increase in total alkanes, suggesting that alkane content, rather than total wax content, might be a better indicator for drought resistance (Ni et al. 2012). Similarly, soybean showed a 28–34% increase in alkanes (Kim et al. 2007b), while in Arabidopsis specifically C29, C31 and C33 alkanes were shown to accumulate and be an indicator of drought adaptation (Panikashvili et al. 2007, Kosma et al. 2009). Collectively, these findings underscore the role of alkanes in managing drought responses among species.

In this study, we show a significant increase in the concentration of alkene in response to drought, and following detailed chemical characterization it was apparent that aldehydes (C18 and C20) were only present in accessions that accumulate alkenes, suggesting a possible biosynthetic connection (Chen et al. 2023). The pattern of aldehyde accumulation and their chain lengths implies that these aldehydes may be degradation products of alkenes, likely formed through oxidative cleavage (Chen et al. 2023). Notably, 22 genotypes did not produce alkenes when grown under control conditions, but did so after exposure to drought. If poplar is unable to substitute alkenes with other wax hydrocarbons, this suggests alkenes may be integral to drought responses. Alternatively, if wax component compensation occurs, then drought-responsive genotypes may rely on different strategies to maintain the overall wax composition to enhance drought resilience.

Furthermore, 127 (37%) genotypes lacked detectable levels of alkene when grown in either condition (see Table S3). These genotypes displayed significant differences in total wax and alkane concentrations across both treatments. Notably, there were distinct differences in total wax (Figure 4A) and alkane (Figure 4B) abundance between AP and AM genotypes under both growth conditions. This implies that certain genotypes may lack the necessary enzymes or pathways, such as those involving PtKCS1 that specifically elongate mono-unsaturated fatty acids to recruit unsaturated substrates for wax synthesis (Gonzales-Vigil et al. 2017). Previously, it was shown that P. trichocarpa genotypes lacking alkenes in their leaves exhibit downregulated PtKCS1 expression and a reduction in total wax load compared with genotypes that produce alkenes, suggesting that the absence of alkenes is not compensated for by the production of other wax constituents (Gonzales-Vigil et al. 2017). Here, we show that AM genotypes displayed a significant decrease in total wax load under both drought and control conditions when compared with AP genotypes grown on the same sites (Figure 4A), further suggesting that alkenes play a critical role in maintaining overall wax production.

In addition, AP genotypes exhibited higher levels of alcohols (Figure 4D) when grown on the control site. However, under drought treatment, AP genotypes showed an increase in alkenes (Figure 4C) and a decrease in alcohols (Figure 4D) and esters (Figure 4E). Similarly, AM genotypes consistently displayed lower abundance in alcohols (Figure 4D) and esters (Figure 4E).

Mapping AP and AM plants to their origin (Figure 3) showed no clear geographical separation, although there was some clustering of AM genotypes in drier regions. This observation aligns with our earlier findings showing no definitive geographic distribution for AM poplar (Gonzales-Vigil et al. 2017). The lack of clear geographical separation likely stems from poplars’ inherent wind-dispersal mechanisms, which promote genetic mixing across environments (Gornall and Guy 2007, McKown et al. 2014).

Primary alcohols (C22–C30) also varied between growing conditions. Among the P. trichocarpa genotypes examined, 1-tetracosanol (C24 alcohol) was the most prevalent. Variations in primary alcohols can affect water retention and drought resilience, and drought treatment significantly reduced alcohol abundance (Cameron et al. 2002). This is consistent with findings in alfalfa, which showed an ~30% reduction in total alcohols (Ni et al. 2012), while in our study, poplar trees displayed ~18% reduction in alcohol abundance when subject to drought.

Populus trichocarpa leaves were also found to contain C44, C46 and C48 wax esters, which were decreased in response to drought; in fact, 43 genotypes produced no esters when water-stressed. Since wax esters are synthesized through the esterification of primary alcohols and fatty acids, a decrease in alcohols directly reduces the available substrates for ester formation. Therefore, the reduction in alcohol availability likely contributes to the observed decline in wax esters under drought stress. These observations contrast findings in Arabidopsis, where it has been reported that C40, C42 and C44 esters increase when the plants were subjected to drought (Patwari et al. 2019).

Significant changes were observed in the abundance of the C28 aldehyde (increase) and benzoic acid octadecyl ester, among other identified compounds. This trend has also been observed in sesame leaves when subjected to water-deficit for 15 days (Kim et al. 2007a). Romero and Lafuente (2020) further supported these observations, as they highlighted a correlation between decreased aldehyde presence and an amplified rate of cuticular transpiration, ultimately rendering plants more susceptible to drought.

Overall, while total wax load remained stable across treatments, individual wax components exhibited significant shifts in response to drought. This was particularly evident for wax esters and alkenes, suggesting that the stability in total wax load masks these underlying changes in individual wax constituents. Furthermore, in contrast to the analysis of plants grouped by site, distinct variations were observed between the AP and AM groups regarding how their wax compositions shifted under different conditions (Figure 4).

GWAS sheds light on genes and pathways influencing wax composition in P. trichocarpa under drought stress

GWAS was completed using 9,751,445 SNP/InDels for 27 leaf cuticular wax components for both drought and control treatments among 339 paired P. trichocarpa genotypes. The GO analysis indicated that protein phosphorylation and protein binding had the highest number of SNPs in drought-treated genotypes (Table 2). Additionally, several traits were shown to be significantly correlated with a number of known genes involved in lipid biosynthesis, including CER1 and CER3, FATB, FAB1, FAR4 and a homologue of SOH1. These genes are known to be associated with the alcohol-forming very long-chain fatty acyl-CoA reductase activity, thioester hydrolase activity and fatty acid biosynthesis (Table 2). Notably, the genes highlighted are associated with the production of C24 fatty acid, C28 aldehyde and C31 alkane in leaves of drought-treated plants, and C48 ester in non-drought plants (Table 1).

Fatty acid hydroxylase superfamily, specifically the CER1 (Potri.014G152900) gene, exhibited a significant association with C28 aldehyde. Additionally, CER3 (Potri.018G099400 and Potri.006G177500) was associated with C24 fatty acid, C31 alkane under drought treatment and C48 ester for non-droughted leaf traits. CER1 and CER3 work together to facilitate VLCFA-CoA conversion to alkanes through the decarbonylation pathway (Kunst and Samuels 2003, Bernard et al. 2012). In Arabidopsis, they, along with cytochrome b5 (CYTB5s), facilitate the alkane-forming pathway by oxidizing VLC-acyl-CoAs into VLC fatty aldehydes and decarbonylating even-numbered VLC fatty aldehydes into odd-numbered VLC alkanes (Schneider-Belhaddad and Kolattukudy 2000, Bourdenx et al. 2011, Bernard et al. 2012). However, in this study, only C28 aldehyde was detected, possibly due to variations in aldehyde-forming enzyme specificity. CER3 may favour C28 aldehyde, and differences in decarbonylation efficiency could convert other aldehydes to alkanes, leaving only C28 aldehyde detectable in the final wax composition (Hegebarth and Jetter 2017). These genes have previously been closely linked to responses to both biotic and abiotic stresses (Bourdenx et al. 2011, Yeats and Rose 2013, Hernández et al. 2024).

For example, overexpressing Poa pratensis ECERIFERUM1 (PpCER1) in Brachypodium distachyon increased alkane abundance, and was accompanied by a decrease in both primary alcohols and total wax (Wang et al. 2021). The relative abundance of C29 and C31 alkane, as well as C28 aldehyde, showed a decline, whereas the corresponding amounts of C25, C27 and C26 alkanes, along with C26 aldehyde, exhibited a significant increase. PpCER1–2 overexpression lines also displayed enhanced drought tolerance and reduced cuticle permeability (Wang et al. 2021). This observation aligns with our findings, which showed that drought-affected genotypes had considerably different levels of C28 aldehyde. In Populus simonii, the expression of CER1 was shown to increase when subject to drought (Chen et al. 2013), while in P. euphratica CER1 overexpression has been shown to trigger cuticle synthesis in response to drought (Xu et al. 2016).

Wax production is influenced by the responses of the CER1 and CER3 genes to ABA, drought and osmotic stressors (Bourdenx et al. 2011). Arabidopsis cer1 and cer3 mutants show reduced wax content and drought susceptibility, while CER1 overexpression increases alkanes, enhancing resilience (Kurata et al. 2003, Bourdenx et al. 2011, Chen et al. 2013). Additionally, the Arabidopsis cer1cer3 double mutant accumulates C24 fatty acid (678% increase) but lacks longer chain fatty acids, highlighting their role in elongating C24 acyl-CoA (Goodwin et al. 2005, Skvortsova 2013). Our findings also indicate notable variations in the abundance of aldehydes, alkanes and esters among genotypes impacted by drought, consistent with these studies.

We identified Potri.015G146600, a homolog of SOH1, which encodes an NAD(P)-binding Rossmann-fold superfamily protein belonging to the short-chain dehydrogenase/reductase family protein. In Arabidopsis, SOH1 functions as a putative aldehyde reductase, facilitating the conversion of aldehydes into 1-alcohols and playing a critical role in the balance between alkane- and alcohol-forming pathways (Li et al. 2025). The SOH1–CER3–CER1 module has been shown to mediate wax precursor partitioning in response to environmental conditions. It is possible that the poplar homologue may play a similar role in regulating wax composition under drought stress. Notably, this gene is linked to C48 ester production, potentially affecting the conversion of aldehydes into alcohols or esters and overall wax composition in response to drought.

Potri.018G072700 is annotated as fatty acyl-ACP thioesterase B (FATB). Acyl-ACP thioesterases are classified depending on their substrate preferences; with FATA thioesterases favouring oleoyl-ACP and FATB thioesterases favouring saturated acyl-ACP substrates of different chain lengths (Kunst and Samuels 2003). The FATB gene is known to have a role in the transport of C16:0 and C18:0 saturated free fatty acids outside of the plastid (Bonaventure et al. 2003, Kunst and Samuels 2003). We detected a SNP in the regulatory region within the 5′ UTR of FATB, which may influence gene expression levels and potentially affect fatty acid availability for wax biosynthesis. The Arabidopsis FATB loss-of-function mutant has decreased total wax load in stems (by ~50%) and leaves (by ~20%) (Bonaventure et al. 2003). In barley, FATB expression was shown to be higher in the drought-tolerant plants, promoting fatty acid elongation and alcohol-forming pathways (Daszkowska-Golec et al. 2020). Our findings suggest that FATB is linked to aldehyde accumulation in drought-stressed poplar genotypes, promoting wax synthesis under drought conditions. In this study, fatty acid biosynthesis 1 (FAB1-Potri.003G069801) was also found associated with C28 aldehyde accumulation. The FAB1 gene encodes a plastidic beta-ketoacyl-ACP synthase II, essential for fatty acid elongation (Li-Beisson et al. 2013). Mutations in fab1 affect membrane structure, freezing stress tolerance and photosynthesis (Wu et al. 1997, Carlsson et al. 2002, Gao et al. 2015, Su et al. 2018).

In the context of this study, the fatty acid reductase-4 gene (FAR4-Potri.009G145100 and Potri.019G075200) also appear to play a significant role in modifying the composition of leaf cuticular wax in response to drought. FAR4 is a member of the alcohol-forming fatty acyl-CoA reductases family (FARs) (Domergue et al. 2010, Kosma et al. 2012). We propose that FAR4 may influence the abundance of the C28 aldehyde intermediate in drought-stressed poplars, consistent with observations by Doan et al. (2012). Fatty acyl-CoA reductases (FARs) catalyze the production of fatty alcohols from acyl-CoA, with the formation of an aldehyde as a proposed intermediate (Kolattukudy 1970, Vioque and Kolattukudy 1997, Doan et al. 2012). In vivo, fatty acyl-CoA is usually converted to fatty alcohol without the production of an intermediate aldehyde by a NADPH-dependent FAR enzyme (Kolattukudy 1970, Domergue et al. 2010). Our findings support the need to comprehensively understand the mechanism(s) behind this link, which may contribute to a better understanding of variations in leaf cuticular wax composition during drought events.

A second independent GWAS completed on AP and AM genotypes (Table 3) revealed two shared candidate genes involved in fatty acid synthesis: very long-chain 3-oxoacyl-CoA synthase/very-long-chain beta-ketoacyl-CoA synthase (KCS1) and long-chain-fatty-acyl-CoA reductase/acyl-CoA reductase (CER4 or FAR3). The KCS1 gene encodes a 3-ketoacyl-CoA synthase that plays a crucial role in the synthesis of VLCFAs required for wax biosynthesis (James Jr et al. 1995, Haslam and Kunst 2013), and influences the levels of alcohols, aldehydes, alkanes, alkenes and ketones (Todd et al. 1999, Gonzales-Vigil et al. 2017). PtKCS1, a homologue in P. trichocarpa, is downregulated in non-AP genotypes and is responsible for elongating monounsaturated fatty acids, thus contributing to the cuticular wax composition (Blacklock and Jaworski 2006, Gonzales-Vigil et al. 2017). PtKCS1 showed a preference towards monounsaturated VLCFAs to yield alkenes via a decarboxylation reaction (Chen et al. 2023). Our study identified an SNP in the coding sequence (CDS) of PtKCS1 that could affect its enzymatic functionality, potentially influencing fatty acid elongation and alkene production.

The CER4/FAR3 gene encodes an alcohol-forming fatty acyl-CoA reductase that is vital for making cuticular wax (Vioque and Kolattukudy 1997). Arabidopsis cer4 mutant plants show reductions in primary alcohols and wax esters with increases in aldehydes, alkanes, secondary alcohols and ketones (Rowland et al. 2006). CER4 is one of eight FAR-like genes in Arabidopsis, and it is very similar to an alcohol-forming FAR found in jojoba seeds, and when expressed in yeast, it led to an accumulation of C24 and C26 primary alcohols, highlighting its preference for VLCFAs (Metz et al. 2000, Rowland et al. 2006). In poplar, PtFAR isoforms (including Potri.009G144900) are upregulated under drought conditions, and have been implicated in synthesis of primary alcohols in bark suberin-associated waxes (Rains et al. 2018).

The differences in alcohol levels observed between AP and AM genotypes may stem from CER4/FAR3 activity, since FAR3 plays a key role in the biosynthesis of primary alcohols and could affect the balance between different wax components. Drought-induced alkene synthesis could reduce alcohol abundance due to the nature of the shared precursors, implicating CER4/FAR3 in enhancing drought tolerance by modulating alkene and alcohol production.

Conclusion

In summary, the total wax load did not significantly change when poplar genotypes were exposed to drought for two consecutive years. While an increase in wax concentration might have been anticipated due to its inherent hydrophobic properties that aid in reducing water loss, the concentrations of specific wax components—alkenes, alcohols, esters, and aldehyde—showed significant variation in response to drought. This variation illustrates that while total wax load was unaffected, the composition of waxes is more dynamic and sensitive to environmental stress. It should be noted that although we only examined the changes in cuticular wax in this study, trees can respond to drought in a variety of ways, and thus, the responses are likely to be more complex and require a whole-tree level investigation to fully understand the drought response in poplar. Analyzing multiple drought responses in combination may provide a more accurate and comprehensive picture of the drought tolerance mechanism(s) of poplar trees. The mechanism behind drought responses will be critical in predicting how ecologically and economically important poplars will respond to future climate change.

Supplementary Material

Supplemental_tpaf060
supplemental_tpaf060.zip (2.4MB, zip)

Acknowledgments

We thank Dr Jay Chen and Foster Hart for their assistance during sample collection, and Green Wood Resources Inc. for providing plant samples from their research field site in Boardman, Oregon, USA. We also thank Dr Gerald A. Tuskan, Dr Brian Stanton, and Dr Biruk A. Feyissa for providing the coordinates of the original sample locations, Dr Amith R. Devireddy for providing the weather data, and Dr Feng Kai and Dr Mengjun Shu for assisting with the GWAS data.

Contributor Information

Melike Karaca-Bulut, Department of Wood Science, University of British Columbia, Faculty of Forestry, 2424 Main Mall, Vancouver, BC V6T 1Z4, Canada.

Eliana Gonzales-Vigil, University of Toronto Scarborough 1265 Military Trail, Toronto, ON M1C 1A4, Canada.

Wellington Muchero, Biosciences Division, Oak Ridge National Laboratory, 1 Bethel Valley Road, Oak Ridge, TN 37831-6309, USA.

Shawn D Mansfield, Department of Wood Science, University of British Columbia, Faculty of Forestry, 2424 Main Mall, Vancouver, BC V6T 1Z4, Canada; Department of Botany, University of British Columbia, Faculty of Science, 6270 University Blvd, Vancouver, BC V6T 1Z4, Canada.

Author contribution

S.D.M. designed the project and M.K.-B. conducted the research. S.D.M. and M.K.-B. drafted the manuscript. W.M. performed the GWAS analysis. All authors reviewed the manuscript and provided valuable feedback.

Conflict of interest

The authors declare that they have no competing interests.

Funding

The authors would like to acknowledge funding from the Great Lakes Bioenergy Research Centre, US Department of Energy, Office of Science, Biological and Environmental Research Programme under Award Number DE-SC0018409 to S.D.M., as well as the Centre for Bioenergy Innovation, U.S. Department of Energy, Office of Science, Biological and Environmental Research Programme under Award Number ERKP886 to W.M. M.K.-B. was partially supported by the Republic of Turkiye Ministry of National Education through the Graduate Study Abroad Selection and Placement Programme.

Data availability

Raw data can be provided upon request to the corresponding author.

Dedication

The authors would like to dedicate this work to our friend and co-author, Wellington Muchero, who left us too early in life—you will be missed, our friend.

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