Abies species, including A. koreana, are sensitive to drought stress [2, 3, 4, 5]. A. koreana has been categorized as an endangered species owing to its severe decline since the early 2000s, primarily due to the drought induced by global warming [8, 9, 10]. Thus, to ensure the sustained presence and positive contribution of Abies species to forest ecosystems, conservation efforts are imperative. However, the drought stress response of A. koreana at the molecular level remains unknown. Transcriptomic analysis has been used to understand the molecular response of A. koreana to elevated CO2 and temperature [13, 14] as well as to identify key genes crucial for drought tolerance in other tree species [16, 17, 18, 19, 20, 21, 22]. Therefore, in this study, we analyzed the transcriptome of A. koreana plants exposed to drought stress. Our results reveal key A. koreana genes associated with drought tolerance, which could be used to aid tree breeding programs for the enhancement of forest management in the face of climate change-induced drought challenges.
Three-year-old A. koreana plants were initially planted in pots containing soil with 50% moisture content. Subsequently, watering was withheld for 14 d to induce water deficit. A reduction in SWC was observed from the 7th day of drought treatment compared with the control (0 d). Additionally, the drought-treated plants exhibited delayed growth, a sign of water deficit in plants (Fig. 1). To investigate the transcriptomic changes in A. koreana plants under drought conditions, RNA was extracted from the leaves of plants treated with drought stress for 0 (control), 7, 10, and 14 d. The transcriptomic analysis yielded 85,403 contigs, with an average length of 1,364 bp and an N50 value of 2,087 bp (Table 2). Notably, the average length and N50 value of A. koreana contigs obtained under drought conditions surpassed those of contigs assembled in other tree species facing similar conditions. The contigs of A. koreana were longer than those of Tree peony (average length: 825 bp, N50: 1,368 bp), Aleppo pine (average length: 906.47 bp, N50: 1,269 bp), Masson pine (average length: 695 bp, N50: 1,227 bp), Ammopiptanthus mongolicus (average length: 304 bp, N50: 471 bp), and Prosopis juliflora (average length: 428 bp, N50: 714 bp) [17, 18, 19, 20, 21]. The longer average length and N50 value of A. koreana contigs obtained in this study strongly suggest that the RNA-seq data represent a complete set of transcripts and a high-quality transcriptome assembly. Based on the GO classification, unigenes involved in cellular processes, metabolic processes, response to stimuli, cell, cell parts, organelles, binding, and catalytic activities were found to be strongly regulated in response to drought stress (Fig. 2). This suggests the significant regulation of unigenes associated with various biological processes, cellular components, and molecular functions in response to drought stress, indicating their substantial involvement in the plant response to drought. Similar observations were reported in other tree species exposed to drought conditions [16, 17, 18, 19, 20, 21, 22], indicating a commonality in the regulatory responses to drought stress across different tree species.
In this study, the number of upregulated genes was higher than that of downregulated genes on day 14 but than that of downregulated genes on days 7 and 10 (Fig. 4a). The observed changes in the number of upregulated and downregulated genes at different time points during the drought treatment can be explained by the dynamic nature of the plant response to drought stress [23, 24]. This dynamic response involves the activation and repression of different sets of genes at different stages of stress. The higher number of downregulated genes on days 7 and 10 could reflect an early response mechanism where the plant initially downregulates certain processes to cope with the immediate impact of drought stress. With the continuation of stress, the plant may switch to upregulating specific genes to initiate more advanced adaptive responses, leading to a greater impact on its gene expression profiles. Therefore, the observed increase in the number of upregulated genes on the 14th day of drought may reflect the activation of specific regulatory pathways that mediate the acclimation of plants to prolonged water deficit [23, 24].
Several studies have shown that the response to drought stress is a complex process involving the regulation of a large number of genes, including those encoding TFs that play critical roles in the adaptation to abiotic stresses [25, 26]. For example, TFs belonging to the ERF, MYB, WRKY, NAC, and bHLH families have been reported to be involved in the regulation of gene expression in response to drought stress in various plant species, including A. koreana [13, 14, 25, 26]. These TFs are known to directly regulate the expression of stress-responsive genes, and their upregulation is associated with enhanced tolerance to drought stress in tress species [27, 28, 29, 30, 31, 32].
Therefore, a substantial number of TF genes were identified in this study, including those encoding bZIP, HD-ZIP, bHLH, ERF, MYB, WRKY, LBD, and NAC TFs, which have been implicated in various physiological processes, such as stomatal movement and hormone signal transduction. Notably, the number of ERF TFs was found to be greater than that of other TFs, and a large number of ERF TFs were upregulated under drought stress (Fig. 7). A similarly high number of upregulated ERF genes was observed in A. koreana under other stresses such as heat and CO2 stress [13, 14], strongly indicating the significant role of ERF TFs in the adaptation of A. koreana to different abiotic stresses. MYB, ERF, NAC, and bHLH TF families are known to be large and functionally diverse. Different members of each TF family may play distinct roles and functions in response to stress [25, 26]. The observed mix of upregulated and downregulated TF genes across the different drought treatments (7, 10, and 14 d) suggests that A. koreana employs a complex and dynamic regulatory network to respond to drought stress. In addition, the observed upregulation or downregulation of specific MYB, ERF, NAC, and bHLH TF genes could be related to their functions in distinct signaling pathways or cellular processes activated at different stages of drought stress [25, 26]. The functions of TFs in conferring tolerance to drought stress in woody crops have been extensively analyzed. Overexpression of ERF genes has been shown to enhance tolerance to drought stress in woody crops [33, 34]. Additionally, MdHB-7, which encodes an HD-Zip TF, was found to promote drought tolerance in apple [35].
Furthermore, recent studies show that the PtrbZIP3 (basic leucine zipper) TF as well as NAC, WRKY, bHLH, LBD, and MYB TFs play essential roles in the response to drought stress, and overexpression of genes encoding these TFs increases drought tolerance in woody crops [27, 28, 29, 30, 31]. In this study, we confirmed the reliability of our RNA-seq data by analyzing the expression patterns of six genes encoding TFs (BES1, HD-ZIP, bHLH, ERF, MYB, and LBD) at different time points using qRT-PCR (Fig. 8). Overall, the expression of stress-responsive genes, including those encoding TFs, was induced in a time-dependent manner in response to drought stress. Taken together, this study revealed several candidate genes, including those encoding ERF, MYB, LBD, and NAC TFs, associated with drought tolerance in A. koreana. Although further investigation is needed to gain valuable insights into the role of these genes in the adaptation of A. koreana plants to drought stress, we predict that the results of this study will aid tree breeding programs and facilitate the enhancement of forest management in the face of climate change-induced drought challenges.