Structural and functional characterization of genes PYL‑PP2C‑SnRK2s in the ABA signalling pathway of Cucurbita pepo

Iglesias‑Moyaetal. BMC Genomics (2024) 25:268 https://doi.org/10.1186/s12864‑024‑10158‑9 RESEARCH Open Access © The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecom‑ mons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. BMC Genomics Structural andfunctional characterization ofgenes PYL‑PP2C‑SnRK2s intheABA signalling pathway ofCucurbita pepo Jessica Iglesias‑Moya1, Álvaro Benítez1, María Segura1, Sonsoles Alonso1, Dolores Garrido2, Cecilia Martínez1* and Manuel Jamilena1* Abstract Background The core regulation of the abscisic acid (ABA) signalling pathway comprises the multigenic families PYL, PP2C, and SnRK2. In this work, we conducted a genome‑wide study of the components of these families in Cucurbita pepo. Results The bioinformatic analysis of the C. pepo genome resulted in the identification of 19 CpPYL, 102 CpPP2C and 10 CpSnRK2 genes. The investigation of gene structure and protein motifs allowed to define 4 PYL, 13 PP2C and 3 SnRK2 subfamilies. RNA‑seq analysis was used to determine the expression of these gene families in different plant organs, as well as to detect their differential gene expression during germination, and in response to ABA and cold stress in leaves. The specific tissue expression of some gene members indicated the relevant role of some ABA signal‑ ling genes in plant development. Moreover, their differential expression under ABA treatment or cold stress revealed those ABA signalling genes that responded to ABA, and those that were up‑ or down‑regulated in response to cold stress. A reduced number of genes responded to both treatments. Specific PYL‑PP2C‑SnRK2 genes that had potential roles in germination were also detected, including those regulated early during the imbibition phase, those regulated later during the embryo extension and radicle emergence phase, and those induced or repressed during the whole germination process. Conclusions The outcomes of this research open new research lines for agriculture and for assessing gene function in future studies. Keywords Zucchini squash, Response to ABA, Cold stress response, Germination, Tissular expression Background Plant growth and development, ranging from seed germination to fruit growth and maturation requires the orchestration of a multitude of factors, including phytohormones. Abscisic acid (ABA) plays an essential role in response to environmental stresses, regulating the relocation of resources to cope with stress, even at the expense of reducing plant growth [1]. Stress-induced ABA causes multiple changes at physiological and developmental levels, including stomatal closure, cuticular wax accumulation, leaf senescence, bud dormancy, growth inhibition, and the control of seed development, desiccation *Correspondence: Cecilia Martínez cmar[email protected] Manuel Jamilena [email protected] 1 Department of Biology and Geology. Agri‑food Campus of International Excellence (CeiA3) and Research Center CIAIMBITAL, University of Almería, 04120 Almería, Spain 2 Department of Plant Physiology. Faculty of Science, University of Granada, 18021 Granada, Spain Page 2 of 19 Iglesias‑Moyaetal. BMC Genomics (2024) 25:268 tolerance, dormancy and germination, among others [2]. In addition to its master role in stress response, ABA is a positive or negative regulator of developmental processes such as root growth and architecture [3], hypocotyl growth [4], and fruit growth, and maturation [5]. These functions are accomplished by itself or in interplay with other hormones. Therefore, understanding the genes involved in the ABA signalling pathway is essential not only to unravel the complex system that controls plant responses under stress, but also the role of this hormone in vegetative and reproductive development. The ABA signalling pathway comprises three main components: the ABA receptors PYRABACTIN RESISTANCE 1/PYR1-LIKE/REGULATORY COMPONENTS OF ABA RECEPTORS (PYR1/PYL/RCARs) [6–8], the PROTEIN PHOSPHATASE TYPE-2C (PP2C) coreceptors [9–14], and SUCROSE NON-FERMENTING 1-RELATED SUBFAMILY 2 KINASES (SnRK2) [15– 17]. In the absence of ABA, PP2Cs bind to and repress SnRK2s, thereby blocking the ABA signalling pathway [6–8]. In the presence of ABA, ABA binds to a PYR1/ PYL/RCAR receptors (hereafter referred as PYLs), which undergoes conformational rearrangements that lead to the formation of PYL-PP2C heterodimer [6–8, 18, 19]. The interaction between PYL-ABA and PP2Cs provokes the dissociation of the PP2C-SnRK2, inhibiting the phosphatase activities of PP2Cs, which results in the autophosphorylation and the activation of SnRK2, and in the stimulation of ABA response [7, 8]. How the main components of the core ABA signalling system are regulated determines the response to ABA and is essential for maintaining plant growth under nonoptimal environments [2]. Several studies have identified genes that encode crucial components of ABA signalling in different species. The PYL family comprises 14 genes in Arabidopsis, 13 in rice, 24 in banana, 15 in tomato, and 14 in cucumber [20–23]. In Arabidopsis, PYL members have redundant functions in the regulation of PP2Cs, but differ in their ABA binding properties and their temporal and spatial expression [19, 24]. All of these are soluble proteins with STAR-RELATED LIPID-TRANSFER (START) domains that are distributed in the cytoplasm and nucleus [6, 7]. Although the phylogeny of PYLs leads to the establishment of three subfamilies [6], they can be classified into two major classes according to their oligomeric nature. Dimeric PYLs belong to subfamily III (PYR1 and PYL1– 3, although PYL3 may have a faster equilibrium between dimer and monomer) [19], and monomeric PYLs belong to subfamilies I and II (PYL4–13, except for untested PYL7) [19, 25]. Dimeric ABA receptors have lower ABA binding affinity for dimer dissociation and inhibition of PP2C [19, 25], while monomeric forms have higher ABA binding affinity and can achieve complete inhibition of PP2C at much lower ABA concentration, or even in the absence of ABA [19]. Arabidopsis PYL13 is the only protein identified that does not respond to ABA and inhibits several PP2Cs in an ABA-independent manner [26, 27]. Genetic variability between PYL members provides explanation of their different basal activity and roles in plant development and stress responses [19, 25]. PP2Cs are Mg2+/Mn2+-dependent serine/threonine phosphatases that are closely related to the phosphoprotein phosphatases (PPP) family. They have, however, no sequence homology with PPP, and form a single cluster in the phosphoprotein metal phosphatases (PPM) family [28]. The PP2C family is highly conserved throughout evolution, having been found in archaea, bacteria, fungi, plants, and animals [29]. A total of 80 PP2Cs have been identified in Arabidopsis, 78 in rice, 87 in banana, 92 in tomato and 56 in cucumber [21, 28, 30, 31]. In Arabidopsis they have been divided into 13 subfamilies (A-L), with the exception of seven that could not be clustered [30]. Subfamily A contains proteins that have been characterized as key factors in the transduction of ABA signal, including ABI1, ABI2 (AT5G57050), AHG1 (AT5G51760), AHG3 (AT3G11410), HAB1 (AT1G72770), and HAB2 (AT1G17550) [32]. A-type PP2Cs inactivate SnRK2 by dephosphorylation, a function that is inhibited by PYL receptors in an ABAdependent manner [30]. Subfamily B participates in the mitogen-activated protein kinase (MAPK) signalling pathway during salt stress or wounding [33]. Some genes in subfamily C, such as AtPOL (AT2G46920) and AtPLL1 (AT2G35350), are involved in flower development and maintenance of stem cell polarity [34, 35]. Subfamily D members respond to salt and alkali stress [36] and are also involved in the regulation of seed germination in the dark, seed growth, and the ABA signalling pathway by mediating the activity of the plasma membrane H+-ATPase in cells [30, 37]. Subfamily E is involved in the regulation of stomatal closure [38]. Another member of PP2C of subfamily F, WIN2, (AT4G31750) is involved in the response of the plant to bacterial stress [39]. Only a few genes within each subfamily have been characterized, and information on the functions of PP2Cs from other subfamilies is not yet available. The SnRK family comprises three major subfamilies, SnRK1, SnRK2, and SnRK3. SnRK1 is homologous to yeast SUCROSE NON-FERMENTING 1 (SNF1) kinase and mammalian AMP-ACTIVATED PROTEIN KINASES (AMPKs) [40], and is involved in cellular responses to nutritional signals [41], while SnRK2 and SnRK3 are specific to plants. SnRK3s are characterized by their ability to interact with Ca2+ sensor CALCINEURIN B-LIKE PROTEIN (CBL), while SnRK2 are Page 3 of 19 Iglesias‑Moyaetal. BMC Genomics (2024) 25:268 the main drivers of ABA-triggered responses [42, 43]. SnRK2 kinases are monomeric serine/threonine protein kinases composed of a well-conserved N-terminal catalytic domain, and a regulatory C-terminal domain consisting of two subdomains: domain I and domain II [44]. Domain I, also known as the SnRK2 box, is conserved in all SnRK2s and is required for ABA-independent activation in response to osmotic stress. Domain II, also known as the ABA box, is required for ABA-dependent activation [44]. SnRK2 has been clustered into three subfamilies: subfamily I comprises kinases that are not activated by ABA; subfamily II comprises kinases that are not activated or are very weakly activated by ABA; and subfamily III comprises kinases strongly activated by ABA [45]. The SnRK2 family has been identified in many plant genomes, including 10 SnRK2 genes in Arabidopsis [46], 11 in banana [21], 10 in rice [47], 11 in maize [48], and 11 in cucumber [49]. Gene expression data and mutant characterization in several species have shown that the SnRK2 genes are an essential part of the ABA signalling pathway, at multiple stages of development and in response to abiotic stresses [50–52]. SnRK2s catalyze the phosphorylation of various downstream targets, including ABA INSENSITIVE 5 (ABI5), which plays an essential role in the post-germinative development arrest checkpoint [53–55], and ABA-RESPONSIVE ELEMENT BINDING FACTOR (ABF), which are transcription factors that finally induce the expression of ABA-responsive genes [7, 8, 14]. The squash, Cucurbita pepo, is an important vegetable crop with significant production and economic value around the world. Its genome was sequenced in 2018, which revealed a duplication of the whole genome associated with the origin of Cucurbita species [56]. We have identified 19 PYLs, 102 PP2Cs and 10 SnRK2s in the C. pepo genome, and determined their phylogenetic relationships, protein motifs, and gene structure. Their spatial gene expression patterns and transcriptional regulation during germination, as well as in response to ABA and cold treatments, were also investigated. This study will enhance our understanding of the core components of the ABA signalling pathway and offers a potential new perspective for squash breeding programs. Results Identification andclustering ofC. pepo PYL‑PP2C‑SnRK2 proteins A total of 19 PYL, 102 PP2C, and 10 SnRK2 genes were identified by analyzing the C. pepo reference genome [56]. Tables S1-S3 include information on gene annotation for each PYL, PP2C, and SnRK2 gene in the CuGenDBv2 and NCBI databases. Given that the current C. pepo genome derived from a complete genomic duplication, most of the identified ABA signalling genes had a paralogue on another chromosome. Only 4 out of 19 CpPYLs, 26 out of 102 CpPP2Cs and 2 out of 10 CpSnRK2s did not show the expected paralogous in the genome (Tables S1-S3). To explore the evolutionary relationships and functional diversity of the CpPYL, CpPP2C, and CpSnRK2 proteins, we constructed phylogenetic trees using multiple sequence alignments of the PYL-PP2C-SnRK2 proteins from C. pepo and the model species Arabidopsis (Tables S1-S4). According to the phylogenetic analyses, the PYL, PP2C and SnRK2 families were divided into 4 (I-III), 13 (A-L, and one unclassified subfamily U), and 3 (I-III) subfamilies, respectively (Figs. 1 and 2). The CpPYLs were named on the basis of their homology with Arabidopsis PYL members [6]. The PYL subfamily I was found to be the largest, having 7 CpPYLs clustered together with AtPYL7–10, subfamily II-a included 6 CpPYL together with AtPYL4–6, and subfamily II-b only presented a single CpPYL protein (Cp4.1LG09g07940) that clustered with AtPYL11–13. Finally, subfamily III consisted of five CpPYL proteins clustered with AtPYR1 and AtPYL1–3 (Fig.1A). The phylogenetic tree of PP2C proteins of C. pepo and Arabidopsis showed the conserved diversity of this family of receptors (Fig.2). From the 13 subfamilies found, subfamilies D and E are the largest ones, each one comprising 16 proteins, whereas subfamily J is the smallest, comprising only one protein. The AtPP2C proteins that were previously found unclustered [30], were here grouped in the subfamily A (AT4G11040), subfamily F (AT3G23360), subfamily J (AT2G40860), subfamily K (AT4G33500), and subfamily U (AT1G18030, AT4G27800, and AT5G19280), the later with the squash PP2C proteins Cp4.1LG14g06750 and Cp4.1LG09g00180 (Fig.2). For the SnRK2 family, subfamilies II and III clustered four squash CpSnRK2 proteins each one, whereas only two squash proteins were found clustered in subfamily I, although this subfamily displayed the highest number of AtSnRK2s (Fig.1B). Gene structure andconserved protein motifs ofC. pepo PYL‑PP2C‑SnRK2s The gene structure of the C. pepo PYL-PP2C-SnRK2 families was analyzed using the GSDS database (Figs.3 and 4). The structure of the CpPYL gene subfamilies was in accordance with the phylogenetic analysis based on the sequence (Fig.1A). Three to five exons were found within the genes of subfamily I. Members of subfamily II-a and II-b had one single exon, except for Cp4.1LG13g07430 that presents two exons (Fig.3A). The genes of subfamily III showed two exons with the sole exception of Cp4.1LG05g00020, which had only one (Fig.3A). The conserved motifs in CpPYLs also supported their sequence homology (Fig.3A). A total Page 4 of 19 Iglesias‑Moyaetal. BMC Genomics (2024) 25:268 Fig. 1 Phylogenetic analysis of C. pepo PYL (A) and SnRK2 (B) proteins. The circles represent squash proteins, and the triangles represent Arabidopsis proteins used for comparison. The phylogenetic tree was built with Mega X using the Maximum Likelihood method and 1000 bootstrap replications. The subfamily number is established according to the Arabidopsis classification adapted to this analysis [6, 45]. Members of Arabidopsis and squash included in the same clade were represented with the same color Page 5 of 19 Iglesias‑Moyaetal. BMC Genomics (2024) 25:268 of 10 motifs were identified in members of the CpPYL family. All CpPYLs presented motifs 1, 2, and 3, while motif 7 was found to only be present in subfamily I (Fig.3A). This is consistent with the annotation of the conserved domain PYR/PYL/RCAR_like (cd07821) in members of the CpPYL family (Table S1). High diversity was found in the exon/intron structure of the CpPP2C genes (Fig. 4A). The number of exons within each subfamily was variable, although subfamilies E (5 exons), G (4 exons), I (11 exons), J (14 exons), and U (11 exons) displayed a fixed number of exons. All genes in the E, J, U, and H subfamilies, as well as some genes from subfamilies A, G, B, and F, Fig. 2 Phylogenetic analysis of C. pepo PP2C proteins. Circles represent squash CpPP2C proteins, and triangles represent Arabidopsis AtPP2C proteins used for comparison. Black asterisks indicate AtPP2C proteins that were unclustered in the phylogenetic analysis performed by Xue et al. [30]. The phylogenetic tree was built with Mega X using the Maximum Likelihood method and 1000 bootstrap replications. Subfamily letter is established in base to Arabidopsis classification adapted to this analysis [30]. Members of AtPP2C and CpPP2C included in the same clade were represented with the same color Page 6 of 19 Iglesias‑Moyaetal. BMC Genomics (2024) 25:268 presented annotated 3′ or 5′ untranslated regions (UTR) sequences, while the genes of the subfamilies K, L, C, and D did not have annotated 3′ or 5′ UTR sequences (Fig.4B). The analysis of conserved motifs in this family led to the identification of as many as 20 (Fig. 4B). The presence/absence and the distribution of motifs within proteins are specific for each subfamily, validating the subfamilies established by phylogenetic analysis (Fig.2). Motifs 1, 2, and 3 were significantly detected in almost all CpPP2C proteins except Cp4.1LG02g02090 and Cp4.1LG02g11150 of subfamily K and Cp4.1LG12g08540 of subfamily A. Most members of CpPP2C contained more than seven motifs, while some members had only four motifs, such as Cp4.1LG12g01250, Cp4.1LG17g02300, and members of subfamily K. Different subfamilies have their own specific motifs, probably in association with the functional divergence of each subfamily. So, all members of subfamily E comprised motifs 12 and 13, those belonging to subfamily L had specifically motifs 17 and 19, the subfamily D proteins contained motifs 8 and 14, and subfamily H was the only one containing motif 20 (Fig.4B). The gene structure of the CpSnRK2 family is shown in Fig.3B. Members of the three subfamilies had nine exons, except for Cp4.1LG10g12230 and Cp4.1LG05g11310, which had 10 and 12 exons, respectively (Fig.3B). As observed for CpPYLs and CpPP2Cs, CpSnRK2 genes in the same subfamily also showed a similar exon-intron organization. The CpSnRK2 proteins comprised 10 motifs. All motifs but Nr. 9 were identified in all subfamilies, while motif 9 was only detected in three proteins in subfamily III (Fig.3B). This is consistent with the conserved domains annotated in NCBI (Table S3). All members of the family, but three, had the STKc_SnRK2 (cd14662) domain. The remaining three CpSnRK2, belong to subfamily III and presented STKc_SnRK2–3 (cd14665) or PKc_like (cl21453) domains. STKc_SnRK2s are domains involved in plant response to abiotic stresses and ABA-dependent plant development, while STKc_ SnRK2–3 domains are representative of kinases strongly activated by ABA (Table S3). Fig. 3 Gene structure and motif analysis of gene families CpPYL (A) and CpSnRK2 (B). 1 Exon–intron structures of genes inferred with GSDS. The black lines and green boxes indicate introns and exons, respectively. The blue boxes indicate upstream and downstream untranslated regions (UTRs). 2 Distribution of protein motifs identified by MEME software. Each colored box represents a conserved motif sequence Page 7 of 19 Iglesias‑Moyaetal. BMC Genomics (2024) 25:268 Fig. 4 Gene structure and motif analysis of gene family CpPP2C. A Exon–intron structures of CpPP2C genes inferred with GSDS. The black lines and green boxes indicate introns and exons, respectively. The blue boxes indicate upstream and downstream untranslated regions (UTRs). B Distribution of all protein motifs identified by MEME software. Each colored box represents a conserved motif sequence Page 8 of 19 Iglesias‑Moyaetal. BMC Genomics (2024) 25:268 The expression profiles ofPYL‑PP2C‑SnRK2 genes indifferent plant organs To gain insight into the role of each PYL-PP2C-SnRK2 gene in the ABA signalling pathway, an RNA-seq analysis was performed in both vegetative and reproductive organs, including young roots and leaves, apical shoots, male and female flowers, and ovaries, fruits, and seeds. The number of raw reads and the percentage of reads after cleaning are listed in Table S5. Figure5 shows the tissular expression patterns for each gene family. CpPYLs Most CpPYLs showed a low transcription level in the tissues under study. In general, the lowest FPKM values were found among CpPYLs in subfamily II-a (Cp4.1LG19g02690, Cp4.1LG01g05500, Cp4.1LG10g07810, Cp4.1LG13g07430) and the single gene of subfamily II-b (Cp4.1LG09g07940), which showed no or reduced expression in most tissues analyzed (Fig.5A). Five CpPYLs of the subfamily III, and 4 CpPYLs of the subfamily I showed intermediate FPKM values (Log2 FPKM < 4.5), and the highest expression values were found for the genes Cp4.1LG09g00400, Cp4.1LG03g16100, and Cp4.1LG08g00920 in subfamily I (Fig.5A), which, together with their constitutive expression in the different organs, could indicate an essential role of these three CpPYLs in different physiological and developmental processes. The upand down-regulation of some CpPYLs in specific organs may be related to their functions. Thus, the specific up-regulation of Cp4.1Lg09g07940 (Log2 FPKM > 5) in dry seed (Fig.5A) suggests a potential association with the maintenance of desiccated and metabolically quiescent mature embryos. In the fruit, a high abundance of transcripts (Log2 FPKM > 4.5) of Cp4.1LG03g16100, Cp4.1LG08g00920, and Cp4.1LG09g00400, but also the specific activation of Cp4.1LG17g10330 and Cp4.1LG06g02330 was found (Fig.5A), which may indicate the involvement of these genes in fruit growth and development. Similarly, roots were characterized by the highest transcript accumulation of Cp4.1LG08g00920 (Log2 FPKM > 6.8), and the specific activation of Cp4.1LG19g02690 gene (Fig.5A). CpPP2Cs Figure5B shows the expression patterns of 102 CpPP2Cs in the eight analyzed plant organs. Most genes had values of Log2 FPKM < 4.5. In fact, 20 CpPP2C show Log2 FPKM < 3 in all tissues except in seed (Fig. 5B). Genes from different subfamilies of PP2C were clustered together based on gene expression, suggesting that their expression was not dependent on their phylogenetic origin. Female and male flowers presented similar transcription patterns for CpPP2Cs. Therefore, Cp4.1LG07g08850, Cp4.1LG04g08500, and Cp4.1LG18g04070 were highly expressed (Log2 FPKM > 5) in both male and female flowers, and only a few genes, such as Cp4.1LG01g04510 and Cp4.1LG02g14360 were more expressed in male flowers than in female flowers (Fig.5B). Interestingly, the largest number of CpPP2C with a high expression level was found in the fruit, with a total of eight genes displaying Log2 FPKM > 6. Among them, Cp4.1LG04g02760, Cp4.1LG06g06790, and Cp4.1LG02g1366 showed Log2 FPKM > 6.6; Cp4.1LG18g04070 had Log2 FPKM > 7.3; and Cp4.1LG04g10270 had a transcription value of Log2 FPKM > 8.5 (Fig. 5B). Regarding ovarian tissue, Cp4.1LG02g11790 (subfamily B) had the highest abundance of transcripts (Log2 FPKM = 6.7), a gene that was also highly transcribed in fruits (Log2 FPKM = 6.2) (Fig.5B). In roots and apical shoots, the highest expression of the PP2C genes was found for the genes Cp4.1LG12g06400 (subfamily G) and Cp4.1LG02g11790 (subfamily B), showing Log2 FPKM > 6.7 (Fig. 5B). Finally, the leaf and seed showed a distinctive expression pattern with respect to other plant organs. Therefore, the genes Cp4.1LG02g11790 (subfamily B) and Cp4.1LG12g06400 (subfamily G) are specifically negatively regulated in leaves, while Cp4.1LG04g08500 (subfamily H) and Cp4.1LG014g06750 (subfamily U) (Log2 FPKM > 5) were specifically positively regulated in leaves (Fig.5B). In dry seeds, the transcripts of Cp4.1LG13g11070 (Log2 FPKM = 8.3), Cp4.1LG02g13660 (Log2 FPKM > 6), and Cp4.1LG09g01270 (Log2 FPKM = 5.7) were specifically and highly accumulated (Fig.5B), suggesting that they were specifically expressed during seed maturation. CpSnRK2s The CpSnRK2 genes were clustered in two groups according to their tissue transcription profiles (Fig.5C), although they were not related to the phylogenetic subfamilies established by sequence homology. One of the clusters comprised genes with Log2 FPKM < 3.9, including Cp4.1LG08g07070, Cp4.1LG14g09100, Cp4.1LG05g11310, Cp4.1LG05g12260 and Cp4.1LG19g09940. The other group showed values of Log2 FPKM between 2.5 and 7.2 and includes Cp4.1LG19g00190, Cp4.1LG10g01490, Cp4.1LG16g00530, Cp4.1LG04g08220 and Cp4.1LG10g12230. Among the CpSnKR2 genes, the gene Cp4.1LG19g00190 showed the highest levels of expression in the different plant organs studied (Fig.5C). A low level of transcription was found for most of CpSnRK2 in leaf, even for the most transcribed genes, Cp4.1LG10g01490 (subfamily II) and Cp4.1LG19g00190 (subfamily I), which showed transcription values of Page 9 of 19 Iglesias‑Moyaetal. BMC Genomics (2024) 25:268 Fig. 5 Expression profiles of CpPYL-CpPP2C‑CpSnRK2 genes in different plant organs. A Heatmap of CpPYL genes. B Heatmap of CpPP2C genes. C Heatmap of CpSnRK2 genes. Data were normalized using log2 FPKM and TBtools was used to draw the expression heatmap. The expression values assigned to a color gradient from low log2 FPKM (green) to high log2 FPKM (red) are shown on the right of each figure Page 16 of 19 Iglesias‑Moyaetal. BMC Genomics (2024) 25:268 each condition. Germination of each seed was considered complete when rupture of the seed coat and radicle protrusion were observed (> 1 mm). Three biological replicates for dry, soaked, and germinated seeds were sampled, each consisting of 30 seeds. The seed coat was removed before being pulverized in liquid nitrogen and stored at − 80 °C. To select the ABA concentration, we conducted two separate experiments. First, we analyzed the percentage of germination at various concentrations of ABA in the MUCU16 inbred line. The results of the dose-response curve are illustrate in Fig. S3. We chose the ABA concentration of 100 μM because it resulted in a 35% reduction in germination compared to seeds germinated in water (Fig. S3). At higher concentrations, germination was completely blocked. Following the selection of the ABA concentration for germination, we investigated whether the application of 100 μM ABA could also impact the water loss in the leaves of MUCU16. Our findings revealed that treatment with 100 μM of ABA reduced water loss in ABA-treated seedlings at 1 and 4 h after starting treatment (Fig. S4). Additionally, cold stress also decreased the loss of water compared to the control from 1 to 24 h after the onset of treatment (Fig. S4). Water loss assays were conducted on 14-days-old seedlings. Twelve plants per treatment were sprayed with distilled H2O or 100 μM ABA and placed in a growth chamber with a photoperiod of 16/8 h of light/dark at 24 °C and 60% RH. For cold treatment, the aerial parts of the seedlings were incubated at 4 °C in a growth chamber with a photoperiod of 16/8 h of light/dark and 60% RH. The weights of the aerial parts of the plants were measured at 1, 4, 6, and 24 h after the initiation of treatment. The percentage of water loss was calculated using the following formula: where WT0 is the initial weight (g) and WTx is the weight at each recorded point (g). RNA extraction andsequencing Frozen tissue at − 80 °C was ground using stainless steel beads, previously cooled with dry ice. For RNA extraction, the E.Z.N.A® Plant RNA Kit (Omega Bio-tek) was used following the manufacturer’s protocol. After extraction, RNA was eluted in nuclease-free water and immediately prepared for sequencing on the BGI DNBseq Sequencing Platform, generating 150 pb pair-end reads. All raw reads generated were made publicly % Waterloss = (W T0− W Tx ) W T0 x 100 available in the NCBI database (https:// www. ncbi. nlm. nih. gov/) under project number PRJNA1042934 and PRJNA1019290. Bioinformatic analysis oftranscriptomic data The quality of the sequenced reads was checked by the FastQC tool [86]. SOAPnuke [87] were used to delete and trim low quality bases within the data. Mapping of highquality reads and transcriptome assembly were carried out by HISAT2 [88] and STRINGTIE [89, 90]. To evaluate the expression patterns of the PYL-PP2CSnRK2 genes in different tissues, gene expression levels were calculated as fragments per kilobase million (FPKM). Subsequently, a heatmap was created for each family of genes using TBtools [91]. Data were normalized using log2 (FPKM), and values of FPKM < 1 were considered as 1. Differential expression analysis was performed using the total count matrix, using edgeR ver. 3.28 [92, 93] and limmavoom ver. 3.42.2 [94, 95] packages in R [96]. Voom function, available in the limma package, was applied during data treatment. The adjusted p-value for each gene was calculated using the Benjamini & Hochberg (BH) method [97]. Counts per million (CPM) values were calculated and used for multidimensional scaling (MDS) of the expression data using glimmaR ver. 2.10 [98]. To determine differentially expressed genes (DEG) under different treatments, only genes with adjusted P.value < 0.05 were considered. Supplementary Information The online version contains supplementary material available at https:// doi. org/ 10. 1186/ s12864‑ 024‑ 10158‑9. Supplementary Material 1. Supplementary Material 2. Authors’ contributions MJ and CM designed and coordinated the research. JI‑M conducted most of the experiments and data analysis. AB, MS, SA, and DG collaborated in data analysis. JI‑M, CM, and MJ wrote the first version of the manuscript, and the other authors contributed later to improve it and approved the final version for submission. All authors contributed to the article and approved the submitted version. Funding This work was supported by grants PID2020‑118080RB‑C21, UAL18‑BIO‑ B017‑B, and P20_00327, funded by the Spanish Ministry of Science and Innovation, the University of Almería and Junta de Andalucía. JI‑M gratefully acknowledges the FPI Scholarship Program from the Spanish Ministry of Sci‑ ence and Innovation. MS acknowledges D.I scholarship programmer from MCI (DIN2018–010127) with the company Green Breeding Biotech S. L. Availability of data and materials All sequence information regarding C. pepo is available from CuGenDBv2 (http:// cucur bitge nomics. org/ v2/) and the accession numbers are listed in Tables S1‑S3. The PYL, PP2C, and SnRK2 protein sequences from Arabidopsis are available from TAIR (https:// www. arabi dopsis. org/) and the accession numbers Page 17 of 19 Iglesias‑Moyaetal. BMC Genomics (2024) 25:268 are listed in Table S4. The transcriptomic data were deposited in NCBI‑SRA database (https:// www. ncbi. nlm. nih. gov/ sra/) (Project number: PRJNA1042934 and PRJNA1019290). Declarations Competing interests The authors declare no competing interests. Received: 27 September 2023 Accepted: 24 February 2024 References 1. Brookbank BP, Patel J, Gazzarrini S, Nambara E. Role of basal ABA in plant growth and development. Genes. 2021;12. 2. Chen K, Li GJ, Bressan RA, Song CP, Zhu JK, Zhao Y. Abscisic acid dynam‑ ics, signaling, and functions in plants. J Integr Plant Biol. 2020;62:25–54. 3. McAdam SAM, Brodribb TJ, Ross JJ. Shoot‑derived abscisic acid promotes root growth. Plant Cell Environ. 2016;39:652–9. 4. Humplík JF, Bergougnoux V, Jandová M, Šimura J, Pěnčík A, Tomanec O, et al. 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