Abstract
Knowledge about the genetic potential of wild Prunus species is important for recognizing gene pools in germplasm and developing effective strategies for germplasm conservation and breeding. In this study, cultivated sweet cherry (Prunus avium L.) and relative wild Cereus subgenus genotypes belonging to P. microcarpa Boiss and P. incana (Pall.) Batsch. species were assayed in controlled drought conditions performing an integrated morphological, physiological, genomic and transcriptomic analysis of the stress response. These analyses were performed under well-watered and water-deficient conditions analysing morphology of leaves, gas exchange parameters and chlorophyll content differently affected by drought, especially under long-term water deficiency. From a genomic point of view, these genotypes were characterized using simple sequence repeat (SSR) markers. Finally, in the analysis of the transcriptional responses, drought tolerance candidate genes were analysed by quantitative PCR (qPCR). From a morphological point of view, P. microcarpa showed less leaf area, height and diameter in comparison to P. incana and mainly P. avium. The highest rate of photosynthesis in genotypes differently was observed in the control treatment and the lowest rate viewed during stress. The analysed genes showed different responses to drought in the genotypes, and more detailed analysis techniques to explain the molecular mechanisms of drought tolerance are needed in these genotypes. Our results provide a significant contribution to the understanding of how P. incana and P. microcarpa respond to drought stress, which may help to explain molecular and physiological mechanisms associated with the response to the drought of cherries. Additionally, these results show that wild cherries can be considered as a genetic source of drought resistance in breeding programs.
Similar content being viewed by others
Avoid common mistakes on your manuscript.
Introduction
In the last few decades, the biggest challenge especially in agriculture sector that the world faces is climate change and global warming. Following climate change, drought has become likely the most important constraint limiting the productivity crop and finally food security worldwide. In some countries, climate change has been so high that it can compensate for improvements in yield resulting from technology, fertilization and other factors through negative effects on plant growth and reproduction. Reduce precipitation and change of rainfall patterns causes repeated droughts around the world (Lesk et al. 2016; Lobell et al. 2011; Yordanov et al. 2000).
To study drought response, several parameters and their relations such as stomatal conductance, net photosynthetic rate leaf turgor synthesis of abscisic acid or water-use efficiency have been studied in fruit trees (Martínez-García et al. 2020). On the other hand, extensive studies suggest that plant strategies to reduce drought effects include those that enable plants to avoid and tolerate low water potentials. In the avoid strategy, water loss and water uptake maintain balanced and preserved the plant water status by tapping ground water with deep roots, stomatal closure and small leaves. With the onset of drought, plants tolerate water stress through osmotic or elastic adjustment or the accumulation of osmoprotective substances such as cyclitols (Pirasteh-Anosheh et al. 2016; Roychoudhury et al. 2013). Therefore, activation of these processes enables maintenance of cellular homeostasis through lipid and carbohydrate metabolism.
Based on the current comprehension from drought-responsive genes that included regulatory and effector genes, the identification of these genes and understanding their functional are necessary for improvement of drought tolerance in crops of economic importance (Shinozaki and Yamaguchi-Shinozaki 2007). In Prunus species, several water-deficit resistance genes dependent and independent of abscisic acid (ABA) biosynthesis have been identified mainly transcription factors (TF) such as basic helix-loop-helix (bHLH) (Bianchi et al. 2015). Due to ABA multiple roles in seed germination and dormancy infliction (Rodríguez-García et al. 2009), fruit ripening (Teribia et al. 2016) and induction of drought resistance (Balint and Reynolds 2016; Li et al. 2012) in plants, each of these genes is expected to have specific functions. ABA receptors promote drought resistance in Arabidopsis and rice by limiting the loss of transpiration water and creating similar reactions to summer dormancy, such as old leaves senescence and growth prevention in young tissues in continuous drought conditions (Zhao et al. 2016; Lau et al. 2021). HD-Zip (homeobox-leucine zipper) genes have been detected in a vast variety of plant species and have a numerous functional range. Chen et al. (2010) showed that the 12 HD-Zip genes are responsive to the drought and salt stressors. ATHB-12 is one of the HD-Zip families that is induced by water-deficit and acts as a negative primary regulator of the ABA response mechanism in Arabidopsis. In addition, AFP3/ABI (ninja-family protein/five-binding protein) regulates stress response by the downregulation of ABA responses (García et al. 2008).
Given the ABA role in abiotic stresses, it is important that its receptors play important role in responses to these stresses. In apple (Malus domestica Borkh.), the activation of the ABA signal pathway mediated by γ-aminobutyric acid (GABA) improves drought resistance (Liu et al. 2021; Yao et al. 2020). In addition, it is known that several TF families including bHLH, MYB, WRKY, bZIP, AUX/IAA, dehydration-responsive element binding protein (DREB) and peroxidase (Pd) are involved in drought stress in plants (Alimohammadi et al. 2013; Feng et al. 2017). However, few previous studies have performed on the function of these genes under drought stress in cherry. Recently, Xu et al. (2023) evidenced the role of ABA-related genes and transcription factors (PavWRKY and PavMYB), in response to drought of cherry rootstock.
On the other hand, Prunus is one of the genera belonging to the Rosaceae family with high diversity and economic significance. It is native to temperate regions of the Northern Hemisphere specially Europe and Asia (Mozaffarian 2002). While most commercial cultivars are sensitive to drought, wild genotypes grow well in water-limited areas and play an important role in the ecological environment. Research on molecular mechanisms underlying the drought tolerance of these plants is scarce, and we can almost say that wild species of cherries have not been investigated in relation to the molecular basis of their drought stress tolerance (Mozaffarian 2002). Although P. incana and P. microcarpa two wild species are resistant to drought, so far, no research has been conducted on their resistance to water scarcity (Nazari et al. 2012). Only recent evidence in in vitro assays has been described (Sevgin 2021). These wild species not only can be used as a source of new genes or alleles, but also have the potential to breeding of rootstocks for dwarfing, cold and drought-tolerant.
Iran is one of the countries of origin of Cerasus subgenus plants, and there has been no study on this subgenus about drought tolerance; in this study, we investigated the integrated morphological, physiological and transcriptional response of wild cherries to drought stress in order to provide a comprehensive analysis of adaptation in cultivated cherry (P. avium L.) and wild P. microcarpa Boiss and P. incana (Pall.) Batsch. subgenera Cerasus species under control and drought stress. The information obtained may provide new insights into the underlying molecular mechanisms of the response to drought stress in Prunus species.
Material and Methods
Plant Materials
In this study, different species were assayed including the cultivated P. avium L. [‘Avi-Ala’] and the wild species P. microcarpa Boiss [‘Mic-Kor’] and P. incana (Pall.) Batsch [‘Inc-Kho’]. One accession of P. mahaleb L. [‘Mah’] was also assayed as out group in the phylogeny analysis (Table 1; Fig. 1). Seeds from the different accessions collected in different parts of Iran were stratified and germinated. Seedlings were grown under irrigation in glasshouse with 48–55% relative humidity and day/night temperature ~ 30/18 °C. One-year-old seedlings were incorporated in this study. Thirty pots of each species were randomly selected and divided into two groups, one group was use for drought treatment, and the other was used as control. The drought treatment was applied by withholding water and stopping irrigation during 15 days, while the control treatment was continued irrigation (Fig. 2). This period of 15-day no watering used as drought treatments to compare control vs non-irrigated seedlings will allow the monitoring of the response to water stress of seedlings as described before by Centritto (2005) and ˇCerekovi´c et al. (2013) and Bnikkou et al. (2021).
Evaluation of Morphological and Physiological Parameters
Five biological replicates were assayed to evaluate each morphological and physiological parameter. Morphological parameters included leaf area (mm2), height (cm) and diameter (mm). In addition, all physiological measurements were performed after 0, 7 and 15 days at 09:00 to 11:00 during the process of drought stress assaying five replications per assayed accession. The net photosynthetic rate (Pn) was measured using a portable photosynthesis (LICOR 6400, LI-COR Inc., Lincoln, NB, USA). Chlorophyll index was measured using a Minolta SPAD-502 m (Karimpour et al. 2021). The SPAD-502 m is a non-destructive measuring device initially developed for the chlorophyll content of leaves widely used to optimize the timing and quantity of fertilizer to improve crop yield. Chlorophyll content is in general one indicator of plant health also affected by other physiological factors including growth regulators, photorespiration and oxidative stress (Ling et al. 2011). At the same time, plant stress meter was used to measure chlorophyll fluorescence. After a dark-adapted period (20 min) with dark leaf clip, the minimum fluorescence (F0), maximum fluorescence (Fm), variable fluorescence (Fv) and maximum photochemical efficiency (Fv/Fm) were measured (Turner 1988). Relative water content (RWC) was measured as per previously published method and calculated using the following equation: RWC (%) = [(FW − DW)/(TW − DW)] × 100.
DNA Extraction and SSR Analysis
DNA was isolated by CTAB method from several seedlings from Prunus avium, Prunus microcarpa and Prunus incana and Prunus mahaleb used as out group using a modified procedure of the described by Doyle and Doyle (1989). The genomic DNA was quantified at 260 nm, and its purity was measured at 260/280 nm absorbance ratio using a NanoDrop One Spectrophotometer (Thermo Fisher Scientific). Isolated DNA wax analysed using a set of 20 SSR codominant markers specific to Prunus species (Table 2). PCRs were performed in 15 µl mix containing approximately 5 ng of genomic DNA, 0.2 µM of each primer, 1 × Taq buffer (Biolabs), 1 mM MgCl2, 0.2 mM of dNTPs mix and 1 unit of Taq polymerase (Biolabs). The amplification program was carried out according to the following: an initial melting step at 94 °C for 4 min followed by 35 cycles (of 95 °C for 30 s, annealing temperature (53.8, 55 and 57 °C) for 30 s and 72 °C for 1 min) and then by a final elongation step at 72 °C for 7 min and hold at 10 °C. Amplified products were resolved by electrophoresis in TBE buffer using 3% MetaPhor agarose gel with a 1 kbp DNA ladder as a molecular standard. Polymorphic alleles were scored as present (1) or absent (0). The band scoring was analysed with the ImageJ gel analysis software (Wayne Rasband, National Institutes of Health, USA). The genetic diversity characterization was estimated by the number of heterozygote alleles per locus SSRs, and farther genetic information of the codominant SSR markers was determined by the observed heterozygosity (H) and by power of discrimination (PD) (Kloosterman et al. 1993). In addition, the mean character difference distances were calculated for all pairwise comparisons with the (ImageJ gel analysis software), which was used to construct UPGMA dendrograms (cluster analysis) (Nei and Li 1979) depicting the phonetic relationship among different ecotypes. Relative support for the branches in each dendrogram was assessed by UPGMA bootstrap analysis (2000 replicates). Finally, association studies of SSR polymorphisms were carried out with mixed linear model (MLM) considering both Q and K matrices as covariates in TASSEL software (Yu and Buckler 2006).
RNA Extraction and qRT-PCR Analysis
Total RNA was extracted from leaves of selected seedlings ‘Avi-Ala 12’ (sensitive), ‘Mic-Kor 3’ (tolerant) and ‘Inc-Kho’ (tolerant) by the method of Le Provost et al. (2007). The most important genes related to the drought response previously described in different Prunus species including PdDREB2c, BHLH71, PdP40, ATHB-12, ABI and AUX_IAA were analysed by qPCR (García et al. 2008; Li et al. 2012; Balint and Reynolds 2016; Yao et al. 2020; Liu et al. 2021). Several housekeeping reference genes were assayed using two as internal controls: RNA polymerase II (RPII) and ubiquitin 10 (UBQ10) (Tong et al. 2009). Specific primers for all genes were designed based on Prunus sequences using Primer3 software (Table 3). CDNA was synthetized using SSIII Reverse Transcriptase (Thermo Fisher Scientific). To investigate the expression pattern of candidate genes in samples after 15 days under drought stress compared to that in control samples, real-time qPCR experiments were executed with One-Step Plus Real-Time PCR system (Applied Biosystems) assaying three biological replicates and two technical replicates. Primers designed on almond (P. dulcis (Mill.) D.A. Webb) sequences were validated by the standard curve method. For all real-time qPCR reactions, a 10 μl mix was made including 5 μl Power SYBR® Green PCR Master Mix (Applied Biosystems), 20 ng of cDNA and 0.5 μl of each primer (5 μM). The experiments were employed in the following conditions: 95 °C for 10 min, 40 cycles of 95 °C for 15 s and 60 °C for 1 min. The melting temperature of these experiments was set to 60 ~ 95 °C and rising in 0.3 °C/s. Each biological sample was implemented in duplicate. RPII and actin were used as reference genes for data normalization, and the levels of relative expression were calculated by the method proposed by Pfaffl (2001). Data following a normal distribution were subjected to ANOVA single factor (p ≤ 0.05) to test for significant differences between treatments and genotypes in terms of gene expression.
Results
Evaluation of Morphological and Physiological Parameters
According to the results of vegetative trait evaluation, P. avium, P. microcarpa and P. incana control genotypes showed high diversity in terms of morphological leaf traits (Fig. 2). Interestingly, ‘Mic-Kor 2’ had significantly the smallest leaf area, diameter and the lowest height with short nodes among genotypes that can be interesting for breeders. The largest leaf area and the highest height belonged to the ‘Avi-Ala 11’, and the largest trunk diameter was found in the ‘Avi-Ala 24’ genotypes (Table 1). In terms of an overview of genotypes, from a morphological point of view, ‘Mic-Kor’ showed less leaf area, height and diameter in comparison to ‘Inc-Kho’ and mainly ‘Avi-Ala’. In addition, Mic-Kor genotypes tended to recumbent growth habit with more leaf serration than other genotypes (Fig. 2). For ‘Inc-Kho’ and ‘Mic-Kor’, the leaves have pubescence on the lower surface which was easily visible with the naked eye. Finally, ‘Inc-Kho’ on the contrary with ‘Mic-Kor’ has longer and darker leaves.
On the other hand, compared means showed that the physiological parameters differently affected by drought, especially under long-term water deficiency. After 15 days of drought stress, all physiological parameters in the genotypes were significantly reduced compared to controls (Fig. 3). But P. incana and P. microcarpa drought-tolerant genotypes showed higher photosynthetic stability in contract with the more drought susceptible P. avium genotypes. This effect was obviously in the appearance of P. avium genotypes as opposed to P. incana and P. microcarpa genotypes. ‘Avi-Ala 12’ showed the greatest reduction in photosynthesis rate (AN) and ‘Inc-Kho’ and ‘Mic-Kor 3’ the minimal. This is the highest photochemical efficiency (Fv/Fm) observed in ‘Avi-Ala 7’ and ‘Avi-Ala 2’. ‘Inc-Kho’ and ‘Mic-Kor 2’ also showed high photochemical efficiency versus Avi-Ala 1 and 3 that showed greatest reduction.
RWC is considered as an important criterion of plant water status. The leaf RWC decreased significantly in drought-stressed plants at 15 days of experiment in comparison with the control plants. But significant differences were observed between genotypes, so that ‘Avi-Ala 18’ reached a minimum value of RWC (47.17%) in front of ‘Inc-Kho’ that showed the maximum (71.48%) (Fig. 3).
Leaf Chl concentration was significantly affected by drought after 15 days of stress (Fig. 2). So ‘Inc-Kho’, ‘Mic-Kor 2’, ‘Mic-Kor 3’ and ‘Avi-Ala 2’ and ‘Avi-Ala 18’ showed the most leaf Chl concentration than the other genotype. ‘Avi-Ala 14’ showed the lowest leaf Chl concentration. The general aspects of the whole plants of studied genotypes from wild Cerasus subgenus species after the drought stress treatment including cultivated P. avium and wild P. microcarpa and P. incana corroborated this reduction Chl concentration in P. avium (Fig. 3).
Genomic Characterization
The evaluation of the 20 SSR markers in the 4 studied Prunus species generated a total of 86 alleles (Table 2). High level of polymorphism was detected among the studied species, and thus, they were useful for fingerprinting study in Cerasus subgenera germplasm (Fig. 4). The most of the amplified allele sizes ranged between 100 and 180 bp. Some of these alleles in different species were shared. The highest number of alleles (7) was observed at PceGA34, UDAp471, UDAp456 and UDP98-410 loci. The lowest number of alleles was obtained in the CPDCT044 and CPPCT023 loci (Table 2).
‘Inc-Kho’ within species showed the highest number of alleles with 10 alleles, while the least number of alleles was showed in Avi-Ala 1 with 4 alleles. Notably, ‘Inc-Kho’ was placed in a group with ‘Mah-Urm’. Within the species, the most polymorphic samples were those corresponding to ‘Inc-Kho’ and ‘Mic-Kor’ species which were polymorphic in all the SSRs and showed more polymorphisms than other species (Fig. 4).
According to the results, CPPCT-008 showed the lowest heterozygosity value (0.08) and UDAp471, UDAp456 and PACITA6 the highest (0.72). ‘Avi-Ala 22’ and ‘Avi-Ala 23’ genotypes were showed the highest and lowest heterozygosity, respectively. Conforming to genetic matrix distance, ‘Avi-Ala 11’ with ‘Avi-Ala 14’ and ‘Avi-Ala 16’ with ‘Inc-Kho’ were, respectively, the closest and the furthest genotypes (Fig. 4). However, no SSR markers showed a good degree of linkage with the drought response of the assayed genotypes and the evaluated gene expression.
Transcriptomic Analysis
The assayed genes related to drought response auxin-responsive protein IAA1-like (AUX_IAA), PdDREB2c, BHLH71, PdP40, ATHB-12 and ABI were analysed by qPCR (Fig. 5). AUX/IAA expression in drought-treated leaves decreased compared to control levels in all genotypes, significantly in P. incana after 15 days of treatment. DREB2C expression in sensitive genotype (P. avium ‘Avi-Ala 12’) had no significant difference with control but in the tolerant genotype (P. microcarpa ‘Mic-Kor 3’) has notably increased. P. incana had been meaningfully reduced in the treatment. AFP3 (ninja-family protein) levels in sensitive genotype (P. avium ‘Avi-Ala 12’) significantly increased compared to control. P40 (peroxidase 40) belongs to the peroxidase family that plays a key role in oxidative metabolism. Unlike Mic-Kor 3, P40 expression levels in Inc-Kho significantly decreased under drought stress; ATHB-12 (Arabidopsis thaliana homeobox-leucine zipper protein) expression did not significantly varied with respect to control in any of the genotypes studied. ATHB12 expression decreased non-significantly in all genotypes. BHLH71 levels in sensitive genotypes (P. avium ‘Avi-Ala 12’) significantly decreased compared to the control. Its expression increased in tolerant species (P. incana ‘Inc-Kho’) towards the control (Fig. 5).
Discussion
Evaluation of Morphological and Physiological Parameters
Iran is one of the origins of cultivated cherries (P. avium) and has a significant share of their production in the world (258,691 hg/ha). Due to climatic conditions and the strong germplasm of these plants in Iran, most rootstocks used in Iran originated from Mazard, with significant difference in resistance to abiotic and biotic stresses versus commercial varieties. Meanwhile, P. incana and P. microcarpa are unknown to some extent, and no study has yet been conducted on the resistance of these species to environmental stresses as the valuable source of genes for future breeding programs (Nazari et al. 2012). To face environmental stresses and climate change, native genotypes with interesting physiological traits need to be investigated and protected for development of improved rootstock varieties. In this regard, we appraised various Iranian cherries in their native habitats for resistance to drought stress. Morphological data in this study showed that P. incana and P. microcarpa can be used in breeding programs as dwarf and drought-resistant rootstocks in sweet and sour cherry in agreement with the recent in vitro results published by Sevgin (2021). P. incana and P. microcarpa species are able to grow in rocky and dry soils having small leaves with many pubescence which indicate their resistance to drought conditions previously reported by Mozaffarian (2002).
Plants can successfully use complex physiological and molecular strategies to cope with environmental pressures. In this study, physiological measurements confirmed that plants are specifically affected by water stress and that plants provide physiological responses to deal with it. Photosynthetic processes, including Pn and chlorophyll, decreased drastically as the drought continued; similar results on Prunus response to water stress have been reported by several studies (Jiménez et al. 2013; Escobar-Gutiérrez et al. 1998). Photosynthesis is the most important plant metabolism for energy supply. It can be said that stomatal conductance is the most important factor affecting photosynthesis than during the drought period; its reduction can be ascribed to variability in soil moisture. With decreasing Gs and chlorophyll, photosynthesis decreases during drought stress in plants, according to the findings of other studies (Wang et al. 2015; Monakhova and Chernyadev 2002).
The effect of drought stress on photosynthesis could be direct by photosynthetic metabolism and Calvin cycle enzyme activity or indirect by increased oxidative stress. In wild almonds (P. mongolica Maxim.), low expression of transcripts related to photosynthetic routines resulted in reduced efficiency of photosystem I, photosystem II, light-harvesting chlorophyll protein complex and ultimately reduced photosynthesis (Wang et al. 2015; Fahad et al. 2017). Damage to photosynthetic systems and reduced leaf chlorophyll can also be due to reduced CO2 uptake due to drought stress. RWC was found at the lowest level in seedlings after 15 days of water stress. Leaf RWC indicates metabolic activity in tissues that its reduction due to water stress can be attributed to the unavailability of water in the soil and/or the inability of root systems to compensate for water lost through transpiration (Martínez-García et al. 2020; García et al. 2007; Shalhevet 1993; Gadallah 2000). In all genotypes, a decrease in Fv/Fm indicates photochemical damage in photosystem II and reduces absorption, which can be caused by a disturbance in the photosynthetic electron transfer system or damage to the thylakoid membranes (García-Sánchez et al. 2007). The reduction in the number of chloroplasts and their deformation due to drought in tobacco has been reported by Yang et al. (2017).
In Japanese plum (Prunus salicina L.), the decrease in shoot growth was also more pronounced in more drought-tolerant cultivars in comparison with the less tolerant. The application of moderate water stress caused an intense drop in the midday water potential and RWC in stressed trees of assayed cultivars (Hajlaouli et al. 2022). In addition, regulated deficit irrigation (RDI) caused a reduction in gas exchange parameters (photosynthetic assimilation and stomatal conductance) in the three assayed plum cultivars (Hajlaouli et al. 2022; Hamdani et al. 2023) in agreement with the drought response found in our tolerant and sensitive wild and cultivated cherry species.
Genomic Characterization
SSR markers have been selected as important markers for studies of genetic diversity in sweet cherry (Stanys et al. 2012). In this study, SSR was applied to evaluate the genetic variability and relationships of selected genotypes.
The observed diversity of germplasm was good and can greatly aid in breeding programs and rootstock selection. Geographical diversity and possibly open pollination of these genotypes have led to high genetic diversity in wild species of this subgenus. P. incana placed in a group and P. microcarpa genotypes were grouped separately with P. avium genotypes which were probably due to pollination between them. Contrary to assumption, all genotypes of P. avium were not included in a same cluster. This could be because of the different locations of the collection and probably cross-pollination. In addition, in most cases, tested species indicated 2–4 alleles in each example according species which is consistent with findings of Khadivi-Khub et al. (2014).
This close genetic relationship allows the use of this wild species in the breeding of the cultivated P. avium species as has been described in almond (P. dulcis) and plum (P. salicina) (Paudel et al. 2019; Hajlaouli et al. 2022).
Regarding the linkage of SSR markers with the drought response of the assayed genotypes and the evaluated gene expression, the lack of results indicated the need to increase number of DNA markers in these association studies with the use of the most abundant single-nucleotide polymorphism (SNP) markers (Salazar et al. 2017).
Transcriptomic Analysis
On the other hand, the results of transcriptomic analysis were remarkable. The reduction of IAA (involved in auxin signal transduction) was more pronounced in ‘Inc-Kho’ under drought stress. One of the plants’ responses to drought is to reduce the synthesis of growth-promoting hormones such as auxin and cytokinin. By reducing these hormones, cell division and enlargement are reduced, and thus, the plant survives by diminishing growth in drought conditions (Wang et al. 2015). The AUX/IAA family has various functions in regulating plant growth and development such as root development, shoot growth and fruit ripening (Luo et al. 2018). It seemed that the inhibition of response to the auxin hormone IAA (iIndole-3-acetic Acid) is an adaptive survival strategy to reduce lateral root emergence as their maintenance requires metabolic investment that may slow the axial root elongation in deep soil. This is important for the acquisition of water which availability is higher in deep soils. During drought stress, the auxin receptor TIR1 (transport inhibitor response1) levels are kept low by the upregulated miR393 resulting in a decrease auxin response factors (ARFs) mediated from AUX/IAA heterodimerization, which, as growth slows, may increase the plant’s tolerance to stress (Singh et al. 2017).
DREB2C expression in sensitive species (‘Avi-Ala 12’) had no significant difference with control but in the tolerant genotype (‘Mic-Kor 3’) has notably increased. ‘Inc-Kho’ had been meaningfully reduced in the treatment. This could be because of the different mechanisms of species to confronting with drought stress taking in account that P. incana and P. microcarpa genotypes showed higher photosynthetic stability in contract with P. avium genotypes. DREB family plays important role in adjusting plant responses to abiotic stresses. Lee et al. (2010) showed that DREB2C interacts with ABF2, a bZIP protein regulating abscisic acid-responsive gene expression, and its overexpression affects abscisic acid sensitivity. AFP3 (ninja-family protein/ABI five-binding protein 3) levels in sensitive species (‘Avi-Ala 12’) significantly increased compared to control. AFP3 regulates stress response by the downregulation of ABA responses and stress responses (Zhao et al. 2016). AFPs are caused by ABA and dehydration stresses in seedlings. But their induction time is different. In addition, they negatively regulate jasmonic acid signalling as a part of repressor complex. Previous studies have shown different roles of some of them, such as seedling growth, germination control, carbohydrate metabolism, root growth and flowering (Huang and Wu 2007; Lopez-Molina et al. 2003). Recently, in agreement with our results, Xu et al. (2023) also evidenced the role of ABA-related genes in response to drought of cherry rootstock.
PdP40 belongs to the peroxidase family that plays a key role in oxidative metabolism. Unlike ‘Inc-Kho’ and ‘Avi-Ala 12’, its levels in ‘Mic-Kor 3’ had been significantly increased under drought stress that it might be due to differences in drought management mechanisms in genotypes. Plants have different enzymatic and non-enzymatic antioxidant systems to respond to environmental stresses that control the overproduction of reactive oxygen species under stress (Mittler 2002; Moller 2001). Drought stress like other stresses induces production reactive oxygen species (ROS) in plants. Some drought-resistant plant produces secondary metabolites such as antioxidant enzymes under stress conditions that scavenge reactive oxygen species (ROS) (Foyer et al. 1997; Jogawat et al. 2021). Recently, antioxidant enzyme genes (PavGST and PavPOD) were identified as differentially expressed by using RNA-Seq in relation to drought resistance in cherry rootstock (Xu et al. 2023), highlighting the role of antioxidant enzymes under stress conditions that scavenge ROS (Fig. 6).
BHLH71 levels in sensitive species (‘Avi-Ala 12’) significantly decreased compared to control. Its expression had been increase in tolerant species (‘Inc-Kho’) towards control. Filiz and Kurt (2021) reported that 72% of the BHLH family found under drought stress in potatoes were upregulated that some of them are thought to be involved in root hair development under drought stress. According to studies, BHLH71 may be involved in the differentiation of stomatal guard cells (Nadeau 2009). In apple, most of the BHLH TFs are crucial regulators of drought stress by involving in ABA signalling pathways (Mao et al. 2017). Xu et al. (2023) also evidenced the role of transcription factors (PavWRKY and PavMYB), in response to drought of cherry rootstock.
Finally, ATHB-12 expression had not been significantly different towards control in all three genotypes. There was a non-significant decrease in ATHB12 expression in all three genotypes during drought stress. In Arabidopsis thaliana, leaf growth was promoted by ATHB12 mainly along the cell expansion stage, and suppression of its expression leads to reduced leaf growth and development (Hur et al. 2019). In our study, the reduced expression of ATHB12 in drought treatments especially in ‘Inc-Kho’ was due probably to the interaction with ABA signalling. It has been shown that there is an interaction between ABA signalling and ATHB12 and HTHB7, due to their downregulation by ABI (Olsson et al. 2004). Valdés et al. (2012) also reported that ATHB12 transcription factor which is an ABA-induced gene was induced by drought conditions and ABA signal in Arabidopsis. According to studies, HD-Zip family l is involved in regulating growth and stress responses. The overexpression of the Oshox22 increases the ABA content and reduces the drought tolerance in plants (Zhang et al. 2012). However, no studies have been performed on the ATHB12 under drought stress in wild cherry cultivars.
Our study starts up the evaluation of drought tolerance of P. incana and P. microcarpa for the first time and put out a significant portion to the understanding of how their response to drought stress which may help to illuminate the molecular mechanisms was associated with the drought response of Prunus. With the base of a different phenotype response to drought, P. microcarpa showed less leaf area, height and diameter in comparison to P. incana; a differential gene expression response was observed (Fig. 6). In order to better understand the relationships between genes during drought stress, biochemical analysis is also required that we will be doing in the near future. The future implementation of new omics approaches across time points will help the dissection of this response in cultivated and wild Prunus L. subgenus Cerasus, determining when gene express or switch off in response to drought.
Conclusions
P. incana and P. microcarpa are widely established in mountains of Iran and show extreme tolerance to drought. No research has been conducted to studies the genomic resources of these species and the molecular mechanisms underlying their drought tolerance. Therefore, to check the mechanisms that authorize these plants to maintain growth in extremely dry environments, the response of these wild seedlings to drought stress was analysed using physiological and molecular approaches. After 15 days of drought stress, although the all physiological parameters in the genotypes were significantly reduced compared to controls, P. incana and P. microcarpa genotypes showed higher photosynthetic stability in contrast with P. avium genotypes. Due to the changes in the levels of hormones (such as auxins and abscisic acid) and their roles in closing the stomata and reducing growth for promoting plant survival in the arid environments, measuring their level is recommended in this study. Expression analysis of candidate genes related to drought stress in studied Prunus species showed that depending on the gene, the expression pattern can change between genotypes in response to drought stress. In this study, our hypothesis of high tolerance of P. incana and P. microcarpa to drought stress in comparison with the cultivated sweet cherry species (P. avium) was confirmed, and this study showed an overview of the function of the studied genes in these wild genotypes under drought stress. However, the genotypes showed opposite trends and different expression in the studied genes. So that contrary to our expectations, wild species showed low expression in some of the studied genes, including ATHB12.
Data Archiving
No data archiving.
Data Availability
Data available on request from the authors. The data that support the findings of this study are available from the corresponding author, upon reasonable request.
References
Alimohammadi A, Shiran B, Martínez-Gómez P, Ebrahimie E (2013) Identification of water-deficit resistance genes in wild almond Prunus scoparia using cDNA-AFLP. Sci Hort 159:19–28
Aranzana MJ, García-Mas J, Carbó J, Arús P (2002) Development and variability analysis of microsatellite markers in peach. Plant Breed 121:87–92
Balint G, Reynolds AG (2016) Abscisic acid and its catabolites in leaves and berries of chardonnay are affected by water status. Plant Growth Reg J 35:1126–1140
Bnikkou S, Laknifli A, Majourhat K, Jalili S, Hernández M-Gómez P, Martínez-García PJ (2021) Molecular characterization using SSR markers and biochemical analysis of Moroccan and Spanish argan [Argania spinosa (L.) Skeels] ecotypes under water stress and rewatering. Biologia 76:799–808
Bianchi VJ, Rubio M, Trainotti L, Verde I, Bonghi C, Martínez-Gómez P (2015) Prunus transcription factors: breeding perspectives. Front Plant Sci 6:443
Centritto M (2005) Photosynthetic limitations and carbon partitioning in cherry in response to water deficit and elevated CO2. Agri Ecosys Enviro 106:233–242
Čereković N, Pagter M, Kristensen HL, Pedersen HL, Brennan R, Petersen KK (2013) Effects of drought stress during flowering of two pot-grown blackcurrant (Ribes nigrum L.) cultivars. Sci Hort 162:365–373
Chen H, Hwang JE, Lim CJ, Kim DY, Lee SY, Lim CO (2010) Arabidopsis DREB2C functions as a transcriptional activator of HsfA3 during the heat stress response. Biochem Biophys Res Comm 401:238–244
Cipriani G, Lot G, Huang WG, Marrazzo MT, Peterlunger E, Testolin R (1999) AC/GT and AG/CT microsatellite repeats in peach [Prunus persica (L) Batsch]: isolation, characterisation and cross-species amplification in Prunus. Theor Appl Genet 99:65–72
Dirlewanger E, Cosson P, Tavaud M, Aranzana MJ, Poizat C, Zanetto A, Laigret F (2002) Development of microsatellite markers in peach [Prunus persica (L.) Batsch] and their use in genetic diversity analysis in peach and sweet cherry (Prunus avium L.). Theor Appl Genet 105:127–138
Downey SL, Iezzoni AF (2000) Polymorphic DNA markers in black cherry (Prunus serotina) are identified using sequences from sweet cherry, peach, and sour cherry. J Am Soc Hort 125:76–80
Doyle JJ, Doyle JL (1989) A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem Bull 19:11–15
Escobar-Gutiérrez AJ, Zipperlin B, Carbonne F, Moing A, Gaudillére JP (1998) Photosynthesis, carbon partitioning and metabolite content during drought stress in peach seedlings. Funct Plant Biol 25:197–205
Fahad S, Bajwa AA, Nazir U, Anjum SA, Farooq A (2017) Crop production under drought and heat stress. Plant Resp Manag Opt 8:1–16
Feng Y, Liang C, Li B, Wan T, Liu T, Cai Y (2017) Differential expression profiles and pathways of genes in drought resistant tree species Prunus mahaleb roots and leaves in response to drought stress. Sci Hort 226:75–84
Filiz E, Kurt F (2021) Expression and co-expression analyses of WRKY, MYB, bHLH and bZIP transcription factor genes in potato (Solanum tuberosum) under abiotic stress conditions: RNA-seq data analysis. Potato Res 2:1–21
Foyer CH, López-Delgado H, Dat JF, Scott IM (1997) Hydrogen peroxide- and glutathione-associated mechanisms of acclimatory stress tolerance and signalling. Physiol Plant 100:241–254
Gadallah MAA (2000) Effects of indole-3-acetic acid and zinc on the growth, osmotic potential and soluble carbon and nitrogen components of soybean plants growing under water deficit. Arid Enviro J 44:451–467
García AL, Marcelis L, García-Sánchez F, Nicolas N, Martínez V (2007) Moderate water stress affects tomato leaf water relations in dependence on the nitrogen supply. Biol Planta 51:707–712
García ME, Lynch T, Peeters J, Snowden C, Finkelstein R (2008) A small plant-specific protein family of ABI five binding proteins (AFPs) regulates stress response in germinating Arabidopsis seeds and seedlings. Plant Mol Biol 67:643–658
García-Sánchez F, Versluesrtsen JP, Gimeno V, Botía P, Perez-Perez JG (2007) Responses to flooding and drought stress by two citrus rootstock seedlings with different water-use efficiency. Physiol Planta 130:532–542
Hajlaoui H, Maatallah S, Guizani M, Boughattas NEH, Guesmi A, Ennajeh M, Dabbou S, Lopez-Lauri F (2022) Effect of regulated deficit irrigation on agronomic parameters of three plum cultivars (Prunus salicina L.) under semi-arid climate conditions. Plants 22:1545
Hamdani A, Hssaini L, Bouda S, Charafi J, Boutagayout A, Razouk R (2023) Agronomic and physiological response of various Japanese plums (Prunus salicina L.) to severe water stress. Vegetos. https://doi.org/10.1007/s42535-022-00544-7
Huang MD, Wu WL (2007) Overexpression of TMAC2, a novel negative regulator of abscisic acid and salinity responses, has pleiotropic effects in Arabidopsis thaliana. Plant Mol Biol 63:557–569
Hur YS, Kim J, Kim S, Son O, Kim WY, Kim GT, Ohme-Takagi M, Cheon CI (2019) Identification of TCP13 as an upstream regulator of ATHB12 during leaf development. Genes 10(9):644. https://doi.org/10.3390/genes10090644
Jiménez S, Dridi J, Gutiérrez D, Moret D, Irigoyen JJ, Moreno MA, Gogorcena Y (2013) Physiological, biochemical and molecular responses in four Prunus rootstocks submitted to drought stress. Tree Physiol 33:1061–1075
Jogawat A, Yadav B, Lakra N, Singh AK, Narayan OP (2021) Crosstalk between phytohormones and secondary metabolites in the drought stress tolerance of crop plants: a review. Physiol Planta 172:1106–1132
Karimpour S, Davarynejad GH, Zaki-Aghl M, Safarnejad MR, Martínez-Gómez P, Rubio M (2021) Rapid assessment of sanitary and physiological state of thermotherapy-treated apple shoots by chlorophyll content evaluation. Eur J Hortic Sci 86:205–211
Khadivi-Khub A, Zamani Z, Fattahi R, Wünsch A (2014) Genetic variation in wild Prunus L. subgen. Cerasus germplasm from Iran characterized by nuclear and chloroplast SSR markers. Trees Struc Fun 28:471–485
Kloosterman AD, Budowle B, Daselaar P (1993) PCR- amplification and detection of the human DIS80 VNTR locus. Amplification conditions and application in forensic analysis. Integrat J Legal Medec 105:257–264
Lau SE, Hamdan MF, Pua TL, Saidi NB, Tan BC (2021) Plant nitric oxide signaling under drought stress. Plants 10:360
Le Provost G, Herrera R, Paiva JA, Chaumeil P, Salin F, Plomion CA (2007) Micromethod for high throughput RNA extraction in forest trees. Bio Res 40:291–297
Lee SJ, Kang JY, Park HJ, Kim MD, Bae MS, Choi HI, Kim SY (2010) DREB2C interacts with ABF2, a bZIP protein regulating abscisic acid-responsive gene expression, and its overexpression affects abscisic acid sensitivity. Plant Physiol 153:716–727
Lesk C, Rowhani P, Ramankutty N (2016) Influence of extreme weather disasters on global crop production. Nature 529:84–87
Li Q, Li P, Sun L, Wang Y, Ji K, Sun Y, Leng P (2012) Expression analysis of β-glucosidase genes that regulate abscisic acid homeostasis during watermelon (Citrullus lanatus) development and under stress conditions. Plant Physiol J 169:78–85
Ling Q, Huang W, Jarvis P (2011) Use of a SPAD-502 meter to measure leaf chlorophyll concentration in Arabidopsis thaliana. Photosynth Res 107:209–214
Liu C, Wang H, Zhang X, Ma F, Guo T, Li C (2021) Activation of the ABA signal pathway mediated by GABA improves the drought resistance of apple seedlings. Int J Mol Sci 22:12676
Lobell DB, Schlenker W, Costa-Roberts J (2011) Climate trends and global crop production since 1980. Science 333:616–620
Lopes MS, Sefc KM, Laimer M, Machado ADC (2002) Characterization of microsatellite loci in apricot. Mol Ecol Notes 2:24–26
Lopez-Molina L, Mongrand S, Kinoshita N, Chua NH (2003) AFP is a novel negative regulator of ABA signaling that promotes ABI5 protein degradation. Genes Dev 17:410–418
Luo J, Zhou JJ, Zhang JZ (2018) Aux/IAA gene family in plants: molecular structure, regulation, and function. Int J Mol Sci 19:259
Mao K, Dong Q, Li C, Liu C, Ma F (2017) Genome wide identification and characterization of apple bHLH transcription factors and expression analysis in response to drought and salt stress. Front Plant Sci 8:480
Martínez-García PJ, Hartung J, Pérez de los Cobos F, Martínez-García P, Jalil S, Sánchez-Roldán JM, Rubio M, Dicenta F, Martínez-Gómez P (2020) Temporal response to drought stress in several Prunus rootstocks and wild species. Agronomy 10:1383
Messina R, Lain O, Marrazzo MT, Cipriani G, Testolin R (2004) New set of microsatellite loci isolated in apricot. Mol Eco Notes 4:432–434
Mittler R (2002) Oxidative stress, antioxidants and stress tolerance. Trends Plant Sci 7:405–410
Mnejja M, García-Mas J, Howad W, Badenes ML, Arús P (2004) Simple-sequence repeat (SSR) markers of Japanese plum (Prunus salicina Lindl.) are highly polymorphic and transferable to peach and almond. Mol Ecol Notes 4:163–166
Møller IM (2001) Plant mitochondria and oxidative stress: electron transport, NADPH turnover, and metabolism of reactive oxygen species. Ann Review Plant Biol 52:561–591
Monakhova OF, Chernyadev II (2002) Protective role of kartolin-4 in wheat plants exposed to soil drought. App Biochem Microbiol 38:373–380
Mozaffarian V (2002) Studies on the flora of Iran, new species and new records. Pak J Bot 34:391–396
Nadeau JA (2009) Stomatal development: new signals and fate determinants. Curr Opin Plant Biol 12:29–35
Nazari SA, Zamani Z, Fatahi MR, Sofla HS (2012) Morphological characterization of Prunus incana Pall. by multivariate analysis. Plant Syst Evol 298:29–35
Nei M, Li WH (1979) Mathematical model for studying genetic variation in terms of restriction. Proc Nat Acad Sci USA 76:5269–5273
Olsson A, Engström P, Söderman E (2004) The homeobox genes ATHB12 and ATHB7 encode potential regulators of growth in response to water deficit in Arabidopsis. Plant Mol Biol 55:663–677
Paudel I, Gerbi H, Wagner Y, Zisovich A, Sapir G, Brumfeld V, Klein T (2019) Drought tolerance of wild versus cultivated tree species of almond and plum in the field. Tree Physiol 40:454–466
Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT–PCR. Nucleic Acids Res 29:e45
Pirasteh-Anosheh H, Saed-Moucheshi A, Pakniyat H, Pessarakli M (2016) Stomatal responses to drought stress. In: Ahmad P (ed) Water stress and crop plants: a sustainable approach, 1st edn. John Wiley & Sons, Hoboken, NJ, USA, pp 24–40
Rodríguez-Gacio MDC, Matilla-Vázquez MA, Matilla AJ (2009) Seed dormancy and ABA signaling: the breakthrough goes on. Plant Signal Behav 4:1035–1048
Roychoudhury A, Paul S, Basu S (2013) Cross-talk between abscisic acid-dependent and abscisic acid-independent pathways during abiotic stress. Plant Cell Reprod 32:985–1006
Salazar JA, Pacheco I, Shinya P, Zapata P, Silva C, Ruiz D, Martínez-Gómez P, Infante R (2017) Genotyping by sequencing for SNP-based linkage analysis and identification of QTLs linked to fruit quality traits in Japanese plum (Prunus salicina Lindl.). Front Plant Sci 8:476
Sevgin N (2021) Drought stress responses of Prunus microcarpa C. A. Mey. subsp. tortusa rootstocks under in vitro conditions. Legume Res 44:LR–589
Shalhevet J (1993) Plants under salt and water stress. Plant Adapt Enviro Stress 1:133–154
Shinozaki K, Yamaguchi-Shinozaki K (2007) Gene networks involved in drought stress response and tolerance. J Exp Bot 58:221–227
Singh R, Parihar P, Singh S, Singh MPVB, Singh VP, Prasad SM (2017) Micro RNAs and nitric oxide cross talk in stress tolerance in plants. Plant Growth Regul 83:199–205
Stanys V, Frercks B, Siksnianiene JB, Stepulaitiene I, Gelvonauskiene D, Staniene G, Bobinas C (2012) Identification of sweet cherry (Prunus avium L.)cultivars using AFLP and SSR markers. Zemdirbyste 99:437–444
Teribia N, Tijero V, Munné-Bosch S (2016) Linking hormonal profiles with variations in sugar and anthocyanin contents during the natural development and ripening of sweet cherries. New Biotech 33:824–833
Testolin R, Marrazzo T, Cipriani G, Quarta R, Verde I, Dettori MT, Sansavini S (2000) Microsatellite DNA in peach (Prunus persica L. Batsch) and its use in fingerprinting and testing the genetic origin of cultivars. Genome 43:512–520
Tong Z, Gao Z, Wang F, Zhou J, Zang Z (2009) Selection of reliable reference genes for gene expression studies in peach using real-time PCR. BMC Mol Biol 10:71
Turner NC (1988) Measurement of plant water status by the pressure chamber technique. Irrig Sci 9:289–308
Valdés AE, Övernäs E, Johansson H, Rada-Iglesias A, Engström P (2012) The homeodomain-leucine zipper (HD-Zip) class I transcription factors ATHB7 and ATHB12 modulate abscisic acid signalling by regulating protein phosphatase 2C and abscisic acid receptor gene activities. Plant Mol Biol 80:405–418
Wang J, Zheng R, Bai S, Gao X, Liu M, Yan W (2015) Mongolian almond (Prunus mongolica Maxim): the morpho-physiological, biochemical and transcriptomic response to drought stress. PLoS ONE 10:e012442
Xu Y, Wang Y, Xu J, Zhengxin L, Manzoor MA, Mao J, Zhang X, Liu R, Whiting MD, Jiu S, Zhang C (2023) Strigolactone and salicylic acid regulate the expression of multiple stress-related genes and enhance the drought resistance of cherry rootstocks. Sci Hort 313:111827
Yang H, Zhao L, Zhao S, Wang J, Shi H (2017) Biochemical and transcriptomic analyses of drought stress responses of LY1306 tobacco strain. Sci Rep 246:1–10
Yao T, Zhang J, Xie M, Yuan G, Tschaplinski TJ, Muchero W, Chen JG (2020) Transcriptional regulation of drought response in Arabidopsis and woody plants. Front Plant Sci 8:11
Yordanov I, Velikova V, Tsonev T (2000) Plant responses to drought, acclimation, and stress tolerance. Photosynthetica 38:171–186
Yu JM, Buckler ES (2006) Genetic association mapping and genome organization of maize. Curr Opin Biotech 17:155–160
Zhang S, Haider I, Kohlen W, Jiang L, Bouwmeester H, Meijer AH, Ouwerkerk PB (2012) Function of the HD–Zip I gene Oshox22 in ABA-mediated drought and salt tolerances in rice. Plant Mol Biol 80:571–585
Zhao Y, Chan Z, Gao J, Xing L, Cao M, Yu C, Zhu JK (2016) ABA receptor PYL9 promotes drought resistance and leaf senescence. Proc Natl Acad Sci USA 113:1949–1954
Acknowledgements
The authors do appreciate the Iran National Science Foundation (INSF) and CEBAS-CSIC for all the financial support and valuable contributions of each members of the CEBAS-CSIC in this study.
Funding
Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. This study has been supported by the Iran National Science Foundation (INSF) and Grant Nº 19879/GERM/15 of the Seneca Foundation of the Region of Murcia and the Apricot Breeding project of the Spanish Ministry of Economy and Competiveness and the project ‘Selection and characterization of drought-resistant almond cultivars from the Mediterranean basin with high nutraceutical values’ (Nut4Drought) of the ARIMNET-2 European Program.
Author information
Authors and Affiliations
Contributions
SJ and ASP did the qPCR experiments. SJ, JAS and PJMG did the SSR experiment. KA and PMG designed the experiments. SJ, KA, NB and PMG wrote the paper.
Corresponding authors
Ethics declarations
Competing Interests
The authors declare no competing interests.
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Key Message
Our study starts up for the first time the evaluation of drought tolerance of wild cherry species Prunus incana (Pall.) Batsch. and P. microcarpa Boiss in comparison with the cultivated one (P. avium L.) to the understanding of how they respond to drought stress which may help to illuminate the molecular mechanisms associated with the drought response of Prunus.
Rights and permissions
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/.
About this article
Cite this article
Jalili, S., Arzani, K., Prudencio, A.S. et al. Integrated Morphological, Physiological and Molecular Analysis of the Drought Response in Cultivated and Wild Prunus L. Subgenera Cerasus Species. Plant Mol Biol Rep 41, 440–453 (2023). https://doi.org/10.1007/s11105-023-01379-5
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11105-023-01379-5