Integrated Morphological, Physiological and Molecular Analysis of the Drought Response in Cultivated and Wild Prunus L. Subgenera Cerasus Species

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.


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 droughtresponsive 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.

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.Oneyear-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  1) 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 (mm 2 ), 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% Meta-Phor 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 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).
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.

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 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).

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, lightharvesting 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 CO 2 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  1) based on UPGM original tree analysis of SSR polymorphisms 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 droughttolerant 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 Fig. 5 qPCR analysis of candidate genes in genotypes of 'Avi-Ala 12' (sensitive) and 'Mic-Kor 3' and 'Inc-Kho' (tolerant) after 15 days under drought stress compared to control (see codes in Table 1).Three biological replicates and two technical replicates were assayed.Standard deviations are indicated with vertical bars 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 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.

Fig. 2
Fig. 2 Overview of the assayed whole plants and detail of leaves showing the studied morphological traits (leaf area, height and diameter) of the studied genotypes from cultivated and wild Cerasus subgenus species in control conditions in pots

Fig. 3
Fig.3Photosynthesis rate (An), maximum photochemical efficiency (Fv/ Fm), relative water content (RWC) and SPAD in control and droughtstressed of studied P. avium accessions and the related species (P.micro-

Fig. 4 A
Fig. 4 A Methaphor ® agarose gels showing SSR polymorphism of the assayed UDAp-412 and PACITA6 markers in the cultivated and wild cherry samples analysed.M, molecular ladder assayed 1 kb DNA ladder.B Dendrogram of the assayed accessions of Prunus

Fig. 6
Fig. 6 Schematic representation of the drought tolerance response at biochemical and molecular level in wild cherry species Prunus microcarpa and P. incana

Table 2
SSR markers assayed, number of alleles detected, observed heterozygosity and power of discrimination (PD)

Table 3
Specific/degenerate primers used for amplification of genes related to drought response from the assayed cultivated and wild cherry species.RNA polymerase II (RPII) and Ubiquitin 10 (UBQ10) have been assayed as housekeeping reference genesGeneOligonucleotides