Chinese Cherry (Cerasus pseudocerasus Lindl.) ARF7 Participates in Root Development and Responds to Drought and Low Phosphorus

Abstract

In this paper, an auxin-responsive transcription factor, CpARF7, was isolated from the roots of Chinese cherry (Cerasus pseudocerasus Lindl. Cv. “Manao Hong”). CpARF7 is highly homologous to AtARF7 or AtARF19 in Arabidopsis, and PavARF1 or PavARF14 in sweet cherry. However, in the phenotype of transgenic tomatoes, the root morphology changed, the main root elongated, and the lateral root increased. Both drought treatment and low-phosphorus conditions can elongate the roots of transgenic tomatoes. In addition, the drought resistance and low-phosphorus tolerance of the transgenic lines are improved, and the POD, SOD, and CAT activities under drought and low-phosphorus environments are increased. There is an effect on the tomato somatotropin suppressor gene, SlIAAs, in which SlIAA1/14/19/29 are up-regulated and SlIAA2/11/12/16 are down-regulated. These results indicate that CpARF7 plays an essential regulatory role in root formation and abiotic stress response, and deepens the understanding of auxin-responsive genes in root growth and abiotic stress.

1. Introduction

The Chinese cherry (Cerasus pseudocerasus Lindl.) is a perennial fruit tree, and belongs to the Rosaceae family, which is widely distributed in the north and south of China [1]. Chinese cherries have bright colors and an excellent taste, and have been widely promoted and cultivated in recent years. In Guizhou Province (a province in southwest China), cherries are grown on a large scale as an economic fruit tree, which has brought a certain increase in local economic benefits. After years of cultivation, a cherry variety suitable for growing in Guizhou was discovered, namely the “Manao Hong” cherry. However, due to its location in the plateau region, defined by poor soil and a chronic lack of sunlight, the increase in cherry production has not been significant.

The foundation for plants to be fixed in the soil is the root system. The roots play an essential role in water uptake and material transportation, environmental communication, and nutrient absorption. The formation of roots is the basic condition for the normal development of plants [2]. Auxin is of great significance, participating in plant root development and regulating plant root structure, which is often reflected in auxin synthesis, transport, and auxin signal transduction pathways [3,4]. Auxin response factors (ARFs) are a type of gene family with many members, and they are a type of transcription factor widely found in land-based plants [5]. The ARF contains three conserved domains, which are divided into transcriptional activators and transcriptional repressors according to the difference in amino acid richness in the middle region [5]. When there is a high auxin level in the plant, auxin/indole 3-acetic acid (AUX/IAA) is bound by the transport inhibitor response 1/auxin signaling F-box protein (TIR1/AFB), and the ARF recognizes and binds to a sequence called the auxin response element (TGTCTC) to initiate gene transcription; when the auxin level is low, the combination of AUX/IAA and the ARF prevents the ARF from binding to the target gene promoter and achieves the purpose of inhibiting gene transcription [5,6]. There are 23 ARF members in Arabidopsis thaliana, 19 of which have complete structures, and only 5 have transcriptional activators [6]. After years of research, the functions of these ARFs have been initially confirmed, and most of them involve plant growth and development [7].

The importance of ARFs in mediating auxin-related growth and development has been discovered by both reverse and forward genetics approaches in diverse plant species. In recent years, all ARF members in plant species such as rice [8], maize [9], sweet orange [10], peaches [11] and sweet cherries [12] have been identified. These members are widely involved in regulating the life processes of flower formation, leaf morphology, fruit development, root system formation, and seed development [5]. ARF3 plays a function in gynoecium patterning and leaf polarity specification. AtARF3 plays a significant regulatory role in the decisiveness of floral meristems, and has a complex dynamic expression pattern in the primordium of floral organs [13]. SlARF2 in tomatoes has a higher transcription level in the stamen, ovary, sepal, and petal, and mediates auxin and ethylene to regulate lateral root formation and the senescence of floral organs [14]. In rice, OsARF19 is highly expressed in the cortex and root primordium; it interacts with IAA13 to regulate the number of lateral roots, which is of great significance for the auxin signal to regulate the development of rice roots [15]. ARF7 and ARF19 play important roles in auxin-mediated plant growth and development in Arabidopsis thaliana, and their functions overlap, and mutant arf7 and arf19 have strong auxin-related phenotypic changes [16,17].

In the present research, the transcriptome sequencing of the root system of “Manao Hong” after biochar treatment revealed that CpARF7 has a significant expression. Based on the significant role of ARFs in plants, CpARF7 was cloned, a plant overexpression vector was constructed, and a functional analysis was performed after the genetic transformation in accordance to the soil environment of Guizhou Province, to design drought and low-phosphorus experiments to study the performance of genetically modified materials under different treatments. This study can provide a certain theoretical basis for the excellent breeding of Chinese cherries in plateau areas, and help us to reveal how ARFs participate in the drought and low-phosphorus response.

2. Materials and Methods

2.1. Plant Materials and Growth Conditions

“Manao Hong” cherries were picked from the standard greenhouse of the Institute of Agricultural Bioengineering of Guizhou University, with a daily temperature regime of 22–26 °C (day) and 17–20 °C (night), 14 h/10 h, light/dark photoperiod under 40% relative humidity. Cherry saplings are 2-year-old seedlings treated with biochar. The root-soil is then washed and quickly frozen in liquid nitrogen before being stored at −80 °C for later use. Sterile tomato seedlings are grown in MS (Murashige and Skoog) medium and stored in a sterile plant tissue culture room, with a temperature of 25 °C under 50% relative humidity and 14 h/10 h (day/night) 250 μmol m⁻² s⁻¹ intense luminosity.

2.2. RNA Extraction and Gene Isolation

Total RNA was extracted from the samples using a Plant Polysaccharide Polyphenol Total RNA Extraction Kit (SENO, Zhangjia Kou, China) following the manufacturer’s instructions. The ORF (Open Reading Frame) full-length sequence of CpARF7 was RT-PCR amplified using the primers listed in Table S1. Primers were designed using SnapGene and Primer Premier 5. The ORF of CpARF7 was ligated into Blunt vector, transformed into DH5α E. coli (TransGen Biotech, Beijing, China), and sent to Sangon (Shanghai, China; https://www.sangon.com/; accessed on 6 December 2021) for sequencing using Sanger method.

2.3. Sequence Analysis of the CpARF7

In order to understand the sequence characteristics and homology of CpARF7 protein, 23 ARF protein sequences of Arabidopsis thaliana were downloaded from TAIR (https://www.arabidopsis.org/; accessed on 11 January 2022), 16 ARF protein sequences of sweet cherry [12] were downloaded from GDR (https://www.rosaceae.org/; accessed on 18 January 2022), and phylogenetic trees were constructed with CpARF7, respectively. The conserved domains of CpARF7 were searched using the NCBI (https://www.ncbi.nlm.nih.gov/cdd/; accessed on 24 October 2021) and Pfam (http://pfam.xfam.org/; accessed on 7 October 2021) databases. Multiple sequence alignment of ARF proteins was performed using Clustal-W. The phylogenetic tree was performed for the protein sequences in MEGA 11.0 using the neighbor-joining method with 1000 bootstrap. Amino acids in the middle region of CpARF7 were counted with DNAMAN software. We used online tools, PHYRE 2 (http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index; accessed on 17 August 2021) and SWISS-MODEL (https://swissmodel.expasy.org/; accessed on 17 August 2021), to predict the three-dimensional structure of CpARF7 protein, and used PyMOL software to view the results.

2.4. Genetic Transformation of Tomato

The coding sequence of CpARF7 was inserted into pBWA(V)KS-GUS vector to construct 35S:CpARF7 plant overexpression vector. After the constructed overexpression plasmid was verified by restriction enzyme digestion, the heat shock method was used to transform Agrobacterium GV3101. Agrobacterium-mediated genetic transformation of tomato refers to Wang’s method [18]. Transgenic strains were first selected using kanamycin, and then PCR and GUS staining were used for selection.

2.5. Vegetative Growth and Root System Parameter Analyses

The difference of vegetative growth phenotypes, such as total length of the root system, total surface area of root, total volume of root, total number of root tips, total branch number of root, and average diameter of root, were investigated between 35S:CpARF7 lines and wildtype on the 30th day. The root system was scanned and analyzed using EPSON Expression 1200XL (Nagano, Japan) and WinRHIZO software. The transgenic tomato plants with the highest and lowest expression levels were chosen for drought and low-phosphorus treatment. Tomato roots were measured after 30 days of treatment at 0, 0.1, 0.3, 0.5, and 0.8 mg/L IBA. Drought stress was simulated using PEG treatment [19]. For drought treatment, wild-type plant and germinated and transgenic lines (OE5) were incubated in MS medium (20% PEG 6000 with 5.8 pH) for 20 days, and five plants were chosen for each treatment, assay water count, and enzyme activity [20]. The low-phosphorus stress treatment was carried out in the phosphorus-deficient MS medium (pH 5.8). We set up a gradient of four concentrations (0 mmol/L, NP; 0.125 mmol/L, LP; 1.25 mmol/L, MP; 12.5 mmol/L, HP) and observed the phenotypic changes and physiological and biochemical measurements after 20 days of treatment. Peroxidase (POD) Assay Kit (BC0090), Superoxide Dismutase (SOD) Assay Kit (BC0170), Malondialdehyde (MDA) Assay Kit (BC0020), Catalase (CAT) Assay Kit (BC4780), and Proline (Pro) Content Assay Kit (BC0290) were used to detect the content of POD, SOD, MDA, CAT, and Pro after drought and low-phosphorus treatment (Solarbio, Beijing, China). Use Tissue Inorganic Phosphorus Content Assay Kit and Total Phosphorus Assay Kit to detect inorganic phosphorus and tissue total phosphorus content respectively (Solarbio, Beijing, China).

2.6. Real-Time Quantitative PCR and GUS Staining

The expression patterns of CpARF7 in genetically modified tomatoes were investigated using real-time qPCR experiments. The PrimeScript^TM RT reagent Kit with gDNA Eraser (TaKaRa, Dalian, China) reverse transcription kit was used to synthesize the first strand of cDNA, and the internal reference gene SlEF1α was used to test the cDNA [21]. The CpARF7- and SlIAA-specific primers [22] are listed in Table S1. The expression levels were analyzed by qRT-PCR with SYBR Green Mix (Biomarker, Beijing, China) according to the manufacturer’s protocol in the CFX Connect^TM Real-Time System instrument (BIO-RAD, Hercules, CA, USA). The reaction volume is 10 μL (1.0 μL cDNA (70 ng/μL), F/R primer 0.5 μL (10 μmol), SYBR green mix 5 μL, and ddH₂O (RNase-free) 3 μL). The amplification reactions were as follows: incubated at 94 °C for 4 min, followed by 35 cycles of 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 45 s. The relative expression level was calculated using the 2^-ΔCt method [23]. Three biological replicates were taken for each sample.

For b-glucuronidase (GUS) histochemical analysis, we put the roots and leaves (20 d) of genetically modified tomato into the X-Gluc (Solarbio, Beijing, China) dye solution for staining. After vacuuming, it was incubated at 37 °C for 24 h. Following GUS staining, samples were then washed several times to extract chlorophyll using ethanol solutions.

2.7. Statistical Analysis

The experiments were repeated at least three times, and the resulting data were analyzed using SPSS 21.0. Significant difference for the treatments were compared using Duncan’s test (p ≤ 0.05).

3. Results

3.1. CpARF7 in the ARF Family

In the current study, the full-length coding sequence of CpARF7 was amplified from the cherry genome. The coding sequence of CpARF7 is 3498 bp and encodes 1165 amino acids. Compared with the 23 AtARF members of Arabidopsis thaliana, CpARF7 is highly homologous to AtARF7/19; compared with the sweet cherry’s 16 PavARF members, CpARF7 is highly homologous to PavARF1/14 (Figure 1). Based on sequence features and the evolutionary relationship of these genes, it is suggested that these genes may have similar biological functions (Figure 1 and Figure 2a,b).

To understand the sequence characteristics of CpARF7, the homologous AtARF7/19 and PavARF1/14 were subjected to multiple sequence alignment and tertiary structure predictions. Previous studies have shown that AtARF7/14 has similar biological functions and overlapping functions [16]. CpARF7 has three conserved domains: DBD, Auxin_resp, and AUX_IAA (PB1) domains (Figure 2a). The results of the multiple sequence alignment showed that these genes are highly homologous to CpARF7 in Arabidopsis thaliana and sweet cherry, especially their domains (Figure 2a,b). The PB1 domain contains two regions, Domain III and Domain IV, which are homologous to AUX/IAA (Figure 2b,c). In addition, the tertiary structure showed that CpARF7 has typical ARF structural features (Figure 2c). This shows that ARFs and AUX/IAA may have a wide range of interactions, and it also shows that AUX/IAA in species may be derived from the ARF in evolution, because some of the ARF gene members that have been found in other species are not completely structured, such as ARF3/13 in Arabidopsis thaliana [6]. The Q content in the middle region of CpARF7 is relatively high, indicating that this gene may act as a transcriptional activator according to the amino acid sequence alignments. (Figure 2d).

3.2. CpARF7 Promotes Root Growth

In order to understand the biological function of CpARF7 and its regulatory role in plant growth and development, the coding sequence (CDS) of CpARF7 was cloned and a plant overexpression vector was constructed. After the genetic transformation of the tomato, the phenotypic changes were analyzed. After the PCR verification, two transgenic lines (OE1 and OE5) were obtained and the expression level of OE5 was higher than that of OE1. According to the phenotypic comparison, the overexpression lines and systems were more developed, and the number of lateral roots was significantly higher than that of the wild type. (Figure 3a,b). The root system was statistically analyzed with the EPSON root scanner. The main root length was significantly longer than the WT, and OE5 was 1.6-fold longer than the WT after the IBA treatment (Figure 4a). Statistics on the average root diameter, root volume, surface area, root tip number, and total branch number showed that the transgenic plants all increased significantly compared to the WT. The average diameters of OE1 and OE5 were increased by 1.53-fold and 1.66-fold, respectively, compared with the WT. The volumes of OE1 and OE5 were increased by 1.39-fold and 1.69-fold, respectively, compared with the WT. The total surface area was also increased by 1.16-fold and 1.35-fold, respectively (Figure 4b–g).

To gain insight into the expression of CpARF7 in plants, the CpARF7 was fused to the GUS reporter gene sequence, and the construct obtained (CpARF7:GUS) was used to stably transform the tomato plants. GUS staining was performed on the roots and leaves of OE1 and OE5, and the results showed that CpARF7 was expressed in these tissues, and also suggested that CpARF7 is widely involved in plant growth and development (Figure 3c–e). To understand the response of CpARF7 to exogenous auxin in overexpressing lines, OE5 was treated with different concentrations of IBA. The phenotype, upon treatment, improved in roots compared to the WT, and the number of taproots was significantly higher than the WT.

3.3. CpARF7 Can Improve the Drought Resistance of Transformed Tomato

There have been previous reports on the involvement of the ARF family in drought response [24,25,26]. Different concentrations of PEG 6000 were used to treat OE1 and OE5 for 20 d to better understand CpARF7’s ability to respond to drought in transgenic tomatoes. The results showed that under drought stress, wild-type plants have a dwarf phenotype, their main roots were shorter, and their leaves turned yellow (Figure 5a). This indicates that the WT is more susceptible to drought stress, resulting in slower growth. Compared with the wild type, OE1 and OE5 grow well, the main roots maintain elongation, and the shoots remained green. Meanwhile, the water content of OE1 and OE5 are both higher than that of the WT, suggesting that the overexpression of CpARF7 can promote plant water maintenance (Figure 5b).

After drought stress, the activities of CAT, POD, and SOD were determined, and the results revealed that there were no significant differences between the three enzyme activities in the control group (Figure 5c–e). After drought treatment, the activities of SOD, POD, and CAT increased significantly in OE1 and OE5. In addition, the MDA and Pro of the transgenic plants were determined (Figure 5f,g). The results showed that MDA decreased and the Pro content increased in the transgenic lines after drought treatment. These results suggest that the overexpression of CpARF7 can enhance the activity of antioxidant enzymes in tomatoes, thereby improving the drought resistance of plants.

3.4. Overexpression of CpARF7 Improves the Phosphorus Absorption Capacity of Roots

In the process of plant growth and development, phosphorus is a crucial chemical substance. Previous studies have shown that ARFs have a regulatory effect on phosphorus homeostasis in plants [27]. In order to explore the potential function of CpARF7 in phosphorus absorption and assimilation, four different gradients of phosphorus concentrations were set up to treat transgenic tomatoes. The results illustrated that under low-phosphorus treatment, the main roots of the transgenic lines elongated and the lateral root number decreased (Figure 6a). The lateral roots of the transgenic tomatoes increased after high-phosphorus treatment. Furthermore, the biomass was tested. Under NP, the weight of the root was reduced and the main root was elongated. As the phosphorus content increases, the root weight increases (Figure 6b).

The detection of the phosphorus content of the tomatoes displayed that the phosphorus content in the shoots and roots of OE5 increased by 1.50-fold and 1.55-fold, respectively, and the inorganic phosphorus content increased by 2.21-fold and 4.56-fold after NP treatment. Likewise, phosphorus in OE1 also increased by 1.25-fold and 1.54-fold, and inorganic phosphorus increased by 1.72 and 3.95-fold. Under LP conditions, the phosphorus content in the branches and stems of OE5 increased by 1.46-fold and 1.41-fold, and OE1 increased by 1.24-fold and 1.38-fold (Figure 6c,d). These findings suggest that when a plant is in a low-phosphorus environment, it may adjust its phosphorus content to maintain an adequate balanced state of phosphorus in the plant, to achieve the goal of high-efficiency phosphorus-use. It may also indicate that the phosphorus absorbed in the roots is quickly transported to the shoots. Under HP treatment, the phosphorus content in the roots of OE1 and OE5 accumulated more. This implies that when there is excess phosphorus in the environment, the roots absorb more, but the excess is not transferred to the stems.

Similarly, we detected the content of CAT, POD, and SOD after treatment with different phosphorus. The results showed that under low-phosphorus treatment, CAT, SOD, and POD all increased significantly (Figure 6e–g). The overexpression of CpARF7 may promote the utilization efficiency of phosphorus, or promote phosphorus absorption through root elongation. These results suggest that overexpression plants have higher resistance to stress when auxotrophic conditions occur.

3.5. The Effect of Overexpression of CpARF7 on Downstream Gene Expression

As a key gene in the auxin response pathway, ARFs have extensive interactions with the auxin response inhibitor, AUX/IAA. In order to understand the effect of the cherry CpARF7 gene on IAA expression in two independent transgenic tomatoes, 19 IAA genes were selected for a qPCR analysis [22]. The results showed that among the 19 SlIAA genes, eight genes showed varying degrees of expression changes compared to the WT (Figure 7). Among them, SlIAA1/14/19/29 up-regulated expression, and SlIAA2/11/12/16 down-regulated expression.

4. Discussion

The evolution of plant root systems is of great significance for plants to adapt to the terrestrial environment [28]. The root system serves as a gateway between the soil and the atmosphere, as well as a vital component of material exchange and water circulation [29]. Plants are organisms that are connected to the ground, and the root system is among the most vital components of the plant, since it ensures the plant’s stability. Root formation is also an important part of plant growth and development. The formation of plant roots is affected by factors such as plant hormone balance, gravity, water, light, and nutritional conditions, which are very complex biological problems [2,30]. As we all know, the synergistic effect of auxin and cytokinin plays a vital role in the size of the root meristem and in ensuring root growth [31]. The auxin response factor, which is functionally divided into transcription activator and transcription repressor, has an important regulatory role in this process [32]. From lower moss to higher angiosperms, ARFs are widely present in land plant species, and have produced huge gene families during the long evolutionary process [33,34]. In microalgae, incomplete ARF domain sequences have been found, and these short-sequence domain ARFs or AUX/IAAs have conserved structures and high homologies [33]. The above results indicate that auxin may have regulated a signal network in the early stages of plant evolution. In the current research, the CpARF7 gene, which may regulate the growth of the cherry root, was cloned from the cherry. Members of sweet cherry and Arabidopsis have a homologous relationship with this transcription factor (Figure 1), implying that these genes may have evolved from the same ancestral gene [35]. A large number of studies have shown that the PB1 domain in the CTD of ARFs has homology with the domain III/IV of AUX/IAA, and has a high degree of sequence similarity [36,37]. CpARF7 in this study is also similar to some AUX/IAA member sequences in sweet cherry or Arabidopsis (Figure 2b), which is consistent with previous research reports. According to function and evolution, ARF family members can be divided into three distinct categories, namely A, B, and C. Among them, the Q-rich middle region is classified as class A [6,38]. The center zone of CpARF7 in this study was revealed to be Q-rich, indicating that CpARF7 belongs to class A (Figure 2d). Furthermore, it can be verified based on yeast two-hybrid assays and protoplast transfection experiments [39,40].

The ARF transcription factor is involved in root growth regulation. According to recent research, ARF7 plays an important regulatory role in plants’ adaptation to the soil environment, which is accomplished through a series of post-translational modifications of ARF7 that alter the root architecture [41]. SlARF2 is linked to the senescence of floral organs in tomatoes and regulates the formation of lateral roots [14]. ARF8 can affect the length of hypocotyls and the root growth of Arabidopsis thaliana, and may play an important role in the regulation of auxin balance in light response [42]. Potato StARF10 and StARF16 are key regulators in the formation of root structure. They are targeted by miR160a/b, and change the level of auxin and the expression of related genes [43]. OsARF12 in rice acts as a transcriptional activator to regulate root elongation. In OsARF12-deficient mutants, the auxin in the elongation zone of the root is reduced, and the root length is significantly shorter than that of the wild type [44]. Overexpression of cherry CpARF7 in tomatoes can also promote root elongation and increase lateral roots according to the current study. In the overexpression lines, CpARF7 was expressed in roots, suggesting that this gene is related to the formation of the root structure (Figure 3e,f).

In the response to abiotic stress, the ARF plays a crucial role. The function of CpARF7 in response to drought was investigated in this study using a drought treatment. It was found that CpARF7 can increase the length of roots in an arid environment, which may be to absorb more water. Apples’ drought tolerance depends heavily on MdARF17. Apples with low MdARF17 expression are more drought resistant, while those with high MdARF17 expression are more drought susceptible [45]. Furthermore, the above gene is associated with the formation of adventitious roots in sweet potato. IbARF5 is involved in the biosynthesis of carotenoids and can improve the drought resistance and salt tolerance of transgenic Arabidopsis thaliana, suggesting that this gene plays an important role in abiotic stress [26]. The transgenic lines in this study grew better under drought treatment, and the enzyme activities, such as SOD and POD, increased, which improved the drought tolerance of the transgenic plants (Figure 4). There are 15 ARF members in the birch (Betula platyphylla) genome, among which BpARF1 has been significantly induced by drought; the activities of POD and SOD increase in plants inhibited by RNAi, indicating that silencing BpARF1 can increase the drought tolerance of plants [46]. Previous studies have shown that when plants are in a drought environment, chlorophyll fluorescence will change, which can be used as a reference for tracking the impact of drought on plant growth [47,48]. In the current study, overexpressed tomatoes treated with PEG6000 were greener than treated wild-type phenotypes, suggesting a difference in chlorophyll fluorescence between the WT and OE.

Phosphorus is essential for the normal growth of plants. Under the present study, transgenic tomatoes were treated with four distinct phosphorus concentration gradients. The overexpression strains grew faster and had a more developed root system according to the findings (Figure 5). Studies have shown that after OsARF12 is mutated in rice, genes related to phosphorus response accumulate in large amounts under phosphorus-rich or phosphorus-free conditions, and the root system is changed. These results indicate that ARFs can participate in the regulation of phosphorus levels [27]. The removal of OsARF16 in rice will result in the loss of the response of tap roots, lateral roots, and root hairs to auxin and phosphorus; under -Pi conditions, the above-ground biomass is slightly reduced [49]. High-throughput sequencing was performed on soybeans after low-phosphorus treatment, and it was identified that some of the microRNAs were targeted to the ARF family, suggesting that the ARF is involved in complex regulatory processes in the low-phosphorus response [50]. In the current work, the inorganic phosphorus content and total phosphorus content have increased after the low-phosphorus treatment. The overexpression lines increased phosphorus absorption and had a more reasonable utilization and phosphorus balance in the plants after the low-phosphorus treatment.

As a plateau province in western China, the problems of drought and a lack of soil nutrients have not been effectively solved. However, from the point of view of the plant itself, it may be a new solution according to local conditions. With the study of drought resistance genes and low-phosphorus response genes, these genes have also become potential sites for genetic engineering and breeding. In this study, an auxin-responsive factor, CpARF7, was discovered from cherries. Through genetic transformation studies, it was found that CpARF7 may have potential functions in drought resistance and responses to low-phosphorus environments. This result can lay a foundation for the future molecular genetic breeding of cherries.