Transcriptome-wide identification of walnut PP2C family genes in response to external stimulus

Walnut is an important economic tree species while confronting with global environmental stress, resulting in decline in quality and yield. Therefore, it is urgent to elucidate the molecular mechanism for the regulation of walnut response to adversity. The protein phosphatase 2C (PP2C) gene family participates in cellular processes in eukaryotes through reversible phosphorylation of proteins and signal transduction regulation. However, the stress response function of PP2C genes was far to be clarified. Therefore, to understand the stress response mechanism of walnut tree, in this study, a total of 41 PP2C genes with complete ORFs were identified from Juglans regia, whose basic bio-information and expression patterns in response to multiple stresses and ABA were confirmed. The results showed that the ORFs of JrPP2Cs were 495 ~ 3231 bp in length, the predicted JrPP2C proteins contained 164 to 1076 amino acids and the molecular weights were 18,581.96 ~ 118,853.34 Da, the pI was 4.55 ~ 9.58. These JrPP2C genes were unevenly distributed on 14 chromosomes, among which Chr11 and Chr13 contained the most genes. Phylogenetic analysis found that these JrPP2C proteins were classed into 9 subfamilies, among which group F covered most JrPP2Cs. The JrPP2Cs in the same subfamily exhibited similarities in the composition of conserved domains, amino acid sequences of motifs and exon/intron organization in DNA sequences. Each JrPP2C includes 4 ~ 10 motifs and each motif contained 15 ~ 37 amino acids. Among the motifs, motif1, motif2, motif3 and motif8 were most abundant. Most of the JrPP2C genes diversely response to osmotic, cadmium, and Colletotrichum gloeosporioide stress as well as ABA treatments, among which JrPP2C28, JrPP2C17, JrPP2C09, JrPP2C36 were more obvious and deserves further attention. All these results indicated that JrPP2C genes play potential vital roles in plant response to multiple stimulus, and are possibly involved in ABA-dependent signaling pathway.


Introduction
Walnut is an important tree species for nut and timber production in the world, and its values of economic, ecological and social have been widely concerned [1]. In China, walnut has a wide range of planting areas and rich varieties. It has gradually become a large-scale agricultural and forestry industry with a wide range of fields, a long industrial chain and an increasingly prominent economic status. It plays an important role in the economic development of the vast mountainous area. However, while the walnut planting area is increasing, it also encounters various problems: the selection of varieties is not necessarily suitable, the yield and quality are unstable, the development of characteristic resources is insufficient, the plantation management is inappropriate and Open Access *Correspondence: yangguiyan@nwsuaf.edu.cn the plants are exposed to various environmental factors (such as drought, high temperature, pests and diseases). These factors restrict the healthy development of the walnut industry. One of the main reasons for these phenomena is that the mechanism of walnut response to adversity is unknown, and walnut cultivation and management measures cannot be effectively formulated. Therefore, in order to provide genetic resources for revealing the stress-resistant response mechanism of walnut and the selection of new germplasm for stress-resistant rootstocks, it is necessary to identify the key genes of walnut in response to stress and then to reveal the stress-resistant regulation mechanism of walnut on this basis.
Reversible phosphates of protein kinases and protein phosphatase-mediated proteins are widely present in organisms and involve in a variety of physiological processes. Protein translation modification can change the physiological and biochemical properties of important functional molecules in the signaling pathway. So it is of great significance for plants to regulate cell cycle, growth and development, hormone and other environmental stimulation [2,3]. According to the modification function, protein phosphatases (PPs) were divided into three major classes: tyrosine phosphatases (PTPs), serine/threonine phosphatases (PSPs), and dual-specificity phosphatases (DSPTPs) [4]. Among them, PSPs were categorized into three subfamilies-phosphoprotein phosphatases (PPPs), metal-dependent protein phosphatases (PPMs), and aspartate-based phosphatases (APPs). Representative members of the PPP subfamily include PP1, PP2A, PP2B, PP4, PP5, PP6, and PP7. The PPM subclass covered protein phosphatases dependent on manganese/magnesium ions (Mn 2+ /Mg 2+ ), such as PP2C and pyruvate dehydrogenase phosphatase [5,6]. The PP2C subfamily protein is an important branch of the PP family, whose C-terminus has a conserved catalytic domain and the N-terminus is an extension region with different functions; the dephosphorylation of PP2C depends on Mn 2+ and Mg 2+ when participating in phosphorylation [7]. The PP2C genes are widely present in animals, microorganisms and plants [8], and play regulatory roles in various biological processes. For instance, in mice, PP2Cβ played a crucial role during gametogenesis, fertilization, and early stages of embryonic development [9]. In microorganisms, Ptc6 was believed to be involved in virulence and MAPK signaling in Fusarium oxysporum [10]. In plants, Arabidopsis PP2C49 negatively regulated salt tolerance through inhibition of AtHKT1;1 [11], wheat TaPP2C-a10 negatively modified plant drought resistance through ABA signaling [12].
The PP2C genes response to plant stress via ABA signaling. ABA receptor PYR/PYL/RCAR in plants receives ABA molecular signals to inhibit protein phosphatase activity, reduce or eliminate the inhibition of PP2Cs on downstream kinases (eg, SnRK2s, OST1), and enhance kinase phosphorylation of substrate proteins to participate in plant growth and stress modulation [13,14]. Arabidopsis PP2CG1 positively regulates salt stress in an ABA-dependent manner [15]. PeHAB1 could interact with the ABA receptor PYL4 in an ABA-independent manner to reduce tolerance to drought in poplar [16]. In maize, ZmPP2C26 has a negative regulatory effect against drought stress [17]. Tomato SlPP2C gene family that encoding the core component of ABA signaling could regulate tomato fruit development and be induced by drought [18]. Due to the extensive role of PP2Cs in plant stress resistance, it has received some attention in recent years. However, it is far to be enough, especial in woody plants, which limits the whole and deep understanding of the target plant in many aspects of life process, such as stomatal switching, growth and development, and stress response. Therefore, in the present study, in order to identify candidate genes for revealing walnut stress response mechanism, walnut PP2C genes were selected from Juglans regia according to chromosome distribution, gene structure, protein motifs and phylogeny. Meanwhile, five stresses (drought, salt, cadmium, ABA and anthrax) were applied to assess the expression activity of the selected PP2C genes. The results of this study will supply new evidence for subsequent study of JrPP2Cs respond to stimulus.

Plant materials and treatments
The plant material used in this study was the 3-year-old 'Xiangling' walnut (a phenotype widely grown in China) that grown in a greenhouse (22 ± 2 °C, relative humidity 70 ± 5%, light cycle 14 h light/10 h dark) [19]. 20% (w/v) PEG 6000 , 0.3 mol/L NaCl, and 0.2 mmol/L CdCl 2 were watered to the roots of the seedlings, respectively, and the leaves were collected at 0 and 6 d and saved in − 80 °C refrigerator for further RNA isolation. For ABA treatment, 30 μmol/L ABA was used and sampling time is 0, 6 and 9 d. For walnut anthracnose treatment, Colletosporum gloeosporioide colonies with conidia were rinsed with sterile water and cultivated to the concentration of 10 5 ~ 10 6 cells/mL. After the walnut leaves are slightly damaged by friction, spray the prepared anthracnose spore re-suspension on the leaves for treatment and then the leaves were sampled at 0 and 9 d. Each treatment contained 6 seedlings. For tissue expression analysis, the tissues of 6 years old grafted 'Xiangling' including leaves, tender stems, old stems, male flowers, and female flowers were collected on April 13th, 2019, and three biological replicates were applied for each test sample [20].

Identification of PP2C genes in walnut
'Protein phosphatase' was used to search for the walnut transcriptomes (sequenced by our research group) under treatments of drought, salt, cadmium, ABA and C. gloeosporioide. Then the PP2C protein sequences of Arabidopsis were obtained from the TAIR database (https:// www. arabi dopsis. org/) [21] and used for homology alignment to screen the walnut PP2C members. The ORF Finder (https:// www. ncbi. nlm. nih. gov/ orffi nder/) was used to find the open reading frame (ORF) of potential walnut PP2C genes, whose protein sequences were further queried and verified in the NCBI protein database by BLASTp (https:// blast. ncbi. nlm. nih. gov/ Blast. cgi? PROGR AM= blast p& PAGE_ TYPE= Blast Searc h& LINK_ LOC= blast home). The molecular weight (MW), theoretical isoelectric point (pI) and amino acid composition were predicted using the ExPASy server (https:// web. expasy. org/ protp aram/). The corresponding gene accession numbers were blast from NCBI. The PP2C domains (PF00481) of all of the walnut were analyzed using HMM (Hidden Markov Model) by searching PFAM (Protein family: http:// pfam. sanger. ac. uk/ search) and HMMER (https:// www. ebi. ac. uk/ Tools/ hmmer/ search/ phmmer) [22]. Those proteins lack PP2C domain were removed. NCBI-Conserved domain database (CDD) (https:// www. ncbi. nlm. nih. gov/ Struc ture/ cdd/ wrpsb. cgi) was applied for domain composition analyses.

Analysis of evolutionary relationship, gene structure and chromosomal locations
To analyze the evolutionary relationship of walnut PP2C genes, 78 Arabidopsis PP2C protein sequences were download from the TAIR database (https:// www. arabi dopsis. org/) and 28 Populus PP2C protein sequences were obtained from Phytozome v13 (https:// phyto zome-next. jgi). The PP2C protein sequences of walnut, Arabidopsis, and Populus trees were compared using the Clustal W2 program [23] and the phylogenetic tree was constructed using the neighborjoining method in MEGA7 [24]. The evolutionary tree was beautified using the online software itol (https:// itol. embl. de/) [25]. JrPP2Cs were classified into subgroups according to the topology of the phylogenetic tree and referring to the previous studies on A. thaliana [26].
The gene structure map of the exon-intron of JrP-P2Cs was determined by Gene Structure Display Server 2.0 (GSDS 2.0: http:// gsds. gao-lab. org/). The MEME online tools (http:// alter nate. meme-suite. org/) were used to analyze the conservative motifs with the following parameters: the number of motifs is 12, allowing any number repetitions, motif width is from 15 to 36. The chromosomal location information of 41 JrPP2Cs in the walnut genome was confirmed from NCBI (https:// www. ncbi. nlm. nih. gov/), the walnut genome data refer to J. regia (assembly Walnut 2.0) [27][28][29]. The motif domain and chromosomal location were visualized by TBtools [30].

Expression analysis of JrPP2Cs
The total RNA of all samples were extracted by CTAB (cetyltrimethylammonium ammonium bromide) method [31]. The RNA concentration was determined and reverse-transcribed to cDNA by PrimeScript ™ RT reagent Kit (CWBIO, Beijing, China) after treated by DNA digestion enzyme. The cDNA was diluted 10 times and used as the template of quantitative real-time PCR (qRT-PCR). QRT-PCR was performed using the SYBR Green Real time PCR Master mix (CWBIO) with an internal reference gene of walnut 18S rRNA (HE574850) [32]. The primers used are shown in Table S1. The instrument used for the quantitative reaction is the StepOne ™ Real Time PCR system produced by Applied Biosystems. The reaction procedures were: 94 °C for 30 s, 45 cycles of 94 °C for 12 s, 60 °C for 45 s, 72 °C for 45 s; 81 °C for 1 s, 3 replicates per sample. The quantitative results were analyzed by 2 -ΔΔCT method [33]. The data were analyzed using the SPSS package (SPSS, Chicago, Illinois, USA). Sample variability is expressed as standard deviation. Expression differences between different time points and 0 d were analyzed by T test (P < 0.05). The results were visualized in Tbtools software and Origin 2017 and the results are represented using heat maps [30].

Sequence characteristics and chromosomal locations of walnut PP2C genes
A total of 44 putative JrPP2C genes were screened from walnut transcriptome, among these 44, 3 lacked PP2C catalytic domain confirmed by PFAM and SMART tools. Therefore, 41 genes in J. regia were identified as PP2C family members. These 41 PP2C genes were anchored to corresponding chromosomes and designated as JrPP2C1 to JrPP2C41 according to their order on the chromosomes (Table 1, Fig. 1). The ORFs of 41 JrPP2C genes were 495 ~ 3231 bp in length, the molecular weights of the deduced peptides were 18,581.96 ~ 118,853.34 Da with 164 ~ 1076 amino acids, and the theoretical isoelectric point (pI) was 4.55 ~ 9.58 (Table 1).
41 JrPP2C genes were dispersed on 14 chromosomes. On each chromosome, the number of JrPP2C varies drastically, ranging from 1 to 7, the largest number of JrPP2C members was observed on chromosomes 11 with 7 genes, followed by chromosome 13 with 6 genes, whereas the least numbers were revealed on chromosomes 2, 3, 4 and 9, each contains only 1 gene (Fig. 1), suggesting the uneven distribution of JrPP2C genes on chromosomes.

Gene structure of JrPP2Cs
Exon-intron structural diversity within a gene family is an important clue for the evolutionary and functional analyses. To know the components of the JrPP2C gene structure, the exons and introns, including their amount and distribution among JrPP2C genes were examined. The results revealed that most members in the same subfamily shared similar exon numbers and different exon and intron lengths. The number of introns and exons of these 41 JrPP2Cs ranges from 1 to 15, and 2 to 16, respectively (Fig. 5B) (Fig. 5B).

Tissue expression specificity of JrPP2Cs
To investigate the potential role of JrPP2Cs, female flowers (FL), male flowers (ML), old stems (ST, stems of 2 years old and older branches), tender stems (SH, stems of the new shoots in the current year) and leaves (LE) were collected and the transcription level of 41 JrPP2C genes were confirmed using qRT-PCR method. The results showed that most JrPP2Cs were expressed in all tissues with various profiles and could group into following types (Fig. 6).

Expression activity of JrPP2Cs to biotic and abiotic stresses as well as ABA treatment
To explore the potential function of JrPP2Cs in response to common stresses and whether involving in ABA signalling, the expression of 41 JrPP2Cs were analyzed under stresses of drought, salt, heavy metal, and C. gloeosporioides as well as treatment of ABA (Figs. 7, 8 and S1).

Under drought stress
The expression of these 41 JrPP2Cs were showed the same trend under PEG 6000 stress. After 6 d of PEG 6000 stress, their relative expression was increased, and the average expression value was 2.86. The transcription of nine genes (JrPP2C28, JrPP2C22, JrPP2C29, JrPP2C23, JrPP2C36, JrPP2C09, JrPP2C10, JrPP2C38, JrPP2C37) exceeded 4.00, among them JrPP2C28 displayed the highest induction (4.87). The relative expression level of 10 genes (JrPP2C30, JrPP2C41, JrPP2C05, JrPP2C16, JrPP2C33, JrPP2C24, JrPP2C15, JrPP2C02, JrPP2C19, JrPP2C20) were less than 2.00. In which, JrPP2C20 was the one that induced with lowest expression level, the transcription of JrPP2C28 is 3.78-fold of JrPP2C20 ( Fig. 7), suggesting that JrPP2C28 may be the most potential candidate gene for walnut drought stress regulation in these 41 JrPP2Cs.

Under salt stress
The expression of 41 JrPP2Cs under NaCl stress could class to three groups: (i) Genes with relative expression levels greater than 1 that covered 20 genes (JrPP2C17,   (Fig. 7), indicating that JrPP2C17 may be a salt stress response gene that the worthiest one for further study.

Under heavy metal stress
Under the treatment of CdCl 2 , the relative expression levels of 41 JrPP2Cs genes changed obviously, 68% of the genes was up-regulated, of which JrPP2C17 (6.57) was induced to the highest level, JrPP2C09 (6.04) was ranked at the second site, followed by JrPP2C29 ( (Fig. 7). These results tell us that JrPP2C17 may be also the Cd response candidate.

Expression to C. gloeosporioides stress
73% of the 41 JrPP2C genes were up-regulated and other 27% were down-regulated under C. gloeosporioides stress. All genes can be classified into three groups based and JrPP2C15 were down-regulated to a level below − 1, whose mean value was − 2.34. Notably, JrPP2C24 was suppressed most obviously, and the value is − 4.40 (Fig. 8). These results suggested that if we want to understand the molecular mechanism of walnut resistance to C. gloeosporioides, JrPP2C36 is an important candidate gene.

Under ABA treatment
All JrPP2Cs were up-regulated by ABA with varied expression profiles that could be classified into two categories: (i) Genes whose peak relative expression levels appeared at 6 d, including JrPP2C23, JrPP2C07, JrPP2C28, JrPP2C40, JrPP2C38, JrPP2C36, JrPP2C25, JrPP2C21, JrPP2C34, JrPP2C15, JrPP2C13, JrPP2C04, JrPP2C20, JrPP2C19, JrPP2C11, JrPP2C14, JrPP2C12, JrPP2C24. Among them, JrPP2C28, JrPP2C40, and JrPP2C38 were induced to a level exceed 4.00. (ii) Genes those induced by ABA to maximum level at 9 h contained the genes apart those in subgroup (i) and JrPP2C27. In sub-family ii, the top two genes in expression level were JrPP2C17 (4.85) and JrPP2C09 (4.77); then JrPP2C10, JrPP2C36, JrPP2C29, JrPP2C22, and JrPP2C39 were also up-regulated to a level higher than 3.00. While JrPP2C16, JrPP2C20, JrPP2C27, and JrPP2C33 were the genes with little change at 6 and 9 d and their expression were close to 0 (Figs. 7, 8 and S1), implying the varied relation between the JrPP2Cs and ABA.

Discussions
The PP2C gene family is one of the largest families of plant and has been identified as important members playing crucial roles in phytohormone signaling, developmental processes, biotic and abiotic stress responses [8,17,36], however, PP2C genes from walnut trees was still not reported. In order to reveal the adversity adaptation mechanism of walnuts then to provide a basis for walnut cultivation and management to ensure the yield and quality, in this study, 41 walnut PP2C genes those may have potential functions in stress response were identified ( Table 1). The sequence characteristics (ORF length, amino acid number, molecular weight, and pI) of JrPP2Cs (Table 1) were ranged similarly as other species, for instance, the molecular weights of PP2C proteins from Pyrus bretschneideri, tomoto and current walnut were 7.5 ~ 243 [37], 6.7 ~ 120 [26], and 18.6 ~ 119 kDa, respectively. In terms of evolutionary relationship, the 41 JrPP2C proteins shared a high similarity with the members of PP2C family of Arabidopsis and poplar, and could be classified into eleven subfamilies with reference to the classification in Arabidopsis [38], and wild soybean [39] ( Fig. 2). Meanwhile, except the PP2C conserved domain, PP2C proteins usually contain other domains which might bind potential functional sites thereby activating their function [40]. JrPP2C proteins in this study all have PP2C domain as well as other one or more conserved domains (Pkinas, cNMP_binding, Pkinase_Tyr, PP2C_2, SpoiIE) with differential domain segment duplications ( Fig. 5A and Table S2). Multi-sequence comparisons show that 41 JrPP2Cs are highly conserved, and most JrPP2Cs included motif1, motif2, motif3, motif8 (Fig. 3, Table 2), in which motif3 (AFFGVFDGHGGPDAA) presumed to be a marker of PPM phosphatase [8], confirming that these 41 JrPP2Cs belong to PP2C protein family and shared potential varied functions. Gene structure is also a cue for functions. The 41 JrPP2C genes were located in different chromosomes at different sites ( Fig. 1) with changeable numbers and distributions of exon and intron (Fig. 5B). In organisms, exons perform phenotypic regulation by encoding protein regions throughout the organism's genome, so the length and location of exons contain important biological information. The loss/gain of intron position and length is slow, so intron positions can often retain information about gene homology [41]. Many studies of exon/ intron structure have shown that most members in the same subfamily have similar exon numbers and different exon and intron lengths [42,43]. In this study, we found that the number of exons/introns in group A, B, and C was exactly equal, and most of the gene structures in group F, G, and H were similar, while some genes (such as JrPP2C04, JrPP2C07, and JrPP2C41) are quite different from other genes (such as JrPP2C37, JrPP2C19, and JrPP2C25) (Figs. 2, 5B), indicating the functional similarity and specificity of these JrPP2C genes. Moreover, the gene and protein structural features of JrPP2Cs were similar to those of PP2C in Glycine max [44], Gossypium hirsutum [45], Brassica rapa [46] and Brachypodium distachyon [47]. Soybean PP2Cs could control plant growth and development [44]. Cotton PP2C gene family plays critical role in organ and fiber development, as well as abiotic stress tolerance [45]. BraPP2Cs has been demonstrated potential ability to regulate biotic and abiotic stress, and BdPP2CA6 was involved in ABA and stress signaling pathways [34]. Therefore, we speculate these JrPP2C genes may relate to the life activity and adversity response of walnut.
To understand whether these JrPP2Cs are involved in growth and development or tissue expression specificity, the transcription levels of 41 JrPP2Cs were detected in various tissues, and the results showed that all JrPP2Cs displayed strong expression in leaves, tender stems, old stems, male flowers, and female flowers (Fig. 6). This observation was similar to the expression pattern of other gene families in walnut and PP2Cs in other species. For instance, five MYB genes could express in a varied pattern in walnut leaves, tender stems, old stems, male flowers, and female flowers and believed to be important candidates for walnut breeding [20]. JrWRKY2 and JrWRKY7 displayed obvious expression level in walnut pistil, terminal leaf, other leaves and stems, implying the potential involvement in metabolic processes leading to nut formation [48]. Most wheat TaPP2C genes exhibited a wide range of transcription in leaf, stem, root, spike, and grain tissues those related to different developmental stages [8]. 29 B. rapa PP2C paralogous gene pairs were detected from various tissues (root, stem, leaf, flower, and silique) [46]. According to the current results and other reports on PP2C genes, we believe that JrPP2Cs genes are correlated with walnut growth and development. Meanwhile, JrPP2C08 has the highest tissue expression activity (Fig. 6), therefore, it may have the most research potential in the regulation of walnut tissue development.
Considering that the adversity of drought, salt stress, heavy metal pollution and diseases as well as pathogens will affect the growth and yield of walnut, to confirm whether JrPP2Cs might be related to the stress response of walnut, the transcriptional activities of 41 JrPP2C genes were analyzed under abiotic stress (PEG 6000 , NaCl, CdCl 2 ) and biotic stress (C. gloeosporioide). The results showed that all JrPP2Cs could response to above stresses with various degrees, the relative expression levels of 11 genes were increased under above stresses, among which JrPP2C09 and JrPP2C17 were induced more obviously than other genes, especially in response to NaCl and CdCl 2 stress. Under drought stress, all JrPP2C genes were induced. In response to C. gloeosporioide stress, the most obvious induction was JrPP2C36 and JrPP2C17 (Figs. 7, 8, S1), implying the potential different response ability of these JrPP2Cs to specific adversity, and may play vital and wide role in drought response. Gene expression is an important and basic way for gene function prediction, for example, RsHSFs were judged to play a crucial role in the biological process of salt stress response by analyzing the relatively high expression levels of RsHSF-11 and RsHSF-22 [49]. JrWRKY2 and JrWRKY7 were found to be induced by drought, salt and cold, which were further confirmed as drought tolerance regulators [48]. Therefore, we believe that JrPP2Cs are important candidate genes of walnut in response to drought, salt, Cd and anthracnose, and the genes with large changes in expression activity deserve further attention.
Protein phosphatases alter protein function by removing phosphate groups from phosphorylated proteins. Studies have shown that ABA plays an important role in plant protein phosphorylation and that some PP2Cs are involved in plant stress regulation through the ABA pathway [36,50,51], and that ABA receptors (PYR/ PYL/RCAR: pyrabactine resistance/PYR-like/regulatory components of ABA response) receive ABA signals and selectively interact with evolved branch A PP2Cs and regulate downstream SnRK2s-type kinases, which in turn regulate the expression of other transcription factors through multiple phosphorylations in response to various stresses [52,53]. To clarify whether the response of JrPP2Cs to adversity was related to ABA, walnut was treated with ABA for the same duration as each adversity treatment (6 and 9 d), and the expression of each JrPP2C was analyzed and found that all JrPP2C genes could be induced to different degrees after treatment with ABA (Figs. 7, 8, S1). Moreover, the genes that were significantly up-regulated by ABA were also significantly up-regulated by above stresses. For example, JrPP2C28, JrPP2C40, JrPP2C38, JrPP2C17, JrPP2C36, and JrPP2C09, which had higher relative expression levels under ABA for 6 d, were up-regulated more obviously by PEG 6000 , NaCl, and CdCl 2; JrPP2C28 even had the highest relative expression levels under both drought and ABA treatments. JrPP2C19 and JrPP2C20 were transcribed lowly under PEG 6000 , NaCl, CdCl 2 as well as ABA treatment for 6 d. JrPP2C36, JrPP2C17, JrPP2C09, JrPP2C22, JrPP2C10, JrPP2C29, whose expression levels were prominent under C. gloeosporioide stress, also showed higher expression levels at 9 d of ABA treatment (Fig. 8). It can be seen that the involvement of walnut PP2C in stress regulation correlated with ABA. This is similar to other reported ABA-related genes. For example, JrWRKY2 was induced to a similar expression patter under ABA and drought stress, further, JrWRKY2 was believed to regulate JrG-STU23 and JrVHAc4 in plant drought tolerance via ABA signal pathway [19,48]. JrVHAG1 was induced by CdCl 2 and further confirmed that its Cd-responsive function is also achieved through the ABA signal pathway [54]. PbrPP2C10, PbrPP2C11, PbrPP2C15, and PbrPP2C18 were up-regulated by exogenous ABA, as a presumption that PbrPP2C is related to ABA [37]. Therefore, based on the performance of JrPP2Cs under different stress and ABA in this study and other previous reports, we believe that the response of walnut PP2C family genes to stress is related to ABA signal.
Moreover, the PP2C family has many members with different functions achieved by various ways. For example, BdPP2CA6 positively regulates salt tolerance in transgenic Arabidopsis via interacting with BdPYLs and BdSnRK2 [47]. The SlPP2C gene contributes to tomato resistance to bacterial blight and may be regulated by many light-response elements in the promoter region [26]. Betula platyphylla BpPP2C1 regulates salt stress tolerance involving in ABA signaling pathway, flavonoid biosynthetic pathway, reactive oxygen species (ROS) metabolism, oxidative stress and anion transport [7]. Cold-response elements were found in the promoter region of 31 Broussonetia papyrifera BpPP2Cs; Bp01g0320 was found to act as a hub protein; Bp01g0512 and Bp09g1278 played key roles relating to ABA-signaling and MAPK cascades, respectively [55]. ZmPP2C-A10 gene negative regulated maize response to drought stress linking endoplasmic reticulum (ER) stress signaling [56]. In the current study, based on basic biological information, tissue expression and expression analysis under different stresses, the potential functions of the walnut JrPP2C family genes were clarified, and several potential members (JrPP2C09, JrPP2C28, JrPP2C17, JrPP2C36) were identified. In the follow-up research on walnut stress resistance and characteristic germplasm breeding, we will combine the above possible pathways (such as interaction, upstream regulatory elements, ABA signaling, flavonoid biosynthetic pathway).

Conclusions
A total of 41 JrPP2C genes were identified and their distribution on chromosomes, gene structure, conserved motif, and evolutionary relationships were analyzed. The results show that JrPP2Cs are highly conserved, and the protein structure contains special sequences of the PP2C family. JrPP2Cs could express in most tissues, and JrPP2C08 is transcript most obviously. Under the stresses of drought, salt, heavy metals, ABA and anthrax bacteria, the relative expression of most walnut JrPP2C genes changed significantly, among which JrPP2C09, JrPP2C28, JrPP2C17, and JrPP2C36 were relatively obvious, which deserve further attention and research, and these results implyed that walnut JrPP2C genes may resist drought, salt stress, heavy metals, and anthrax. This study provides useful information for further study of the function and response mechanism of the JrPP2C genes.