Identification and Functional Characterization of Apple MdCKX5.2 in Root Development and Abiotic Stress Tolerance

Abstract

Cytokinin oxidase/dehydrogenases (CKXs) are the key enzymes in cytokinin degradation and have been widely studied in model plants. Little is known about apple’s (Malus×domestica) CKX genes. Here, using genome-wide analysis, we identified 10 MdCKX genes in apple. The phylogenetics, chromosome locations, and genome structures were then tested. Expression analysis showed that MdCKX genes had different expression profiles in apple, pointing to the different roles. Meanwhile, relative expression analysis showed that these genes have different expression patterns in response to several exogenous cytokinin factors, including trans-zeatin (ZT), thidiazuron (TDZ), and N6-furfuryladenine (KT). Finally, we introduced the MdCKX5.2 gene into Arabidopsis to evaluate its functions, and the results suggested the transgenic Arabidopsis displayed phenotypes related to promoting primary root and lateral root development, response to exogenous ZT, and conferring to drought and salt tolerant. Taken together, our results provide insights on the possible application of the MdCKX5.2 gene for molecular breeding in apples.

1. Introduction

Cytokinins (CTKs) play several essential roles in plant development, such as seed germination, leaf senescence, root and shoot branching, photosynthesis, flower, and fruit development, and act in response to environmental signaling [1,2,3,4]. The fine-tuned control of CTKs is properly maintained by their biosynthesis and metabolism [5]. The enzyme isopentenyl transferase (IPT) catalyzes CTKs synthesis in plants [4]. The irreversible degradation of CTKs is catalyzed by cytokinin oxidases/dehydrogenases (CKXs), which are the only known enzymes that specifically degrade CTKs and are encoded in plants by a small gene family [6]. The CKX enzyme, a flavor enzyme that contains a FAD-binding domain and a cytokinin-binding domain, contributes to transferring the electrons of CTKs to electron acceptors by the flavine cofactors [5,7] and catalyzing the degradation of CTKs [8,9], respectively.

The first CKX gene family has been identified in maize [10,11]. Several CKX genes have been identified and characterized in tobacco [12], Dendrobium orchid [13], Arabidopsis thaliana [14,15], Hordeum vulgare [7], Oryza sativa [16,17], Lotus japonicas [18], Vitis vinifera L. [19], and Medicago truncatula [20]. Therefore, it is well demonstrated that CKX family genes regulate plant growth and development by influencing the activity of CTKs. AtCKX1 overexpression results in cytokinin-deficient phenotypes, including the enhancement of root growth and inhibition of shoot development [14,15]. Correspondingly, the ckx3/ckx5 double mutant containing a higher concentration of CTKs leads to larger flowers, more flower primordia and siliques, and increased seed yield compared with wild type [21]. Further studies on natural rice variants, in which expression of OsCKX2 is reduced, found that CTK’s accumulation in inflorescence meristems results in the number of reproductive organs being increased and the grain yield being improved. However, decreasing the CKX enzymes leads to an increase in root mass and plant productivity in barley [22].

Besides the impact on developing plant organs, CKX genes also engage in the plant’s environmental responses. Most AtCKX genes are upregulated with drought treatment or salt treatment [23,24,25]. Several studies have shown that CKXs respond to abiotic stress. Ectopic expression of the AtCKX1 gene in tobacco increased drought tolerance [26,27]. Introducing the AtCKX1/2/3/4 overexpression vectors driven by the 35S promoter confers various degrees of resistance to Arabidopsis [23]. Further, the CKX family genes present different expression profiles under treatment with exogenous phytohormones. CKXs are almost upregulated in response to exogenous 6-benzylaminopruine (6-BA) in apple [28], while the expression of CKX is increased when treated with abscisic acid (ABA) in Medicago sativa [29]. Taken together, these studies showed that CKX genes play a vital role in plant development and response to environmental factors. Whole-genome analyses of the CKX gene family have also been processed, following the release of the full genome sequences in many plants. There are 7 CKX family members in Arabidopsis [6], 13 in maize [30], 11 in rice [16], 12 in Chinese cabbage [31], 5 in potato [32], and 8 in grape [19]. Despite the roles of the CKX gene in some model plants being widely studied, there is little information regarding the biofunctions of the apple CKX family genes. Here, we identified 10 MdCKX family genes by the apple genome-wide search. The distribution of MdCKXs in the genome, evolutionary relationships, motif characteristics, organ-specific expression patterns, and response to exogenous CTKs are also analyzed in detail. Finally, the MdCKX5.2 gene was characterized after being introduced into Arabidopsis to evaluate its functions.

2. Materials and Methods

2.1. Plant Materials and Growth Conditions

For gene expression analysis of different tissues in apple (Malus×domestica), 5-year-old ‘Royal Gala’ cultivar trees derived from an apple somatic clone were used. Stems, leaves, flowers, fruits, and roots were bagged and quickly frozen in liquid nitrogen, and then stored in a refrigerator at −80 °C separately.

For gene expression analysis of different CTKs and gene cloning, seedlings of the one-month-old live seedling ‘tea crabapple’ were applied. Before treatment, the seedlings were placed at 25 °C with a continuous 16-h-light/8-h-dark photoperiod for four weeks. The seedlings were then hydroponically cultured in solutions containing 100 µM ZT, TDZ, and KT separately for 0 h, 3 h, 6 h, and 12 h. Finally, the whole plants were collected and frozen in liquid nitrogen for RNA extraction.

The Arabidopsis materials were cultured on MS medium containing 0.7% agarose under a short-day (8-h-light/16-h-dark) photoperiod for 6 days and then grown in long-day conditions (16-h-light/8-h-dark) at 20–22 °C in the greenhouse.

2.2. Genome-Wide Identification of MdCKXs in Apple

Based on BLASTP (with E-value less than 10 × 10⁻⁵), apple CKX proteins were identified in the Malus×domestica_v1.0_consensus_peptide dataset (downloaded from https://www.rosaceae.org/tools/jbrowse (accessed on 18 April 2019)) using 7 AtCKX proteins as search sequences. Further, SMART (http://smart.embl.de/smart/batch.pl (accessed on 19 April 2019)) and Pfam databases (http://pfam.xfam.org/search#tabview=tab1 (accessed on 19 April 2019)) were used for filtering sequences that had incomplete domains and inconsistent lengths. Candidate genes of MdCKXs were named according to homologous AtCKXs. The information of molecular weight and theoretical pI (isoelectric point) of MdCKXs were obtained from the ProtParam tool of Expasy (https://web.expasy.org/protparam/ (accessed on 22 April 2019)). The information on the genomic distribution of MdCKXs was obtained from the GDR database. MdCKXs and other CKX proteins from other plant species were used for building the evolutionary tree. Multiple-sequence alignment of CKX proteins was carried out with Clustal X, and the neighbor-joining tree was constructed in MEGA 5.0 with 1000 replicates.

2.3. Vector Construction and Arabidopsis Transformation/Hybridization

The full-length MdCKX5.2 ORF was cloned from the ‘tea crabapple’ and was then cloned into the pBI121 vector. The primers used for gene amplification and vector construction were 5′-ATTTCCTCTCACTCTGTCCCTC-3′ (forward) and 5′-CAGTATATGTACAAGACTAACCC-3′ (reverse). MdCKX5.2 was then introduced into Arabidopsis Columbia (Col-0) through the floral dip method using Agrobacterium strain GV3101 [33]. The MdCKX5.2-transgenic Arabidopsis were individually harvested in the T1 generation, and then positive plants which showed 3:1 separation in T2 generation were used to produce T3 generation. Finally, the T3 generation of homozygous transgenic lines was detected and applied for further research.

The ARR5::GUS/35S::MdCKX5.2 and DR5::GUS/35S::MdCKX5.2 transgenic lines were generated by crossing the MdCKX5.2 lines with ARR5::GUS and DR5::GUS lines, respectively. The seeds were surface sterilized and sown in MS medium after stratification treatment for 3 days at 4 °C. In addition, the seeds were germinated under a 16-h-light/8-h-dark photoperiod at 22 °C before treatment.

2.4. Analysis of Exogenous ZT Treatment

Six-day-old seedlings, including wild-type (WT) lines and transgenic (L1, L8, and L11) lines, were placed in MS medium containing different concentrations (0.1/1/10 μM) of ZT. The seedlings were grown vertically for several days. The primary root (PR) length and lateral root (LR) number were then measured.

2.5. GUS Staining

The GUS staining buffer (0.1 M NaH₂PO₄ (pH = 7.0), 10 mM EDTA, 0.5 mM ferricyanide, 0.5 mM ferrocyanide, 1.9 mM X-glucuronide, and 0.1% Triton X-100) [34] was used to immerse individual representative plants at 37 °C for 10 h in the dark. Then, 95% ethanol was used to dissolve the chlorophyll of stained seedlings, and the seedlings were then imaged.

2.6. RNA Extraction, cDNA Synthesis, and Gene Expression Analysis

The method of total RNA extraction of apple organs was carried out as described [35], and Trizol reagent (Invitrogen, Waltham, MA, USA) was used to extract Arabidopsis total RNA. The PrimeScript First Strand cDNA Synthesis Kit (Takara, Beijing, China) was used to synthesize the first-strand cDNA. Semiquantitative RT-PCR was carried out to detect the gene expression pattern of different tissues. qRT-PCR was conducted with the UltraSYBR mixture (SYBR Green I) (Tiangen, Beijing, China) and calculated using the 2^−ΔΔCt method. There were 40 cycles, and the annealing temperature was 60 °C. Apple and Arabidopsis Actin genes were used as internal controls. The primers used for gene expression analysis, including melting temperature, R², and efficiency of each primer, are listed in Table S1. In all experiments, three independent replicates were used to calculate the values for the mean expression and standard deviation (SD).

2.7. Stress Tolerance Assays

Salt and drought stress tolerance experiments were carried out, as described before [36]. For drought stress treatment, the plants were pre-cultured on normal condition for 2 weeks and subsequently transferred to incubators (22 °C, 16-h-light/8-h-dark photoperiod, 65 mmol m^–2 s^–1 photon flux density and 50–60% relative humidity) for an additional 20 days for drought stress treatment until most of the plants undergo lethal effect on dehydration. After re-watering for 2 days, the survival rates were calculated.

For salt tolerance analysis, plants were grown on MS medium for 6-day-old and then were transferred into MS medium plates containing 0, 100, and 150 mM of sodium chloride (NaCl) for 9 days. More than 12 seedlings of each line were used for one experiment in different conditions. PR length, LR numbers, and fresh weight were measured based on the results from three independent experiments.

2.8. Chlorophyll Extraction

Leaves were cut from treated plants, and then immersed in tubes containing 5 mL of 96% ethanol. The tubes wrapped in tin foil were set in a shaker overnight in dark. Until leaves were completely discolored, their solution absorbance was measured at 665 nm and 649 nm. The total chlorophyll content was calculated by 6.63 × (A665) + 18.08 × (A649).

2.9. Statistical Analysis

The software R (3.0.2) with the R Commander package was used to perform the statistical analysis. p < 0.05 and ** p < 0.01 were taken as statistically significant differences. The small letters (a, b, c, and d) produced by the analysis of variance (ANOVA) indicate statistically significant differences (p < 0.05) between different groups. Each experiment was repeated at least three times, and three parallel technical replicates were performed.

3. Results

3.1. Whole-Genome Identification of the Apple CKX Genes

To identify all apple CKX genes, the seven AtCKXs proteins were employed to perform blastp searches against the GDR database (http://www.rosaceae.org/ (accessed on 18 April 2019)). As a result, 10 MdCKXs proteins, containing a conserved FAD domain and a cytokinin binding motif, were identified (Table S2). These genes were named based on their homology with Arabidopsis CKXs. The gene identifier, length of amino acid, protein mass, isoelectric point (pI), and genomic position are noted in Table S2. As shown in these results, the identified apple CKX genes encoded proteins that ranged from 472 to 577 amino acids, with the protein mass ranging from 52.9 kD (MdCKX6.1) to 62.5 KD (MdCKX1.2) and protein pI from 5.52 (MdCKX5.2) to 8.31 (MdCKX1.1). The evolutionary relationships between the CKXs proteins in different plants were then identified based on the full-length amino acid sequences. To construct the unrooted phylogenetic tree using MEGA 5, multiple-sequence alignment files were used. Based on the similarity and topology of the protein sequence, the CKXs were divided into four groups (group I to group IV) (Figure 1). The locations of the MdCKXs in apple chromosomes were then performed. The results revealed that these 10 MdCKX genes were mapped on 7 of 17 apple chromosomes, and 2 sister pairs of paralogous CKXs (MdCKX6.1/6.2, MdCKX7.1/7.2) were found on the duplicated blocks (Figure S1), proving that a scale segmental duplication event occurred in the apple genome. The conserved domains of MdCKX proteins were then identified and an alignment within all the MdCKXs and AtCKXs proteins was performed. Like the AtCKXs proteins, the results showed that all 10 MdCKXs proteins had a deeply conserved FAD-binding domain in the N-terminus and a cytokinin-binding domain in the C-terminus. Furthermore, a conserved GHS motif, which is involved in maintaining the structural stability of FAD [8,37], was also found in the FAD-binding domain (Figure S2).

3.2. Organ Expression and CTKs Response Analysis of the MdCKX Genes

CKX-family genes have various expression patterns in Arabidopsis and other species [13,14,38]. To gain some insights into the potential role of MdCKXs, we analyzed the spatial expression patterns by semiquantitative RT-PCR. As shown in Figure S3, MdCKX genes had different spatial transcription patterns. MdCKX1.1, MdCKX5.2, and MdCKX7.1 were expressed mainly in roots. MdCKX6.1 was mostly accumulated in leaves, while MdCKX6.2 was mainly expressed in flowers. MdCKX3, MdCKX4, and MdCKX7.2 were expressed in most organs. Additionally, MdCKX3 had a stronger band in the lanes of leaves and MdCKX7.2 in the fruits (Figure S3).

CKXs are differentially upregulated when exposed to benzyl aminopurine (BA) [28,39]. The expression of CKX is induced when treated with 6-benzylaminopurine (BAP) in D. huoshanense [14] and is also activated in response to various CTKs in Dendrobium orchid [13]. To detect the roles of MdCKX-family genes in response to exogenous CTKs (ZT, TDZ, and KT), qRT-PCR was then utilized to analyze the gene expression levels of one-month-old apple seedlings when treated with different kinds of CTKs for 0, 3, 6, and 12 h (Figure 2). The results showed that the transcriptional levels of MdCKX3/5.1/5.2/6.1/6.2/7.1 were significantly upregulated after treatment with several CTKs, while the opposite was true for other MdCKXs. ZT and TDZ had a remarkable effect on the induction of expression of MdCKX3/5.1/5.2/6.1/7.1. In contrast, KT had a weak effect on these genes. Additionally, in most cases, the inhibitions were well established after 6 h, while the inductions were completed after 3 h. In addition, the expression level of MdCKX7.1 was upregulated and then downregulated; and the downregulation of MdCKX1.1/4/7.2 was most significant after 3 h in KT treatment. Additionally, we found that the expression level of MdCKX3 was significantly upregulated after TDZ and ZT treatment compared with other genes. These results indicated that MdCKX-family genes may play a complex role in controlling cytokinin metabolism in apple.

3.3. MdCKX5.2 Transgenic Arabidopsis Affect Lateral Root Development and Activity of Cytokinin and Auxin

Several studies have focused on CKXs in model species [14,26], and apple CKX-family genes in axillary buds have also been studied well [28]. However, analyzing the role of stable apple CKX overexpressor in plant development could further provide a deeper understanding of those genes. To explore the roles of MdCKX in root development, we selected the MdCKX5.2 gene which was detected mostly in roots in our study, constructed the overexpression vector 35S::MdCKX5.2, and then introduced it into Col-0 by the floral dip method. Consequently, three individuals homozygous T3 lines (L1, L8, and L11) were selected for further functional analysis based on their higher expression levels than WT (Figure S4). After preculturing in MS medium, it was found that the average primary root (PR) lengths of transgenic lines were 90 mm, but WT lines were just 70 mm (Figure 3a,c). Additionally, we found that the number of lateral roots (LRs) in the transgenic strains was 3.25 to 4 times higher than the wild type (Figure 3a,b). These results suggested that MdCKX5.2 expression promotes PR elongation and LRs development.

CKXs engage in the irreversible degradation of CTKs [5]. To test whether 35S::MdCKX5.2-transgenic Arabidopsis lines influence cytokinin conditions, we introduced the cytokinin-responsive ARR5::GUS lines into the MdCKX5.2-overexpressing and WT background. The cytokinin activity can be reflected by GUS signaling after staining in these lines. It was found that GUS activity in the WT was higher than in the overexpressed background. GUS signaling was detected at the primary root tips and lateral root primordia regions in the WT background. GUS signaling was also detected at the tip of PRs in the overexpression background but was barely present at the lateral root primordia regions (Figure 4a). These results demonstrated that overexpression of MdCKX5.2 diminishes CTKs activity in Arabidopsis root.

Previously, a study showed that CTKs cause auxin redistribution and cell differentiation [40]. Combined with the phenotypes of the overexpressor roots, we speculated that MdCKX5.2 may also engage in this. Therefore, the auxin-responsive marker lines, i.e., DR5::GUS lines, were crossed with the MdCKX5.2-overexpressing and WT lines for further experiments. Auxin signaling is displayed after GUS buffer staining. The results showed that GUS signals accumulated at the tips of LRs primordia during the initiation of LRs, while also being assembled at PR tips. Additionally, the stronger signals can be more distinctly observed in the overexpression compared with the WT background (Figure 4b). In combination with the results that ARR5::GUS activity was reduced in the MdCKX5.2 transgenic Arabidopsis, it was concluded that MdCKX5.2 influenced the activity of cytokinin and auxin in the roots. To be more specific, it reduced the signaling of cytokinin as well as activated the auxin.

3.4. 35S::MdCKX5.2 Plants in Response to Exogenous ZT

It has previously been reported that cytokinins repress root development [14,15]. As described above, the MdCKX5.2-overexpressing lines exhibited cytokinin-deficient-like phenotypes in regulating developing LRs and PR. Then, the response of the MdCKX5.2-overexpressing lines to exogenous cytokinin was examined. The transgenic MdCKX5.2 and WT lines were treated with exogenous ZT in various levels (0/0.1/1/10 μM). The result showed that increasing ZT concentrations, primary root length, and lateral root number were notably inhibited both in MdCKX5.2-overexpressing lines and wild-type plants. In other words, when growing in a medium without ZT, the overexpression lines have longer PR length (average 86.3 mm) and a higher LR number (average 12) than WT lines, which agrees with Figure 3. The LRs number of L1/L8/L11 was higher than WT lines in 0/0.1/1 μM ZT conditions. Additionally, PR length between transgenic and WT lines progressively becomes no significantly different when ZT concentration increased to 1 μM (Figure S5). These results suggested that root development was influenced when exposed to exogenous ZT and that MdCKX5.2-overexpressing lines could degrade exogenous ZT; however, this effect was diminished when the concentration was increased to 10 μM. To better assess the inhibitory effect of exogenous ZT on root development, obtained data were expressed as a percentage of the 0-μM condition. The percentage of LR number was almost similar regardless of ZT concentration in all lines, while the percentage of PR length in transgenic lines was lower than WT when ZT concentration was up to 0.1 μM (Figure 5a–c). These results suggested the LR development was equally sensitive to exogenous ZT in transgenic and WT lines. Meanwhile, the overexpression of MdCKX5.2 increases the sensitivity to exogenous ZT during PR elongation.

3.5. Ectopic Expression of MdCKX5.2 Enhanced Drought Tolerance in Arabidopsis

CKXs play a positive role in responding to abiotic stress [26,27,29,41]. To investigate the role of MdCKX5.2 in response to water stress, the transgenic lines (L1, L8 and L11) and WT lines were well irrigated until they were 2 weeks old. They were then deprived of water for 20 days and re-watered after 2 days. WT plants showed severe wilting symptoms (withered and yellow leaves, dried-up stems), while all three transgenic lines were relatively normal, as shown in Figure 6a. The survival rates of transgenic lines (average 60% to 66%) were significantly higher than WT lines (average 7%) after being re-watered (Figure 6b). These results indicated that the overexpression of MdCKX5.2 in Arabidopsis confers drought tolerance.

3.6. Ectopic Expression of MdCKX5.2 Enhanced Salt Tolerance in Arabidopsis

To gain more insight into the role of MdCKX5.2 in abiotic stress tolerance, the transgenic (L1, L8, and L11) and wild-type (WT) seedlings were pre-germinated on MS medium for 6 days and then transferred to media containing 100 mM and 150 mM of NaCl. As shown in Figure 7a, the transgenic and WT lines grew normally on MS medium, but WT lines were relatively smaller than transgenic lines. When exposed to 100 mM and 150 mM NaCl for 10 days, the leaf discolored and wilted in all lines, but the symptoms were less severe in transgenic lines. The primary root length, lateral roots number, and fresh weight of transgenic lines were significantly lower than that of WT lines in all conditions (Figure 7b–d). The chlorophyll content was also detected, which showed that the content of transgenic lines was significantly higher than WT lines in salt conditions, while there was no difference in normal MS medium. These results showed that overexpression of MdCKX5.2 in Arabidopsis improves resistance to high salinity stress during early seedling development.

4. Discussion

Based on Arabidopsis CKX genes, we identified 10 MdCKXs in Malus×domestica. MdCKXs are distributed on seven chromosomes. Previously, some studies found that CKX genes in Arabidopsis and other species showed diverse expression patterns in different organs [14,20,24,28], which was also confirmed in apple using RT-PCR analysis (Figure 3). In this result, the MdCKX genes with special expression patterns are noteworthy. For example, MdCKX7.2 has stronger bands in fruits lanes and MdCKX3 has stronger bands in leaves lanes, suggesting that these genes may play a major role in fruit and leaf development, respectively. MdCKX5.2 and MdCKX7.2 are mainly expressed in roots, which suggests the essential functions of these genes in roots development. Several studies have found that CKX genes respond to various CTKs [20,29]. In our study, MdCKX genes have different CTK sensitivity, of which most expressions were highly upregulated or downregulated in response to TDZ and ZT, except for KT (Figure 4). This is an interesting finding that promotes exploration, of which CTKs are best for growing apples. Some CKX genes were upregulated when treated with CTKs, which indicated that they are involved in the metabolism of matching CTKs. Further, MdCXK1.1/1.2/4/7.2 were downregulated, while the CKX genes were induced in Arabidopsis when treated with CTK [24]. We believe that these genes are involved in the regulation of negative feedback, to maintain the balance of CTK levels in apple.

CKX overexpressors showed a cytokinin-deficient phenotype, such as elongation of primary root or increased root branching [14,26,27,30,38]. A previous study focused on the CKX expression profiles during apple axillary bud outgrowth, which provides a great foundation for further analyzing the role of these genes in shoot branching [28]. In our study, ectopic expression of MdCKX5.2 in Arabidopsis showed the cytokinin-deficient phenotype, which provides a direct insight into CKX’s role in root development.

In Figure 4a, ARR5::GUS lines in the MdCKX5.2-overexpressing background display lower GUS activity, indicating that the concentration of CTKs was downregulated in the root. This has also been confirmed in 35S::AtCKX1 and 35::AtCKX7 lines [14]. It is suggested that MdCKX5.2 is a classical CKX enzyme, influencing CTKs in the conserved way, similar to other CKX species. The DR5::GUS lines were used to study the auxin condition in root. The GUS signals were distinctly detected during LRs initiation (Figure 4b), and L1/L8/L11 transgenic lines exhibited more LRs than WT lines (Figure 3b). These results suggest that the increased concentration of auxin in LR primordia regions is essential for LRs development in Arabidopsis, thus contributing more LRs in the overexpressed lines. Additionally, in Figure 4, the GUS signals were significantly accumulated at PR tips in DR5::GUS/35S::MdCKX5.2 lines, but not ARR5::GUS/35S::MdCKX5.2 lines, which is consistent with a longer PR length than the WT phenotype. Based on these results, we assumed that MdCKX5.2 plays an important role in the architectural construction of apple roots.

Drought is one of the most important abiotic stresses affecting the yield of most crops [42]. To enhance the drought tolerance of crops, genes that controlled root traits, including root volume, root length, and the root/shoot ratio, were thought to be valuable for genetic engineering breeding [43,44]. CKXs play a positive regulator in root growth and response to drought stress [45]. Therefore, reducing the endogenous cytokinin levels of plants by enhancing CKXs expression levels may be a promising aspect for gene engineering breeding to cope with drought stress. Several studies showed that enhancing cytokinin oxidase activity in Arabidopsis and tobacco significantly increases root biomass and root/shoot ratio, which results in improved drought stress resistance [26,27]. In our results, the MdCKX5.2 overexpressor showed relatively stronger drought resistance than the WT lines. One reason for this is perhaps that the root system of the overexpression lines is relatively more robust (Figure 3).

Salt and drought stress signaling are closely related, and their mechanisms overlap with each other [46]. Salt stress reduces water uptake efficiency and decreases osmotic pressure, leading to water-deficient symptoms [47]. Therefore, robust root systems facilitate uptake water [44] and help to cope with the salt stress. In our results, the transgenic lines showed robust root systems and exhibited tolerance to 100-mM and 150-mM NaCl treatments. Besides, the overexpression of AtCKX1/2/3/4 genes showed that its survival rate significantly increased compared with WT lines. Along these lines, ABA biosynthesis-related genes decreased, implying that CKX synergistically regulates salt stress with ABA [23]. Ectopic expression of the MsCKX gene from Medicago sativa in Arabidopsis enhanced salt tolerance by maintaining a higher chlorophyll content, decreasing the levels of ROS, and increasing the expression levels of stress-related genes [30]. In our results, transgenic lines keep higher chlorophyll content than WT lines. Moreover, the expression of stress-associated genes and ROS levels need to be further studied. Taken together, our results showed that the MdCKX5.2 gene may be valuable for breeding drought and salt resistance.

5. Conclusions

We identified 10 apple CKX genes in the apple genome by using Arabidopsis CKXs and found that they had FAD-binding and cytokinin-binding domains. The expression pattern suggested that CKX genes, with a variety of expression profiles in several organs, are responsive to different CTKs. Subsequently, we demonstrated that the MdCKX5.2-overexpressing plants change the activity of CTK and auxin, regulate PR elongation and LRs development, and respond to exogenous ZT. Besides, the overexpressor showed higher drought and salt stress tolerance than WT plants.