Previous studies showed that the members of the CBL family in plants could be divided into four groups. A phylogenetic analysis based on the protein sequences of the 11 MdCBLs and 10 AtCBLs was performed to investigate the relationship between these 11 CBL family members in apples. The phylogenetic tree showed that the 11 MdCBL proteins could be divided into four groups, similar to the grouping results in *Arabidopsis* and many other plant species (Figure 2A).

**Table 1.**Characterization of the CBL family genes in apple.

Best hits in *Arabidopsis* were determined by local blastp method. Sequence length, mass weight, pI, and charge at PH 7.0 of the revised proteins (MdCBL10.1, MdCBL10.2, and MdCBL3.2) were listed in parentheses.

**Figure 1.** Genome locations of CBL family genes in apple. Purple lines and red font indicate the segmental and tandem duplication genes, respectively. Collinear blocks are represented by grayish lines. Chr00 represents the unassembled genomic scaffolds.

**Figure 2.** Phylogenic relationships (**A**), gene structure (**B**), and conserved motifs (**C**) for CBL family proteins in apples and *Arabidopsis*. Green and red dots indicate CBL proteins in *Arabidopsis* and apple, respectively. Red lines show that the fifth exon of *AtCBL7* and *AtCBL3* could be further differentiated into two shorter exons. Introns are represented by black line segments of the same length to facilitate the comparison of the exon–intron composition patterns of these genes.

Gene structure is one of the factors that reflects the evolution of a multigene family. Thus, the exon–intron composition patterns of these *MdCBL* and *AtCBL* genes were analyzed. Although the length of introns varied significantly (Figure S2), genes that belonged to the same group exhibited the same exon–intron composition patterns, with the largest variation in the N-terminal region of these genes (Figure 2B). Based on gene structure analysis, three *MdCBL* genes with partial exons missing compared with other *CBL* genes in the same group, namely *MdSOS3.2*, *Md10.1*, and *MdCBL3.2*, were also identified. This result suggested that the predicted coding sequences of these three genes in apple genome (GDDH13) might be wrong. The conserved motifs of the CBL family were further explored using the online software MEME, and nine conserved motifs were found (Figure S3). The motifs 1–5 were conserved in almost all CBL proteins except MdSOS3.2 and MdCBL3.2 (Figure 2C). Besides, MdCBL10.1 was the missing part of the N-terminal compared with other proteins in group B (Figure 2C). These results further supported the presence of errors in the prediction of coding sequences of these three genes.

Gene-specific primers were designed based on the predicted sequences in the apple genome (GDDH13) and used for gene cloning to confirm the coding sequence of *Md-CBL* genes. Using total RNA extracted from the leaves of "Golden Delicious" apple as the template, the coding sequences of these *MdCBL* genes were finally obtained by polymerase chain reaction (PCR) amplification, except for *MdSOS3.2* (Supplementary File S2). The sequence alignment showed that the predicted protein sequences of MdCBL10.1 (MD09G1194600) and MdCBL3.2 (MD11G1037200) were missing a segment of N-terminal and C-terminal, respectively (Figure S4A). Although the coding sequence of *MdSOS3.2* was not obtained, the sequence comparison between MD01G1081000 and MdSOS3 indicated an incorrect fragment deletion and an incorrect fragment insertion in the N- and C-terminals of MD01G1081000, respectively (Figure S4B).

#### *3.3. Promoter Analysis of MdCBL Genes*

CBL family genes are involved in plant response to various environmental stresses. The promoter regions (upstream 1500 bp of the start codon ATG) of these genes were obtained from the apple genome (GDDH13) and submitted to the online software PlantCARE for *cis*-acting element analysis to explore the possible response of *MdCBL* genes to abiotic stresses. Various *cis*-elements related to abiotic stresses (hypoxia, cold, and drought) and plant hormone (ABA, auxin, MeJA, ethylene, GA, and SA) responsiveness were found (Figure 3 and Supplementary File S3), suggesting that these *MdCBL* genes played an important role in apple stress response. Many of these *cis*-elements appeared multiple times in the promoter region of the same gene (Figure 3). Besides, more hormone response–related *cis*-elements were present in the promoter regions of *MdCBLs* in groups A and B, especially *cis*-elements related to ABA and MeJA, while the *cis*-elements in the promoter regions of *MdCBLs* in groups C and D were mostly related to abiotic stress response (Figure 3). This indicated the functional differentiation of *MdCBL* genes between different groups.

**Figure 3.** *Cis*-element analysis of the *MdCBL* gene promoter regions in apple. Arrows and triangles indicate the *cis*-elements related to abiotic stress response and hormone response, respectively. Positive and negative directions indicate whether the motif existed in the plus or minus strand of the *cis*-acting elements, respectively.

#### *3.4. Functional Identification of Mdcbls in Regulating Salt Tolerance in Yeast*

Previous studies demonstrated that CBL proteins affected plant salt tolerance through the SOS pathway. The yeast mutant strain ∆*ena*1−4 that lacked Na+ -ATPase and was sensitive to high [Na+ ] was used to identify which CBL proteins in apples had a significant regulatory effect on salt tolerance. The full-length coding sequences of the ten *MdCBL* genes were cloned into the pDR196 vector and then transformed into the yeast mutant ∆*ena*1−4. The yeast strain W313-1B and positive transformants of the empty vector pDR196 were used as positive and negative controls. All of these strains were cultured in a YPD medium containing different concentrations of NaCl for three days. The growth of all strains was almost the same in a normal YPD medium (0 NaCl) (Figure 4). The addition of NaCl significantly inhibited the growth of these strains. The growth of the yeast mutant ∆*ena*1−4 was almost completely inhibited at a 200 mM NaCl concentration. The overexpression of any one of the seven *MdCBL* genes (MdCBL5, MdCBL10.1, MdCBL10.2, MdCBL1.1, Md-CBL1.2, MdCBL3.1, and MdCBL3.2) could significantly inhibit the sensitivity of ∆*ena*1−4 to a high NaCl concentration (Figure 4). These results indicated that these seven *MdCBL* genes played a positive role in modulating salt tolerance in yeast. The growth of *MdCBL5* transgenic yeast was also completely inhibited with the further increase in the NaCl concentration (Figure 4), indicating a stronger capacity of the other six *MdCBL* genes than *MdCBL5* in enhancing yeast salt tolerance.

**Figure 4.** Functional identification of *MdCBLs* in the Na+ -sensitive yeast mutant. Aliquots (10 µL) of serial dilutions (100 , 101 , 102 , 103 , and 104 ) were dotted onto the YPD medium in the presence of 0, 100, 150, 200, 300, and 500 mM NaCl and grown for 3 days. W303-1B was the positive control. ∆*ena1*−*4* and ∆*ena1*−*4* transformed with the pDR196 empty vector were used as negative controls.

#### *3.5. Overexpression of MdCBL10.1 Improved Salt Tolerance of Cisgenic Apple Calli*

The SOS3/CBL10-SOS2-SOS1 and CBL10-CIPK8-SOS1 signaling pathways are the paramount regulatory mechanisms for facilitating Na+ extrusion and are critical to the ability of plants to adapt to high salinity [31,41]. Based on the functional identification results in yeast, *MdCBL10.1* was selected for the subsequent transgene and functional identification in apple. Full-length CDS of *MdCBL10.1* was cloned into the pBI121 vector and transformed into apple calli. PCR identification and quantitative reverse transcription (qRT)-PCR expression analysis demonstrated that several cisgenic lines with high *MdCBL10.1* expression were obtained (Figure S5). Three lines (OE-3, OE-4, and OE-7) with high *MdCBL10.1* expression levels were selected for NaCl treatment.

After 20 days of culture, no significant difference was found between OE lines and wild type (WT) on the normal MS medium. The growth of all lines was inhibited under NaCl treatment, with significantly reduced fresh weight compared with the apple calli cultured in MS medium (Figure 5A,B). However, the growth of *MdCBL10.1* cisgenic lines was significantly better, and the fresh weight was significantly higher than that of the WT under salt treatment. These results suggested that the overexpression of *MdCBL10.1* could significantly improve the salt tolerance of cisgenic apple calli (Figure 5A,B). In *Arabidopsis*, CBL10 participates in the SOS pathway and promotes the Na+ efflux [31,41]. Therefore, the Na+ content of the apple calli was measured. No significant difference was observed between different lines cultured in the normal MS medium. Under NaCl treatment, the Na+ contents of three cisgenic lines were significantly lower than that of the WT (Figure 5C), suggesting that the overexpression of *MdCBL10.1* could inhibit the excessive accumulation of Na+ in cisgenic apple calli.

**Figure 5.** Functional identification of *MdCBL10.1* in cisgenic apple calli. (**A**) Phenotypes of the cisgenic (OE) and wild-type (WT) apple calli treated with NaCl stress. The scale bar represents 1 cm. Fresh weight (**B**) and Na+ content (**C**) in cisgenic and WT apple calli. For (**B**) and (**C**), error bars represent the SD of three independent biological replicates, with each biological repeat having at least three dishes. Bars labeled with \* in each panel are significantly different from the WT (*p* < 0.05, Student *t* test).

#### *3.6. Overexpression of MdCBL10.1 in Roots Enhanced the Salt Tolerance of Apple Plants*

The use of *A. rhizogenes* K599 to obtain plants with cisgenic roots provides a convenient way for studying the function of *MdCBL10.1* in apple plants because of the low genetic transformation efficiency [39,40]. Full-length CDS of *MdCBL10.1* was cloned into the pCAMBIA2300 vector fused with a GFP tag, and a selected inhibitory fragment of *MdCBL10.1* was cloned into the RNA-interference vector pK7GWIWG2D. Through GFP fluorescence detection and expression analysis (Figure S6), 20 plants with high *MdCBL10.1* expression (MdCBL10.1-OE) and 20 plants with *MdCBL10.1* expression significantly inhibited (MdCBL10.1-RNAi) were selected for subsequent salt treatment. Plants with their roots transformed with empty vectors pCAMBIA2300-GFP (OE-EV) and pK7GWIWG2D (RNAi-EV) were used as controls.

Under normal conditions (control group), no significant difference was observed between different types of cisgenic plants. After ten days of 150 mM NaCl irrigation, the RNAi plants were severely damaged by salt treatment, with their leaves showing obvious yellowing, wilting, and even death. Compared with the RNAi and control lines, the OE plants exhibited a better growth state, with their leaves still bright green and vigorous (Figure 6A). The measurements of relative ion leakage (REL) and MDA content in the leaves of these plants also indicated that the OE plants suffered less, whereas the RNAi plants suffered more stress damage caused by NaCl treatment (Figure 6B,C).

**Figure 6.** Phenotypic analysis of *MdCBL10.1* cisgenic apple plants under 150 mM NaCl treatment. (**A**) Growth phenotypes of cisgenic apple plants after NaCl treatment. Scale bars represent 3 cm. (**B**) Relative electrolyte leakage (REL) of apple leaves. (**C**) Malondialdehyde (MDA) content in apple leaves. Representative chlorophyll fluorescence images (**D**), Fv/Fm ratios (**E**), and net photosynthetic rate (Pn) (**F**) of cisgenic apple plants under normal and NaCl stress conditions. (OE)-EV and (RNAi)-EV represent roots transformed with the empty vector pCambia2300 and pK7GWIWG2D, respectively. The values of each index for cisgenic plants of the same type are the average values from all lines. Values are means of 20 replicates ± SD (each plant acts as a biological replicate). \* in each panel denotes values significantly different from the corresponding control lines (*p* < 0.05, Student *t* test).

Besides REL and MDA content, the maximum quantum yield of PSII (Fv/Fm) is also an appropriate indicator for the early identification of the degree of damage in plants. Under normal conditions, the leaves of all lines were healthy and maintained high Fv/Fm ratios (Figure 6D,E). After NaCl treatment, the Fv/Fm of leaves of the RNAi plants decreased significantly, while that of the OE plants remained high (Figure 6D,E). The damage to photosynthetic units directly affected photosynthesis. After NaCl treatment, the net photosynthetic rate (Pn) of RNAi plants was significantly lower, while that of the OE plants was significantly higher than that of the control lines (Figure 6F). This was consistent with the performance of Fv/Fm and further supported that the overexpression of *MdCBL10.1* alleviated the stress damage to apple plants caused by NaCl treatment.

Salt stress triggers the accumulation of reactive oxygen species (ROS), which is harmful to plant growth. The accumulation of ROS in the roots of these NaCl-treated apple plants was determined to evaluate the damage caused by salt stress to root systems. The results showed that the roots of the OE lines accumulated less H2O2 and O2 −, while the roots of the RNAi plants accumulated more ROS than controls (Figure 7A,B). Since *MdCBL10.1* overexpression could inhibit Na+ accumulation in apple calli, the Na+ content in these apple plants after NaCl treatment was measured. As expected, the Na+ content in the roots and leaves of OE plants was significantly lower than that of the controls, while the RNAi plants showed the opposite trend (Figure 7C,D). These results indicated that *MdCBL10.1* overexpression in roots could reduce the Na+ content in both roots and leaves, thereby alleviating salt stress-induced damage to apple plants.

**Figure 7.** Overexpression of *MdCBL10.1* in apple roots inhibited the ROS and Na+ accumulation in cisgenic apple plants. (**A**) Hydrogen peroxide (H2O2 ) content in roots. (**B**) Superoxide anion (O2 −) content in roots. (**C**) Na+ content in roots. (**D**) Na+ content in leaves. The values of each index are the average values of all lines in cisgenic plants of the same type. Values are means of 20 replicates ± SD. \* in each panel denotes values significantly different from the corresponding control lines (*p* < 0.05, Student *t* test).

#### **4. Discussion**

Plants are inevitably exposed to a variety of adverse environmental conditions, such as water shortage, low temperature, high salinity, and so forth, due to the sessile lifestyle. These abiotic stresses are increasingly harmful to crop yield and quality. Calcium and its signaling pathway play an important role in plant stress response. As specific Ca2+ sensors, CBL proteins play vital roles in calcium signaling and stress resistance regulation [7]. Apple is one of the most economically important fruits in the world. Its cultivation and extension are restricted by various abiotic stresses. The study of CBL proteins is thus important for the resistance breeding of apple. To date, the CBL family proteins in many plant species have been identified at the genome-wide level. However, no detailed characterization of apple CBL family proteins has been reported, and little is known about their functions in abiotic stress response.

Ca2+ is an essential element for plant growth and survival, and Ca2+ signals are an important regulator of growth, development, and biotic and abiotic stresses in plants [7,38,42]. Studies in various fruit tree crops also have shown that Ca2+ plays an important role in regulating fruit development, ripening, quality, and storage [43–47]. The CBL family has been identified and systematically studied in many plant species, such as dicotyledons *Arabidopsis* (10 AtCBLs) [33,48], eggplant (5 SmCBLs) [36], cotton (13 GaCBLs, 13 GrCBLs, 22 GhCBLs) [1], Cassava (9 MeCBLs) [12], pepper (9 CaCBLs) [10], tea plant (7 CsCBLs) [32], and pigeon pea (9 CcCBLs) [49], as well as the monocotyledon rice (10 OsCBLs) [33,48], due to the important role of CBL proteins in Ca2+ signaling. The CBL family has also been identified in some fruit trees such as grapevine (8 VvCBLs) [8], banana (11 MaCBLs) [50], and pineapple (8 AcCBLs) [11]. Although the genomes of these species are significantly larger than that of *Arabidopsis*, the number of *CBL* genes in most of these species is not significantly higher than that in *Arabidopsis* [48]. In this study, 11 *MdCBL* genes were

identified from apples (Table 1), only one more than that from *Arabidopsis*, suggesting that the CBL family in apples had not expanded significantly during evolution. This was different from other gene families that were previously identified in apple, such as the bHLH [37], Lhc [51], and CaCA [38] families. Tandem and segmental duplication events are fundamental mechanisms of gene family expansion. Considering the two genome-wide duplication events that occurred during apple evolution [52], collinear analysis between these *MdCBLs* was performed. The results showed that only two segmental duplication events occurred (Figure 1), indicating that the CBL family did not greatly expand during apple evolution. The evolutionary analysis of many plant species also suggested that the number of CBL members was independent of their genome size [48].

Although the number of CBL members varied, the phylogenetic analysis of CBL proteins in various plant species indicated that this family should be divided into four groups. In this study, the MdCBL proteins were divided into four groups (groups A to D), as in *Arabidopsis* (Figure 2A). This grouping result was also supported by the gene structure analysis and conserved motif prediction. The intron/exon and motif composition patterns of genes within the same group were consistent, whereas significant differences were observed between different groups (Figure 2B,C). In addition, based on the comparison of gene structure and conserved motifs of genes within the same group, three *MdCBL* genes (*MdSOS3.2*, *MdCBL10.1*, and *MdCBL3.2*) that might have errors in their predicted CDS in the apple genome were identified. The gene cloning results confirmed this speculation (Table 1 and Figure S4) and further suggested that this comparative analysis method could help people identify genes in a gene family whose coding sequences were incorrectly predicted. On the contrary, the high similarity of gene structure, conserved motifs, and *cis*-acting elements (Figures 2 and 3) suggested functional redundancy among *MdCBL* genes, especially *MdCBL* genes in the same group. Similar results were also found in *Arabidopsis*, such as SOS3 and CBL10 [31].

Stress stimulation causes a transient increase in the intracellular Ca2+ concentration. CBL proteins can sense and interact with intracellular increased Ca2+. The binding of Ca2+ promotes the interaction between CBL and CIPK proteins, which is crucial for the activation of the kinase activity of CIPKs. Then, the activated CIPKs phosphorylate downstream substrates, further triggering a range of response mechanisms [7,33,42]. Decades of research have revealed extensive and complex interaction networks between CBL and CIPK family proteins. As Ca2+ sensors, each CBL has three or more EF-hand domains and Ca2+-binding sites. Besides, the CBL proteins harbor a conserved FPSF domain in their C-terminal, which is the target of phosphorylation by CIPK. Moreover, many of the CBL proteins also contain conserved MGCXXS/T motifs in their N-terminal, which contribute to the anchorage of CBLs in the membrane to transduce a Ca2+ signal [48–50,53]. Several studies proved that different CBLs could be localized to the plasma or vacuole membrane and could regulate plant salt tolerance by promoting the Na+ efflux or sequestration [7,42]. In this study, all MdCBL proteins contained the FPSF domain in their C-terminal (Figures 2, S3 and S4), suggesting complex interactions between MdCBLs and CIPK family proteins in apple. For example, four MdCBL proteins were found to interact with the CIPK family protein MdSOS2L1 [54]. Many of these MdCBLs contained the conserved MGCXXS/T domain in their N-terminal. Further, the distribution of this domain showed obvious group specificity, which was found only in groups A and C of the MdCBL family (Figures 2, S3 and S4). These results suggested that MdCBL proteins in these two groups might be more likely to function on the membrane and also indicated the functional and subcellular differentiation of CBL proteins between different groups. More studies on the subcellular localization and functional identification of apple CBL proteins are needed to confirm this hypothesis.

The CBL-CIPK model is reported to characterize many forms of abiotic stress responses. The first identified CBL-CIPK pathway was the SOS pathway, which plays vital roles in plant salt tolerance regulation [55]. It contains three key components: SOS1 (Na+/H+ antiporter), SOS2 (CIPK24), and SOS3 (CBL4). Under salt stress, the SOS2–SOS3 complex activates the transport properties of SOS1 to promote the Na+ efflux, thus en-

hancing plant salt tolerance. Subsequent studies found that CBL10 (SCABP8) could also interact with SOS2 and activate SOS1, suggesting the functional redundancy between SOS3 and CBL10 [31]. Studies in apples also showed that MdCBL10 could interact with MdSOS2L1, an SOS2-like protein that played positive roles in regulating plant salt tolerance [54]. In addition, a recent study found that the CBL10–CIPK8 complex regulated plant salt tolerance through its interaction with SOS1 [41]. These results, combined with the functional identification results in yeast (Figure 4), led to the selection of *MdCBL10.1* for further genetic transformation and functional identification in apple. The phenotypic comparison and measurement of stress-related physiological indicators showed that *Md-CBL10.1* overexpression significantly enhanced the salt tolerance of cisgenic apple materials (Figures 5–7). Moreover, the significantly reduced Na+ content in transgenic and cisgenic materials indicated that *MdCBL10.1* overexpression could significantly inhibit Na+ accumulation under salt stress. These results suggested that MdCBL10.1 enhanced plant salt tolerance by inhibiting Na+ accumulation, and this was probably achieved through the SOS pathway.

In conclusion, 11 *MdCBL* genes were identified from apple. CDSs of these genes were cloned, and the gene structure and conserved motifs were analyzed. Several *MdCBLs* that played positive roles in salt tolerance were identified using the Na+ -sensitive yeast mutant. The function of *MdCBL10.1* in regulating salt tolerance was also identified in cisgenic apple materials in detail. This study provided a foundation for future research examining the function and mechanism of CBL proteins in regulating apple salt tolerance.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/ijms222212430/s1. Figure S1: Phylogenetic analysis of proteins in HMMER screening results and CBL family members in Arabidopsis; Figure S2: Comparison of gene structure of the CBL family genes in apples and Arabidopsis; Figure S3: Putative conserved motifs identified in the sequences of apple CBL family proteins. Red dots indicate the conserved "FPSF" and "MGCxxS/T" domains; Figure S4: Sequence comparison between the predicted MdCBLs in the apple genome and the MdCBLs that were actually cloned in this study; Figure S5: Identification of cisgenic apple calli. (**A**) PCR identification of the MdCBL10.1 transgene based on genomic DNA extracted from apple calli. (**B**) Expression level of MdCBL10.1 in cisgenic apple calli. MdMDH served as an internal reference gene. Bars labeled with different letters in each panel are significantly different (*p* < 0.05, one-way ANOVA and Duncan's test); Figure S6: Identification of the MdCBL10.1 transgene in the roots of apple plants. (**A**) GFP expression in cisgenic apple roots. (**B**) qRT-PCR determination of MdCBL10.1 expression in cisgenic apple roots. (OE)-EV and (RNAi)-EV represent roots transformed with the empty vector pCambia2300 and pK7GWIWG2D, respectively. MdMDH served as an internal reference gene. Bars labeled with different letters in each panel are significantly different (*p* < 0.05, one-way ANOVA and Duncan's test). Supplementary File S1: HMMER screening results. Supplementary File S2: CDS and protein sequences of the cloned MdCBL genes. Supplementary File S3: Cis-elements identified in the promoter regions of MdCBL genes. Plus and minus signs indicate whether the cis-element was located in the plus or minus strand.

**Author Contributions:** P.C., J.Y., K.M. and F.M. conceived the project; P.C., J.Y. and K.M. designed the research plan; P.C., J.Y., Q.M., H.L. and Y.C. carried out the experiments; P.C., J.Y. and Q.M. analyzed the data; J.Y., K.M. and F.M. wrote the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by National Key Research and Development Program of China (2018YFD1000300/2018YFD1000301), the National Natural Science Foundation of China (31701894), the Key S&T Special Projects of Shaanxi Province (2020zdzx03–01-02), and the China Agriculture Research System of MOF and MARA (CARS-27).

**Institutional Review Board Statement:** No applicable.

**Informed Consent Statement:** No applicable.

**Data Availability Statement:** All data supporting the findings of this study are available within the paper and within its supplementary data published online.

**Acknowledgments:** We thank Huazhong Shi (Texas Tech University) and Jiafu Jiang (Nanjing Agricultural University) for providing the Na+ -sensitive yeast mutants. Apple calli ('Orin') and the 'Gala' (GL-3) seedlings were kindly provided by Yujin Hao (Shandong Agricultural University) and Zhinghong Zhang (Shenyang Agricultural University), respectively.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

## *Article* **Genome-Wide Identification and Characterization of Wheat 14-3-3 Genes Unravels the Role of TaGRF6-A in Salt Stress Tolerance by Binding MYB Transcription Factor**

**Wenna Shao 1,2,†, Wang Chen 3,† , Xiaoguo Zhu 2,† , Xiaoyi Zhou 1,2 , Yingying Jin 1 , Chuang Zhan 1 , Gensen Liu 1 , Xi Liu 1 , Dongfang Ma 1, \* and Yongli Qiao 2, \***

**Citation:** Shao, W.; Chen, W.; Zhu, X.; Zhou, X.; Jin, Y.; Zhan, C.; Liu, G.; Liu, X.; Ma, D.; Qiao, Y. Genome-Wide Identification and Characterization of Wheat 14-3-3 Genes Unravels the Role of TaGRF6-A in Salt Stress Tolerance by Binding MYB Transcription Factor. *Int. J. Mol. Sci.* **2021**, *22*, 1904. https://doi.org/10.3390/ ijms22041904

Academic Editors: Mirza Hasanuzzaman and Masayuki Fujita

Received: 29 January 2021 Accepted: 9 February 2021 Published: 14 February 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

**Abstract:** 14-3-3 proteins are a large multigenic family of general regulatory factors (GRF) ubiquitously found in eukaryotes and play vital roles in the regulation of plant growth, development, and response to stress stimuli. However, so far, no comprehensive investigation has been performed in the hexaploid wheat. In the present study, A total of 17 potential 14-3-3 gene family members were identified from the Chinese Spring whole-genome sequencing database. The phylogenetic comparison with six 14-3-3 families revealed that the majority of wheat *14-3-3* genes might have evolved as an independent branch and grouped into ε and non-ε group using the phylogenetic comparison. Analysis of gene structure and motif indicated that 14-3-3 protein family members have relatively conserved exon/intron arrangement and motif composition. Physical mapping showed that wheat *14-3-3* genes are mainly distributed on chromosomes 2, 3, 4, and 7. Moreover, most *14-3-3* members in wheat exhibited significantly down-regulated expression in response to alkaline stress. VIGS assay and protein-protein interaction analysis further confirmed that TaGRF6-A positively regulated slat stress tolerance by interacting with a MYB transcription factor, TaMYB64. Taken together, our findings provide fundamental information on the involvement of the wheat 14-3-3 family in salt stress and further investigating their molecular mechanism.

**Keywords:** 14-3-3 gene family; *Triticum aestivum* L.; bioinformatics analysis; salt tolerance; proteinprotein interactions

#### **1. Introduction**

General regulatory factor (GRF) proteins, also known as 14-3-3 proteins, are found among all eukaryotic organisms [1]. The 14-3-3 protein, first discovered in the bovine brain as a soluble acidic protein, acquired its name based on the fraction number in diethylaminoethyl (DEAE) cellulose chromatography and migration position on starch-gel electrophoresis [2]. Initially, 14-3-3 proteins were believed to be unique to animals. However, further research showed that these proteins are found in almost all eukaryotes [1,3,4]. The 14-3-3 proteins are a class of highly conserved regulatory proteins that can form homoor heterodimers, and each monomer in the dimer can interact with a separate target protein. This dimeric property of 14-3-3s allows them to serve as scaffolds for bringing together different regions of a protein or two different proteins in close proximity [5,6]. Therefore,

14-3-3 proteins regulate activities of numerous target proteins via protein-protein interactions, specifically by binding to the phosphoserine/phosphothreonine residues of target proteins. Evidence suggests that 14-3-3s are the most important phosphopeptide-binding proteins that play a regulatory role in various biological processes including the regulation of cell cycle, metabolism, apoptosis, development, protein trafficking, stress response, and gene transcription [7–15].

In plants, the first plant 14-3-3 protein was isolated from *Arabidopsis thaliana* as a component of the G-box complex and was named GF14 [16]. Subsequently, 14-3-3 proteins were detected in many plant species and were named GF or GRF, followed by Arabic numerals [17]. With the development of new high-throughput sequencing technologies and modern bioinformatics tools, more and more plant genomes were sequenced, which dramatically accelerated the genome-wide survey of 14-3-3 proteins in plants. To date, the 14-3-3 gene family has been identified in *Arabidopsis* (13), rice (*Oryza sativa* L.) (8), tomato (*Solanum lycopersicum* L.) (12), soybean (*Glycine max* L.) (18), cotton (*Gossypium hirsutum* L.) (25), banana (*Musa acuminata* L.) (25), grape (*Vitis vinifera* L.) (11), and many other plant species [18].

Wheat (*Triticum aestivum* L.) is the most widely cultivated crop in the world and one of the primary grains consumed by humans [19]. Drought, extreme temperatures, and salinity are the major abiotic stresses that reduce wheat production throughout the growing season [20]. The 14-3-3 proteins act as key regulators of signaling networks and abiotic stress responses [21]. For example, in rice, OsGF14e interacts with OsCPK21 to promote the salt stress response [22]. Additionally, studies show that OsGF14f has a negative effect on grain development and filling, while OsGF14e negatively impacts cell death and disease resistance [23,24]. In tomato, four 14-3-3 genes (*TFT1*, *TFT4*, *TFT7*, and *TFT10*) are significantly up-regulated under salt stress [25]. Ectopic expression of the wheat *TaGF14b* gene in tobacco (*Nicotiana tabacum* L.) enhanced the tolerance of mature tobacco plants to drought and salt stresses, which are related to the abscisic acid (ABA) signaling pathway, and improved their growth and survival compared with the control [26]. Overexpression of the soybean *GsGF14o* gene in *Arabidopsis* showed that GsGF14o regulates stomata size, root hair development, and drought tolerance [27].

Recently, Guo et al. [28] analyzed wheat *TaGF14* genes based on the TGACv1 reference genome and found that five of these genes were up-regulated under drought stress, and all of the analyzed *TaGF14s* were down-regulated during heat stress. These results suggest that some of the *TaGF14* genes play a vital role in combating drought stress, and all *TaGF14s* are negatively associated with heat stress. Additionally, the study showed that TaGF14s might be involved in starch biosynthesis. However, no information is available on the structure of 14-3-3 genes and their role in response to salt tolerance in wheat.

In this study, we conducted an in-depth analysis of the wheat 14-3-3 gene family members based on the newly released Chinese Spring reference genome (IWGSC RefSeq v1.1, https://urgi.versailles.inra.fr/download/iwgsc/IWGSC\_RefSeq\_Annotations/v1.1/ (accessed on 13 February 2021)). We performed a comprehensive analysis of the structure, evolutionary relationship, *cis*-acting elements, Gene Ontology (GO) annotations, and expression profiles of the *14-3-3* genes, and deciphered the biological roles of these genes in salt stress tolerance using the virus-induced gene silencing (VIGS) assay and proteinprotein interaction analysis. It provided an updated view of the wheat 14-3-3 family and lays the foundation for studying the molecular mechanism of *TaGRF6-A* in regulating salt tolerance in wheat.

#### **2. Results**

#### *2.1. Genome-Wide Identification and Characterization of 14-3-3 Family Members in Wheat*

A total of 32 14-3-3 protein sequences of wheat were identified using the basic local alignment search tool, BLASTp, and validated using the Hidden Markov Model (HMM) search tool, HMMER. These sequences were further confirmed using SMART and NCBI-CDD online tools, which revealed that all sequences contained conserved 14-3-3 protein domains. These protein sequences were encoded by 17 genes, including 12 genes showing alternative splicing, and splice variants with complete domains were chosen as representatives (Table 1). Multiple sequence alignment and secondary structure analysis revealed that the 14-3-3 family members contained nine typical α-helices. But the *TraesCS4A02G167100* gene lacked the first α-helix, making it the first 14-3-3 family member that does not contain nine α-helices (Figure S1). The 14-3-3 amino acid sequences were highly conserved, indicating that these proteins may perform similar functions to the 14-3-3 proteins of other plant species. The C-terminal end of 14-3-3 proteins was relatively divergent, which explains the functional diversity observed among these proteins. Genes with a 1:1:1 correspondence in all three sub-genomes (A, B, and D) of wheat are called triads [29]. We identified five triads based on the results of Ramírez-González et al. (Table S1). Because of the peculiarity of *TraesCS4A02G167100*, this gene was named *TaGRF-like1*. The remaining 16 genes were named according to their order on various homologous chromosomes (Table 1).

A detailed description of *TaGRFs* is summarized in Table 1. The deduced wheat 14-3-3 proteins contained 244–282 amino acid residues, and their molecular weight (MW) ranged from 27.25 to 31.93 kDa. The predicted isoelectric point (pI) of these proteins ranged from 4.67 to 6.32, implying that these proteins were acidic in nature. Three-dimensional modeling clearly showed the presence of α-helices and indicated that higher structures of these proteins were very similar (Figure S2).

#### *2.2. Gene Structure and Conserved Motif Analysis*

Phylogenetic analysis revealed that *TaGRFs* were divided into two groups (ε and non-ε) and three corresponding members of each triad were closely clustered together (Figure 1A). To examine the structural diversity of *TaGRFs*, we manually extracted the exonintron structure from the GFF3 annotation file (Table S2). All *TaGRF* genes, especially those within the same phylogenetic group, shared a similar exon-intron structure (Figure 1B). The number of exons varied from 4 to 6 and the genes within each phylogenetic group exhibited nearly identical exon lengths. To understand the motif composition, a search was implemented using the MEME website, with the maximum number of motifs set at 10 (Figure 1C). Functional analysis using SMART revealed that 3 of these 10 motifs (motifs 1–3) were annotated as 14-3-3, which were included in all members (Table S3). This result suggests that motifs 1–3 represent the main domains that determine the function of 14-3-3 proteins. At the same time, this result also indicates the functional conservation of the *14-3-3* genes during evolution. In addition, motif composition was highly similar within each phylogenetic group but was relatively divergent between groups. All members of the non-ε group contained six main motifs (1–6), whereas members of the ε group contained two unique motifs (9 and 10), which indicates the potentially different roles during plant growth and response to various stresses.

**Table 1.**Detailed information of putative TaGRFs.

AS a (Number of alternative splices); CDS b (Length of the coding sequence, bp); Protein c (Amino acid length, aa); MW d (Molecular weight, kD); pI e (Isoelectric point); Chr f (represents Chromosome); (−) g (represents antisense strand); (+) h (represents sense strand); reference#.

**Figure 1.** Structural analysis of *TaGRFs*. (**A**) The phylogenetic tree of *TaGRFs*. (**B**) Gene structure analysis of *TaGRFs* was conducted using the GSDS database. All introns are shown the same length. (**C**) Motif analysis of *TaGRFs* was identified by MEME. Motif components in the amino acid sequence of *TaGRFs* are displayed using MAST. Different motifs were represented in different color blocks.

#### *2.3. Chromosomal Distribution and Evolutionary Analysis of TaGRF Genes*

To clarify the chromosome position of *TaGRFs*, a schematic diagram was constructed. The *TaGRF* genes were mainly distributed on homoeologous chromosomes 4A/4B/4D, and only one gene was located on each of the remaining chromosomes (Figure 2A).

To trace the evolutionary origin of wheat *TaGRFs*, we performed a synteny analysis of family members in common wheat (AABBDD) and its progenitor species *Aegilops tauschii* L. (DD), *Triticum urartu* L. (AA), and *T. dicoccoides* L. (AABB) (Figure 2B). Given that the B sub-genome donor has not been sequenced, wild emmer wheat (*T. dicoccoides* L.) was used as the source of the B sub-genome. The results showed that *TaGRFs* had seven, twelve, and five orthologs in *T. urartu*, *T. dicoccoides*, and *Ae. tauschii*, respectively (Table S4). Interestingly, the origin of *TaGRF* genes was traceable. Our results indicated that the *TaGRF* genes in common wheat were entirely derived from the sub-genome donors and have the same number on the corresponding chromosomes. Wheat chromosomes 4A, 4B, and 4D harbored three, three, and two *TaGRF* genes, respectively. The same number of *GRF* homologs were detected on chromosomes 4A and 4B of *T. dicoccoides* and on chromosome 4D of *Ae. tauschii*. However, the corresponding gene was not detected on chromosome 2A of *T. urartu*. We speculated that the genes on chromosome 2A of common wheat evolved from *T. dicoccoides* or the corresponding gene on *T. urartu* used to exist but was later lost. This implied that members of the *TaGRF* gene family did not experience large fluctuations. The only polyploidization doubles the number of genes twice. In addition, the non-synonymous-to-synonymous substitution ratio (Ka/Ks) of *TaGRF* gene duplicates was <1 (Figure 2C, Table S3), indicating that these genes are under negative selection pressure to maintain the protein sequence.

ε

ε ε

ε

ε ε **Figure 2.** Evolutionary analysis of TaGRFs (**A**) Chromosomal locations and repeated events of TaGRFs. Orange bars represent chromosomes. The black rectangular dots above is the centromere. The scale indicates the corresponding physical distance. Chr 2A is the second chromosome on sub-genome A. The ε group genes are shown in red font, and non-ε group genes are shown in black font. The segmentally duplicated homologous genes were marked with blue lines. (**B**) Orthologous relationships analysis among T. aestivum (Ta, AABBDD, pink box) and progenitor species Ae. tauschii (Ae, DD, blue box), T. urartu (Tu, AA, orange box), and T. dicoccoides (Td, AABB, green box). The lines represent collinearity between chromosomes. (**C**) Ka/Ks value of TaGRF repeat gene pair. (**D**) Phylogenetic analysis of TaGRF proteins and other species such as *A. thaliana*, *O. sativa*, *G. max*, *P. trichocarpa*, *M. truncatula*, and *V. vinifera*. ClustalW2 was used to align the protein sequences, and the phylogenetic tree was constructed by the Neighbor-Joining (NJ) method of MEGA 7 with 1000 bootstrap replicates. Then the tree was modified by ITOL online tool. The two major groups were marked with different colors.

To understand the evolutionary relationships of wheat 14-3-3 proteins with those of other plant species, a phylogenetic tree of full-length 14-3-3 amino acid sequences from seven plant species, including *T. aestivum*, *A. thaliana*, *O. sativa*, *G. max*, *Populus trichocarpa*, *Medicago truncatula*, and *V. vinifera*, was constructed using the maximum likelihood (ML) method (Figure 2D, Table S5). Consistent with the previous classification [30], all 14-3-3 proteins were divided into two main evolutionary branches, namely, ε and non-ε. Only *TaGRF-like1* and *TaGRF5-B* were grouped into the ε group, and the remaining *TaGRFs* clustered in the non-ε group. The proteins from wheat, *Arabidopsis*, and rice were closely related, suggesting that these proteins may perform similar functions, which provides clues for functional analysis.

#### *2.4. Cis-Element Analysis and Functional Annotation of TaGRF Genes*

To examine *cis*-acting elements in *TaGRF* promoter sequences, we searched 1.5 kb sequence upstream of the start codon (ATG) of all *TaGRF* genes. A total of 1789 *cis*-acting elements were identified (Table S6). These included elements involved in biotic and abiotic stress responses, plant growth and development, and phytohormone response processes. The number of elements involved in the first two processes was significantly more (Figure 3A,B). Motifs such as TGACG and CGTCA (involved in responding to methyl jasmonate [MeJA]) and ABA-responsive element (ABRE; involved in the response to ABA) were commonly found in *TaGRF* gene promoters, suggesting that these genes are involved in hormone regulation. In addition, stress-related *cis*-elements, such as Myb, STRE, Sp1, G-box, Myc, as-1, LTR, and GC-motif, were also identified, implying that *TaGRFs* are important players in response to adverse conditions.

**Figure 3.** Analysis of *cis*-acting elements in the promoter of *TaGRFs*. (**A**) Different numbers and shades of color in the grid indicated the number of different promoter elements. (**B**) The histogram showed the sum of the cis-acting elements in different classes of each gene.

> To further predict the function of *TaGRFs*, we performed GO annotation analysis. The results showed that *TaGRFs* were divided into three categories (molecular function, cell composition, and biological process) (Figure S3). In the molecular function category, GO terms such as "ATP binding" and "protein domain specific binding" were highly enriched, indicating that TaGRF proteins may bind to other proteins to perform various cellular functions. In the cell component category, GO terms such as nucleus, cytosol, plasma membrane, and organelles, such as mitochondrion, were highly enriched, suggesting that *TaGRFs* perform a wide range of functions. In the biological process category, *TaGRFs* play a prominent role in the response to cadmium ions. Both *TaGRF5-B* and *TaGRF-like1* were annotated only under the GO term "protein domain specific binding", suggesting that these proteins play a limited role in wheat.

#### *2.5. Expression Profiling of TaGRF Genes*

The results of *cis*-element and GO annotation analyses suggested that *TaGRFs* are potentially involved in abiotic stress responses. To better understand the function of *TaGRFs*, we examined their expression patterns using RNA-seq data. The expression profiles of 16 *TaGRFs* in different wheat varieties under salt stresses are shown in Figure 4A. In general, the expression of *TaGRFs* decreased after the salt treatment. Furthermore, the expression levels of *TaGRF4-A*, *TaGRF4-B*, and *TaGRF5-B* were extremely low. In the wheat variety Kharchia Local, *TaGRFs* showed different expression profiles in different tissues; for example, *TaGRF1-B*, *TaGRF1-D*, and *TaGRF2-A* showed low expression in roots, while *TaGRF6-A*, *TaGRF6-B*, and *TaGRF6-D* showed high expression in leaves, which indicated that these genes may perform a function mainly in leaves. Compared with Kharchia Local, QM6 and Chinese Spring showed greater differences in gene expression after the salt treatment, suggesting that genotype is an important factor affecting the differences in abiotic stress responses.

**Figure 4.** Expression patterns of *TaGRFs*. (**A**) Transcriptome analysis of *TaGRFs* across salt stress. The expression data were generated from NCBI and viewed in RStudio software. The relative expression level of a particular gene in each column was normalized against the mean value by log2 transformation. The color scale represents relative expression levels. (**B**) qRT-PCR analyses of two *TaGRF* genes in leaves under NaCl treatment. The asterisks indicate statistical significance between CK and NaCl treatments (\*\*, *p* < 0.01; \*, *p* < 0.05; Student's *t*-test).

> To verify the reliability of the transcriptome data, we analyzed the expression of two genes (*TaGRF4-D* and *TaGRF6-A*) in 1-week-old salt-treated wheat seedlings by quantitative real-time PCR. Both the *TaGRFs* responded to salt stress, but with different rates and intensities (Figure 4B). Compared with the control, the expression of *TaGRF4-D* did not change significantly, while the expression of *TaGRF6-A* was significantly up-regulated at 12 h post salt treatment, and then decreased sharply at 24 h post salt treatment. This trend suggests that *TaGRF6-A* can be induced by salt stress at a certain stage, and may play a role during this period.

#### *2.6. TaGRF6-A Positively Regulates Salt Tolerance in Wheat*

The VIGS assay is used to silence specific genes in plants, thus enabling rapid characterization of gene function [31]. To further understand the role of *TaGRF6-A* in salt stress, we performed a virus-induced gene silencing assay. Two fragments of the *TaGRF6-A* gene were chosen for silencing (Figure S3). At 10 days post-inoculation (dpi), wheat leaves inoculated with BSMV showed mild chlorotic mosaic symptoms while leaves inoculated with BSMV: *TaPDS* showed bleaching symptoms, indicating that the BSMV-induced gene

silencing system was working (Figure 5A). When treated with 300 mM NaCl for 12 days, both fragments of *TaGRF6-A* were well silenced by qRT-PCR (Figure 5C), and at this time, the leaf curling degree of plants carrying BSMV: *TaGRF6-A-1/2* was stronger than that of the control group carrying the empty vector (Figure 5B). According to the statistical results, the number of leaf curls of plants carrying BSMV: *TaGRF6-A-1/2* was significantly more than that of the empty carrier group (Figure 5D), indicating that *TaGRF6-A* contributes to salt tolerance in wheat.

**Figure 5.** The silencing of *TaGRF6-A* decreases salt tolerance in wheat. (**A**) Phenotypic characteristics of wheat leaves after *TaGRF6-A* silenced by VIGS technology. (**B**) The curling phenotype of wheat leaves after 12 days of 300 mM NaCl treatment. Bars, 1 cm. (**C**) Silencing efficiency assessment of *TaGRF6-A* in the *TaGRF6-A*-knockdown plants treated with 12 days of 300 mM NaCl. (**D**) Statistics of leaf curls rate of *TaGRF6-A* in the *TaGRF6-A*-knockdown plants treated with 12 days of 300 mM NaCl. Error bars represent the SD of three biological replicates. The asterisks indicate statistical significance using Student's *t*-tests. (\*\*, *p* < 0.01; \*, *p* < 0.05).

#### *2.7. TaGRF6-A Interacts with TaMYB64 In Vitro and In Vivo*

Since *TaGRF6-A* expressed salt tolerance, we started to look for its internal mechanism. We focused on the expression levels of stress-related genes and their potential targets (Figure 6A). It is worth noting that *TaMYB64* is significantly down-regulated, suggesting that there may be a synergistic effect to enhance abiotic stress tolerance together with *TaGRF6-A*. Previous studies in soybean and rice showed that interactions between MYB transcription factors and 14-3-3 proteins enhance plant growth and stress toler-

ance [32,33]. These findings, combined with our qRT-PCR results, prompt us to investigate the association between TaMYB64 and TaGRF6-A. Before that, we analyzed the subcellular localization of TaGRF6-A and TaMYB64 by generating a C-terminal fusion of gene and yellow fluorescent protein (YFP) gene. After transient expression in *N. benthamiana* leaves using *Agrobacterium*-mediated transformation, compared with the YFP empty vector, confocal microscopy showed that TaGRF6-A has a similar subcellular localization and was detected in both the nucleus and the cytoplasm (Figure 6B), and TaMYB64 was detected in the nucleus, indicating the place where it functions. When exploring the interaction of TaMYB64 and TaGRF6-A, we first performed yeast two-hybrid (Y2H) assays. Although yeast transformants expressing both TaMYB64 and TaGRF6-A could grow on high stringency media, the number of yeast cells was less (Figure 6C). To further verify the interaction between TaMYB64 and TaGRF6-A, we performed bimolecular fluorescence complementation (BiFC) and co-immunoprecipitation (Co-IP) assay. After transiently co-expressing TaGRF6-A labeled with the N-terminal half of YFP (nYFP), together with TaMYB64 labeled with the C-terminal half of YFP (cYFP) in *N. benthamiana* leaves, the YFP signal was detected in the nucleus and cytoplasm of *N. benthamiana* leaf epidermal cells. While no signal was detected in the control combinations (Figure 6D). The TaMYB64-YFP-3×FLAG fusion proteins were co-expressed with TaGRF6-A-YFP-HA or YFP-HA in *N. benthamiana*. After immunoprecipitation using the anti-HA antibody, Western blot was performed using the anti-FLAG antibody to detect TaMYB64 proteins. The TaMYB64-YFP-3×FLAG protein could be coimmunoprecipitated by TaGRF6-A-YFP-HA, but not the YFP-HA control, revealing a specific interaction between TaMYB64 and TaGRF6-A in vivo (Figure 6D). These results proved that TaGRF6-A could physically interact with TaMYB64.

#### *2.8. TaGRF6-A and TaMYB64 Work Together to Cope with Salt Stress*

To clarify whether TaGRF6-A interacts with TaMYB64 to respond to salt stress, we chose to silence the *TaMYB64* gene. Ten days post-inoculation (dpi), wheat leaves inoculated with BSMV showed mild symptoms of chlorosis, while leaves inoculated with BSMV: *TaPDS* showed symptoms of bleaching stripes, indicating that BSMV-induced gene silencing was effective (Figure 7A). qRT-PCR results revealed that both fragments of *TaMYB64* were successfully silenced (Figure 7C). After treated with 300 mM NaCl for 12 days, the plants carrying BSMV: *MYB64-1/2* had stronger curls and a significantly higher number of rolled leaves compared with the control group carrying the empty vector (Figure 7B,D). These results indicated that *TaMYB64* was also involved in response to salt stress.

×

×

**Figure 6.** Analysis of protein interaction between TaGRF6-A and TaMYB64. (**A**) Relative expression levels of potential targets and stress-related genes in the leaves of *TaGRF6-A*-knockdown plants treated with 12 days of 300 mM NaCl. Error bars represent the SD of three biological replicates. The asterisks indicate statistical significance using Student's *t*-tests. (\*\*, *p* < 0.01; \*, *p* < 0.05). (**B**) Subcellular localization of TaGRF6-A in *N. benthamiana*. Free YFP and TaGRF6-A-YFP and TaMYB64-YFP fusion proteins were transiently injected into *N. benthamiana* leaves by *Agrobacterium* (GV3101) transformation. Subcellular localization observed by laser confocal microscopy after 48 h post-infiltration. Scale bars, 40 µm. (**C**) Yeast two-hybrid interaction results of TaGRF6-A and TaMYB64 proteins. -L-W refers to media lacking leucine and tryptophan, -L-W-H-A refers to media lacking leucine, tryptophan, histidine, and adenine. (**D**) BiFC assay showing interactions of TaGRF6-A and TaMYB64 proteins. Scale bars, 40 µm. (**E**) Co-immunoprecipitation experiments show that TaGRF6-A interacts with TaMYB64 in planta. The total proteins of TaMYB64 with FLAG tag and TaGRF6-A with HA tag were extracted from *N. benthamiana* leaves. The immune complex was pulled down using anti-HA agarose gel, and the co-precipitation of TaGRF6-A and TaMYB64 was detected by Western blotting.

**Figure 7.** Silencing of *TaMYB64* decreases salt tolerance in wheat. (**A**) Phenotypic characteristics of wheat leaves after *TaMYB64* silenced by VIGS technology. (**B**) The curling phenotype of wheat leaves after 12 days of 300 mM NaCl treatment. Bars, 1 cm. (**C**) Silencing efficiency assessment of *TaMYB64* in the *TaMYB64*-knockdown plants treated with 12 days of 300 mM NaCl. (**D**) Statistics of leaf curls rate of *TaMYB64* in the *TaMYB64*-knockdown plants treated with 12 days of 300 mM NaCl. Error bars represent the SD of three biological replicates. The asterisks indicate statistical significance using Student's *t*-tests. (\*\*, *p* < 0.01; \*, *p* < 0.05).

#### **3. Discussion**

14-3-3 genes are ubiquitous in many species, and the study of its gene function is very necessary. Recent studies have shown that the four 14-3-3 proteins (MdGF14a, MdGF14d, MdGF14i, and MdGF14j) of apples interact with the flower integrator TFL1/FT and participate in flowering regulation [34]. Under the condition of nitrate deficiency, the interaction of MdGRF11 and MdBT2 in apples can induce the accumulation of anthocyanins and provide a new mechanism for anthocyanin biosynthesis [35]. Under sugar starvation, MYBS2 interacted with 14-3-3 protein to inhibit the expression of α-amylase, thereby to improve rice plant growth, stress resistance, and grain weight [32]. As the hub of different signal pathways, 14-3-3 protein can transmit and integrate different hormone signals in plant signal transduction [36]. 14-3-3 interacts with AtWRI1, and the biosynthesis of oil increased in transgenic tobacco leaves co-expressing these two genes [37]. 14-3-3λ and k are important salt tolerance regulators. When there is no salt stress, 14-3-3λ and k interact with SOS2 and inhibit the kinase activity of SOS2; while in the presence of salt stress, the interaction of them weakens and activates SOS2 kinase activity [38].

In the present study, we performed a genome-wide characterization and in silico analysis of the *14-3-3* gene family in wheat. A total of 17 *14-3-3* genes were identified in wheat based on the genome sequence of Chinese Spring. However, this result is inconsistent with previous studies, which identified 20 *14-3-3* genes based on the TGACv1 wheat genome assembly [28]. We found that previous results contained duplicate genes and several other genes that do not exist in our newly released genome sequencing data. For example, Traes\_2DL\_1D912517E and Traes\_2DL\_21639EB55, which were previously identified as two distinct genes, represent the same gene in the new genome. In addition, TraesCS2D02G325600 and Traes\_3B\_3BF024700350CFD\_c1 are not identified in the present study. We speculate that this difference between studies is caused by differences in genome sequencing depth, genome analysis, and gene annotations. We also cannot rule out the possibility of wheat germplasm resource differences.

The 14-3-3 proteins are highly conserved and usually contain nine α-helices, which constitute the conserved core region of each monomer in the dimer [13,39]. In the current study, we showed that *TaGRF-like1* lacks the first α-helix, which may affect the binding of phosphorylated target proteins. while the secondary structure of TaGRF proteins was conserved except in the C-terminal region (Figure S1). Previous studies have shown that the variable C-terminus is the key to dimer formation, and it can interact with different ligands to show the target specificity of 14-3-3 protein [40,41].

Phylogenetic analysis of 89 14-3-3 proteins belonging to wheat and six other plant species showed that these proteins were clustered into two major groups (ε and non-ε), which is consistent with previous reports [18,34]. The result indicates that the formation of these two isoforms is a basic and ancient difference (Figure 2D). As *TaGRF6-A* was adjacent to *OsGF14d*, which is expressed under salt, heat, and cold stress [42], the function of TaGRF6-A may be similar to that of *OsGF14d*. According to previous studies, the ε group genes usually contain a greater number of exons and motifs than the non-ε genes. However, this finding was not in agreement with our results; although the ε group genes contained more motifs, not all ε group genes contained more exons than non-ε group genes (Figure 1B,C). For instance, the ε group gene *TaGRF-like1* contained four exons, whereas the non-ε group gene *TaGRF2-B* contained six exons. This difference may be due to the insertion and deletion of introns over the long-term evolution of wheat. Nevertheless, gene structure and motif distribution were diverse between the two groups but similar within a group, which supports the results of the phylogenetic analysis (Figure 1B,C).

Common wheat is an allohexaploid with a large and complex genome, which makes its genome research difficult. Genome sequencing of common wheat and its sub-genome donors has been completed, which provides an important reference for the evolutionary analysis of wheat. The chromosome map of common wheat shows that the distance between genes is much greater than 200 kb (Figure 2A), which means that these genes were not generated by tandem duplication [43]. Collinearity analysis of common wheat and its ancestors revealed the direct source of the wheat 14-3-3 sub-genomic donor (Figure 2B), indicating that the entire family of wheat 14-3-3 proteins originated by polyploidization. The Ka/Ks ratio of 14-3-3 genes was <1 (Figure 2C), implying that wheat 14-3-3 proteins may remain unchanged during the process of long-term domestication. Analysis of *cis*-acting elements in the promoter region of *TaGRFs* indicated that these genes may be involved in biotic and abiotic stress responses, growth and development, and phytohormone response (Figure 3A). Previously, there have been some reports showing the involvement of 14-3-3 proteins in abiotic stress tolerance [44,45]. Considering the potential function of 14-3-3 proteins in abiotic stress, we performed transcriptome analysis of wheat plants under salt stress. Different *TaGRF* genes showed different expression profiles under salt stress. Most *TaGRF* genes were down-regulated after the salt treatment, suggesting that these genes may negatively regulate the response to salt stress (Figure 4A). However, in rice, tomato, *Brachypodium distachyon*, and other plant species, members of the 14-3-3 family are involved in salt stress tolerance [22,25,46]. In addition, wheat *TaGF14b* (named as *TaGRF1-B* in this article) also enhanced salt stress tolerance when ectopically expressed in tobacco [26]. In

the current study, we selected two genes for the salt treatment and found that *TaGRF6-A* was changed greatly responding to salt stress (Figure 4B). To further verify the role of 14-3-3 family genes in salt stress, we used VIGS technology to silence the *TaGRF6-A* gene. The results showed that the level of leaf curling and number of BMSV: *TaGRF6-A* were more than the wild type (Figure 5B–D), indicating that *TaGRF6-A* plays a positive role in response to salt stress.

To elucidate the possible mechanism of action of *TaGRF6-A* under salt stress, we examined the expression of seven stress-related genes or potential interacting partners of *TaGRF6-A*. Interestingly, the expression level of *TaMYB64* was significantly down-regulated after *TaGRF6-A* silencing (Figure 6A). Since the interaction between MYBS2 and 14-3- 3 protein has been reported in rice and soybean [32,33,47], we cloned the homolog of OsMYBS2 in wheat and performed Y2H, BiFC, and CoIP assays. The results showed that TaMYB64 and TaGRF6-A did interact in wheat (Figure 6C–E). Thus, we have the question of whether they use this interaction to improve salt tolerance. Next, we used the same method to silence *TaMYB64* in wheat, and the results proved our deduction. Compared with the control, the silenced plants also have a higher degree and amount of curling (Figure 7B,D). In summary, the results in the present study revealed the function of *TaGRF6-A* in salt stress, and initially elucidated its salt tolerance mechanism. Moreover, our study improved our understanding of the biological functions of the wheat *14-3-3* gene family.

#### **4. Materials and Methods**

#### *4.1. Plant Material and Salt Treatment*

Wheat (*T. aestivum* L.) cultivar Emai 170 was used in this study. Seeds of uniform size were selected and soaked in clear water for 24 h. Then, the seeds were sown in a small pot filled with nutrient-rich soil, and the pots were placed in a greenhouse maintained at 23 ± 2 ◦C, 16 h light/8 h dark photoperiod, and 200 lux light intensity. At the 2–3-leaf stage, wheat plants were treated with either double-distilled water (control) or 300 mM NaCl solution. Leaves were collected at 2, 4, 6, 12, 24, and 96 h after the treatment, frozen in liquid nitrogen and stored at −80 ◦C.

#### *4.2. Mining and Phylogenetic Analysis of Wheat 14-3-3 Genes*

To identify potential members of the wheat 14-3-3 gene family, published 14-3-3 amino acid sequences of *A. thaliana*, *O. sativa*, and *G. max* were used as queries in BLASTp searches against the wheat genome IWGSC RefSeq v.1.1, with an *E*-value cut-off <10−10 . The HMM profile of the conserved domain of 14-3-3 (PF00244) was downloaded from the PFAM 32.0 database [48] and used to examine all wheat protein sequences with the HMMER search tool [49]. According to the sequence ID of the target candidate 14-3-3 protein, genomic sequences were isolated. Then, all matching sequences were validated using the SMART website (Simple Modular Architecture Research Tool, http://smart. embl-heidelberg.de/ (accessed on 13 February 2021)) and NCBI-CDD (National Center for Biotechnology Information-Conserved domain database, https://www.ncbi.nlm.nih. gov/Structure/bwrpsb/bwrpsb.cgi (accessed on 13 February 2021)) search to identify the conserved 14-3-3 protein domain with default parameters, and proteins lacking the 14-3-3 domain were discarded [50,51]. Previously known 14-3-3 protein sequences from *A. thaliana* (13), *O. sativa* (8), and *G. max* (18) used in this study were retrieved from the Phytozome v.12 database (http://phytozome.jgi.doe.gov (accessed on 13 February 2021)) [52].

#### *4.3. Amino Acid Sequence Alignment and Characterization of TaGRF Proteins*

Multiple sequence alignments were performed using DNAMAN software (version 7.212, Lynnon Corp., Quebec, QC, Canada). Secondary structures and three-dimensional models were constructed using Protein Homology/analogY Recognition Engine v.2.0 (Phyre2, http://www.sbg.bio.ic.ac.uk/phyre2/html/ (accessed on 13 February 2021)) [53]. The basic physicochemical properties of the 14-3-3 proteins, including MW, pI, and the

number of amino acid residues, were examined using ProtParam on the ExPASy website ( https://www.expasy.org/vg/index/protein (accessed on 13 February 2021)) [54].

#### *4.4. Gene Structure Analysis and Conserved Motif Prediction*

The GFF3 annotation file was obtained from the wheat reference genome IWGSC Ref-Seq v.1.1. Gene structure analysis was conducted using the Gene Structure Display Server 2.0 (GSDS, http://gsds.cbi.pku.edu.cn (accessed on 13 February 2021)) [55]. Conserved motifs in protein sequences were analyzed by MEME (http://meme-suite.org/tools/meme (accessed on 13 February 2021)) [56], with the maximum number of motifs set to 10.

#### *4.5. Chromosome Distribution, Synteny, Ka/Ks, and Phylogenetic Analysis of TaGRFs*

Information about the start and end of *TaGRFs* was extracted from the GFF3 file. A physical map of *TaGRFs* was constructed using MapInspect software Version 1.0 ( http://www.softsea.com/review/MapInspect.html (accessed on 13 February 2021)) [57]. The TBtools software (https://github.com/CJChen/TBtools/ (accessed on 13 February 2021)) was used to determine the Ka and Ks values of *TaGRFs*, based on their coding sequences (CDSs) [58]. Reference genome sequences of wheat sub-genome donors were downloaded from NCBI, and *14-3-3* genes of each species were identified using the same methods as those used for determining the *TaGRFs*. To determine the paralogous or orthologous relationship between wheat *TaGRFs* and *14-3-3* genes of its sub-genome donors, the general tool "all against all BLAST searches" was used, with an *E*-value of 1 × 10−10 and sequence similarity > 75% [59]. The "circlize" package of the R program was used to draw the relationship between wheat *TaGRFs* and *14-3-3* genes of its sub-genome donors [60]. Phylogenetic analysis was conducted using the ML method of MEGA7, based on the aligned *14-3-3* sequences of *T. aestivum*, *A. thaliana*, *O. sativa*, *G. max*, *P. trichocarpa*, *M. truncatula*, and *V. vinifera*, with 1000 bootstrap replications [61]. The phylogenetic tree file was then uploaded to the Interactive Tree of Life (https://itol.embl.de/ (accessed on 13 February 2021)) for adjustment and modified [62].

#### *4.6. Cis-Acting Element Analyses*

The 1.5 kb genomic DNA sequences upstream of the start codon (ATG) of each *TaGRFs* genes were extracted from the wheat genome sequence. *Cis*-regulatory elements in the promoters were identified using the PlantCARE database (http://bioinformatics.psb.ugent. be/webtools/plantcare/html/ (accessed on 13 February 2021)) [63].

#### *4.7. Expression Analysis of TaGRF Genes under Abiotic Stress Conditions*

To determine the expression patterns of 14-3-3 genes in wheat under salt stress, wheat transcriptome data were downloaded from the NCBI Short Read Archive (SRA) database and mapped onto the reference genome of wheat using hisat2. The FPKM (fragments per kilobase of transcript per million) values obtained after "cufflinks" assembly were log-transformed, and a heatmap was drawn using the RStudio software "pheatmap" to display the expression profiles of *TaGRFs* [64,65].

#### *4.8. RNA Isolation and qRT-PCR Analysis*

Total RNA was extracted from leaf and root tissues of wheat plants treated with salt or water (control) using the RNAprep Pure Plant Kit (Invitrogen). The isolated total RNA was reverse transcribed to synthesize cDNA using the HiScript® II 1st Strand cDNA Synthesis Kit (Vazyme) for qRT-PCR analysis. The cDNA was diluted to 100 ng/µL with RNase-free water, and qRT-PCR was performed in a 10 µL reaction volume containing 5 µL ChamQ Universal SYBR qPCR Master Mix, 0.5 µL each forward and reverse primer (10 µM), and 4 µL cDNA template. The following conditions were used for PCR: initial denaturation at 95 ◦C for 3 min, followed by 40 cycles of denaturation at 95 ◦C for 10 s, and annealing at 60 ◦C for 30 s. Fluorescence signals were collected after each cycle, and the temperature was increased from 60 ◦C to 95 ◦C after each cycle for melting curve analysis. The EF-1α gene (GeneBank accession: BT009129.1) was used as a reference gene. The relative expression level of genes was calculated using the 2−∆∆CT method [66]. Three technical repeats were performed for each sample, and three independent replicates were carried out. Primers used for qRT-PCR are listed in Table S7.

#### *4.9. Virus-Induced Gene Silencing (VIGS) Assay in Wheat*

There are four kinds of vectors involved in the VIGS test: α, β, γ, and γ-PDS. The gene fragments were cloned into the γ vector to obtain a recombinant vector. The vector (α, β, γ, γ-PDS, and recombinant vector) were linearized, and the linearized plasmid was treated with RiboMAX ™ Large Scale RNA Production System-T7 and the Ribom7G Cap Analog (Promega) to obtain capped in vitro transcription products. VIGS inoculation was carried out at the 3-leaf stage [67]. Steps are as follows: Mix equal volumes of in vitro transcription products α, β, γ (or γ-PDS /recombinant γ), dilute with DEPC water, add 1 × FES buffer (0.1 M glycine, 0.06 M K2HPO4, 1% *w*/*v* tetrasodium pyrophosphate, 1% *w*/*v* bentonite, and 1% *w*/*v* celite, pH 8.5), and then rubbed onto wheat leaves. BSMV: γ-PDS (PDS: wheat phytoene desaturase gene) and BSMV: γ were used as controls for BSMV infection. After inoculation, when BSMV: γ-PDS showed bleaching and yellowing phenomenon (about 10 days later), ddH2O and 300 mmol NaCl solution were used for irrigation. After 12 days of irrigation, the curl phenotype of the fourth leaf was recorded and the curl rate was counted. Curl rate is the percentage of curled leaves in all leaves.

#### *4.10. Subcellular Localization of the TaGRF6-A Protein*

The CDS of *TaGRF6-A* was cloned into the pQBV3 Gateway entry vector and then cloned into the pEarlyGate101 destination expression vector [68]. The resulting TaGRF6-A-YFP fusion construct was transformed into *Agrobacterium tumefaciens* strain GV3101, which was grown on LB (add antibiotics: kanamycin, rifampicin, gentamicin) solid medium for 2 days. The positive colonies were verified by PCR and transferred to LB (add antibiotics: kanamycin, rifampicin, gentamicin) liquid medium. The culture was grown for 16 h at 28 ◦C on a shaker until the optical density of the culture (measured at 600 nm absorbance; OD600) reached 1.5–1.8. The cells were harvested by centrifugation at 4000× g for 15 min and resuspended in acetosyringone (AS) culture solution. Then, 1 mL culture (OD600 = 0.8) was injected into the abaxial surface of the leaves of 3–4-week-old *N. benthamiana* plants using a needleless syringe, followed by incubation in the dark for 4 h. At 48 h post-inoculation, the distribution of the YFP signal in leaf epidermal cells was observed under a confocal laser scanning microscope (Zeiss LSM710) [69].

#### *4.11. Y2H, BiFC, and CoIP Assays*

The CDSs of *TaGRF6-A* and *TaMYB64* were cloned into pGBKT7 (BD) and pGADT7 (AD) vectors, respectively. According to the Yeast Protocols Handbook (Clontech, Mountain View, CA, USA), the recombinant plasmids were transformed into the yeast AH109 strain (*Saccharomyces cerevisiae*) and plated on an SD/-LW selection medium. The plates were incubated at 30 ◦C for 3–5 days until the appearance of colonies. Single colonies were picked using an inoculation ring and streaked onto SD/-LW and SD/-LWHA solid media. Plates were incubated at 30 ◦C for 3–5 days, and photographs were taken to record the growth of yeast colonies. T + P53 and T + lam served as positive and negative controls, respectively. Full-length cDNA sequences of *TaGRF6-A* and *TaMYB64* minus the stop codon were PCR amplified using the Pfu polymerase (NEB). The PCR products were ligated into the pQBV3 vector and then cloned into pEarleyGate201-YN and pEarleyGate202-YC vectors using the LR enzyme (Gateway LR Clonase II Enzyme mix, Invitrogen). The resulting plasmids were transformed into *A. tumefaciens* strain GV3101 and then transiently expressed in *N. benthamiana* leaves using the method described above. For Co-IP analysis, the PCR products were constructed on pEarleyGate100 and pEarleyGate104 vectors. Using the method of Qiao et al. [69], the protein was transiently expressed in *N. benthamiana* and the total protein was extracted, and then incubated with HA magnetic beads (MBL, Tokyo, Japan) at 4 ◦C

and enriched with magnetic beads on ice. The precipitated protein was then separated by SDS-PAGE electrophoresis. Use anti-FLAG and anti HA antibody (MBL, Japan) to detect the coprecipitation signal of TaMYB64-YFP-3FLAG and TaGRF6-A-YFP-HA.

#### *4.12. Statistical Analysis*

Student's *t*-test were applied to test differences among treatments.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/1422-006 7/22/4/1904/s1.

**Author Contributions:** X.Z. (Xiaoguo Zhu), Y.Q., and D.M. conceived this research. W.S. and W.C. designed the experiment and wrote the manuscript. Y.J., C.Z., G.L., and X.L. collected and analyzed public data sets, and W.S., X.Z. (Xiaoyi Zhou) conducted bioinformatics analysis and experiments. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by National Key R&D Program of China [2018YFD0200506]; the Key Project of Hubei Provincial Department of Education [D20191305].

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data is contained within the article or supplementary material.

**Acknowledgments:** The authors thank Zhensheng Kang (Northwest A & F University, Yangling, China) for providing vector of VIGS.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

## *Article* **Heterologous Expression of the Melatonin-Related Gene** *HIOMT* **Improves Salt Tolerance in** *Malus domestica*

**Kexin Tan † , Jiangzhu Zheng † , Cheng Liu, Xianghan Liu, Xiaomin Liu, Tengteng Gao, Xinyang Song, Zhiwei Wei, Fengwang Ma \* and Chao Li \***

> State Key Laboratory of Crop Stress Biology for Arid Areas/Shaanxi Key Laboratory of Apple, College of Horticulture, Northwest A & F University, Yangling, Xianyang 712100, China<EMAIL_ADDRESS>(K.T<EMAIL_ADDRESS>(J.Z<EMAIL_ADDRESS>(C.L<EMAIL_ADDRESS>(X.L<EMAIL_ADDRESS>(X.L<EMAIL_ADDRESS>(T.G<EMAIL_ADDRESS>(X.S<EMAIL_ADDRESS>(Z.W.)

**\*** Correspondence<EMAIL_ADDRESS>(F.M<EMAIL_ADDRESS>(C.L.)

† These authors contribute equally in this study (co-first author).

**Abstract:** Melatonin, a widely known indoleamine molecule that mediates various animal and plant physiological processes, is formed from N-acetyl serotonin via N-acetylserotonin methyltransferase (ASMT). ASMT is an enzyme that catalyzes melatonin synthesis in plants in the rate-determining step and is homologous to hydroxyindole-O-methyltransferase (HIOMT) melatonin synthase in animals. To date, little is known about the effect of *HIOMT* on salinity in apple plants. Here, we explored the melatonin physiological function in the salinity condition response by heterologous expressing the homologous human *HIOMT* gene in apple plants. We discovered that the expression of melatonin-related gene (*MdASMT*) in apple plants was induced by salinity. Most notably, compared with the wild type, three transgenic lines indicated higher melatonin levels, and the heterologous expression of *HIOMT* enhanced the expression of melatonin synthesis genes. The transgenic lines showed reduced salt damage symptoms, lower relative electrolyte leakage, and less total chlorophyll loss from leaves under salt stress. Meanwhile, through enhanced activity of antioxidant enzymes, transgenic lines decreased the reactive oxygen species accumulation, downregulated the expression of the abscisic acid synthesis gene (*MdNCED3*), accordingly reducing the accumulation of abscisic acid under salt stress. Both mechanisms regulated morphological changes in the stomata synergistically, thereby mitigating damage to the plants' photosynthetic ability. In addition, transgenic plants also effectively stabilized their ion balance, raised the expression of salt stress–related genes, as well as alleviated osmotic stress through changes in amino acid metabolism. In summary, heterologous expression of *HIOMT* improved the adaptation of apple leaves to salt stress, primarily by increasing melatonin concentration, maintaining a high photosynthetic capacity, reducing reactive oxygen species accumulation, and maintaining normal ion homeostasis.

**Keywords:** hydroxyindole-O-methyltransferase gene; melatonin; ROS; ABA; ion homeostasis; amino acids

#### **1. Introduction**

Apple (*Malus domestica* Borkh.) is one of the main fruits in the world, the cultivation in China is mainly concentrated in bohai Bay and Loess Plateau, which are two dominant producing areas. Soil salinization in these areas is one of the main obstacles to the expansion of apple eugenic cultivation areas [1]. The sustainable development of apple industry urgently needs the support of apple salt tolerance strategy and technology.

Soil salinity is a progressively serious issue worldwide, as it hinders plant development and causes yield losses [1]. The detrimental influence of NaCl stress on apple plants has two primary aspects [2]. First, the excessive sodium ions accumulation results in a significant decrease in the effective use of water [3], second, Na + and Cl − ions toxic effects

**Citation:** Tan, K.; Zheng, J.; Liu, C.; Liu, X.; Liu, X.; Gao, T.; Song, X.; Wei, Z.; Ma, F.; Li, C. Heterologous Expression of the Melatonin-Related Gene *HIOMT* Improves Salt Tolerance in *Malus domestica*. *Int. J. Mol. Sci.* **2021**, *22*, 12425. https://doi.org/ 10.3390/ijms222212425

Academic Editor: Setsuko Komatsu

Received: 23 October 2021 Accepted: 15 November 2021 Published: 17 November 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

lead to ion imbalances [4]. The resulting disturbances in most physiological processes include growth inhibition, reduction in photosynthetic capacity, destruction of membranes, changes in enzymatic activities, and ionic imbalances [5]. These phenomena emphasize the urgency of improving apple plants salt stress tolerance.

Photosynthesis supplies the energy source for plant normal growth, while the photosynthetic apparatus are sensitive to external stress [6]. Salinity stress reduces leaf photosynthetic capacity through both stomatal and non-stomatal mechanisms [7]. Stomatal limitations result from exposure to high salt concentrations; plants become dehydrated due to a reduction of osmotic potential and turgor pressure, causing a decrease in stomatal aperture and reducing CO2 assimilation capacity, which in turn reduce net photosynthesis [8]. Non-stomatal limitations are related to damage to photosystem II (PSII), CO2 assimilation rate, and electron transport chain (ETC) efficiency [9].

Plants take up specialized approaches to cope with high salt concentrations. Reactive oxygen species (ROS) production is one of the initial reactions caused by salinity [10], and excessive ROS accumulation can initiate lipid peroxidation processes and increase the concentration of malondialdehyde (MDA), ultimately increasing the plasma membrane permeability, causing intracellular electrolytes leakage [11]. To mitigate ROS damage to various tissues, plants trigger salt-induced antioxidant systems, increasing the activity of superoxide dismutase (SOD), peroxidase (POD), ascorbate peroxidase (APX), and related enzymes. Together, these antioxidant enzymes interact to scavenge ROS and improve plant salt tolerance [12]. Studies have shown that ROS accumulation can act as a positive modifier to mediate abscisic acid (ABA) signal and response. As an abiotic stress phytohormone, ABA has a central function in salt stress defense [13]. Under salt stress, the CLAVATA3/ESR-RELATED 25 (CLE25) peptide may be secreted and moved to the shoots where it is identified by the receptors BARELY ANY MERISTEM 1 (BAM1) and BAM3, thus promoting the expression of the ABA biosynthesis gene *NCED3* [14]. This is one of the mechanisms of plant root from the dehydration signal under salinity condition.

The sequestration of toxic ions and the biosynthesis of osmotic substances in plants under salt stress are related to improved osmotic stress tolerance [15]. The intracellular ion balance does not allow toxic ions to accumulate in the cytoplasm and requires net Na+ and Cl− uptake and subsequent vacuolar separation [16]. Appropriate Na+/K+ homeostasis maintains the normal physiological metabolism of plants [17], and the absorption, biosynthesis, and transformation of amino acids under stress can alleviate associated damage [18]. As a crucial mechanism for plants response to salt stress, SOS transports excess Na+ to the extracellular space using the plasma membrane proton gradient [15,19]. NHX family members act on the tonoplast membrane, and the activity of NHX1, NHX2, and NHX4 hinges on the proton gradient to achieve Na+ compartmentalization [20]. The activity of low-affinity K channel (AKT) is also closely associated with salt tolerance. Free amino acid concentrations also respond sensitively to salinity stress. For example, it is common that physiological response of different plants accumulating Pro under salinity condition [21]. Salt stress results in upregulation of genes in the aromatic amino acid (AAA) biosynthetic pathway, increasing the AAA concentration and the production of related secondary metabolites such as auxins, alkaloids, and flavonoids, thereby improving abiotic stress tolerance [22].

Multitude studies have demonstrated that melatonin is an indole molecule with multiple regulatory functions that has a considerable role in protecting plants from abiotic stresses, including salinity–alkalinity, drought, temperature extremes, oxidative stress, and UV radiation [23–27]. The biosynthetic pathway of plant melatonin is well characterized. First, tryptophan decarboxylase (TDC) promotes the conversion of tryptophan to tryptamine. Then serotonin is produced by the hydroxylation reaction of tryptophan 5-hydroxylase (T5H), and finally, arylalkylamine-N-acetyltransferase (AANAT) and Nacetylserotonin methyltransferase (ASMT) catalyze the synthesis of plant melatonin [26,27]. At present, there are abundant researches show that exogenous melatonin can improve plant resistance under salt-alkali stress [24–26]. Among them, Li et al. [24] found that

exogenous melatonin by enhancing the activity of antioxidant enzymes and stabilizing the ion balance to enhance the salt tolerance of *Malus hupehensis*. Recently, several studies have documented the physiological functions of melatonin in plants with heterologous overexpression of melatonin biosynthetic genes [28]. For example, Huang et al. [29] demonstrated that transfer ovine *AANAT*, *HIOMT* genes into switchgrass improved its growth and salinity endurance under salt stress. Droxyindole-O-methyltransferase (*HIOMT*), encodes the last enzyme catalyzes the synthesis of animal melatonin, is a homolog of *ASMT* in apple plants, plays a rate-limiting role in the melatonin synthesis pathway. Its function is to convert N-acetyl serotonin into melatonin [30,31]. In our research team, Liu et al. [31] previously found that ectopic expression *AANAT* and *HIOMT* in apple plants mainly by scavenging reactive oxygen species and increasing total phenolic content to improve resistance to UV-B stress. Although the regulation of *HIOMT* on the UV-B stress response of apples has been described, the mechanism by which *HIOMT* acts in *Malus domestica* under salinity condition has not yet been known clearly. Here, we used hydroponic methods to explore in detail the mechanisms by which *HIOMT* heterologous expression impacts the salinity response of *Malus domestica*.

#### **2. Results**

#### *2.1. Heterologous Expression of HIOMT Improved Apple Salt Stress Tolerance and Increased Melatonin Concentration in Apple Leaves*

In GL-3 (*Malus domestica* Borkh.) plants, salinity induced the heterologous expression of the *HIOMT* homolog *MdASMT*; its expression was highest at 9 h, when it was upregulated 5.39-fold relative to unstressed controls (Figure 1A). To explore the possible physiological role of *HIOMT* in the apple salinity response, we exposed heterologous expression of the *HIOMT* lines previously obtained to salt stress under hydroponic conditions [31]. No notable deviations between the transgenic lines and the wild type (WT) under normal nutritional conditions. By contrast, all genotypes exhibited damage after NaCl treatment for 15 d. Nonetheless, all leaves of the WT exhibited poorly wilting and necrosis, while only the upper leaves of the transgenic lines exhibited brown spots or chlorosis (Figure 1B). These findings demonstrated that heterologous expression of *HIOMT* conferred improved apple plants salt stress tolerance.

To study variations in melatonin metabolism under salinity condition, we surveyed the transcript levels of four core melatonin synthesis genes. Under 1/2 nutrient solution culture condition, their expression levels were low. Under salinity conditions, all melatoninrelated genes were induced, and the levels in transgenic lines were higher than those in the WT significantly (Figure 1C). We also measured melatonin concentration by LC-MS and found that it was significantly lower in WT plants in than in transgenic lines under normal and salinity conditions. The melatonin concentration upregulated by salinity and was highest in the H5 lines, reaching 1.81 ng g−1 FW (Figure 1D). These findings showed that heterologous expression of *HIOMT* enhanced remarkably increases in the endogenous melatonin concentration of apple leaves under salinity condition.

#### *2.2. Heterologous Expression of HIOMT Promoted Better Growth and Development of Plants under Salinity Stress*

After salt treatment, the shoot height (SH) did not differ significantly among lines, but there were differences in root length (RL), total fresh weight (TFW), and total dry weight (TDW). RL increased under salt stress, and transgenic lines had greater RL than the WT. We also measured leaf number (LN), leaf fresh weight (LFW), and leaf dry weight (LDW) (Table 1). Transgenic lines continued to grow new leaves under salt stress, and their LNs were higher than that of the WT; this explains why total plant weight was lower in WT that in the transgenic lines. In comparation with control conditions, the TFW of WT plants after salinity treatment decreased to 47.9%, whereas that of H1 decreased to 65.9%, that of H2 decreased to 68.2%, and that of H5 decreased to 65.0% (Table 1). These findings illustrated that heterologous expression of *HIOMT* may effectively moderate some of the defective influences of salinity on the growth of apple lines.

**Figure 1.** Heterologous expression of *HIOMT* confers enhanced salinity tolerance to apple. (**A**) The expression of *MdASMT* after salt treatment in GL-3 apple; (**B**) phenotypes of WT and transgenic lines under control and 120 mM NaCl condition. Bars: 50 mm; (**C**) The expression of MT-related synthesis genes under control and salinity conditions; (**D**) the concentration of melatonin after salinity treatment. Values are noted as means of 3 replicates ± SE. Based on Tukey' s multi-range test (*p* < 0.05), not the same lowercase letters were used to represent statistically significant differences. WT, wild type. H1, heterologous expression of *HIOMT* line 1. H2, heterologous expression of *HIOMT* line 2. H5, heterologous expression of *HIOMT* line 5. CK, control group. ST, salt stress group.

**Table 1.** Stem height (SH), root length (RL), leaf number (LN), leaf fresh weight (LFW), leaf dry weight (LDW), total fresh weight (TFW), total dry weight (TDW), relative growth of heterologous expression of *HIOMT* apple plants after 15 d under control, and salinity conditions.

Values are noted as means of 6 replicates ± SE. Based on Tukey' s multi-range test (*p* < 0.05), not the same lowercase letters were used to represent statistically significant differences.

#### *2.3. Heterologous Expression of HIOMT Changed REL, Root Vitality, and MDA Concentration in Apple*

Plant abiotic stress tolerance be assessed by basic physiological indicators such as relative electrolyte leakage rate (REL), root vitality and MDA concentration. No clearly differences in the three indicators among the control lines. After salt stress, REL was 21.76–28.41% lower in transgenic lines than in WT (Figure 2A). Root vitality increased, and transgenic lines showed a greater increase (Figure 2B). Finally, the MDA concentration of

the WT was higher than that of the transgenic lines significantly, which was 1.11-fold that of the unstressed control (Figure 2C). These results showed that heterologous expression of *HIOMT* could improve the endurance of apple plants to salinity.

**Figure 2.** Heterologous expression of *HIOMT* had an impact on REL, Root vitality and MDA concentration under control and salinity conditions. (**A**) Leaf relative electrolyte leakage (REL); (**B**) root vitality and (**C**) leaf MDA concentration. Values are noted as means of 3 replicates ± SE. Based on Tukey' s multi-range test (*p* < 0.05), not the same lowercase letters were used to represent statistically significant differences.

#### *2.4. Heterologous Expression of HIOMT Enhanced the Antioxidant Activity of Apple Plants under Salinity Stress*

According to the histochemical staining results after salt stress, the WT apple leaves showed darker blue and yellow colors after staining with 3,3′ -diaminobenzidine (NBT) and nitro blue tetrazolium (DAB) (Figure 3A,B). We further confirmed it by quantitative analysis. The transgenic lines accumulated significantly fewer ROS than the WT (Figure 3C,D). In addition, after salt treatment, the SOD, POD, and APX activities of all plants were significantly upregulated and were higher in the transgenic lines than in the WT obviously (Figure 3E–G). Therefore, under salinity condition, heterologous expression of *HIOMT* enhanced the activity of antioxidant enzymes, enabling apple plants to avoid excessive ROS accumulation.

**Figure 3.** Accumulation of active oxygen and antioxidant enzyme in *HIOMT* transgenic and WT plants under control and salinity conditions. (**A**,**B**) Chemical staining of O2 − and H2O2 in transgenic lines and WT apple leaves under control and salinity condition; (**C**,**D**) O2 − and H2O2 concentrations in apple leaves with and without salinity treatment; the activity of (**E**) SOD; (**F**) POD and (**G**) APX in transgenic Lines and WT apple leaves under control and salinity condition. Values are noted as means of 3 replicates ± SE. Based on Tukey' s multi-range test (*p* < 0.05), not the same lowercase letters were used to represent statistically significant differences.

#### *2.5. Heterologous Expression of HIOMT Enabled Apple Plants to Maintain Higher Photosynthetic Capacity under Salinity Stress*

We measured the photosynthetic parameters of the plants every three days throughout the entire salinity stress process. Under control conditions, no significant differences among the genotypes in Pn (net photosynthetic rate), Ci (intercellular CO2 concentration), gs (stomatal conductance), or Tr (transpiration rate). Under salt treatment, all four parameters had decreased rapidly in all lines by the third day, but those of the WT had decreased more (Figure 4A–D). The downward trend slowed after day 3, but the values of all parameters were significantly lower in the WT than in the transgenic plants. The Pn of the transgenic plants was about 1.7 times that of the WT. Total chlorophyll concentration also reduced in answer to salt stress, but *HIOMT* heterologous expression alleviated this decrease (Figure 4E).

**Figure 4.** Influences in photosynthetic capacity of heterologous expression of *HIOMT* in apple under control and salinity conditions. (**A**) Changes in Pn; (**B**) gs; (**C**) Ci and (**D**) Tr; (**E**) the concentration of total chlorophyll; (**F**) chlorophyll fluorescence images; (**G**) Fv/Fm ratios of WT and transgenic lines treated with and without salinity. Values are noted as means of 5 replicates ± SE. Based on Tukey' s multi-range test (*p* < 0.05), not the same lowercase letters were used to represent statistically significant differences.

Photosystem II (PSII) is considered to be the primary site of photoinhibition in plants. We measured its maximum photochemical efficiency (Fv/Fm) and found that under salinity condition, the Fv/Fm values of the three transgenic lines decreased by about 3.9%, 4.5%, and 5.6%, whereas that of the WT was reduced to 0.6774, a decrease of 13.6% (Figure 4F,G). These data show that the heterologous expression of *HIOMT* could alleviate damage to plant photosynthetic capacity under salt treatment and enhance plant salt tolerance to some extent.

#### *2.6. Heterologous Expression of HIOMT Alleviated Stomatal Closure in Apple under Salinity Stress*

Plant stomata undergo a series of changes under salt stress. We found that stomatal length, width, and aperture of apple leaves decreased after salt treatment compared with normal conditions. Stomatal contraction was most obvious in the WT (Figures 5 and 6A–D). The stomatal density of all lines increased under salt stress relative to normal conditions, and there were no distinct differences among the lines. ABA mediates the response

of stomatal aperture to changes in the external environment. We measured leaf ABA concentration after salt treatment and found that under normal conditions, little difference in ABA concentration among genotypes. The ABA concentration increased in all lines after salt treatment, but this increase was greatest in the leaves of WT plants; their ABA levels were 1.2 times those of the transgenic plants (Figure 6F). We measured the transcript levels of *MdNCED3*, a key gene that induces ABA biosynthesis under stress. We found that salt treatment significantly increased its expression, but its expression was significantly lower in transgenic lines than in the WT (Figure 6G). These findings indicated that heterologous expression of *HIOMT* could alleviate the ABA concentration increasement in and the decrease in stomatal aperture under salinity condition.

**Figure 5.** Changes in stomatal morphology, scanning electron microscopy (SEM) images of stomata from *HIOMT* transgenic and WT plants under control and salinity conditions. (**A**–**H**) Control, changes in stomatal morphology of WT, H1, H2, H5 under control condition, (**A**–**D**) magnification 300×, scale bars = 50 µm; (**E**–**H**) magnification 3000×, scale bars = 5 µm. (**I**–**P**) Salt, changes in stomatal morphology of WT, H1, H2, H5 under salt condition, (**I**–**L**) magnification 300×, scale bars = 50 µm; (**M**–**P**) magnification 3000×, scale bars = 5 µm.

**Figure 6.** Changes in ABA concentration and ABA synthesis genes expression of WT and *HIOMT* plants under control and salinity conditions. Changes in (**A**) stomatal density, (**B**) stomatal length, (**C**) stomatal width and (**D**) stomatal aperture under salt stress. (**E**) the concentration of ABA; Changes in expression of (**F**) *MdNCED3*. Values are noted as means of 3 replicates ± SE. Based on Tukey' s multi-range test (*p* < 0.05), not the same lowercase letters were used to represent statistically significant differences.

#### *2.7. Heterologous Expression of HIOMT Reduced the Na+ /K+ Ratio in Apple Plants under Salinity Stress*

Under normal conditions, levels of Na+ ions were very low in the leaves of transgenic and WT plants, and no significant differences among the lines. The K+ ion concentration was 23.4–26.5 mg g−1 DW. Plants were under NaCl stress for 15 d, the Na+ ion concentration of all plant leaves increased significantly, but that of the three transgenic lines was significantly lower (74.8%, 93.7%, and 82.5%) than that of the WT. The K+ ion concentration declined over time, and the final Na+/K+ ratio of the WT was much higher than that of the transgenic lines (Figure 7A–C). We selected key salt stress–related genes for quantitative analysis, and we found that the expression levels of *MdSOS1*, *MdSOS2*, and *MdSOS3* in the SOS pathway were all upregulated in leaves under salinity condition (Figure 7D–F). The expression levels of *MdNHX1*, *MdNHX2*, and *MdNHX4* also showed the same trend (Figure 7G–I). At the same time, the expression of a membrane protein involved in K+ ion transport in leaves (AKT potassium transport protein) was also significantly upregulated (Figure 7J). These results showed that heterologous expression of *HIOMT* stabilized the leaf Na+/K+ ratio under salinity condition.

**Figure 7.** The changes of Na+ and K+ of WT and *HIOMT* plants leaves and the expression of saltrelated genes of WT and *HIOMT* plants leaves under control and salinity conditions. (**A**) Changes in concentrations of Na+ ; (**B**) concentrations of K+ and (**C**) Na+/K+ ratios in the leaves of WT and transgenic lines. Changes in expression of (**D**) *MdSOS1*; (**E**) *MdSOS2*; (**F**) *MdSOS3*; (**G**) *MdNHX1*; (**H**) *MdNHX2*; (**I**) *MdNHX4*; (**J**) *MdAKT1*; Values are noted as means of 3 replicates ± SE. Based on Tukey' s multi-range test (*p* < 0.05), not the same lowercase letters were used to represent statistically significant differences.

#### *2.8. Heterologous Expression of HIOMT Mediated Amino Acid Metabolism under Salinity Stress*

Under control conditions, there were no clear diversities in the concentration of amino acid between the lines (Figure 8), but there were significant differences between the transgenic and WT plants under salt stress. The concentrations of alanine (Ala), aspartic acid (Asp), and threonine (Thr) were lower after stress than under control conditions. The concentration of Ala and Thr in the transgenic lines had dropped more, whereas the concentration of Asp was slightly higher in the transgenic lines than in the WT (Figure 8A,B,G). After stress, the concentration of most amino acids increased; these included lysine (Lys), phenylalanine (Phe), proline (Pro), tyrosine (Thr), and tryptophan (Tyr). Their concentration increased more in the WT and were 18.5, 7.8, 12.4, 13.1, and 10.9 times those of the unstressed control; proline concentration increased to 149.4 µg/g. The amino acid concentration of the transgenic lines were lower than those of the WT significantly (Figure 8D–F,H,I). The leucine (Leu) concentration of the WT and H5 was higher after stress than in the unstressed control; the Leu concentration of H5 increased less, the Leu concentration of H1 and H2 was reduced, and the Leu concentration of WT was the highest (Figure 8C). These results showed that heterologous expression of *HIOMT* in apples altered amino acid metabolism under salt stress.

**Figure 8.** Changes in the concentration of amino acids in the leaves of WT and *HIOMT* plants after salt stress. The concentration of (**A**) Alanine, (**B**) Aspartic acid, (**C**) Leucine, (**D**) Lysine, (**E**) Phenylalanine, (**F**) Proline, (**G**) Threonine, (**H**) Tryptophan, (**I**) Tyrosine. Values are noted as means of 3 replicates ± SE. Based on Tukey' s multi-range test (*p* < 0.05), not the same lowercase letters were used to represent statistically significant differences.

#### **3. Discussion**

There is abundance evidence conducted studies the important roles of melatonin in plant tolerance to stressors [23,27,28]. HIOMT is an enzyme that has a rate-limiting effect in the process of human melatonin synthesis. The genes encoding HIOMT enzymes was transferred into GL-3 apple, which increased endogenous melatonin concentration in plants significantly [31]. The ability of exogenous melatonin to improve *Malus hupehensis* endurance to salinity has been reported previously [24]. However, barely known about the specific function of *HIOMT* in the apple salinity response. At this respect, we discovered that heterologous expression of *HIOMT* induced melatonin-related genes up-regulated under salt stress (Figure 1A,C), and we then used hydroponic salt treatment to understand of the mechanisms by which HIOMT functions in response to salinity stress.

We discovered that the melatonin concentration enhanced in all lines under salinity condition, but it was much lower in WT than in transgenic lines under both normal and salinity conditions (Figure 1D). This result is in accordance with antecedent research showing that changes in the external environment cause plants to activate the endogenous melatonin regulatory pathway and change their hormone levels to improve stress tolerance under various stresses, including salt stress [32–34]. A number of other studies have also confirmed that transgenic plants with foreign genes can exhibit increased melatonin production and improved stress tolerance [2,35]. Basic salt stress phenotypes include plant growth inhibition, dysplasia, metabolic disorders, and ion toxicity [36]. The clearest manifestation of salt stress is chlorosis and wilting of leaves (Figure 1B). Electrical conductivity and MDA concentration can also reflect the severity of stress damage [1]. These two indicators were lower in the transgenic lines than in the WT significantly (Figure 2A,C), and transgenic lines showed higher levels of growth, root activity, and other physiological indicators (Table 1 and Figure 2B). These findings show that the heterologous expression

of *HIOMT* increased melatonin concentration, reduced the inhibitory effect of salinity on plant development, and alleviated the symptoms of salt damage.

Salinity leads to damage for the photosynthetic system, inhibits photosynthetic electron transfer, and promotes excessive ROS accumulation [11]. The antioxidant enzyme system has vital function in removing excess ROS from plant tissues [10]. Chen et al. [37] discovered that exogenous melatonin improved the activity of antioxidant enzymes in corn seeds under salinity condition, thereby increasing the salt tolerance of corn. They also discovered that after salinity stress, the concentration of H2O2 and O2 − was lower in apple leaves that overexpressed *HIOMT* than in the WT, and the activities of the ROS scavenging enzymes SOD, POD, and APX were significantly higher (Figure 3A–G). These results indicated that *HIOMT* heterologous expression could increase the antioxidant enzyme activity in apple leaves, removing excess ROS and preventing plants from oxidative damage under salt stress.

Reductions in Pn under salt stress are related to changes in leaf anatomical characteristics [5]. The combination of stomatal closure, chlorophyll loss, and other metabolic changes may lead to a decline in photosynthetic capacity [2]. Here, we found that *HIOMT* heterologous expression reduced stomatal contraction (Figure 5A–P and Figure 6A–D) and total chlorophyll loss under salt stress (Figure 4E), and this may interpret the stronger photosynthetic capacity of the transgenic lines (Figure 4A–D). Some degree of stomatal closure under salt stress is a self-protective mechanism [38]. In a high-salt environment, stomatal closure prevents the water lost through transpiration from exceeding the water absorbed by the roots, thereby preventing dehydration. An increase in stomatal density enhances the capacity for CO2 assimilation and improves the stress resistance of plants [39]. Reductions in chlorophyll concentration and chlorophyll fluorescence parameters occur simultaneously, and chlorophyll fluorescence can provide useful information on changes in photosynthetic performance under stress [40]. Here, we found that transgenic lines also had higher Fv/Fm data than the WT under salt stress (Figure 4F,G). Wei et al. [41] proved that exogenous melatonin appliance helped apple leaves to maintain a higher Fv/Fm value when subjected to photooxidative stress, a result consistent with our present findings.

ABA is closely related to plant stress response [42]. ABA accumulation can cause stomatal closure to prevent water loss while also mediating related genes to facilitate leaf osmotic regulation [43]. To explore stomatal function under salt stress, we measured leaf ABA concentration and found that it was consistent with the changes in stomatal aperture of each line (Figure 6E,F), suggesting that heterologous expression of *HIOMT* can alter the plant's endogenous ABA concentration to control changes in stomatal behavior. Sato et al. [44] reported that *NCED3* may be a key enzyme that promotes ABA biosynthesis in Arabidopsis. Our results show that *MdNCED3* was significantly upregulated under salt stress, but heterologous expression of *HIOMT* inhibited its expression, compliance with the discoveries of Li et al. [45], in which exogenous melatonin restrained the expression of the ABA synthesis gene *MdNCED3* under drought.

Under salinity condition, the absorption and regulation of Na+ and K+ ions is an essential ingredient of plant salt tolerance [17,46]. Here, we gauged the concentrations of Na+ and K+ in the leaves and found that the transgenic lines had strong salt tolerance (Figure 7A–C). This is related to their restriction of Na+ absorption, stable K+ concentration, and maintenance of Na+/K+ balance under salinity condition [24]. The SOS pathway uses the driving force of the plasma membrane proton gradient to discharge excess Na+ , thereby reducing the toxic effect of intracellular Na+ [47]. The NHX pathway acts mainly in concert with the H+ -ATPase and H+ -PPase proton pumps on the tonoplast membrane, sequestering Na+ from the cytoplasm into the vacuole and thereby reducing its potential toxicity [48]. Here, we discovered that salt treatment improved the expression of *SOS* genes apple plants leaves (Figure 7D–F). The expression of *NHX* genes showed a similar trend in all lines (Figure 7G–I), indicating that the Na+ efflux ability of the transgenic lines was stronger, enabling them to effectively alleviate the accumulation of Na+ in leaves under salinity condition. AKT1 is an internal K+ ion current channel, and overexpression of *SsAKT1*

in *Suaeda salsa* promoted the accumulation of potassium ions under salinity condition and enhanced plant salt endurance [49]. Therefore, we also measured the expression levels of *MdAKT1* genes and found that their upregulation under salt stress was higher in transgenic lines than in the WT (Figure 7J). Based on these results, we hypothesize that the heterologous expression of *HIOMT* helps to maintain Na+/K+ homeostasis by mediating the expression of genes connected with ion transport, thereby alleviating symptoms of leaf salt damage. The specific regulatory mechanism requires further exploration.

As the basic unit of biologically functional proteins, amino acids have vital functions in plant signal transduction and stress resistance [50]. Here, we found that the increase in Pro under salinity condition was much more in the WT than in the transgenic lines (Figure 8F). This result is consistent with the review by Mansour and Ali [51], who reported that Pro overproduction is positively correlated with the salt stress pressure experienced by plants. For example, Kanawapee et al. [52] obviously showed that the proline accumulation under salt stress showed a trend opposite to that of salt tolerance level and was consistent with changes in the Na+/K+ ratio. Here, we suspect that the slight increase in Pro concentration in transgenic lines under salt stress occurred because the 120 mM salt concentration caused a lighter stress, and the transgenic plants were able to adjust their osmotic potential, whereas the WT could not. In addition to proline, we also focused on three AAAs (Trp, Tyr, and Phe) whose concentration showed consistent changes with salt stress (Figure 8E,H,I). Studies have shown that diverse secondary metabolites are derived from these AAAs [22]. The synthesis of auxins and alkaloids requires Trp as a precursor material, Tyr is the precursor of betaine, and anthocyanins, flavonoids, and others are derivatives of Phe; all of these secondary metabolites play important roles in protecting plants from stress [53]. Trp, a precursor for melatonin synthesis, can be converted to tryptamine via TDC to synthesize melatonin [54]. We found that the expression level of *MdTDC1* was much higher in transgenic lines than in the WT (Figure 1B), while the Trp concentration of them was lower than WT (Figure 8H). This result may be due to Trp in the transgenic lines be converted to melatonin by *MdTDC1* and other melatonin synthases, thereby further improving the resistance of transgenic lines to salt stress. In summary, heterologous expression of *HIOMT* can directly or indirectly mediate amino acid metabolism to increase plant stress adaptability.

#### **4. Materials and Methods**

#### *4.1. Plant Materials and Treatments*

Tissue-cultured apple plants of *M. domestica* 'GL-3' lines (WT) and transgenic GL-3 plants (heterologous expression *HIOMT* lines) were acquired from the study in our laboratory previously, the heterologous expression levels of *HIOMT* (Accession#M83779) lines were increased by 52.06, 203.05 and 90.92-fold in the H1, H2 and H5 lines, respectively [31]. After 30 d of subculturing and 40 d of rooting, tissue culture seedlings were transplanted to 12 × 12 cm2 nutrition bowls filled with the mixture of soil, vermiculite, perlite (v1:v2:v3 = 4:1:1). The bowls were placed in a constant temperature light incubator. After the seedlings had grown about 7–8 fully expanded true leaves, they were moved to hydroponic containers (35 × 28 × 15 cm3 ) that were wrapped in black plastic and contained 6.5 L half concentration Hoagland's nutrient solution. The pH of the nutrient solution was adjusted to 6.0 ± 0.2 with H3PO4, and it was changed every 5 d. The culture system was designed and improved according to Li et al. previous experiments [24]. The experiments performed in a hydroponic laboratory in Northwest A & F University Yangling (34◦20′ N, 108◦24′ E), Shaanxi Province, China. Plants growth with condition included a 14 h photoperiod (the light intensity was 160 µmol m−2 ·s −1 ), 24 ± 2 ◦C/16 ± 2 ◦C day/night and 60 ± 5% relative humidity [55]. After 2 weeks of pre-adaptation, plants of consistent size and healthy were picked for treatment. The seedlings were divided into two groups, each containing 45 plants: (1) the control group (CK) was grown in half concentration Hoagland nutrient solution as the control, and (2) the salt stress group (ST) was grown in half concentration Hoagland nutrient solution supplemented with 120 mM

NaCl. Both group with a pH of 6.5 ± 0.2. The nutrient solution was updated at an interval of 3 d and treated it for 15 d.

After salt application, control and treated plant leaf tips were taken for *MdASMT* expression analysis. At the end of the treatment, the 4th to 6th leaves of the shoot tips were collected from 12 plants per line and stored at −80 ◦C for subsequent index determination.

#### *4.2. Determination of Melatonin*

The extraction steps of melatonin from leaves referred to the description of Pothinuch and Tongchitpakdee [56]. In brief, a 0.3-g frozen leaf tissue was weighed, each treatment was set up with 5 biological replicates. 5 mL methanol was added; the mixture was sonicated at 4 ◦C and 40 Hz for 40 min. The centrifuge was set to 10,000 g and 4 ◦C for 15 min of centrifugation. All the supernatant was collected, placed in a new centrifuge tube, and dried under nitrogen. After reconstitution in pure methanol, the melatonin concentration in the reconstituted solution was determined by high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS), the above steps refer to Zhao et al. [57]. Three analytical replicates were selected from the measurement results for analysis.

#### *4.3. Growth Measurements*

Six plants of each genotype were selected for growth measurements after 15 d of hydroponic treatment. The height from the junction of the rhizome to the top bud of the plant was measured with an iron ruler; the leaves number was accurately counted; and the fresh and dry weights of each plant were recorded [58]. The formula for calculating the relative growth rate was as follows: the average weight of the treated plants divided by the average weight of the control plants.

#### *4.4. Measurement of Relative Electrolyte Leakage, Root Vitality, and MDA Concentration*

The REL of control leaves, plant leaves under salt stress, and double distilled water were recorded as S1, S2, S0, respectively, refer to the methods of Dionisio-Sese and Tobita for relative conductivity measurement, and calculate REL = S1−S0/S2−S0 × 100%. [59]. After 15 d, white roots were harvested from 5 individual plants and stained with triphenyltetrazolium chloride as described by Gong [34]. Malondialdehyde (MDA) concentration was measured using a test kit (Suzhou Coming Biotechnology) according to the instructions of manufacturer. Three analytical replicates were selected from the measurement results for analysis.

#### *4.5. Qualitative and Quantitative Determination of H2O2 and O2* −

At the end of the treatment, 5–8 fully developed mature leaves were obtained from 5 plants per line for histochemical staining with DAB and NBT. Tissue was decolorized thoroughly to observe the accumulation of H2O2 and O2 − as described previously [60]. Quantitative analysis of each concentration was performed with a test kit (Suzhou Comin Biotechnology). Three analytical replicates were selected from the measurement results for analysis.

#### *4.6. Determination of Antioxidant Enzyme Activity*

After 15 days of treatment, the frozen sample of the leaves was ground into powder, weighed 0.1 g into a 2 mL centrifuge tube, each treatment was set up with 3 biological replicates. Then used a test kit (Suzhou Comin Biotechnology) according to the instructions of manufacturer to determine the enzyme activities of SOD, POD, and APX, three replicates for each treatment [61]. Three analytical replicates were selected from the measurement results for analysis.

#### *4.7. Quantification of Photosynthetic Parameters*

During the whole process of salt treatment, every three days we measured Pn, Ci, gs, and Tr using a CIRAS-3 portable photosynthesis system (CIRAS-3, PP Systems, Amesbury, MA, USA) between 9:00 and 11:00. The instrument parameter setting referred to the description of Liu et al. [55]. For each treatment, 5–8 functional leaves at the same position are selected for the measurement of photosynthetic parameters. Five analytical replicates were selected from the measurement results for analysis.

#### *4.8. Chlorophyll Concentration and Fv/Fm Measurements*

Chlorophyll concentration was measured using the method of Arnon [62]. In brief, 8 mL 80% acetone was added to 0.1 g fresh leaves in the dark for at least 24 h to extract pigments, and the mixture was shaken 3–4 times during this period until the leaves turned white. Each treatment was set up with 3 biological replicates. The optical density values were measured at 663, 645, and 470 nm with a UV-2250 spectrophotometer (Shimadzu, Kyoto, Japan).

To measure chlorophyll fluorescence, fully expanded leaves with minimal salt damage from the same position on 5 plants were wrapped in tin foil. After 20 min of dark adaptation, the leaves were cut and placed into the chlorophyll fluorescence imaging system (IMAGING-PAM, Heinz Walz, Effeltrich, Germany) to monitor the maximum quantum efficiency of photosystem II (Fv/Fm). The parameters of the imaging system were set as follows: meas. light 3, act. light 5, ext. light 3, int 10, and FoFm 6 [63]. Five analytical replicates were selected from the measurement results for analysis.

#### *4.9. Stomatal Observations by Scanning Electron Microscopy (SEM)*

The measurement of stomatal characteristics refered to Bai et al. [64]. After treatment, three upper leaves were cut at the same leaf position for each treatment, avoiding the main and lateral veins; the leaves were cut into 5 mm × 5 mm squares, stored in pre-cooled 25% glutaraldehyde prepared in advance with 0.2 M phosphate buffered saline (PBS) (pH 7.4). The vacuum was applied with a syringe until the leaf sunk to the bottom of the liquid. The sample was wrapped with tin foil and placed in the refrigerator at 4 ◦C. It was then washed with PBS (pH 6.8), dehydrated with an ethanol gradient, and placed in 1 mL of isoamyl acetate. After drying and spraying with gold, leaf stomata were discerned and photographed with the scanning electron microscope at 300 and 3000 magnifications. Five pictures were taken for each treatment at different magnifications. Finally, the stomatal density, size, and degree of opening were analyzed using ImageJ software.

#### *4.10. Determination of ABA Concentration*

Determination of ABA concentration based on the description by Zhang et al. [65]. In brief, endogenous ABA was extracted using extraction liquid (methanol: isopropanol: acetic acid = 20:79:1). A 0.1-g frozen tissue was combined with 1 mL of extraction liquid, vortexed for 5 min, and then put in refrigerator at −20 ◦C for 12 h. The centrifuge was set to 4 ◦C and 12,000 rpm for 10 min of centrifugation. All the supernatant was aspirated and filtered into a sample bottle with a 0.22-µm filter, each treatment was set up with 3 biological replicates, and the ABA concentration of the supernatant was measured by HPLC-MS/MS. Three analytical replicates were selected from the measurement results for analysis.

#### *4.11. Measurements of Sodium and Potassium Ions*

At the end of the stress proceeding, leaves from 10 plants were randomly picked out for each treatment and washed thoroughly. The leaves were wiped dry with folded blotting paper. Samples were dried at 105 ◦C for 20 min and then transferred to a 65 ◦C oven for at least 72 h. After the dried tissue was ground with a mortar and a tissue lyser, it was stored in a 10-mL centrifuge tube for determination of mineral element concentration after sieving by flame spectroscopy (M410; Sherwood Scientific, Cambridge, UK). The specific steps wrote in Liang et al. [58]. Three analytical replicates were selected from the measurement results for analysis.

#### *4.12. Measurements of Amino Acids*

Amino acids were extracted and the concentration of them were measured as accounted by Huo et al. [61] with improvements. In brief, 0.1 g of frozen leaf sample was weighed, soaked in 1 mL 50% ethanol, shaken at 4 ◦C for 5 min, the centrifuge was set to 12,000 rpm, and the time was 10 min, each treatment was set up with 3 biological replicates. Impurities were filtered out, the supernatant was diluted with methanol, and stored in a sample bottle. Amino acids in the sample were measured by liquid chromatography-mass spectrometry (LC-MS, LC: AC, Exion LC; MS: QTRAP 5500, AB SCIEX), and standard curves were used to calculate amino acid concentration. Three analytical replicates were selected from the measurement results for analysis.

#### *4.13. qRT–PCR Analysis*

Samples were thoroughly ground in liquid nitrogen. Roughly 0.05 g of the resulting powder was weighed for RNA extraction with the Wolact Plant RNA Extraction Kit (Wickband, Hong Kong, China) according to the instructions. Then the synthesize of cDNA by the Prime Script RT reagent Kit with gDNA Eraser (Perfect Real Time). The cDNA was uniformly diluted to 200 ng/µL, and qRT-PCR quantitative analysis was performed. Each gene set three biological replicates, and each replicate used 10 µL SYBR Premix Ex Taq (TaKaRa). The ∆Ct value was calculated using *EF* as the internal reference gene [66]. Primer sequences are listed in Table S1. At 0, 3, 6, 9, and 12 h and 3, 6, 9, 12 and 15 d after the initiation of salt stress, the top leaves of the plants were randomly selected for measurement. Three analytical replicates were selected from the measurement results for analysis.

#### *4.14. Statistical Analysis*

All values were statistically analyzed using SPSS 25.0 software, and Tukey's test was used for multiple comparisons (*p* < 0.05). Results are represented as mean ± SE.

#### **5. Conclusions**

Here, we showed that heterologous expression of *HIOMT* enhanced the tolerance of apple leaves to salt stress (Figure 9). Heterologous expression of *HIOMT* increased the expression of melatonin synthase genes under salinity condition and promoted an increase in endogenous melatonin concentration. *HIOMT*-mediated resistance to salinity was associated with reductions in ROS accumulation, maintenance of a strong photosynthetic capacity, regulation of osmotic pressure, and stabilization of the sodium–potassium balance. The heterologous expression of *HIOMT* increased the endogenous melatonin concentration, thereby improving the activity of antioxidant enzymes and inhibiting ROS accumulation. In addition, stomatal aperture could be adjusted by regulating ABA metabolism to restrain water loss and enhance carbon dioxide absorption, thereby improving plant photosynthetic capacity. *HIOMT* heterologous expression also helped to maintain the dynamic balance of intracellular ions by balancing the ratio of sodium and potassium ions and altering amino acid metabolism to alleviate osmotic stress. Our research provides evidence for melatonin-mediated salt tolerance and has crucial applications for improving the growth of horticultural crops on salinity soils.

**Figure 9.** A scheduled model for accounting regulator function of *HIOMT* in physiological reaction of salinity in apple. Under salinity stress, heterologous expression of *HIOMT* enhanced the activity of antioxidant enzymes and inhibited ROS production; inhibited the increase of ABA, maintained stomatal aperture, and improved photosynthetic capacity; mediated amino acid metabolism; stabilized the balance of Na+ , K+ and maintains ion homeostasis in cells. These were all through the heterologous expression of *HIOMT* resulted in an increase in endogenous melatonin. Therefore, heterologous expression of *HIOMT* improved the salt tolerance in transgenic apple plants.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/ijms222212425/s1, Table S1: Primers used in this study.

**Author Contributions:** C.L. (Chao Li) and K.T. designed the research. Z.W. provided the material. K.T., J.Z., C.L. (Cheng Liu), X.L. (Xianghan Liu) and X.S. performed the experiments. K.T., X.L. (Xiaomin Liu) and T.G. analyzed the data. Assisted by J.Z. and X.L. (Xiaomin Liu), K.T. drafted the manuscript. C.L. (Chao Li) revised the manuscript. F.M. and C.L. (Chao li) provided financial support for the study. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the National Key Research and Development Program of China (2018YFD1000303), the National Natural Science Foundation of China (31972389), and the earmarked fund for the China Agricultural Research System (CARS-27). The authors are grateful to Dr. Jason Qee for editing the language of the article.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

## *Article* **Functional Characterization of** *PsnNAC036* **under Salinity and High Temperature Stresses**

**Xuemei Zhang 1 , Zihan Cheng 1 , Wenjing Yao 1,2 , Kai Zhao 1 , Xueyi Wang 1 and Tingbo Jiang 1, \***

	- Nanjing Forestry University, Nanjing 210037, China

**\*** Correspondence<EMAIL_ADDRESS>
**Abstract:** Plant growth and development are challenged by biotic and abiotic stresses including salinity and heat stresses. For *Populus simonii* × *P. nigra* as an important greening and economic tree species in China, increasing soil salinization and global warming have become major environmental challenges. We aim to unravel the molecular mechanisms underlying tree tolerance to salt stress and high temprerature (HT) stress conditions. Transcriptomics revealed that a *PsnNAC036* transcription factor (TF) was significantly induced by salt stress in *P. simonii* × *P. nigra*. This study focuses on addressing the biological functions of *PsnNAC036*. The gene was cloned, and its temporal and spatial expression was analyzed under different stresses. *PsnNAC036* was significantly upregulated under 150 mM NaCl and 37 ◦C for 12 h. The result is consistent with the presence of stress responsive *cis*-elements in the *PsnNAC036* promoter. Subcellular localization analysis showed that *PsnNAC036* was targeted to the nucleus. Additionally, *PsnNAC036* was highly expressed in the leaves and roots. To investigate the core activation region of *PsnNAC036* protein and its potential regulatory factors and targets, we conducted trans-activation analysis and the result indicates that the C-terminal region of 191–343 amino acids of the *PsnNAC036* was a potent activation domain. Furthermore, overexpression of *PsnNAC036* stimulated plant growth and enhanced salinity and HT tolerance. Moreover, 14 stress-related genes upregulated in the transgenic plants under high salt and HT conditions may be potential targets of the *PsnNAC036*. All the results demonstrate that *PsnNAC036* plays an important role in salt and HT stress tolerance.

**Keywords:** *Populus simonii* × *P. nigra*; *PsnNAC036*; transcription factor; salt stress; HT tolerance

#### **1. Introduction**