*2.2. Phylogenetic Relationship Analysis of MAPK Gene in Kiwifruit*

In order to evaluate the evolutionary relationships among the MAPK proteins, a phylogenetic tree was constructed with amino acid sequences of 18 putative *AcMAPKs* from kiwifruit, 20 *AtMAPKs* from Arabidopsis, and 14 *VvMAPKs* from grapevine. In plants, MAPK proteins have diverged into four major subfamilies (A, B, C, and D) [3], as shown in Figure 1. The phylogenetic analysis showed that the 18 putative *AcMAPKs* could be divided into five distinct groups (groups A, B, C, D, and E) together with their MAPK orthologs in *Arabidopsis* and grapevine, which are more groups than identified in previous reports [42]. *AcMAPKs* belonging to the A, B, C, and E subfamilies all possess a TEY motif, except for *AcMAPK18*, which harbors a TDY motif, whereas the D subfamily possesses a TDY motif at the activation site (Table 1).

*AcMAPK5*, *AcMAPK12*, *AcMAPK15* and *AcMAPK16* genes are clustered in Group A, which contains well-characterized *MAPK* genes including *AtMPK3*, *AtMPK6*, *VvMPK12*, and *VvMPK14* genes. *AcMAPK1*, *AcMAPK3*, *AcMAPK4*, *AcMAPK8*, and *AcMAPK11* genes belong to Group B, which ncludes *AtMPK4*, *AtMPK5*, *AtMPK11*, *AtMPK12*, *VvMPK9*, and *VvMPK11* genes. Group C contained three genes: *AcMAPK2*, *AcMAPK7*, and *AcMAPK9* genes. Group D includes *AcMAPK10*, *AcMAPK14*, and *AcMAPK17* genes of the kiwifruit MAPKs (Figure 1), which have a TDY motif, consistently found in members of the other MAPK subfamily. *AcMAPK6*, *AcMAPK13*, and *AcMAPK18*, genes belonging to group E, were separated from other groups (Figure 1).

**Figure 1.** Phylogenetic relationship of putative *MAPK* genes in *Arabidopsis chinensis*, *V. vinifera*, and *A. thaliana*. The phylogenetic tree was created using MEGA5.0 program with the neighbor-joining (NJ) method. Bootstrap values for 1000 replicates are indicated at each branch. Letters A–E indicate different groups of MAPKs. **Figure 1.** Phylogenetic relationship of putative *MAPK* genes in *Arabidopsis chinensis*, *V. vinifera*, and *A. thaliana*. The phylogenetic tree was created using MEGA5.0 program with the neighbor-joining (NJ) method. Bootstrap values for 1000 replicates are indicated at each branch. Letters A–E indicate different groups of MAPKs.

#### *2.3. Gene Structure Analysis of MAPK Gene in Kiwifruit 2.3. Gene Structure Analysis of MAPK Gene in Kiwifruit*

The identification of exon-intron structures for each *AcMAPK* gene was determined by aligning corresponding genomic DNA sequences. The exon/intron structures of putative *AcMAPK* genes could also be divided into five subgroups based on their phylogenetic relationship (Figure 2). We found that *AcMAPK* genes in different groups have strikingly different exon/intron structures, but that the gene structures of putative *AcMAPK* members in the same group were highly conserved in kiwifruit (Figure 2). The putative *AcMAPK* members were composed of exons varying from five to seven in Group A. Group B contains exons varying from six to eight, whereas those of Group C only had two or three exons. Nine to 11 exons were present in the *AcMAPK* genes in Group D, and Group E had a larger number of exons with variable exon lengths than other groups; in this group, the number of exons varied from 16 to 29 (Figure 2). The identification of exon-intron structures for each *AcMAPK* gene was determined by aligning corresponding genomic DNA sequences. The exon/intron structures of putative *AcMAPK* genes could also be divided into five subgroups based on their phylogenetic relationship (Figure 2). We found that *AcMAPK* genes in different groups have strikingly different exon/intron structures, but that the gene structures of putative *AcMAPK* members in the same group were highly conserved in kiwifruit (Figure 2). The putative *AcMAPK* members were composed of exons varying from five to seven in Group A. Group B contains exons varying from six to eight, whereas those of Group C only had two or three exons. Nine to 11 exons were present in the *AcMAPK* genes in Group D, and Group E had a larger number of exons with variable exon lengths than other groups; in this group, the number of exons varied from 16 to 29 (Figure 2).

Supplementary Material File 3).

**Figure 2.** The phylogenetic analysis and intron/exon structures of putative *MAPK* genes in *A. chinensis*. The phylogenetic tree (left panel) was created using MEGA5.0 program with the neighborjoining (NJ) method. Exon/intron structures of the *MAPK* genes are shown in the right panel. The green boxes indicate the exons, whereas the single lines indicate introns. Gene models were drawn to scale as indicated on bottom. **Figure 2.** The phylogenetic analysis and intron/exon structures of putative *MAPK* genes in *A. chinensis*. The phylogenetic tree (left panel) was created using MEGA5.0 program with the neighbor-joining (NJ) method. Exon/intron structures of the *MAPK* genes are shown in the right panel. The green boxes indicate the exons, whereas the single lines indicate introns. Gene models were drawn to scale as indicated on bottom.

#### *2.4. The Conserved Motifs Domain and Promoter Regions Analysis of MAPK Gene in Kiwifruit 2.4. The Conserved Motifs Domain and Promoter Regions Analysis of MAPK Gene in Kiwifruit*

To explore the structural diversity of the *AcMAPK* genes, we submitted the 18 putative AcMAPK protein sequences to the online MEME program to search for conserved motifs (Figure 3, Supplementary Material File 2) [43]. As shown in Figure 3, 20 conserved motifs were identified. Specifically, all the identified AcMAPKs contained motifs 1, 3 (contained the TXY signature motif), and 5 (Figure 3), indicating that all the kiwifruit MAPKs were typical of the MAPK family. Additionally, the majority of AcMAPKs contained the 13 protein kinase motifs (motifs 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, and 15) (Figure 3). We found all the members identified in the same subfamily shared similar conserved motifs. For instance, along with all the conserved motifs, most MAPK proteins in Groups A and B had specific motif 11 at the N-terminal region, whereas 18 motifs only existed in most MAPKs in Group C. MAPKs in group D contained specific motif 19 at the N-terminal region as well as motif 16 at the C-terminal region, and motifs 13 and 17 only existed in Group E of the MAPK To explore the structural diversity of the *AcMAPK* genes, we submitted the 18 putative AcMAPK protein sequences to the online MEME program to search for conserved motifs (Figure 3, Supplementary Material File 2) [43]. As shown in Figure 3, 20 conserved motifs were identified. Specifically, all the identified AcMAPKs contained motifs 1, 3 (contained the TXY signature motif), and 5 (Figure 3), indicating that all the kiwifruit MAPKs were typical of the MAPK family. Additionally, the majority of AcMAPKs contained the 13 protein kinase motifs (motifs 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, and 15) (Figure 3). We found all the members identified in the same subfamily shared similar conserved motifs. For instance, along with all the conserved motifs, most MAPK proteins in Groups A and B had specific motif 11 at the N-terminal region, whereas 18 motifs only existed in most MAPKs in Group C. MAPKs in group D contained specific motif 19 at the N-terminal region as well as motif 16 at the C-terminal region, and motifs 13 and 17 only existed in Group E of the MAPK proteins (Figure 3).

proteins (Figure 3). To further investigate the potential functions and transcriptional regulation of these putative *AcMAPK* genes, we identified the *cis*-regulatory elements by the transcriptional start site (ATG) using 1500 bp upstream regions. We found a large amount of pathogen-related, stress-related, and hormone-related *cis*-elements in the putative promoter regions of the putative *AcMAPK* genes in kiwifruit. Some genes contain more *cis*-elements, and some genes contain less (Figure S3, To further investigate the potential functions and transcriptional regulation of these putative *AcMAPK* genes, we identified the *cis*-regulatory elements by the transcriptional start site (ATG) using 1500 bp upstream regions. We found a large amount of pathogen-related, stress-related, and hormone-related *cis*-elements in the putative promoter regions of the putative *AcMAPK* genes in kiwifruit. Some genes contain more *cis*-elements, and some genes contain less (Figure S3, Supplementary Material File 3).

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**Figure 3.** The conserved motifs of kiwifruit putative MAPKs according to the phylogenetic relationship. All motifs were identified online with the MEME program with the complete amino acid sequences of the 18 MAPKs. Different colors of the boxes represent different motifs in the corresponding position of each AcMAPK proteins. Detailed information of the 20 motifs is provided in Supplementary Material File 2. **Figure 3.** The conserved motifs of kiwifruit putative MAPKs according to the phylogenetic relationship. All motifs were identified online with the MEME program with the complete amino acid sequences of the 18 MAPKs. Different colors of the boxes represent different motifs in the corresponding position of each AcMAPK proteins. Detailed information of the 20 motifs is provided in Supplementary Material File 2.

#### *2.5. Expression Profiles of AcMAPK Genes in Response to Hormone Treatments 2.5. Expression Profiles of AcMAPK Genes in Response to Hormone Treatments*

To investigate the contribution of *AcMAPK* to various hormone treatments, we subjected fourweek-old seedlings of Jinkui (*A. chinensis* var. *deliciosa*) to examine the expression patterns of 18 *AcMAPK* genes using quantitative real-time PCR. In order to obtain a comprehensive view and compare the effects of different treatments on a given gene, the produced heat-map graphic of the expression profiles for all genes and all hormone treatments is provided in Figure 4. It was interesting that the transcript levels of almost all genes were down-regulated in response to hormone treatments (Figures 4, S4, and S5). In our work, the transcript levels of all *AcMAPK* genes were down-regulated after abscisic acid (ABA) treatment (Figures 4 and S4A). *AcMAPK5*, *AcMAPK 9*, *AcMAPK15*, and *AcMAPK16* genes were up-regulated at four hours. *AcMAPK17* showed obvious up-regulation at 4 and 48 h after 1-aminocyclopropanecarboxylic acid (ACC) treatment (Figures 4 and S4B). These genes (*AcMAPK4*, *AcMAPK5*, and *AcMAPK9*) were significantly up-regulated at 12 h after salicylic acid (SA) treatment, and *AcMAPK5*, *AcMAPK9*, *AcMAPK15*, *AcMAPK16*, and *AcMAPK17* were induced by jasmonic acid (JA) treatment (Figures 4 and S5). In these genes, *AcMAPK5* demonstrated significantly higher induction after the hormone treatments than other genes. To investigate the contribution of *AcMAPK* to various hormone treatments, we subjected four-week-old seedlings of Jinkui (*A. chinensis* var. *deliciosa*) to examine the expression patterns of 18 *AcMAPK* genes using quantitative real-time PCR. In order to obtain a comprehensive view and compare the effects of different treatments on a given gene, the produced heat-map graphic of the expression profiles for all genes and all hormone treatments is provided in Figure 4. It was interesting that the transcript levels of almost all genes were down-regulated in response to hormone treatments (Figure 4, Figure S4, and Figure S5). In our work, the transcript levels of all *AcMAPK* genes were down-regulated after abscisic acid (ABA) treatment (Figure 4 and Figure S4A). *AcMAPK5*, *AcMAPK 9*, *AcMAPK15*, and *AcMAPK16* genes were up-regulated at four hours. *AcMAPK17* showed obvious up-regulation at 4 and 48 h after 1-aminocyclopropanecarboxylic acid (ACC) treatment (Figure 4 and Figure S4B). These genes (*AcMAPK4*, *AcMAPK5*, and *AcMAPK9*) were significantly up-regulated at 12 h after salicylic acid (SA) treatment, and *AcMAPK5*, *AcMAPK9*, *AcMAPK15*, *AcMAPK16*, and *AcMAPK17* were induced by jasmonic acid (JA) treatment (Figure 4 and Figure S5). In these genes, *AcMAPK5* demonstrated significantly higher induction after the hormone treatments than other genes.

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**Figure 4.** Hierarchical clustering of the expression profiles of *AcMAPK* genes in response to different hormones treatments in kiwifruit leaves. ABA: treatments with abscisic acid, ACC: treatments with 1-Aminocyclopropanecarboxylic Acid, SA: treatments with salicylic acid; JA: treatments with jasmonic acid, details of the treatments are reported in Materials and Methods. The heat-map demonstrates the relative fold-change expression for all *AcMAPK* genes in response to the different hormone treatments in comparison to their respective controls. Red and green colors represent increased or decreased expression levels, respectively, in comparison to controls, as reported by the scale. Genes were clustered according to phylogenetic relationships in expression profiles. Relative expression values for each gene and each treatment are provided in Figures S4 and S5. **Figure 4.** Hierarchical clustering of the expression profiles of *AcMAPK* genes in response to different hormones treatments in kiwifruit leaves. ABA: treatments with abscisic acid, ACC: treatments with 1-Aminocyclopropanecarboxylic Acid, SA: treatments with salicylic acid; JA: treatments with jasmonic acid, details of the treatments are reported in Materials and Methods. The heat-map demonstrates the relative fold-change expression for all *AcMAPK* genes in response to the different hormone treatments in comparison to their respective controls. Red and green colors represent increased or decreased expression levels, respectively, in comparison to controls, as reported by the scale. Genes were clusteredaccording to phylogenetic relationships in expression profiles. Relative expression values for each geneand each treatment are provided in Figures S4 and S5.

#### *2.6. Expression Patterns of AcMAPK Genes under Abiotic and Biotic Stresses 2.6. Expression Patterns of AcMAPK Genes under Abiotic and Biotic Stresses*

We also investigated the expression of *AcMAPK* genes in response to various abiotic and biotic stress responses with different hormone treatments (Figures 5, S6, and S7). In response to cold stress, the expression of five *AcMAPK* genes (*AcMAPK5*, *AcMAPK9*, *AcMAPK10*, *AcMAPK11*, and *AcMAPK12*) were significantly up-regulated throughout the treatment process, and *AcMAPK4* was up-regulated at 48 h of treatment; whereas *AcMAPK2*, *AcMAPK6*, *AcMAPK7*, *AcMAPK13*, and *AcMAPK18* genes were down-regulated at all treated time points (Figures 5 and S6A). After heat treatment, nine *AcMAPK* genes (*AcMAPK1*, *AcMAPK5*, *AcMAPK10*, *AcMAPK11*, *AcMAPK14*, *AcMAPK15*, *AcMAPK16*, *AcMAPK17* and *AcMAPK18*) were up-regulated after four hours of heat stress treatment at 48 °C. The *AcMAPK11* gene was significantly up-regulated (Figures 5 and S6B). With salt treatment, the expression of *AcMAPK4*, *AcMAPK5*, *AcMAPK9*, and *AcMAPK12* genes were significantly up-regulated at all treatment time points, and *AcMAPK10*, *AcMAPK13* and *AcMAPK17* genes were up-regulated at several treated time points, whereas the remaining genes were almost down-regulated under salt treatment (Figures 5 and S7A). Almost all the *AcMAPKs* genes (except *AcMAPK2*, *AcMAPK3* and *AcMAPK9*) were down-regulated after *Pseudomonas syringae* pv. *actinidiae* (Psa) treatment (Figures 5 and S7B). We also investigated the expression of *AcMAPK* genes in response to various abiotic and biotic stress responses with different hormone treatments (Figure 5, Figure S6, and Figure S7). In response to cold stress, the expression of five *AcMAPK* genes (*AcMAPK5*, *AcMAPK9*, *AcMAPK10*, *AcMAPK11*, and *AcMAPK12*) were significantly up-regulated throughout the treatment process, and *AcMAPK4* was up-regulated at 48 h of treatment; whereas *AcMAPK2*, *AcMAPK6*, *AcMAPK7*, *AcMAPK13*, and *AcMAPK18* genes were down-regulated at all treated time points (Figure 5 and Figure S6A). After heat treatment, nine *AcMAPK* genes (*AcMAPK1*, *AcMAPK5*, *AcMAPK10*, *AcMAPK11*, *AcMAPK14*, *AcMAPK15*, *AcMAPK16*, *AcMAPK17* and *AcMAPK18*) were up-regulated after four hours of heat stress treatment at 48 ◦C. The *AcMAPK11* gene was significantly up-regulated (Figure 5 and Figure S6B). With salt treatment, the expression of *AcMAPK4*, *AcMAPK5*, *AcMAPK9*, and *AcMAPK12* genes were significantly up-regulated at all treatment time points, and *AcMAPK10*, *AcMAPK13* and *AcMAPK17* genes were up-regulated at several treated time points, whereas the remaining genes were almost down-regulated under salt treatment (Figure 5 and Figure S7A). Almost all the *AcMAPKs* genes (except *AcMAPK2*, *AcMAPK3* and *AcMAPK9*) were down-regulated after *Pseudomonas syringae* pv. *actinidiae* (Psa) treatment (Figure 5 and Figure S7B).

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**Figure 5.** Hierarchical clustering of the expression profiles of *AcMAPK* genes in response to various biotic and abiotic stresses. Cold: treatment at 4 °C; Heat: treatment at 48 °C and 24 °C; Salt: treatment with NaCl; Psa: *Pseudomonas syringae* pv. *actinidiae* infection. Details of the treatments are reported in Materials and Methods. The heat-map depicts the fold-change of the relative expression of all *AcMAPK* genes in response to the various treatments in comparison to their respective controls. Red and green colors represent increased or decreased expression levels, respectively, in comparison to controls, as reported by the scale. Genes were clustered according to phylogenetic relationships in expression profiles. Relative expression values for each gene and each treatment are provided in **Figure 5.** Hierarchical clustering of the expression profiles of *AcMAPK* genes in response to various biotic and abiotic stresses. Cold: treatment at 4 ◦C; Heat: treatment at 48 ◦C and 24 ◦C; Salt: treatment with NaCl; Psa: *Pseudomonas syringae* pv. *actinidiae* infection. Details of the treatments are reported in Materials and Methods. The heat-map depicts the fold-change of the relative expression of all *AcMAPK* genes in response to the various treatments in comparison to their respective controls. Red and green colors represent increased or decreased expression levels, respectively, in comparison to controls, as reported by the scale. Genes were clustered according to phylogenetic relationships in expression profiles. Relative expression values for each gene and each treatment are provided in Figures S6 and S7.
