**3. Discussion**

Figures S6 and S7.

**3. Discussion**  In recent years, the characterization of gene families has been useful for studying their function [44]. The accuracy and reliability of gene family evolutionary characterization analysis depend on the genomic sequences. The availability of the complete kiwifruit genome sequence has made it possible to identify all the MAPK family members in this plant species for the first time. In this study, we identified 18 putative *MAPK* genes in the *A. chinensis* genome. The numbers are comparable to those in *A. thaliana* genome, where 20 MAPK members have been identified [3], but the genome size of *A. chinensis* (~616.1 Mb) is approximately four times that of the *A. thaliana* genome (~125 Mb). The 18 members in kiwifruit is a larger number than found in grapevine (14 members) and strawberry (12 members), but smaller than in apple (26 members) and banana (25 members) in fruit. The full-length sequences of putative *AcMAPK* ranged from 336 to 1056 amino acids. Variation in the length of the entire *MAPK* gene is usually due to differences in the length of the MAPK domain or the number of introns [18]. We found most members in the same group share a similar exon/intron structure, which was similar to other plants, including Arabidopsis, tomato, and poplar [1,7,15]. So, the exon/intron structures of each gene cluster originated from tandem or segmental duplication events in the *MAPK* gene family and tended to share similar structure organizations, except for tiny differences. The In recent years, the characterization of gene families has been useful for studying their function [44]. The accuracy and reliability of gene family evolutionary characterization analysis depend on the genomic sequences. The availability of the complete kiwifruit genome sequence has made it possible to identify all the MAPK family members in this plant species for the first time. In this study, we identified 18 putative *MAPK* genes in the *A. chinensis* genome. The numbers are comparable to those in *A. thaliana* genome, where 20 MAPK members have been identified [3], but the genome size of *A. chinensis* (~616.1 Mb) is approximately four times that of the *A. thaliana* genome (~125 Mb). The 18 members in kiwifruit is a larger number than found in grapevine (14 members) and strawberry (12 members), but smaller than in apple (26 members) and banana (25 members) in fruit. The full-length sequences of putative *AcMAPK* ranged from 336 to 1056 amino acids. Variation in the length of the entire *MAPK* gene is usually due to differences in the length of the MAPK domain or the number of introns [18]. We found most members in the same group share a similar exon/intron structure, which was similar to other plants, including Arabidopsis, tomato, and poplar [1,7,15]. So, the exon/intron structures of each gene cluster originated from tandem or segmental duplication events in the *MAPK* gene family and tended to share similar structure organizations, except for tiny differences. The results were consistent with those of domain and phylogenetic analyses performed.

results were consistent with those of domain and phylogenetic analyses performed. In plants, *MAPK* genes have diverged into four subfamilies based on the conserved residues of the TEY/TDY motifs in the activation loop region (T-loop) [3]. However, phylogenetic analysis showed that the 18 putative *AcMAPKs* were divided into five distinct groups (A, B, C, D, and E), together with their MAPK orthologs in *Arabidopsis* and grapevine, which is more than previously reports [42]. *AcMAPK5*, *AcMAPK12*, *AcMAPK15*, and *AcMAPK16* belong to Group A, which contains *AtMPK3* and *AtMPK6* (Figure 1). It has been well-characterized that *AtMPK3* is activated in response to pathogens and abiotic stresses, and *AtMPK6* can be activated by various abiotic and biotic stresses [1]. *AcMAPK1*, *AcMAPK3*, *AcMAPK4*, *AcMAPK8*, and *AcMAPK11* belong to Group B, which includes *AtMPK4*, *AtMPK5*, *AtMPK11*, *AtMPK12*, *VvMPK9*, and *VvMPK11* (Figure 1). The MAPKs in Group B In plants, *MAPK* genes have diverged into four subfamilies based on the conserved residues of the TEY/TDY motifs in the activation loop region (T-loop) [3]. However, phylogenetic analysis showed that the 18 putative *AcMAPKs* were divided into five distinct groups (A, B, C, D, and E), together with their MAPK orthologs in *Arabidopsis* and grapevine, which is more than previously reports [42]. *AcMAPK5*, *AcMAPK12*, *AcMAPK15*, and *AcMAPK16* belong to Group A, which contains *AtMPK3* and *AtMPK6* (Figure 1). It has been well-characterized that *AtMPK3* is activated in response to pathogens and abiotic stresses, and *AtMPK6* can be activated by various abiotic and biotic stresses [1]. *AcMAPK1*, *AcMAPK3*, *AcMAPK4*, *AcMAPK8*, and *AcMAPK11* belong to Group B, which includes *AtMPK4*, *AtMPK5*, *AtMPK11*, *AtMPK12*, *VvMPK9*, and *VvMPK11* (Figure 1). The MAPKs in Group B are involved in both abiotic stress responses and cell division in Arabidopsis. *AtMPK4* and its upstream

are involved in both abiotic stress responses and cell division in Arabidopsis. *AtMPK4* and its upstream *AtMKK2* can be activated by biotic and abiotic stresses [31]. Group C contained three genes: *AtMKK2* can be activated by biotic and abiotic stresses [31]. Group C contained three genes: *AcMAPK2*, *AcMAPK7*, and *AcMAPK9* (Figure 1). Members from this group in other plant species are known to be regulated by both biotic and abiotic stresses. For example, *AtMPK1* in Group C is regulated by salt stress treatment [4], and *AtMPK1* and *AtMPK2* are activated by ABA [45]. In addition, the rice *BWMK1* and alfalfa *TDY1* genes in Group C are activated by wounding and pathogens [46]. Group D includes *AcMAPK10*, *AcMAPK14*, and *AcMAPK17* of the kiwifruit MAPKs (Figure 1), which have the TDY motif in their T-loop, which are consistently found in members of the other MAPK groups. We found that Group D is the largest group of MAPKs in most plant species. *AcMAPK6*, *AcMAPK13*, and *AcMAPK18*, belonging to Group E, were separated from other groups (Figure 1). The *AcMAPKs* genes of Group E are found only in the grapevine genome among other plant species; there were no orthologs of *AtMAPK* in *A. thaliana*.

The result of our examination of the conserved motifs domain found 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. Above, we stated that all 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. The MAPKs of Group D contained the 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). This suggests functional consistency among the MAPK members in the same group. Moreover, motifs in each group were diverse, in accordance with the intron/exon structure of each group. Thus, the composition and the sequential order of these motifs in the same group showed high similarity. A large amount of stress-, pathogen-, and hormone-related *cis*-elements were found in the putative promoter regions of the *AcMAPK* genes in kiwifruit as shown by *cis*-regulatory elements analysis. The existence of these *cis*-elements suggested that these *AcMAPK* genes might have potential functions in various stress signaling pathways. Similar *cis*-elements were found in *MAPK* genes of tomato [15] and *B. distachyon* [17].

A large number of reports demonstrated the involvement of *MAPK* genes in response to various biotic and abiotic stresses and hormone signaling [4]. AtMAPK3 and AtMAPK6 of Group A are the most prominent kinases, which have been widely studied and have been strongly associated with various environmental stresses in *Arabidopsis* [11]. In this study, he transcription level of the *AcMAPK5* gene, which is the kiwifruit orthologue of the *AtMAPK3* gene, showed an obvious up-regulation response to cold, heat, salt, ACC, SA, and JA treatments, which indicates that *AcMAPK5* might be an important regulator in response to abiotic stresses and hormone signaling molecules. Notably, the gene expression down-regulation of *AcMAPK12* observed in any hormone treatment was induced transcriptionally by cold and salt stress, suggesting that activation of AcMAPK12 protein kinase activities might be not correlated with their transcript levels, similar to AtMAPK6. However, the expression of *AcMAP15* and *AcMAP16* genes was repressed by most hormone (except for JA) and heat treatments; these results are similar to previous reports in which the MKK3/MPK6 module was proposed to participate in JA signaling [47]. The MAPKs of Group B (*AtMAPK4* and *AtMAPK11*) have been implicated in pathogen defense and abiotic stress responses [48]. The relationship of MAPK signaling pathways and SA in plant abiotic stress responses was recently characterized [49]. The *AcMAPK11* gene showed an obvious up-regulation response to cold and heat stress. The *AcMAPK4* gene was induced by cold, salt, SA, and ABA treatments, suggesting the involvement of these genes in abiotic stress tolerance and hormone signal transduction in kiwifruit. The expression of *AcMAPK* genes from Group B was repressed by most stresses and hormone treatments, suggesting that *AcMAPK* genes of Group B may function in an early stage of stress signaling transduction as negative regulators in kiwifruit. The MAPKs of Group C in *Arabidopsis* are activated by ABA, providing evidence for a role in an ABA-induced MAPK pathway in plant stress signaling [50]. However, the transcription level of *AcMAPK2*, *AcMAPK7*, and *AcMAPK9* from Group C were down-regulated by ABA treatment in this study, which suggests that the involvement of these genes in ABA signaling might be regulated

at the level of translation. The *AcMAPK9* gene was induced by cold, heat, salt, *P. syringae*, ACC, SA, and JA treatments, which suggests that this gene might also have important functions in abiotic stress and hormone signaling. *AtMPK7* was significantly up-regulated in response to cold stress [17]. *AcMAPK9*, which showed the highest homology to *AtMPK7*, showed strong activation by cold stress, suggesting a similar function. The *MAPK* genes of Group D have not been as well studied as those of Groups A and B. *AcMAPK10* and *AcMAPK17* genes in Group D were induced by cold, heat, salt, and ACC treatments. It is interesting that expression of *AcMAPK14* was up-regulated by all biotic and abiotic stresses, whereas down-regulation induced by all hormone treatments. Together, these results indicate possible roles of the *MAPK* genes of Group D in abiotic stress responses and hormone signaling. The *AcMAPK*s genes of Group E are found only in the grapevine genome among other plant species; there were no orthologs of *AtMAPK* in *Arabidopsis*. The expression of Group E gene members was repressed by most biotic and abiotic stresses, similar to their response to hormone treatments, except for heat treatment. However, more research is needed to determine the specific functions of the MAPK family of genes by additional experiments.
