**1. Introduction**

Plants are often challenged by different biotic and abiotic stresses in nature, including pathogen infection, cold, drought, salt, and oxidative stresses; thus, they have developed some sophisticated signaling networks to sense and transmit environmental stimuli at the molecular or cellular levels [1]. A series of highly elaborate signaling networks are composed of some stress-activated molecular pathways [2]. Mitogen-activated protein kinase (MAPK) cascades play an important role in protein phosphorylation of signal transduction events and are one of the major mechanisms in controlling intracellular response to extra cellular signals in plants [3,4].

MAPK cascades are involved in the protein phosphorylation of signal transduction events that contribute to signaling [5], and MAPK cascades are classically composed of three protein kinases: MAPK (MAPK/MPK), MAPK kinase (MAPKK/MKK), and MAPK kinase kinase (MAPKKK/MAP3K/MEKK), but sometimes contain a MAPK kinase kinase kinase (MAPKKKK/MAP4K) that phosphorylates the corresponding downstream substrates [6–8]. MAPK can catalyze the phosphorylation of a substrate protein by chemically adding phosphate groups from adenosine triphosphate (ATP) [9]. MAP3Ks are the first component of this phosphorelay cascade, which phosphorylates two serine/threonine residues in a conserved S/T-X3-5-S/T motif of the MKK activation loop. Then, MKKs are dual-specificity kinases that activate the downstream MAPK through TDY or TEY phosphorylation motif in the activation loop (T-loop) [3,4,10]. The activated MAPK ultimately phosphorylates various downstream substrates, including transcription factors and other signaling components that regulate the expression of downstream genes [11]. MAPK proteins contain 11 evolutionary conserved kinase domains that may be involved in substrate specificity or protein–protein interaction [1,12].

Compared with MAPKKs and MAP3Ks, MAPKs act at the bottom of MAPK cascades in much greater numbers and show more complexity and sequence diversity. MAPK cascade proteins have TEY or TDY phosphorylation motifs in their activation loops between kinase domains VII and VIII, which provide protein-binding domains for the activation of MAPKs [3,6]. Plant MAPKs can be separated into four groups (A, B, C, and D) based on the phylogenetic relationships of the amino acid sequence and the phosphorylation motif. Members of the A, B, and C subfamily have the TEY motif at its phosphorylation site, and members of the D subfamily possess the TDY motif [3,4].

The MAPK proteins belong to a complex gene family in plants [13]. The identification and characterization of different members of the MAPK cascades have been revealed by genome sequencing projects in various plant species. The model plants that have been most studied are *Arabidopsis thaliana* and rice; there are 20 MAPKs in the *A. thaliana* genome [3], whereas the rice genome contains 17 MAPKs [14]. Recent research has reported that a total of 16, 19, 16, 14, 12, 17, 10, and 15 homologs in *MAPK* family genes have been identified from tomato (*Solanum lycopersicum*) [15], maize (*Zea mays*) [16], purple false brome (*Brachypodium distachyon*) [17], grapevine [13,18] and strawberry (*Fragaria vesca*) [19], tobacco (*Nicotiana tabacum*) [20], mulberry (*Moraceae morus*) [21], wheat (*Triticum aestivum*) [22] genomes, and as many as 21, 26, and 25 putative *MAPK* genes were identified in poplar (*Populus trichocarpa*) [23], apple (*Malus domestica*) [24], and banana (*Musa acuminata*), respectively.

In plants, MAPKs are involved in cellular responses to the regulation of the cell cycle, plant growth and development, hormones, and responses to biotic and abiotic stresses [7,25]. To date, several plant MAPK signaling cascades have been characterized in detail. The MEKK1-MKK4/5-MPK3/6 cascade was the first characterized signaling module in Arabidopsis, which up-regulated the expression of the transcription factors of WRKY22/29 and then increased resistance to both fungal and bacterial pathogens [25,26]. In addition, *AtMPK3* and *AtMPK6* are involved in the anther, embryo, inflorescence development, and stomatal distribution on the leaf surface [27,28]. The MEKK1-MKK1/2-MPK4 cascade was shown to positively regulate defense responses against necrotrophic fungi while negatively regulating defenses against biotrophic pathogens [29,30], also shown to be activated by drought, cold, and salt stresses [31]. *MAPK* genes in other important crops have also attracted considerable attention. For example, *OsMAPK3* and *OsMAPK6* are induced by a chitin elicitor in rice [32], *OsMPK5* is activated by pathogens and abiotic stresses [1], and overexpression of *OsMAPK33* enhances sensitivity to salt stress in rice through unfavorable ion homeostasis as negative regulators [33]. *ZmMPK3*, *ZmMPK5*, and *ZmMPK17* genes in maize are involved in signal transduction pathways associated with different environmental stresses [34–36]. Overexpression of *BnMAPK4* enhances resistance to *Sclerotinia sclerotiorum* in transgenic *Brassica napus* [37]. *GhMPK7* (*Gossypium hirsutum*) is induced by pathogen infection, and may be an important regulator in broad spectrum disease resistance and plant growth and development [38]. The expression of *VvMAPK3* and *VvMAPK6* genes were induced by salinity and drought [18].

Kiwifruit (*Actinidia chinensis*) is a nutritionally and commercially important and valuable fruit, well known for its remarkably high vitamin C content. For example, the Hongyang kiwifruit, which is derived from *A. chinensis* var. *chinensis* [39], is becoming a favorite of consumers, growers, and breeders due to its unique phenotype and high premium price at market. To date, systematic investigations

and functional analyses of the MAPK gene family have not been reported for *A. chinensis*, despite the importance of MAPK proteins in multiple biological processes. Recently, the genome of a heterozygous kiwifruit cultivar "Hongyang" (*A. chinensis* var. *chinensis*) was sequenced [40], suggesting that kiwifruit has potential as a model organism for fruit trees. As such, it has become an imperative to compare the functions of gene families, particularly those having vital functions with the gene families characterized from *Arabidopsis* [41], which provides an opportunity for systematic analysis of *MAPK* in the kiwifruit species. With the rapid development of molecular biology and bioinformatics, the mining and positioning of functional genes in plant genome-wide data have become research hotspots. Due to the importance of MAPKs in diverse biological and physiological processes as well as their potential application to the development of improved stress tolerant transgenic plants, we performed the classification and phylogeny of the *MAPK* gene family of kiwifruit through bioinformatics analysis. Additionally, we conducted a comprehensive analysis of all the identified *AcMAPK* genes to determine which of these genes contribute to stress and hormone responses using quantitative real-time polymerase chain reaction (qRT-PCR) analysis. These data further provide information about the relationship between MAPK function and growth and development, disease resistance, and stress response of kiwifruit. The results of our identification and comprehensive investigation of the MAPK gene family in kiwifruit provide a theoretical basis for future gene cloning and expression, especially for the genetic improvement in the breeding of kiwifruit.
