*4.5. Total RNA Isolation and qRT-PCR Expression Analysis*

Total RNA was extracted from the collected samples as described previously with some modifications [5]. Reverse transcription of mRNA was synthesized with a Prime Script™ RT Reagent Kit (Perfect Real Time, TaKaRa, Ostu, Japan) with 1 µg total RNA. The cDNA samples were diluted 1:10 with sterile double-distilled water and stored at −20 ◦C before being used.

The expressions of *AcMAPKs* were examined by qRT-PCR using a SYBR Green method on an ABI 7300 Real-time PCR System (Applied Biosystems, Waltham, MA, USA). The primer sequences used were designed based on gene sequences and the Beacon designer software (NJ, USA), as shown in Supplementary Material File 4 in this study. Kiwifruit actin was used as the housekeeping gene to monitor cDNA abundance [56]. qRT-PCR was carried out as described previously [55]. The relative gene expression level was calculated according to the 2−∆∆*C*<sup>t</sup> method, where ∆∆*C*<sup>t</sup> = (*C*t target gene <sup>−</sup> *C*t actin)treatment − (*C*t target gene − *C*t actin)ck [5,57]. To visualize the relative expression levels data, 0 h at each treatment was normalized as "1", which are presented as the mean fold changes between treated and control samples at each time point ± standard deviations (SDs). The expression data of the 18 *AcMAPK* genes were transformed in log<sup>2</sup> values and used for heat map generation. The heat map was created with MeV4.8 software (Boston, MA, USA) (http://www.tm4.org/mev/).
