Fruit treatment
Kiwifruit (Actinidia deliciosa cv. Xuxiang) were harvested at commercial maturity with the uniformity of shape and size from a commercial orchard in Fuyang District, Hangzhou, China. Treatment was based on Han et al. [9]. The surface was sterilized with 0.5% (v/v) NaClO solution for 3 min, washed with sterile water and air-dried naturally. Artificial wound was made by cutting the fruit into halves lengthwise. Fruit halves were treated respectively with 0.5 mmol L-1 ABA (≥ 90%, Aladdin Industrial Inc., China) and sterile water (control) by vacuum infiltration. Afterwards, fruit halves were stored in a sterile incubator at 20 ℃ and 85% relative humidity for wound healing under darkness. Suberized tissue was separated from the scarred outmost layer of the wound surface after incubating for 2, 3 and 4 days and stored at -80 °C until further analysis.
RNA extraction
The cetyltrimethylammonium bromide (CTAB) method was carried out to extract the total RNA [18]. The implementation details referred to Han et al. [9]. Briefly, 2% CTAB extraction buffer and LiCl solution (12 mol L-1) were applied to extract and denature the RNA on the first day. On the second day, the SSTE buffer (containing 1.0 mM EDTA, 10 mM Tris-HCl pH 8.0, 0.5% (m/v) SDS and 1.0 M NaCl), chloroform and ethanol were added to dissolve, purify and precipitate the RNA, respectively. Finally, wash the RNA pellet with pre-chilled 75% ethanol for twice and dissolve the RNA pellets again using RNase-free water. The quality of the RNA samples was measured using a NanoDrop 2000 (Thermo Fisher Scientific, USA).
DNA extraction
The total DNA was extracted by implementing the CTAB method [19]. The implementation details referred to Han et al. [9]. Briefly, 2% CTAB buffer, the solution of phenol: chloroform: isoamylol (25:24:1) and the solution of chloroform: isoamylol (24:1) were applied to extract and purify the DNA. After centrifuging, NaAc solution and isopropanol were added to precipitate the DNA. Afterwards, wash and dissolve the DNA precipitate respectively with 75% (v/v) ethanol and TE buffer. The quality of DNA samples was measured by a NanoDrop 2000.
Molecular cloning
The gene sequence of transcription factor AchnbZIP29 (Achn340751) and AchnMYB70 (Achn117821) were identified based on the Cornell University kiwifruit database (http://bioinfo.bti.cornell.edu/cgi-bin/kiwi/home.cgi). The cloning conditions were according to Han et al. [9]. Based on the primers in Supplementary Table 1 (AchnbZIP29-Full and AchnMYB70-Full), both genes of AchnbZIP29 and AchnMYB70 were cloned from reverse transcribed cDNA. And the promoter of AchnKCS was cloned from the extracted total DNA using the corresponding AchnKCS-Pro primers. After linking the amplified product with pEASY-T1 simple vector and transferring it into Escherichia coli, the test of white spot screening was carried out to obtain the recombinant plasmid.
Subcellular localization of AchnbZIP29 and AchnMYB70
After cloning the CDS of AchnbZIP29 and AchnMYB70, the sequence with no stop codon was amplified and inserted into the 1300-35S-eGFP vector. The obtained AchnbZIP29-GFP and AchnMYB70-GFP fusion expression vectors were respectively transferred into Agrobacterium strain. The preparation of the infection buffer of Agrobacteria and the inoculation of tobacco (Nicotiana benthamiana) leaves were according to Han et al. [9]. After inoculation for 48 h, a confocal microscope (Leica SP8, Leica Microsystems Co., Germany) was applied to observe the GFP fluorescence of the leaf discs at 488 nm excitation.
Yeast one-hybrid assay (Y1H)
In order to test the protein-DNA interaction of AchnbZIP29, AchnMYB70 and AchnKCS promoter, Y1H was carried out. And according to the Matchmaker® Gold Yeast One-Hybrid Library Screening System (Cat. No. 630491, TaKaRa,Dalian, China), the Y1H assays were performed. Auto-activation analysis of AchnKCS promoter was conducted at first and the minimum inhibitory concentration of aureobasidin A (AbA, a yeast toxin) was determined. The recombinant plasmid of AchnKCS-Pro-pABAi was transferred into Y1H Gold through PEG/LiAc after linearizing. The full-length regions of AchnbZIP29 and AchnMYB70 were cloned into pGADT7 vector (AD) via restriction enzyme cutting sites (EcoRI and XhoI sites, SmaI and SacI sites, respectively). Transformed Y1H Gold harboring both AchnKCS-Pro-pABAi and AchnbZIP29-pGADT7 or AchnMYB70-pGADT7 were cultured to test the interaction on SD/-Leu with AbA at 30 °C for 3 days. And Y1H Gold co-transformed with p53-promoter and pGADT7-Recwere used as positive control. Y1H Gold co-transformed with AchnKCS-Pro-pABAi and empty pGADT7 was used as negative control.
Dual luciferase assay
Dual-luciferase assay was carried out to determine the trans-activation role of AchnbZIP29 and AchnMYB70 on target AchnKCS promoter. The implementation details referred to Tao, et al. [20]. The promoter sequence of AchnKCS was inserted into LUC vector (pGreen II 0800-LUC, cut by HindIII and BamHI). The CDSs of AchnbZIP29 and AchnMYB70 were amplified and inserted into pGreen II 0029 62-SK vector (SK) (cut by HindIII and BamHI), respectively. And the ClonExpress II One Step Cloning Kit (C112-01, Vazyme, China) was applied to drive the connection reactions. The procedures of Agrobacterium tumefaciens transformation and the preparation of the infection buffer of Agrobacteria were according to Han et al [9]. Afterwards, the Agrobacteria culture mixtures of AchnbZIP29 or AchnMYB70 and AchnKCS promoter (v/v 10:1) were prepared to infect tobacco leaves with needleless syringes. After 72 h for infiltration, the Dual-Luciferase Reporter Assay System (E1910, Promega, USA) with Modulus Luminometers (Promega, USA) was performed to detect the activities of firefly luciferase (LUC) and renilla luciferase (REN). Six biological replicates were considered to determine the results.
Real-time quantitative reverse transcription PCR analysis (qRT-PCR)
The first-strand cDNA was obtained by RNA reverse transcription according to the manufacturer’s instructions of PrimeScriptTM RT reagent Kit (Perfect Real Time, TaKaRa Bio Inc., China). The CFX96-TouchTM Deep Well Sequence Detection system (Bio-Rad Laboratories, Inc. CA, USA) was applied to detect gene transcription levels with SYBR® Premix Ex TaqTMII (TliRNaseH Plus, TaKaRa Bio Inc., China). Each gene was analyzed in triplicate and Actin was used as reference gene. The relative expression levels of genes were calculated by the 2-△△CT method [21] and presented in multiples relative to the initial value without any treatment (normalized to 1).
Statistical analysis
Data represented the mean value minus or plus standard deviation (± SD). SPSS software (version 20.0) was used to analyze the significance of the differences by Least significant difference (LSD) test, and Origin 9.0 software was used to draw figures. The difference was considered to be statistical significance when the p value was less than 0.05 and extremely significant difference when less than 0.01, and expressed with different letters or “*”, “**” in figures.