Plant materials and treatments
The experimental research on all plants complied with institutional, national and international guidelines. All plants were grown in a glasshouse at the Guangdong Key Laboratory for Innovative Development and Utilization of Forest Plant Germplasm, South China Agricultural University. The tissue culture plantlets of E. camaldulensis cv. 103 (a gift from Dr. Qian Zhang, Guangdong Academy of Forestry, China) were performed as described previously  and 30-day-old rooting plantlets were used in this study. No permission was needed for obtaining and using the tissue culture plantlets of E. camaldulensis cv. 103.
Tobacco (Nicotiana benthamiana, a gift from Prof. Aimin Wu, South China Agricultural University, China) plants were cultured in a growth chamber at 25 °C with 16/8 h light/dark photoperiod, and 5-week-old plants were used for further subcellular localization and BiFC analysis.
The A. thaliana wild type (Col-0) used as the control group was provided by Prof. Aimin Wu from South China Agricultural University. The AtICE1 knockout mutants (ice1, SALK_003426) were purchased from The Arabidopsis Information Resource (TAIR; http://www.arabidopsis.org/). Arabidopsis plants were cultured at 23 °C with 16/8 h light/dark photoperiod.
RNA isolation, gene cloning and sequence analysis
Total RNA was extracted as described previously , and treated with DNase I (Promega, Madison, WI, USA). 1 μg DNA-free total RNA was used the template for synthesizing the first strand cDNA ( PrimeScript II 1st Strand cDNA Synthesis Kit; Takara, Dalian, China). The primers of EcaICE1 and EcaHOS1 (listed as table 1) were used for amplifying the aim genes with the first strand cDNA as the template. The aim genes were sequenced at Beijing genomics Institute (BGI, China). The coding sequence (CDS) of EcaICE1 and EcaHOS1 were predicted using FGENESH 2.6 software ( http://linux1.softberry.com/berry.phtml?topic=fgenesh&group=programs&subgroup=gfind ), and further confirmed by BLASTP program on the NCBI website (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The protein secondary domains were predicted by Motif scan (https://myhits.isb-sib.ch/cgi-bin/motif_scan). Finally, sequence alignments with other plants were performed with CLUSTALX software.
The full-length CDS of EcaICE1 was amplified by RT-PCR using primers (listed as Supplemental table S1) and fused into the 5′-terminus of the yellow fluorescent protein (YFP) of the pEarleyGate101 vector, driven by CaMV 35S promoter. The recombinant plasmid 35S::EcaICE1-YFP was transferred into Agrobacterium tumefaciens strain GV3101 by heat shock method. The strain GV3101 harboring plasmid 35S::EcaICE1-YFP were cultured in the liquid yeast extract broth medium at 28 °C on a shaker at 220 rpm until the absorbance of the OD measurement at 660 nm reached 1.0~1.5, and then the culture were centrifuged at 8000 × g for 5 min. The thallus was mixed with the infecting solution containing 1% MES, 1% MgCl2 and 0.1% acetosyringone, and cultured for 3 h at 28°C and then injected into the abaxial surfaces of 5-week-old tobacco leaves via a syringe with an incubator for 48-72 h. The YFP fluorescence in the tobacco leaves was visualized using a confocal microscopy (Zeiss LSM 710, Carl, Germany) with excitation and emission at 513 and 527 nm, respectively.
Bimolecular fluorescence complementation (BiFC) assay
To perform BiFC assays, the whole CDS of EcaICE1 and EcaHOS1 (without their stop codons) were subcloned into pUC-pSPYNE or pUC-pSPYCE vectors as described previously  using primers (listed as Supplemental table S2). Expressions of each target gene alone were used as negative controls. The recombinant vectors were used for transient assays of tobacco leaves as described earlier. The transformed tobacco leaves were then kept in an incubator at 22 °C for 24-48 h. YFP signal was examined using a confocal microscope (Zeiss LSM 710, Carl, Germany).
Yeast two-hybrid (Y2H) assay
Yeast two-hybrid assays were carried out using the Matchmaker™ gold Yeast two-Hybrid Systems (Clontech, USA). Different truncated CDS of EcaICE1 without transcriptional activation activity were subcloned into pGBKT7 (BD) to form the bait vector. The full-length CDS of EcaHOS1 was amplified and inserted into pGADT7 (AD) to construct the prey vector (AD-EcaHOS1). The primers are listed in Supplemental table S3. The bait and prey vectors were co-transformed into yeast strain gold Y2H using lithium acetate (LiAc) method. Then yeast cells were plated on SD/-LW medium (minimal media double dropouts, SD basal medium without Leu and Trp) according to the manufacturer’s protocol (Clontech, USA) for 72 h. Transformed colonies were sprayed on SD/-LWHA medium (minimal media quadruple dropouts, SD medium with -Leu/-Trp/-Ade/-His) containing 125-μM Aureobasidin A (AbA), to test for possible protein-protein interactions, according to the yeast cell growth status. Each experiment replicated three technological repeats in separate experiments.
Phosphorylation sites prediction and site-direct mutagenesis
The phosphorylation sites within the key region of EcaICE1 for its interaction with EcaHOS1 were predicted using NetPhos 3.1 server ( http://www.cbs.dtu.dk/services/NetPhos/ ), and further analyzed by ProtParam program on the expasy website (http://web.expasy.org/protparam). The potential phosphorylation sites were used for substitution into alanine by site-direct mutagenesis. Site-direct mutagenesis experiments were carried out with KOD-Plus Mutagenesis Kit (TOYOBOCO, China). The BD-EcaICE1T3 plasmid was used as the temple for the site-saturation mutagenesis. The primers were listed in Supporting Information Table S4. All mutants were confirmed by sequencing at Beijing Genomics Institute (BGI, China). The BD-EcaICE1T3 and its mutants were used for further Y2H and β-galactosidase activity assays.
Assay of β-galactosidase activity
The β-galactosidase activity was measured based on protocols from the yeast β-galactosidase assay kit manual (Thermo, USA). Single yeast colonies grown on SD/-LW medium of BD-EcaICE1T3 and its mutants with AD-EcaHOS1 were picked and transferred into 5 mL YPDA liquid medium, and incubated at 30 °C with 200 rpm shaker for 10-14 h, and the OD measurement at 660 nm was recorded. 1.0 mL of the culture medium was centrifuged at 13,000 g for 1 min, and the supernatant was removed. Then, 250 μL Y-PER and 250 μL ONPG solution were added, and immediately incubated at 37° C. When the mixed solution turned yellow, 200 μL stop solution was added to stop the reaction, and recorded the reaction time. After centrifugation at 13,000 g for 30 s, 200 μL supernatant was measured for the OD measurement at 420 nm. Each assay replicated three technological repeats in separate experiments. β-galactosidase activity was calculated as follows:
β-Galactosidase (units)=(1000×OD420)( T×V×OD660)
where OD420, T, V, and OD660 were the OD measurement at 420 nm, reaction time (min), reaction solution volume (mL), and the OD measurement at 660 nm, respectively.
Plasmid construction and plant transformation
The coding regions of EcaICE1 and its mutants were cloned into the binary vector pEarleyGate 101 driven CaMV 35S promoter. The constructed plasmids were transformed into the Arabidopsis through floral dipping method  using Agrobacterium tumefaciens GV3101, respectively. The transgenic lines were obtained by antibiotic selection and confirmed by PCR and qRT-PCR analyses. Homozygous T4 generation plants from transgenic lines were used for subsequent experiments.
RNA isolation and qRT-PCR analysis
Total RNAs were extracted from Arabidopsis by using plant RNA Kit (OMEGA, Beijing, China). qRT-PCR was performed on a Bio-Rad CFX96TM real-time thermal PCR system (Bio-Rad, Hercules, CA, USA) by using Takara SYBR Premix EX Taq mix kit (Takara, Japan) according to the manufacturer’s instructions. The relative expression level was calculated by method . Results were obtained using three biological replicates from each sample. Four genes (CBF3, COR15A, COR47 and KIN1) were used for gene expression analysis and actin was used as reference gene for Arabidopsis. The primers used for the qRT-PCR are listed in Table S1.
Measurements of POD, SOD and MDA
Two-month-old tobacco plantlets were cold-acclimated at 15 C for 3 days, and then treated at 4 C for 1, 3, or 5 days. 1 g leaves were ground in extraction buffer to determine SOD, and POD activities and MDA content described previously . One unit of SOD activity was defined as the amount of enzyme inhibiting photochemical reduction of Nitrotetrazolium Blue chloride (NBT) by 50 %. One unit of POD activity was defined as the amount of enzyme causing a 0.01 increase in OD470 per min under assay conditions. MDA content was measured by the thiobarbituric acid reaction. Results were obtained using three biological replicates from each sample.
Data statistical analysis
The data values presented were the means ± standard errors (SE) of three replicates through statistical analysis via R software (v 3.5.1), and further analyzed by ANOVA and Duncan's multiple range test to compare the differences between treatments at the P < 0.05 level.