Characterization of the Key Region and Key Phosphorylation Sites of EcaICE1 for its Molecular Interaction with EcaHOS1 Protein in Eucalyptus camaldulensis

ICE1 (inducer of CBF expression 1), a MYC-like bHLH transcriptional activator, plays an important role in plant under cold stress via regulating transcriptional expression of downstream cold-responsive genes. Ubiquitination-proteasome pathway mediated by high expression of osmotically responsive gene1 (HOS1) can effectively induce the degradation of ICE1 and decrease the expression of expression of CBFs and their downstream genes under cold stress response in Arabidopsis , but the knowledge about ubiquitination regulation of ICE1 by HOS1 is still unknown in woody plants. The complete EcaICE1 gene and a new E3 ubiquitin ligase gene EcaHOS1 were amplified from the tissue culture seedlings of Eucalyptus camaldulensis . Yeast two-hybrid (Y2H) and BiFC assay results showed that EcaICE1 can interact with EcaHOS1 protein in the nucleus, and further Y2H assay demonstrated that the 126-185 amino acid region at the N-terminus of EcaICE1 protein was indispensable for its interaction with EcaHOS1 protein. Moreover, we found that the amino acids at positions 143, 145, 158 and 184 within the key interaction region were the potential phosphorylation sites of EcaICE1 based on bioinformatics analysis, and that only the substitution of Serine (Ser) 158 by Alanine (Ala) blocked the protein-protein interactions between EcaICE1 and EcaHOS1 by Y2H and β-galactosidase assays using site-direct mutagenesis. Overexpression of EcaICE1 and its mutations in Arabidopsis could significantly increase POD and SOD activities with a reduction for MDA content and up-regulate four cold-responsive genes ( CBF3 , KIN1 , COR15 and COR47A ) in the transgenic lines. ,


Results
The complete EcaICE1 gene and a new E3 ubiquitin ligase gene EcaHOS1 were amplified from the tissue culture seedlings of Eucalyptus camaldulensis . Yeast two-hybrid (Y2H) and BiFC assay results showed that EcaICE1 can interact with EcaHOS1 protein in the nucleus, and further Y2H assay demonstrated that the 126-185 amino acid region at the N-terminus of EcaICE1 protein was indispensable for its interaction with EcaHOS1 protein. Moreover, we found that the amino acids at positions 143, 145, 158 and 184 within the key interaction region were the potential phosphorylation sites of EcaICE1 based on bioinformatics analysis, and that only the substitution of Serine (Ser) 158 by Alanine (Ala) blocked the protein-protein interactions between EcaICE1 and EcaHOS1 by Y2H and βgalactosidase assays using site-direct mutagenesis. Overexpression of EcaICE1 and its mutations in Arabidopsis could significantly increase POD and SOD activities with a reduction for MDA content and up-regulate four cold-responsive genes ( CBF3 , KIN1 , COR15 and COR47A ) in the transgenic lines.

Conclusion
We first reported that EcaICE1 could interact with EcaHOS1 protein in Eucalyptus , and 3 identified Ser 158 of EcaICE1 as the key phosphorylation site for its interaction with EcaHOS1 protein.

Background
Cold stress is a major environmental factor that adversely affects plant growth and development, as well as the yield, product quality and geographic distribution of crops [1].
Plants must adjust various physiological and biochemical processes in response to cold stress by reprogramming gene expression [2]. Currently, the most well-understood cold signaling pathway is the ICE-CBF-COR transcriptional regulatory cascade [3,4]. CBF transcription factors (TFs) recognize the CRT/dehydration-responsive element (DRE) elements in the promoters of certain cold-responsive (COR) genes and regulate their expressions and functions [5]. Inducer of CBF expression 1 (ICE1), a MYC-like bHLH transcriptional activator, acts upstream as a positive regulator of CBFs by binding to MYC recognition elements in the CBF promoters in cold-responsive signaling [4,6,7]. In addition, CBF genes also appear to be negatively regulated by ICE1 via its interactions with MYB15 [8] and HOS1 [9], while positively by ICE1 via their interactions with SIZ1 [10] and OST1 [11]. Recently, Li et al. reported that MPK3/MPK6 could interact with and phosphorylate ICE1, reducing the stability of ICE1 as well as its transcriptional activity, thus negatively regulating CBF expression and freezing tolerance in Arabidopsis [12]. Therefore, ICE1 is the key regulators of ICE-CBF-COR transcriptional regulatory cascades in the cold-responsive signaling pathway. ICE-like genes have been isolated and characterized in some woody plants as Populus suaveolens [13], Malus domestica [14], Vitis amurensis [15], E. camaldulensis [16], Pyrus ussuriensis [17], Poncirus trifoliata [18] and Hevea brasiliensis [19]. However, the positive and negative regulation pathway of ICE1 in relation to cold stress response still remains poorly understood in woody plants.
Eucalyptus species are the important commercial woody plants because of rapid growth, broad adaptability, and the source of wood pulp. However, even widely distributed, its plantation is mainly restricted to tropical and subtropical regions because of freezing sensitivity, especially for the commercial Eucalyptus species. Therefore, it is necessary to discover the molecular regulation mechanism of cold response and carry out genetic improvement on freezing tolerance in Eucalyptus. Although CBF genes have been isolated and characterized from Eucalyptus [20][21][22][23][24][25], the knowledge about its upstream regulator ICE1 and its positive and negative regulation pathway needs to be investigated. Our precious studies have revealed that ectopic expression of EcaICE1 from E. camaldulensis confers improved cold tolerance and the expression level of downstream genes in transgenic tobaccos [16]. Nevertheless, the factors controlling ICE1 protein stability associated with cold stress response in Eucalyptus are not clearly elucidated. In this study, we cloned the HOS1 gene EcaHOS1 from the E. camaldulensis and analyzed the protein interactions between EcaICE1 and EcaHOS1 using Bimolecular fluorescence complementation (BiFC) and Yeast two-hybrid (Y2H) assays. Subsequently, the key phosphorylation sites of EcaICE1 for its interaction with EcaHOS1 protein were predicted by bioinformatics and characterized by site-direct mutagenesis using Y2H and assays.

Sequence Analysis of EcaICE1 and EcaHOS1
The sequence of EcaICE1 gene was identical to our precious result [16]. The typical conserved domains, such as MYC-like bHLH domain, zipper structure at the C-terminus, Srich (Serine-rich) acidic domain, SUMO binding site and NLS Nuclear localization sequence box, were found in EcaICE1 protein from the multiple alignments ( Figure S1).
Nevertheless, only Eucalyptus ICE1 proteins had an additional Q-rich (Glutamine-rich) domain, suggesting that the characteristics of ICE1 proteins might be different between Eucalyptus and the other plants. 5 The sequence of EcaHOS1 gene was 2889 bp long and encoded a complete coding frame consisting of 962 amino acids. BLAST analysis revealed that EcaHOS1 shared a high sequence identity with other plant HOS1-like proteins, such as A. thaliana (52%, OAP11605), Vitis viinifera (60%, NP_001268014) and Poncirus trifoliate (58%, XP_024445010). The multiple alignments of plant HOS1 protein sequences ( Fig. 1) showed that the EcaHOS1 protein had a conserved RING finger domain and ELYS domain, similar to other plant HOS1 proteins. The conserved RING finger domain is the crucial functional region of E3 ubiquitin ligases [26]. These results showed that EcaHOS1 was the HOS1 protein and a new E3 ubiquitin ligase from E. camaldulensis, which may have a functional role in the ubiquitination pathway.

Subcellular localization of EcaICE1
The multiple alignments (Additional file: Fig. S1) showed that there was an NLS box in the EcaICE1, implying that it may be nuclear localized protein. To confirm the result, the subcellular localization assay of EcaICE1 was carried out using a fusion protein of EcaICE1 and YFP reporter gene, driven by 35S promoter. The yellow fluorescence of EcaICE1-YFP fusion protein was detected in the nucleus (Fig. 2). These results indicated that EcaICE1 was a nuclear protein, similar to other woody plant ICE1 proteins [17,18,19].

EcaICE1 could interact with EcaHOS1
Previous report showed that HOS1 could interact with ICE1 and mediate the ubiquitination of ICE1 both in vitro and in vivo in A. thaliana, which attenuated cold stress response by the ubiquitination/proteasome pathway [9]. To elucidate whether EcaHOS1 could also interact with EcaICE1 in E. camaldulensis, a BiFC assay was performed to identify the protein-protein interactions in tobacco leaves. Microscopic visualization results (Fig. 3) revealed that there was no YFP fluorescent signal for the negative controls including EcaICE1-pSPYCE co-expressed with unfused pSPYNE or EcaHOS1-pSPYNE co-expressed with 6 unfused pSPYCE whereas the YFP fluorescence was observed exclusively in the nucleus for the EcaICE1-pSPYCE co-transfected with EcaHOS1-pSPYNE. These results showed that EcaICE1 could interact with EcaHOS1 to form heterodimers at the nucleus.

Protein-protein interaction region between EcaICE1 with EcaHOS1
We perform the Y2H assay to further confirm the protein-protein interaction between EcaICE1 with EcaHOS1 and discover the key interaction region of EcaICE1 for the interaction. Unfortunately, both EcaICE1 and EcaHOS1 had autoactivation activity (Fig.   S3). Then, transcriptional activation assay demonstrated that amino acids from positions 84 to 125 in EcaICE1 were critical for the transactivation activity of EcaICE1 (Fig. S2). Now the truncated EcaICE1 protein without transcriptional activation activity was constructed into vector pGBKT7, and co-transformed with AD-EcaHOS1 into yeast strain separately to find the protein-protein interaction region. The results showed that only the EcaICE1 T3 interacted with EcaHOS1 and the other regions did not (Fig. 4A), while all of these four truncated EcaICE1 proteins could interact with AtHOS1( Fig. 4B), indicating that the Nterminus region of EcaICE1(126-185aa) was the key region for its interaction with EcaHOS1 protein, quite different from A. thaliana [27].

Effects of key phosphorylation site of EcaICE1 for its interaction with EcaHOS1 protein
We searched the phosphorylation sites within the N-terminus region (126-185 aa) of EcaICE1 by bioinformatics software NetPhos 3.1 and found that Ser 143 (Ser at 143 aa), Thr 145(Thr at 145 aa), Ser 158(Ser at 158 aa) and Ser 184(Ser at 184 aa) were predicted as the potential phosphorylation sites. After the substitution of these four sites by Alanine (S143A, T145A, S158A and S184A, respectively) using site-direct mutagenesis based on the EcaICE1 T3 (abbreviated as T3), Y2H results ( Fig. 5A) showed that only T3(S158A) blocked its interaction with EcaHOS1, while the residual three mutants and T3 still worked.
It is interesting that both T3 and its mutants of EcaICE1 could interact with AtHOS1( Fig.   5B), indicating that the ubiquitination pathway of ICE1 by HOS1 may be different between Eucalyptus and Arabidopsis. β-galactosidase assay (Table 2) revealed that β-galactosidase activity of T3(S158A) was not significant from negative control at P<0.05. The βgalactosidase activity of T3(T145A) and T3(S184A) was not significant from wild type T3 at P<0.05, while that of T3(S143A) was significantly higher than wild type T3 (P<0.05). The β-galactosidase activity assay also confirmed that only T3(S158A) blocked the protein interactions between EcaICE1 and EcaHOS1. These results suggested that Ser 158 was the key phosphorylation site of EcaICE1 for its interaction with EcaHOS1.

Overexpression of EcaICE1 and its mutations in Arabidopsis
We further transformed EcaICE1 and its mutations into Arabidopsis plants to reveal their functions. The morphological performance results (Fig. S4) showed that there was no significant difference between the phenotype of each transgenic lines and the wild type (WT) line and the ice1 mutant in Arabidopsis. After 24 h treatment at 4 °C, physiological index measurement results ( Fig. 6) showed that the activities of both POD and SOD of all five transgenic lines were significantly higher than those of WT and ice1 mutant (P<0.05), while the MDA contents were significantly lower than those of WT and ice1 mutant (P<0.05). Nevertheless, the measured physiological indexes were not significantly different among these five transgenic lines. Moreover, real-time PCR results (Fig. 7) showed that the expression levels of all tested four cold-responsive genes (CBF3, KIN1, COR15 and COR47A) in the transgenic lines were significantly up-regulated (P<0.05), indicating that EcaICE1 and its mutants can positively regulate downstream coldresponsive genes and may improve the stress tolerance in Arabidopsis.

Discussion
Eucalyptus can increase freezing tolerance by cold acclimation as well as other plant 8 species. CBF genes have been cloned from Eucalyptus [20][21][22]25] and their overexpression in cold-sensitive Eucalyptus could improve the freezing tolerance [23]. Cao et al. further reported that there were 17 CBF orthologs in the E. grandis genome and that 14 CBF genes were located on the scaffold 1 within a cluster of about 117 Kb [24]. We first cloned an ICE1 gene EcaICE1 from E. camaldulensis and found that ectopic expression of EcaICE1 could confer improved cold tolerance and increase the expression level of downstream genes in transgenic tobaccos [16]. However, the factors controlling ICE1 protein stability associated with cold stress response in Eucalyptus, that is, whether there is similar ubiquitination or SUMOylation pathway of ICE1 to Arabidopsis, are still lack of empirical studies. In this current study, we further researched the ubiquitination pathway of ICE1 mediated by HOS1 in Eucalyptus and characterized EcaICE1 protein having a direct physical interaction with EcaHOS1 in vivo (Fig. 3, 4A, and 5A).
E3-ubiquitin-ligases-mediated ubiquitination are emerging as major regulators in plants' response to abiotic stress, circadian rhythm control, cell cycling and plant-microbe interactions [28][29][30]. High expression of osmotically responsive genes1 (HOS1), one of the RING finger-containing E3 ubiquitin ligase, was initially described as a cold-signaling attenuator of ICE1 [9,31], and also involved in ethylene signal transduction [32] and photoperiodic flowering [33] in Arabidopsis. It is reported that most of the plant species just have a single copy of the HOS1 gene [34], suggesting that HOS1 would have important roles in various plant species. Based on the multiple alignments of EcaHOS1 with other plant HOS1 proteins (Fig. 1), the RING finger domain is highly conserved among the presented plants, and the first residue in the RING finger of all seven HOS1 proteins is Leu, which is Cys in animal inhibitor of apoptosis (IAPs) [35]. The RING-finger domain is the crucial player in the ubiquitin-dependent protein degradation system [36], indicating that plant HOS1 proteins are highly conserved E3 ubiquitin ligase. In addition, all seven 9 HOS1 proteins have a highly conserved embryonic large molecule derived from yolk sac (ELYS) domain, which is required for the protein recruitment of the nuclear pore complex (NPC), implying that HOS1 also has nonproteolytic roles such as mRNA export and chromatin remodeling [34]. These analyses indicate that EcaHOS1 is the HOS1 gene from E. camaldulensis, and its encoded protein may have E3 ubiquitin ligase activity and mediate ubiquitination in the nucleus.
BiFC assay showed that EcaICE1 could interact with EcaHOS1 in nucleus, similar to A. thaliana [9], but which region of EcaICE1 was the key region for the protein-protein interactions is still unknown. Therefore, using different truncated EcaICE1 without autoactivation activity as bait protein, the N-terminal 126-185 amino acid of EcaICE1 protein was further identified as the key region by Y2H assay. Interestingly, the key region of EcaICE1 (126-185 aa) is not located within the conserved bHLH domain, the zipper motif or ACT-like domain at the C-terminus region of EcaICE1. However, in Arabidopsis, AtHOS1 interacted with the C-terminus of AtICE2 containing the zipper motif [27]. Surprisingly, all truncated EcaICE1 could interact with AtHOS1, indicating that the zipper motif at Cterminus of ICE protein is indeed necessary for its interaction with AtHOS1, different from that in EcaHOS1.
A number of evidences show that non-lysine residues, such as serine, threonine and cysteine, are ubiquitylation sites of many E3 ubiquitin ligases [37][38][39]. Miura et al. reported that substitution of Ser 403 by Alanine (Ala) in Arabidopsis ICE1 could inhibit the polyubiquitylation of ICE1 in vivo, but did not affect the degradation of ICE1 [40]. They argued that although Ser 403 is not the main target residue for ubiquitylation or SUMOylation, it is a key residue for the attenuation of cold-stress responses by HOS1mediated degradation of ICE1 [40]. Here, we further found that Ser 143, The 145, Ser 158 and Ser 184 within 126-185 aa of EcaICE1 were predicted as the potential phosphorylation sites based on bioinformatics method. Only substitution of Ser 158 by Ala (S158A) using site-direct mutagenesis blocked the protein-protein interactions between EcaICE1 and EcaHOS1 using Y2H assay (Fig. 5A), and β-galactosidase activity assay (Table 2) also confirmed this result. Surprisingly, all of EcaICE1 mutants could interact with AtHOS1 again (Fig. 5B), indicating that the conserved domains at the C-terminus of ICE are exactly essential for its interactions with AtHOS1 and that the ubiquitylation site of ICE for AtHOS1 maybe the Ser 403.
To further elucidate the molecular function of EcaICE1 and its mutants in Arabidopsis, relative mRNA transcription levels of stress responsive genes of Arabidopsis were also measured. It is well known that overexpression of ICE and ICE-like genes can intensely induced the expression of CBF3 in plants [41]. Here, CBF3, KIN1, COR15, and COR47A were strongly expressed not only in the transgenic lines of EcaICE1, similar to our precious studies [42], but also in those of its mutants, than the WT and ice1 mutant after cold treatment (Fig. 6), showing that the site-direct mutagenesis of EcaICE1 did not affect its functions in Arabidopsis. Meanwhile, ROS-scavenging enzymes SOD, POD, and CAT are mainly involved in ROS detoxification under cold stress [43]. In our study, transgenic plants of EcaICE1 and its mutants exhibited higher activities of POD and SOD with lower MDA content than the WT and ice1 mutant after cold treatments, indicating that the sitedirect mutagenesis of EcaICE1 did not affect the efficiency of ROS-scavenging system in transgenic Arabidopsis plants under cold stress.
Our results suggested that ubiquitination pathway of ICE1 mediated by HOS1 may be different between Eucalyptus and Arabidopsis and that Ser 158 was the key phosphorylation site of EcaICE1 for its interaction with EcaHOS1. As for whether EcaHOS1 acts as an E3 ubiquitin protein ligase and whether it could mediate the degradation of EcaICE1 for playing a role in cold stress response in Eucalyptus, including whether Ser 158 11 affects the EcaHOS1-mediated ubiquitination of ICE1, need further experiments to investigate.

Conclusions
Herein, we amplified the gene EcaICE1 and EcaHOS1 from E. camaldulensis, and the EcaICE1 sequence was the same as the previous result, and the deduced EcaHOS1 protein was highly conserved with other plant HOS1 proteins. EcaICE1 was located in nucleus, and it could interact with EcaHOS1 in nucleus by BiFC assay. Moreover, Y2H assays confirmed the interaction between EcaICE1with EcaHOS1 and revealed that the N-terminal region from position 126 to 185 in EcaICE1 was the key region for its interaction with EcaHOS1 protein. Ser 158 was the key phosphorylation site of EcaICE1 for its interaction with EcaHOS1 by Y2H and β-galactosidase assay using site-direct mutagenesis. Finally, sitedirect mutagenesis of EcaICE1 did not affect efficiency of ROS-scavenging system and the molecular functions of EcaICE1 in transgenic Arabidopsis plants under cold stress. Taken together, we made a foundation on the ubiquitination regulation mechanism of EcaICE1 mediated by EcaHOS1.

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

RNA isolation, gene cloning and sequence analysis
Total RNA was extracted as described previously [16], and treated with DNase I (Promega,

13
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

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 [44]

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-EcaICE1 T3 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-EcaICE1 T3 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 βgalactosidase assay kit manual (Thermo, USA). Single yeast colonies grown on SD/-LW medium of BD-EcaICE1 T3 and its mutants with AD-EcaHOS1 were picked and transferred where OD 420 , T, V, and OD 660 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 [45] 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 [46]. 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 [42]. 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 OD 470 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.

Authors' contributions
LC and WZ constructed vectors and performed Y2H and BiFC assays. JH and ZZ did the genetic transformation and measured the physiological indexes. JH and YL did qRT-PCR assay. YL designed the project and oversaw the project management. LC, WZ, and YL wrote the manuscript. All authors contributed to project conception and data analysis. All authors have read and approved the final version of the manuscript.

Funding
This research was supported by the National Natural Science Foundation of China (No.31470673).

Availability of data and materials
The data sets supporting the results of this article are included within the article and its additional files.

Ethics approval and consent to participate
The experimental research on all plants complied with institutional, national and international guidelines. The researchers only collected the tissue culture plantlets of E. camaldulensis from the cultivated variety 103 developed by Guangdong Academy of Forestry and didn't harm the tree natural resources. The collected behavior through the consent and supervision of the local protection agency and the forestry bureau. In this study, we get the help from Guangdong Academy of Forestry. Hereby declare.

Supplementary Files
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