Antifreeze protein from Ammopiptanthus nanus functions in temperature-stress through domain A

Temperature stress restricts plant growth and development. Antifreeze protein (AFP) can improve plants antifreeze ability. In our previous study, the AnAFP gene cloned from Ammopiptanthus nanus was confirmed to be an excellent candidate enhancing plant cold resistance. But, AnAFP protein shared similar structures with KnS type dehydrins including K, N and S domains except ice crystal binding domain A. Here, we generated AnAFPΔA, AnAFPΔK, AnAFPΔN and AnAFPΔS, and transformed them into ordinary and cold sensitive strains of E. coli, and Arabidopsis KS type dehydrin mutant to evaluate their function. Expression of AnAFPΔA decreases cold and heat tolerance in E. coli, meanwhile, AnAFP enhances heat tolerance in Arabidopsis, suggesting that domain A is a thermal stable functional domain. AnAFP, AnAFPΔA and AnAFPΔS localize in whole cell, but AnAFPΔK and AnAFPΔN only localizes in nucleus and cytoplasm, respectively, exhibiting that K and N domains control localization of AnAFP. Likewise, K domain blocks interaction between AnAFP and AnICE1. The result of RT-qPCR showed that expression of AnAFP, AnICE1 and AnCBF genes was significantly induced by high-temperature, indicating that the AnAFP is likely regulated by ICE1-CBF-COR signal pathway. Taken together, the study provides insights into understanding the mechanism of AnAFP in response to temperature stress and gene resource to improve heat or cold tolerance of plants in transgenic engineering.

Temperature stress including low-and high-temperature stress restricts plant growth and development. Low temperature inhibits enzyme activity and destroys membrane permeability resulting in physiological disorder, metabolic obstruction and even cell death 1,2 . Similarly, high temperature leads to wilting, accumulation of reactive oxygen species (ROS), and destruction of membrane system [3][4][5] . After perceiving temperature stress, plants approach genes expression change, physiological and biochemical response via signal transduction [5][6][7][8] . The ICE1-CBF-COR pathway is the most studied signaling pathway under temperature stress in plants. Under low temperature conditions, a MYC-like basic helix loop helix (bHLH) transcription factor ICE1 (Inducer of CBF expression 1) induces expression of the CBF gene. The CBF transcription factor binds to the CRT/DRE (C-repeats/dehydration responsive) element (CCGAC) and activates the expression of cold-responsive genes (COR) 9,10 . In response to high temperature stress, the energy releasing from cell physiological disorder such as membrane fluidity increasing, DNA unwinding and protein subunit dissociation triggers transcriptional changes to restore homeostasis, promoting cell survival, and elaborating longer-term responses for adaptation, growth, and development 11 .
Antifreeze proteins (AFPs) were firstly found in polar fishes, as well as insects living in freeze-zone. AFPs prevent water from freezing by adsorbing to the ice surface and stopping the growth of minute ice crystals to large crystals in a non-colligative manner, and help the organisms survive in subzero temperature environments. The distribution of AFPs in different species appears to be the outcome of a combination of independent evolutionary events, which is probably the convergent evolution or horizontal gene transfer 12 . These AFPs genes were introduced into crops to improve their tolerance to low temperature stress. Expression of antifreeze protein of winter flounder or insect (Microdera puntipennis dzungarica) confers the cold tolerance to transgenic spring www.nature.com/scientificreports/ wheat at subzero temperatures, tobacco or tomato, respectively [13][14][15] . Meanwhile, the transgenic plants did not demonstrate significant tolerance improvement when compared to wild-types [13][14][15] . The non-colligative manner of the heterologous animal AFPs, as well as their expression rate, localization, and stability, might not be suitable for cellular environments in transgenic plants 16 . However, the AFPs from overwintering plants including Lolium perenne, Loliurn perenne and Ammopiptanthus nanus showed higher inhibitory effect on ice growth and recrystallization than that of AFPs of fishes and insects [17][18][19][20] .
Ammopiptanthus nanus (A. nanus) is a tertiary relict plant and evergreen broad-leaved shrub distrusted in deserts in Central Asia, exhibits excellent tolerance to abiotic stress including drought, low and high temperature. In our previous study, the AnAFP gene was cloned from xerophyte A. nanus, and evaluated its cold tolerance function by ectopic expression in Escherichia coli (E. coli), tobacco and maize 21,22 . Bioinformatics analysis showed that AnAFP shared high similarity with some members of KnS type dehydrins 22 . In addition to the ice crystal binding domain (A domain) of AFPs, AnAFP also has three conserved domains of dehydrins including K, S and N (Nuclear localization sequence) domains 22,23 . In order to evaluate the function of these domains, four mutants of AnAFP deleting A (AnAFPΔA), K (AnAFPΔK), S (AnAFPΔS) and N (AnAFPΔN) domain were generated by overlapping PCR, respectively. In this study, these four mutants and AnAFP were introduced into cold-sensitive and ordinary strains of E. coli, as well as Arabidopsis mutant of KnS type dehydrin gene AtHIRD11 to identify thermal stability of each domain under low-and high-temperature stress, respectively. Together with their subcellular localization, interacting proteins and induced endogenous expression, the regulation mechanism of the AnAFP protein in response to temperature stress was elucidated.

Results
Sequence of mutant genes. Through overlap PCR, the sequences of AnAFPΔA, AnAFPΔK, AnAFPΔN and AnAFPΔS were amplified from AnAFP ( Figure S1a). Their encoding putative proteins were deleted of crystal binding domain A, and K, NLS and S domains of dehydrin, respectively ( Figure S1b).
Expression of AnAFPΔA increases cold sensitivity of BX04. After low temperature treatment at 17 °C for 9 days, the cold-sensitive BX04 containing pINIII-AnAFPΔA showed growth defect and failed to form colonies with an average survival rate < 10%, which was similar to BX04 cells contain pINIII. However, the BX04 containing pINIII-AnAFPΔK, pINIII-AnAFPΔS and pINIII-AnAFPΔN grew vigorously and formed more colonies with an average survival rate > 30%, respectively, which was similar to BX04 cells with pINIII-AnAFP (Fig. 1). These results suggest that domain A is a functional domain of AnAFP protein related to cold tolerance.
Expression of AnAFPΔA increases heat sensitivity of BL21. After IPTG induction, the expression of candidate genes in BL21 was confirmed and analyzed by SDS-PAGE ( Figure S2). After 30 min of heat treatment at 50 °C, the colony growth of each dilution time of the E. coli BL21 transformed by pET28a was inhibited, and the average survival rate was only 14.46%. Whereas the BL21 cells with pET28a-AnAFP grew better, with an average survival rate of 85.29%. Notably, the BL21 cells with pET28a-AnAFPΔA was similar to BL21 with pET28a,

Expression of mutant genes increase heat sensitivity of Arabidopsis.
After screening with 50 mg/L kanamycin, the five homozygous lines for every gene were identified by PCR amplification of AnAFPΔA, AnAFPΔK, AnAFPΔN and AnAFPΔS, respectively ( Figure S4), indicating that these genes were integrated into the genome of Arabidopsis. Subsequently, the ORF of AnAFPΔA, AnAFPΔK, AnAFPΔN and AnAFPΔS were amplified by RT-PCR from the T 3 lines of transgenic lines, but not in untransformed mutant. Likewise, the specific fragment of AtActin was amplified both in transgenic lines and untransformed mutant (Figs. 3 and S5), indicating the ectopic expression of candidate genes in transgenic plants.
As shown in Fig. 4, before heat treatment, the transgenic lines and untransformed mutants grew vigorously. After 46 °C treatment for 3 h and then recovered 2 weeks, the transgenic lines transformed by AnAFPΔA, AnAFPΔK, AnAFPΔN and AnAFPΔS and untransformed mutants all died. However, a few plants of transgenic line transformed by AnAFP still survived. This result suggests that ectopic expression of AnAFP improves heat tolerance of heat sensitive Arabidopsis mutant. However, deletion of any one of A, K, N and S domains will lead to loss of heat resistance function of AnAFP protein.

Deletion of domains K and N alters subcellular localization. Subcellular localization results showed
that green fluorescence signals were observed both in cytoplasm and nucleus of onion epidermal cells transformed by 35S-eGFP, 35S-AnAFP-eGFP, 35S-AnAFPΔA-eGFP, and 35S-AnAFPΔS-eGFP, respectively. However, green fluorescence was observed only in the nucleus of onion epidermal cells with 35S-AnAFPΔK-eGFP, and only in the cytoplasm of onion epidermal cells with 35S-AnAFPΔN-eGFP (Fig. 5). These results suggest that the deletion of domains K and N changes the subcellular localization of AnAFP protein. Fig. 6, the yeast Y2H Gold cotransformed by bait vector and trap vector could grow normally on SD/-Leu-Trp plates, but not grow and not turn blue on SD/-Leu-Trp-His-Ade with X-α-Gal plates except positive control (pGADT7-T + pGBKT7-53), showing that these proteins have no toxicity and no autoactivation in yeast cell. Likewise, the yeast cells with pGBKT7-AnAFPΔK and pGADT7-AnICE1 could grow and be stained to blue on the auxotroph SD/-Leu-Trp-His-Ade with X-α-gal plates, while the cells co-transformed by pGBKT7-AnAFP, pGBKT7-AnAFPΔA, pGBKT7-AnAFPΔN, pGBKT7-AnAFPΔS, and pGADT7-AnICE1 did not grow, suggesting that AnAFPΔK interacts with AnICE1. This result indicates that domain K of AnAFP protein blocks its interaction with AnICE1 protein.   (Fig. 7). This result suggests that the

Discussion
All AFPs are conserved for their ice crystal binding sites, although difference in their structural characteristics due to their different evolution 24,25 . Under low temperature stress (17 °C for 9 days), the colony growth and average survival rates of the cold-sensitive E. coli BX04 with AnAFPΔA were significantly poorer and lower than the strains transformed with AnAFP, as well as AnAFPΔK, AnAFPΔN and AnAFPΔS mutants (Fig. 1). Under high temperature stress (50 °C for 30 min), the colony growth and average survival rate of E. coli BL21 (DE3) transformed by AnAFPΔA were also significantly poorer and lower than the strains transformed by AnAFP, as well as AnAFPΔK, AnAFPΔN and AnAFPΔS (Fig. 2). These results confirmed the conserved thermostable function of ice crystal binding domain (domain A) in AnAFP proteins. However, the function of each domain of AnAFP is not indispensable. All T 3 plants of Arabidopsis mutant of KnS type dehydrin athird11 transformed by AnAFPΔA, AnAFPΔK, AnAFPΔN and AnAFPΔS mutants died after high temperature stress (46 °C for for 3 h), while a few T 3 plants of the same mutant transformed by the AnAFP gene survived (Fig. 4). Subcellular localization showed that the mutants without domains A and S were localized in the cytoplasm and nucleus, which was same as AnAFP protein. However, the mutant without domain K was only localized in the nucleus, while the mutant without domain N was localized only in the cytoplasm (Fig. 5). This result suggests that domains K and N determine the subcellular localization of AnAFP protein and affect its function. Similar phenomenon was observed for KnS-type dehydrin ZmDHN13 in maize 26 .
The Y2H result indicates that domain K of AnAFP protein blocks its interaction with AnICE1 proteins (Fig. 6). This result can be explained by the molecular shield model of dehydrin to protect functional proteins 27,28 . The internal disordered structure of dehydrin protein occupies the space between the target proteins and reduces their collision. This kind of shield forms a loose structure around the target protein but not interact with it like the classical molecular chaperone.
In our previous study, the AnAFP was found to be a KnS type dehydrin and the AnAFP gene was identified as a member of COR genes 22 . Dehydrin and COR genes can be activated by CBF participating ICE1-CBF-COR pathway [29][30][31][32] In the present study, the RT-qPCR result showed that the endogenous expression of AnAFP, AnICE1 and AnCBF genes was significantly upregulated by high temperature stress (Fig. 7). It suggests that high temperature stress also induces the expression of COR genes. In response to low and high temperature stress, the endogenous expression of AnAFP is probably induced by signaling pathway of ICE1-CBF-COR. In Arabidopsis, the expression of heat shock proteins was also found to be extensively overlapped with non-heat stress response pathways 33 . Therefore, the regulation of the endogenous expression of AnAFP in A. nanus can be concluded as signaling pathway sketched in Fig. 8.

Conclusion
In the presents study, we generated AnAFPΔA, AnAFPΔK, AnAFPΔN and AnAFPΔS, and transformed them into ordinary and cold sensitive strains of E. coli, and Arabidopsis KS type dehydrin mutant to evaluate their function. It's confirmed that domain A is a thermal stable functional domain, K and N domains control localization of AnAFP. Likewise, K domain blocks interaction between AnAFP and AnICE1. The expression of AnAFP, AnICE1 and AnCBF genes was significantly induced by high-temperature, indicating that the AnAFP is likely regulated by ICE1-CBF-COR signal pathway.

Materials and methods
Evaluation of AnAFP domains in cold resistance of E. coli. Two pairs of specific primers (Table S1) with restriction sites of EcoRI/BamHI were designed by using CE Design V1.04 (http:// www. downcc. com/ soft/ 281907. html), and used to amplify open reading frame (ORF) of AnAFP and four mutants deleting domain A (AnAFPΔA), K (AnAFPΔK), S (AnAFPΔS) and N (AnAFPΔN) created in our previous study, respectively. The products were cloned into the EcoRI/BamHI site of prokaryotic expression vector pINIII to generate pIN- www.nature.com/scientificreports/ III-AnAFP, pINIII-AnAFPΔA, pINIII-AnAFPΔK, pINIII-AnAFPΔS and pINIII-AnAFPΔN, and confirmed by sequencing, respectively. The re-constructed plasmids were transformed into competent cells of E. coli cold-sensitive strain BX04. After confirming by screening with 50 mg/L ampicillin, PCR amplification and sequencing, the positive colonies were transferred into LB liquid medium and incubated at 37 °C until OD6 00 = 0.5-0.6. According to the methods of Yang et al. 34 and Deng et al. 21 with minor modification, the cultured cells were treated at 17 °C for 9 days with three replicates. All samples were diluted by 10 0 to 10 7 times, respectively. The 5 μL and 100 μL of them were plated onto LB plates with 50 mg/L ampicillin, incubated at 37 °C for 12 h and photographed, respectively. Before treatment, 5 μL of them were plated onto LB plates with 50 mg/L ampicillin and incubated at 37 °C for 12 h for control. The colonies number with spraying 100 μL cells were counted and used to calculate average survival rates under cold stress. (Table S2) with restriction sites of NdeI/HindIII were designed, and used to amplify ORF of AnAFP, AnAFPΔA, AnAFPΔK, AnAFPΔS and AnAFPΔN using the above plasmids as template, respectively. The products were cloned into Nde I/Hind III site of prokaryotic expression vector pET28a to generate pET28a-AnAFP, pET28a-AnAFPΔA, pET28a-AnAFPΔK, pET28a-AnAFPΔS and pET28a-AnAFPΔN, and confirmed by sequencing, respectively.

Evaluation of AnAFP domains in heat resistance of E. coli. Two pairs of specific primers
The recombined plasmids were transferred into competent cells of E. coli BL21 (DE3). After confirming by screening with 50 mg/L kanamycin, and PCR amplification and sequencing, the positive colonies were transferred into LB liquid medium and incubated at 37 °C until OD 600 = 0.5-0.6. The ectopic expression of AnAFP, AnAFPΔA, AnAFPΔK, AnAFPΔS and AnAFPΔN was induced by 0.5 mmol/L IPTG, detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). As described by Li et al. 35 with minor modification, the induced cells were splinted into six centrifuge tubes, three of them were treated at 50 °C for 30 min, while others were incubated at 37 °C for 30 min, respectively. All samples were diluted by 100 to 104 times, respectively. The 5 μL and 100 μL of them were plated onto LB plates with 50 mg/L kanamycin, incubated at 37 °C for 12 h, and photographed. The colonies number with spraying 100 μL cells were counted and used to calculate average survival rate under heat stress. (Table S3) with restriction sites of BspI/PstI were designed, and used to amplify ORF of AnAFP, AnAFPΔA, AnAFPΔK, AnAFPΔS and AnAFPΔN, respectively. The products were cloned into Bsp I/Pst I site of plants expression vector pCAMBIA2300-35S-eGFP to generate 35S-AnAFP-eGFP, 35S-AnAFPΔA-eGFP, 35S-AnAFPΔK-eGFP, 35S-AnAFPΔS-eGFP and 35S-AnAFPΔN-eGFP, and confirmed by sequencing, respectively. These plasmids were transferred into competent cells of Agrobacterium tumefaciens GV3101 by freeze-thaw method.

Plasmids reconstruction and Arabidopsis transformation. Two pairs of specific primers
After confirming by screening with 50 mg/L rifampicin and 50 mg/L kanamycin on YEB plates, PCR amplification and sequencing, the positive colonies were transferred into liquid medium YEB and incubated at 28 °C until OD6 00 = 1.0-1.5. The cells were collected by centrifugation at 4 °C and 4000 r/min for 10 min, resuspended and adjusted to OD 600 = 1.0 with 5% (wt/vol) fresh sucrose solution, added surfactant Silwet L-77 to a concentration of 0.02% (vol/vol), and used to transform Arabidopsis mutant of the KnS type dehydrin gene AtHIRD11 (AT1G54410) by floral-dip method.
Heat tolerance of transgenic Arabidopsis. As described by Sun et al. 36 , T 1 seeds were surface-sterilized with 75% ethanol for 1 min and 10% NaClO for 10 min, and plated onto 1/2 MS plates with 50 mg/L kanamycin (Sigma, USA) for screening of transgenic plants, which were used to produce T 2 generation. The T 2 plants with 3:1 segregating-ratio to resistance/susceptibility of kanamycin were self-pollinated to generate T 3 . The homozy- www.nature.com/scientificreports/ gous lines without segregation were collected from T 3 , and were identified by PCR amplification using the primers (Table S4) for the specific fragments of AnAFP, AnAFPΔA, AnAFPΔK, AnAFPΔN and AnAFPΔS. The total RNA of every line was extracted by RNA extractor kit (Sangon, China), reacted with RNase-free DNase I, and used to reverse transcribed into cDNA using PrimeScript RT Reagent Kit (TaKaRa, Dalian). The ectopic expression of the transformed genes was identified by reverse transcription PCR (RT-PCR) using the above primers. The AtActin gene was amplified and used as reference. Five T 3 lines were planted in pots, and grown in green house at 22 ℃ and 60-70% relative humidity under a 10 h light/14 h dark photoperiod. One-month-old seedlings were used for heat-shock treatment at 46 °C for 3 h, and recovered for 2 weeks at 22 ℃, and investigated for phenotype. Yeast two-hybrid. Two pairs of specific primers (Table S5) with restriction sites of NdeI/BamHI were designed, and used to amplify ORF of AnAFP, AnAFPΔA, AnAFPΔK, AnAFPΔS and AnAFPΔN, respectively. The products were cloned into NdeI/BamHI sites of yeast two hybrid (Y2H) bait vector pGBKT7 to generate pGBKT7-AnAFP, pGBKT7-AnAFPΔA, pGBKT7-AnAFPΔK, pGBKT7-AnAFPΔS and pGBKT7-AnAFPΔN, and confirmed by sequencing, respectively. Another pair of specific primers (Table S6) with restriction sites of NdeI/ BamHI were designed and used to amplify ORF of AnICE1 from the cDNA of A. nanus seedlings. The products were cloned into Y2H trap vector pGADT7 to generate pGADT7-AnICE1 and confirmed by sequencing. The yeast strain Y2H Gold was transformed with every plasmid using Yeastmaker Yeast Transformation System 2 (Clotech, Japan), and used to test of self-activation and toxicity of AnAFP, AnAFPΔA, AnAFPΔK, AnAFPΔN, AnAFPΔS, and AnICE1 proteins. Subsequently, the yeast strain Y2H Gold was co-transformed by each pair of bait vector pGBKT7-AnAFP, pGBKT7-AnAFPΔA, pGBKT7-AnAFPΔK, pGBKT7-AnAFPΔN, pGBKT7-AnAFPΔS, and trap vector pGADT7-AnICE1, respectively. The transformants were screened on the auxotroph SD/-Trp-Leu plates at 30 °C for 3-5 days. The mono-colonies were transferred onto the auxotroph SD/-Leu-Trp-His-Ade plates, incubated at 30 °C for 3-5 days, and strained with X-α-Gal.

Subcellular localization. The
Real-time quantitative PCR. The seeds of A. nanus were surface-sterilized with 75% ethanol for 10 min, and planted in soil and grown in green house at 25 °C 12 h light/ 20 °C 12 h dark and 60-70% relative humidity. At the six-leaf stage, the seedlings with same size were treated at high temperature of 45 °C for 0 (negative control), 3, 6, 12, and 24 h with three biological replicates, as described by Yu et al. 37 . The total RNA was extracted, and reverse transcribed into cDNA as above.
For pairs of specific primers (Table S7) were designed, and used to amplify a 150-250 bp fragment for AnAFP, AnAFPΔA, AnAFPΔK, AnAFPΔN and AnAFPΔS, as well as the internal reference gene AnGAPDH. The RT-qPCR was performed using SYBR Green Super Mix (Bio-Rad, USA) by two step real-time PCR cycles (95 °C 30 s; 95 °C 5 s, 50-65 °C 30 s, 39 cycles) in CFX96 Real-Time System (Bio-Rad, USA). The 2 −ΔΔCT method was used to normalize the expression differentiation between reference gene and investigated genes 38 .

Statistical analysis.
All experiments were conducted with three replicates. The data are presented as the mean values ± standard deviation (SD). Statistical difference was analyzed using Student's t tests. License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.