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

Abstract

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.

Figure 1

Phenotype of cold sensitive BX40 strain of E. coli with AnAFP, AnAFPΔA, AnAFPΔK, AnAFPΔN and AnAFPΔS gene under low temperature stress (17 °C) for 9 days. (a) Colony growth of different dilution times. (b) Monoclone of transformed strains. (c) Average survival rate of transformed strains. Lower case letter indicates the significant difference at p < 0.05 level in student’s t-test. The experiment was performed with three replicates. The data were presented as the mean values ± SD.

Figure 2

Thermotolerant phenotype of E. coli BL21strains transformed by AnAFP, AnAFPΔA, AnAFPΔK, AnAFPΔN and AnAFPΔS gene under heat stress (50 °C) for 30 min. (a) Colony growth of different dilution times. (b) Monoclone of transformed strains. (c) Average survival rate. Lower case letter indicates the significant difference at p < 0.05 level in student’s t-test. The experiment was performed with three replicates. The data were presented as the mean values ± SD.

Figure 3

Ectopic expression of AnAFP (a), AnAFPΔA (b), AnAFPΔK (c), AnAFPΔN (d), AnAFPΔS (e) genes in T₃ transgenic Arabidopsis by RT-PCR. M: DNA molecular weight marker DL2000; –: Untransformed mutant; 1, 2, 3, 4, 5 indicates independent transgenic line.

Figure 4

Phenotype of T₃ transgenic lines and untransformed mutant under heat treatment. 1, 2, 3, 4, 5 indicates independent transgenic line. Five T₃ 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.

Figure 5

Subcellular localization of AnAFP and its deletion mutants in onion epidermal cells. (a) The diagram of vector for transient expression. (b) Green fluorescence observed by confocal microscopy. Scale bars = 50 µm.

Figure 6

Protein interaction by Y2H. (a) The toxicity and autoactivation assay. (b) Y2H between AnICE1 and AnAFP, as well as its deletion mutants.

Figure 7

Relative expression levels of AnAFP, AnICE1 and AnCBF genes under high temperature (45 °C) treatment. The AnGAPDH gene was used as internal reference. The 2^−ΔΔCT method of the CFX Manger™ software version 2.0 (Bio-Rad, USA) was used to normalize the expression differentiation between reference gene and investigated genes. The experiment was performed with three replicates. The data were presented as the mean values ± SD.

Figure 8

Signaling model of AnAFP in A. nanus in response to temperature stress.