MbICE1 Confers Drought and Cold Tolerance through Up-Regulating Antioxidant Capacity and Stress-Resistant Genes in Arabidopsis thaliana

Abstract

Malus baccata (L.) Borkh is an apple rootstock with good drought and cold resistance. The ICE gene is a key factor in the molecular mechanisms of plant drought and cold resistance. In the present research, the function of drought- and cold-induced MbICE1 of Malus baccata was investigated in Arabidopsis. According to GFP fluorescence images, MbICE1 was determined to be a nuclear protein. The MbICE1 was transferred to Arabidopsis, showing enhanced tolerance to drought and cold stresses. Under drought and cold treatments, the transgenic Arabidopsis had higher chlorophyll content and free proline content than WT plants, but the Malondialdehyde (MDA) content and electrolyte leakage (EL) were lower than those of WT plants. In addition, drought and cold led to a large accumulation of ROS (H₂O₂ and O^2-) content in Arabidopsis, while overexpression of MbICE1 enhanced the antioxidant enzyme activity in Arabidopsis and improved the plant's resistance to stresses. Moreover, the accumulation of MbICE1 promoted the expression of AtCBF1, AtCBF2, AtCBF3, AtCOR15a, AtCOR47 and AtKIN1 genes in Arabidopsis. These data indicate that MbICE1 is a key regulator of drought and cold and can be used as a backup gene for breeding Malus rootstocks.

Keywords: Malus baccata (L.) Borkh; MbICE1; cold stress; drought stress.

Figure 1

Multi-sequence alignment and evolutionary relationship analysis of MbICE1 protein. (A) Multiple sequence alignment. (B) Evolution tree. The target protein is indicated with a red line. GenBank ID: MdICE1 (NP_001280967.1), PbICE1 (XP_009337514.1), PuICE1 (APC57593.1), PaICE1 (XP_021814716.1), PdICE1 (XP_034216895.1), PmICE1 (XP_008239552.1), PpICE1 (XP_007209976.2), FvICE1 (XP_004297495.2), RcICE1 (XP_024168837.1), JcICE1 (NP_001306859.1), PvICE1 (XP_031277289.1), ChICE1 (ADZ48234.1), MrICE1 (KAB1217526.1), QlICE1 (XP_030939307.1), QsICE1 (XP_023877763.1), AtICE1 (NP_001030774.1), AtICE2 (NP_172746.2).

Figure 2

Structure and domain prediction of MbICE1. (A) Secondary structure of MbICE1. (B) Functional domain of MbICE1. (C) Tertiary structure of MbICE1.

Figure 3

Subcellular localization of MbICE1. The transient vector harboring 35S:GFP and 35S:MbICE1-GFP vectors were transformed into onion epidermal cells by gene gun bombardment. (A,D) Bright field of vision. (B,E) GFP signals in dark field. (C,F) Effects after DAPI dyeing. The white scale bar represents 5 μm.

Figure 4

The expression of MbICE1. (A) Expression of MbICE1 gene in different tissues of Malus baccata under normal growing conditions. The new leaf was selected as the control group. (B) The relative expression of MbICE1 gene in Malus baccata new leaf and (C) root under control conditions (CK, no stress treatment was performed) and stress conditions. The value is the average of three repeats, and the standard deviation is represented by the error bar. The asterisk indicates the difference between treatment and control conditions (* p ≤ 0.05).

Figure 5

Overexpression of MbICE1 improves drought resistance of plants. (A) PCR was used to verify the expression of MbICE1 in WT, VL, and transgenic Arabidopsis. The actin gene (AB638619.1) was selected as the control group. (B) Phenotypes of WT, VL (vector line), and overexpression-MbICE1 (S1, S8, and S9) under control, drought, and recovery growth. The white scale bar represents 3 cm. (C) Survival rates of WT and overexpression-MbICE1 lines after Drought 0 d and Drought 7 d. The value is the average of three repeats. The standard deviation is represented by the error bar. The asterisk indicates the difference compared to WT (** p ≤ 0.01).

Figure 6

Evaluation of physiological indices responsive to drought stress. (A) Proline content (Pro); (B) electrolyte leakage (EL); (C) malondialdehyde content (MDA); (D) chlorophyll content; (E) peroxidase activity (POD); (F) catalase activity (CAT); (G) superoxide dismutase activity (SOD); (H) O2⁻ content; (I) H₂O₂ content in WT, VL (vector line), and overexpression-MbICE1 under control and drought. The value is the average of three repeats, and the standard deviation is represented by the error bar. The asterisk indicates difference among WT, VL, and overexpression-MbICE1 (** p ≤ 0.01). Each index of WT in each treatment was used as the control group.

Figure 7

Overexpression of MbICE1 improves cold resistance of plants. (A) Phenotypes of WT, VL, (vector line) and overexpression-MbICE1 under Cold 0 h, Cold 12 h, and recover growth. The white scale represents 3 cm. (B) Survival rates of WT, VL, and overexpression-MbICE1 after Cold 0 h and Cold 12 h. The value is the average of three repeats, and the standard deviation is represented by the error bar. The asterisk indicates the difference compared to WT (** p ≤ 0.01).

Figure 8

Evaluation of physiological indices responsive to cold stress. (A) Proline content (Pro); (B) electrolyte leakage (EL); (C) malondialdehyde content (MDA); (D) chlorophyll content; (E) peroxidase activity (POD); (F) catalase activity (CAT); (G) superoxide dismutase activity (SOD); (H) O2⁻ content; (I) H₂O₂ content in WT, VL (vector line), and overexpression-MbICE1 under Cold 0 h and Cold 12 h. The value is the average of three repeats, and the standard deviation is represented by the error bar. The asterisk indicates difference among WT, VL, and overexpression-MbICE1 (** p ≤ 0.01). Each index of WT in each treatment was used as the control group.

Figure 9

Analysis of downstream gene expression of MbICE1 in WT, VL (vector line), and overexpression-MbICE1 under drought and cold stresses. Relative expression level of (A) AtCBF1; (B) AtCOR15a; (C) AtCBF2; (D) AtCOR47; (E) AtCBF3; (F) AtKIN1 by q-PCR. The value is the average of three repeats, and the standard deviation is represented by the error bar. The asterisk indicates the difference compared to WT (** p ≤ 0.01).

Figure 10

A model for drought or cold adaptation mediated by MbICE1. MbICE1 receives the signals of drought and cold and directly combines with MYC elements in the CBF promoter to activate the expression of CBFs, thus positively regulating the expression of AtCOR15a and AtCOR47 and improving the survival ability of Arabidopsis under drought and cold stress. In addition, the accumulation of MbICE1 may regulate the ABA signal pathway by activating the RING type E3 ubiquitin ligase AtPPRT3, thus increasing the expression of AtKIN1 and enhancing the plant’s adaptation to drought and cold.