Overexpression of MdZAT5, an C2H2-Type Zinc Finger Protein, Regulates Anthocyanin Accumulation and Salt Stress Response in Apple Calli and Arabidopsis

Abstract

Zinc finger proteins are widely involved and play an important role in plant growth and abiotic stress. In this research, MdZAT5, a gene encoding C2H2-type zinc finger protein, was cloned and investigated. The MdZAT5 was highly expressed in flower tissues by qRT-PCR analyses and GUS staining. Promoter analysis showed that MdZAT5 contained multiple response elements, and the expression levels of MdZAT5 were induced by various abiotic stress treatments. Overexpression of MdZAT5 in apple calli positively regulated anthocyanin accumulation by activating the expressions of anthocyanin biosynthesis-related genes. Overexpression of MdZAT5 in Arabidopsis also enhanced the accumulation of anthocyanin. In addition, MdZAT5 increased the sensitivity to salt stress in apple calli. Ectopic expression of MdZAT5 in Arabidopsis reduced the expression of salt-stress-related genes (AtNHX1 and AtABI1) and improved the sensitivity to salt stress. In conclusion, these results suggest that MdZAT5 plays a positive regulatory role in anthocyanin accumulation and negatively regulates salt resistance.

Keywords: MdZAT5; anthocyanin; apple; salt tolerance.

Figure 1

Basic information about the MdZAT5 sequence. (A) Conserved sequence of MdZAT5 protein. The blue rectangle indicates the zinc finger domain. The numbers represent the length of amino acids. (B,C) predicted the secondary and tertiary protein structures of MdZAT5, respectively. The numbers denote the length of amino acids.

Figure 2

Phylogenetic tree, amino acid sequence alignment, and conserved motifs analysis. (A) The phylogenetic tree of ZAT5 proteins from 18 different plants. (B) Comparison of amino acid sequences of ZAT5 proteins from 10 different plants. The red triangle represents MdZAT5. The red box represents a conserved domain. They all have two conserved zinc finger domains and an EAR motif.

Figure 3

Tissue expression analysis of MdZAT5. (A) The relative expression level of MdZAT5 in different tissues (roots, stems, leaves, flowers, and fruits) by qRT-PCR. (B) Tissue expression analysis of MdZAT5 via GUS staining in transgenic Arabidopsis. Different lowercase letters represent a significant difference (p < 0.05). Data are the mean ± SD of three independent replicates.

Figure 4

The expression pattern of MdZAT5 under different treatment conditions. The relative expression of MdZAT5 in 150 mM NaCl (A), 10% PEG6000 (B), 4 °C (C), and 100 μM ABA (D), respectively. Different lowercase letters represent a significant difference (p < 0.05). Data are the mean ± SD of three independent replicates.

Figure 5

GUS staining in ProMdZAT5::GUS transgenic calli and Arabidopsis. The ProMdZAT5::GUS transgenic Arabidopsis (A) and calli (B), treated with 24 °C, 150 mM NaCl, 6% PEG, 4 °C, 100 μM ABA, and high light. (C) The GUS activity of MdZAT5 of (B). Different lowercase letters represent a significant difference (p < 0.05). Data are the mean ± SD of three independent replicates.

Figure 6

Overexpression of MdZAT5 in apple calli and Arabidopsis promoted anthocyanin accumulation. The phenotypes (A) and anthocyanin content (B) of WT and MdZAT5-OVX. Expression analysis of MdZAT5 (D) and genes involved in anthocyanin biosynthesis-related genes (MdANR, MdCHI, MdCHS, MdDFR, MdF3H, and MdUFGT) (C) in WT and MdZAT5-OVX. The phenotypes (E) and anthocyanin content (G) of Col-0 and MdZAT5-OE. (F) Expression analysis of MdZAT5 in Col-0 and MdZAT5-OE. Different lowercase letters represent a significant difference (p < 0.05). Data are the mean ± SD of three independent replicates.

Figure 7

MdZAT5 enhanced the sensitivity to salt in apple calli. (A) The phenotypes of WT and MdZAT5-OVX with 100 mM NaCl. Fresh weight (B), MDA content (C), relative electronic conductivity (D) of WT, and MdZAT5-OVX. Different lowercase letters represent a significant difference (p < 0.05). Data are the mean ± SD of three independent replicates.

Figure 8

MdZAT5 enhanced the sensitivity to salt in transgenic Arabidopsis. (A) The phenotypes of Arabidopsis seedlings treated with MS medium, 150 mM NaCl treatment. Lateral root numbers (B) and primary root length (C) in Col-0 and MdZAT5-OE. (D) Phenotypes of Arabidopsis treated with 150 mM NaCl after 14 days and MDA content (E). The expression level of AtNHX1 (F) and AtABI1 (G) in Col-0 and MdZAT5-OE. Different lowercase letters represent a significant difference (p < 0.05). Data are the mean ± SD of three independent replicates.

Figure 9

Ectopic expression of MdZAT5 enhances ROS buildup under salt treatment. DAB staining for H₂O₂ (A) and NBT staining for O₂⁻ (B) in Col-0 and MdZAT5-OE Arabidopsis leaves after 14 days of salt treatment. H₂O₂ content (C) and O₂⁻ generation rates (D) in Col-0 and MdZAT5-OE Arabidopsis after 14 days of salt treatment. Different lowercase letters represent a significant difference (p < 0.05). Data are the mean ± SD of three independent replicates.