A High-K+ Affinity Transporter (HKT) from Actinidia valvata Is Involved in Salt Tolerance in Kiwifruit

Abstract

Ion transport is crucial for salt tolerance in plants. Under salt stress, the high-affinity K⁺ transporter (HKT) family is mainly responsible for the long-distance transport of salt ions which help to reduce the deleterious effects of high concentrations of ions accumulated within plants. Kiwifruit is well known for its susceptibility to salt stress. Therefore, a current study was designed to decipher the molecular regulatory role of kiwifruit HKT members in the face of salt stress. The transcriptome data from Actinidia valvata revealed that salt stress significantly induced the expression of AvHKT1. A multiple sequence alignment analysis indicated that the AvHKT1 protein contains three conserved amino acid sites for the HKT family. According to subcellular localization analysis, the protein was primarily present in the cell membrane and nucleus. Additionally, we tested the AvHKT1 overexpression in 'Hongyang' kiwifruit, and the results showed that the transgenic lines exhibited less leaf damage and improved plant growth compared to the control plants. The transgenic lines displayed significantly higher SPAD and Fv/Fm values than the control plants. The MDA contents of transgenic lines were also lower than that of the control plants. Furthermore, the transgenic lines accumulated lower Na⁺ and K⁺ contents, proving this protein involvement in the transport of Na⁺ and K⁺ and classification as a type II HKT transporter. Further research showed that the peroxidase (POD) activity in the transgenic lines was significantly higher, indicating that the salt-induced overexpression of AvHKT1 also scavenged POD. The promoter of AvHKT1 contained phytohormone and abiotic stress-responsive cis-elements. In a nutshell, AvHKT1 improved kiwifruit tolerance to salinity by facilitating ion transport under salt stress conditions.

Keywords: A. valvata; HKT; ion transport; overexpression; salt tolerance.

Figure 1

The phylogenetic analysis of AvHKT1 in kiwifruit and HKT family members from other plant species. Nt: Nicotiana tabacum, Sl: Solanum lycopersicum, Pp: Prunus persica, Av: Actinidia valvata, Cs: Citrus sinensis, At: Arabidopsis thaliana, Ts: Thellungiella salsuginea, Os: Oryza sativa subsp. Japonica, Ta: Triticum aestivum.

Figure 2

Multiple sequence alignments for HKT proteins from different plant species. Nt: Nicotiana tabacum, Sl: Solanum lycopersicum, Pp: Prunus persica, Av: Actinidia valvata, Cs: Citrus sinensis, At: Arabidopsis thaliana, Ts: Thellungiella salsuginea, Os: Oryza sativa subsp. Japonica, Ta: Triticum aestivum. The conserved Ser/Gly residues in the P_A–D region are indicated by the arrowhead. The identical residues are marked by different letters.

Figure 3

The prediction of the AvHKT1 trans-membrane domain. The different numbers represent the five transmembrane topologies.

Figure 4

The expression pattern of AvHTK1 in A. valvata under salt stress. (a) Analysis of AvHKT1 expression pattern at different time points of salt stress treatment and (b) tissue-specific expression of AvHKT1 under salt stress for 72 h. Different letters (a–c) represent significant mean differences at p < 0.05.

Figure 5

Subcellular localization analysis of the AvHKT1 protein. GFP: Green fluorescence protein; BF: Bright field; Merged: Merged field for GFP and BF. The scale bar was set at 10 µm.

Figure 6

RT-PCR identification analysis of AvHKT1 transgenic lines. (a) Identification of full-length AvHKT1 sequence in overexpression lines and (b) detection of GFP locus fragments in overexpression vector sequences. M, 2000 bp marker; +, the recombinant plasmid containing AvHKT1 (positive control); WT, wild-type kiwifruit; OE1, OE2, OE3, overexpression transgenic lines.

Figure 7

Functional validation of AvHKT1 in kiwifruit under salt stress. (a) Phenotypes of WT (A. chinensis cv. ‘Hongyang’) and overexpression kiwifruit plants under salt stress conditions. (b) MDA content, (c) POD activity, and (d) SPAD value of wild-type and overexpressed plants before and after salt stress treatment. (e) Expression of AvHKT1 in transgenic lines and WT plants after salt stress for 1 d. **, p < 0.01, (student’s t-test). Different letters in the same picture indicate significant differences at p < 0.05 level.

Figure 8

The effect of salt stress on ion content in WT and overexpressed lines. The content of Na⁺ and K⁺ in leaf (a,b) and shoot (c,d), and Na⁺/K⁺ ration in leaf (e) and shoot (f) before and after salt stress treatment. Different letters in the same picture indicate significant differences at p < 0.05 level.