Characterization of the Heavy-Metal-Associated Isoprenylated Plant Protein (HIPP) Gene Family from Triticeae Species

Abstract

Heavy-metal-associated (HMA) isoprenylated plant proteins (HIPPs) only exist in vascular plants. They play important roles in responses to biotic/abiotic stresses, heavy-metal homeostasis, and detoxification. However, research on the distribution, diversification, and function of HIPPs in Triticeae species is limited. In this study, a total of 278 HIPPs were identified from a database from five Triticeae species, and 13 were cloned from Haynaldia villosa. These genes were classified into five groups by phylogenetic analysis. Most HIPPs had one HMA domain, while 51 from Clade I had two, and all HIPPs had good collinear relationships between species or subgenomes. In silico expression profiling revealed that 44 of the 114 wheat HIPPs were dominantly expressed in roots, 43 were upregulated under biotic stresses, and 29 were upregulated upon drought or heat treatment. Subcellular localization analysis of the cloned HIPPs from H. villosa showed that they were expressed on the plasma membrane. HIPP1-V was upregulated in H. villosa after Cd treatment, and transgenic wheat plants overexpressing HIPP1-V showed enhanced Cd tolerance, as shown by the recovery of seed-germination and root-growth inhibition by supplementary Cd. This research provides a genome-wide overview of the Triticeae HIPP genes and proved that HIPP1-V positively regulates Cd tolerance in common wheat.

Keywords: Cd tolerance; HIPP; Haynaldia villosa L.; gene family; subcellular localization.

Figure 1

The total number of heavy-metal-associated isoprenylated plant protein (HIPP) genes in the nine species of T. aestivum, T. dicoccoides, T. urartu, Ae. tauschii, H. vulgare, H. villosa, B. distachyon, A. thaliana, and Oryza sativa.

Figure 2

The phylogenetic analysis of the HIPP gene family from nine plant species. Species abbreviations: Ta: T. aestivum; Tu: T. urartu; Aet: Ae. tauschii; Td: T. dicoccoides; Hv: H. vulgare; Bd: B. distachyon; Ath: A. thaliana; Osa: O. sativa; -V: H. villosa. The red asterisks indicate the HIPP genes from H. villosa.

Figure 3

Chromosomal distribution of the HIPPs in the five Triticeae species. The chromosomal distribution of the HIPP genes in T. aestivum (a), T. dicoccoides (b), Ae. tauschii (c), T. urartu (d), and H. vulgare (e). The identities of the chromosomes are indicated at the top of each chromosome—the number in brackets corresponds to the number of genes located on the corresponding chromosome, while the HIPP gene names are shown to the right of each chromosome. Ta: T. aestivum; Tu: T. urartu; Aet: Ae. tauschii; Td: T. dicoccoides; Hv: H. vulgare.

Figure 4

Heat map of the expression profiling of common wheat HIPPs in different tissues and under various stresses: the expression of HIPPs in different tissues (a), and their response to powdery mildew (b), yellow rust (c), drought (d), and heat treatment (e). The expression signal of each gene was based on the Z-score-normalization value. Abbreviations: Bgt: powdery mildew; Pst: yellow rust.

Figure 5

Subcellular localization of HIPPs in the epidermal cells of Nicotiana benthamiana: subcellular localization of GFP (a), GFP-HIPP1-V (b), GFP-HIPP2-V (c), GFP-HIPP3-V (d), GFP-HIPP4-V (e), GFP-HIPP5-V (f), GFP-HIPP6-V (g), GFP-HIPP7-V (h), GFP-HIPP8-V (i), GFP-HIPP9-V (j), GFP-HIPP10-V (k), GFP-HIPP11-V (l), GFP-HIPP12-V (m), and GFP-HIPP13-V (n). GFP was used as the control. The localization of mCherry-SYP122 is shown in red, and the localization of GFP and its fusion proteins are shown in green. Scale bar = 10 µm.

Figure 6

Tolerance of HIPP1-V transgenic plants to Cd. Time-course expression profiling of HIPP1-V of H. villosa in response to Cd treatment in the roots (a), stems (b), and leaves (c). (d) The plant height between OE-HIPP1-T₃ and Yangmai158 plants exposed to different Cd concentrations. (e) The root length between OE-HIPP1-T₃ and Yangmai158 plants in different Cd concentrations. (f–i) OE-HIPP1-T₃ and Yangmai158 phenotypes under treatments of different Cd concentrations (0, 100 μM, 10 mM, and 20 mM). *p < 0.05. hai: hours after induction.