Arbuscular mycorrhiza mitigates zinc stress on Eucalyptus grandis through regulating metal tolerance protein gene expression and ionome uptake

Abstract

Arbuscular mycorrhizal (AM) fungi are symbionts of most terrestrial plants and enhance their adaptability in metal-contaminated soils. In this study, mycorrhized and non-mycorrhized Eucalyptus grandis were grown under different Zn treatments. After 6 weeks of treatment, the growing status and ionome content of plants as well as the expression patterns of metal tolerance proteins and auxin biosynthesis-related genes were measured. In this study, mycorrhized E. grandis showed higher biomass and height at a high level of Zn compared with non-mycorrhized plants. In addition, AM plants accumulated P, Mg, and Mn in roots and P, Fe, and Cu in shoots, which indicate that AM fungi facilitate the uptake of ionome nutrients to promote plant growth. In addition, mycorrhiza upregulated the expression of EgMTP1 and EgMTP7, whose encoding proteins were predicted to be located at the vacuolar membrane. Meanwhile, Golgi membrane transporter EgMTP5 was also induced in AM shoot. Our results suggest that AM likely mitigates Zn toxicity through sequestrating excess Zn into vacuolar and Golgi. Furthermore, the expression of auxin biosynthesis-related genes was facilitated by AM, and this is probably another approach for Zn tolerance.

Keywords: Eucalyptus grandis; arbuscular mycorrhiza; auxin biosynthesis–related genes; metal tolerance protein; nutrient uptake; zinc stress.

Figure 1

Effects of Zn and AM fungi on the growth of E. grandis. (A) Root fresh weight; (B) shoot fresh weight; (C) plant height; (D) the phenotype of E. grandis with or without AM fungi under 5, 50, and 150 μM Zn treatment. NM, non-mycorrhizal plants; AM, mycorrhizal plants. Values were indicated as mean ± SE of six biological replicates. Different letters above bars indicated significant differences at P < 0.05.

Figure 2

Total mineral element concentrations. The microelements (Zn, Fe, Cu, and Mn) and macroelements (P and Mg) of roots and shoots were analyzed by ICP-OES. NM, non-mycorrhizal plant; AM, mycorrhizal plant. (A) Total mineral concentration in root. (B) Total mineral concentration in shoot. Values were indicated as mean ± SE. Different letters above the bars indicated significant differences between treatments (P < 0.05).

Figure 3

Phylogenetic relationships of MTP. The neighbor-joining tree was generated using MEGA 7.0 with 1,000 bootstrap replicates. E. grandis MTP proteins were in bold. At, Arabidopsis thaliana; Os, Oryza sativa; Gm, Glycine max; Pt, Populus trichocarpa; Cit, Citrus sinensis; Eg, Eucalyptus grandis.

Figure 4

Expression profiles of EgMTP. The heat map was generated using the expression fold changes of EgMTP family. Relative gene expression was calculated by the 2^−ΔΔCT method using the EgUBI3 as a normalizer. For each gene, the expression level in NM roots with 5 μM Zn was defined as 1. (A) The expression profiles of EgMTPs in root. (B) The expression profiles of EgMTPs in shoot.

Figure 5

Expression profiles of auxin biosynthesis–related genes. The relative expression of EgAAO3 (A), EgYUC2 (B), EgYUC3 (C), and EgAMI1 (D) was calculated by the 2^−ΔΔCT method using the EgUBI3 as a normalizer. For each gene, the expression level in NM roots with 5 μM Zn was defined as 1. NM-Root, the root of non-mycorrhizal plants; AM-Root, the root of mycorrhizal plants. Values were indicated as mean ± SE. Different letters on the histograms indicated significant differences (P < 0.05).

Figure 6

Mycorrhizal colonization. (A) Mycorrhizal frequency, (B) mycorrhizal intensity, and (C) arbuscule abundance were quantified using the MYCOCALC program. Values were indicated as mean ± SE. Different letters on the histograms indicated that the means significant differences (P < 0.05). (D) a, arbuscule; h, hyphae; v, vesicles; s, septa; bar, 50 μm.

Figure 7

Schematic representation of the major changes induced by Zn in EgMTP expression in mycorrhizal and E. grandis. Red transporters, upregulated EgMTPs by mycorrhizal; ER, endoplasmic reticulum; V, vacuole; G, golgi; Chl, chloroplast; M, mitochondrion.