Bacteria-zinc co-localization implicates enhanced synthesis of cysteine-rich peptides in zinc detoxification when Brassica juncea is inoculated with Rhizobium leguminosarum

Abstract

Some plant growth promoting bacteria (PGPB) are enigmatic in enhancing plant growth in the face of increased metal accumulation in plants. Since most PGPB colonize the plant root epidermis, we hypothesized that PGPB confer tolerance to metals through changes in speciation at the root epidermis. We employed a novel combination of fluorophore-based confocal laser scanning microscopic imaging and synchrotron based microscopic X-ray fluorescence mapping with X-ray absorption spectroscopy to characterize bacterial localization, zinc (Zn) distribution and speciation in the roots of Brassica juncea grown in Zn contaminated media (400 mg kg(-1) Zn) with the endophytic Pseudomonas brassicacearum and rhizospheric Rhizobium leguminosarum. PGPB enhanced epidermal Zn sequestration relative to PGBP-free controls while the extent of endophytic accumulation depended on the colonization mode of each PGBP. Increased root accumulation of Zn and increased tolerance to Zn was associated predominantly with R. leguminosarum and was likely due to the coordination of Zn with cysteine-rich peptides in the root endodermis, suggesting enhanced synthesis of phytochelatins or glutathione. Our mechanistic model of enhanced Zn accumulation and detoxification in plants inoculated with R. leguminosarum has particular relevance to PGPB enhanced phytoremediation of soils contaminated through mining and oxidation of sulphur-bearing Zn minerals or engineered nanomaterials such as ZnS.

Keywords: Brassica juncea; X-ray absorption spectroscopy; Zn detoxification; Zn speciation; bacterial localization; cysteine; plant growth promoting bacteria; zinc (Zn) accumulation.

Figure 1

(a, c, e) Root length, shoot length and seedling biomass in un-inoculated Brassica juncea (B), B. juncea inoculated with Pseudomonas brassicacearum (P), Rhizobium leguminosarum (R) and combinations (RP) in uncontaminated treatments (o) and under zinc (Zn), and (b, d, f) root, shoot and seedling biomass tolerance indexes under Zn showing better root and biomass tolerance in treatment inoculated with R. leguminosarum and its combination with P. brassicacearum. Bars are means of root length, shoot length, seedlings biomass and % TI(s) from six Petri dishes each containing 12 seedlings. Error bars show standard errors. Different letters indicate significant (P < 0.05) differences in plant growth parameters between treatments.

Figure 2

3-D reconstruction of confocal laser scanning microscopy (CLSM) images of (a) Brassica juncea root un-inoculated, (b) inoculated with Pseudomonas brassicacearum, (c) Rhizobium leguminosarum and (d) both bacterial strains, 14 d after exposure to 400 mg kg⁻¹ zinc (Zn). Green fluorescent bodies are bacteria cells and the orange spots are areas of Zn localization. Note that the strains in the co-inoculation treatment (d) could not be distinguished since they both appear green. Areas of high bacteria localization along the root epidermis are circled. Figure shows absence of bacteria cells in the un-inoculated root.

Figure 3

Confocal laser scanning microscopy (CLSM) imaging of (a) Pseudomonas brassicacearum and (b) Rhizobium leguminosarum colonization of Brassica juncea root. ε shows endophytic (interior) colonization and s shows rhizospheric (outer sphere) colonization of zinc (Zn) embedded root strand in (a) and (b), respectively. Numbers (1–3) show sequence of image acquisition across the root depth. Green bodies are bacteria cells and orange spots indicate the presence of Zn.

Figure 4

Synchrotron micro X-ray fluorescence (μXRF) imaging of zinc (Zn) in the root of (a) Brassica juncea un-inoculated (BZn), (b) inoculated with Pseudomonas brassicacearum, (c) Rhizobium leguminosarum and (d) combinations of the two bacterial strains, 14 d after seed planting in 400 mg kg⁻¹ Zn. Figure shows that the plant growth promoting bacteria (PGPB) significantly enhance Zn sequestration at the epidermis. Symbols o and × represent spots in the root epidermis and endodermis, respectively, that were subjected to microfocus X-ray absorption near edge structure (μXANES) analysis.

Figure 5

Zinc (Zn) compound compositions (%) in the root (a) epidermis and (b) endodermis of Brassica juncea. Zn SUL, zinc sulphate; Zn OXA, zinc oxalate; Zn PHY, zinc phytate; Zn CYS, zinc cysteine. Bars are means of zinc compound compositions from six and three Zn X-ray absorption near edge structure (XANES) collected from the epidermis and endodermis, respectively, of two replicate root samples per treatment. Different alphabets and symbols (upper and lower cases) show significant differences (P < 0.05) between treatments.

Figure 6

Schematic model of zinc (Zn) speciation and detoxification mechanisms deduced from this research for Brassica juncea roots (BZn) inoculated with Rhizobium leguminosarum (RZn), Pseudomonas brassicacearum (PZn) and a combination of the two bacterial strains (RPZn) upon exposure to 400 mg kg⁻¹ Zn contamination as zinc sulphate. Figure depicts the native presence of phytate, oxalate and O-acetylserine, with uptake of sulphate enhancing cysteine synthesis from O-acetylserine in BZn roots. Although all three ligands are capable of zinc complexation, induction of additional cysteine synthesis by R. leguminosarum in RZn increases cysteine concentration and increases endodermal zinc chelation as cysteine bound peptide compared with PZn, whereas the combination of the two bacteria in RPZn leads to a more balanced distribution between phytate and cysteine complexation in root endodermis. Better root growth was observed in RZn and RPZn which correlated with higher proportion of cysteine bound zinc in root endodermis compared with BZn and PZn treatments.