Knocking-Out OsPDR7 Triggers Up-Regulation of OsZIP9 Expression and Enhances Zinc Accumulation in Rice

doi:10.1016/j.rsci.2022.05.004

Abstract

Abstract:

Zinc (Zn) is an essential trace mineral that is required for plant growth and development. A number of protein transporters, which are involved in Zn uptake, translocation and distribution, are finely regulated to maintain Zn homeostasis in plant. In this study, we functionally characterized an ATP-binding cassette (ABC) transporter gene, OsPDR7, which is involved in Zn homeostasis. OsPDR7 encodes a plasma membrane-localized protein that is expressed mainly in the exodermis and xylem in the rice root. ospdr7 mutants resulted in higher Zn accumulation compared with the wild type. Heterogeneous expression of OsPDR7 in a yeast mutant rescued the Zn-deficiency phenotype, implying transport activity of OsPDR7 to Zn in yeast. However, no ZIP genes except for OsZIP9 showed change in expression profile in the ospdr7 mutants, which suggested that OsPDR7 maintains cellular Zn homeostasis through regulating OsZIP9 expression. RNA-Seq analysis further revealed a set of differentially expressed genes between the wild type and ospdr7 mutants that allowed us to propose a possible OsPDR7-associated signaling network involving transporters, hormone responsive genes, and transcription factors. Our results revealed a novel transporter involved in the regulation of Zn homeostasis and will pave the way toward a better understanding of the fine-tuning of gene expression in the network of transporter genes.

Key words: OsPDR7, OsZIP9, zinc, metal accumulation, rice, ATP-binding cassette transporter

Meng Lu, Tang Mingfeng, Zhu Yuxing, Tan Longtao. Knocking-Out OsPDR7 Triggers Up-Regulation of OsZIP9 Expression and Enhances Zinc Accumulation in Rice[J]. Rice Science, 2023, 30(1): 36-49.

Figures/Tables 5

Fig. 1. Expression profile of OsPDR7 and subcellular localization of OsPDR7. A, Tissue specific gene expression in various tissues at different growth stages. Rice plants were grown under field conditions. OsUBC was used as the internal standard for gene expression. I, II and III represent the serial numbers of node, leaf blade, leaf sheath and internode from top to bottom of rice plants. Data are Mean ± SD (n = 3). B, Relative expression levels of OsPDR7 in the roots of seedlings exposed to different metal concentrations. The 21-day-old wild type seedlings were transferred to 0.5× Kimura B (KB) solution deficient in Zn, Fe, Cu and Mn (-Zn, -Fe, -Cu and -Mn) or supplemented with 50 μmol/L Zn (Zn-exposed, ++Zn) for 7 d. Data were separately compared with that from seedlings grown in normal 0.5× KB (CK). Values represent Mean ± SD (n ≥ 3). Statistical comparisons were performed using the Tukey’s HSD mean-separation test at the 0.05 level and no significant difference was observed. C, Histochemical analysis of transgenic plants expressing the GUS gene driven by the OsPDR7 promoter (a and c) and wild type plants (b and d). (a), ProOsPDR7::GUS seedling. (b), Wild type seedling. (c), Primary root of ProOsPDR7::GUS transgenic plant. (d), Primary root of wild type plant. (e-i), Immunostaining with anti-GUS antibody was performed in roots of ProOsPDR7::GUS transgenic rice seedlings (f-i) and wild type plant (e). Blue indicates autofluorescence emitted from the cell wall due to 4,6-diamidino-2-phenylindole (DAPI) staining. Red indicates the anti-GUS antibody-specific fluorescent signal. (e) Representative transverse sections of (d). (f) and (g), Representative transverse sections of (c). (h) and (i), Representative regions magnified from (g). P, Pericycle; St, Stele; Ep, Exodermis. Scale bars, 1 mm in (a-d), 50 μm in (e-g) and 20 μm in (h) and (i). D, Subcellular localization of OsPDR7 protein determined in rice protoplasts. For each localization experiment, ≥ 20 individual cells were examined using a Zeiss LSM880 confocal laser scanning microscope (Carl Zeiss, Germany). Scale bars, 2 μm. GFP, Green fluorescent protein.

Fig. 2. Zn profiles of OsPDR7 knock-out lines and over- expression lines at vegetative and reproductive growth stages. A, Total uptake of 67Zn by wild type (WT) and three OsPDR7 knock-out lines (ospdr7-1 to -3) at the vegetative stage. B, 67Zn concentration in the shoots and roots at the seedling stage. C, 67Zn distribution ratio between the roots and shoots at the seedling stage. D and E, Zn accumulation in brown rice (D) and in husk, rachis, node, leaf blade and leaf sheath (E) at the reproductive stage. F and G, Zn accumulation in brown rice (F) and in husk, rachis, node, leaf blade and leaf sheath (G) in OsPDR7 over-expression (OsPDR7ox-1 and -2) lines at the reproductive stage. I and II in E and G represent the serial numbers of node, leaf blade and leaf sheath from top to bottom of rice plants.Seedlings were grown in 0.5× Kimura B (KB) nutrient solution for 21 d, then transferred to 0.5× KB containing 0.4 μmol/L 67ZnSO4 for 2 d in A-C. Plants were grown in a paddy field until the grains were ripe in D-G. Values represent Mean ± SD (n ≥ 3). Statistical comparisons against WT were performed with the Tukey’s HSD mean-separation test (*, P < 0.05; **, P < 0.01).

Fig. 3. Complementation assay of OsPDR7 in yeast mutant strain. A, Zn and Cd transport activity assay of OsPDR7 expressed in yeast. The empty vector pYES2C or OsPDR7 was introduced into Zn uptake-deficient mutant strain ?ZHY3 or Cd-sensitive mutant strain ?ycf on the synthetic defined-Ura (SD-U) medium. The yeast strains were cultured on the plate with different Zn or Cd concentrations at 30 for 2-4 d. Zn was chelated with 50 μmol/L ethylenebis (oxyethylenenitrilo) tetraacetic acid (EGTA). B and C, Growth rates of yeast cells. Yeast mutant strain ?ZHY3 expressing OsPDR7 or the empty vector was cultured in SD-U medium containing galactose with 2.5 μmol/L Zn (B) or 500 μmol/L Zn (C). D, Concentration of Zn in the mutant yeast strain ?ZHY3 expressing the empty vector pYES2C or OsPDR7. Data are Mean ± SD (n ≥ 3). In B-D, significant differences were determined by one-way analysis of variance (*, P < 0.05; **, P < 0.01).

Fig. 4. OsZIP9 expression in response to exogenous Zn supplied via root culture or foliar spray. A and B, Expression levels of OsZIP9 in roots and shoots of wild type (WT) and three OsPDR7 knock-out lines (ospdr7-1 to -3) (A), as well as in WT and OsPDR7 over-expression line (OsPDR7ox-1) (B). The 28-day-old seedlings were used. C-E, OsZIP9 relative expression levels in local system (C), foliar spray with 0.4 μmol/L 67ZnSO4 (D), and in control treatments (E) in WT and OsPDR7 knockout lines. The 21-day-old seedlings were cultivated in Zn-deficient 0.5× Kimura B (KB) medium for 7 d, followed by 0.4 μmol/L 67Zn re-supply to the leaves via foliar spray or 0.5× KB containing 0.4 μmol/L 67Zn as the local Zn system for 1 h. The control was 0.01% Trition X-100. Values represent Mean ± SD (n ≥ 3). Statistical comparison was performed with the Tukey’s HSD mean-separation test (*, P < 0.05; **, P < 0.01).

Fig. 5. Differentially expressed genes (DEGs) between wild type (WT) and OsPDR7 mutant (ospdr7-4). A, Number of DEGs between WT and ospdr7-4. B, Gene Ontology enrichment analysis of DEGs (P ≤ 0.05). C, A heat map showing relative expression of transcription factor, transporter, and hormone responsive-related genes. JA, Jasmonic acid; IAA, Indole-3-acetic acid; ABA, Abscisic acid. The roots of WT and ospdr7-4 seedlings (28-day-old) were collected for transcriptome analysis. To identify DEGs, the expression level of each transcript was calculated using the transcripts per million reads method (n = 3).

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