Os03g0571900

The rice gene Os03g0571900 was reported as pez1 in 2011. PEZ1 belongs to the Multidrug and Toxic compound Extrusion (MatE) transporter family, a group of proteins with 12–14 transmembrane domains transporting small organic compounds.

Annotated Information

Function

• PEZ1 is responsible for an increase of PCA concentration in the xylem sap, and is essential for the utilization of apoplasmic precipitated iron in the stele. rice seems to transport PCA through PEZ1 for the solubilization of precipitated apoplasmic Fe in the root xylem, which may contribute to the long-distance transport of Fe.

• The PCA efflux activity of PEZ1 was investigated in Xenopus laevis oocytes with radiolabeled PCA as a substrate. Results confirmed that PEZ1 transports PCA (Fig. 3). PEZ1 did not rescue the growth defect of a Cd sensitive yeast mutant confirming that PEZ1 does not transport Cd.

Fig. 3. PCA efflux activity of PEZ1. Oocytes injected with water, or PEZ1 cRNA were loaded with 1.0 mM 14C labelled PCA. Columns (means ± SD) with different letters are significantly different from each other according to a one-way ANOVA followed by a Downloaded from http://www.jbc.org/ by guest on August 4, 2016 15 Student–Newman–Keuls test: P < 0.01; n = 8 each. .

• PEZ1 might also play a major role in Al tolerance.

Mutation

• The researchers isolated two Cd-accumulating mutants that were related to phenolic secretion, and named them phenolics efflux zero1 (pez1) -1, and -2. When grown in soil, these mutants accumulated higher Cd amounts in leaves and seeds (Fig. 1A, B) whereas no difference was observed for leaf dry weight per plant, SPAD value (Fig. 1C, D), the concentration of other metals in seed, as well as yield. When grown in hydroponic solution, pez1-1 and pez1-2 also showed higher Cd concentrations in roots and leaves compared to the WT (Fig. 1E, F).

Fig. 1. The pez1 mutants accumulated Cd. Cd concentration in the leaves (A) and seeds (B) of WT, pez1-1 (1-1), and pez1-2 (1-2) grown in soil. Leaf dry weight (C) and SPAD value (D) of WT, pez1-1 (1-1), and pez1-2 (1-2) grown in soil. Cd concentration in the leaves (E) and roots (F) of WT, pez1-1 (1-1), and pez1-2 (1-2) grown in hydroponic culture solution. Columns (means ± SD) with different letters are significantly different from each other according to a one-way ANOVA followed by a Student–Newman–Keuls test: P < 0.05; n = 5 each. .

• To identify the substrates of PEZ1, LC/MS data profiles of the xylem sap of pez1-1 and pez1-2 mutants were compared to that of WT. In pez1-1 and pez1-2, a peak at 22.8 min with m/z 153.03 was not detected in the xylem sap of the mutants (Fig. 2A-D). We searched KNApSAcK, a comprehensive species–metabolite relationship database (http://kanaya.naist.jp/KNApSAcK/), and found that this peak corresponds to protocatechuic acid (PCA; http://kanaya.naist.jp/knapsack_jsp/result.jsp?sname=organism&word=oryza). In addition to this peak, a peak at 22.7 min with m/z 179.04 was not detected in the xylem sap of both mutants, and a database search suggested that this corresponds to CA (Fig. 2E-H). Spiking the xylem of pez1-2 with purified PCA and CA confirmed that these peaks correspond to PCA and CA (Fig. 2I, J).

Fig. 2. PCA was decreased in the xylem sap of pez1 mutants. Mass chromatograms [PCA-H]- m/z 152.5–153.5 of the xylem sap of WT (A), pez1-1 (B), pez1-2 (C), and pez1-2 + 500 μM PCA (D). A peak in PCA appeared at 22.8 min. Mass chromatograms [CA-H]- m/z 178.5–179.5 of the xylem sap of WT (E), pez1-1 (F), pez1-2 (G) and pez1-2 + 500 μM caffeic acid (CA) (H). Mass spectrometry of PCA (I) and CA (J). .

• In the pez1-2 mutant, Fe accumulation was observed in the roots and not in leaf, and the differences were higher in the presence of Cd (Fig 6A, B). There was no significant difference in Zn, Mn and Cu concentration between the WT and pez1-2, both in roots and shoots, with or without Cd (Fig. S4A-F). On the other hand, Fe concentration in the xylem sap was lower than in the WT both with and without Cd, whereas there was no significant difference in xylem Cd and Mn concentration (Fig. 6C-E). Moreover, the expression of OsIRT1 was up-regulated in the mutant roots compared to the control when grown with or without Cd (Fig. 6F). Leaf samples were stained with Perl’s solution to check the localization of Fe and significant differences were found between WT and pez1-2 mutant for the localization of insoluble Fe(III) (Fig. 6G, H).

Fig. 6. PEZ1 is essential for solubilizing apoplasmic Fe. Fe concentrations in the leaf (A) and the root (B) of WT, and pez1-2 grown with or without Cd. Fe (C), Cd (D) and Mn (E) concentrations in xylem sap. Expression of OsIRT1 in WT and pez1-2 (F). Perl’s staining of WT (G) and pez1-2 (H). Columns (means ± SD) with different letters are significantly different from each other according to a one-way ANOVA followed by a Student–Newman–Keuls test: P < 0.05; n = 3. .

• To further understand the role of PEZ1, the PEZ1 over-expression lines were developed and characterized for phenotype and metal concentration. Real time RT PCR analysis confirmed that the expression of PEZ1 was significantly high in PEZ1 over-expression lines both in roots and shoots. In PEZ1 over-expression lines, the Fe concentration in roots and leaves was higher compared to WT plants under normal nutrient conditions (Fig. 7A, B). The growth of over-expression lines was severely restricted in terms of plant height and root length and many necrotic, dark-brown spots were observed on old leaves (Fig. 7C, D). Root and shoot dry weight was significantly reduced in PEZ1 over-expression lines (Fig. S5C, D). Moreover significant differences were observed for root Zn and leaf Mn concentrations, while there was no difference for the accumulation of other metals in roots and leaves (Fig. S5E-J). Differences were also observed for SPAD value between PEZ1 over-expression lines and WT (Fig. S5K). In a medium containing higher concentrations of Fe, precipitated Fe(III) was not observed in PEZ1 over-expression lines, while Fe was precipitated at the WT root surface (Fig. S6). In a calcareous soil, where plants cannot absorb enough Fe from the rhizosphere owing to high pH, PEZ1 over-expression lines grew better in terms of plant height and leaf chlorophyll content than the WT plants and dark-brown spots were not observed in PEZ1 over-expression lines (Fig. 7E and Fig. S7A, B).

Fig. 7. PEZ1 over-expression lines accumulated higher Fe. Fe concentrations in leaves (A) and roots (B) of WT, over-expression (OX) 1, OX2 and OX3 plants under normal nutrient conditions. Columns (means ± SD) with different letters are significantly different from each other according to a one-way ANOVA followed by a Student–Newman–Keuls test: P < 0.05; n = 3. (C, D) Phenotype of WT and over-expression (OX1, OX2 and OX3) plants under normal nutrient conditions. (E) WT and over-expression (OX1, OX2 and OX3) plants grown in calcareous soil. .

Subcellular localization

• PEZ1 was localized to the plasma membrane in rice roots cells, in rice root hairs and onion epidermal cells (Fig. 4A-G).

Fig. 4. Subcellar localization of 35Sp-PEZ1-GFP in the rice root. Fluorescence (A), differential interference contrast (DIC) (B), and overlay (C) image of rice root epidermal cells. Scale bars = 20 μm. (D) Fluorescence image of rice root. Fluorescence, (E) DIC (F) and overlay (G) image of rice root hair cells during plasmolysis when the samples were flooded with 20% sucrose. .

Expression

• To further understand its role, the PEZ1 promoter was used to drive the expression of β-glucuronidase (GUS) in rice. Histochemical analysis revealed that PEZ1 was expressed in the stele at the base of roots (Fig. 5A). In roots transverse sections taken from around 2.5 mm from root tip, the expression was observed around the xylem vessels, while in transverse sections taken at a distance of 5 mm from root tip the expression was mainly observed in stele (Fig. 5B, C).

Fig. 5. Histochemical observation of GUS activity in PEZ1 promoter GUS transgenic plants. (A) Longitudinal section. (B) Transverse section from the base of root (~2.5 mm form root tip). (C) Transverse section of the root (~5 mm from root tip). Scale bars = 500 μm (A), 100 μm (B, C). Rice plants were grown for six weeks after germination on +Fe MS medium. .

Labs working on this gene

• Department of Global Agricultural Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan
• Genome Resource Center, National Institute of Agrobiological Sciences, Kannondai 2-1-2, Tsukuba, Ibaraki 305-8602, Japan
• Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Aobayama 6-6-07, Sendai 980-8579, Japan
• Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University, Suematsu 1-308, Nonoichi-machi, Ishikawa 921-8836, Japan