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.

• 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. .

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). .

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