MOESM1 of Extrahepatic cytochrome P450s play an insignificant role in triptolide-induced toxicity

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OsNRAMP3 Is a Vascular Bundles-Specific Manganese Transporter That Is Responsible for Manganese Distribution in Rice

<div><p>Manganese (Mn) is an essential trace element for plants. Recently, the genes responsible for uptake of Mn in plants were identified in <i>Arabidopsis</i> and rice. However, the mechanism of Mn distribution in plants has not been clarified. In the present study we identified a natural resistance-associated macrophage protein (NRAMP) family gene in rice, <i>OsNRAMP3</i>, involved in Mn distribution. <i>OsNRAMP3</i> encodes a plasma membrane-localized protein and was specifically expressed in vascular bundles, especially in phloem cells. Yeast complementation assay showed that OsNRAMP3 is a functional Mn-influx transporter. When <i>OsNRAMP3</i> was absent, rice plants showed high sensitivity to Mn deficiency. Serious necrosis appeared on young leaves and root tips of the <i>OsNRAMP3</i> knockout line cultivated under low Mn conditions, and high Mn supplies could rescue this phenotype. However, the necrotic young leaves of the knockout line possessed similar levels of Mn to the wild type, suggesting that the necrotic appearance was caused by disturbed distribution of Mn but not a general Mn shortage. Additionally, compared with wild type, leaf Mn content in <i>osnramp3</i> plants was mostly in older leaves. We conclude that OsNRAMP3 is a vascular bundle-localized Mn-influx transporter involved in Mn distribution and contributes to remobilization of Mn from old to young leaves.</p></div

Histochemical staining of GUS activity in rice plants transformed with the construct <i>OsNRAMP3</i>-promoter:<i>GUS</i>.

<p>(A) Root tip; (B) mature root at 2 cm from tip; (C) lateral root; (D) leaf sheath, ligule and auricle; (E) leaf blade; (F) partly enlarged view of E; (G) hull at heading stage; (H) endosperm and hull at 25 d after heading; (I) transverse section of root, as described in B; (J) high-magnification of I; (K) transverse section of leaf blade, as described in E; (L) detail of a large vascular bundle from K; (M) enlarged view of phloem region of L; and (N) high-magnification of a small vascular bundle from K. All samples except hulls and endosperm were harvested from rice plants grown hydroponically for three weeks under normal conditions. CV: commissural vein; SV: small vascular bundle; LV: large vascular bundle; xy: xylem; ph: phloem; cc: companion cells.</p

Metal analysis of different leaves in wild type and <i>osnramp3</i> plants.

<p>Different leaves were sampled from wild type and <i>osnramp3</i> plants grown for two weeks at 0.08 (A) or 8 (B) µM Mn supplies after two weeks of normal conditional cultivation by hydroponics and elemental analysis was performed by ICP-MS. The 1–4 leaves were harvested from the same tiller of wild type or <i>osnramp3</i> plants, and represented leaves from oldest to youngest respectively. Data are means ± SD (n = 3). One and two asterisks indicate values are significantly different from wild type at P<0.05 and P<0.01, respectively (by <i>t</i>-test).</p

Subcellular localization of OsNRAMP3 protein.

<p>Subcellular localization of OsNRAMP3 protein was determined in <i>Arabidopsis</i> protoplasts. The confocal images were acquired using a confocal laser scanning microscope (TCS SP2; Leica).</p

Complementation assay of <i>OsNRAMP3</i> on Mn-uptake deficient yeast mutant strain.

<p>(A) Yeast mutant <i>Δsmf1</i> and its wild type (WT) cells containing <i>pYES2</i> (vector), <i>OsNRAMP3</i> or <i>AtNRAMP1</i> (positive control) grown on synthetic defined (SD)-Ura plates with different EGTA supplies and 2% galactose. (B) Metal determination in <i>Δsmf1</i> and its wild-type cells containing <i>pYES2</i>, <i>OsNRAMP3</i> or <i>AtNRAMP1</i> grown in liquid SD-Ura culture with 0.2 µM Mn supplies and 2% galactose. Data are means ± SD (n = 4).</p

Expression analysis of <i>OsNRAMP3</i> by real-time RT-PCR.

<p>(A) The relative expression level of <i>OsNRAMP3</i> in different rice tissues. C1: first culm; CII: second culm; CIII: third culm; B: leaf blade; P: panicle; SH: leaf sheath; R: root. (B) Transcripts of <i>OsNRAMP3</i> of different leaves under different Mn conditions. The leaves detected here were sampled from the same tiller of rice plants – with the first leaf the oldest and the fourth leaf the youngest and partly wrapped in the leaf sheath. The plants were cultivated hydroponically under normal conditions for two weeks and then shifted to Mn-replete or Mn-free conditions for an additional one week. ZH11: cv. Zhonghua 11. (C) Expression analysis of <i>OsNRAMP3</i> in different regions of the fourth leaf. (D and E) Kinetics of the response of <i>OsNRAMP3</i> to Mn or Fe deficiency in shoot (D) and root (E) of rice plants. The plants were cultivated hydroponically under normal conditions for two weeks and then shifted to different conditions for treatment. Data are means ± SD (n = 3).</p

Determination of Mn concentration in wild type and <i>osnramp3</i> plants.

<p>Elemental analysis was performed by ICP-MS on roots (A) and shoots (B) of wild type or <i>osnramp3</i> plants grown for two weeks at three different Mn supplies after two weeks of normal conditional cultivation by hydroponics. Data are means ± SD (n = 3). One and two asterisks indicate values that are significantly different from wild type at P<0.05 and P<0.01, respectively (by <i>t</i>-test).</p

Identification of the knockout line of <i>OsNRAMP3</i>.

<p>(A) The structure of <i>OsNRAMP3</i>. <i>OsNRAMP3</i> contains 13 exons and 12 introns, and a T-DNA inserted into the 12^th exon of osnramp3 mutant plants. (B) The determination of accumulation of <i>OsNRAMP3</i> in <i>osnramp3</i> plants by RT-PCR. (C) Phenotypic analysis of knockout line of <i>OsNRAMP3</i>. The plants were cultivated hydroponically under normal conditions for two weeks and then shifted to different Mn supplies for an additional two weeks. Three levels of Mn were applied: 0.08 µM (a), 8 µM (b) and 800 µM (c). The general condition for growth of plants in the three Mn treatments was photographed (d). Careful observations were performed on roots (e) and leaves (f) of wild type and <i>osnramp3</i> plants at 0.08 µM Mn supply. The red arrow indicates the necrotic area that appeared in <i>osnramp3</i> roots. In (f), the left leaves are from wild type and the right leaves from <i>osnramp3</i>; 1–4 leaves were from the same tillers, with 1-leaf the oldest and 4-leaf the youngest.</p

Effects of different Mn supplies on the growth of knockout plants of <i>OsNRAMP3</i>.

<p>The plants were cultivated hydroponically under normal conditions for two weeks and then shifted to three different Mn supplies for an additional two weeks. The plant heights (A) and root lengths (B) were investigated. Then the roots (C) and shoots (D) were harvested separately and their dry weights recorded. Data are means ± SD (n = 15). One and two asterisks indicate values that are significantly different from wild type at P<0.05 and P<0.01, respectively (by <i>t</i>-test).</p