OsPIE1, the Rice Ortholog of Arabidopsis PHOTOPERIOD-INDEPENDENT EARLY FLOWERING1, Is Essential for Embryo Development

orthologs in monocots remains unknown., respectively).Taken together, our results suggest that OsPIE1 is the rice ortholog of Arabidopsis PIE1 and plays an essential role in rice embryo development

Biomonitoring of Non-Dioxin-Like Polychlorinated Biphenyls in Transgenic Arabidopsis Using the Mammalian Pregnane X Receptor System: A Role of Pectin in Pollutant Uptake

<div><p>Polychlorinated biphenyls (PCBs) are persistent organic pollutants damaging to human health and the environment. Techniques to indicate PCB contamination <i>in planta</i> are of great interest to phytoremediation. Monitoring of dioxin-like PCBs in transgenic plants carrying the mammalian aryl hydrocarbon receptor (AHR) has been reported previously. Herein, we report the biomonitoring of non-dioxin-like PCBs (NDL-PCBs) using the mammalian pregnane X receptor (PXR). In the transgenic Arabidopsis designated NDL-PCB Reporter, the <i>EGFP-GUS</i> reporter gene was driven by a promoter containing 18 repeats of the xenobiotic response elements, while PXR and its binding partner retinoid X receptor (RXR) were coexpressed. Results showed that, in live cells, the expression of reporter gene was insensitive to endogenous lignans, carotenoids and flavonoids, but responded to all tested NDL-PCBs in a dose- and time- dependent manner. Two types of putative PCB metabolites, hydroxy- PCBs and methoxy- PCBs, displayed different activation properties. The vascular tissues seemed unable to transport NDL-PCBs, whereas mutation in <i>QUASIMODO1</i> encoding a 1,4-galacturonosyltransferase led to reduced PCB accumulation in Arabidopsis, revealing a role for pectin in the control of PCB translocation. Taken together, the reporter system may serve as a useful tool to biomonitor the uptake and metabolism of NDL-PCBs in plants.</p></div

Mutation in a gene required for pectin synthesis led to PCB resistance.

<p>(a) A screened <i>pcb1</i> mutant resistant to the bleach effects of 10 µM PCB 18. Six-d-old Arabidopsis seedlings were transferred to plates containing 10 µM PCB 18 and grown for 2 weeks. (b) Map-based cloning of <i>pcb1</i> revealed it was a new allele of <i>quasimodo1</i> and had a G- to A- transition in its second exon. The positions of each locus in chromosome 3 of Arabidopsis and the recombinants in relation to each marker are shown. (c) The PCB1/QUA1 protein had a transmenbrane (TM) domain in the N-terminus and a glycosyltransferase 8 (GT8) domain in the C-terminus, while <i>pcb1</i> had a mutation in the conserved GT8 domain. (d) The arabinose (Ara), xylose (Xyl), and galactose (Gal) contents in 12-d-old seedlings of <i>pcb1</i> were comparable to the wild-type, but the GalA content showed a ∼20% reduction.</p

Dose- and time-dependent GUS activities in NDL-PCB Reporters in response to PXR ligands.

<p>(a) Induced GUS activity in three transgenic lines in response to 5 µM PCB 18. (b) Dose-dependent GUS activity upon treatment with various concentrations of PCB 18. (c) Time-dependent GUS activity in response to 5 µM PCB 18. (d) GUS activity was responsive to NDL-PCBs but insensitive to DL-PCBs. (e) Response of GUS activity to typical PXR agonists. (f) Methoxyl- PCBs could induce low GUS activity, while hydroxyl-PCBs could not. Six-d-old seedlings were transferred to plates with indicated chemicals and grown for 4 d before GUS activity analysis. Chemicals in (d) to (f) were at concentrations of 10 µM.</p

Reduced cell adhesion and PCB accumulation in the <i>pcb1</i> mutant.

<p>(a) Cell wall staining in a transverse section of a wild-type root. (b) Cell wall staining in a transverse section of a <i>pcb1</i> root showed dramatically reduced cell adhesion in epidermal, cortical and endodermal cells of roots. (c) GUS activity in the wild-type and the <i>pcb1</i> mutant induced by PCB 18. (d) Comparison of PCB 18 accumulation in wild-type and <i>pcb1</i>. Six-d-old Arabidopsis seedlings were transferred to plates containing 10 µM PCB 18 and grown for 6 d. Bars: 12.5 µm.</p

The expression of the reporter gene in NDL-PCB (non-dioxin-like polychlorinated biphenyl) Reporters was insensitive to endogenous secondary metabolites in living cells.

<p>(a) Histochemical assay of GUS activity in young seedlings of NDL-PCB Reporters. Three-day-old seedlings were transferred to plates without (control) or with 50 nM PCB 18 (+ PCB 18) and grown for 3 days. The right root under PCB 18 treatment was stained blue. (b) Histochemical assay of GUS activity in 12-day-old NDL-PCB Reporters. Six-day-old seedlings were transferred to plates with 50 nM PCB 28 and grown for 6 days. Roots under PCB 28 treatment were stained blue. (c) NDL-PCB Reporters were insensitive to endogenous auxin in root tips as no GFP signals were observed by confocal microscopy. The red PI (propidium iodide) indicated the cell wall. (d) The DR5::GFP line reported auxin peaks (GFP signals) in roots. (e) Naringenin chalcone (yellow fluorescence) in distal elongation zones and meristems of 10-d-old Arabidopsis roots. Meristematic cells were small in size. (f) Kaempferol and quercetin (gold fluorescence) in mature elongation zones of 10-day-old Arabidopsis roots. Bars for (c) to (f), 30 µm.</p

Construction of T-DNA vectors carrying the mouse PXR (pregnane X receptor) system.

<p>(a) pGypsy-TATA-TL-Su(Hw) was a destination vector for enhancer analysis which was constructed based on pGypsy-TL-Su(Hw) (She <i>et al.</i>, 2010). The positions and orientations of the selectable marker gene <i>HPT</i> (hygromycin phosphotransferase) and the reporter gene <i>EGFP-GUS</i> (Enhance Green Fluorescent Protein plus β-glucuronidase) are shown with respect to the left border (LB) and right border (RB) of the T-DNA regions. The <i>ccdB</i> region was expected to be replaced by an enhancer sequence by a LR reaction. A <i>35S</i> CaMV mini promoter (−46 to 0, TATA) together with a translation leader (TL) region from pH 7WG2D (Karimi <i>et al.</i>, 2002) were inserted upstream the reporter gene. (b) Schematic overview of the xenobiotic response elements (XREs) used in this study. The sequence was derived from the proximal region of <i>CYP3A11</i> mouse promoter and modified with the PXR DNA-binding signatures reported recently (Cui <i>et al.</i>, 2010). (c) and (d) Mouse <i>PXR</i> and <i>RXR</i> (retinoid X receptor) were co-expressed using the vectors pK7WG2D (Karimi <i>et al.</i>, 2002) and pEarleyGate303 (Earley <i>et al.</i>, 2006), respectively.</p

The vascular tissues of Arabidopsis seemed unable to transport NDL-PCBs actively.

<p>(a) Histochemical assay of GUS activity in root epidermal and cortical cells. (b) Histochemical assay of GUS activity in root vascular tissues. (c) Under transport assay, H-TAM (4-hydroxyl-tamoxifen) was able to induce GUS activity in the vascular tissues of hypocotyls. (d) PCB 18 was unable to induce GUS activity in the vascular tissues of hypocotyls in the transport assay. (e) Under an uptake assay, the vascular tissues of hypocotyls displayed very weak GUS activity. Six-d-old seedlings were transferred to plates with indicated chemicals and grown for 4 d before histochemical assay. Uptake assay: plates were placed vertically (90°), thus, some parts of cotyledons and hypocotyls were in contact with the media. Transport assay: plates were placed at 100°, thus, only the roots were in contact with the media. Bars, 30 µm. The orientations of roots and hypocotyls were indicated.</p