Functional Analysis of NtZIP4B and Zn Status-Dependent Expression Pattern of Tobacco ZIP Genes

Tobacco is frequently considered as a plant useful for phytoremediation of metal-contaminated soil, despite the mechanisms for regulation of uptake and accumulation being largely unknown. Here we cloned and characterized a new tobacco Zn and Cd transporter NtZIP4B from the ZIP family (ZRT-IRT-Like proteins). It complemented the Zn-uptake defective yeast mutant zrt1zrt2, and rendered the wild type DY1457 yeast more sensitive to Cd. Bioinformatic analysis and transient expression of the NtZIP4B-GFP fusion protein in tobacco leaves indicated its localization to the plasma membrane. Real-time q-PCR based analysis showed that it is expressed in all vegetative organs with the highest level in leaves. The Zn status determined transcript abundance; NtZIP4B was upregulated by Zn-deficiency and downregulated by Zn excess. At the tissue level, in roots NtZIP4B is expressed in the vasculature of the middle part of the roots and in surrounding tissues including the root epidermis; in leaves primarily in the vasculature. Bioinformatic analysis identified two copies of ZIP4 in tobacco, NtZIP4A and NtZIP4B with 97.57% homology at the amino acid level, with the same expression pattern for both, indicating a high degree of functional redundancy. Moreover, the present study provides new insights into the coordinated function of NtZIP1, NtZIP2, NtZIP4, NtZIP5, NtZIP8, NtIRT1, and NtIRT1-like in response to low-to-high Zn status. Leaves were the major site of NtZIP4, NtZIP5, and NtZIP8 expression, and roots for NtZIP1, NtZIP2, NtIRT1, and NtIRT1-like. Contrasting expression level in the apical and basal root parts indicates distinct roles in root-specific processes likely contributing to the regulation of Zn root-to-shoot translocation. In summary, new insight into the role of ZIP genes in Zn homeostasis pointing to their overlapping and complementary functions, offers opportunities for strategies to modify Zn and Cd root/shoot partition in tobacco

Prostaglandin signalling regulates ciliogenesis by modulating intraflagellar transport

Cilia are microtubule-based organelles that mediate signal transduction in a variety of tissues. Despite their importance, the signalling cascades that regulate cilium formation remain incompletely understood. Here we report that prostaglandin signalling affects ciliogenesis by regulating anterograde intraflagellar transport (IFT). Zebrafish leakytail (lkt) mutants show ciliogenesis defects, and the lkt locus encodes an ATP-binding cassette transporter (ABCC4). We show that Lkt/ABCC4 localizes to the cell membrane and exports prostaglandin E2 (PGE2), a function that is abrogated by the Lkt/ABCC4T804M mutant. PGE2 synthesis enzyme cyclooxygenase-1 and its receptor, EP4, which localizes to the cilium and activates the cyclic-AMP-mediated signalling cascade, are required for cilium formation and elongation. Importantly, PGE2 signalling increases anterograde but not retrograde velocity of IFT and promotes ciliogenesis in mammalian cells. These findings lead us to propose that Lkt/ABCC4-mediated PGE2 signalling acts through a ciliary G-protein-coupled receptor, EP4, to upregulate cAMP synthesis and increase anterograde IFT, thereby promoting ciliogenesis

NO₃⁻/H⁺ antiport in the tonoplast of cucumber root cells is stimulated by nitrate supply: evidence for a reversible nitrate-induced phosphorylation of vacuolar NO₃⁻/H⁺ antiport.

Studies in the last few years have shed light on the process of nitrate accumulation within plant cells, achieving molecular identification and partial characterization of the genes and proteins involved in this process. However, contrary to the plasma membrane-localized nitrate transport activities, the kinetics of active nitrate influx into the vacuole and its adaptation to external nitrate availability remain poorly understood. In this work, we have investigated the activity and regulation of the tonoplast-localized H(+)/NO₃(-) antiport in cucumber roots in response to N starvation and NO₃(-) induction. The time course of nitrate availability strongly influenced H(+)/NO₃(-) antiport activity at the tonoplast of root cells. However, under N starvation active nitrate accumulation within the vacuole still occurred. Hence, either a constitutive H(+)-coupled transport system specific for nitrate operates at the tonoplast, or nitrate uses another transport protein of broader specificity to different anions to enter the vacuole via a proton-dependent process. H(+)/NO₃(-) antiport in cucumber was significantly stimulated in NO₃(-)-induced plants that were supplied with nitrate for 24 hours following 6-day-long N starvation. The cytosolic fraction isolated from the roots of NO₃(-)-induced plants significantly stimulated H(+)/NO₃(-) antiport in tonoplast membranes isolated from cucumbers growing on nitrate. The stimulatory effect of the cytosolic fraction was completely abolished by EGTA and the protein kinase inhibitor staurosporine and slightly enhanced by the phosphatase inhibitors okadaic acid and cantharidin. Hence, we conclude that stimulation of H(+)/NO₃(-) antiport at the tonoplast of cucumber roots in response to nitrate provision may occur through the phosphorylation of a membrane antiporter involving Ca-dependent, staurosporine-sensitive protein kinase

The properties of H⁺-coupled transport of sulphate and chloride in tonoplast vesicles isolated from the roots of cucumbers grown under different nitrate supply.

<p>The time-course of acridine orange absorbance change after the addition of different concentration of K₂SO₄ (A) or KCl (C) to the reaction media containing ΔpH-energized tonoplast vesicles obtained from NO₃⁻-grown (grey bars), NO₃⁻-induced (dark bars) or N-deprived (white bars) plants. The effect of sulphate (B) or chloride (D) concentration on the acridine orange absorbance ΔpH-energized, tonoplast obtained from NO₃⁻-grown (squares), NO₃⁻-induced (triangles), or N-deprived (circles) plants. Values are the means ±SE (<i>n</i> = 5–6 measurements from 4–6 independent tonoplast preparations). Asterisks indicate a significant difference (<i>P</i><0.05) between H⁺-coupled SO₄²⁻ and Cl⁻ transport in tonoplast isolated from different plants. The Km and R² values were calculated using GraphPrism Software. The −1/Km values are indicated by red arrows. V represents the ΔA₄₉₅×min⁻¹×mg⁻¹ protein.</p

V-ATPase activity and quantity in tonoplast vesicles isolated from roots of cucumber plants grown under different nitrate supply.

<p>A–B. The rate of MgATP-dependent, bafilomycin-sensitive proton transport in tonoplast under constant (squares, grey bars) or temporary (triangles, dark bars) NO₃⁻ supply and under N-deprivation (circles, white bars). Figure A is representative for the results obtained in six independent experiments. Figure B presents the average values ± SD of four independent experiments. Asterisks indicate a significant difference (<i>P</i><0.05) between transport activity of V-ATPase under different nitrate supply. C. NO₃⁻-sensitive hydrolytic activity in membranes obtained from NO₃⁻-grown (light grey bars), NO₃⁻-induced (dark grey bars) and N-deprived (white bars) plants. Data are the average values ± SD of three independent experiments. Asterisks indicate a significant difference (<i>P</i><0.05) between V-ATPase activity under different nitrate supply. D. A representative Coomassie blue-stained gel of total tonoplast protein isolated from plants grown under different nitrate regime. 15 µg of tonoplast proteins were separated by SDS-PAGE on a 10% linear acrylamide gel. The positions of PAGE molecular mass markers are shown in kilodaltons on the right of the gel image. E. Immunoblot of V-ATPase subunit a on tonoplast membranes obtained from plants grown under different nitrate regime. Presented picture is representative for the results obtained in three to four independent experiments.</p

The activity of proton-coupled nitrate transport in the tonoplast membranes isolated from cucumbers grown under different nitrate supply.

<p>50 µg of tonoplast membrane protein was incubated with 20 mM Tris-Mes 0,25 M sucrose, 1 mM DTT and acridine orange until a stable baseline was reached (3–5 min). The increase in acridine orange absorbance in membranes isolated from N-deprived (A), NO₃⁻-grown (B) and NO₃⁻-induced (C) plants was initiated by the addition of different concentrations of KNO₃ into the reaction media (indicated by the arrows) and monitored during the following 3 min. Presented values are representative for the results obtained in three to four independent experiments with each experiment done in triplicate. D. The rate of nitrate accumulation in tonoplast vesicles isolated from N-deprived (white bars), NO₃⁻-grown (light grey bars) and NO₃⁻-induced (dark bars) plants determined by HPLC. The reaction mixture for HPLC assay was deprived of pH-sensitive probe. Values are the means ±SE (<i>n</i> = 5–6 measurements from 4–6 independent tonoplast preparations). Asterisks indicate a significant difference (<i>P</i><0.05) between proton-coupled nitrate transport activities in tonoplast membranes isolated from different plants.</p

The effect of soluble fractions (A) and soluble fraction isolated from NO₃

<p>⁻-<b>induced roots, protein kinase and phosphatase inhibitors or EGTA (B) on proton-coupled nitrate transport in tonoplast membranes obtained from NO₃</b>⁻-<b>grown plant roots.</b> Cytosolic soluble fraction (supernatant 120 000 g, 50 µl) alone or with other compounds was added to the reaction media containing 0.25 M sucrose, 1 mM DTT, 10 µM acridine orange and tonoplast membranes (50 µg of protein). After 5-min long incubation, 10 mM KNO₃ was introduced into the membranes to initiate proton efflux from the vesicles observed as the acridine orange absorbance increase. At first, the rate of H⁺-coupled nitrate antiport was measured in the presence of KNO₃ (light grey bars) and cytosolic fractions isolated from NO₃⁻-grown (dark grey bars), NO₃⁻-induced (dark bars) or N-deprived (white bars) plants (A). In further experiments, H⁺/NO₃⁻ activity was also determined in the presence of KNO₃, cytosolic fraction obtained from NO₃⁻-induced plants and phosphatase inhibitors (black bars) or kinase inhibitor (white bars) or EGTA (striped bars) (B). Protein kinase inhibitor, staurosporine and phosphatase inhibitors, okadaic acid (OA) and cantharidin were used at 5 µM and 2 µM concentration, respectively, whereas EGTA was applied to the media at final 5 mM concentration. In control assays, equal amounts of water or DMSO was used instead of cytosolic fraction/EGTA or inhibitors, respectively (light grey bars). Values are the means ±SE (<i>n</i> = 5–6 measurements from 4–6 independent tonoplast preparations). Asterisks indicate a significant difference (<i>P</i><0.05) between H⁺-coupled NO₃⁻ transport in tonoplast isolated from different plants.</p

CsCLCs expression in roots of cucumber grown under different nitrate supply.

<p>The level of <i>CsCLCa</i>, <i>CsCLc</i> and <i>CsCLCg</i> transcript was analyzed by Real-time PCR (A) and PCR (B) using reversed transcribed total RNA isolated from plant roots. For the real-time PCR assay, total RNA was prepared from NO₃⁻-grown (grey bars), NO₃⁻-induced (dark bars) and N-deprived (white bars) plants. The cDNA synthesized from RNA isolated from NO₃⁻-grown plants was also used for the PCR assay (B). The gene encoding clathrin adaptor complex subunit (CACS) was used as internal control. Values are the means ±SE (<i>n</i> = 5–6 measurements from 3 independent experiments). Asterisks indicate a significant difference (<i>P</i><0.05) between the expression of <i>CsCLCs</i> under different availability of nitrate in nutrient solution (A). Presented picture (B) is representative for the results obtained in three to four independent experiments.</p

NtZIP11, a new Zn transporter specifically upregulated in tobacco leaves by toxic Zn level

Understanding the molecular mechanisms governing the uptake and accumulation of Zn in the leaves of tobacco plants exposed to high Zn concentrations is important from the perspective of phytoremediation of metal contaminated soil. This study identifies a new candidate gene, NtZIP11, which may contribute to Zn accumulation in tobacco leaves. NtZIP11 encodes a protein of 346 amino acids, with characteristic conserved sequences of the ZIP family of proteins. Phylogenetic analysis shows that NtZIP11 forms a distinct clade with other ZIP11 proteins. Transient expression of GFP-tagged ZIP11 in the abaxial epidermis of tobacco leaves demonstrates localization at the plasma membrane. Yeast complementation tests and growth assays indicate that NtZIP11 is involved in Zn but not Fe, Mn and Cd uptake. NtZIP11 complements the zrt1zrt2 mutant deficient in Zn uptake, but not the fet3fet4 mutant deficient in Fe uptake. Nor does it modify the sensitivity of wild-type yeast, DY1457, to increasing concentrations of Fe, Mn and Cd. NtZIP11 is expressed in both roots and leaves, with transcript abundance increasing with age. Noteworthy, the expression level of NtZIP11 is not modulated by Zn-deficiency but is highly upregulated especially in the older leaves by high Zn concentrations (50 and 200 µM). Our data indicate that the primary role for NtZIP11 is in the uptake of Zn by the cells of tobacco leaves specifically when experiencing high Zn concentrations. It also likely contributes to maintaining a basal supply of Zn to cells at the level of the whole plant. Thus the present study contributes to broadening our understanding of Zn homeostasis mechanisms in tobacco, and clarifying the role of ZIP11 genes