Overexpression of the auxin binding PROTEIN1 modulates PIN-dependent auxin transport in tobacco cells

Background: Auxin binding protein 1 (ABP1) is a putative auxin receptor and its function is indispensable for plant growth and development. ABP1 has been shown to be involved in auxin-dependent regulation of cell division and expansion, in plasma-membrane-related processes such as changes in transmembrane potential, and in the regulation of clathrin-dependent endocytosis. However, the ABP1-regulated downstream pathway remains elusive. Methodology/Principal Findings: Using auxin transport assays and quantitative analysis of cellular morphology we show that ABP1 regulates auxin efflux from tobacco BY-2 cells. The overexpression of ABP1can counterbalance increased auxin efflux and auxin starvation phenotypes caused by the overexpression of PIN auxin efflux carrier. Relevant mechanism involves the ABP1-controlled vesicle trafficking processes, including positive regulation of endocytosis of PIN auxin efflux carriers, as indicated by fluorescence recovery after photobleaching (FRAP) and pharmacological manipulations. Conclusions/Significance: The findings indicate the involvement of ABP1 in control of rate of auxin transport across plasma membrane emphasizing the role of ABP1 in regulation of PIN activity at the plasma membrane, and highlighting the relevance of ABP1 for the formation of developmentally important, PIN-dependent auxin gradients

AUXIN BINDING PROTEIN 1 (ABP1) and its role in the auxin management in plant cells

Conclusions The role of AUXIN BINDING PROTEIN 1 (ABP1) in the auxin management in plant cells was followed using simplified model material of suspension-cultured cells of tobacco BY-2 line. ABP1 is a putative auxin receptor considered to mediate fast non-genomic responses to auxin and it can be involved in every aspect of the regulation of auxin responses, metabolism and transport. There are four major conclusions that could be made based on the results presented in this thesis: 1) Auxin binding protein 1 mediates both cell division and expansion in tobacco BY-2 cells. In standard cultivation conditions or at lower concentrations of 2,4-D in culture medium, ABP1 overexpression had no detectable impact on cell division, cell elongation or cell growth.. 5- times increased 2,4-D concentration stimulated weakly cell elongation. . Antisense suppression of ABP1 expression resulted in disturbance in both cell expansion and cell division intensity, suggesting that ABP1 is essential for the control of balance between cell division and cell elongation during the growth cycle. ABP1 is localized in endoplasmic reticulum of cells cultivated in standard medium supplemented with 1 μM 2,4-D and it appeared also at the plasma membrane following the IAA application. 2) ABP1 mediates intercellular auxin transport. Cells..

AUXIN BINDING PROTEIN 1 (ABP1) a jeho role v regulaci hladin auxinu v rostlinných buňkách

Závěry Úloha AUXIN BINDING PROTEIN 1 (ABP1) v hospodaření rostlinných buněk s auxinem byla studována s využitím zjednodušeného modelového materiálu suspenzní kultury tabáku BY-2. ABP1 je předpokládaným receptorem pro auxin, který zprostředkovává rychlé odpovědi k auxinu nezávislé na expresi genů, a může být součástí veškerých procesů souvisejících s regulací odpovědí na auxin, jeho metabolismu a transportu. Z výsledků uvedených v této práci plynou čtyři hlavní závěry: 1) Auxin binding protein 1 je nezbytný pro dělení i růst buněk suspenzní kultury BY-2. Za běžných kultivačních podmínek ani za podmínek sníženého obsahu 2,4-D v kultivačním médiu nemá zesílení expresse ABP1 v buňkách BY-2 žádný měřitelný vliv na dělení buněk nebo na jejich prodlužování. Pěstování buněk se zesílenou expresí ABP1 v médiu s pětinásobně zvýšenou koncentrací 2,4-D vedlo k mírnému zvýšení buněčné elongace. Buňky s potlačenou expresí ABP1 nejsou schopné prodlužování a intenzita buněčného dělení je výrazně zpomalena. ABP1 je tedy důležitý pro řízení buněčného cyklu a je nezbytný pro kontrolu rovnováhy mezi buněčným dělením a buněčnou expanzí. ABP1 je lokalizován v endoplazmatickém retikulu u buněk kultivovaných ve standardním médiu obsahujícím 2,4_D, ale pokud byly buňky vystaveny IAA namísto 2,4-D, ABP1 protein se objevil i na...Conclusions The role of AUXIN BINDING PROTEIN 1 (ABP1) in the auxin management in plant cells was followed using simplified model material of suspension-cultured cells of tobacco BY-2 line. ABP1 is a putative auxin receptor considered to mediate fast non-genomic responses to auxin and it can be involved in every aspect of the regulation of auxin responses, metabolism and transport. There are four major conclusions that could be made based on the results presented in this thesis: 1) Auxin binding protein 1 mediates both cell division and expansion in tobacco BY-2 cells. In standard cultivation conditions or at lower concentrations of 2,4-D in culture medium, ABP1 overexpression had no detectable impact on cell division, cell elongation or cell growth.. 5- times increased 2,4-D concentration stimulated weakly cell elongation. . Antisense suppression of ABP1 expression resulted in disturbance in both cell expansion and cell division intensity, suggesting that ABP1 is essential for the control of balance between cell division and cell elongation during the growth cycle. ABP1 is localized in endoplasmic reticulum of cells cultivated in standard medium supplemented with 1 μM 2,4-D and it appeared also at the plasma membrane following the IAA application. 2) ABP1 mediates intercellular auxin transport. Cells...Katedra experimentální biologie rostlinDepartment of Experimental Plant BiologyPřírodovědecká fakultaFaculty of Scienc

ABP1 inhibits fluorescence recovery after photobleaching (FRAP) of PIN1-GFP.

<p>(A–D) FRAP in the three-day-old tobacco PIN1-GFP/GVG-<i>At</i>ABP1 and PIN1-GFP cells. (A) Transversal plasma membranes decorated by PIN1-GFP in the PIN1-GFP/GVG-<i>At</i>ABP1 cells showing the situation before, immediately after and 170s after the photobleaching. Scale bars, 10 µm. (B) FRAP measured in the PIN1-GFP control cells 170 s after photobleaching. Cells were treated with DEX (1µM) in DMSO or DMSO only. Error bars, SEM, n=15. (C) Kinetics of FRAP in non-induced and induced PIN1-GFP/GVG-<i>At</i>ABP1 cells. Error bars, SEM, n=6. (D) Comparison of FRAP after 170 s in cells pre-treated with BFA (20 µM for 30 min), tyrphostin A23 (Tyr A23, 50 µM for 30 min), NAA (5 µM for 60 min), or NPA (10 µM for 25 min). Error bars, SD, Ctrl, n=6; BFA, n=6; Tyr A23, n=6; NAA, n=10; NPA, n=7. FRAP for the PIN1-GFP/GVG-<i>At</i>ABP1 non-induced cells, 100%. Asterisks indicate significant difference between <i>At</i>ABP1 non-expressing (non-induced) and expressing (induced) cells within given treatment. *P < 0.001, **P = 0.080 using independent samples t-test. The differences for Tyr A23 (P = 0.800), NAA (P = 0.412), and NPA (P = 0.332) treatments are not statistically significant. (E) Schematic depiction of a dual action of ABP1 in regulation of PIN dynamics and activity, resulting in control of auxin levels in a cell. In brackets, experimental intervention is presented. Under low intracellular auxin level, e.g. after overexpression of PIN efflux carriers, ABP1 promotes PIN endocytosis to reduce undesirable auxin export. Under high auxin level, e.g. after external addition of auxin or after inhibition of the active auxin efflux by NPA, ABP1 counteracts the endocytosis of PINs and leaves them on the PM thus promoting the active auxin efflux.</p

ABP1 prevents tobacco cells from PIN-dependent auxin starvation phenotype, and from excessive auxin efflux.

<p>(A–D) Morphology of three-day-old GVG-PIN7 and GVG-PIN7/<i>Nt</i>ABP1. (A,B) Nomarski DIC images. Scale bars, 20 µm. (C, D) Cell length of non-induced and induced cells. 100%, value for non-induced cells. Error bars, SEM, n~250. Asterisk indicates significance using two sample t-test assuming unequal variances; (C) *P < 0.001, df = 347; (D) the difference is not statistically significant, P = 0.736, df = 444. (E) Categorized cell-length distribution and (F,G) growth curves of non-induced and induced GVG-PIN7 and GVG-PIN7/<i>Nt</i>ABP1 cells. Error bars, SEM, n=10 in E and n = 4 in F, G. (H,I) Accumulation of [³H] NAA as an indicator of the auxin efflux, measured 25 min after the addition of [³H] NAA to one-day-old cells treated with NPA (10 µM, applied immediately after the addition of [³H] NAA, 2 nM). Data are shown as differences in [³H] NAA accumulation between induced and non-induced cells (zero level = non-induced/non-treated line). (H) GVG-PIN7 and GVG-PIN7/<i>Nt</i>ABP1 cells. Error bars, SEM, n=3. Asterisks indicate significantly different means between cells expressing only the endogenous Nt<i>ABP1</i>(GVG-PIN7) and over-expressing the Nt<i>ABP1</i> gene (GVG-PIN7/<i>Nt</i>ABP1). Paired samples t-test, *P < 0.001, **P = 0.006. (I) PIN1-GFP/GVG-<i>At</i>ABP1 cells. Error bars, SEM, n=2. The asterisk indicates significant difference between induced and non-induced NPA-treated cells, P = 0.013, paired samples t-test. There was no significant difference between cell lines without NPA treatment. (J, K) qRT-PCR of Nt<i>ABP1</i> and At<i>PIN7</i> genes in the GVG-PIN7/<i>Nt</i>ABP1 cell line (J), and <i>AtABP1</i> and <i>PIN1-GFP</i> genes in PIN1-GFP/GVG-<i>At</i>ABP1 cell line (K). Expression verified at 24 hours after application of 1 µM DEX. Error bars, SEM, n=6. Inset in (K) shows PIN1-GFP fluorescence in both non-induced and induced cells.</p

The effects of 35S-driven expression of <i>Nt</i>ABP1 in 35S-<i>Nt</i>ABP1 tobacco BY-2 cells, and treatment with the inhibitor of auxin efflux NPA.

<p>(A–E) Morphology of three-day-old non-induced and induced cells, control and NPA-treated (10µM for three days). (A,B,D,E) Nomarski DIC images. Scale bars, 20 µm. (C) Cell length of non-treated and NPA-treated (10 µM for three days) cells. 100%, value for non-treated BY-2 cells. Error bars, SEM, n~300. Asterisks indicate significantly different means between cells non-expressing and expressing 35S-driven Nt<i>ABP1</i> gene. Two sample t-test assuming unequal variances; *P < 0.001, df = 465; **P < 0.001, df = 320. (F) Growth curves for BY-2 and 35S-<i>Nt</i>ABP1cells. Error bars, SEM, n=4. (G) Accumulation of [³H] NAA as an indicator of the auxin efflux. Difference in [³H] NAA accumulation between cells non-treated and treated with NPA is shown for one-day-old BY-2 (control) and 35S-<i>Nt</i>ABP1 cells. Radioactivity was measured 25 min after addition of radioactively labelled auxin (2 nM) without or together with NPA (10 µM). Error bars, SEM, n=3. The asterisk denotes statistical significance of difference (P = 0.018 in paired samples t-test). (H,I) Relative expression of Nt<i>ABP1</i> gene in control BY-2 and 35S-<i>Nt</i>ABP1 cell lines. (H) qRT-PCR from cDNA 24 hours after inoculation of cells into the fresh medium. Error bars, SEM, n=6. (I) PCR of Nt<i>ABP1</i> using genomic DNA (702bp fragment of endogenous gene, 256bp fragment of transgenic cDNA).</p

The effects of inducible expression of <i>At</i>ABP1 in GVG-<i>At</i>ABP1 tobacco BY-2 cells, and treatment with the inhibitor of auxin efflux NPA.

<p>(A–E) Morphology of three-day-old non-induced and induced cells, control and NPA-treated (10µM for three days). (A,B,D,E) Nomarski DIC images. Scale bars, 40 µm. (C) Cell length of non-treated (Ctrl) and NPA-treated (10 µM for three days) cells. 100%, value for non-induced control. Error bars, SEM, n~300. Asterisks indicate significantly different means between cells non-expressing and expressing the At<i>ABP1</i> gene, two sample t-test assuming unequal variances; *P < 0.005, degrees of freedom (df) = 581; **P < 0.001, df = 573). (F) Growth curves for non-induced and induced cells, non-treated and treated with NPA (10 µM for three days). Error bars, SEM, n=4. (G) Accumulation of [³H] NAA as an indicator of the auxin efflux. One-day-old GVG-<i>At</i>ABP1 cells were treated with [³H] NAA (2 nM) alone (Ctrl) or in combination with NPA (10 µM), and radioactivity was measured after 25 min. Data values are percentages of non-induced, non-treated control (100%). Error bars, SEM, n=3. The differences in [³H] NAA accumulation between non-induced and induced GVG-<i>At</i>ABP1 cells either without or after NPA application are not statistically significant (P = 0.707 and P = 0.328, respectively, paired samples t-test). (H) Relative expression of the At<i>ABP1</i> gene in the GVG-<i>At</i>ABP1 cell line. qRT-PCR at 24 hours after induction with dexamethasone (DEX, 1 µM). Error bars, SEM, n=6. (I) Accumulation of [³H] NAA in BY-2 cells transformed with an empty pTA7002 vector, measured 25 min after the addition of [³H] NAA (2 nM). Data are expressed as percentages of non-treated control (100%), and represent the mean of four technical repetitions. Error bars, SEM, n=4.</p