Transcription of specific auxin efflux and influx carriers drives auxin homeostasis in tobacco cells

Abstract

Summary Auxin concentration gradients are informative for the transduction of many developmental cues, triggering downstream gene expression and other responses. The generation of auxin gradients depends significantly on cell‐to‐cell auxin transport, which is supported by the activities of auxin efflux and influx carriers. However, at the level of individual plant cell, the co‐ordination of auxin efflux and influx largely remains uncharacterized. We addressed this issue by analyzing the contribution of canonical PIN‐FORMED (PIN) proteins to the carrier‐mediated auxin efflux in Nicotiana tabacum L., cv. Bright Yellow (BY‐2) tobacco cells. We show here that a majority of canonical NtPINs are transcribed in cultured cells and in planta. Cloning of NtPIN genes and their inducible overexpression in tobacco cells uncovered high auxin efflux activity of NtPIN11, accompanied by auxin starvation symptoms. Auxin transport parameters after NtPIN11 overexpression were further assessed using radiolabelled auxin accumulation and mathematical modelling. Unexpectedly, these experiments showed notable stimulation of auxin influx, which was accompanied by enhanced transcript levels of genes for a specific auxin influx carrier and by decreased transcript levels of other genes for auxin efflux carriers. A similar transcriptional response was observed upon removal of auxin from the culture medium, which resulted in decreased auxin efflux. Overall, our results revealed an auxin transport‐based homeostatic mechanism for the maintenance of endogenous auxin levels. Open Research Badges This article has earned an Open Data Badge for making publicly available the digitally‐shareable data necessary to reproduce the reported results. The data is available at http://osf.io/ka97b/

Intrinsic Cell Polarity Coupled to Growth Axis Formation in Tobacco BY-2 Cells

“…One way of addressing these questions is to study the localization of polarity proteins in cell cultures or protoplasts where cell neighbors can be reduced or eliminated. Expression of polarity proteins PIN1 (auxin transporter) and BOR1 (borate transporter) in tobacco BY-2 cell filaments gives signal at cross-walls between cells but no obvious polarity from one end of the cell to the other, except for terminal cells, which have only one cross-wall [12][13][14][15][16][17][18]. In protoplasts, PIN and SOSEKI do not exhibit polarity [1,14,19], and it has been proposed that PIN polarity is maintained though association with domains connected to the cell wall, preventing lateral diffusion [20].…”

“…There is no strong divergence between minor and major axis lengths (N). Protoplast E, polarized BASL appears at 5 o'clock at t = 7 and then progressively changes orientation to 1 o'clock (t = [7][8][9][10][11][12][13][14][15][16][17][18][19]. The orientation of the major axis is relatively fixed.…”

Correlative Light-Environmental Scanning Electron Microscopy of Plasma Membrane Efflux Carriers of Plant Hormone Auxin

“…In our previous work, estradiol-inducible NtPIN3b-GFP have been shown to be one of the main vegetative auxin efflux carriers in tobacco, being present in the PM of tobacco BY-2 cells and performing an auxin-transporting role [30]. The heterogeneity in the distribution of this protein within the PM of protoplasts, which we show here by TIRFM, is in good agreement with several recently published reports on the distribution of Arabidopsis thaliana PIN proteins within PM, including nanodomains of PIN3 in the hypocotyl epidermal cells shown by Airyscan confocal laser scanning microscopy [17] and clusters of PIN2-GFP in the root epidermal cells shown by transmission electron microscopy on immunostained platinum replicas [35].…”

“…Liquid Murashige and Skoog (MS) medium (pH 5.8) containing 3% sucrose, 4.3 g/L MS salts, 100 mg/L myo-inositol, 1 mg/L thiamine, 0.2 mg/L 2,4-dichlorophenoxyacetic acid, and 200 mg/L KH 2 PO 4 (Sigma-Aldrich Inc., St. Louis, MO, USA) was used for wild type cells. BY-2 cells transformed with XVE-NtPIN3b-GFP [30] were cultured in MS medium supplemented with 40 µg/mL hygromycin and 100 µg/mL cefotaxime. To induce NtPIN3b-GFP expression, MS medium was supplemented with 1 µM β-estradiol (10 mM stock solution in DMSO, Sigma-Aldrich Inc., St. Louis, MO, USA) at the beginning of the subculture interval.…”

“…Tobacco BY-2 cells were induced with 1 µM β-estradiol at the beginning of the subculture interval and immuno-stained 48 h after the induction. Only cell populations that showed homogeneous NtPIN3b-GFP fluorescence at the PM [30] immediately before the…”