Auxin protects Arabidopsis thaliana cell suspension cultures from programmed cell death induced by the cellulose biosynthesis inhibitors thaxtomin A and isoxaben

Abstract

Background: Thaxtomin A (TA) is a natural cellulose biosynthesis inhibitor (CBI) synthesized by the potato common scab-causing pathogen Streptomyces scabies. Inhibition of cellulose synthesis by TA compromises cell wall organization and integrity, leading to the induction of an atypical program of cell death (PCD). These processes may facilitate S. scabies entry into plant tissues. To study the mechanisms that regulate the induction of cell death in response to inhibition of cellulose synthesis, we used Arabidopsis thaliana cell suspension cultures treated with two structurally different CBIs, TA and the herbicide isoxaben (IXB).

Results: The induction of cell death by TA and IXB was abrogated following pretreatment with the synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D) and the natural auxin indole-3-acetic acid (IAA). The addition of auxin efflux inhibitors also inhibited the CBI-mediated induction of PCD. This effect may be due to intracellular accumulation of auxin. Auxin has a wide range of effects in plant cells, including a role in the control of cell wall composition and rigidity to facilitate cell elongation. Using Atomic Force Microscopy (AFM)-based force spectroscopy, we found that inhibition of cellulose synthesis by TA and IXB in suspension-cultured cells decreased cell wall stiffness to a level slightly different than that caused by auxin. However, the cell wall stiffness in cells pretreated with auxin prior to CBI treatment was equivalent to that of cells treated with auxin only.

Conclusions: Addition of auxin to Arabidopsis cell suspension cultures prevented the TA- and IXB-mediated induction of cell death. Cell survival was also stimulated by inhibition of polar auxin transport during CBI-treatment. Inhibition of cellulose synthesis perturbed cell wall mechanical properties of Arabidopsis cells. Auxin treatment alone or with CBI also decreased cell wall stiffness, showing that the mechanical properties of the cell wall perturbed by CBIs were not restored by auxin. However, since auxin's effects on the cell wall stiffness apparently overrode those induced by CBIs, we suggest that auxin may limit the impact of CBIs by restoring its own transport and/or by stabilizing the plasma membrane - cell wall - cytoskeleton continuum.

Keywords: Auxin; Calcium; Cell wall; Isoxaben; Programmed cell death; Thaxtomin A.

Fig. 1

Calcium influx is required for the CBI-induction of cell death. Percentage of dead cells in Arabidopsis suspension cells 48 h after the addition of calcium transport inhibitors. a and c Cells were treated with thaxtomin A (TA; 1 μM), isoxaben (IXB; 1 μM) or lanthanum chloride (LaCl₃; 500 μM) alone, or pretreated with LaCl₃ (500 μM) 30 min before adding TA or IXB. b and d Cells were treated with TA (1 μM), IXB (1 μM) or ruthenium red (RR; 50 nM) alone, or pretreated with 50 nM of RR 30 min before adding TA or IXB. Ctrl = control cells treated with equal volume of methanol as treated samples. Each value is the mean of n = 15 (100 cells each) ± SD. Statistically different values (t-test followed by Holm-Šídák method, p < 0.05) are indicated by different letters

Fig. 2

Auxin protects Arabidopsis cells from CBI-induced cell death. Percentage of dead cells in Arabidopsis suspension-cultured cells 48 h after addition of auxin and/or CBI. a Cells were treated with thaxtomin A (TA; 1 μM), 2,4-dichlorophenoxyacetic acid (2,4-D; 50 μM), or 2,4-D 30 min before adding TA. b Cells were treated with isoxaben (IXB; 1 μM), IAA (1 μM), or IAA (1 μM) 30 min before adding IXB. Ctrl = control cells treated with equal volume of methanol as treated samples. Each value is the mean of n = 15 (100 cells each) ± SD. Statistically different values (t-test followed by Holm-Šídák method, p < 0.05) are indicated by different letters

Fig. 3

Inhibition of auxin transport (efflux) protects from CBI-induced cell death. Percentage of dead cells in Arabidopsis cell cultures 72 h after addition of auxin transport inhibitor and/or CBI. a Cell death was evaluated after treatment with thaxtomin A (TA; 1 μM) and in cells pretreated for 30 min with 10 μM of different auxin transport inhibitors: 2,3,5-triiodobenzoic acid (TIBA) and 1-N-naphthylphthalamic acid (NPA) as efflux inhibitors, 2-naphthoxyacetic acid (NOA) and 3-chloro-4-hydroxyphenylacetic acid (CHPAA) as influx inhibitors prior to TA treatment. b Cell death was evaluated after treatment with isoxaben (IXB; 1 μM) and in cells pretreated for 30 min with the different auxin transport inhibitors prior to IXB addition. Ctrl = control cells treated with equal volume of methanol as treated samples. Each value is the mean of n = 15 (100 cells each) ± SD. Statistically different values (t-test followed by Holm-Šídák method, p < 0.05) are indicated by different letters

Fig. 4

CBIs and auxin decrease cell wall stiffness. Atomic force microscopy (AFM) was used to measure cell wall stiffness profiles of Arabidopsis suspension-cultured cells treated with: a methanol (control), b thaxtomin A (TA; 1 μM), c 2,4-dichlorophenoxyacetic acid (2,4-D; 50 μM), d 2,4-D and TA, e isoxaben (IXB; 1 μM), f IAA (1 μM), g IAA and IXB for 24 h. Force curves were recorded at the surface of 3 cells per condition in order to extract Young’s modulus (MPa) values (see Material and Methods). Each histogram bar represents the relative proportion of Young’s modulus values measured on the cell surface in each condition. Because of the low proportion of values measured above 1 MPa, a scale change was used after 1 MPa. h Average median values of Young’s modulus were calculated for each condition from 3 different experiments (3 to 4 cells per experiment). Error bars represent SD