Thaxtomin A affects CESA-complex density, expression of cell wall genes, cell wall composition, and causes ectopic lignification in Arabidopsis thaliana seedlings

Abstract

Thaxtomin A, a phytotoxin produced by Streptomyces eubacteria, is suspected to act as a natural cellulose synthesis inhibitor. This view is confirmed by the results obtained from new chemical, molecular, and microscopic analyses of Arabidopsis thaliana seedlings treated with thaxtomin A. Cell wall analysis shows that thaxtomin A reduces crystalline cellulose, and increases pectins and hemicellulose in the cell wall. Treatment with thaxtomin A also changes the expression of genes involved in primary and secondary cellulose synthesis as well as genes associated with pectin metabolism and cell wall remodelling, in a manner nearly identical to isoxaben. In addition, it induces the expression of several defence-related genes and leads to callose deposition. Defects in cellulose synthesis cause ectopic lignification phenotypes in A. thaliana, and it is shown that lignification is also triggered by thaxtomin A, although in a pattern different from isoxaben. Spinning disc confocal microscopy further reveals that thaxtomin A depletes cellulose synthase complexes from the plasma membrane and results in the accumulation of these particles in a small microtubule-associated compartment. The results provide new and clear evidence for thaxtomin A having a strong impact on cellulose synthesis, thus suggesting that this is its primary mode of action.

Fig. 1.

Thaxtomin A alters ¹⁴C incorporation in cell wall fractions. ¹⁴C incorporation in 6-d-old, liquid culture-grown, etiolated Arabidopsis seedlings treated with 200 nM thaxtomin A for 2 d (black bars) and control seedlings (white bars) is shown for different cell wall fractions as follows: (i) chloroform fraction, (ii) ammonium-oxalate fraction, (iii) 0.1 M KOH fraction, (iv) 4 M KOH fraction, (v) acetic/nitric acid soluble fraction, and (vi) acid insoluble fraction. ¹⁴C incorporation in each fraction is expressed as a percentage of total ¹⁴C uptake. Each bar represents the mean ±SE from four biological replicates.

Fig. 2.

Changes in gene expression in thaxtomin A and isoxaben-treated seedlings. (A) Cell wall genes, (B) genes involved in lignin synthesis, and (C) genes involved in defence responses. The expression levels are given expressed as ΔΔC_T (i.e. a logarithmic scale), where ΔΔC_T is the difference in normalized qRT-PCR threshold cycle number of the respective gene between thaxtomin- or isoxaben-treated seedlings and control seedlings. Negative and positive numbers thus represent repression and induction in thaxtomin-treated seedlings, respectively. Normalization was preformed using the UBQ10 (At4g05320) as a reference. Data for thaxtomin A-treated seedlings are shown as grey bars, data for seedlings treated with isoxaben as slightly displaced white bars at the back. Each value represents the mean of four biological replicas from two independent experiments. Standard errors of C_T values are small and not shown.

Fig. 3.

Thaxtomin A causes ectopic lignification in etiolated Arabidopsis seedlings. Lignification was analysed in 6-d-old, liquid culture-grown, etiolated Arabidopsis seedlings treated with 200 nM thaxtomin A for 2 d (A, D), methanol-treated control seedlings (B, E) or seedlings treated with 5 nM isoxaben (C, F). Typical staining patterns are shown. Insets in (A) and (F) show magnifications of strongly lignified regions.

Fig. 4.

Thaxtomin A affects CESA-C abundance and motility. (A, B) Optical sections of plasma membrane in upper hypocotyl cells of 3-d-old etiolated cesa3^je5:GFP-CESA3 seedlings. Images were acquired by spinning disc confocal microscopy. Average projections of 39 and 54 frames acquired in 15 s intervals in the plane of the cell membrane of control (A) and thaxtomin A-treated seedlings (B), respectively are shown. Average projections illustrate the movement of labelled particles along linear tracks. (C) Kymograph of the region marked by the dotted line in (A) displaying steady and bidirectional particle translocation. (D) Kymograph of region marked by the dotted line in (B) displaying steady, slow, accelerated or very fast particle translocation. (E) Box plot diagram of particle velocities calculated from 140 or 300 particles of control or thaxtomin A-treated seedlings, respectively. Particle velocity was measured in six cells from four seedlings and was determined by manual tracking.