Plant cell division is specifically affected by nitrotyrosine

Abstract

Virtually all eukaryotic alpha-tubulins harbour a C-terminal tyrosine that can be reversibly removed and religated, catalysed by a specific tubulin-tyrosine carboxypeptidase (TTC) and a specific tubulin-tyrosine ligase (TTL), respectively. The biological function of this post-translational modification has remained enigmatic. 3-nitro-L-tyrosine (nitrotyrosine, NO(2)Tyr), can be incorporated into detyrosinated alpha-tubulin instead of tyrosine, producing irreversibly nitrotyrosinated alpha-tubulin. To gain insight into the possible function of detyrosination, the effect of NO(2)Tyr has been assessed in two plant model organisms (rice and tobacco). NO(2)Tyr causes a specific, sensitive, and dose-dependent inhibition of cell division that becomes detectable from 1 h after treatment and which is not observed with non-nitrosylated tyrosine. These effects are most pronounced in cycling tobacco BY-2 cells, where the inhibition of cell division is accompanied by a stimulation of cell length, and a misorientation of cross walls. NO(2)Tyr reduces the abundance of the detyrosinated form of alpha-tubulin whereas the tyrosinated alpha-tubulin is not affected. These findings are discussed with respect to a model where NO(2)Tyr is accepted as substrate by TTL and subsequently blocks TTC activity. The irreversibly tyrosinated alpha-tubulin impairs microtubular functions that are relevant to cell division in general, and cell wall deposition in particular.

Fig. 1.

Schematic diagram of the determination of the cross wall angle ratio in tobacco BY-2 cell files. The uppermost cell file shows a symmetric cross wall with two equal angles α and β; the ratio is 1. The second cell file shows an asymmetric cross wall with α bigger than β; the ratio derives from 1. The third and fourth cell files correspond to the first and second; the difference is the form of the cell file, which is curved instead of straight.

Fig. 2.

Quantification of the effects of NO₂Tyr on 6-day-old rice seedlings (left column, A, C, E, and G) and BY-2 cell culture (right column, B, D, F, and H). (A) Inhibition (percentage of control) of coleoptile (open circles) and root (open diamonds) growth in rice seedlings (n ≥18, error bar=SE). (B) Inhibition of growth (given as a percentage of packed volume) in BY-2 cells grown with NO₂Tyr (open triangles) or NO₂Phe (filled triangles; n ≥4, error bar=SE). (C) Inhibition of mitosis (given as a percentage of control) in rice root tips, incubated for 1 h (open diamonds) or for 6 d with NO₂Tyr (filled diamonds; n=3 independent experiments with 300 cells each, error bar=SE). (D) Inhibition of mitosis (given as a percentage of control) in BY-2 cell culture treated with NO₂Tyr (filled triangles; n=7 independent experiments with 300 cells each, error bar=SE). (E) Comparison of the inhibition of root growth in rice treated with either Tyr (open diamonds) or NO₂Tyr (filled diamonds; n ≥11, error bar=SE). (F) Comparison of the inhibition of growth (given as a percentage of packed volume) in BY-2 cell culture treated either with tyrosine (open triangles) or NO₂Tyr (filled triangles; n=4, error bar=SE). (G) Comparison of the inhibition of mitosis (given as a percentage of control) in rice root tips treated either with tyrosine (open diamonds) or NO₂Tyr (filled diamonds; n=5 independent experiments with 240 cells each, error bar=SE). (H) Comparison of the inhibition of mitosis (given as a percentage of control) in BY-2 cells treated with either tyrosine (open triangles) or NO₂Tyr (filled triangles; n=5 independent experiments with 260 cells each, error bar=SE).

Fig. 3.

Physiological effects of 24 h treatment with NO₂Tyr on 4-day-old BY-2 cells. (A) Increase of cell length, given as a percentage of control (n=3 independent experiments with 500 cells each, error bar=SE). (B) Frequency of correct (ratio 1.0–1.09) and incorrect (ratio ≥1.1) cross walls (n=2 independent experiments with 150 ratios each, error bar=SE).

Fig. 4.

Inhibition of growth (given as a percentage of packed volume of control) of BY-2 cell culture. White columns represent growth inhibition with oryzalin only (from 0 nM to 50 nM); black columns represent growth inhibition with additional 0.1 μM NO₂Tyr (n ≥3, error bar=SE).

Fig. 5.

Distribution of tyr-tub and detyr-tub in samples derived from total soluble protein extract from 4-day-old BY-2 cells, untreated or treated with 10 μM NO₂Tyr for 24 h. The left panel represents the SDS–gel stained with CBB, showing an equal concentration of the applied proteins (left lane protein marker, middle lane untreated cells, right lane cells treated with 10 μM NO₂Tyr). The middle panel shows the reactions of the antibody TUB-1A2 against tyr-tub in the untreated and treated samples. The right panel shows proteins of untreated and treated cells tested with the antibody DM1A against detyr-tub. The upper signal is a cross-reaction with tyr-tub and the lower signal (only abundant in the untreated sample) corresponds to detyr-tub.