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M. pudica closes its leaves and points its petiole downwards when touched (Fig. 1a,b). Petiole bending is caused by a rapid shrinking of the lower side of motor organs called the main pulvinus6,7,8 (Fig. 1c,d). Cytochalasin B and phalloidin, which both have inhibitory effects on the actin cytoskeleton, inhibit the bending of the petiole, suggesting that actin is involved in bending9. We found that actin filaments in the motor cells at the lower side of the pulvinus, but not at the upper side, become more peripheral after bending (data not shown), although it is not known what molecular mechanism regulates these changes.

Figure 1: Mimosa pudica L. responding to stimulation by touch.
figure 1

a,c, Plant before touching and b,d, after touching; c,d, shows the main pulvinus (arrowheads), with a petiole (arrow)

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We carried out two-dimensional polyacrylamide gel electrophoresis on total proteins from the whole pulvinus before and after bending, and immunoblotted them with the anti-actin antibody N350 and reprobed with the anti-phosphotyrosine antibody PY20. Densitometry indicated that, before movement, about 80% of actin molecules in the pulvinus were tyrosine-phosphorylated ( Fig. 2a,b). After movement, the total amount of actin did not change, but immunoblots with N350 and PY20 on one-dimensional polyacrylamide gel electrophoresis showed that the amount of tyrosine phosphorylation in actin had dropped (data not shown).

Figure 2: Correlation between the amount of actin tyrosine-phosphorylation and bowing in M. pudica.
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a–f, Total proteins from the whole pulvinus before (a–c ) and after (d–f) petiole bending were immunoblotted with the anti-actin antibody N350 (a,d) or anti-phosphotyrosine antibody PY20 (b,e), or were silver-stained (c,f) after two-dimensional polyacrylamide gel electrophoresis (IEF, isoelectric focusing; SDS, denaturing polyacrylamide gel). Four actin spots were detected (numbered): spots 1–3 are tyrosine-phosphorylated, spot 4 is not. g–j, Phosphorylation of actin from petioles before the plant is touched (g and j, lane 1), and after touching (h,i) in the absence (h and j, lane 2) or presence (i and j, lane 3) of phenylarsine oxide (PAO), a protein-tyrosine phosphatase inhibitor. Dotted lines indicate the original position of the petiole; arrowheads, actin bands.

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Consistent with this decrease in tyrosine phosphorylation, we found that phosphorylated actin in spot 1, the most acidic isoform, disappeared (Fig. 2d–f) — probably because it had been dephosphorylated and shifted to a more basic isoform as a result of bending. In contrast, actin molecules in the stem, which does not move, were heavily tyrosine-phosphorylated, and the level of phosphorylation did not change during movement (data not shown). Changes in the levels of tyrosine phoshorylation of actin in the pulvinus therefore coincide with petiole bending.

In Dictyostelium spores, a decrease in actin phosphorylation is a prerequisite for recovering the dynamic actin cytoskeleton involved in spore germination. A specific inhibitor of protein-tyrosine phosphatases, phenylarsine oxide, inhibits the dephosphorylation of actin and the restoration of cell motility during germination4. We investigated the effects of phenylarsine oxide on M. pudica. When touched, the average bending angle of petioles treated with phenylarsine oxide (Fig. 2g–i ) was smaller (37.3±20.24 degrees; n = 25 ) than that of controls (99.0±4.24 degrees). There was no decrease in the amount of actin phosphorylation following stimulation for the pulvinus exposed to phenylarsine oxide (Fig. 2j).

The bending of M. pudica is therefore correlated with reduced actin phosphorylation in the pulvinus. We propose that actin phosphorylation is essential for petiole bending resulting from actin cytoskeletal alterations. Actin may also be phosphorylated during the opening and closing of guard cells, as actin is dynamically arranged in these cells10.