Traps of <i>Dionaea muscipula</i> in response to mechanical stimulation.

<p>An open trap (A), the same trap 1 min after double mechanical stimulation of trigger hairs (B) and 3 hours later after repeated mechanical stimulation in narrowed phase (C).</p

Abundance of Cysteine Endopeptidase Dionain in Digestive Fluid of Venus Flytrap (<i>Dionaea muscipula</i> Ellis) Is Regulated by Different Stimuli from Prey through Jasmonates

<div><p>The trap of the carnivorous plant Venus flytrap (<i>Dionaea muscipula</i>) catches prey by very rapid closure of its modified leaves. After the rapid closure secures the prey, repeated mechanical stimulation of trigger hairs by struggling prey and the generation of action potentials (APs) result in secretion of digestive fluid. Once the prey's movement stops, the secretion is maintained by chemical stimuli released from digested prey. We investigated the effect of mechanical and chemical stimulation (NH₄Cl, KH₂PO₄, further N(Cl) and P(K) stimulation) on enzyme activities in digestive fluid. Activities of β-D-glucosidases and N-acetyl-β-D-glucosaminidases were not detected. Acid phosphatase activity was higher in N(Cl) stimulated traps while proteolytic activity was higher in both chemically induced traps in comparison to mechanical stimulation. This is in accordance with higher abundance of recently described enzyme cysteine endopeptidase dionain in digestive fluid of chemically induced traps. Mechanical stimulation induced high levels of <i>cis</i>-12-oxophytodienoic acid (<i>cis</i>-OPDA) but jasmonic acid (JA) and its isoleucine conjugate (JA-Ile) accumulated to higher level after chemical stimulation. The concentration of indole-3-acetic acid (IAA), salicylic acid (SA) and abscisic acid (ABA) did not change significantly. The external application of JA bypassed the mechanical and chemical stimulation and induced a high abundance of dionain and proteolytic activity in digestive fluid. These results document the role of jasmonates in regulation of proteolytic activity in response to different stimuli from captured prey. The double trigger mechanism in protein digestion is proposed.</p></div

Double trigger mechanism for protein digestion.

<p>Three control points in prey capture and digestion of Venus flytrap, which ensure effective production of digestive enzymes. For detailed description, see discussion. AP – action potential, the increase of phytohormone level is indicated by arrows: no increase (-), small (↑), moderate (↑↑), high (↑↑↑).</p

Narrowing response after different treatments.

<p>As a control wet (distilled H₂O) and dry filter papers were applied. M – mechanical stimulation, Different letters denote significant differences at <i>P</i> < 0.05 (ANOVA, Tukey-test), means ± s.e., n  =  5–9.</p

Electrical activity measured by extracellular electrode on trap surface.

<p>Mechanical irritation delivered in 10 minutes intervals (A), detailed view on action potentials (B) and chemical stimulation with NH₄Cl and KH₂PO₄ did not trigger any AP (C).</p

Enzymatic activities in response to external JA application.

<p>Acid phosphatase activities (A), proteolytic activities (B) and activities measured after inhibition with 10 µM cysteine proteinase inhibitor E-64. Activity without inhibitor of given variant is 100 % (C). ** - significant differences at <i>P</i>  =  0.01 (Student's t-test), means ± s.e., n  =  6–7.</p

Endogenous concentration of phytohormones (JA, JA-Ile, IAA, ABA, <i>cis</i>-OPDA and SA) in trap tissue 36 - 48 hours after stimulation.

<p>Note different scale on y-axis for <i>cis</i>-OPDA and SA. Control (C), mechanical (M), P(K) and N(Cl) stimulation. Different letters denote significant differences at <i>P</i> < 0.05 of particular phytohormone among treatments (ANOVA, Tukey test), means ± s.e., n  =  9–10.</p

Enzymatic activities in response to mechanical and chemical stimulation after 24 (black bars) and 48 hours (white bars).

<p>Acid phosphatase activity (A) and proteolytic activity (B). Different letters denote significant differences at the same time interval at <i>P</i> < 0.05 (ANOVA, Tukey-test), means ± s.e., n  =  7–9.</p

Protein pattern, Western blot and zymogram in response to external application of jasmonic acid.

<p>Silver-stained SDS-polyacrylamide gel containing proteins in <i>D. muscipula</i> digestive fluid after mechanical stimulation (M) and external application of jasmonic acid (JA), (A). The same amount of proteins was electrophoresed in 12% (v/v) SDS-polyacrylamide gel and subjected to Western blot analysis using antibodies against dionain-1 (B). Detection of protease activity in 12% (v/v) SDS-polyacrylamide gel with casein as a substrate (C). The clear bands against background indicate protease activity. Representative gels at least from 4 repetitions are shown.</p

Correlation between endogenous phytohormone level and proteolytic activity.

<p>Correlation between endogenous concentration of JA (A), JA-Ile (B), IAA (C), ABA (D), <i>cis</i>-OPDA (E), SA (F) and proteolytic activity induced by mechanical and chemical stimulation. The linear relationship is significant only for JA (<i>P</i>  =  0.048, significance test for linear regression), means ± s.e.</p