Calcium dynamics during trap closure visualized in transgenic Venus flytrap

Abstract

The leaves of the carnivorous plant Venus flytrap, Dionaea muscipula (Dionaea) close rapidly to capture insect prey. The closure response usually requires two successive mechanical stimuli to sensory hairs on the leaf blade within approximately 30 s (refs. 1,2,3,4). An unknown biological system in Dionaea is thought to memorize the first stimulus and transduce the signal from the sensory hair to the leaf blade2. Here, we link signal memory to calcium dynamics using transgenic Dionaea expressing a Ca2+ sensor. Stimulation of a sensory hair caused an increase in cytosolic Ca2+ concentration ([Ca2+]cyt) starting in the sensory hair and spreading to the leaf blade. A second stimulus increased [Ca2+]cyt to an even higher level, meeting a threshold that is correlated to the leaf blade closure. Because [Ca2+]cyt gradually decreased after the first stimulus, the [Ca2+]cyt increase induced by the second stimulus was insufficient to meet the putative threshold for movement after about 30 s. The Ca2+ wave triggered by mechanical stimulation moved an order of magnitude faster than that induced by wounding in petioles of Arabidopsis thaliana5 and Dionaea. The capacity for rapid movement has evolved repeatedly in flowering plants. This study opens a path to investigate the role of Ca2+ in plant movement mechanisms and their evolution.

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Fig. 1: Cold stimulation induces a [Ca2+]cyt increase in a Dionaea trap leaf.
Fig. 2: A [Ca2+]cyt increase propagates from the sensory hair to the leaf blade after mechanical stimulation.
Fig. 3: Quantitative analysis of temporal fluorescence changes after mechanical stimulation and the putative [Ca2+]cyt threshold at which movement is triggered.

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request. Sequence data for genes and plasmids can be found in GenBank under the following accession numbers: sGFP (ABL09837), pSB1 (AB027255) and pSB11 (AB027256). Sequence of GCaMP6f can be found in Addgene data libraries as catalogue no. 40755. Source data are provided with this paper.

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Acknowledgements

We thank the Model Plant Research Facility of National Institute for Basic Biology for plant cultivation, the Japan Tobacco Plant Innovation Center for providing pSB11 and LBA4404 harbouring pSB1, National BioResource Project for supplying Lotus japonicus POLYUBIQUITIN promoter through Frontier Science Research Center of the University of Miyazaki, T. Murata for instructions of microscopy analyses, H. Narukawa for instructions of vibratome sections and Y. Matsuzaki for technical assistance. This work was supported by the JSPS KAKENHI (grant nos. 17J08569 to H.S., 17H05007 and 18H05491 to M.T., 18H04790 to Y.T. and 16K14761 and 17H06390 to M.H.) and DFG-funded Reinhart Koselleck project (HE 1640/42-1 to R.H.).

Author information

Affiliations

Authors

Contributions

H.S., H.M., M.T., Y.T. and M.H. conceived and designed the research. K.F. established the aseptic culture. M.T. provided the GCaMP6f vector. H.S. and H.M. performed the experiments with advice from M.T., Y.T. and M.H. I.T. and T.M. suggested the experimental design. R.H. provided electrophysiological information on unpublished data. H.S. prepared the figures. H.S., Y.T. and M.H. wrote the manuscript with contributions from all authors.

Corresponding author

Correspondence to Mitsuyasu Hasebe.

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Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature Plants thanks Edward E. Farmer, Andrej Pavlovic and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 GCaMP6f signals in glands by a series of mechanical stimuli.

A bright-field image (upper leftmost) and fluorescence images (others) at the leaf blade. A pictured area of the fluorescence images corresponds to the square area at the proximal side of a leaf blade in the bright-field image. Sensory hairs are indicated by light blue arrow heads. The most distal sensory hair (left blue arrow-head) was stimulated by a needle. Pseudocolour images were extracted from those shown in Supplementary Video 2. Seconds (s) after the first stimulus are indicated. Non-pigmented glands are indicated by white arrows. We could not detect the increase of GCaMP6f signals in pigmented digestive glands by any of the three stimuli, although a previous study28 with FURA-2 calcium indicator showed that the increase of FURA-2 signal was not detected by the first and second mechanical stimuli but was by the third stimulus. Scale bar, 1 mm.

Extended Data Fig. 2 The [Ca2+]cyt increase induced by a mechanical stimulation propagates from the one lobe of a blade to the other.

Fluorescence images of a GCaMP6f Dionaea leaf, after a sensory hair was subjected to the first mechanical stimulus with a needle (yellow arrow). Frames are extracted in pseudocolour from those shown in Supplementary Video 3. Seconds (s) after the first stimulus is indicated. Maximum intensity was measured at the whole pictured area. Scale bar, 1 mm.

Extended Data Fig. 3 Propagation velocity of the [Ca2+]cyt increase.

a–c, Propagation velocity of the [Ca2+]cyt increase from a stimulated sensory hair (a and b) or from a wounded tissue (c). Fluorescence were measured after the first stimulus (a) (n = 8, leaves from two biologically independent plants), the second stimulus (b) (n = 3, leaves from two biologically independent plants), and the wounding stimulus by needle at the central area (c) (n = 4, leaves from two biologically independent plants) were applied. The most distal sensory hairs were mechanically stimulated after 5.18, 5.52 and 5.54 s from the first stimuli. Mean values are indicated by reticles. Velocities were measured in four directions from the stimulated site (Figs. 1b and 2f top middle). Two-way analysis of variance without replication with Holm’s sequentially rejective Bonferroni procedure was used to calculate two-tail p-values. d, Propagation velocity of [Ca2+]cyt increase by wounding (n = 5, leaves from two biologically independent plants). The midrib was cut at the proximal end of the leaf blade by scissors. The cut position corresponds to that in the previous study in A. thaliana5. Mean values are indicated by reticles. Velocities were measured in the proximal direction from the stimulated site (Extended Data Fig. 5). Source data

Extended Data Fig. 4 The [Ca2+]cyt increase induced by a wounding stimulus to the leaf blade.

Bright-field image (upper leftmost) and fluorescence images (others) of a GcaMP6f Dionaea leaf wounded by a needle (yellow arrow). Frames were extracted with pseudocolour from those shown in Supplementary Video 4. Seconds (s) after the wounding stimulus is indicated. Scale bar, 2 mm.

Extended Data Fig. 5 [Ca2+]cyt increase induced by a wounding stimulus to the midrib propagates along the midrib.

Bright-field image (upper leftmost) and fluorescence images (others) of a GCaMP6f Dionaea leaf wounded by cutting with scissors (yellow arrow) the midrib at the proximal end of the leaf blade. Frames were extracted with pseudocolour from those shown in Supplementary Video 5. Seconds (s) after the wounding stimulus is indicated. Scale bar, 2 mm.

Extended Data Fig. 6 Leaf areas exhibiting a [Ca2+]cyt increase following the first and second stimulation overlapped.

Bright-field image (a) and the cpEGFP fluorescence changed area at the maximum intensity frame after application of the first (b) and second (c) stimulus. Merged image of (b) and (c) is shown in (d). Maximum intensity was measured across the whole pictured area. White and red lines mark the boundary of the leaf blade and petiole, respectively. Blue lines indicate leaf veins around the lateral boundary of fluorescence. The second stimulus was applied 12.06 s after the first stimulus. Scale bar, 1 mm.

Extended Data Fig. 7 Procedure for the normalization of fluorescence intensity.

a–b, Representative time courses of the average pixel intensities of fluorescence in the lateral (a) and central (b) areas without normalization. First and second stimuli are indicated as “1st” and “2nd”, respectively. After the second stimulus, the leaf closed. The normalized fluorescence intensity Ft was calculated as (ftf0) / (f1f0), where ft, averaged pixel intensity of fluorescence at a given time “t”; f0, averaged ft of 50 frames 1 s before the first stimulus; f1, maximum ft after the first stimulus before the second stimulus.

Extended Data Fig. 8 Temporal fluorescence changes of GCaMP6f Dionaea leaves that did not close by the first and the second mechanical stimuli but closed by the third stimulus.

a–b, Representative time courses of the normalized fluorescence intensity at time t [Ft] in the lateral (a) and the central (b) areas (n = 5, leaves from two biologically independent plants). Time at the max intensity after the first mechanical stimulus (red arrow-head) is set as 0 seconds for each experiment. The second and the third stimuli are indicated by arrow heads and arrows, respectively. The colour of arrow heads in the second and the third stimuli correspond to that of the plots. Time points when leaf movement started are indicated by black dots. Putative thresholds for the movement (red lines) and 95% CIs (red dotted lines) are taken from Table 1.

Extended Data Fig. 9 Temporal fluorescence changes with no mechanical stimuli in a GCaMP6f Dionaea leaf whose petiole base is put in water.

a, b, Representative time courses of the normalized fluorescence intensity at time t [Ft] in the lateral (a) and central (b) areas of a leaf whose petiole base was put in water (n = 4, leaves from two biologically independent plants). Time when fluorescence intensity started to increase without a stimulus is indicated as 0 seconds. Even though leaves were not mechanically stimulated, two or three successive increases of signals were detected. Time points when leaf movement started are indicated by black dots. Putative thresholds for the movement (red lines) and their 95% CIs (red dotted lines) are taken from Table 1.

Extended Data Fig. 10 Temporal fluorescence changes after mechanical stimulation of GCaMP6f Dionaea leaves immersed in the calcium channel blocker LaCl3 solution.

a–b, Representative time courses of the normalized fluorescence intensity at time t [Ft] in the lateral (left) and the central (right) areas 30 minutes after immersion in water (a) (n = 3, leaves from two biologically independent plants) or 20 mM LaCl3 solution (b) (n = 3, leaves from two biologically independent plants). Time at the first mechanical stimulus (red arrow-head) is set as 0 seconds. The second and the third stimuli are indicated by arrow heads and arrows, respectively. The colour of arrow heads in the second and the third stimuli correspond to that of the plots. Time points when leaf movement started are indicated by black dots. Putative thresholds for the movement (red lines) and their 95% CIs (red dotted lines) are taken from Table 1.

Supplementary information

Supplementary Information

Supplementary Figs. 1–8, Table 1, Videos 1–9 and references.

Reporting Summary

Supplementary Video 1

The [Ca2+]cyt increases following mechanical stimulation of a sensory hair of the GCaMP6f-transgenic Dionaea. The video is played at real time speed. Time (seconds, s) after the first mechanical stimulus is indicated. Scale bar, 2 mm.

Supplementary Video 2

[Ca2+]cyt increase at non-pigmented glands but does not at digestive glands following mechanical stimulation of a sensory hair of the GCaMP6f-transgenic Dionaea. The video is played by real time speed. Time (seconds, s) after the first mechanical stimulus is indicated. Scale bar, 1 mm.

Supplementary Video 3

The [Ca2+]cyt increase propagates from one lobe of a blade to the other in GCaMP6f-transgenic Dionaea. The video is played at real time speed. Time (seconds, s) after the first mechanical stimulus is indicated. Scale bar, 2 mm.

Supplementary Video 4

The [Ca2+]cyt increases following wounding at the leaf blade of the GCaMP6f-transgenic Dionaea. The video is played by real time speed. Time (seconds, s) after the wounding stimulus is indicated. Distal and proximal sensory hairs are indicated by white arrows. Scale bar, 2 mm.

Supplementary Video 5

The [Ca2+]cyt increases following wounding at the midrib of the boundary between the leaf blade and petiole of the GCaMP6f-transgenic Dionaea. The video is played at 10 times the speed. Time (seconds, s) after wounding is indicated. Scale bar, 2 mm.

Supplementary Video 6

Leaf movement is triggered by the third stimulus when the leaf does not close by the second stimulus. The video is played at real time speed. Time (seconds, s) after the first mechanical stimulus is indicated. Scale bar, 2 mm.

Supplementary Video 7

[Ca2+]cyt increases at the leaf immersed the basal part of a petiole in water and the leaf closes without mechanical stimulus. The video is played at real time speed. Time (seconds, s) after the first increase of fluorescence intensity is indicated. Scale bar, 2 mm.

Supplementary Video 8

The [Ca2+]cyt increases by successive mechanical stimuli in water immersed leaf as same with untreated leaves. The video is played at real time speed. Time (seconds, s) after the first mechanical stimulus is indicated. Scale bar, 2 mm.

Supplementary Video 9

The [Ca2+]cyt increases by the first stimulus but not additively increases by the second stimulus in LaCl3 solution immersed leaf. The video is played at real time speed. Time (seconds, s) after the first mechanical stimulus is indicated. Scale bar, 2 mm.

Supplementary Data 1

Statistical source data for Supplementary Fig. 4.

Supplementary Data 2

Statistical source data for Supplementary Table 1.

Supplementary Data 3

Statistical source data for Supplementary Fig. 7.

Source data

Source Data Fig. 3

Statistical source data.

Source Data Extended Data Fig. 3

Statistical source data.

Source Data Table 1

Statistical source data.

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Suda, H., Mano, H., Toyota, M. et al. Calcium dynamics during trap closure visualized in transgenic Venus flytrap. Nat. Plants 6, 1219–1224 (2020). https://doi.org/10.1038/s41477-020-00773-1

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