# Actin dynamics contribute to stem straightening responses

Khan Kinza, Jérôme Franchel, Félix Hartmann, Nicole Brunel-Michac, Mélanie Decourteix, Valérie Legué, Jason W Reed, Bruno Moulia, Nathalie Leblanc-Fournier

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## To cite this version:

Khan Kinza, Jérôme Franchel, Félix Hartmann, Nicole Brunel-Michac, Mélanie Decourteix, et al. Actin dynamics contribute to stem straightening responses. Plant Molecular Biology: Plant Communication: Understanding Signal Creation, Transmission, and Perception in Plant Systems, Jun 2024, Holderness, United States. **hal-04877476**

HAL Id: hal-04877476

https://hal.science/hal-04877476v1

Submitted on 9 Jan 2025

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Distributed under a Creative Commons Attribution 4.0 International License
Proprioception defines the ability of a plant organ to sense its local curvature, and drives the progressive establishment of organ straightening (Bastien et al., 2013; Moulia et al., 2019). Combined with gravisensing and light perception, proprioception regulates the dynamics of growth of the aerial organs in order to achieve correct final shape and posture (Figure 1). The specific molecular mechanisms governing this process remain elusive. Under simulated microgravity conditions using a rotating clinostat, Okamoto et al. (2015) showed that myosins XI-K and XI-F, motor proteins that drive movements along actin filaments, were involved in proprioception. Myosin XI-f is highly expressed in interfascicular fibre (IF) cells where the presence of long actin bundles was revealed. F-actin microfilament in these cells may control polarity establishment and cytoplasmic streaming, or may act as tension sensors. To explore this latter hypothesis, we disrupted these actin cables by reducing the gene expression *Villin2* and *Villin3* involved in bundling branching of actin filaments. Through this approach, we seek to elucidate the importance of actin filaments organization in the intricate process of proprioception.

<figure><img src="image_3.png" /><figcaption>**Figure 1.** ① Statocytes: Gravity sensing, statolith rearrangement, auxin transport. ② Light gradient: Guides auxin transport based on angle. ③ Auxin distribution: Integrates light, gravity responses. ④ Propriocytes: Detect elongation, curvature, influence growth. ⑤ Auxin concentration: Drives elongation, curvature. ⑥ Propriocytes feedback: Counter curvature changes. ⑦ Combined drives: Adjust curvature. ⑧ Curvature variations: Induce segment rotation. ⑨ ArC model: Integrates angles, curvature. ⑩ ACÉ-model: Includes elastic bending, slack. ⑪ MMFT: Considers lateral auxin transport, biomechanics. ⑫ Position sensor (Astdm): Models statolith motion, PIN relocalization. ⑬ Moulia et al., 2022</figcaption></figure>

## Experimental procedure:

An original phenotyping approach was developed to evaluate the gravi-perception and proprioception sensitivities (Figure 2). Plants are initially placed horizontally to trigger the gravitropic response, resulting in an upward curvature of the flower stalk. After this first step, horizontal continuous rotation (clinostat) induces a simulated microgravity environment by disrupting statolith sedimentation. Plants rely on curvature sensing (γ) to adjust their growth and tend to rectify the organ by decreasing its curvature.

<figure><img src="image_4.png" /><figcaption>**Figure 2** Phenotypic approach for gravity and proprioceptive sensitivities characterization</figcaption></figure>

Data interpretation is performed using a mathematical model describing that the rate of change of curvature (dC/dt) in stem segment S is directly proportional to the sensitivity (β) to the inclination angle (A) and sensitivity (γ) to its own curvature C (s,t) in space(s) and time (t):

$$ \partial C(s,t)/\partial t = -\beta A(s,t) - \gamma C(s,t) $$

Utilizing time-lapse imaging alongside with software analysis, key parameters are characterized to quantify both β and γ, shedding light on both gravitropic and proprioceptive sensing mechanisms.

## Global Characterization of *vln2vln3RNAi* mutant lines.

* Most significant reduction in *Vln2* and *Vln3* expression was in inflorescence stem of RNAi lines.
* No significant growth rate difference observed.
* RNAi *vln2vln3* lines show fallen branch.

<figure><img src="image_5.png" /><figcaption>**Figure 3.** Comparison of *VLN2* and *VLN3* expression in various organs compared to wild type.</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>**Figure 4.** illustrates the phenotypic characterization, showcasing mutant lines with a fallen branch phenotype.</figcaption></figure>

## Phenotypic characterization of *vln2-vln3* RNAi plants

In Wild-Type plants (WT), 60 minutes after inflorescence stem was placed horizontally, the shoot starts to curve upwards due to gravity perception. When the clinostat is started, due to rotation, sedimentation of statoliths are disturbed and WT senses only its posture. The stem continues to curve during at least 60 min then adjust its posture by curvature decreasing along the growth axis. However, the *vln2vln3* RNAi line does not show any de-curving when exposed to the same conditions and shows proprioceptive insensitivity.

<figure><img src="image_7.png" /><figcaption>**Figure 5.** Time-lapse images comparing WT and *vln2vln3* RNAi lines on a clinostat, demonstrating the insensitivity of the mutant lines.</figcaption></figure>

**Figure 6.** Time-lapse images of both WT and mutant lines were analyzed using the interactive interekt software. (Hartmann *et al.*, 2021).

Final yellow shape reveals mutant lines are unable to correct their posture in the direction of growth axis.

<figure><img src="image_8.png" /><figcaption>yellow: final stem shape
purple: shape at the start of experiment</figcaption></figure>

<figure><img src="image_9.png" /><figcaption>**Figure 7.** Comparison of gravisensitive sensitivity β in WT and mutant lines.</figcaption></figure>

<figure><img src="image_10.png" /><figcaption>**Figure 8.** Comparison of proprioceptive sensitivity γ in wildtype and mutant lines.</figcaption></figure>

## Confocal microscopic analysis of F-actin network in IF cells of vertical inflorescence stalk

Whereas wild-type plants showed long actin bundles localized longitudinally in fiber cells, short actin filaments with altered orientation were observed in *vln2vln3* RNAi and *myosin xif xik* mutants had slightly wavy actin bundles.

<figure><img src="image_11.png" /><figcaption>**Figure 9.** Actin filament organization in the vertical inflorescence stem is shown. Analysis of actin filaments was performed on longitudinal sections of upper part of stem. After fixation, samples were stained with phalloidin AlexaFluor™ 488 and observed with DAPI filter (left) and GFP Filter.</figcaption></figure>

## Actin filament organization undergoes changes in response to positional treatments.

<figure><img src="image_12.png" /><figcaption>**Figure 10.** Confocal observation of F-actin network in inflorescence stems during curving and decurving response phases. Both in WT and *vln2vln3*, during the curving phase, long actin bundles in IF cells deviate from their usual straight alignment and become wavy, where as during the "de-curving" phase controlled by proprioception, actin bundles regain their straight configuration in WT and become oblique in mutant lines.</figcaption></figure>

## Quantitative analysis of Actin filament orientation with Fibril tool(Fiji)

<figure><img src="image_13.png" /><figcaption>**Figure 11.** Illustration showing the frequency trend of actin filament angles during positional treatment in both wild-type (WT) and mutant lines.</figcaption></figure>

<figure><img src="image_14.png" /><figcaption>**Figure 12.** Illustration of the distribution of actin filament angles during positional treatment in both WT and mutant lines.</figcaption></figure>

## References:
1. Bastien, R., Bohr, T., Moulia, B., & Douady, S. (2013). Unifying model of shoot gravitropism reveals proprioception as a central feature of posture control in plants. *Proceedings of the National Academy of Sciences*, 110(2), 755-760.
2. Moulia, B., Bastien, R., Chauvet-Thiry, H., & Leblanc-Fournier, N. (2019). Posture control in land plants: growth, position sensing, proprioception, balance, and elasticity. *Journal of Experimental Botany*, 70(14), 3467-3494.
3. Moulia, B., Badel, E., Bastien, R., Duchemin, L., & Eloy, C. (2022). The shaping of plant axes and crowns through tropisms and elasticity: an example of morphogenetic plasticity beyond the shoot apical meristem. *New Phytologist*, 233(6), 2354-2379.
4. Okamoto, K., Ueda, H., Shimada, T., Tamura, K., Kato, T., Tasaka, M., ... & Hara-Nishimura, I. (2015). Regulation of organ straightening and plant posture by an actin-myosin XI cytoskeleton. *Nature plants*, 1(4), 1-7.
5. Hartmann, F. P., Chauvet-Thiry, H., Franchel, J., Ploquin, S., Moulia, B., Leblanc-Fournier, N., & Decourteix, M. (2022). Methods for a quantitative comparison of gravitropism and posture control over a wide range of herbaceous and woody species. *Plant Gravitropism: Methods and Protocols*, 117-131.

## Conclusion

The modification of actin filaments observed in mutant lines plays a crucial role in proprioception sensing. By examining alterations in actin filament configuration under three distinct positional treatments, it is evident that during the gravitropic response, actin filaments adopt a wavy pattern. This behavior is not only observed in wild-type (WT) cells but also in RNAi-*vln2vln3* lines. Furthermore, in WT cells, actin filaments revert to their original straight configuration during clinostat rotation. In contrast, in RNAi-*vln2vln3* lines, they regain their initial oblique orientation, suggesting a potential reason for the mutant lines' inability to sense their own posture. However, the mechanisms by which decurvature occurs remain to be discovered.