- Research article
- Open Access
Mechanical stress in Arabidopsis leaves orients microtubules in a 'continuous’ supracellular pattern
© Jacques et al.; licensee BioMed Central Ltd. 2013
- Received: 29 November 2012
- Accepted: 9 October 2013
- Published: 18 October 2013
Cortical microtubules form a dynamic network and continuously undergo shrinking (catastrophe), pausing and rebuilding (rescue). The advantage of such a dynamic system is that it may mediate appropriate responses in a short time span. Microtubules are known to play a pivotal role in determining the orientation of the cellulose microfibril deposition in the plant cell wall. The latter is a solid exoskeleton surrounding the protoplast. It forms the physical framework that interconnects most cells and has to bear the tensile stresses within the tissue. Here we describe the effect of externally applied pressure on microtubule organization in growing Arabidopsis leaves.
Confocal microscopy examination of transgenic plants bearing GFP-tagged TUA6 proteins led to the observation that application of an additional mechanical pressure on growing Arabidopsis leaves triggers an excessive bundling of microtubules within the individual cell. Besides, the microtubules seem to align in neighboring cells, creating a 'continuous’ supracellular pattern. This effect occurs within 3 hours after applied external force and is age-dependent, whereby only cells of leaves up to 19 days after sowing (DAS) are susceptible to the applied pressure.
Upon externally applied pressure on developing Arabidopsis leaves, microtubules bundle and rearrange to form seemingly continuous supracellular patterns. As microtubules guide the cellulose synthase complexes, this observed reorganisation pattern probably affects the cellulose deposition, contributing to the reinforcement of the cell wall in a particular position to cope with the extra-applied pressure. The age-effect is reasonable, since younger cells, which are actively shaping their cell walls, are more vulnerable to altered mechanical stresses while in leaves older than 19 DAS, the walls are more robust and therefore can sustain the applied forces.
- Arabidopsis thaliana
- Cell development
- Mechanical stress
Compelling evidence supports the idea that biophysical aspects of the developing tissue are co-determinants of growth [1, 2]. In plants, the cell walls form an interconnecting framework that glues all cells together and that has to bear all encountered stresses . Tensions within the tissue are generated between the different cell layers (tissue stresses) [4–6] and between neighboring cells as the result of differential growth rates . Furthermore, plant cell walls behave like an elastic vessel under pressure. Osmotic-driven water uptake in the vacuole of the protoplast generates a so called 'turgor pressure’ [5, 7] that pushes against the wall.
To endure all these stresses, crystalline arrays of cellulose microfibrils are deposited into the wall. These microfibrils provide tensile strength, which depends on the orientation pattern and the overall matrix composition including other wall components like hemicelluloses and pectins . Importantly, this fortified wall needs to acquire some plasticity during growth to deform and allow expansion. These events are tightly managed on cellular level by the directed guidance of the cellulose synthase (CESA) complexes, determining cellulose microfibril orientation, by modification of the hemicellulosic tethers between adjacent cellulose microfibrils , by targeted delivery/activation of extracellular proteins that modulate the wall properties and by alterations in the apoplastic pH .
Most of the abovementioned processes involve the cytoskeleton, a dynamic network that spans the cortical region and consists of microtubules and actin filaments. The latter deals with the targeted transport of exocytotic vesicles [11–13], while microtubules influence the positioning of newly deposited cellulose microfibrils by guiding the CESA complexes in the membrane [14–18].
Here, we demonstrate the response of microtubules upon applied pressure in pavement cells of the Arabidopsis leaf. The stress response involves a reorganization of the microtubule pattern within 3 hours of applied external force. This is characterized by a pronounced parallel alignment of the microtubules. Moreover, microtubules in adjacent cells seem to form an overarching pattern, creating the impression of a continuous and supracellular pattern. This response occurs in an age-dependent manner whereby only the younger leaves up to 19 days after sowing (DAS) are susceptible to the stress.
Microtubule stress response over time
Trigger of the stress response
Scoring the presence of stress responses in adaxial pavement cells
Normal sample (3 h)
Detached (3 h)
Sample attached to plant (3 h)
Submerged (3 h)
Special chamber (6.5 h)
The benefit of this particular microtubule array could lay in the subsequent modification of the orientation of cellulose microfibril deposition in the cell wall. Since the cells and moreover, the leaf tissue need to maintain their integrity, reinforcement of the wall may be needed to resist the extra endured forces. Indeed, a similar effect is discussed by Hamant et al.  for microtubules in the shoot apical meristem that respond to differential stresses, caused by cell ablation. A similar organization of microfibrils in the cells wall results in the observed anisotropic expansion perpendicular to the influential stress . Upon external applied pressures, the forces in the leaf are altered thus the cells need to respond in a proper way to cope with the new conditions. This may also explain the age-dependent effect. It is reasonable that younger cells, which are actively shaping their cell walls (Jacques et al., unpublished results), are more vulnerable to altered mechanical stresses, while in older leaves, the walls are more robust and therefore can sustain the applied forces.
These results point to an important potential source of artifacts when studying microtuble orientation in leaves, for example for the purpose of phenotyping or developmental studies. The experimental set-up used for imaging microtubules needs to be verified carefully to ensure that the mounting of the leaves does not alter microtubule orientation within the time period of observation. A time window of maximal two hours should be respected to exclude side effects of the sample preparation when observing the Arabidopsis leaf.
Nowadays, the biophysical aspects during morphogenesis are gaining more importance . However, it dates back to the early seventies, when Lintilhac  postulated the possibility that mechanical stresses orient microtubules. Experimental data now confirm this theory ([1, 2, 21, 25], this report). Here, we provide evidence that microtubules in pavement cells of Arabidopsis thaliana are responsive to applied pressures in an age-dependent manner. This encourages looking further into the potential importance of mechanical stresses in the developing leaf. Also the observation that mechanical cues may influence auxin transport  further stimulates research on this, until now, largely unexplored topic.
The microtubules of the adaxial pavement cells in the fourth leaf were visualised using a fusion construct of the green fluorescent protein (GFP) tagged to α-tubulin 6 in Colombia 0 (Col-0) background (glTUA6-GFP) . The leaves were cut off at the petiole from the shoot and placed between a microscope slide and coverslip in distilled water.
To test the age-effect, microtubule patterns of leaves of 15 to 20 and 22 days after sowing (DAS) were monitored during 3 hours. The cortical microtubule pattern was visualized with a Nikon C1 laser scanning confocal microscope (Nikon, Melville, NY) equipped with an argon and a helium/neon laser using a 63x planfluor lens (NA: 1.95) and the automated filter sets for GFP. A z-stack was made and the successive slides were merged into a maximal projection image, which was used for the observations.
Different conditions were tested by cutting off the leaf and placing it for 3 hours on a microscope slide or submerging it in distilled water in a glass petri dish (50 mm in diameter). Moreover, a sample was prepared with the fourth leaf still attached to the plant. Special chambers with spacers in between the microscopic slide and coverslip are prepared as described in Sawchuk et al. to monitor leaves up to 6.5 hours (Figure 4a). A normal sample whereby the leaf was mounted in water between a microscopic slide and coverslip, acted as a positive control. Microtubules were visualized after 3 hours for all treatments with a Nikon C1 laser scanning confocal microscope (Nikon, Melville, NY). The negative control was a freshly made sample. The images were taken at the base, middle and top zone of the leaf. This was performed for leaves of 19 and 20 DAS.
A glass petri dish (60 mm in diameter) was placed on top of the leaf of 19 and 22 DAS to induce local pressure (as shown in Figure 5). The total weight of the glass petri dish was 17 g, meaning that it had a weight/circumference ratio of 0.90 g/cm. For the 22 DAS old leaves, an additional experiment was performed with an extra weight (34 g total weight). Images of the microtubules were taken in regions underneath and neighbouring the applied pressure and at more distal positions in the leaf.
Three leaves were sampled for all conditions tested and scored for the presence of stress patterns by visual observation. At least five images were taken per leaf. When in more than 50% of the images highly bundled microtubules were detected, they were positively scored for the presence of stress patterns.
The authors would like to thank Dr. Robert Kumpf for his artistic input and the Interuniversity Attraction Poles Programme – Belgian State – Belgian Science Policy [IUAP VI/33], the Research Foundation – Flanders (FWO) and the University of Antwerp for funding.
- Hamant O, Heisler MG, Jönsson H, Krupinski P, Uyttewaal M, Bokov P, Corson F, Sahlin P, Boudaoud A, Meyerowitz EM, Couder Y, Traas J: Developmental patterning by mechanical signals in Arabidopsis. Science. 2008, 322: 1650-1655. 10.1126/science.1165594.PubMedView ArticleGoogle Scholar
- Uyttewaal M, Burian A, Alim K, Landrein B, Borowska-Wykret D, Dedieu A, Peaucelle A, Ludynia M, Traas J, Boudaoud A, Kwiatkowska D, Hamant O: Mechanical stress acts via katanin to amplify differences in growth rate between adjacent cells in Arabidopsis. Cell. 2012, 149: 439-451. 10.1016/j.cell.2012.02.048.PubMedView ArticleGoogle Scholar
- Thompson DS: How do cell walls regulate plant growth?. J Exp Bot. 2005, 56 (419): 2275-2285. 10.1093/jxb/eri247.PubMedView ArticleGoogle Scholar
- Peters WS, Tomos AD: The history of tissue tension. Ann Bot. 1996, 77: 657-665. 10.1093/aob/77.6.657.PubMedView ArticleGoogle Scholar
- Kutschera U: The growing outer epidermal wall: design and physiological role of a composite structure. Ann Bot. 2008, 101: 615-621. 10.1093/aob/mcn015.PubMedPubMed CentralView ArticleGoogle Scholar
- Savaldi-Goldstein S, Chory J: Growth coordination and the shoot epidermis. Curr Opin Plant Biol. 2008, 11: 42-48. 10.1016/j.pbi.2007.10.009.PubMedPubMed CentralView ArticleGoogle Scholar
- Somerville C, Bauer S, Brininstool G, Facette M, Hamann T, Milne J, Osborne E, Paredez A, Persson S, Raab T, Vorwerk S, Youngs H: Toward a systems approach to understanding plant cell walls. Science. 2004, 306: 2206-2211. 10.1126/science.1102765.PubMedView ArticleGoogle Scholar
- Crowell EF, Gonneau M, Vernhettes S, Höfte H: Regulation of anisotropic cell expansion in higher plants. CR Biol. 2010, 333: 320-324. 10.1016/j.crvi.2010.01.007.View ArticleGoogle Scholar
- Vissenberg K, Martinez-Vilchez IM, Verbelen J-P, Miller JG, Fry SC: In vivo colocalisation of xyloglucan endotransglycosylase activity and its donor substrate in the elongation zone of Arabidopsis roots. Plant Cell. 2000, 12: 1229-1237.PubMedPubMed CentralView ArticleGoogle Scholar
- Staal M, De Cnodder T, Simon D, Vandenbussche F, Van Der Straeten D, Verbelen J-P, Elzenga T, Vissenberg K: Apoplastic alkalinisation is instrumental for the inhibition of cell elongation in the Arabidopsis thaliana root by the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC). Plant Physiol. 2011, 155: 2049-2055. 10.1104/pp.110.168476.PubMedPubMed CentralView ArticleGoogle Scholar
- Wasteneys GO, Galway ME: Remodelling the cytoskeleton for growth and form: an overview with some new views. Annu Rev Plant Biol. 2003, 54: 691-722. 10.1146/annurev.arplant.54.031902.134818.PubMedView ArticleGoogle Scholar
- Bannigan A, Baskin TI: Directional cell expansion – turning toward actin. Curr Opin Plant Biol. 2005, 8: 619-624. 10.1016/j.pbi.2005.09.002.PubMedView ArticleGoogle Scholar
- Smith LG: Spatial control of cell expansion by the plant cytoskeleton. Annu Rev Cell Dev Bi. 2005, 21: 271-295. 10.1146/annurev.cellbio.21.122303.114901.View ArticleGoogle Scholar
- Paredez AR, Somerville CR, Ehrhardt DW: Visualization of cellulose synthase demonstrates functional association with microtubules. Science. 2006, 312: 1491-1595. 10.1126/science.1126551.PubMedView ArticleGoogle Scholar
- Crowell EF, Bischoff V, Desprez T, Rolland A, Stierhof YD, Schumacher K, Gonneau M, Höfte H, Vernhettes S: Pausing of golgi bodies on microtubules regulates secretion of cellulose synthase complexes in Arabidopsis. Plant Cell. 2009, 21: 1141-1154. 10.1105/tpc.108.065334.PubMedPubMed CentralView ArticleGoogle Scholar
- Gutierrez R, Lindeboom JJ, Paredez AR, Emons AMC, Ehrhardt DW: Arabidopsis cortical microtubules position cellulose synthase delivery to the plasma membrane and interact with cellulose synthase trafficking compartments. Nat Cell Biol. 2009, 11 (7): 797-808. 10.1038/ncb1886.PubMedView ArticleGoogle Scholar
- Li S, Lei L, Somerville CR, Gu Y: Cellulose synthase interactive protein1 (CSI1) links microtubules and cellulose synthase complexes. Proc Natl Acad Sci U S A. 2012, 109: 185-190. 10.1073/pnas.1118560109.PubMedPubMed CentralView ArticleGoogle Scholar
- Bringmann M, Li E, Sampathkumar A, Kocabek T, Hauser MT, Persson S: POM-POM2/CELLULOSE SYNTHASE INTERACTING1 is essential for the functional association of cellulose synthase and microtubules in Arabidopsis. Plant Cell. 2012, 24: 163-177. 10.1105/tpc.111.093575.PubMedPubMed CentralView ArticleGoogle Scholar
- Kitsios G, Alexiou KG, Bush M, Shaw P, Doonan JH: A cyclin-dependent protein kinase, CDKC2, colocalizes with and modulates the distribution of spliceosomal components in Arabidopsis. Plant J. 2008, 54 (2): 220-235. 10.1111/j.1365-313X.2008.03414.x.PubMedView ArticleGoogle Scholar
- Shaw S, Kamyar R, Ehrhardt D: Sustained microtubule treadmilling in Arabidopsis cortical arrays. Science. 2003, 300: 1715-1718. 10.1126/science.1083529.PubMedView ArticleGoogle Scholar
- Hardham AR, Takemoto D, White RG: Rapid and dynamic subcellular reorganization following mechanical stimulation of Arabidopsis epidermal cells mimics responses to fungal and oomycete attack. BMC Plant Biol. 2008, 8: 63-10.1186/1471-2229-8-63.PubMedPubMed CentralView ArticleGoogle Scholar
- Kwiatkowska D, Dumais J: Growth and morphogenesis at the vegetative shoot apex of Anagallis arvensis L. J Exp Bot. 2003, 54 (387): 1586-1595.View ArticleGoogle Scholar
- Uyttewaal M, Traas J, Hamant O: Integrating physical stress, growth, and development. Curr Opin Plant Biol. 2010, 13: 46-52. 10.1016/j.pbi.2009.10.004.PubMedView ArticleGoogle Scholar
- Lintilhac PM: Differentiation, organogenesis and the tectonics of cell wall orientation. II. Separation of stresses in a two-dimensional model. Am J Bot. 1974, 61: 135-140. 10.2307/2441182.View ArticleGoogle Scholar
- Cleary AL, Hardham AR: Pressure induced reorientation of cortical microtubules in epidermal cells of Lolium rigidum leaves. Plant Cell Physiol. 1993, 34 (7): 1003-1008.Google Scholar
- Heisler MG, Hamant O, Krupinski P, Uyttewaal M, Ohno C, Jönsson H, Traas J, Meyerowitz EM: Alignment between PIN1 polarity and microtubule orientation in the shoot apical meristem reveals a th-ight coupling between morphogenesis and auxin transport. PLoS Biol. 2010, 8 (10): e1000516-10.1371/journal.pbio.1000516.PubMedPubMed CentralView ArticleGoogle Scholar
- Ueda K, Matsuyama T, Hashimoto T: Visualization of microtubules in living cells of transgenic Arabidopsis thaliana. Protoplasma. 1999, 206: 201-206. 10.1007/BF01279267.View ArticleGoogle Scholar
- Sawchuk MG, Head P, Donner TJ, Scarpella E: Time-lapse imaging of Arabidopsis leaf development shows dynamic patterns of procambium formation. New Phytol. 2007, 176: 560-571. 10.1111/j.1469-8137.2007.02193.x.PubMedView ArticleGoogle Scholar
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