Keywords

These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

7.1 Basics of Plants Tissue Mechanics

7.1.1 Generalities

From the dawn of humanity, the diversity of mechanical properties exhibited by plant tissues were explored in tool-making, fabrics, houses, furniture, and cutlery. Even the discovery of artificial polymers did not entirely replace plant-derived materials such as linen and cotton. This chapter focuses only on plant tissue mechanics and its link to growth: the interplay between organogenesis and the mechanics of the primary cell wall. The main characteristic of plant cells is the presence of a cell wall, which is a rigid pectocellulose hydrogel encapsulating every single plant cell. The cell wall forms a protective layer and provides structural support for the cell, generating unique properties of the plant tissue as well as strong constraints on cell growth.

7.1.2 Basis of Plant Cell Wall Mechanics

The first thing you see in a plant tissue is the cell wall, as Robert Hook’s historical description in his book Micrographia so remarkably demonstrated. This hydrogel, delimited by a membrane, surrounds the protoplast with its nucleus, mitochondria, chloroplasts, and vacuole (Fig. 7.1). The protoplast exerts a pressure on the cell wall. A good metaphor is a bicycle tire and its tube. If you remove the pressure, cells collapse and the plant loses its shape. In some tissues, a process known as secondary cell wall thickening dramatically increases cell wall rigidity. In such tissues, turgor pressure is not required to maintain organ shape. For more information, read Busse-Wicher et al. (2016). Here, we focus on the primary cell wall, which is able to undertake expansion and growth.

Fig. 7.1
figure 1

Cell wall structure: (a) Organization of a plant cell, showing (a) cell wall, (b) middle lamella, (c) plasma membrane, (d) vacuole, (e) plasmodesmata, (f) chloroplast, (g) Golgi apparatus, (h) mitochondria, (i) nucleus, and (j) endoplasmic reticulum. (b) Structure of the primary cell wall, showing (a) microtubules, (b) plasma membrane, (c) cellulose synthase complex, (d) cellulose microfibril, (e) hemicellulose, (f) xyloglucans, (g) pectin, (h) demethylated homogalacturonan, and (i) methylated homogalacturonan

Full size image

Another metaphor that helps in understanding the growth process in plants is the growing classroom: to expand a classroom in a brick building you need to extend the walls. For that, you must push on the walls and add new bricks or reshuffle the existing bricks into a less compact structure. As with the tire metaphor, it helps in understanding the huge tension exerted on the cell wall and the energy needed to expand the cell wall. If the cell wall loses its integrity or the turgor pressure is too high, the cell bursts and the plant collapses (Fig. 7.2). How the cell wall manages to expand without losing its integrity is an extraordinary biophysical puzzle that is explored in this chapter.

Fig. 7.2
figure 2

Changes in the cell wall during growth: (a) The cell wall expands under the tension of turgor pressure (blue arrows). Local rearrangement of the cell wall along labile like (green) permits rearrangement of the cell wall. Red arrows indicate the direction of expansion. (b) Synthesis in situ and exocytosis of new cell wall components (purple elements) and the change in cell wall links to more rigid ones (red circles) prevent cell wall bursting

Full size image

7.2 What Is Growth?

7.2.1 Definition

First, let us define growth as an irreversible extension of the cell. If we compare plant tissue to an inert material (with no biological activity), the extension can be described as plastic. For a tissue to expand, the cell walls must expand through rearrangement of existing cell wall components or synthesis of new material. To describe the process of growth, we need to measure three parameters simultaneously (Boyer et al. 1985):

  • Turgor pressure: This is the force that pulls the cell walls apart. This pressure originates from the highly concentrated water in the cytoplasm and vesicles of the cell. The hydrophilic molecules in the cytoplasm attract water that flows freely in and out of the cells and from cell to cell through aquaporins (pores in the plasma membrane) or the plasmodesmata (cell-to-cell cytoplasmic junctions).

  • Cell wall mechanics: Here we determine how much energy is needed to expand the existing cell wall (elasticity) and how much it can reshuffle itself (plasticity).

  • Synthesis of new material: Growth involves exocytosis of new cell wall material and cell wall synthetizing enzymes.

To measure all these parameters simultaneously is very difficult and has not been achieved so far (with the exception of pollen tubes). We discuss the technical challenges one by one in the following sections.

7.2.2 Is Growth Really a Mechanical Problem?

For a long time, organogenesis was studied by tracking cells throughout cell division and neglected the cell expansion aspect of organogenesis. New organs correlate with new cell division patterns. For example, new organ formation in the meristem, or later in the root, is always associated with periclinal division in the deep layer of the tissue (Walles 1991). In some cases, the whole process of organogenesis can be described as a series of organized symmetric or asymmetric divisions (Gunning et al. 1978). Studies have demonstrated how important the cell division pattern is for organogenesis. In the early 1990s, a series of experiments measuring the increasing or decreasing cell division rate in elongating tissue showed that the rate had little or no effect on organogenesis (Wyrzykowska et al. 2002). This brought back the old idea that cell mechanics, rather than cell number, controls growth. However, cell division and cell expansion are linked; cell division is under the control of cell expansion. It is possible to predict cell division in the meristem by its increase in size and the mechanical stress it is experiencing (Jones et al. 2017). In other words, cell wall expansion prefigures the division pattern that follows the cell structure achieved by growth. Therefore, we could settle on the idea that growth and organogenesis in plants is driven by cell wall expansion.

Yet, a recent study has shown that the levels of cell wall synthesizing enzyme in the meristem are cell-division controlled (Yang et al. 2016). Thus, there is mutual control: growth-associated changes in cell wall mechanics could be under the control of the cell division process.

Summary

Cell division and growth are linked through the following feedback loop: cell wall mechanics controls cell expansion, which controls cell division, which in turn affects cell wall mechanics.

7.3 Modeling and Mathematical Approaches

The elements that control cell wall expansion are clearly part of a complex process. To grasp a complex problem, it is often helpful to propose a simple mathematical equation with the minimum number of possible variables. This was most clearly stated by Lockhart (1965), who proposed a biophysical equation for the mechanical control of growth of the cell wall:

$$ \mathrm{Rate}=m\ \left(\Psi p-Y\right) $$

The growth rate is proportional to the turgor pressure (Ψp) and extensibility (m) above the yield threshold (Y).

Behind this mathematical statement is the following idea. The pressure is associated with two parameters that describe cell wall mechanics: its capacity to expand irreversibly (m) and a threshold (Y). The existence of a threshold represents the ability of plant tissue to halt growth without stopping synthesis or to reduce the turgor pressure to zero.

Defining these parameters and estimating their numerical values is quite challenging. The extensibility m is a complicated parameter to determine. In Lockhart, m stands for all the parameters that permit expansion: synthesis of new cell wall components and extension of the existing ones. Which of the two parameters is most important is the subject of a debate that is polarizing the scientific community. In creep experiments, which determine cell wall remodeling under tensile stress, m is often reduced to plastic deformation. As discussed above, synthesis of cell wall components should also be considered in irreversible expansion and is a component of the m factor.

Another way of evaluating parameters is through computer simulation. Since the Lockhart publication, a series of models describing organ growth have been proposed. These models always face the geometric problem and a huge number of unknown parameters (e.g., thickness of the cell wall and synthesis rate). To date, some successful models have managed to describe growth in two dimensions (2D).

Anja Geitmann (Parre and Geitmann 2005) was the first to propose a reliable model for pollen tube growth. The pollen tube is a cell presenting very rapid tip growth; its goal is to project the sperm cell situated at the tip of the tube into the ovule and thus grows through the pistil. The most recent models take into account changes in the local geometry of the cell wall over time. They are able to simulate the transient oscillatory growth in different pollen tube species observed in nature. In Geitmann’s work, the minimum number of parameters for the model were measured directly. To best fit reality, “guessing” the different parameters of the equation was associated with real measurement of cell wall elasticity, cell wall synthesis (exocytosis rate), turgor pressure, and growth using high temporal resolution.

Summary

Modeling helps to test and evaluate the importance of different elements in growth. The most informative models are the simplest ones that can describe the observed growth based on the minimum number of variables. The best studies also associate the evaluation of parameters with in situ measurement.

7.4 Measuring Turgor Pressure

Turgor pressure is a crucial parameter (Deri Tomos et al. 1989); yet, its measurement is technically challenging. A series of different methods have been proposed and used, but there have been only a handful of successful experiments on growing organs. The first methods were based on finding the point at which the turgor pressure in the cell is balanced by the pressure in the mounting media. Above a certain threshold, the turgor pressure does not act on the cell wall and the cell is plasmolyzed. It is important that the osmolyte (the ion used in the medium to compete with the cellular ionic concentration) cannot be internalized by the cell, and that water can flow freely out of the tissue (Falk et al. 1958; Nilsson et al. 1958; Stadelmann 1984).

Microscopy observation can be used to observe when the ionic activity of the external solution matches the cell. A classic classroom experiment is often performed on naturally colored cells such as red onion or flower petals. The limitation of this method is the field of view of the microscope. For a full tissue, one can use vibration to determine the plasmolysis point. This technique relies on the fact that the vibration properties of a tissue are related to its rigidity, and the rigidity depends on the turgor pressure (Virgin 1955). The rigidity drops with a drop in pressure until plasmolysis is reached. At this point, the rigidity is not sensitive to plasmolysis and depends only on the cell wall elasticity.

Another approach is to measure the pressure directly by puncturing the cell with a tube. This method works for big cells, but not for the very small cells important for growing tissue (Green 1968; Green et al. 1971; Büchner et al. 1981). The most complete measurement was done on the root, but the authors could not detect any differences in the turgor pressure along the elongating roots. This indicates that, so far, there is no evidence to support the action of turgor pressure on the variability of growth rate observed within the organ.

Summary

The tools available for measuring turgor pressure are not precise enough to measure single-cell turgor pressure in the early stages of organ formation. This is especially true for the model plant Arabidopsis thaliana, which has particularly small cells.

7.5 Cell Wall Rheology

7.5.1 Definition

Rheology studies the deformation and flow of matter; here we review the rheology of a particular hydrogel, the cell wall. Like any hydrogel, the mechanical properties of the cell wall change with the amount of water it contains. Importantly, once dehydrated, the cell wall has irreversibly lost its original mechanical properties.

The mechanical properties of a hydrogel depend on the ionic composition of the solution. Ions influence water activity (i.e., cell wall hydration) through their affinity to water. Monovalent ions intercalate into the gel and affect the distance between the polymers working as plasticizers. In contrast, divalent ions can create bonds between the charged molecules of the polymer mesh. For more details, please refer to Sect. 6.2 on pectin. Thus, the mechanical properties of the primary cell wall can only be determined on the fresh, intact cell wall with a protocol that does not change the ion composition.

How do we measure the mechanical properties? One method is to deform the sample and record the force required over time. Alternatively, a constant force can be applied and the change in shape of the sample recorded over time. Several parameters can be measured in this way, but depend on the type of deformation observed. If the deformation is reversible, elasticity is measured (the cell wall regains its original shape after the force has been removed). The time taken to come back to its original shape is a measure of the viscoelasticity. If extension is irreversible, the viscosity of the material is measured (Braybrook et al. 2012). To measure the change in shape indirectly, one looks at the indentation depth or uses fluorescent probes (Kim et al. 2015).

In plant biophysics, the majority of mechanical measurements are designed to measure the growth capacity of the tissue; thus, a different rheological property of the cell wall is measured, the creep.

7.5.2 Creep

The definition of creep is inconsistent in the literature. In general, creep refers to the growth capacity of the tissue. It could be thought as the m factor in the Lockhart equation (Taiz 1984). If growth occurs mainly as a result of rearrangement of the cell wall network, it can be measured as the energy required to stretch the cell wall (Keegstra et al. 1973). Many components involved in loosening of the cell wall have been characterized with this method. One of the founding fathers of this type of measurement is Paul Green, who worked on giant cells from Characeae green algae (Green 1976). He measured the relative importance of turgor pressure, elasticity, and creep in growth of the cell wall, thanks to measurement of extension at a subcellular resolution and the extension capacity. Green always took a critical view of creep measurement and its inability to separate the contributions of rearrangement of the cell wall and cell wall synthesis (Green and Cummins 1974).

Recently, cell wall rearrangement during creep has been observed thanks to the use of atomic force microscopy (AFM). The studies demonstrated that cell wall rearrangement, at least in the epidermis, is associated with elongation of the tissue and is reflected in the creep experiments (Zhang et al. 2017).

7.5.3 Other Measurements of Cell Wall Rheology

Measurement of the elasticity, viscoelasticity, and viscosity in living tissue using a nanoindenter has recently been developed. Surprisingly, elasticity (reversible deformation of the cell wall) was correlated with growth and not viscosity or viscoelasticity (Peaucelle et al. 2015). This is paradoxical because elasticity is a reversible deformation, whereas growth is an irreversible process. At first glance, the finding is also in opposition to creep experiments that put cell wall remodeling at the heart of the growth process. This can be explained if elasticity correlates with growth through control of cell wall synthesis and not cell wall remodeling. In other words, the synthesis of new material is linked to the elasticity of the cell wall. This correlation was first demonstrated on pollen tubes: Local changes in cell wall elasticity correlated with the position of exocytosis of cell wall components at the tip of a cell. This process was observed to involve cytoplasmic calcium signaling coupled with deformation-sensitive calcium channels (Fayant et al. 2010).

Summary

Creep experiments directly measure the ability of the cell wall to rearrange in association with growth. Elasticity of the cell wall relates to growth in manner that could be related to cell wall synthesis (but has yet to be determined). Therefore, two independent growth processes could relate to different cell wall rheological properties.

7.6 Organogenesis and Polar Growth of Tissue: Contribution of the Cell Wall Component

The turgor pressure that drives cell expansion is isotropic. If it was the only parameter controlling growth, turgor pressure could lead to homogenous elongation (i.e., a sphere). Then, plants would look like a drawing of La Gioconda by Botero (Fig. 7.3). Somehow, this isotropic force is transformed into anisotropic orientated growth. Which component of the cell is responsible? A good candidate is cellulose (Green 1980).

Fig. 7.3
figure 3

Isotropic growth illustrated by (a) representation of La Gioconda of Leonardo da Vinci and (b) La Gioconda of Botero

Full size image

7.6.1 Cellulose

Determination of the structure of cellulose was a long and difficult path. It took 30 years from the first chemical isolation of cellulose to determination of its polymer structure. From the start, cellulose was considered to be the load-bearing component of the cell wall and responsible for anisotropic growth.

The basic idea is that bundles of cellulose fibrils build up an orientated network surrounding the cell and block expansion in one direction. We could compare it to the metal rings around a barrel that prevent it from opening up. Electron microscopy data and cell wall optical imaging support this theory. A series of brilliant images showed orientated microtubules not exactly perpendicular to the cell but organized in sheets like the laminated structure of wood (Fig. 7.4). At the same time, Paul Green observed in giant algae cells that the cellulose fibrils were orientated in a looser way, in a network (Green 1960; Gertel and Green 1977). These two publications mark the point when the scientific community divided into two camps. The first theory supports the laminated organization of cellulose and suggests that orientation of the fibrils in a sheet is stable during growth. Loosening between lamellae leads to progressive reorientation of the whole sheet. In contrast, the network theory, following Green’s observations, suggests that the latest microtubules are deposited in an orientated way, but that growth modifies their orientation and distorts the network. In this theory, only the most recently synthesized cellulose fibrils control anisotropic orientation (Marga et al. 2005). Recent observation by Cosgrove and colleagues (Zhang et al. 2017) of a multinetwork structure and its reorientation supports the network concept. In fact, both visions could be right: The multinetwork structure was observed in the external cell wall of epidermal cells, whereas the laminated structure was mostly in the internal cell wall. Thus, the two concepts of cell wall structure might simply relate to two different types of cell wall.

Fig. 7.4
figure 4

Scheme of a laminated cell wall

Full size image

The key role of cellulose in anisotropic growth was most strikingly demonstrated by the swelling of plants following chemical treatment affecting cellulose or microtubule synthesis and by the phenotype of a mutation affecting cellulose synthase (Ledbetter and Porter 1963; Heath 1974; Mueller and Brown 1982). The similarity of this phenotype to the result of inhibition of microtubule synthesis led to the idea that the orientation of microtubules is generated by the orientation of cellulose (Heath 1974). This concept is supported by the observation that cellulose synthase and microtubules are found in close proximity. A commonly used model states that cellulose synthase polymerizes cellulose directly in the plasma membrane following orientation of the microtubules and is supported by microscopy observations. The microtubule orientation then leads to mechanical anisotropy in the cell wall and anisotropic expansion of the cells.

To complicate this picture, recent work (Peaucelle et al. 2015) has shown that treatments affecting cellulose synthase or microtubule orientation also affect another component of the cell wall, pectin.

7.6.2 Pectin

Pectin forms a fine meshed network surrounding the other components of the cell wall. There are several chemically different components of pectin, but here we focus on the homogalacturonans. This component is a polymer of galacturonan sugar, which presents a lateral carboxyl group that can be methylated or not. In the 1980s, the 3D structure of the two polymers was predicted (Morris et al. 1982). Methylated pectin was predicted to form a very compact structure, with proton-stabilized interaction on the methylated carboxyl (Grant et al. 1973).

Demethylated pectins were anticipated to generate a more hydrated and less packed structure stabilized by calcium electronic interactions through demethylated carboxyl groups. This model was named the egg box structure, where the stability of the conformation would be archived for at least nine successive demethylated homogalacturonans. At first, the calcium bonds found in demethylated pectin were thought to generate strong links in the cell wall and thus limit cell wall remodeling and creep. It was suggested that they slowed down growth. Interactions between methylated pectin were ignored, except in the food industry. The vision of demethylated pectin linked to a rigid cell wall was first challenged when pectin demethylation was shown to lead to organ formation and reduction in cell wall elasticity in the meristem (Peaucelle et al. 2011).

Later, it was found that the anisotropic changes in pectin methylation are required for polarized elongation of the hypocotyl and are associated with a reduction in cell wall elasticity (Peaucelle et al. 2015). This finding led to the proposal of a two-step process for anisotropic elongation of the tissue: Antipodal changes in cell wall elasticity caused by changes in pectin methylation lead to a tenfold anisotropic elongation. This anisotropic growth is followed by alignment of microtubules and cellulose microfibers. Thus, cellulose microfibers are needed for further anisotropic elongation, which can achieve 100- and even 1000-fold anisotropic elongation. Furthermore, these two components interact, as demonstrated by treatments affecting microtubule and microfibril orientation, which also affected the pectin methylation pattern (Peaucelle et al. 2015).

Summary

Polar elongation is a two-step process: First, a change in pectin methylation leads to a change in cell wall elasticity, followed by cell polarity (cell mechanical asymmetry). Microtubules reorient along the elongation axes, leading to orientated cellulose synthesis. This generates cell wall anisotropy.

7.6.3 Xyloglucans

Xyloglucans are components of hemicellulose that have attracted a lot of attention since their strong interaction with cellulose was described. Models predict that reducing the amount of xyloglucans could increase creep by decreasing cellulose microfibril cohesion and helping local rearrangement of the cell wall.

The enzymes that control the structure of the xyloglucan network have been predicted. The genes coding for these proteins are expressed in a tight developmental pattern and are present in sites with strong elongation (Antosiewicz et al. 1997). Unfortunately, multiple mutations in these genes do not present an obvious growth defect phenotype. Are xyloglucans without a function? Certainly not. We have seen that there are multiple mechanisms controlling growth; therefore, it is likely that the absence of xyloglucan remodeling is compensated (Cosgrove 2016).

7.6.4 Expansins

Expansins form a family of cell wall proteins. Their importance in growth was demonstrated when purified expansin proteins were shown to accelerate growth in some tissues (Fleming et al. 1997). They are the only known proteins to promote creep in vitro (Cosgrove 1998; Shieh and Cosgrove 1998). There are two isoforms present in a multigene family found throughout the plant kingdom (Cosgrove 2015). The first isoform interacts with cellulose and the second (found in grasses) interacts with glucuronoarabinoxylan, a grass-specific carbohydrate. Interestingly, only specific cells are sensitive to expansins. This suggests that not all cell walls can respond to expansin, demonstrating multilevel control (McQueen-Mason and Cosgrove 1995).

Summary

Cell wall chemical components have redundant functions in cell wall mechanical properties and growth. This chemistry is very dynamic and is under the control of complex signaling networks that are still to be described. So far, we have seen only the tip of the iceberg of this chemical complexity.

7.7 Input from Growth Measurements

Understanding plant cell wall mechanics and its link to cell wall chemistry is only one part of the problem. It is also important to undertake detailed quantitative measurement of the growth process, in particular plant growth-induced motion.

Observation of plant motion has been at the heart of scientific debate for a long time. First reported in 400 BC, it was also discussed in Hook’s famous publication, which coined the word “cell.” Growth-related motion, in particular circumnutation, fascinated Charles Darwin (Darwin 1880). The first movie of a growing plant dates from the end of the nineteenth century, yet quantification of growth is still difficult because it occurs in three dimensions. Until now, only 2D growth in response to gravity has been fully described (Erickson 1976).

Those early films revealed that plants adapt their shape to external stimuli such as light and gravity. These growth movements are named phototropism and gravitropism, respectively. There also exist lesser known growth movements such as ototropism, also named proprioception (Bastien et al. 2013). Proprioception means that plants are able to sense their own shape and control tissue growth so that they stay straight. The shape of Arabidopsis grown in microgravity at the international space station illustrates proprioception very well. Plants grown in space are almost identical to control plants grown on Earth (Link et al. 2003, 2014). Study of gravitropism in Earth-grown plants has led to the same conclusion. These exciting results reinforce the crucial importance of the feedback loop between growth mechanics and tissue structure, not only at the subcellular level but also at the whole organ and organism level.

Another fascinating thing about plant motion linked to proprioception is oscillatory movement, which reveals complex regulatory networks of growth acting at different time scales. It also explains the redundant functions and parallel growth processes we have discussed so far.

7.8 About the Regulatory Network

The next step is to explore the regulatory networks involved in growth. The study of signaling network in plants is described in other chapters of this book; here, we briefly discuss two aspects. The auxin regulatory network is the most studied aspect. The plant hormone auxin was isolated thanks to its capacity to promote growth. The growth induction capacity of auxin was rapidly associated with the acid form of the molecule. It was proposed that auxin promotes growth through acidification of the cell wall, leading to cell wall rearrangement. This model was rapidly confirmed by the observation that the expanding cell wall has a low pH (Tepfer and Cleland 1979). Intriguing information about the auxin growth network was obtained from study of the meristem and generation of the phyllotactic pattern. Since the work of Stephane Douady and Yves Couder, we have known that generation of the phyllotactic pattern requires a dynamic feedback loop between inhibitory and activating signals in the meristem (Douady and Couder 1992). Isolation of the pin1 mutant and the development of fluorescent reporters enabled the discovery that auxin is the activator molecule necessary and sufficient for induction of organ formation (Okada et al. 1991). The dynamics of the auxin transporter system in the meristem depletes auxin in the areas surrounding new organs and thus inhibits formation of new primordia nearby (de Reuille et al. 2006). The authors suggested that the dynamics of the structure was generated by the auxin concentration itself.

Recently, auxin was shown to induce pectin demethylation in the primordia and that this change was necessary and sufficient for organ formation (Braybrook and Peaucelle 2013). Intriguingly, pectin demethylation is also necessary and sufficient for auxin-induced growth, suggesting that auxin acidity is not sufficient for organ growth and that the acidification of the cell wall commonly associated with growth could instead be attributed to acidification by carboxyl groups formed during pectin demethylation. Regulation of polar auxin transport was also questioned; it could not simply be controlled by auxin concentration because the changes in cell wall chemistry lead to destabilization of polar auxin transport. In parallel, cell ablation experiments in the meristem showed that, like microtubules, auxin polar transport responds to mechanical stimulus (Hamant et al. 2011). These results suggest that polar auxin transport is at least partially under the control of mechanical constraints arising from the differential cell wall elasticity of the growing organ. This feedback loop is at the heart of organ formation. How exactly this feedback is generated is still to be discovered; it could be via chemical or mechanical signals.

How mechanical clues from the cellular environment can be synthetized and transduced to the cell is also an important future research area (Wolf et al. 2012). One important component of this regulation is the transmembrane kinase receptor (THESEUS and FERONIA are the most studied receptors). It is possible that the extracellular domain of this protein can sense the chemical/mechanical stress of the cell wall and feedback through a kinase cascade to the nucleus and affect gene transcription. The beauty of the kinase-signaling cascade is its integrative capacity (for more information, read about the regulatory kinases in animal cell cycles). If the regulatory system of plants is as complicated as that described for mammalian cells, it could be decades before we can grasp all the subtleties of these regulatory networks.

7.9 Conclusions and Perspectives

Clearly, we are far from understanding the mechanics of plant growth. Yet, we are gaining new insights from all directions at an incredible pace. The precise description of several of the key elements regulating growth forms the basis for study of the regulatory network. However, part of the process is still invisible. A complete understanding of the process is currently out of our reach, either because of its complexity or because we lack a crucial aspect. We still do not have a satisfactory answer to our original question: How does the cell wall expand without the cell bursting? New technology and thinking out of the box will certainly help to solve this puzzle.