published: 25 February 2019
Frontiers in Plant Science | www.frontiersin.org 1February 2019 | Volume 10 | Article 173
AgriBio, La Trobe University, Australia
Max-Planck-Institut für Molekulare
Pennsylvania State University,
Department of Forest Genetics and
Plant Physiology, Umeå Plant Science
Centre, Swedish University of
Agricultural Sciences, Umeå, Sweden
This article was submitted to
Plant Biophysics and Modeling,
a section of the journal
Frontiers in Plant Science
Received: 28 November 2018
Accepted: 01 February 2019
Published: 25 February 2019
Verger S, Liu M and Hamant O (2019)
Mechanical Conﬂicts in Twisting
Growth Revealed by Cell-Cell
Front. Plant Sci. 10:173.
Mechanical Conﬂicts in Twisting
Growth Revealed by Cell-Cell
Stéphane Verger*†, Mengying Liu and Olivier Hamant
Laboratoire de Reproduction et Développement des Plantes, ENS de Lyon, UCBL, INRA, CNRS, Université de Lyon, Lyon,
Many plants grow organs and tissues with twisted shapes. Arabidopsis mutants with
impaired microtubule dynamics exhibit such a phenotype constitutively. Although the
activity of the corresponding microtubule regulators is better understood at the molecular
level, how large-scale twisting can emerge in the mutants remains largely unknown.
Classically, oblique cortical microtubules would constrain the deposition of cellulose
microﬁbrils in cells, and such conﬂicts at the cell level would be relaxed at the tissue scale
by supracellular torsion. This model implicitly assumes that cell-cell adhesion is a key step
to transpose local mechanical conﬂicts into a macroscopic twisting phenotype. Here we
tested this prediction using the quasimodo1 mutant, which displays cell-cell adhesion
defects. Using the spriral2/tortifolia1 mutant with hypocotyl helical growth, we found
that qua1-induced cell-cell adhesion defects restore straight growth in qua1-1 spr2-2.
Detached cells in qua1-1 spr2-2 displayed helical growth, conﬁrming that straight growth
results from the lack of mechanical coupling between cells rather than a restoration of
SPR2 activity in the qua1 mutant. Because adhesion defects in qua1 depend on tension
in the outer wall, we also showed that hypocotyl twisting in qua1-1 spr2-2 could be
restored when decreasing the matrix potential of the growth medium, i.e., by reducing the
magnitude of the pulling force between adjacent cells, in the double mutant. Interestingly,
the induction of straight growth in qua1-1 spr2-2 could be achieved beyond hypocotyls,
as leaves also displayed a ﬂat phenotype in the double mutant. Altogether, these results
provide formal experimental support for a scenario in which twisted growth in spr2 mutant
would result from the relaxation of local mechanical conﬂicts between adjacent cells via
global organ torsion.
Keywords: adhesion, twisting, mechanical stress, morphogenesis, arabidopsis
Because complex morphogenesis generally involves diﬀerential growth, mechanical conﬂicts are
widespread in developing organisms. In animals, such conﬂicts can be resolved through cell
rearrangements, as cells are in principle free to move. Yet, cell-cell adhesion often prevents such
outcome and patterns of tension and compression appear. Mechanical conﬂicts can be resolved
through global tissue deformation, as shown for instance in the gut (Savin et al., 2011; Nerurkar
et al., 2019). Interestingly, some of the relevant mechanotransduction factors play a role in cell-cell
adhesion. For instance, cadherins are both central regulators of epithelial cohesions and transducers
Verger et al. Twisting and Adhesion
of mechanical signals inside the cell (Leckband and de Rooij,
2014). Therefore, while mechanical conﬂicts emerge from
diﬀerential growth and cell-cell adhesion, they also in turn
contribute to growth patterns and adhesion through the cell
response to mechanical stress, in a feedback loop.
With few exceptions such as pollen tube and ﬁber cell growth
(Gorshkova et al., 2012; Chebli and Geitmann, 2017; Marsollier
and Ingram, 2018), cell-cell adhesion and thus symplastic growth
is ubiquitous in developing plant organs. The presence of
contiguous cell walls with a pectin-rich middle lamella maintains
adhesion between adjacent cells (Jarvis et al., 2003) (Daher and
Braybrook, 2015) Besides, many reports point at the high degree
of growth heterogeneity in plant tissues (Hong et al., 2018).
It follows that mechanical conﬂicts are widespread in growing
plants. As reported in animals, plant cells are able to sense and
respond to such cues to control cell division plane orientation
(Lintilhac and Vesecky, 1984; Louveaux et al., 2016), growth
direction (Green and King, 1966; Hamant et al., 2008), cell
polarity (Heisler et al., 2010; Nakayama et al., 2012; Bringmann
and Bergmann, 2017) and cell identity (Coutand et al., 2009;
Landrein et al., 2015). In parallel to these active responses to
stress, mechanical conﬂicts may also be resolved through passive
and global tissue deformation (Coen et al., 2004). For instance,
mechanical conﬂicts are thought to play a major role in shaping
complex ﬂoral shapes, such as Antirhinum petals, as a result of
instructive biochemical signals, but without necessarily involving
an active mechanical feedback on cells (Coen and Rebocho, 2016;
Rebocho et al., 2017). One of the challenges for future research in
this area is to understand the relative contributions of passive and
active responses to mechanical stress in morphogenesis. Here we
take the example of organ twisting to explore that question.
Several mutations on microtubule regulators, or even on
tubulins, lead to twisted organs in Arabidopsis (Ishida et al.,
2007b; Smyth, 2016). In such mutants, cells exhibit oblique
cortical microtubule orientations and the handedness of the
microtubule helix is always opposite to the handedness of tissue
growth (Ishida et al., 2007a). For instance, the lefty mutations
in α-tubulins lead to both a left-handed helical growth and
a right-handed cortical microtubule orientations in the root
epidermis (Thitamadee et al., 2002). Such phenotypes are only
What is best known is the relation between microtubule
orientation and growth: except for a few counterexamples
[e.g., (Himmelspach et al., 2003; Sugimoto et al., 2003)],
cortical microtubules generally guide the deposition of cellulose
microﬁbrils; as cellulose microﬁbril stiﬀness constrain cell growth
direction, cortical microtubule orientation becomes a proxy
for the mechanical anisotropy of cell walls. Therefore, right-
handed microtubule orientations would inevitably drive cell
growth direction in a left-handed helix (Thitamadee et al.,
2002; Smyth, 2016). Conversely, organ twisting is aﬀected
when the cellulose synthase—microtubule nexus is impaired
in the csi1 mutant (Landrein et al., 2013). Another cell wall
mutant has recently been shown to have organ twisting without
aﬀecting microtubule organization, but is nevertheless believed
to impact cellulose organization and cell wall mechanical
anisotropy (Saﬀer et al., 2017).
What is least known is 2-fold. First, it is unclear how
microtubule arrays would acquire a stable and oblique
orientation. Reports so far rather suggest that unstable
microtubules tend to acquire a right-handed orientation
(as in the lefty mutants), while stabilized microtubules acquire
a left-handed orientation (Ishida et al., 2007b). This latter
case is typical of the spiral2/tortifolia1 mutant, which exhibits
right-handed helical growth (Buschmann et al., 2004; Shoji et al.,
2004). SPR2 was recently shown to bind and stabilize the minus
end of microtubules to control their depolymerization rate, with
an indirect impact on microtubule severing (Fan et al., 2018;
Nakamura et al., 2018), although this latter point is debated and
might depend on tissue identity (Wightman et al., 2013). In
the end, microtubule dynamics are stimulated in spr2 mutants,
resulting in more stable cortical microtubule alignments. It
remains unclear how aﬀecting microtubules dynamics would
lead to stable and consistent left or right handedness of cortical
microtubule arrays. It has been proposed that the origin of such
handedness lies in the microtubule structure itself. Microtubules
are in general composed of 13 protoﬁlaments and this confers
them a straight structure. However, microtubules can in
principle be composed of 10 to 16 protoﬁlaments, some of these
conﬁgurations conferring them a consistent left or right handed
twisted structure (Pampaloni and Florin, 2008). Such chirality
at the molecular level could be the basis for the consistent tilted
microtubule arrays, however this has not been conﬁrmed in
twisting mutants so far (Ishida et al., 2007b). Second, it is unclear
how local cell wall modiﬁcations would lead to torsion of a
whole organ. Indeed, because they exhibit oblique mechanical
anisotropy in their walls, each cell would simply twist around
their axis as they grow, if they were not attached to one another
(Wada and Matsumoto, 2018). However, because of cell-cell
adhesion, these cells cannot twist independently. It has been
proposed that such local mechanical conﬂicts could be relaxed
by the global torsion of the organ (Wada and Matsumoto, 2018,
see Figures 1A,B). However, the presence of these conﬂicts, and
their role in helical growth, has never been demonstrated in vivo.
This is what we aim at testing here.
MATERIALS AND METHODS
Plant Material and Genotyping
The qua1-1 (WS-4) T-DNA insertion line and the spr2-2 (Col-0)
EMS mutant, were previously reported in Bouton et al. (2002)
and Shoji et al. (2004), respectively. The qua1-1 mutant was
genotyped using the primers described in Bouton et al. (2002)
and the spr2-2 mutant was genotyped by Sanger sequencing using
the following primers: FW_5′-TGTCATCAGCAGCTCAGACA-
Arabidopsis thaliana seeds were sown on solid custom-
made Duchefa “Arabidopsis” medium (DU0742.0025, Duchefa
Biochemie), containing either 1 or 2.5% agarose as gelling agent
(Figures 4K,L, and see Verger et al., 2018).
Seeds were cold treated for 48 h to synchronize germination
and then grown in a phytotron at 20◦C. For hypocotyl etiolation,
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Verger et al. Twisting and Adhesion
FIGURE 1 | Twisting, from the molecular to the organ scale. (A,B) Schematic
representation of the effect of cortical microtubules (represented in green)
orientation at the cell level (small cylinder on the left) and its effect at the whole
organ level (cylinder on the right), explaining straight, and twisting growth in a
cylindrical organ. (A) Transverse cortical microtubules promote the longitudinal
expansion of the cell, which leads to straight cell ﬁles at the organ level, as
observed in wild-type hypocotyls, at least when considering inner cells
(Crowell et al., 2011). (B) Tilted cortical microtubules impose a tilted
mechanical anisotropy of the cell wall leading to the twisting of the cell at the
single cell level. However, because cells are attached to one another they
cannot twist by themselves and the mechanical conﬂict is relaxed through
global organ torsion, as in spr2-2 seedlings.
seeds were exposed to light for 4 h to induce germination. The
plates were then wrapped in three layers of aluminum foil to
ensure skotomorphogenesis, and placed in a phytotron at 20◦C
for 4 days before imaging.
Cell Wall Staining and Confocal
For cell wall staining, plants were immersed in 0.2 mg/ml
propidium iodide (PI, Sigma-Aldrich) for 10 min and washed
with water prior to imaging. For imaging, samples were placed
between glass slide and coverslip separated by 400 µm spacers to
prevent tissue crushing. Images were acquired using a Leica TCS
SP8 confocal microscope. PI excitation was performed using a
552 nm solid-state laser and ﬂuorescence was detected at 600–
650 nm. Stacks of 1024 ×1024 pixels (pixel size of 0.363 ×0.363
micron) optical section were generated with a Z interval of 1 µm.
Twisting Angle Quantiﬁcations and
We quantiﬁed the angle of cell ﬁles of the ﬁrst cortex cell layer
in the hypocotyl (i.e., the layer under the epidermis, Figure 2K).
For each condition/mutant we quantiﬁed the twisting angle of
12 hypocotyls from 3 biological replicates. The angles were
measured relative to the hypocotyl axis. An angle of 0◦reveals
no twisting, while positive and negative angle values mark left-
handed and right-handed twisting, respectively. Twisting angle
measurement was performed with Fiji (https://ﬁji.sc/). Statistical
analyses and data plotting was performed with R (https://www.r-
project.org/). Pairwise Wilcoxon rank sum tests were performed
to test the diﬀerences of twisting angle between the samples.
Loss of Cell-Cell Adhesion Prevents
Hypocotyl Twisting in qua1-1 spr2-2
To reveal the mechanical conﬂicts in mutants exhibiting helical
growth, we reasoned that disrupting cell-cell adhesion would lead
to cell autonomous behavior through the (partial) mechanical
uncoupling of cells, and would possibly aﬀect the helical growth
of organs. To test that hypothesis, we thus analyzed the spr2
phenotype in the presence of cell-cell adhesion defects. The
QUA1 gene encodes a glycosyltransferase and mutation in the
gene impairs pectin synthesis and cell-cell adhesion (Bouton
et al., 2002; Mouille et al., 2007). We generated qua1-1 spr2-2 lines
and observed the hypocotyl phenotype by measuring the twisting
angle θT. An angle of 0◦reveals no twisting, while positive
and negative angle values mark left-handed and right-handed
twisting, respectively. As reported before, hypocotyls exhibit
straight cell ﬁles for both WS-4 (Mean θTof −0.72 ±1.18◦,n=
12 samples, Figures 2A,F) and Col-0 (Mean θTof 0.09 ±1.74◦,n
=12 samples, Figures 2B,G). As expected, in spr2-2, hypocotyls
exhibited a pronounced right-handed helix of cell ﬁles (Mean
θTof −9.98 ±2.85◦,n=12 samples, Figures 2D,I). For qua1-
1, in many cases cell ﬁles could not be properly recognized due
to the presence of major cell-cell adhesion defects (Figure 2C).
However, we could observe cell ﬁles in the cortex layer under
the epidermis, which revealed no twisting for qua1-1 (Mean
θTof −1.23 ±1.63◦,n=12 samples, Figure 2H). Note that,
to allow comparison between genotypes, all quantiﬁcations of
twisting angles were obtained on that cell layer (Figure 2K, see
material and method). Strikingly, we found that in the qua1-1
spr2-2 double mutant, cell ﬁles were straight: spr2-induced helical
growth was suppressed (θTof −0.23 ±2.56◦,n=12 samples,
Figures 2E,J). Pairwise Wilcoxon rank sum test was used to test
the diﬀerences between these genotypes. While WS-4, Col-0,
qua1-1, and qua1-1 spr2-2 were not signiﬁcantly diﬀerent from
one another, only spr2-2 was found to be signiﬁcantly diﬀerent
from all the other genotypes (Figure 2L). This suggests that the
mechanical coupling between adjacent cells is indeed required for
the production of twisted hypocotyls in spr2.
qua1-1 spr2-2 Cells Retain the Ability to
Undergo Helical Growth
To explain the restoration of straight growth in qua1-1 spr2-
2, one may invoke alternative hypotheses. For instance, an
unknown genetic interaction between qua1 and spr2 mutations
may compensate the loss of spr2 activity, e.g., by aﬀecting
microtubule dynamics. As mentioned above, the relation between
microtubule dynamics and the helical behavior of their arrays
is still an open question, so we cannot completely exclude
that scenario. Yet, the mechanical uncoupling of adjacent cells
in qua1-1 and qua1-1 spr2-2 oﬀers the unique opportunity to
reveal the contribution of SPR2 to growth direction in semi-
isolated cells. In qua1-1, detached epidermal cells curled outward
from the hypocotyl, as previously reported (Figures 3A,C,D and
Movies S1,S2). More importantly, we observed that these cells
did not exhibit twisted growth, they detached and curled along
their longitudinal axis, showing that the qua1 mutation does not
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Verger et al. Twisting and Adhesion
FIGURE 2 | Loss of cell-cell adhesion prevents hypocotyl twisting in qua1-1 spr2-2.(A–E) Z-projections (maximal intensity) of confocal stacks from representative (12
samples observed in 3 biological replicates for each genotype/condition), propidium iodide stained, four-day old dark-grown hypocotyls. (F–J) Optical sections from
the corresponding stacks from A–E, revealing the ﬁrst cortex cell layer in the hypocotyl (i.e., the layer under the epidermis), following the yellow line drawn in (K).(K) is
an orthogonal section of an hypocotyl showing the epidermal as well as the two cortex cell layers. (A,F) WS-4, (B,G) Col-0, (C,H) qua1-1,(D,I) spr2-2, and (E,J)
qua1-1 spr2-2, highlight the twisting phenotype of spr2-2 as compared to the straight growth of the other genotypes. (L) Boxplot of twisting angle values,
representing each data point and their distribution for each genotype. An angle of 0◦corresponds to no twisting (straight growth), while positive and negative angle
values mark left-handed and right-handed twisting, respectively. Wilcoxon rank sum test ***p<0.0005. Scale bars, 50µm.
aﬀect cell twisting. In the qua1-1 spr2-2 background, epidermal
cells also detached, but they displayed a clear torsion at the single
cell level (Figures 3B,E–H and Movies S3,S4). This phenotype
could be observed on every qua1-1 spr2-2 samples. Note that cells
had to be suﬃciently detached along their axis to exhibit torsion
(see Figure 3B in which one cell is largely detached and is twisted,
whereas surrounding cells exhibit abnormal morphology but are
not twisting on their own as they are not detached from the
epidermis). We never observed cells curling “straight” along the
longitudinal axis of the hypocotyl in the qua1-1 spr2-2 line. This
strongly suggests that the spr2 mutation still promotes helical
growth in the qua1-1 background. Therefore, the mechanical
uncoupling between adjacent cells in qua1 spr2-2 allows the
relaxation of the local torsional stress by single cell, rather than
whole organ, twisting.
Hypocotyl Twisting in qua1-1 spr2-2 Is
Restored Through the Modulation of
Cell-Cell Adhesion Defects
To further conﬁrm that cell-cell adhesion is indeed required
for hypocotyl twisting in spr2-2, we next undertook to restore
adhesion defects in qua1-1 spr2-2 and check whether hypocotyl
twisting would also be restored in these conditions. To do so,
we grew the seedlings on medium containing 2.5% agarose,
instead of 1% agarose (Figures 4K,L). Indeed, increasing agarose
concentration decreases the matrix potential, which in turn
aﬀects plant cell mechanics: water availability to the plant and
tension in the outer wall are reduced, as previously shown
using atomic force microscopy (Verger et al., 2018). In these
conditions, cell-cell adhesion defects were largely rescued in
qua1-1, as previously shown (Verger et al., 2018), consistent with
a scenario in which cracks between cells occur only if tension
in the epidermis is strong enough to pull cells apart (Figure 4).
Therefore, this strategy allowed us to mechanically rescue the
adhesion defects in the qua1-1 spr2-2 double mutant and test its
impact on hypocotyl shape.
When seedlings were grown on medium containing 2.5%
agarose, hypocotyls still exhibited straight cell ﬁles in WS-4
(Mean θTof 0.11 ±1.36◦,n=12 samples, Figures 4A,F), Col-
0 (Mean θTof −0.48 ±2.07◦,n=12 samples, Figures 4B,G)
and qua1-1 (Mean θTof −0.02 ±2.65◦,n=12 samples,
Figures 4C,H). Similarly the spr2-2 mutant still exhibited a
pronounced right-handed helix of cell ﬁles (Mean θTof −9.47
±2.14◦,n=12 samples, Figures 4D,I). However, twisting
growth was almost fully restored in the qua1-1 spr2-2 background
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Verger et al. Twisting and Adhesion
FIGURE 3 | qua1-1 spr2-2 cells retain the ability to undergo helical growth. (A–H) Z-projections (maximal intensity) of confocal stacks from representative, propidium
iodide stained, four-day old dark-grown hypocotyls from (A,C,D) qua1-1 and (B,E–H) qua1-1 spr2-2. Panel C and E are close-ups from (A,B) respectively. (D,F,G,H)
Are additional close-up views from additional qua1-1 (D) and qua1-1 spr2-2 (F,G,H) samples. qua1-1 cells detach and curl along their longitudinal axis, while in the
qua1-1 spr2-2 background, epidermal cells also detach, but they displayed a clear torsion at the single cell level. Scale bars, 30 µm.
(Mean θTof −6.91 ±3.53◦,n=12 samples, Figures 4E,J).
Pairwise Wilcoxon rank sum tests showed that WS-4, Col-0,
qua1-1 were not signiﬁcantly diﬀerent from one another, whereas
spr2-2 and qua1-1 spr2-2 were both signiﬁcantly diﬀerent from
WS-4, Col-0 and qua1-1. Note that spr2-2 and qua1-1 spr2-2
were also signiﬁcantly diﬀerent from each other. This suggests
that the twisting in qua1-1 spr2-2 is not restored up to the
degree observed in spr2-2 (Figure 4M), also consistent with
the observation that cell adhesion defects of qua1-1 in these
conditions are largely rescued but not fully restored (Figure 4E).
Nevertheless, it remains that the mechanical “re-coupling” of
adjacent cells in qua1-1 is suﬃcient to generate a signiﬁcant
impact on twisted growth.
Cell-Cell Adhesion Defects Suppress
Twisted Growth in qua1-1 spr2-2 Leaves
Because hypocotyl may have a rather speciﬁc growth mode
(Gendreau et al., 1997) and involving strong tissue tension
resulting from mechanical conﬂicts between the epidermis and
inner tissues (Kutschera, 1992; Robinson and Kuhlemeier, 2018),
the restoration of straight growth in qua1-1 spr2-2 may be speciﬁc
to the hypocotyl. Furthermore, hypocotyl elongation in the qua1-
1 spr2-2 line was reduced, when compared to spr2-2 mutants
(Figures 5D,E) and this may also contribute to the degree of
hypocotyl twisting. To test whether the mechanical uncoupling
of adjacent cells is suﬃcient to restore straight growth beyond
hypocotyl cells, we grew the double mutant in the greenhouse,
on soil. Indeed, in vitro plants grow in an atmosphere that
is saturated in water, and unless the matrix potential or the
osmolarity of the medium is changed, growth conditions are very
hypo-osmotic, consistent with the dramatic adhesion defects in
qua1-1 mutants on 1% agar. In fact, in these conditions, viable
adult plants cannot be retrieved as the shoot apical meristem also
experience massive disorganization and rather resembles a callus-
like structure (Verger et al., 2018). Plants that are grown and
watered on soil are likely under less hypo-osmotic conditions,
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Verger et al. Twisting and Adhesion
FIGURE 4 | Hypocotyl twisting in qua1-1 spr2-2 is restored on 2.5% agarose medium. (A–E) Z-projections (maximal intensity) of confocal stacks from representative
(12 samples observed in 3 biological replicates for each genotype/condition), propidium iodide stained, four-day old dark-grown hypocotyls. (F–J) Optical sections
from the corresponding stacks. (A,F) WS-4, (B,G) Col-0, (C,H) qua1-1,(D,I) spr2-2, and (E,J) qua1-1 spr2-2, highlight the restoration of twisting phenotype of
qua1-1 spr2-2 to a degree comparable to that of spr2-2.(K,L) Rescue of qua1-1 cell adhesion defect via the modulation of the medium water potential.
(K) Schematic representation of a dark-grown qua1-1 hypocotyl grown on a 1% agarose medium, and displaying extensive cell adhesion defects (Growth conditions
used in Figure 1). (L) Schematic representation of a dark-grown qua1-1 hypocotyl grown on a 2.5% agarose medium, and displaying reduced cell adhesion defects
(Growth conditions used in this ﬁgure). (M) Boxplot of twisting angle values, representing each data point and their distribution for each genotype. An angle of 0◦
reveals no twisting (straight growth), while positive and negative angle values mark left-handed and right-handed twisting, respectively. Wilcoxon rank sum test ***p<
0.0005. Scale bars, 50 µm.
simply because the atmospheric hygrometry is not saturated
in water. Typically, in our greenhouse, we keep hygrometry
at 70%. In such conditions, cell-cell adhesions defects are still
present in qua1-1, but are not as dramatic as in in vitro
plants grown on 1% agar. This allowed us to explore the qua1-
1 spr2-2 phenotype beyond the opened cotyledon stage, and
in particular in leaves and petioles where tissue twisting is
easily detectable. As expected, greenhouse-grown spr2-2 mutants
exhibited twisted leaves (Figure 5). However, such phenotype
was not observed in the qua1-1 spr2-2 double mutant: leaf aspect-
ratio was slightly aﬀected, but leaves remained ﬂat (Figure 5).
Altogether, these results demonstrate that spr2-2 mutant cells
experience a mechanical conﬂict that is resolved through organ
torsion, via the mechanical coupling of adjacent cells.
Although mechanical conﬂicts are thought to be widespread in
developing organisms, their presence is most often only predicted
through computational modeling, or revealed through invasive
mechanical alterations such as laser ablations. Here, using the
qua1-1 spr2-2 double mutant with naturally occurring cell-
cell adhesion defects and twisted cell growth, we reveal that
individual cells tend to undergo torsion, while the restoration
of adhesion prevents single cell torsion but leads to organ
torsion. Therefore, we provide experimental support for the
theory in which organ torsion relaxes the local mechanical
conﬂicts that emerge between adjacent cells with oblique cortical
microtubules, and arguably, oblique cellulose microﬁbrils (Wada
and Matsumoto, 2018).
Note that the picture is actually slightly more complex than
what is described in Figures 1A,B, notably regarding the actual
organization of cortical microtubules and cellulose microﬁbrils
in the hypocotyl. Cortical microtubules and cellulose microﬁbrils
in the epidermis are initially aligned transversely during early
and accelerating growth phases of the dark-grown hypocotyl.
However they then gradually reorient longitudinally in the outer
wall of the epidermis during the rapid and decelerating growth
phase, arguably to resist growth and stress from internal tissues
(Crowell et al., 2011; Robinson and Kuhlemeier, 2018; Verger
et al., 2018), while they remain transverse on the lateral and
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Verger et al. Twisting and Adhesion
FIGURE 5 | Cell-cell adhesion defects suppress twisted growth in qua1-1 spr2-2 leaves. (A–E) Pictures of 2-week old plants grown on soil. (A) WS-4, (B) Col-0,
(C) qua1-1,(D) spr2-2, and (E) qua1-1 spr2-2, highlight the twisting phenotype of spr2-2 as compared to the straight growth of the petiole and leaves for the other
genotypes. Scale bars, 1 cm.
inner wall faces of the epidermal cells. In fact, such diﬀerential
mechanical anisotropy on the diﬀerent faces of the cell could
explain the curling phenotype of the detached cells in qua1-
1(Figures 3A,C,D) as well as the helical shape (rather than
simply twisted shape) of the detached cells in qua1-1 spr2-
2(Figures 3B,E–H). This particular microtubule and cellulose
organization remains compatible with the twisting growth model
proposed by Wada and Matsumoto (Wada and Matsumoto,
2018). Notably, when a genetic mutation, like spr2-2, imposes
oblique cortical microtubule orientations, the resistance of
longitudinal cellulose microﬁbrils in the outer cell wall becomes
less directional, thus leading to twisting.
Organ twisting is also a good system to analyze the balance
between active and passive mechanical response to mechanical
conﬂicts. Indeed, as adjacent cells become separated following
cell-cell adhesion defects, the supracellular propagation of
mechanical signals also becomes impaired. Typically, tensile
stress direction has been proposed to serve as an instructive
cue that provides consistent cortical microtubule alignments
over several cell ﬁles in several plant tissues (Hejnowicz et al.,
2000; Hamant et al., 2008; Robinson and Kuhlemeier, 2018).
Because the epidermis of aerial organs is under tension in
plants, this comes down to a coordinating role of the outer
wall that embeds all epidermal cells. Cell-cell adhesion defects
generate cracks in that outer wall, disrupting the co-alignment
of cortical microtubules (Verger et al., 2018). Therefore, organ
torsion in mutants with microtubule defects requires adhesion
as a passive mediator of mechanical continuity between adjacent
cells, but it may also require adhesion as an active synchronizer
of microtubule behavior through mechanical stress propagation.
In that respect, the identiﬁcation of interactions between certain
wall receptor kinases and pectin (e.g., Feng et al., 2018),
may open the way for an analysis of the interplay between
mechanoperception and adhesion in morphogenesis.
Cell-cell adhesion defects also destroy plasmodesmata
connections, and thus alter the possibility to have large
symplastic domains with consistent growth properties. In
that scenario, isolated cells in adhesion mutants may grow
independently from their neighbors, as clearly shown by the
detached qua1-1 spr2-2 mutant cell morphology. This may have
two eﬀects: cell growth heterogeneity may increase because
neighboring cells would not mutually constrain their growth
anymore, and this would likely result in distorted organ shapes.
In an alternative scenario, growth heterogeneity may decrease,
either because the presence of adjacent cells rather fuels growth
heterogeneity, as observed in shoot apical meristems (Uyttewaal
et al., 2012), or because the supracellular averaging of individual
cells growing at diﬀerent speed may produce more reproducible
organs than large sectors of cells growing at diﬀerent speed,
as shown in sepals (Hong et al., 2016). The ambivalent nature
of mechanical conﬂicts in growth heterogeneity has recently
been analyzed in computer simulations (Fruleux and Boudaoud,
2019). In a more complex scenario, plasmodemata may have a
direct role in organ twisting. Although this is less likely and still
largely hypothetical, carpels were shown to twist in the quirky
mutant (Trehin et al., 2013) and the QUIRKY protein localizes to
plasmodesmata (Vaddepalli et al., 2014). When confronted to our
results, these alternative scenarios are not exclusive. Yet, the idea
that cell-cell adhesion primarily disrupts the passive relaxation
of local mechanical conﬂicts is by far the most parsimonious in
the case of organ torsion.
Finally, we focused here on the spiral2 mutant with a ﬁxed
handedness, which is usually the case for mutants aﬀected in
microtubule functions. There are other ways to induce organ
twisting in Arabidopsis. In particular, mutants aﬀected in auxin
response or transport can exhibit twisted organs too, although the
handedness is not ﬁxed in such cases (Ishida et al., 2007b). More
generally, organ twisting is widespread in Angiosperms, and
this oﬀers several adaptative and evolutive advantages (Smyth,
2016). For instance, growing organs can rapidly twist in order
to reorient relative to light source or gravity ﬁeld in a process
called “helical tropism” (Borchers et al., 2018). Thin vertical
leaves of Typha sp. tend to twist and this has been associated to
increased stability and reduced bending of the leaf in response
to its own weight (Schulgasser and Witztum, 2004); twisted
awns of wheat seeds contribute to their dispersal (Elbaum et al.,
2007); tendrils twist through contraction of internal tissues,
thereby allowing mechanical support (Gerbode et al., 2012).
Understanding organ twisting may thus also have important
ecological and developmental implications.
Frontiers in Plant Science | www.frontiersin.org 7February 2019 | Volume 10 | Article 173
Verger et al. Twisting and Adhesion
SV and ML performed the experiments. SV analyzed the results.
SV and OH wrote the article. OH secured funding for this project.
This work was supported by the European Research Council
We thank our colleagues for their comments and feedback on this
The Supplementary Material for this article can be found
online at: https://www.frontiersin.org/articles/10.3389/fpls.2019.
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Movie S2 | Cell curling in qua1-1. 360 degree rotation from the sample presented
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Movie S3 | Cell curling in qua1-1 spr2-2. 360 degree rotation from the sample
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Movie S4 | Cell curling in qua1-1 spr2-2. 360 degree rotation from the sample
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Conﬂict of Interest Statement: The authors declare that the research was
conducted in the absence of any commercial or ﬁnancial relationships that could
be construed as a potential conﬂict of interest.
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