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Mechanical Conflicts in Twisting Growth Revealed by Cell-Cell Adhesion Defects

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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.
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ORIGINAL RESEARCH
published: 25 February 2019
doi: 10.3389/fpls.2019.00173
Frontiers in Plant Science | www.frontiersin.org 1February 2019 | Volume 10 | Article 173
Edited by:
Kim Johnson,
AgriBio, La Trobe University, Australia
Reviewed by:
René Schneider,
Max-Planck-Institut für Molekulare
Pflanzenphysiologie, Germany
William Barnes,
Pennsylvania State University,
United States
*Correspondence:
Stéphane Verger
stephane.verger@slu.se
Present Address:
Stéphane Verger,
Department of Forest Genetics and
Plant Physiology, Umeå Plant Science
Centre, Swedish University of
Agricultural Sciences, Umeå, Sweden
Specialty section:
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
Citation:
Verger S, Liu M and Hamant O (2019)
Mechanical Conflicts in Twisting
Growth Revealed by Cell-Cell
Adhesion Defects.
Front. Plant Sci. 10:173.
doi: 10.3389/fpls.2019.00173
Mechanical Conflicts in Twisting
Growth Revealed by Cell-Cell
Adhesion Defects
Stéphane Verger*, Mengying Liu and Olivier Hamant
Laboratoire de Reproduction et Développement des Plantes, ENS de Lyon, UCBL, INRA, CNRS, Université de Lyon, Lyon,
France
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
INTRODUCTION
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
partially understood.
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-
3and RV_5-TGAGAGAGTGGAACCATCGG-3.
Growth Conditions
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 20C. 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 20C
for 4 days before imaging.
Cell Wall Staining and Confocal
Microscopy
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
Statistical Analyses
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 0reveals
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.
RESULTS
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 0reveals 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 0corresponds 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.
DISCUSSION
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
AUTHOR CONTRIBUTIONS
SV and ML performed the experiments. SV analyzed the results.
SV and OH wrote the article. OH secured funding for this project.
FUNDING
This work was supported by the European Research Council
(ERC-2013-CoG-615739 MechanoDevo).
ACKNOWLEDGMENTS
We thank our colleagues for their comments and feedback on this
manuscript.
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found
online at: https://www.frontiersin.org/articles/10.3389/fpls.2019.
00173/full#supplementary-material
Movie S1 | Cell curling in qua1-1. 360 degree rotation from the sample presented
in Figure 3C.
Movie S2 | Cell curling in qua1-1. 360 degree rotation from the sample presented
in Figure 3D.
Movie S3 | Cell curling in qua1-1 spr2-2. 360 degree rotation from the sample
presented in Figure 3E.
Movie S4 | Cell curling in qua1-1 spr2-2. 360 degree rotation from the sample
presented in Figure 3G.
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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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Supplementary resources (4)

... These phenotypic traits are alterations caused by increased environmental selection pressure (Burgess et al., 2019;Langer et al., 2022) or heritable variations (Guo et al., 2022). Some genes involved in twisted growth have been identified using model plants, including SmSPR1 (Liu et al., 2021b), WAVY (Abe et al., 2010), spiral1, spiral2 (Verger et al., 2019), lefty1, lefty2 (Thitamadee et al., 2002), and CSI1 (Bringmann et al., 2012). These genes mainly play a regulatory role in microtubule motility. ...
... (Hellgren et al., 2004;Sundberg et al., 1994;Wilson et al., 1989). Verger et al. (2019) found that Arabidopsis organs also exhibit twisted growth when auxin correspondence or transport is affected. Felten et al. (2018) and Seyfferth et al. (2019) found that ethylene also induces tension wood formation. ...
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Trunk twisting, a special phenomenon observed during plant growth, leads to remarkable changes in plant phenotypic plasticity and adaptability. However, it is a debate whether the trunk twisting belongs to environmental alteration or genetic variation. To understand the mechanisms underlying trunk twisting in Yunnan pine (Pinus yunnanensis Franch.), through a diallel cross experiment, we first determined that trunk twisting was controlled by recessive genes. Anatomical analysis identified that straight and twisty types differed significantly in xylem and phloem. RNA-seq of materials enriched by laser microdissection revealed several metabolic pathways with significant enrichment in twisty pines, including auxin signal transduction, photosynthetic carbon fixation and sucrose metabolism, etc. Application of exogenous auxin and auxin transport inhibitors (TIBA) can only change the growth rate, but cannot change the twisted or straight of the trunk. When auxin signaling in-hibitors (auxinole) were added, the straight pines produce a tendency to twisty growth. Combined with enzyme activity assay and immunohistochemistry, we propose a working model. ARF can not only downregulate POR to block chlorophyll synthesis and photosynthesis but also upregulated Susy expression, and allows a large amount of sucrose to synthesize cellulose. Nevertheless, due to downregulated CBH expression and abnormal cellulolysis, cellulose accumulates and the lignin content decreases, eventually making the trunk highly prone to twisted growth. These results reveal the molecular mechanism of trunk twisting, and suggest that ARF expression method can be vital in trunk shape screening during the early growth stages of Yunnan pines.
... RHM1 is involved in the synthesis of homogalacturonan and rhamnogalacturonan-I, abundant pectic polysaccharides in the cell wall [30]. Also, correct adhesion between cells seems to be important to helical growth at the tissue level [31]. The Arabidopsis quasimodo1 mutant has a putative CAZy membrane-bound protein altered [32], displaying cell-cell adhesion defects, which eventually restore straight growth in Arabidopsis spriral2/tortifolia1 mutants that presents right-handed helical growth [31]. ...
... Also, correct adhesion between cells seems to be important to helical growth at the tissue level [31]. The Arabidopsis quasimodo1 mutant has a putative CAZy membrane-bound protein altered [32], displaying cell-cell adhesion defects, which eventually restore straight growth in Arabidopsis spriral2/tortifolia1 mutants that presents right-handed helical growth [31]. This shows that simultaneous occurrence of cell twisting in the same direction in different tissues leads to the rotation 4 Growth and development ...
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Climbing plants have voluble organs, for example, tendrils and modified stems, which twine up neighboring plants to reach the canopy. These organs perform exaggerated circumnutation, during which they grow towards the shaded areas of the forest (skototropism) to find a host. In response to mechanical stimulus, they grow towards the support (thigmotropism), tailoring their development to firmly attach to it (thigmomorphogenesis). The underlying molecular pathways of these crucial mechanisms are virtually unknown. Here, we review current progress on molecular regulation of the development and growth of climber’s voluble organs. Recent advances in the subject point epigenetics and sensory biology as the emerging frontiers in the study of climbing plant’s growth and functioning. We briefly review new developments on the molecular basis of plants’ mechanosensory system, discussing the findings in the context of the climbing habit.
... Therefore, increase expression of auxin transport-related genes is considered the most important cause of twisted growth (Yu et al., 2016). Verger et al. (2019) found thatArabidopsis organs also exhibit twisted growth when auxin correspondence or transport is affected. Felten et al. (2018) and Seyfferth et al. (2019) found that ethylene also induces tension wood formation. ...
Preprint
It is a debate whether trunk twisting belongs to environmental alteration or genetic variation. Through a diallel cross experiment, we first determined that trunk twisting of Yunnan pines was controlled by recessive genes. Anatomical analysis identified that straight and twisty types differed in xylem and phloem. RNA-seq of materials enriched by laser microdissection revealed three genes involved in auxin signal transduction, photosynthesis, and sucrose metabolism, namely ARF , POR , and CBH. These genes were co-expressed at different growth stages of twisty types, and among them, ARF is crucial regulating trunk twisting formation. The enzyme activities involved in sucrose metabolism, carbon fixation, and glycolysis were significantly increased after exogenous auxin was added to twisty types. When auxin signal transduction inhibitor (auxinole) and transport inhibitor (TIBA) were added, the plant height and related pathways were more obviously reduced in straight types. ARF can not only downregulate POR to block chlorophyll synthesis but also allows abundant sucrose to synthesize cellulose. Nevertheless, due to downregulated CBH expression and abnormal cellulolysis, cellulose accumulates and the lignin content decreases, eventually making the trunk highly prone to twisted growth. This study suggests that ARF can be vital in trunk shape screening during the early growth of Yunnan pines.
... However, the double mutant no longer has curled leaves and the results also suggest that qua2-1 and WAK2cTAP impact common pathways, and this is what one would expect for an allele of a biosynthetic enzyme and a receptor bound to the product of that enzyme. The loss of leaf curling in the double mutant might be linked to a loss of cell adhesion that could suppress the twisting phenotypes, most likely preventing supracellular mechanical coupling of adjacent cells [41]. The levels of ROS and FADlox expression in the double mutant were also measured, and the results are shown in Fig 4C. ...
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Full-text available
Background Many biological processes follow circadian rhythmicity and are controlled by the circadian clock. Predictable environmental changes such as seasonal variation in photoperiod can modulate circadian rhythms, allowing organisms to adjust the timing of their biological processes to the time of the year. In some crops such as rice, barley or soybean, mutations in circadian clock genes have altered photoperiod sensitivity, enhancing their cultivability in specific seasons and latitudes. However, how changes in circadian rhythms interact with the perception of photoperiod in crops remain poorly studied. In tomato, the appearance during domestication of mutations in EMPFINDLICHER IM DUNKELROTEN LICHT 1 ( EID1 , Solyc09g075080) and NIGHT LIGHT-INDUCIBLE AND CLOCK-REGULATED GENE 2 ( LNK2 , Solyc01g068560) delayed both the phase and period of its circadian rhythms. The fact that variation in period and phase are separated in tomato provides an optimal tool to study how these factors affect the perception of photoperiod. Results Here we develop tomato near isogenic lines carrying combinations of wild alleles of EID1 and LNK2 and show that they recreate the changes in phase and period that occurred during its domestication. We perform transcriptomic profiling of these near isogenic lines under two different photoperiods, and observe that EID1, but not LNK2, has a large effect on how the tomato transcriptome responds to photoperiod. This large effect of EID1 is likely a consequence of the global phase shift elicited by this gene in tomato's circadian rhythms. Conclusions Our study shows that changes in phase that occurred during tomato domestication determine photoperiod perception in this species, while changes in period have little effect.
... However, the double mutant no longer has curled leaves and the results also suggest that qua2-1 and WAK2cTAP impact common pathways, and this is what one would expect for an allele of a biosynthetic enzyme and a receptor bound to the product of that enzyme. The loss of leaf curling in the double mutant might be linked to a loss of cell adhesion that could suppress the twisting phenotypes, most likely preventing supracellular mechanical coupling of adjacent cells [41]. The levels of ROS and FADlox expression in the double mutant were also measured, and the results are shown in Fig 4C. ...
Article
Full-text available
Angiosperm cell adhesion is dependent on interactions between pectin polysaccharides which make up a significant portion of the plant cell wall. Cell adhesion in Arabidopsis may also be regulated through a pectin-related signaling cascade mediated by a putative O-fucosyltransferase ESMERALDA1 (ESMD1), and the Epidermal Growth Factor (EGF) domains of the pectin binding Wall associated Kinases (WAKs) are a primary candidate substrate for ESMD1 activity. Genetic interactions between WAKs and ESMD1 were examined using a dominant hyperactive allele of WAK2, WAK2cTAP , and a mutant of the putative O-fucosyltransferase ESMD1. WAK2cTAP expression results in a dwarf phenotype and activation of the stress response and reactive oxygen species (ROS) production, while esmd1 is a suppressor of a pectin deficiency induced loss of adhesion. Here we find that esmd1 suppresses the WAK2cTAP dwarf and stress response phenotype, including ROS accumulation and gene expression. Additional analysis suggests that mutations of the potential WAK EGF O-fucosylation site also abate the WAK2cTAP phenotype, yet only evidence for an N-linked but not O-linked sugar addition can be found. Moreover, a WAK locus deletion allele has no effect on the ability of esmd1 to suppress an adhesion deficiency, indicating WAKs and their modification are not a required component of the potential ESMD1 signaling mechanism involved in the control of cell adhesion. The WAK locus deletion does however affect the induction of ROS but not the transcriptional response induced by the elicitors Flagellin, Chitin and oligogalacturonides (OGs).
... There is now evidence that mechanical conflicts can be much more local, as in between adjacent cells growing at different rates. For instance, twisting in hypocotyls emerges from conflicts in growth patterns between cell files 134 , and these can be resolved by impairing cell-cell adhesion in qua1 135 . In the sepal, fast-growing cells, like trichome precursors, generate circumferential mechanical stress in adjacent cells. ...
Article
Plants produce organs of various shapes and sizes. While much has been learned about genetic regulation of organogenesis, the integration of mechanics in the process is also gaining attention. Here, we consider the role of forces as instructive signals in organ morphogenesis. Turgor pressure is the primary cause of mechanical signals in developing organs. Because plant cells are glued to each other, mechanical signals act, in essence, at multiple scales, through cell wall contiguity and water flux. In turn, cells use such signals to resist mechanical stress, for instance, by reinforcing their cell walls. We show that the three elemental shapes behind plant organs — spheres, cylinders and lamina — can be actively maintained by such a mechanical feedback. Combinations of this 3-letter alphabet can generate more complex shapes. Furthermore, mechanical conflicts emerge at the boundary between domains exhibiting different growth rates or directions. These secondary mechanical signals contribute to three other organ shape features — folds, shape reproducibility and growth arrest. The further integration of mechanical signals with the molecular network offers many fruitful prospects for the scientific community, including the role of proprioception in organ shape robustness or the definition of cell and organ identities as a result of an interplay between biochemical and mechanical signals.
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Plant cells and organs grow into a remarkable diversity of shapes, as directed by cell walls composed primarily of polysaccharides such as cellulose and multiple structurally distinct pectins. The properties of the cell wall that allow for precise control of morphogenesis are distinct from those of the individual polysaccharide components. For example, cellulose, the primary determinant of cell morphology, is a chiral macromolecule that can self-assemble in vitro into larger-scale structures of consistent chirality, and yet most plant cells do not display consistent chirality in their growth. One interesting exception is the Arabidopsis thaliana rhm1 mutant, which has decreased levels of the pectin rhamnogalacturonan-I and causes conical petal epidermal cells to grow with a left-handed helical twist. Here, we show that in rhm1 the cellulose is bundled into large macrofibrils, unlike the evenly distributed microfibrils of the wild type. This cellulose bundling becomes increasingly severe over time, consistent with cellulose being synthesized normally and then self-associating into macrofibrils. We also show that in the wild type, cellulose is oriented transversely, whereas in rhm1 mutants, the cellulose forms right-handed helices that can account for the helical morphology of the petal cells. Our results indicate that when the composition of pectin is altered, cellulose can form cellular-scale chiral structures in vivo, analogous to the helicoids formed in vitro by cellulose nano-crystals. We propose that an important emergent property of the interplay between rhamnogalacturonan-I and cellulose is to permit the assembly of nonbundled cellulose structures, providing plants flexibility to orient cellulose and direct morphogenesis.
Article
Pectin, cellulose, and hemicelluloses are major components of primary cell walls in plants. In addition to cell adhesion and expansion, pectin plays a central role in seed mucilage. Seed mucilage contains abundant pectic rhamnogalacturonan-I (RG-I) and lower amounts of homogalacturonan (HG), cellulose, and hemicelluloses. Previously, accumulated evidence has addressed the role of pectin RG-I in mucilage production and adherence. However, less is known about the function of pectin HG in seed coat mucilage formation. In this study, we analyzed a novel mutant, designated things fall apart2 (tfa2), which contains a mutation in HG methyltransferase QUASIMODO2 (QUA2). Etiolated tfa2 seedlings display short hypocotyls and adhesion defects similar to qua2 and (tumorous shoot development2) tsd2 alleles, and show seed mucilage defects. The diminished uronic acid content and methylesterification degree of HG in mutant seed mucilage indicate the role of HG in the formation of seed mucilage. Cellulosic rays in mutant mucilage are collapsed. The epidermal cells of seed coat in tfa2 and tsd2 display deformed columellae and reduced radial wall thickness. Under polyethylene glycol treatment, seeds from these three mutant alleles exhibit reduced germination rates. Together, these data emphasize the requirement of pectic HG biosynthesis for the synthesis of seed mucilage, and the functions of different pectin domains together with cellulose in regulating its formation, expansion, and release.
Article
Plant morphology emerges from cellular growth and structure. The turgor-driven diffuse growth of a cell can be highly anisotropic: significant longitudinally and negligible radially. Such anisotropy is ensured by cellulose microfibrils (CMF) reinforcing the cell wall in the hoop direction. To maintain the cell’s integrity during growth, new wall material including CMF must be continually deposited. We develop a mathematical model representing the cell as a cylindrical pressure vessel and the cell wall as a fibre-reinforced viscous sheet, explicitly including the mechano-sensitive angle of CMF deposition. The model incorporates interactions between turgor, external forces, CMF reorientation during wall extension, and matrix stiffening. Using the model, we reinterpret some recent experimental findings, and reexamine the popular hypothesis of CMF/microtubule alignment. We explore how the handedness of twisting cell growth depends on external torque and intrinsic wall properties, and find that cells twist left-handedly ‘by default’ in some suitable sense. Overall, this study provides a unified mechanical framework for understanding left- and right-handed twist-growth as seen in many plants.
Article
In plants, although KNOX genes are known to regulate secondary cell wall (SCW) formation, their protein-regulating mechanisms remain largely unknown. Here, we showed that GhKNL1, which regulates SCW formation and fiber development in cotton, could interact with an IQ67 domain containing protein (GhIQD14) in yeast. Confocal observation showed that GhIQD14 was localized to the microtubules. In Arabidopsis, ectopic expression of GhIQD14 caused hypocotyls to be sensitive to microtubule depolymerization agent, organ twisting of seedlings, trichomes, rosette leaves, and capsules, as well as severely irregular xylem vessels and thicker interfascicular fiber cell walls in the inflorescence stem. Furthermore, we found that GhIQD14 interacted with AtKNAT7 in yeast, and instantaneous co-expression of GhIQD14 and AtKNAT7 in tobacco showed that GhIQD14 weakened the distribution of AtKNAT7 in the nucleus, bringing it into the microtubules, thus affecting the SCW formation related genes expression. Our results suggested that GhIQD14 might be involved in the morphological development and SCW formation in cotton.
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The role of mechanical signals in cell identity determination remains poorly explored in tissues. Furthermore, because mechanical stress is widespread, mechanical signals are difficult to uncouple from biochemical-based transduction pathways. Here we focus on the homeobox gene SHOOT MERISTEMLESS (STM), a master regulator and marker of meristematic identity in Arabidopsis. We found that STM expression is quantitatively correlated to curvature in the saddle-shaped boundary domain of the shoot apical meristem. As tissue folding reflects the presence of mechanical stress, we test and demonstrate that STM expression is induced after micromechanical perturbations. We also show that STM expression in the boundary domain is required for organ separation. While STM expression correlates with auxin depletion in this domain, auxin distribution and STM expression can also be uncoupled. STM expression and boundary identity are thus strengthened through a synergy between auxin depletion and an auxin-independent mechanotransduction pathway at the shoot apical meristem.
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The embryonic gut tube is a cylindrical structure from which the respiratory and gastrointestinal tracts develop¹. Although the early emergence of the endoderm as an epithelial sheet2,3 and later morphogenesis of the definitive digestive and respiratory organs4–6 have been investigated, the intervening process of gut tube formation remains relatively understudied7,8. Here we investigate the molecular control of macroscopic forces underlying early morphogenesis of the gut tube in the chick embryo. The gut tube has been described as forming from two endodermal invaginations—the anterior intestinal portal (AIP) towards the rostral end of the embryo and the caudal intestinal portal (CIP) at the caudal end—that migrate towards one another, internalizing the endoderm until they meet at the yolk stalk (umbilicus in mammals)1,6. Migration of the AIP to form foregut has been descriptively characterized8,9, but the hindgut is likely to form by a distinct mechanism that has not been fully explained¹⁰. We find that the hindgut is formed by collective cell movements through a stationary CIP, rather than by movement of the CIP itself. Further, combining in vivo imaging, biophysics and mathematical modelling with molecular and embryological approaches, we identify a contractile force gradient that drives cell movements in the hindgut-forming endoderm, enabling tissue-scale posterior extension of the forming hindgut tube. The force gradient, in turn, is established in response to a morphogenic gradient of fibroblast growth factor signalling. As a result, we propose that an important positive feedback arises, whereby contracting cells draw passive cells from low to high fibroblast growth factor levels, recruiting them to contract and pull more cells into the elongating hindgut. In addition to providing insight into the early gut development, these findings illustrate how large-scale tissue level forces can be traced to developmental signals during vertebrate morphogenesis.
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Mechanical forces have emerged as coordinating signals for most cell functions. Yet, because forces are invisible, mapping tensile stress patterns in tissues remains a major challenge in all kingdoms. Here we take advantage of the adhesion defects in the Arabidopsis mutant quasimodol (qual) to deduce stress patterns in tissues. By reducing the water potential and epidermal tension in planta, we rescued the adhesion defects in qua1, formally associating gaping and tensile stress patterns in the mutant. Using suboptimal water potential conditions, we revealed the relative contributions of shape- and growth-derived stress in prescribing maximal tension directions in aerial tissues. Consistently, the tension patterns deduced from the gaping patterns in qual matched the pattern of cortical microtubules, which are thought to align with maximal tension, in wild-type organs. Conversely, loss of epidermis continuity in the qual mutant hampered supracellular microtubule alignments, revealing that coordination through tensile stress requires cell-cell adhesion.
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Although rather inconspicuous, movements are an important adaptive trait of plants. Consequently, light- or gravity-induced movements leading to organ bending have been studied intensively. In the field, however, plant movements often result in organ twisting rather than bending. This study investigates the mechanism of light- or gravity-induced twisting movements, coined “helical tropisms.” Because certain Arabidopsis cell expansion mutants show organ twisting under standard growth conditions, we here investigated how the right-handed helical growth mutant tortifolia1/spiral2 (tor1) responds when stimulated to perform helical tropisms. When leaves were illuminated from the left, tor1 was capable of producing left-handed petiole torsions, but these occurred at a reduced rate. When light was applied from right, tor1 plants rotated their petioles much faster than the wild-type. Applying auxin to the lateral-distal side of wild-type petioles produced petiole torsions in which the auxinated flank was consistently turned upwards. This kind of movement was not observed in tor1 mutants when auxinated to produce left-handed movements. Investigating auxin transport in twisting petioles based on the DR5-marker suggested that auxin flow was apical-basal rather than helical. While cortical microtubules of excised wild-type petioles oriented transversely when stimulated with auxin, those of tor1 were largely incapable of reorientation. Together, our results show that tor1 is a tropism mutant and suggest a mechanism in which auxin and microtubules both contribute to helical tropisms.
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Significance The two hands of most humans almost superimpose. Likewise, flowers of an individual plant have similar shapes and sizes. This is in striking contrast with growth and deformation of cells during organ morphogenesis, which feature considerable variations in space and in time, raising the question of how organs and organisms reach well-defined sizes and shapes. To link cell and organ scales, we built a theoretical model of growing tissue with fiber-like structural elements that may account for animal cytoskeleton or extracellular matrix, or for the plant cell wall. We show that the response of fibers to growth-induced mechanical stress may enhance or reduce cellular variability of growth, making it possible to modulate the robustness of morphogenesis.
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Plant cells are enclosed in cell walls that weld them together, meaning that cells rarely change neighbours. Nonetheless, invasive growth events play critical roles in plant development and are often key hubs for the integration of environmental and/or developmental signalling. Here we review cellular processes involved in three such events: lateral root emergence, pollen tube growth through stigma and style tissues, and embryo expansion through the endosperm (Figures 1-3). We consider processes such as regulation of water fluxes and cell turgor (driving growth), cell wall modifications (e.g. cell separation) and cell death (for creating space) within these three contexts with the aim of identifying key mechanisms implicated in providing a chemical and biophysical environments permitting invasive growth events.
Chapter
One of the fundamental problems in plant morphogenesis is the molecular and cellular basis of left-right asymmetry that often leads to various chiral structures such as the coils of tendrils and twisted leaves. The twisting mutants of the Arabidopsis roots and hypocotyl exhibit a helical pattern of epidermal cell files with a handedness that is opposite to that of the underlying cortical microtubule arrays in the epidermis. These mutants offer the unique opportunity to investigate the genetic basis of twisting in plants, particularly in the context of cortical microtubules. In this chapter, we address the importance of large-scale mechanical forces to understand the mechanism of this hierarchical helical order, with a particular emphasis on the role of tissue tension combined with the stresses generated by differential growth. Physical processes such as elasticity and geometry might be important factors to coordinate the chirality across different length scales and to organize an entire plant body.
Article
Plants are able to sense external mechanical stress, such as those due to gravity or obstacles, and alter their growth accordingly [1–8]. Like animals [9, 10], plants can also sense internal mechanical stress that plays a role in regulating their development [11–19]. The internal mechanical stresses also known as tissue stress can result from geometry, cell type, or differential growth [19–21]. In a number of tissues, microtubules have been observed to align with mechanical stress predicted from their geometry. In the unidirectionally growing hypocotyl, the predicted tissue stresses do not reflect its cylindrical geometry. The epidermal layer experiences and resists the tensile stress coming from the expansion of the inner layers [22, 23]; this is known as the epidermal-growth-control hypothesis. Here, we use our recently developed automated confocal micro-extensometer (ACME) [24] to apply relative compressive or tensile stresses to the intact Arabidopsis hypocotyls while monitoring growth and microtubule orientation in the different layers. A finite element model revealed that under relative tension, the pattern of tissue stresses was similar to that in the intact growing hypocotyl, while when relative compression was applied, the pattern of tissue stresses was overcome and the maximum stress direction in the epidermis changed to reflect what one would predict based on the geometry of the hypocotyl. Consistent with this, the microtubules in the epidermis changed orientation under relative compression. Once the direction of stress in the epidermis was altered, the growth of the organ increased. Seedling growth may be constrained by obstructions. Robinson and Kuhlemeier mimic this by applying compressive stresses to seedlings. They show that the normal pattern of tissue stress is such that the outer layer restricts growth. When constrained, the direction of tensile stress in the outer layer reverses, and growth is promoted.
Article
The contribution of microtubule tip dynamics to the assembly and function of plant microtubule arrays remains poorly understood. Here, we report that the Arabidopsis SPIRAL2 (SPR2) protein modulates the dynamics of the acentrosomal cortical microtubule plus and minus ends in an opposing manner. Live imaging of a functional SPR2-mRuby fusion protein revealed that SPR2 shows both microtubule plus- and minus-end tracking activity in addition to localization at microtubule intersections and along the lattice. Analysis of microtubule dynamics showed that cortical microtubule plus ends rarely undergo catastrophe in the spr2-2 knockout mutant compared to wild-type. In contrast, cortical microtubule minus ends in spr2-2 depolymerized at a much faster rate than in wild-type. Destabilization of the minus ends in spr2-2 caused a significant decrease in the lifetime of microtubule crossovers, which dramatically reduced the microtubule-severing frequency and inhibited light-induced microtubule array reorientation. Using in vitro reconstitution experiments combined with single-molecule imaging, we found that recombinant SPR2-GFP intrinsically localizes to microtubule minus ends, where it binds stably and inhibits their dynamics. Together, our data establish SPR2 as a new type of microtubule tip regulator that governs the length and lifetime of microtubules. Fan et al. show that SPIRAL2 (SPR2) localizes at the acentrosomal cortical microtubule plus and minus ends and modulates their dynamics in an opposing manner, thus influencing the lifetime of microtubule crossovers and severing frequency. In vitro reconstitution experiments show that SPR2-GFP binds stably to minus ends and inhibits their dynamics.
Article
Development is remarkably reproducible, producing organs with the same size, shape, and function repeatedly from individual to individual. For example, every flower on the Antirrhinum> stalk has the same snapping dragon mouth. This reproducibility has allowed taxonomists to classify plants and animals according to their morphology. Yet these reproducible organs are composed of highly variable cells. For example, neighboring cells grow at different rates in Arabidopsis leaves, sepals, and shoot apical meristems. This cellular variability occurs in normal, wild-type organisms, indicating that cellular heterogeneity (or diversity in a characteristic such as growth rate) is either actively maintained or, at a minimum, not entirely suppressed. In fact, cellular heterogeneity can contribute to producing invariant organs. Here, we focus on how plant organs are reproducibly created during development from these highly variable cells. Expected final online publication date for the Annual Review of Plant Biology Volume 69 is April 29, 2018. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.