Current Biology 24, 931–940, May 5, 2014 ª2014 Elsevier Ltd All rights reserved
http://dx.doi.org/10.1016/j.cub.2014.03.025
Article
Programmed Cell Death Controlled
by ANAC033/SOMBRERO Determines
Root Cap Organ Size in Arabidopsis
Fendrych,1,2,7 Tom Van Hautegem,1,2,7
Matyás
Matthias Van Durme,1,2 Yadira Olvera-Carrillo,1,2
Marlies Huysmans,1,2 Mansour Karimi,1,2
Saskia Lippens,3,4,5 Christopher J. Guérin,3,4,5
Melanie Krebs,6 Karin Schumacher,6
and Moritz K. Nowack1,2,*
1Department of Plant Systems Biology, VIB, 9052 Ghent,
Belgium
2Department of Plant Biotechnology and Bioinformatics,
Ghent University, 9052 Ghent, Belgium
3Bio Imaging Core, VIB, 9052 Ghent, Belgium
4Inflammation Research Center, VIB, 9052 Ghent, Belgium
5Department of Biomedical Molecular Biology, Ghent
University, 9052 Ghent, Belgium
6Plant Developmental Biology, Centre for Organismal Studies,
University of Heidelberg, 69120 Heidelberg, Germany
Summary
Background: The root cap is a plant organ that ensheathes the
meristematic stem cells at the root tip. Unlike other plant organs, the root cap shows a rapid cellular turnover, balancing
constant cell generation by specific stem cells with the
disposal of differentiated cells at the root cap edge. This
cellular turnover is critical for the maintenance of root cap
size and its position around the growing root tip, but how
this is achieved and controlled in the model plant Arabidopsis
thaliana remains subject to contradictory hypotheses.
Results: Here, we show that a highly organized cell death program is the final step of lateral root cap differentiation and that
preparation for cell death is transcriptionally controlled by
ANAC033/SOMBRERO. Precise timing of cell death is critical
for the elimination of root cap cells before they fully enter the
root elongation zone, which in turn is important in order to
allow optimal root growth. Root cap cell death is followed by
a rapid cell-autonomous corpse clearance and DNA fragmentation dependent on the S1-P1 type nuclease BFN1.
Conclusions: Based on these results, we propose a novel
concept in plant development that recognizes programmed
cell death as a mechanism for maintaining organ size and tissue homeostasis in the Arabidopsis root cap.
Introduction
Programmed cell death (PCD) comprises different molecular
pathways of actively controlled, genetically encoded cellular
suicide. Plants make use of PCD processes during development and immune responses, just as animals do. The plant’s
hypersensitive response, accompanied by PCD, is part of the
plant’s immune system and is thought to act against invading
pathogens [1]. Furthermore, PCD is part of a plant’s reaction
to diverse abiotic environmental stresses [2]. Finally, developmental PCD is part of the genetically controlled differentiation
7Co-first
author
*Correspondence: [email protected]
program of a number of tissues, disposing of unwanted, superfluous, or no longer required cells in a controlled way [3].
In doing so, correctly executed PCD is often essential for the
functionality of certain tissues or cell types (e.g., the watertransporting xylem or the pollen-nurturing tapetum layer [4,
5]). There is only limited conservation in the molecular control
of animal and plant PCD, and homologs of Bcl-2 proteins or
caspases have not been identified in plant genomes [6]. Given
the presence of cell walls and the lack of apoptotic regulators
and phagocytic processes in plants, PCD and the subsequent
removal of the cell corpse have to be executed in a distinctive,
plant-specific way. A number of cytological events, such as
DNA fragmentation, plasma membrane (PM) permeabilization,
and collapse of the tonoplast-lined central vacuole, have
been associated with developmental PCD, but, in comparison
with animal apoptosis, we still know little about the molecular
network controlling plant PCD [7].
The root cap is a specialized organ surrounding the growing
root tip. It has important functions for root growth and root
system architecture, including gravity sensing and protection
of the stem cell pool in the root tip [8]. In Arabidopsis thaliana,
the root cap consists of two distinct tissues: the centrally situated columella root cap and the peripherally located lateral
root cap (LRC), which flanks both the columella and the root
meristem (see Figure 1A) [9]. By formative division, a ring of
specific stem cells continually generates both new LRC cells
and root epidermis cells [9]. However, although cell proliferation is generally known to increase plant organ size [10], the
size of the LRC remains constant [8]. To maintain LRC organ
size, LRC cells that reach the distal edge of the organ continually have to be eliminated. Conflicting hypotheses exist on
how this elimination is achieved in Arabidopsis: some recent
reports assume that differentiated LRC cells are sloughed
off into the rhizosphere [11, 12], whereas others imply the
participation of a cell death process [13, 14]. Nevertheless,
no detailed analysis of the ultimate fate of LRC cells in
Arabidopsis exists to date.
Here, we show that a highly organized PCD process is the
final step of LRC development and that it is transcriptionally
controlled by ANAC033/SOMBRERO. Timely PCD execution
is decisive for optimal root growth because it eliminates root
cap cells before they fully enter the root elongation zone.
Root cap PCD comprises a rapid cell-autonomous corpse
clearance characterized by DNA fragmentation, which is mediated by the S1-P1 type nuclease BFN1.
Results
Cell Death Links LRC Organ Size with Root Meristem Size
In order to test the hypothesis that LRC cells undergo cell
death, we compared publicly available Arabidopsis transcriptome profiles of the LRC with those of maturing xylem [15, 16],
which is known to undergo developmental PCD [17].
Two genes were highly coregulated in both tissues: the established senescence- and cell-death-associated S1-P1 nuclease
BFN1 [18] and the poorly characterized aspartic protease
PASPA3 [19]. By analyzing promoter-reporter constructs, we
found specific PASPA3 expression in cell types and tissues
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the root periphery. LRC cells first divide and then differentiate
and elongate until the LRC reaches the transition zone (TZ) at
the end of the meristem [13] (Figure 1A). The most-distal cell
death zone (PCD site I; Figures 1A and 1C) stably remained
in the TZ, tightly linking LRC size to meristem size (Figure 1B).
As if they were on a conveyor belt, the cells of each LRC file
steadily approached the end of the TZ while increasing
PASPA3 expression and then finally died (Figure S1H; Movie
S2). The constant recruitment of new LRC cells into the
PASPA3 expression zone initially compensated for the loss
of distal LRC cells, creating a stable zone of PASPA3 expression over the TZ (Movie S2). Eventually, however, due to the
limited number of LRC cells in a file, cell death progressed
cell by cell toward the root tip (PCD site II; Figures 1A and
1C). By then, the underlying, younger LRC layer had reached
the TZ and, in turn, initiated PASPA3 expression and cell
death, reestablishing PCD site I through an iterative turnover
of LRC layers. When cell death within the oldest LRC layers
finally approached its proximal end at the columella, the last
dying LRC cells lost contact with the root (Figure S1E). This
detachment of entire packets of columella cells with some
attached LRC cells has been frequently observed [12], explaining the widespread notion of a general shedding of living
LRC cells in A. thaliana. The turnover of LRC layers created
a PASPA3 expression pattern that became more complex
with increasing plant age (Figure S1E). Although the PASPA3
marker was not expressed in the embryonic root, it initially
marked only a single PCD zone in young roots; however, in
older roots, progressively more cell death sites appeared.
We observed a similar pattern in emerging lateral roots, with
PASPA3 expression only appearing when the lateral root had
grown about 500 mm in length (Figures S1F and S1G).
Figure 1. LRC Cells Die on the Surface of the A. thaliana Root
(A) New LRC layers (bright yellow) are generated by stem cells (gray) close to
the quiescent center (QC), displacing older layers (dark yellow) to the root
periphery. Within each LRC layer, PASPA3 marker expression (green) precedes cell death (red), which starts when the cells reach the end of the transition zone (TZ; PCD site I) and then moves toward the root tip (PCD site II).
(B) The position of PCD site I is correlated with the end of the TZ in roots of
different ages. The distance of PI-positive cells from the QC is plotted
against the distance of the first elongated epidermal cell from the QC. See
also Figure S1.
(C and D) The proPASPA3::ToIM marker labels the cytoplasm with GFP
and the vacuole with mRFP. Tonoplast rupture causes merging of the two
signals (arrowheads). Medial optical section in (C) and maximal z section
projection in (D) are generated from the same root.
Scale bar represents 50 mm. das, days after sowing.
undergoing developmentally controlled cell death (Figures
S1A–S1D available online). Both proBFN1 and proPASPA3 reporter constructs were also expressed in specific zones of the
LRC, suggesting the existence of a cell death process (Figures
1C, 1D, and S5). To monitor vacuolar collapse as a hallmark of
plant PCD, we designed a genetically encoded tonoplast
integrity marker driven by proPASPA3 (proPASPA3::ToIM).
This marker targets GFP to the cytoplasm and monomeric
red fluorescent protein (mRFP) to the vacuole, and, after
tonoplast rupture, the two signals merge. Live-cell imaging
of proPASPA3::ToIM revealed an abrupt vacuolar collapse in
the most-distal cells of the PASPA3 expression zones (Figures
1C and 1D) and a subsequent rapid disintegration of these
cells on the root surface (Movie S1).
These data suggest that cell death is an inherent part of
A. thaliana LRC development. During this process, new LRC
layers repeatedly displace the next, older LRC layers toward
LRC Cell Death Is a Highly Organized and Rapidly Executed
Cellular Process
The predictability of LRC cell death and its occurrence at the
actual root surface enabled us to analyze subcellular processes during cell death in situ and in vivo with high temporal
resolution. The first sign of cell death preparation was the
onset of PASPA3 expression more than 10 hr before cell death
(Figures 2A and 2B). Vacuolar collapse causing cytoplasm
acidification and release of hydrolases is believed to execute
cell clearance in plant cells [20]. To monitor the cytoplasmic
pH of LRC cells during cell death, we analyzed a pH-sensitive
GFP variant, pHGFP [21]. Surprisingly, we detected a sharp
drop in cytoplasmic pH well before vacuolar collapse (at about
15 min before PM permeabilization was visualized by propidium iodide [PI] entry into the cell) (Figures 2C and 2D). To
test the significance of the pH drop in cell death progression,
we manipulated the extracellular and intracellular pH and
quantified the cell death frequency of LRC cells. An increase
of extracellular or intracellular pH to 7 inhibited cell death,
whereas a decrease to pH 5.8 increased the frequency of
LRC cell death (Figures 2E, S2A, and S2B). This demonstrated
that cytoplasmic acidification was both sufficient and necessary to trigger cell death specifically in terminally differentiated
LRC cells, hinting at the presence of a pH-activated cell death
mechanism in these cells. Following the pH drop, the PM
became permeable to PI, which we arbitrarily designated as
the point to mark cellular death. Surprisingly, the central vacuole collapsed 12 min after PI entry on average (Figures 2F, 2G,
and S2C), which stands in contrast to established plant PCD
systems that are thought to be initialized, rather than followed,
by vacuolar collapse [22]. The actual LRC cell death execution
Arabidopsis Root Cap Cell Death
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Figure 2. LRC Cells Die in an Organized Succession of Cellular Events
(A) Kymograph of two proPASPA3>>H2A-GFPexpressing LRC cells. GFP intensity increases
and the cells elongate and finally die, followed
by tonoplast rupture (arrowheads).
(B) Quantification of proPASPA3 activity (normalized GFP intensity) in three LRC cells over time.
(C) Kymograph showing the cytosolic pH drop
(green arrowhead) and the PI entrance (red arrowhead) into an LRC cell; the 405 nm and 488 nm
excitations of pHGFP are shown in green and
red, respectively, and the PI is shown in white.
(D) Quantification of the cytosolic pH and PI accumulation during cell death, with the 405:488 nm
emission ratio of pHGFP (green) and PI intensities
(red); time 0 = moment of pH drop; n = 10 LRC
cells.
(E) Quantification of PI-positive LRC cells in a
45 min time-lapse experiment. Adjusting extracellular (e7) and intracellular (c7) pH to 7 decreases the frequency of LRC cell death, whereas
an extracellular (e5.8) and intracellular (c5.8) pH
of 5.8 increases the frequency of cell death.
ANOVA-Poisson distribution model, *p < 0.01,
***p < 0.0001; n = 11–17 time-lapse experiments
per treatment. See also Figure S2.
(F) Kymograph of proPASPA3>>H2A-GFP
showing PI entry (red arrowhead) about 12 min
before tonoplast rupture (white arrowhead). See
also Figure S2.
(G) Quantification of H2A-GFP showing that GFP intensity drops prior to PI entry, probably due to quenching of GFP fluorescence by the low pH. The boxplot
indicates the time of tonoplast rupture; time 0 = moment of PI entry. n = 8 LRC cells for the graph; n = 22 LRC cells for tonoplast rupture.
In all graphs, error bars show SD. a.u., arbitrary unit.
proceeded remarkably fast, taking less than 30 min to turn an
apparently intact cell into a cell corpse.
LRC Cell Death Is Followed by Rapid Cell Corpse Clearance
Following vacuolar collapse, PI-positive LRC cell corpses
were rapidly degraded in a cell-clearance zone located distally
to the cell death zone (Figures 1A, 3A, and 3B). We used serial
block face-scanning electron microscopy (SBF-SEM) that has
the capability for 3D electron microscopic imaging [23]. SBFSEM revealed a sharp difference between the last living cell
and the first dead cell, in which most of the cell content was
already degraded (Figure S3E). In contrast to remnants of
apoptotic animal cells that are taken up by phagocytes [24],
plant cells have to organize their breakdown after PCD autonomously [7]. This autolysis might be achieved through the
accumulation of inactive or compartmentalized hydrolytic enzymes that are only activated or released during cell death [17].
Because their promoter activities suggested accumulation
of BFN1 and PASPA3 hydrolases in LRC cells, we isolated
paspa3 and bfn1 null mutants for functional analysis. Whereas
the paspa3 mutant showed no deviation from the wild-type
(data not shown), the bfn1 mutant root surface evidenced
numerous PI-positive nuclear remnants of the LRC cells
(Figure 3A). Time-lapse imaging and SBF-SEM (Figure S3E)
confirmed a significant delay in the degradation of bfn1 LRC
nuclei (Figures 3B and 3C). A functional proBFN1::BFN1VENUS construct that complemented the bfn1 phenotype
(Figures S3A and S3B) allowed us to analyze the subcellular
localization of BFN1. BFN1 first localized to the ER, as suggested before [25], but was then released from the disintegrating ER during cell death (Figures 3E, 3F, S3C, and S3D). This
dynamic localization would allow an accumulation of BFN1 in
the ER as a cellular compartment devoid of the nucleic acids
that this bifunctional nuclease targets. The release of BFN1
during cell death would then initiate an irreversible fragmentation of RNA and DNA species in the entire cell. To follow DNA
degradation in situ, we used terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), which highlighted
nuclei and cytoplasms in the cell death zones in wild-type
LRCs (Figures 3D and S4). Interestingly, weak TUNEL-positive
nuclei already appeared in cells prior to cell death, i.e., before
BFN1 release from the disintegrating ER, suggesting a BFN1independent initiation of DNA fragmentation (Figure 3D). BFN1
has been characterized as a single-strand-specific nuclease
in vitro [26], suggesting that double-stranded DNA would
have to be preprocessed by an unknown DNA-nicking endonuclease to enable efficient BFN1-mediated DNA fragmentation during and after cell death. Indeed, TUNEL was not absent
in bfn1 mutants but revealed a strong delay of LRC DNA degradation, indicated by abundant TUNEL-positive cell fragments
on the bfn1 root surface (Figure 3D). Although BFN1 has
been intensively studied [18, 25, 27], no bfn1 mutant phenotype has been described in the LRC, the xylem, or the senescing leaves. Our LRC data now give unambiguous evidence
about BFN1 function as a key enzyme for the cell-clearance
process during cell death in planta.
Programmed Cell Death Is the Ultimate Step of LRC
Differentiation
To test whether cell death is indeed an integral part of the LRC
development, representing its terminal differentiation step,
we first analyzed the tornado2 (trn2) mutant. TRN2 encodes
a transmembrane tetraspanin-type protein of uncertain molecular function. Lack of TRN2 function causes groups
of epidermal cells to adopt an ectopic LRC cell fate. These
misspecified cells die in the elongation zone, resulting in
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Figure 3. BFN1 Is Responsible for Removal of Nuclei in Autolytic LRC Cells
(A) Only few PI-positive LRC nuclei are present on PI-stained wild-type roots, whereas nuclear remnants are abundant on the surface of bfn1 roots.
(B and C) Kymograph showing the delay of nuclear degradation in bfn1 (B) and its quantification in wild-type and bfn1 mutant roots (C). ANOVA on log10
transformed data, ***p < 0.0001; n = 10 cells for each genotype.
(D) TUNEL-positive LRC cells (in green) correspond to two PCD zones in the wild-type, whereas abundant TUNEL-positive particles are spread over the bfn1
root. A weak TUNEL signal labels nuclei of LRC cells preparing for cell death (arrowheads). DAPI staining of nuclei is shown in the red channel. Details of each
genotype show colocalization of DAPI and TUNEL signals in the LRC cells (t, TUNEL; d, DAPI; m, merge; DAPI intensity was increased to show details). See
Figure S4 for TUNEL controls.
(E) BFN1-VENUS (proBFN1::BFN1-VENUS) accumulates in the ER of LRC cells prior to cell death (the region outlined in green shows a detail of an LRC
nucleus). Upon cell death, BFN1-VENUS is released into the entire cell, including the nucleus (the region outlined in red shows detail of a DAPI-positive
LRC nucleus). Nuclei of dead cells are stained with DAPI (in red). y, VENUS channel; d, DAPI channel; m, merge.
(F) An LRC-expressed ER-GFP marker (J3411) has an identical appearance to BFN1-VENUS (right cell) and, after cell death, is also released into the entire
cell (left cell), indicating ER rupture during cell death. g, GFP channel; d, DAPI channel; m, merge.
Panels show maximal z section projections, except for the insets in (E). WT, wild-type. Scale bars in (A) and (D) represent 50 mm; scale bars in (E) and (F)
represent 20 mm and 5 mm in the insets. See also Figure S3.
gaps in the epidermis tissue [28]. By introducing the
proPASPA3>>H2A-GFP marker in the trn2 mutant, we found
expression in files of epidermal cells in the elongation zone
(Figure 4A). These ectopic PASPA3-positive cells underwent
a rapid cell death reminiscent of LRC cell death (Figure 4B;
Movie S3). Strikingly, the dynamic pattern of GFP loss and PI
accumulation was identical to that of regular LRC cells (Figure 4C). We conclude that LRC cell death is an inherent program of differentiated LRC cells that does not depend on its
specific cellular context at the end of the TZ.
In a complementary approach, we investigated the mutant
anac033/sombrero (smb). SMB is a root-cap-specific NAC
Arabidopsis Root Cap Cell Death
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Figure 4. Cell Death Is the Ultimate Fate of LRC
Cells
(A) The trn2-1 mutants develop epidermal cells
with ectopic LRC identity (arrowheads) that express the cell death marker proPASPA3>>H2A:
GFP. See also Movie S3.
(B and C) Cell death progression is identical in the
LRC cells and the ectopic cells with the LRC identity, as determined by the decline in GFP intensity
prior to cell death; PI accumulation and subsequent cell clearance are demonstrated by PI
disappearance. Kymographs of LRC and ectopic
cells are shown in (B) and quantification of the
GFP and PI intensities in (C). The graph shows
the mean intensity value of n = 6 and n = 12 cells
for the LRC and ectopic position, respectively.
Time 0 = moment of PI entry.
Scale bar in (A) represents 50 mm. a.u., arbitrary
unit.
domain transcription factor (Figures 5A and 5B) [29], and smb
mutants show a delay in LRC differentiation [11]. Strikingly,
PASPA3 and BFN1 expression levels were strongly reduced
in smb root tips and entirely absent from the distal cell death
zone (Figures 5C, 5E, and S5; Movie S4), confirming that the
preparation of cell death is the ultimate part of the LRC differentiation program. Additionally, the distal smb LRC cells did
not show the weak TUNEL signal prior to death as observed
in the wild-type (Figure 5D). In contrast to the bfn1 mutant,
PM permeabilization and cell death were clearly delayed in
smb, with the first LRC cells dying only when progressing far
into the root elongation zone (Figure 5C). Moreover, smb cell
death differed fundamentally from wild-type cell death: PM
permeabilization was not preceded by PASPA3 expression,
and after PM permeabilization, LRC cell corpses persisted
without any signs of the rapid autolysis characteristic of
wild-type cells. Consequently, nondegraded cell corpses
covered large parts of the root surface (Figures 5C and 5F–
5H; Movie S5). Because most living smb LRC cells remained
connected with the elongating epidermal cells, they massively
expanded to over 300% of the terminal wild-type LRC cell
length (Figure 6A). It is possible that this massive physical
strain led to a passive loss of cell viability. To test this hypothesis, we used brassinazole (Brz) to reduce the brassinosteroid-dependent cell expansion in the root elongation zone
[30, 31]. Although a 10 hr treatment with 10 mM Brz caused a
clear reduction of epidermal cell length, it did not influence
regular LRC cell death in the wild-type (Figure S6). On the contrary, Brz treatment did reduce the aberrant cell death in smb
mutants: whereas 24 of 27 mock-treated smb plants showed
dead LRC cells, only 5 of 25 Brz-treated smb plants showed
dead LRC cells (Figure 6F). These results suggest that LRC
cell death in smb mutants represents a passive form of cell
death, which is caused by the sheer mechanical strain that is
generated by the underlying elongating epidermis cells. This
passive smb cell death impressively contrasts the highly organized, genetically controlled cell death in the wild-type and
convincingly characterizes LRC cell death as developmental
PCD by definition.
PCD Controls LRC Organ Size and Is
Crucial for Optimal Root Growth
The delayed LRC cell death in the smb
mutant also uncoupled the tight link of
LRC size to meristem size, causing an
autonomous increase in LRC organ size and cell number (Figures 6B and 6C). These results demonstrate the importance of
timely executed cell death for LRC organ size control in the
wild-type. Strikingly, the accurate timing of PCD is crucial to
the disposal of LRC cells before they enter the elongation
zone, thus preventing a passive cell disruption and accumulation of unprocessed cell corpses on the root surface, as seen in
the smb mutant (Figure 6G). We found that smb roots were
shorter and had less elongated epidermal cells than the wildtype (Figures 6D and 6E). Because SMB expression is
restricted to the LRC, and smb mutants do not show a xylem
differentiation phenotype [11], we propose that mechanical
hindrance of cell elongation by ectopic LRC cells and cell corpses affects the growth of the entire smb mutant root. Hence, by
restricting LRC size to the length of the root meristem, PCD
facilitates cell expansion in the elongation zone, which is critical for optimal root growth.
Discussion
Several cases of developmental PCD have been investigated
in various developmental contexts of a number of different
plant species [3, 32]. Evidence for developmentally regulated
PCD was found, for instance, during gametophyte development, pollination, and fertilization [33–35]; in the endosperm
and seed coat during seed development and germination
[36, 37]; in the embryonic suspensor [38–40]; during tracheary
element [7, 41] and anther tapetum [42, 43] differentiation; and
in some cases, during leaf shape development [44] and organ
abscission [45]. Phytohormones [2], transcription factors [41],
and several protein hydrolases [46] have been implicated in the
regulation and execution of PCD in a developmental context;
however, to date, we are still far from having a comprehensive
view on the intricate network of molecular regulation of PCD
during plant development.
The exposed situation of dying Arabidopsis LRC cells on the
root periphery allowed us to define the sequence of cellular
PCD hallmarks with high spatial and temporal resolution.
The first evident subcellular alteration was a sharp drop in
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Figure 5. smb Mutant LRC Cells Are Unable to Perform PCD and to Clear Their Contents
(A and B) proSMB is active only in the cells of the root cap; a medial optical section (A) and a maximal z section projection (B) are generated from the same
root.
(C) A zone of proPASPA3 expression (green) precedes wild-type LRC cell death (PI-stained cells, arrowhead). Rapid clearance of cell corpses leaves a clean
root surface in distal parts of the root (top panel). Cell death occurs more distally in smb (PI-stained cells, arrowhead) and without preceding PASPA3
expression, whereas differentiating protoxylem still expresses PASPA3 (asterisk). smb cell death is not followed by cell clearance, and cell corpses cover
the entire root surface. Maximal z section projections are shown.
(D) A TUNEL assay does not show the characteristic DNA degradation pattern in the smb mutant (compare to Figure 3D). Instead, the cytoplasm of LRC cells
first becomes TUNEL positive, and only then are the nuclei highlighted. Also note the lack of weak TUNEL staining in the cells prior to cell death (arrowhead).
A tile scan image is shown as maximal z section projection.
(E) PCD marker expression is decreased in the root tips of smb mutants compared to wild-type, as determined by qRT-PCR. See also Figure S5. The error
bars show SEM of three biological replicas (three technical repeats each). t test; ***p < 0.0001.
(F) Kymograph of a dying wild-type (top panel) and smb (bottom panel) distal LRC cell. Unlike the wild-type cell, the smb cell does not express
proPASPA3>>H2A:GFP prior to PI entry and does not proceed into cell autolysis.
(G) Transversal (x-y) SBF-SEM sections showing largely degraded LRC cells on the root surface in the wild-type TZ (white arrowheads). Next to smb LRC cell
corpses containing collapsed nondegraded cytoplasm (white arrowheads), there are living smb LRC cells in the elongation zone (EZ; black arrowheads).
(H) 3D views of an orthogonal and oblique slice of SBF-SEM 75 nm sections of the wild-type TZ and the smb EZ revealing the difference in LRC cell degradation. E, epidermis; C, cortex. Note that the smb cells are more elongated than the wild-type because the death of smb LRC cells occurs later than in the
wild-type.
Scale bars in (A)–(D) represent 50 mm; arrows in (G) represent 5 mm (for each dimension), and arrows in (H) represent 10 mm (for each dimension). See also
Movie S4 and Movie S5.
cytoplasmic pH. Cytoplasmic acidification has been observed
in several developmental PCD systems in plants [20, 47] but
has been generally interpreted to be a consequence of tonoplast rupture [22]. During LRC PCD, however, cytoplasmic
acidification occurred clearly before PM permeabilization
and subsequent tonoplast rupture, arguing for a vacuolarcollapse-independent mechanism. In both animal and plant
cells, alterations in cytoplasmic pH have been shown to
activate PCD-associated enzymes [47–49]. Furthermore, the
cytoplasmic pH has been shown to regulate signaling pathways [50], indicating that cytoplasmic acidification could be
part of an active mechanism triggering PCD in the LRC. Future
research will be necessary in order to identify the mechanisms
responsible for the pH drop and to identify how such a signal
could be translated into a regulatory response leading to PCD.
One of the defining hallmarks of animal apoptosis is the
rapid cleanup of dying cell fragments by neighboring cells or
macrophages via phagocytosis [51]. In plants, a rapid cellular
Arabidopsis Root Cap Cell Death
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Figure 6. The Absence of PCD in the Roots of
smb Has an Adverse Effect on Root Growth
(A) LRC cells of the smb reach a dramatically
higher length prior to PI accumulation than wildtype LRC cells. ANOVA on log10 transformed
data, ***p < 0.0001; n = 40 cells for each genotype.
(B and C) LRC organ length is increased in the
smb mutant, whereas the meristem length is not
altered.
(D and E) The smb mutant root length reaches
83% of that of the wild-type root (D). Accordingly,
the length of mature smb epidermal cells reaches
83% of that of the wild-type (E).
(F) A 10 hr treatment of 3–4 das smb roots with
10 mM Brz decreases the elongation of cells (vertical abscissae), and in turn, LRC cells (arrowheads) survive on the root surface throughout
the elongation zone and cover the epidermis
(asterisk). Median optical sections of PI-stained
roots are shown. Scale bars represent 50 mm.
See also Figure S6.
(G) A schematic model of the LRC fate in the smb
mutant. smb LRC cells reach the elongation zone
without being able to perform PCD. PCD markers
are not expressed in the longest LRC layer, and
the LRC cells are stretched and eventually die
(passive cell death zone). This death is not followed by clearance of the cells. In the most proximal part of the root tip, the LRC cells eventually
reach terminal differentiation and start to express
PCD markers (green). These cells then undergo
PCD and autolysis (see also Movie S4).
In (B)–(E), ANOVA, ***p < 0.0001; meristem size
and LRC length n = 47 WT and n = 25 smb roots;
root length n = 72 WT and n = 23 smb roots; trichoblast length n = 22 WT and n = 21 smb roots.
corpse clearance is characteristic for several developmental
PCD types [52]. Similar to tracheary elements [17], maturing
LRC cells accumulate hydrolytic enzymes prior to cell death.
These enzymes are thought to prepare plant cells for PCD as
the final differentiation step in a cell-autonomous fashion.
Thus, the preproduced and potentially lethal enzymes have
to be kept inactive by inhibitors or inhibiting cellular conditions, by producing inactive zymogens, or by being sequestered into specific subcellular compartments [53]. In the case
of BFN1, protein accretion to high levels is facilitated by
compartmentalization in the ER. Upon BFN1 release, rapid
and irreversible degradation of nuclear and cytoplasmic RNA
and DNA species is executed by this bifunctional nuclease.
TUNEL assays showed a delay in nucleic acid degradation in
both the nuclei and the cytoplasms of bfn1 mutants. The cytoplasmic TUNEL signal could be produced by genomic DNA
fragments leaking from the nucleus or by diverse degraded
RNA species in the cytoplasm because terminal deoxynucleotidyl transferase has been shown to act on free 30 hydroxyl
termini of both DNA and RNA in vitro [54].
By analyzing cell death in the smb and trn2 mutants, we
found two independent indications that LRC PCD is an integral
part of LRC differentiation and the ultimate step of LRC development. In smb mutants, LRC differentiation is delayed by the
lack of SMB transcriptional activity [29]. PASPA3 and BFN1
expression were absent from cells prior
to the delayed smb LRC cell death. The
hydrolases PASPA3 and BFN1 are thus
representatives of a PCD-associated
gene class that is transcriptionally controlled during LRC
development. In addition to PCD, other features of LRC differentiation are delayed in the smb mutant; thus, we assume that
SMB does not directly control PCD but rather activates a set of
PCD-specific downstream transcription factors. Intriguingly,
systemic SMB overexpression leads to growth arrest and
vessel-like cell-wall modifications [55, 56]. It is tempting to
speculate that these phenotypes are accompanied by system-wide cell death induced by the upregulation of primary
and secondary SMB target genes. Extensive transcriptional
regulation of developmental cell death has also been
described for the expression of the key proapoptotic protein
egl-1 in Caenorhabditis elegans [57]. It is possible that analogous mechanisms have independently evolved in animals
and plants to organize developmentally controlled PCD.
The maintenance of tissue homeostasis and organ size
through a balance of cell production by stem cells and cell
degeneration by PCD is well documented in animals [58].
In Drosophila and mammals, the Hippo tumor suppressor
pathway has been implicated in organ size control via several
mechanisms, including cell proliferation and apoptosis [59].
Also, during skin development, continual proliferation of stem
cells in the basal layer is compensated by a specific nonapoptotic keratinocyte cell death to maintain skin homeostasis [60].
Organogenesis differs fundamentally in animals and plants.
Current Biology Vol 24 No 9
938
Plants generally have two types of organ formation: determinate (e.g., leaves or flowers) and indeterminate (e.g., roots or
stems) [61]. The root cap, however, represents a divergent
mode of plant development: it keeps a constant size in spite
of continuous stem cell activity, implying a cellular turnover
system [8]. Some plants (e.g., maize) have border cells that
are constantly sloughed off the main root and are viable for
extended periods of time in the rhizosphere [62]. Here, we
show that in Arabidopsis, shedding of cells at the very root tip
and PCD in the more distal part of the root cap both contribute
to root cap organ size maintenance. If PCD is inhibited, as it is in
the smb mutant, the LRC length increases to reach far up into
the elongation zone. If cell shedding and PCD are inhibited
simultaneously, as they are in the brn1-brn2-smb triple mutant,
dramatic accumulation of root cap tissue occurs around the
columella [11]. To our knowledge, our results implicate PCD in
controlling plant organ size for the first time.
A strict control of LRC organ size is important for two main
reasons. First, it enables the root cap to stay localized at the
root tip, where it can fulfill its functions [63]. Second, it ensures
that root cells are eliminated before they reach the elongation
zone. The smb mutant impressively demonstrates the significance of this PCD-mediated organ size control: if PCD is
delayed, LRC cells get overstretched and die in a distinct,
passive fashion. The elongating LRC cells and nondegraded
cell corpses might impose a direct physical obstruction for
full epidermal cell elongation and thus root growth. Hence,
tight control of PCD initiation and execution, followed by rapid
and thorough cell corpse degradation, is crucial for optimal
root growth and development of Arabidopsis and, likely, other
plants with a similarly organized root meristem.
Experimental Procedures
Plant Material
Unless otherwise indicated, 5-day-old wild-type plants, marker line plants
(J3411 [uNASC] in the C24 background), and mutant plants (paspa3-3
[SALK_056711C], bfn1-1 [GK-197G12], trn2-1, and smb-3 [SALK_143526C])
in the Col-0 background were used. The absence of the respective transcripts was shown by qRT-PCR for bfn-1 and paspa3-3 lines. The accession
numbers are BFN1 (At1g11190), PASPA3 (At4g04460), TRN2 (At5g46700),
and SMB3 (At1g79580).
Imaging
Confocal images were acquired using Zeiss710 and Zeiss780 CLSM microscopes. The pH treatments were done as described in [64], and the TUNEL
assays were done by using the In Situ Cell Death Detection Kit, Fluorescein
(Roche Applied Science). smb seedlings that were 3 to 4 days old were
analyzed after 10 hr of Brz treatment (10 mM, TCI Europe). SBF-SEM samples were imaged by a Zeiss Merlin microscope equipped with Gatan
3View2. Images were analyzed and figures prepared by using the ImageJ
(http://imagej.nih.gov/ij/), Fiji [65], Inkscape (http://inkscape.org/en/), and
Adobe Photoshop programs.
Statistical Analysis
The ANOVA GLM (family = Poisson) and t test functions (R environment,
http://www.r-project.org/) were used for statistical analysis. The data from
[15] were analyzed by the VisuaLRTC [16] method to identify coregulated
genes.
For growth conditions, transgenic line preparation, qRT-PCR, microscope settings, pH treatments, TUNEL, SBF-SEM, and meta-analysis, see
Supplemental Experimental Procedures.
Supplemental Information
Supplemental Information includes Supplemental Experimental Procedures, six figures, and five movies and can be found with this article online
at http://dx.doi.org/10.1016/j.cub.2014.03.025.
Author Contributions
M.F., T.V.H, and M.K.N. designed the experiments, analyzed the data, and
wrote the manuscript. M.F. and T.V.H. performed all experiments, except
for the following: establishment of BFN1 reporter and complementation
lines was done by M.V.D.; cloning of the proPASPA3 constructs and parts
of the transcriptome meta-analysis was done by Y.O.-C.; establishment of
the ToIM lines was done by M.H.; cloning of the gateway transactivation
destination vectors was done by M. Karimi; design and performance of
SBF-SEM was done by S.L. and C.J.G.; and establishment of the
proUBQ10:pHGFP lines was done by M. Krebs and K.S.
Acknowledgments
We thank Lorenzo Frigerio for the sp-mRFP-AFVY seeds, Mieke Van Lijsebettens for providing the trn2-1 seeds, Veronique Storme for assistance
with statistical analysis, and Tom Beeckman and Annick Bleys for help
with the manuscript. We are grateful for technical support by Freya De
Winter, for help with the meta-analysis by Boris Parizot, and experimental
advice by Wei Xuan. We gratefully acknowledge funding by the Marie
Curie-Cofund grant omics@VIB program for M.F., from the IWT and the
FWO for M.V.D. and S.L., and from the BELSPO and CONACyT for Y.O.-C.
Received: December 25, 2013
Revised: January 29, 2014
Accepted: March 7, 2014
Published: April 10, 2014
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