Download Calpain-Mediated Positional Information Directs Cell Wall

Calpain-Mediated Positional Information Directs Cell Wall
Orientation to Sustain Plant Stem Cell Activity, Growth and
Development
1
Department of Plant Sciences, Norwegian University of Life Sciences, PO Box 5003, N-1432 Ås, Norway
Department of Biology, University of Louisiana, Lafayette, LA 70504, USA
3
Plant Gene Expression Centre, USDA/UC-Berkeley, Albany, CA 94710, USA
4
Present address: Department of Biological Sciences, National University of Singapore, 117543, Singapore.
2
*Corresponding author: E-mail, [email protected]
(Received October 10, 2014; Accepted July 17, 2015)
Eukaryotic development and stem cell control depend on
the integration of cell positional sensing with cell cycle control and cell wall positioning, yet few factors that directly
link these events are known. The DEFECTIVE KERNEL1
(DEK1) gene encoding the unique plant calpain protein is
fundamental for development and growth, being essential to
confer and maintain epidermal cell identity that allows development beyond the globular embryo stage. We show that
DEK1 expression is highest in the actively dividing cells of
seeds, meristems and vasculature. We further show that
eliminating Arabidopsis DEK1 function leads to changes in
developmental cues from the first zygotic division onward,
altered microtubule patterns and misshapen cells, resulting
in early embryo abortion. Expression of the embryonic
marker genes WOX2, ATML1, PIN4, WUS and STM, related
to axis organization, cell identity and meristem functions, is
also altered in dek1 embryos. By monitoring cell layerspecific DEK1 down-regulation, we show that L1- and 35Sinduced down-regulation mainly affects stem cell functions,
causing severe shoot apical meristem phenotypes. These results are consistent with a requirement for DEK1 to direct
layer-specific cellular activities and set downstream developmental cues. Our data suggest that DEK1 may anchor cell
wall positions and control cell division and differentiation,
thereby balancing the plant’s requirement to maintain totipotent stem cell reservoirs while simultaneously directing
growth and organ formation. A role for DEK1 in regulating
microtubule-orchestrated cell wall orientation during cell
division can explain its effects on embryonic development,
and suggests a more general function for calpains in microtubule organization in eukaryotic cells.
Keywords: Calpain Cell cycle Cell wall positioning Embryo development Microtubule organization Stem
cell and meristem regulation.
Abbreviations: 35S, Cauliflower mosaic virus 35S promoter;
ATML1, Arabidopsis thaliana MERISTEM LAYER1; BET, basal
endosperm transfer; CLV, CLAVATA; CMT, cortical microtubule; CR4, CRINKLY4; DEK1, DEFECTIVE KERNEL1; DMSO,
dimethylsulfoxide; GUS, b-glucuronidase; LTP1, LIPID
TRANSFER
PROTEIN
1;
MAP65,
MICROTUBULEASSOCIATED PROTEIN 65; MT, microtubule; PIN,
PIN-FORMED; PPB, pre-prophase band; RAM, root apical
meristem; RbcS2b, RUBISCO SMALL SUBUNIT 2B; RNAi,
RNA interference; SAM, shoot apical meristem; STM,
SHOOT MERISTEMLESS; TF, transcription factor; WOX,
WUSCHEL-RELATED HOMEOBOX; WUS, WUSCHEL.
Introduction
The evolution of embryophytes required the production and
maintenance of stem cell pools to drive continuous organogenesis from root and shoot apical meristems, which gradually
generates the characteristic architecture of each plant species.
Plant architecture also depends on the ability of cells to sense
their position within the organism, and on precise cell divisions
that occur antiparallel (anticlinal) and parallel (periclinal) to the
outer surface (Murray et al. 2012), yet how this is achieved
remains a puzzle. The fundamental nature of cellular positional
sensing may point to the existence of an underlying universal
mechanism capable of registering the position of all cells until
identity and differentiation cues are secured. In that case, the
cell wall–cytoskeleton continuum is ideally positioned to sense
position and thereby to regulate the orientation and frequency
of cell divisions during development by directing the positioning of cell wall division planes.
Shoot and root stem cell reservoirs in plants are organized
following the initial patterning events that occur during embryonic development. In Arabidopsis embryos, the establishment of apical–basal polarity depends on an asymmetric
anticlinal division of the zygote. This allows further development of the apical embryo and the basal suspensor, where the
WUSCHEL-RELATED HOMEOBOX (WOX) transcription factor
(TF) genes WOX2 and WOX8 are expressed in the apical and
basal cells, respectively (Takada and Jurgens 2007, Breuninger
et al. 2008). Additional divisions yield an embryo with outer
protodermal (L1) and internal hypodermal (L2) cell layers. The
cells in these layers maintain their clonal identity by dividing
strictly anticlinally, whereas the innermost L3 cells that develop
later divide in varying orientations. The embryonic protoderm
Plant Cell Physiol. 56(9): 1855–1866 (2015) doi:10.1093/pcp/pcv110, Advance Access publication on 27 July 2015,
available online at www.pcp.oxfordjournals.org
! The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.
All rights reserved. For permissions, please email: [email protected]
Regular Paper
Zhe Liang1,4, Roy C. Brown2, Jennifer C. Fletcher3 and Hilde-Gunn Opsahl-Sorteberg1,*
Z. Liang et al. | DEK1 directs cell wall positioning and stem cell functions
generates the meristematic L1 layer, and signaling from it to the
underlying layers plays a vital role in post-embryonic shoot
apical meristem (SAM) function (Bemis and Torii 2007,
Savaldi-Goldstein et al. 2007, Knauer et al. 2013). However,
the molecular mechanisms that control L1 cell layer specification and maintenance are still poorly understood (Javelle et al.
2011). One key player is Arabidopsis thaliana MERISTEM
LAYER1 (ATML1), which encodes a class IV HD-ZIP TF that is
thought to specify the L1 layer (Abe et al. 2003) and sufficient to
confer epidermal cell traits (Takada et al. 2013). The WOX
family gene WUSCHEL (WUS) and the distantly related class I
KNOX homeodomain TF gene SHOOT MERISTEMLESS (STM) are
crucial to maintain the pluripotent stem cell reservoir within the
SAM, but the factors that constrain the expression of these key
developmental genes at early stages are yet to be identified (Friml
et al. 2002, Breuninger et al. 2008, Yadav et al. 2011).
DEFECTIVE KERNEL1 (DEK1) is a candidate to integrate developmental signaling cues in the control of precise cell wall
orientation, possibly by controlling cell cycle progression (Xu
et al. 2008, Roeder et al. 2012). DEK1 encodes a calpain protein
(Lid et al. 2002) that is a member of a highly conserved family of
cysteine proteases (Croall and Ersfeld 2007). DEK1 consists of 21
predicted transmembrane segments containing at least one
potential extracytosolic loop/Ca2+ channel region, and an internal calpain domain, which is likely to be active after cleavage
and to localize to various cellular membranes (Lid et al. 2002,
Tian et al. 2007, Johnson et al. 2008). These observations are
consistent with a role for DEK1 in intercellular signaling.
DEK1 expression is detected throughout the plant (Lid et al.
2005, Johnson et al. 2008), although native promoter-driven
reporter studies have not yet been reported. Importantly,
Arabidopsis dek1 mutants undergo abnormal cell division patterns and globular stage embryo arrest with lack of epidermal
L1 cells (Johnson et al. 2005, Lid et al. 2005). dek1 embryos
reportedly fail to express the protodermal markers ATML1
and ARABIDOPSIS CRINKLY4 (ACR4) (Johnson et al. 2005),
yet the underlying cause of these cellular defects has not
been determined. Reducing DEK1 function by RNA interference
(RNAi) results in seedlings with a flat SAM that may develop a
few radialized rosette leaves before growth terminates. In addition, the cotyledons are partially fused and their epidermal cells
are disorganized. Dominant-negative mutations induced by
overexpressing the DEK1 membrane anchor or down-regulating
the calpain domain generate similar phenotypes (Tian et al.
2007, Johnson et al. 2008). These data show that DEK1 function
depends on the tight control of and balance between its membrane binding and calpain activities. DEK1 is also needed for
trichome formation during vegetative development (Lid et al.
2005, Tian et al. 2007, Robinson and Roeder 2015). Additionally,
DEK1 expression can be uncoupled from HvCR4 regulation and
secondary cell wall development in the des5 barley mutant
(Olsen et al. 2008), suggesting that DEK1 is upstream or independent of CR4 (Wisniewski and Rogowsky 2004, Becraft and Yi
2011) and is connected to primary, not secondary, cell wall
formation (Yi et al. 2011).
DEK1 is found as a single copy in plants, and is a member of a
multigene family of cysteine proteases that are evolutionarily
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highly conserved across kingdoms (Lid et al. 2002, Sorimachi
et al. 2011, Zhao et al. 2012, Liang et al. 2013). Mammalian
calpains mediate signal transduction in response to changes
in intracellular calcium concentrations and have been linked
to a wide variety of functions, including remodeling of the cytoskeleton, apoptosis, cell cycle regulation, stem cell renewal and
differentiation, spindle function and chromosome orientation
(Conacci-Sorrell and Eisenman 2011, Bergounioux et al. 2012,
Santos et al. 2012). However, whether the single plant calpain,
DEK1, affects the cytoskeleton is currently unknown. Although
several genes that control cell division planes and maintain cell
wall positions have been identified, few mutants exhibiting
disorganized division planes and/or cell wall positions while
also possessing an intact cytoskeletal machinery have been
described (Muller et al. 2009, Crowell et al. 2011, Oda and
Fukuda 2012).
To better understand DEK1 function at the cellular level, we
monitored cell wall formation and the vegetative microtubule
(MT) systems during early dek1 embryo development.
Formation of the embryonic body axes and cell identity were
also examined, using molecular markers for different tissue domains, to determine potential effects on cell identity control.
We find that dek1 embryos appear to establish initial apical–
basal polarity correctly but display disrupted cell division, cell
wall positioning and cortical microtubule (CMT) organization,
ultimately leading to failure of the stem cell pools. We further
demonstrate that the ATML1, STM and WUS gene expression
domains are altered in developing dek1 embryos, indicating
that DEK1 is necessary to establish proper radial polarity and
cell identity in the SAM. The underlying cause of dek1 cellular
defects has not previously been determined, and we present
data suggesting that the various DEK1 functions can be linked
to the control of cell division patterns via MT and cell wall
positioning.
Results
DEK1 is expressed in dividing cells
Because DEK1 function is linked to L1 identity in maize and
Arabidopsis, it has been a puzzle that it is expressed in all cell
types studied, and this has been explained by a need to be ready
to secure positional reaction whenever needed (Lid et al. 2002,
Wang et al. 2003, Lid et al. 2005). Previous studies provide a
mosaic picture of Arabidopsis DEK1 gene expression involving
only some plant parts, a few selected cell types, different techniques, and reporter gene analysis using a promoter expected
to be similar to DEK1 (Johnson et al. 2005, Lid et al. 2005, Tian
et al. 2007, Johnson et al. 2008). To improve the understanding
of the DEK1 expression pattern, we monitored the activity of
the native DEK1 promoter, the expression domain of which has
not previously been reported, during early Arabidopsis development. Among 25 independent lines generated, eight lines
expressing GUS (b-glucuronidase) were studied further, all of
which showed the same activity pattern. In seeds, pDEK1::uidA
activity is strongest in actively dividing early embryos and putative basal endosperm transfer (BET)-like cells (Fig. 1A), a
Plant Cell Physiol. 56(9): 1855–1866 (2015) doi:10.1093/pcp/pcv110
Fig. 1 DEK1 (pDEK1::uidA) promoter directs expression mainly to dividing cells. (A) Seed showing strong promoter activity especially in young
embryo (yellow arrow) and BET-like cells. pDEK1::uidA activity in (B) SAM, (C) RAM, (D) lateral root mersitem (RM) and (E) root vasculature.
Vascular activity in (F) phloem/cambium in cotyledon, (G) rosette leaf, (H) leaf petiole and (I) vascular branch, (J) whole seedling vasculature, (K)
adaxial and (L) abaxial side of rosette leaf, and (M) all flower organs except petals. White arrows indicate pDEK1::uidA activity in the epidermal
layer. Scale bars = 20 mm (A–I), 500 mm (J–M).
region not previously recognized as existing in Arabidopsis seed
development (Frederic Berger personal communication).
Following germination, DEK1 promoter activity is observed in
stem cells and vasculature throughout the plant. pDEK1::uidA
activity is observed throughout the seedling SAM (Fig. 1B), the
RAM (Fig. 1C), the lateral root meristem (Fig. 1D), and in the
vascular phloem and cambium of the cotyledons and rosette
leaves (Fig. 1E–L). Consistent with previous studies,
pDEK1::uidA activity is detected in the epidermal cell layer of
vegetative tissues (Fig. 1H, I arrows), as well as in internal layers.
In flowers, DEK1 promoter activity is detected in the vasculature of the sepals, stamens and carpels, but not the petals
(Fig. 1M). These data enhance the previous partial picture of
DEK1 activity, and show that the native DEK1 promoter is
mainly active in dividing cells throughout plants. Additionally,
we show strong DEK1 promoter activity in BET-like cells with
specialized cell walls, a link not previously reported.
DEK1 regulates microtubule-organized cell wall
orientation during embryo cell division
To analyze the involvement of Arabidopsis DEK1 in cell division
and its effect on early embryo development, we studied the cell
division patterns and cytoskeleton in wild-type and dek1 embryos. Wild-type globular stage embryos have regularly positioned cell walls, uniformly sized cells and a suspensor
consisting of a column of single cells (Fig. 2A, D). In contrast,
dek1 embryos exhibit variable morphological architecture, irregular cell division patterns, unequal cell sizes, and suspensors
with multiple cell files (Fig. 2B, E), consistent with previous
findings (Johnson et al. 2005, Lid et al. 2005). In more severely
affected dek1 embryos, nearly all of the cell walls are incorrectly
positioned (Fig. 2C). Rather than forming an elongate radial
structure like wild-type embryos (Fig. 2F), dek1 embryos generally form a mace-like structure in a series of globoid cell clusters (Fig. 2G–I). It appears that the randomly oriented dek1
embryo cell walls prevent the development of well-defined cell
layers and cell types (Fig. 2B, C, E, G–I). Our data show that
dek1 embryos have defective cell division patterns and planes,
indicating that DEK1 is required to organize early embryo cell
division patterns and to restrict suspensor cells to a single cell file.
The cytoskeleton in embryo development was monitored
using confocal laser scanning microscopy and immunostaining.
In wild-type plants, MTs (Fig. 3A) play key roles in cell morphogenesis, positioning the cell wall by the pre-prophase bands
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Fig. 2 dek1 embryos exhibit disorganized cell division planes and microtubule (MT) arrangements. (A) Longitudinal section through a wild-type
globular stage embryo. (B) Section through a dek1 embryo showing disorganized cell division planes (arrowheads). (C) Section through a severely
affected dek1 embryo. (D) Wild-type globular stage embryo showing regularity of cell arrangement and hoop-like cortical MTs, particularly in the
epidermis. (E) dek1 globular stage embryo with disorganized cell wall deposition and a multiseriate suspensor (arrow). (F) Wild-type embryo and
surrounding endosperm. (G and H) dek1 embryos with MTs that appear randomly oriented and thickly bundled (arrows), surrounded by
endosperm that lack radial MT arrangements. (I) dek1 embryo with the appearance of cell differentiation in tiers (arrows) and possible ablated
epidermal cell (asterisk). Scale bars = 10 mm.
(PPBs) (Fig. 3B). During seed development in most plants, the
PPB cycle drives embryo formation and the radial microtubule
(RMT) system cycle drives the common nuclear type of endosperm development from the coenocytic/syncytial stage until
cytokinesis and cellularization (Brown and Lemmon 2001).
During embryo development a PPB marks the plane of the
first and all subsequent cell divisions, resulting in the precise
control of the division pattern (Smith et al. 2001, Chan et al.
2005). The phragmoplast that mediates deposition of the new
cell plate after mitosis follows the former path of the PPB with
great fidelity.
In dek1 embryo cells, the PPB is present (Fig. 3C) and the
cells go through the cell cycle with all components of the cell
division machinery present (Supplementary Fig. S1): PPBs
(Fig. 3C), spindles (Fig. 3F; Supplementary Fig. S1D-F) and
phragmoplasts (Fig. 3G; Supplementary Fig. S1F). Actin is also
unaffected in dek1 cells (Fig. 3H). However, whereas the MTs of
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wild-type tabular protoderm cells are strictly hoop-like, anticlinal to the surface (Fig. 3A, D), the MTs in dek1 protoderm-like
cells are found in more random orientations (Fig. 3E;
Supplementary Fig. S1B, C). In addition, the cells in the surface
layer of dek1 embryos become rounder and protuberant (Fig.
2E, G–I), a feature never seen in the protoderm of wild-type
embryos, suggesting a defect in primary wall deposition. The
MTs of these protuberant dek1 cells appear to have a more
radiate pattern (Fig. 3E). Similarly, the MTs in the dek1 endosperm have lost the organized patterning of wild-type (Fig. 2F)
and appear bundled (Fig. 2H). Some dek1 embryo cells are of
unequal sizes (Fig. 3I), whereas others, particularly those in the
outer protoderm-like layer, ultimately appear to undergo cell
death (Fig. 3J). These data show that DEK1 is involved in the
arrangement of the CMT systems during early embryo development, which in turn may affect cell wall deposition and subsequent alignment of the PPB. Our results demonstrate a
Plant Cell Physiol. 56(9): 1855–1866 (2015) doi:10.1093/pcp/pcv110
Fig. 3 Microtubule changes in dek1 embryo cells. (A) Wild-type MTs in two foci (top) and surrounding nuclei (bottom). (B) Wild-type
protoderm cell showing the pre-prophase bands (PPB). (C) dek1 protoderm cell showing the PPB. (D) Wild-type cell with cortical MTs arranged
in a hoop-like band. (E) dek1 cell with cortical MTs in a random array (arrowheads). (F and G) dek1 cell MT/nuclei showing (F) phragmoplast and
(G) co-localized telophase nuclei. (H) dek1 phragmoplast immunostained for F-actin. (I) The same pair of dek1 cells (left and right) showing
unequal sizes and randomly orientated, misplaced new cell walls. (J) dek1 cells undergoing apoptosis (arrows). Scale bars = 10 mm.
central role for DEK1 in CMT functions that are associated with
cell wall deposition, activities that are important for initially
setting cell shape and organizing the developing embryo.
Analysis of embryo gene expression to monitor
cell identity changes
The effects of lack of specificity in cell identity during development in dek1 embryos were investigated by mRNA in situ hybridization and monitoring of the expression of genes essential
for early embryo patterning. WOX8 expression is correctly restricted to the suspensor after the 16-cell stage in dek1 embryos
(Supplementary Fig. S2A, B), indicating that basal identity is
unaffected and that the embryos develop beyond the 16-cell
stage. ATML1 specifies L1 cell identity from the 16-cell stage
onward (Abe et al. 2003), and we confirm that ATML1 is restricted to the L1 layer in wild-type globular stage embryos
(Fig. 4A, B). In contrast to previous reports (Johnson et al.
2005, Johnson et al. 2008), we detect ATML1 mRNA in dek1
globular stage embryos (Fig. 4C, D). However, ATML1 expression is not restricted to the protodermal cells, but is patchy
throughout the embryo and the suspensor. Consistent with
these results, we also detect pATML1::GUS reporter activity in
all dek1 embryo cells (data not shown). Thus dek1 embryos
have the cues to activate ATML1 transcription but fail to restrict its expression to the protoderm/L1 layer. Together our
data indicate that DEK1-mediated effects on downstream gene
expression play important roles in establishing radial polarity/
L1 identity.
DEK1 is required to establish a functional vegetative SAM
(Tian et al. 2007). To investigate further the role of DEK1 in SAM
organization, we performed in situ hybridization to examine the
expression domains of the key meristem-promoting genes WUS
and STM in dek1 embryos. WUS encodes a WOX family TF
(Mayer et al. 1998) that maintains the stem cell reservoir of
the SAM (Lenhard et al. 2002). In turn, WUS transcription is
negatively regulated by CLAVATA3 (CLV3)-mediated signalling
from the stem cells (Brand et al. 2000, Schoof et al. 2000). WUS
mRNA is expressed in the four inner apical cells of the 16-cell
embryo, and later becomes restricted to the innermost cells of
the subprotodermal layer as expected (Fig. 4E; Supplementary
Fig. S2C). However, in dek1 embryos, WUS expression expands
throughout the embryo, into the basal and outer cells (Fig. 4F;
Supplementary Fig. S2D). pCLV3::GUS activity is detected in
wild-type embryos by the torpedo stage (Supplementary Fig.
S2E), whereas pCLV3::GUS activity is absent from dek1 embryos
(Supplementary Fig. S2F). This is most probably due to their
arrest prior to the stage at which CLV3 expression initiates.
These results indicate that DEK1 activity restricts the WUS expression domain to the interior cell layers during early embryogenesis, before CLV3 is activated.
STM encodes a class I KNOX homeodomain TF that is crucial for establishing the embryonic SAM (Long et al. 1996)
and for maintaining the SAM cells in a proliferative state
(Byrne et al. 2000, Byrne et al. 2002). STM expression initiates
in the presumptive embryonic SAM region beginning at the late
globular stage (Long et al. 1996). Indeed, we observe STM
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Fig. 4 Altered embryonic gene expression patterns and molecular marker distribution in dek1 embryos. (A–D) ATML1, (E, F) WUS and (G, H)
STM expression in wild-type (A, E, G) and dek1 (B–D, F, H) embryos. (I–K) pWOX2::uidA activity in the wild-type apical daughter cell after the first
zygotic division (I), the embryo proper at the 8-cell stage (J) and undetectable at the heart stage (K). (L) pWOX2::uidA activity is absent in dek1
embryos. (M, N) pPIN1::uidA activity in the apical part of the embryo proper of globular stage wild-type (M) and dek1 (N) embryos. (O, P)
pPIN4::uidA activity in the basal domain of the RAM of wild-type torpedo stage embryos, but absent from heart stage embryos (inset). (Q, R)
Premature pPIN4::uidA activity in the basal part of the dek1 globular stage embryo proper and the upper suspensor cells. Scale bars = 20 mm.
expression in the apical and central domain of wild-type globular stage embryos (Fig. 4G). However, in dek1 embryos, the STM
expression domain is broader and expands outward across the
top half of the embryo (Fig. 4H). Thus DEK1 activity restricts
the STM expression domain to the central, presumptive apical
meristem region during early embryogenesis. The factors that
constrain WUS and STM expression are still poorly understood,
and our results show that DEK1 acts as a negative regulator that
limits their domains to the interior cell layers during early embryogenesis. The first division divides the zygote into an apical
and a basal domain. After this initial asymmetric division, the
WOX family TF gene WOX2 is specifically expressed in the apical
cells until it disappears by the early heart stage (Fig. 4I–K). We
do not detect pWOX2::GUS activity in dek1 embryos (Fig. 4L),
indicating that DEK1 is required to activate WOX2, possibly
by directing asymmetric cell divisions. We also examined
pPIN1::uidA activity, which in wild-type globular stage embryos
is detected throughout the embryo proper, most strongly
across the apical half in the provascular cells (Fig. 4M). PIN1
mainly functions to facilitate auxin transport in the L1 layer,
which is not identifiable in dek1 plants. Yet pPIN1::uidA activity
is unaffected in dek1 embryos (Fig. 4N). This observation indicates that L1 identity is not a prerequisite for PIN1 activation,
which is consistent with both PIN1 and DEK1 already being
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expressed in the zygote (Nodine and Bartel 2012). In contrast,
PIN4 regulation is drastically altered in dek1 embryos.
pPIN4::uidA activity is initially detected at the late torpedo
stage in wild-type embryos, with the strongest activity occurring in the RAM region (Fig. 4O, P). Surprisingly, the PIN4
promoter is already strongly active in globular stage dek1 embryos, and is mislocalized to the embryo proper and the apical
part of the suspensor (Fig. 4Q, R). This result shows that DEK1
represses premature PIN4 activation and prevents ectopic PIN4
induction in apical embryo cells. These data suggest that DEK1
may affect PIN4-mediated auxin efflux signaling or polarity
events during embryo development, although it remains to
be shown if DEK1 acts directly on auxin efflux or indirectly by
affecting auxin signaling partners.
DEK1 is required for vegetative shoot apical
meristem function
We next determined whether DEK1 has non-cell-autonomous
effects on vegetative development when down-regulated either
throughout the plant or in specific cell layers (Fig. 5;
Supplementary Fig. S3). To monitor effects of DEK1 downregulation following embryogenesis, the baseline carried the
minimal promoter UAS-TATA controlling either the DEK1RNAi construct or the dominant-negative membrane anchor
Plant Cell Physiol. 56(9): 1855–1866 (2015) doi:10.1093/pcp/pcv110
Fig. 5 Vegetative phenotypes caused by dexamethasone-induced DEK1 down-regulation driven by constitutive or cell layer-specific promoters.
(A) Wild-type seedling, (B) p35S::DEK1RNAi seedling, (C) pRbcS2b::DEK1MEM seedling, (D) pSuc2::DEK1MEM seedling, (E) pLTP1::DEK1MEM
seedling and (F) pATML::DEK1MEM seedling. Longitudinal sections through (G) a wild-type seedling SAM, (H) a pLTP1::DEK1MEM seedling shoot
apex and (I) a pATML1::DEK1MEM seedling shoot apex. Longitudinal sections through (J) a p35S::DEK1RNAi leaf, (K) a pLTP1::DEK1MEM leaf and
(L) a pATML1::DEK1MEM leaf. Scale bar = 100 mm (G–L).
region lacking the calpain domain DEK1-MEM. We have previously shown that both constructs cause equivalent arrested
SAM phenotypes (Tian et al. 2007). The transgenic lines segregating for the dexamethazone-inducible DEK1 knock-down
constructs were driven either by the constitutively active 35S
promoter, the L2 layer-specific pRbcS2b promoter or the L3
layer-specific pSuc2 promoter.
Wild-type seedlings are dark green and form rounded cotyledons and rosette leaves (Fig. 5A). In contrast, stably transformed seedlings from all lines exhibit retarded growth hours
after dexamethasone induction. p35S::DEK1RNAi seedlings
arrest growth after a few rosette leaves are formed (Fig. 5B).
These previously reported growth defects of p35S::DEK1RNAi
lines were important to re-establish in order to investigate
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further the effects of selective silencing of DEK1 in specific cell
types following seed maturation and germination.
pRbcS2b::DEK1MEM seedlings produce small leaves and
become pale green from chlorophyll (chl) reduction (Fig. 5C),
a phenotype not previously reported, before arresting growth.
pSuc2::DEK1MEM seedlings also become slightly pale and exhibit some retarded growth before arrest (Fig. 5D), due to
reduced DEK1 expression in interior L3 cells that previously
had not been regarded as requiring DEK1 function. These
data demonstrate that DEK1 activity is required to achieve
normal seedling growth and light-induced chl production. In
addition, our observation that dexamethasone-induced DEK1
reduction in either the L2 or the L3 cell layer causes phenotypic
effects shows that DEK1 function is vital for sustained growth in
all cell layers, not only in epidermal cells as has been the existing
supposition (Johnson et al. 2005, Lid et al. 2005, Johnson et al.
2008). To test whether reducing DEK1 activity specifically in L1
cells would be sufficient to generate dek1 seedling phenotypes,
we generated additional inducible DEK1 knock-down lines
driven by the L1-specific LTP1 or ATML1 promoters. We
observed that the pLTP1::DEK1MEM and pATML1::DEK1MEM
seedlings exhibit the strongest growth effects, with only a few
leaves forming before SAM activity ceases (Fig. 5E, F). These
data show that reducing DEK1 in the L1 layer is sufficient to
confer severe meristem phenotypes comparable with those displayed by p35S::DEK1RNAi plants. This shows that DEK1 is especially important to L1 functions as previously reported, and
might further suggest that controlled cell wall positioning is a
central regulator of L1 identity and function.
Longitudinal sections indicate that, compared with domeshaped wild-type SAMs (Fig. 5G), both pLTP1::DEK1MEM (Fig.
5H) and pATML1::DEK1MEM shoot apices (Fig. 5I) are flattened and consist of large vacuolated, differentiated cells with
misorientated cell walls. Reducing DEK1 activity in
pLTP1::DEK1MEM seedlings results in longitudinally positioned
cells organized in several layers (Fig. 5H), whereas
pATML1::DEK1MEM seedlings develop larger, perpendicularly
positioned cells (Fig. 5I). The small, densely cytoplasmic stem
cells found at the very tip of the wild-type SAM (Steeves and
Sussex 1989) are absent in these transgenic plants, consistent
with the absence of CLV3 expression in dek1 embryos and confirming the critical role of DEK1 in securing the stem cell reservoir. In addition, the termination of stem cell activity in these
DEK1 knock-down plants despite the presence of WUS and
STM transcripts in the embryo suggests that DEK1 may promote shoot stem cell maintenance independently of the WUS
and STM pathways. Finally, sections of leaves from plants with
p35S-, pLTP1- or pATML1-driven DEK1 down-regulation also
show effects on epidermal and vascular tissues, with the
leaves exhibiting a reduced distinction between outer and
inner cells as well as reduced vasculature (Fig. 5J–L).
Discussion
DEK1 is essential for seed, embryo, epidermis, trichome and
SAM development, and subsequent plant growth. This
1862
unique plant calpain is also linked to syncytial nuclear divisions
prior to the cellularization of several cell types, including aleurone and giant sepal cells (Brown et al. 1994, Roeder et al. 2012),
yet little is known about how DEK1 acts at the cellular and
molecular levels. The altered expression of the initial asymmetric zygotic division marker WOX2, the epidermal marker
ATML1, the SAM-promoting genes WUS and STM, and the
auxin transporter gene PIN4 presented here demonstrates a
fundamental role for DEK1 during development. Induced
down-regulation of DEK1 during seedling growth further indicates that SAM function depends on the activity of DEK1 in all
three cell layers. In addition, our data show that dek1 embryos
exhibit aberrant cell wall positions, disorganized CMTs and
misplaced phragmoplasts, supporting a fundamental role for
DEK1 in cytoskeletal control.
This unique plant calpain links cell wall position
to stem cell functions
The orientation of cell walls is key to body patterning and
probably to the creation of multicellular organisms. In plants
with fixed cell walls, cells can divide either anticlinally or periclinally to the outer surface (Murray et al. 2012). Initial anticlinal
divisions generate an outer L1 cell layer, while periclinal divisions from these L1 cells create new internal cell layers and new
cell identities. How this cell division orientation is initially established according to positional and developmental information is still unknown.
DEK1 affects cell divisions in both the embryo and endosperm during Arabidopsis seed development (Johnson et al.
2005, Lid et al. 2005), and is a plausible candidate for setting
up cell position and downstream cues during development and
growth via effects on the cytoskeleton and associated cell wall
deposition. Lack of WOX2 activation in dek1 embryos indicates
that DEK1 may play a role in directing the first zygotic asymmetric anticlinal division. DEK1 is further required to obtain
epidermal identity by restricting ATML1 expression to the L1
layer; we suggest that this might be due to dependency of
controlled anticlinal divisions (Takada et al. 2013).
Subsequent cell divisions throughout the embryo are irregular,
causing developmental arrest at the globular stage, possibly by
failure to identify cell positioning and correct organization of
anticlinal and periclinal divisions. We suggest that this leads to a
failure to establish and maintain epidermal cell fate specification and dependent downstream cues in embryos and meristems throughout plants.
Our study provides new insights into stem cell regulation in
the developing embryonic SAM. Expression of the stem cellpromoting gene WUS in wild-type embryos initiates at the 16cell stage, followed by STM transcription at the globular stage.
In dek1 embryos, both WUS and STM are induced, but their
expression is not properly restricted to the central region. These
results identify DEK1 as a key negative regulator of early meristem gene expression domains and reveal that DEK1 is necessary to restrict the WUS expression domain prior to CLV3
induction at the heart stage. The failure of the DEK1 knockdown plants to sustain SAM function during vegetative development despite the presence of WUS and STM transcripts in
Plant Cell Physiol. 56(9): 1855–1866 (2015) doi:10.1093/pcp/pcv110
the embryo suggests that DEK1 may promote shoot stem cell
maintenance independently of the WUS and STM pathways.
Signaling from the cells in the L1 layer of the SAM is necessary to keep the underlying cells indeterminate and thus in a
proliferative state. We find that pATML1::DEK1MEM and
pLTP1::DEK1MEM plants with reduced DEK1 activity in the L1
cell layer terminate during early seedling development, with the
cells at the shoot apex taking on a differentiated, vacuolated
appearance. These data demonstrate that reducing DEK1 activity in the L1 layer alone is sufficient to obtain a severe shoot
stem cell phenotype, suggesting that DEK1 could be part of this
signaling mechanism.
DEK1 functions in cytoskeletal control
Evolutionary studies have shown that a variety of different
calpains exist in unicellular species, whereas one highly conserved single copy is found across land plant species (Liang
et al. 2013). The plant DEK1 protein is evolutionarily related
to the multigene calpain family in animals, which includes 15
members in humans (Sorimachi et al. 2011, Zhao et al. 2012).
The DEK1 loop/channel membrane region is necessary to
confer dominant-negative mutant effects, whereas the calpain
domain can rescue the loss-of-function phenotypes (Tian et al.
2007, Johnson et al. 2008). How the membrane region might be
involved in regulating DEK1 function is still not understood,
neither is it known how the loop/channel might be transmitting information. It could take place through non-specific permeability or specific transport of alternative external signals
such as Ca2+, auxin, etc. We interpret the irregular cell divisions
that occur in dek1 embryos as resulting from the inability of the
cells to sense their position and to activate the intracellular
calpain. This causes the failure of the cells to organize their
division planes during cytokinesis and to sustain cell cycle progression, thereby losing their specification and arresting developmental cues, ultimately leading to early embryo abortion.
Our data indicate that early dek1 embryo arrest is not
caused by non-functional mitotic machinery, but instead correlates with loss of controlled cell wall positioning and shaping.
Similarly, Galletti et al. (2015) concluded that altered CMT arrangement in weak dek1-4 mutant plants was due to an alteration in cell shape, although they provided no rationale for this
link. The expression of TONNEAU, which affects the cortical
cytoskeleton of plant cells and MT organizers in cells of other
eukaryotes, is not regulated by DEK1 (Supplementary Table
S2), suggesting they function via alternative mechanisms
(Spinner et al. 2013). However, DEK1 does regulate the expression of AtSAC9, a mutation causing extreme cell wall abnormalities within meristems regardless of cell type and position
(Vollmer et al. 2011). MT organization, cell division plane control and cell wall shaping are all critical for the normal pattern of
plant cell division, yet how this is controlled has not been
determined. Several MT organization genes with functions in
cell division plane control and cell wall orientation have been
characterized (MAP65, MIDD1 and NEK5) that further potentially interact with auxin (Xu et al. 2008, Oda and Fukuda 2012,
Uyttewaal et al. 2012, Dhonukshe et al. 2013). DEK1 affects the
expression of these genes as well as of many auxin-related genes
(Supplementary Table S2). Auxin and/or PLT2 (PLETHORA2),
MAP65 (MICROTUBULE-ASSOCIATED PROTEIN65) and
CLASP (CLIP-ASSOCIATED PROTEIN) control formative cell
division planes, periclinal cell wall orientation and epidermal
cell identity via mechanisms that are still only partly understood (Becraft and Yi 2011, Dhonukshe et al. 2013). MAP65 is
further sufficient to trigger cell division plane switches, and
DEK1 down-regulation of MAP65 (Supplementary Table S2)
suggests that DEK1 may set cell wall positions via its regulation
of this and possibly other genes involved in MT organization.
Higher eukaryotes depend on nuclear membrane degradation
to enter mitosis prometaphase, and calpains are known to function in nuclear envelope permeability and breakdown (Concha
et al. 2005, Bano et al. 2010, Burke and Stewart 2013).
Interestingly, yeast nuclear divisions take place within the nuclear
membrane, showing that yeasts’ dependence on their single-copy
calpain-like cysteine protease for these divisions is not linked to its
activity in the nuclear envelope. However, their dependence on
the gene product could be linked to related functions controlled
by MTs to allow cell divisions and thereby sporulation.
Alternatively it could be due to another function of calpain in
cell cycle progression (Futai et al. 1999). Given the association of
calpains with the nuclear envelope in animal cells, it may be that
the membrane-anchored DEK1 in plant cells transmits surface
information to the nuclear envelope via the internal calpain that
is important for establishing the division plane. Removal of DEK1
function in Physcomitrella using gene replacement has recently
been shown to cause abnormal cell divisions in the emerging
buds, resulting in developmental arrest and the absence of
three-dimensional growth (Demko et al. 2014, Perroud et al.
2014). Whether it is the division plane and/or the cell wall deposition that is affected remains to be addressed.
We conclude that DEK1 affects MT organization, cell division pattern, cell shaping and asymmetric daughter cell generation. Our results indicate that these functions sustain plant
stem cell reservoirs as well as overall growth and development.
Thus DEK1 represents the first likely initial positional sensor in
dividing cells. Future work is needed on the roles of DEK1 as a
component of the interactive system linking surface information in walls with the cell interior. Given that calpain functions
are still poorly understood despite their effects on human and
animal health connected to stem cell functions, cancer and the
cell cycle (Chakraborti et al. 2012), this work may lead to new
insights and progress in understanding calpain functions in all
kingdoms, with possible implications for the general understanding of cell division regulation via the CMT system.
Further experimental and computational work will also increase our insight into the control of stem cell regulation and
developmental cues, with their correlated applications to
plants, animals and mankind.
Materials and Methods
Plant materials
Arabidopsis plants in the Columbia (Col) background were grown under longday conditions with 16 h d1 of cool-white fluorescent light (85 mol m2 s1) at
23 C. Homozygous dek1-1 plants carrying a T-DNA insertion in intron 11 and
1863
Z. Liang et al. | DEK1 directs cell wall positioning and stem cell functions
homozygous dek1-2 plants carrying a T-DNA insertion in exon 3 (Lid et al. 2005,
Tian et al. 2007) were used for morphological and molecular analysis. Seeds
were sown on soil and stratified at 4 C for 2 d. Transgenic plants were generated
by the floral dip method (Clough and Bent 1998), using Agrobacterium strain
C58 pCV2260. Glucocorticoid-inducible GVG lines were selected on Murashige
and Skoog (MS) medium containing 15 mM hygromycin before being transferred to dexamethazone induction medium (Aoyama and Chua 1997), and
transgenic T2 plants were grown on agar plates containing 5 mM dexamethazone for 1–2 weeks.
Promoter studies
The uidA gene (Opsahl-Sorteberg et al. 2004) was amplified by PCR with primers
GUS-F1-SalI and GUS-R1-SpeI (Supplementary Table S1), cloned into TOPTA2.1 (Invitrogen) then subcloned into pSEL1-NOS (Lid et al. 2005) to generate
pSEL1-GUS-NOS. The Arabidopsis DEK1 promoter (2.4 kb upstream of the ATG)
was amplified with primers Aradekpromoter-F-BamHI and Aradekpromoter-RATG-XmalI (Supplementary Table S1), cloned into TOPO-TA2.1 and then subcloned into pSEL1-GUS-NOS to generate pSEL1-Arapromoter-GUS-NOS. Plants
carrying the WOX2 (Haecker et al. 2004), PIN1 (Friml et al. 2003) or PIN4 (Friml
et al. 2002) marker constructs were crossed to heterozygous DEK1/dek1 plants
containing either the dek1-1 or dek1-2 allele, such that the selective marker for the
mutant allele was different from that of the promoter construct. The
ATML1::GUS plasmid pAS103 (Sessions et al. 1999) and the pCLV3::GUS plasmid
(Wurschum et al. 2006) were also separately transformed into heterozygous
DEK1/dek1 plants. All plants were genotyped to select dek1-1 or dek1-2 homozygous individuals carrying the transgenic construct including the uidA marker
gene (Jefferson et al. 1986, Opsahl-Sorteberg et al. 2004). uidA genotyping was
performed using forward uidA-F and reverse uidA-R primers (Supplementary
Table S1) that generated a 583 nucleotide PCR product. For each embryo promoter construct, 62–143 transgenic plants were genotyped after selection.
Among these, 28–64 plants were homozygous for the dek1 allele and 21–52
plants simultaneously carried the promoter construct. Promoter activity was
monitored as uidA reporter gene activity. Dissected embryos were incubated in
X-Gluc, cleared with a 1 : 1 mixture of acetic acid and ethanol, and cleared with
Hoyers solution before being visualized with a Leica microscope using DIC optics.
Images were captured using a CCD camera.
Immunostaining
Arabidopsis seeds from heterozygous DEK1/dek1-1 plants were harvested from
siliques at the desired stages and fixed in 4% paraformaldehyde in PHEM buffer
(60 mM PIPES, 25 mM HEPES, 2 mM MgCl2, 10 mM EGTA; pH 6.9) overnight at
4 C before being rinsed three times for 5 min each in PHEM buffer (as per Tian
et al. 2007). For immunolocalization, wild-type and dek1 embryos were dissected from seeds in a drop of PHEM buffer containing 5% dimethylsulfoxide
(DMSO). Approximately 30 wild-type and 20 dek1 dissected embryos were
dried on cover slips with Meyers’ adhesive (50 ml of fresh egg white, 50 ml of
glycerin). The cover slips were covered with a thin film (0.75% low melting
agarose/0.75% gelatin), and washed three times for 5 min each in PHEM/DMSO
buffer. The embryos were covered in a freshly prepared enzyme mixture (cellulase 0.1%, glucoronidase 0.1%, pectolyase 1%) for 1 h to help digest the cell
walls. The cover slips were washed three times for 5 min each in PHEM/DMSO
buffer before being treated with 1% Triton X to permeabilize the plasma membrane, and then washed again. The cover slips were incubated for 1 h in a humid
chamber at 37 C with rat McAb anti-tubulin primary antibody (Sera-lab) and
monoclonal mouse antibody against pea root actin, before being washed three
times for 5 min each with PHEM/DMSO. The embryos were then incubated
with Rhodamine RedTM-X-conjugated AffiniPure Donkey anti-rat IgG (H+L)
secondary antibody and anti-mouse immunoglobulin G conjugated to fluorescein secondary antibody for 1 h in the dark in a humid chamber at 37 C.
Following several water washes, the nucleic acids were stained with 1 mM ToPro-3 iodide (Molecular Probes). The cover slips were mounted in ProLong antifade mounting media (Invitrogen). Slides were stored in the dark at 4 C until
imaging was performed using a BioRad confocal laser scanning microscope.
Histological sections
Arabidopsis tissues were prepared for morphological examination using light
microscopy as previously described (Lid et al. 2005). Sections were cut at
1864
0.5–1.0 mm thickness, stained with Stevenels Blue dye (Delcerro et al. 1980),
and viewed using a Leitz Aristoplan microscope. Images were captured using a
Leica DC300F digital camera.
In situ hybridization
Sequences for the ATML1 A and B probes were amplified using primers
AtML1_probeA_F,
AtML1_probeA_R,
AtML1_probeB_F
and
AtML1_probeB_R (Supplementary Table S1). Both probes were used for hybridization at the same time. The WUS probe was amplified as described by
Mayer et al. (1998), the STM probe was amplified as described by Long et al.
(1996) and the WOX8 probe was amplified as described by Haecker et al. (2004).
Each PCR product was cloned into the TOPO-TA vector (Invitrogen), linearized
with NotI for the antisense probe or SpeI for the sense probe, and used as a
template for dioxigenin-11-UTP labeling (Roche Molecular Biochemicals) of the
RNA probe. In vitro transcription was performed using T3 or T7 RNA polymerase following the manufacturer’s protocol. Siliques were fixed in 50% ethanol,
5% acetic acid and 3.7% formaldehyde, vacuum infiltrated, dehydrated in a
graded ethanol series to 100%, stained with Eosin Y, passed through an ethanol : histoclear series, and embedded in Paraplast Plus. Tissue sections of 8 mm
thickness were mounted on Superfrost-Plus slides. Pre-hybridization, hybridization and wash steps were performed as described (Jackson 1991). The hybridized probe was detected using the DIG Nucleic Acid Detection Kit (Roche
Molecular Biochemicals). Microscopy was carried out in dark field mode using a
Zeiss Axioplan 2 microscope, and images were captured with an AxioCam HRc
digital camera.
Dexamethazone-inducible DEK1 knock-down
constructs
To produce the transactivating constructs, we generated the vector pRN from
the existing pCAMBIA1300 vector by replacing the short polylinker (50 EcoRI–
HindIII 30 ) with an extended polylinker (50 EcoRI, KpnI, BsrGI, SalI, XbaI, NcoI,
PstI, BglII, MluI, ApaI, SpeI, HindIII 30 ) that allowed us to combine our constructs.
The 35S-GVG-E9 fragment was amplified from pTA7002 using the primers 35SF-KpnI, GVG-R-BamHI, E9-R-SalI and GVG-F-BamHI (Supplementary Table
S1), and then cloned into pCAMBIA2301 to generate pCAMBIA2301-35SGVG-E9. The GVG-E9 cassette from pCAMBIA1300-35S-GVG-E9 was amplified
with 50 BglII and 30 SpeI primers and cloned into the new pRN vector. The
ATML1, AtLTP1, AtSUC2 and AtRbcS2b promoter sequences were amplified and
cloned into the TOPO vector before being cloned into pRN-GVG-E9.
To produce the response constructs, we amplified UAS-TATA-MSC-TA3
from pTA7200 and cloned it into the pUC18 vector. We next inserted a 50 XhoI/
XmaI/SalI/KpnI/SpeI 30 cassette into the vector’s XhoI/SpeI cloning sites, and
this new cassette was cloned into pSEL1-NOS (Lid et al. 2005) to generate
pSEL1-UAS-TATA-NOS. To generate the RNAi response construct, our DEK1RNAi construct (Tian et al. 2007) was digested with XmaI and KpnI, and then
the DEK1-RNAi cassette was cloned into pSEL1-UAS-TATA-NOS. To generate
the MEM response construct, the pSEL1-ProCaMV35-AtDek1-MEM-NOS (Tian
et al. 2007) construct was digested with XmaI and SpeI, and cloned into pSEL1UAS-TATA-NOS. All constructs were confirmed by sequencing. The GVG-E9
baseline was confirmed by crossing it to the positive control basic response
UAS-TATA::GUS line that generated blue plants when induced by dexamethazone (Aoyama and Chua 1997), genetically segregating as expected.
Supplementary data
Supplementary data are available at PCP online.
Funding
This work was supported by the Norwegian Research Council
[project 159031 and IKBM/IPM, NMBU to H-G.O.H.-S. and Z.L.];
and the US Department of Agriculture [Current Research
Information System grants 5335-21000-038-00D and 533521000-041-00D to J.C.F.].
Plant Cell Physiol. 56(9): 1855–1866 (2015) doi:10.1093/pcp/pcv110
Acknowledgments
We thank Tim Nelson’s group and Yale for a great academic
environment; Lene Olsen Hults, Stein Erik Lid and Betty
Lemmon (University of Louisiana Lafayette, USA), Barbro
Sæther and Ellen Andersen (NARC, UiO) for experimental
work and guidance; Ragnhild Nestestog (NARC), Hege Divon,
Kjetil Fosnes, Marten van der Linden, Atiya Rafaqat Ali, Stine
Indrelid and Ingrid Heger (NMBU) for technical assistance;
Thomas Laux, Detlef Weigel and Jiri Friml for providing constructs; Odd-Arne Olsen for initiating the cytoskeletal studies;
and Yrjö Helariutta, Huw D. Jones, Klaus Palme, Ralf Reski, Dirk
Inze and Fred Berger for fruitful discussions.
Disclosures
The authors have no conflicts of interest to declare.
References
Abe, M., Katsumata, H., Komeda, Y. and Takahashi, T. (2003) Regulation of
shoot epidermal cell differentiation by a pair of homeodomain proteins
in Arabidopsis. Development 130: 635–643.
Aoyama, T. and Chua, N.H. (1997) A glucocorticoid-mediated transcriptional induction system in transgenic plants. Plant J. 11: 605–612.
Bano, D., Dinsdale, D., Cabrera-Socorro, A., Maida, S., Lambacher, N.,
McColl, B., et al. (2010) Alteration of the nuclear pore complex in
Ca(2+)-mediated cell death. Cell Death Differ. 17: 119–133.
Becraft, P.W. and Yi, G. (2011) Regulation of aleurone development in
cereal grains. J. Exp. Bot. 62: 1669–1675.
Bemis, S.M. and Torii, K.U. (2007) Autonomy of cell proliferation and
developmental programs during Arabidopsis aboveground organ morphogenesis. Dev. Biol. 304: 367–381.
Bergounioux, J., Elisee, R., Prunier, A.L., Donnadieu, F., Sperandio, B.,
Sansonetti, P., et al. (2012) Calpain activation by the Shigella flexneri
effector VirA regulates key steps in the formation and life of the bacterium’s epithelial niche. Cell Host Microbe 11: 240–252.
Brand, U., Fletcher, J.C., Hobe, M., Meyerowitz, E.M. and Simon, R. (2000)
Dependence of stem cell fate in Arabidopsis on a feedback loop regulated by CLV3 activity. Science 289: 617–619.
Breuninger, H., Rikirsch, E., Hermann, M., Ueda, M. and Laux, T. (2008)
Differential expression of WOX genes mediates apical–basal axis formation in the Arabidopsis embryo. Dev. Cell 14: 867–876.
Brown, R.C. and Lemmon, B.E. (2001) The cytoskeleton and spatial control
of cytokinesis in the plant life cycle. Protoplasma 215: 35–49.
Brown, R.C., Lemmon, B.E. and Olsen, O.A. (1994) Endosperm development in barley: microtubule involvement in the morphogenetic pathway. Plant Cell 6: 1241–1252.
Burke, B. and Stewart, C.L. (2013) The nuclear lamins: flexibility in function.
Nat. Rev. Mol. Cell Biol. 14: 13–24.
Byrne, M.E., Barley, R., Curtis, M., Arroyo, J.M., Dunham, M., Hudson, A.,
et al. (2000) Asymmetric leaves1 mediates leaf patterning and stem cell
function in Arabidopsis. Nature 408: 967–971.
Byrne, M.E., Simorowski, J. and Martienssen, R.A. (2002) ASYMMETRIC
LEAVES1 reveals knox gene redundancy in Arabidopsis. Development
129: 1957–1965.
Chakraborti, S., Alam, M.N., Paik, D., Shaikh, S. and Chakraborti, T. (2012)
Implications of calpains in health and diseases. Indian J. Biochem. Biol.
49: 316–328.
Chan, J., Calder, G., Fox, S. and Lloyd, C. (2005) Localization of the
microtubule end binding protein EB1 reveals alternative pathways of
spindle development in Arabidopsis suspension cells. Plant Cell 17:
1737–1748.
Clough, S.J. and Bent, A.F. (1998) Floral dip: a simplified method for
Agrobacterium-mediated transformation of Arabidopsis thaliana.
Plant J. 16: 735–743.
Conacci-Sorrell, M. and Eisenman, R.N. (2011) Post-translational control of
Myc function during differentiation. Cell Cycle 10: 604–610.
Concha, C., Monardes, A., Even, Y., Morin, V., Puchi, M., Imschenetzky, M.,
et al. (2005) Inhibition of cysteine protease activity disturbs DNA replication and prevents mitosis in the early mitotic cell cycles of sea
urchin embryos. J. Cell Physiol. 204: 693–703.
Croall, D.E. and Ersfeld, K. (2007) The calpains: modular designs and functional diversity. Genome Biol. 8: 218.
Crowell, E.F., Timpano, H., Desprez, T., Franssen-Verheijen, T., Emons, A.M.,
Hofte, H., et al. (2011) Differential regulation of cellulose orientation at
the inner and outer face of epidermal cells in the Arabidopsis hypocotyl.
Plant Cell 23: 2592–2605.
Delcerro, M., Cogen, J. and Cerro, C.D. (1980) Stevenels blue, an excellent
stain for optical microscopical study of plastic embedded tissues.
Microsc. Acta 83: 117–121.
Demko, V., Perroud, P.F., Johansen, W., Delwiche, C.F., Cooper, E.D.,
Remme, P., et al. (2014) Genetic analysis of DEFECTIVE KERNEL1
loop function in three-dimensional body patterning in Physcomitrella
patens. Plant Physiol. 166: 903–919.
Dhonukshe, P., Weits, D.A., Cruz-Ramirez, A., Deinum, E.E., Tindemans,
S.H., Kakar, K., et al. (2013) A PLETHORA–auxin transcription module
controls cell division plane rotation through MAP65 and CLASP. Cell
155: 1189–1189.
Friml, J., Benkova, E., Blilou, I., Wisniewska, J., Hamann, T., Ljung, K., et al.
(2002) AtPIN4 mediates sink-driven auxin gradients and root patterning in Arabidopsis. Cell 108: 661–673.
Friml, J., Vieten, A., Sauer, M., Weijers, D., Schwarz, H., Hamann, T., et al.
(2003) Efflux-dependent auxin gradients establish the apical–basal axis
of Arabidopsis. Nature 426: 147–153.
Futai, E., Sorimachi, H., Jeong, S., Kitamoto, K., Ishiura, S. and Suzuki, K.
(1999) Aspergillus oryzae palB(ory) encodes a calpain-like protease:
homology to Emericella nidulans PalB and conservation of functional
regions. J. Biosci. Bioeng. 88: 438–440.
Galletti, R., Johnson, K.L., Scofield, S., San-Bento, R., Watt, A.M., Murray,
J.A., et al. (2015) DEFECTIVE KERNEL 1 promotes and maintains plant
epidermal differentiation. Development 142: 1978–1983.
Gonzalez-Garcia, M.P., Vilarrasa-Blasi, J., Zhiponova, M., Divol, F., MoraGarcia, S., Russinova, E., et al. (2011) Brassinosteroids control meristem
size by promoting cell cycle progression in Arabidopsis roots.
Development 138: 849–859.
Haecker, A., Gross-Hardt, R., Geiges, B., Sarkar, A., Breuninger, H.,
Herrmann, M., et al. (2004) Expression dynamics of WOX genes mark
cell fate decisions during early embryonic patterning in Arabidopsis
thaliana. Development 131: 657–668.
Jackson, D. (1991) In Situ Hybridization in Plants. Oxford University Press,
Oxford.
Javelle, M., Vernoud, V., Rogowsky, P.M. and Ingram, G.C. (2011) Epidermis:
the formation and functions of a fundamental plant tissue. New Phytol.
189: 17–39.
Johnson, K.L., Degnan, K.A., Walker, J.R. and Ingram, G.C. (2005) AtDEK1 is
essential for specification of embryonic epidermal cell fate. Plant J. 44:
114–127.
Johnson, K.L., Faulkner, C., Jeffree, C.E. and Ingram, G.C. (2008) The
Phytocalpain Defective Kernel 1 is a novel Arabidopsis growth regulator
whose activity is regulated by proteolytic processing. Plant Cell 20:
2619–2630.
Knauer, S., Holt, A.L., Rubio-Somoza, I., Tucker, E.J., Hinze, A., Pisch, M.,
et al. (2013) A protodermal miR394 signal defines a region of stem
cell competence in the Arabidopsis shoot meristem. Dev. Cell 24:
125–132.
1865
Z. Liang et al. | DEK1 directs cell wall positioning and stem cell functions
Lenhard, M., Jurgens, G. and Laux, T. (2002) The WUSCHEL and
SHOOTMERISTEMLESS genes fulfil complementary roles in
Arabidopsis shoot meristem regulation. Development 129: 3195–3206.
Liang, Z., Demko, V., Wilson, R.C., Johnson, K.A., Ahmad, R., Perroud, P.F.,
et al. (2013) The catalytic domain CysPc of the DEK1 calpain is functionally conserved in land plants. Plant J. 75: 742–754.
Lid, S.E., Gruis, D., Jung, R., Lorentzen, J.A., Ananiev, E., Chamberlin, M., et al.
(2002) The defective kernel 1 (dek1) gene required for aleurone cell
development in the endosperm of maize grains encodes a membrane
protein of the calpain gene superfamily. Proc. Natl. Acad. Sci. USA 99:
5460–5465.
Lid, S.E., Olsen, L., Nestestog, R., Aukerman, M., Brown, R.C., Lemmon, B.,
et al. (2005) Mutation in the Arabidopisis thaliana DEK1 calpain gene
perturbs endosperm and embryo development while over-expression
affects organ development globally. Planta 221: 339–351.
Long, J.A., Moan, E.I., Medford, J.I. and Barton, M.K. (1996) A member of
the KNOTTED class of homeodomain proteins encoded by the STM
gene of Arabidopsis. Nature 379: 66–69.
Mayer, K.F.X., Schoof, H., Haecker, A., Lenhard, M., Jurgens, G. and Laux, T.
(1998) Role of WUSCHEL in regulating stem cell fate in the Arabidopsis
shoot meristem. Cell 95: 805–815.
Muller, S., Wright, A.J. and Smith, L.G. (2009) Division plane control in
plants: new players in the band. Trends Cell Biol. 19: 180–188.
Murray, J.A.H., Jones, A., Godin, C. and Traas, J. (2012) Systems analysis of
shoot apical meristem growth and development: integrating hormonal
and mechanical signaling. Plant Cell 24: 3907–3919.
Nodine, M.D. and Bartel, D.P. (2012) Maternal and paternal genomes
contribute equally to the transcriptome of early plant embryos.
Nature 482: 94–97.
Oda, Y. and Fukuda, H. (2012) Initiation of cell wall pattern by a rho- and
microtubule-driven symmetry breaking. Science 337: 1333–1336.
Olsen, L.T., Divon, H.H., Al, R., Fosnes, K., Lid, S.E. and Opsahl-Sorteberg,
H.G. (2008) The defective seed5 (des5) mutant: effects on barley seed
development and HvDek1, HvCr4, and HvSal1 gene regulation. J. Exp.
Bot. 59: 3753–3765.
Opsahl-Sorteberg, H.G., Divon, H.H., Nielsen, P.S., Kalla, R., HammondKosack, B., Shimamoto, K., et al. (2004) Identification of a 49-bp
fragment of the HvLTP2 promoter directing aleurone cell specific
expression. Gene 341: 49–58.
Perroud, P.F., Demko, V., Johansen, W., Wilson, R.C., Olsen, O.A. and
Quatrano, R.S. (2014) Defective Kernel 1 (DEK1) is required for
three-dimensional growth in Physcomitrella patens. New Phytol. 203:
794–804.
Robinson, D.O. and Roeder, A.H. (2015) Themes and variations in cell type
patterning in the plant epidermis. Curr. Opin. Genet. Dev. 32C: 55–65.
Roeder, A.H.K., Cunha, A., Ohno, C.K. and Meyerowitz, E.M. (2012) Cell
cycle regulates cell type in the Arabidopsis sepal. Development 139:
4416–4427.
Santos, D.M., Xavier, J.M., Morgado, A.L., Sola, S. and Rodrigues, C.M.P.
(2012) Distinct regulatory functions of Calpain 1 and 2 during neural
stem cell self-renewal and differentiation. PLoS One 7: e33468.
Savaldi-Goldstein, S., Peto, C. and Chory, J. (2007) The epidermis both
drives and restricts plant shoot growth. Nature 446: 199–202.
Schoof, H., Lenhard, M., Haecker, A., Mayer, K.F., Jurgens, G. and Laux, T.
(2000) The stem cell population of Arabidopsis shoot meristems in
1866
maintained by a regulatory loop between the CLAVATA and
WUSCHEL genes. Cell 100: 635–644.
Sessions, A., Weigel, D. and Yanofsky, M.F. (1999) The Arabidopsis thaliana
MERISTEM LAYER 1 promoter specifies epidermal expression in meristems and young primordia. Plant J. 20: 259–263.
Smith, L.G., Gerttula, S.M., Han, S. and Levy, J. (2001) Tangled1: a microtubule binding protein required for the spatial control of cytokinesis in
maize. J. Cell Biol. 152: 231–236.
Sorimachi, H., Hata, S. and Ono, Y. (2011) Impact of genetic insights into
calpain biology. J. Biochem. 150: 23–37.
Spinner, L., Gadeyne, A., Belcram, K., Goussot, M., Moison, M., Duroc, Y.,
et al. (2013) A protein phosphatase 2A complex spatially controls plant
cell division. Nat. Commun. 4: 1863.
Steeves, T.A. and Sussex, I.M. (1989) Patterns in Plant Development.
Cambridge University Press, New York.
Takada, S. and Jurgens, G. (2007) Transcriptional regulation of epidermal
cell fate in the Arabidopsis embryo. Development 134: 1141–1150.
Takada, S., Takada, N. and Yoshida, A. (2013) ATML1 promotes
epidermal cell differentiation in Arabidopsis shoots. Development 140:
1919–1923.
Tian, Q., Olsen, L., Sun, B., Lid, S.E., Brown, R.C., Lemmon, B.E., et al. (2007)
Subcellular localization and functional domain studies of DEFECTIVE
KERNEL1 in maize and Arabidopsis suggest a model for aleurone cell
fate specification involving CRINKLY4 and SUPERNUMERARY
ALEURONE LAYER1. Plant Cell 19: 3127–3145.
Uyttewaal, M., Burian, A., Alim, K., Landrein, B.T., Borowska-Wykret, D.,
Dedieu, A., et al. (2012) Mechanical stress acts via katanin to amplify
differences in growth rate between adjacent cells in Arabidopsis. Cell
149: 439–451.
Vollmer, A.H., Youssef, N.N. and DeWald, D.B. (2011) Unique cell wall
abnormalities in the putative phosphoinositide phosphatase mutant
AtSAC9. Planta 234: 993–1005.
Wang, C., Barry, J.K., Min, Z., Tordsen, G., Rao, A.G. and Olsen, O.A. (2003)
The calpain domain of the maize DEK1 protein contains the conserved
catalytic triad and functions as a cysteine proteinase. J. Biol. Chem. 278:
34467–34474.
Wisniewski, J.P. and Rogowsky, P.M. (2004) Vacuolar H+-translocating inorganic pyrophosphatase (Vpp1) marks partial aleurone cell fate in
cereal endosperm development. Plant Mol. Biol. 56: 325–337.
Wurschum, T., Gross-Hardt, R. and Laux, T. (2006) APETALA2 regulates
the stem cell niche in the Arabidopsis shoot meristem. Plant Cell 18:
295–307.
Xu, X.F.M., Zhao, Q., Rodrigo-Peiris, T., Brkljacic, J., He, C.S., Muller, S., et al.
(2008) RanGAP1 is a continuous marker of the Arabidopsis cell division
plane. Proc. Natl. Acad. Sci. USA 105: 18637–18642.
Yadav, R.K., Perales, M., Gruel, J., Girke, T., Jonsson, H. and Reddy, G.V.
(2011) WUSCHEL protein movement mediates stem cell homeostasis in
the Arabidopsis shoot apex. Genes Dev. 25: 2025–2030.
Yi, G., Lauter, A.M., Scott, M.P. and Becraft, P.W. (2011) The thick aleurone1 mutant defines a negative regulation of maize aleurone cell fate
that functions downstream of defective kernel1. Plant Physiol. 156:
1826–1836.
Zhao, S., Liang, Z., Demko, V., Wilson, R., Johansen, W., Olsen, O.A., et al.
(2012) Massive expansion of the calpain gene family in unicellular eukaryotes. BMC Evol. Biol. 12: 193.