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First published online 31 March 2009
doi: 10.1242/dev.035535
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Institut de Biologie Moléculaire des Plantes du CNRS, 12 rue du Général Zimmer, 67084 Strasbourg Cedex, France.
Author for correspondence (e-mail:
pascal.genschik{at}ibmp-ulp.u-strasbg.fr)
Accepted 3 March 2009
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Arabidopsis, Cell cycle, Endoreduplication, Ubiquitin, Vasculature
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Pickart,
2001; Smalle and Vierstra,
2004). Degradation via the UPS is a two-step process: the protein
is first tagged by covalent attachment of ubiquitin and subsequently degraded
by a multicatalytic protease complex called the 26S proteasome. The transfer
of ubiquitin to an internal lysine residue in the target protein substrate
requires an ubiquitin protein-ligase (E3). As E3 enzymes specify the
substrates, they play the most important role in the ubiquitylation reaction.
Two E3s dominate DNA duplication and cell division: the SCF and the anaphase
promoting complex/cyclosome (APC/C).
The APC/C is the largest E3 known so far. In vertebrates, it is composed of
12 subunits (Peters, 2002),
whereas, in yeast, 13 subunits have been described
(Yoon et al., 2002). The
functions of individual subunits are still unclear, although a structural
model of its organization has been proposed
(Thornton et al., 2006). The
minimal ubiquitin ligase module of the APC/C comprises APC2, a distant member
of the cullin family and the RING-finger protein APC11. The APC/C also
contains three essential subunits with tetratricopeptide repeat (TPRs)
protein-protein interaction domains: APC3/CDC27, APC8/CDC23 and APC6/CDC16.
One of them (APC3/CDC27) has been implicated in binding the activating subunit
CDH1 (Vodermaier et al., 2003;
Kraft et al., 2005).
Phosphorylation of these subunits during mitosis is required to activate the
APC/C (Rudner and Murray,
2000; Kraft et al.,
2003). Finally, the APC10/DOC1 subunit, which is characterized by
the presence of a DOC domain, is important for substrate recognition and/or
extending the poly-ubiquitin chain on a substrate
(Carroll and Morgan, 2002;
Carroll et al., 2005;
Passmore et al., 2003). In
addition to its core components, the APC/C requires co-activator proteins such
as CDC20/FIZZY or CDH1/FIZZY-RELATED, which activate the APC/C sequentially
during the cell cycle. CDC20 and CDH1 also bind to substrates, suggesting that
they recruit substrates to the catalytic center of the E3 complex
(Vodermaier, 2001).
During the past decade, much attention has been paid on the role of the
APC/C in regulating cell cycle transitions. The APC/C is mainly required to
induce progression and exit from mitosis by mediating proteolysis of different
cell cycle regulators, including PDS1/SECURIN and cyclin B. Degradation of
PDS1/SECURIN is required for sister chromatid separation
(Uhlmann et al., 2000;
Yanagida, 2000), whereas the
destruction of cyclin B triggers the inhibition of CDK1 activity and, as a
consequence, induces different cell processes such as disassembly of the
mitotic spindle, chromosome decondensation, cytokinesis and reformation of the
nuclear envelope (Murray and Kirschner,
1989; Luca et al.,
1991; Gallant and Nigg,
1992; Holloway et al.,
1993; Surana et al.,
1993). In all eukaryotes, the instability of B-type cyclins is
conferred by a small degenerated but conserved motif of nine amino acids
RxxLxxIxN located in the N-terminal region of these proteins and known as the
destruction box (Dbox) (Glotzer et al.,
1991). Deletion or point mutation of the Dbox inhibits cyclin
proteolysis (Brandeis and Hunt,
1996; Yamano et al.,
1998; Genschik et al.,
1998).
In addition to PDS1/SECURIN and mitotic cyclins, many other important cell
cycle proteins were proved to be targets of APC/C in yeast and mammalian
cells. Among them, the kinesin-related protein XKID involved in chromosome
movements (Funabiki and Murray,
2000; Levesque and Compton,
2001), two motor proteins (KIP1 and CIN8)
(Gordon and Roof, 2001;
Hildebrant and Hoyt, 2001) and
the mitotic spindle-associated protein ASE1 are implicated in central spindle
formation and cytokinesis (Juang et al.,
1997; Yamashita et al.,
2005). The APC/C remains also active during G1 to restrain the
accumulation of the mitotic cyclins, but its inactivation is required for
timely S-phase entry (Irniger and Nasmyth,
1997).
More surprising was the discovery that the APC/C is expressed and remains
active in differentiated vertebrate cells, such as neuron
(Gieffers et al., 1999).
Unexpected neurobiological functions orchestrated by APC/CCDH1 have
since been deciphered, ranging from axon growth, neuronal survival and
synaptic functions (reviewed by Kim and
Bonni, 2007). Thus, SnoN, a co-transcriptional repressor of the
TGFβ signaling pathway, which is involved in elongation of neuron axons
in developing cerebellum, is a target of APC/CCDH1
(Stroschein et al., 2001;
Wan et al., 2001;
Stegmüller et al., 2006).
Another transcriptional regulator targeted in a Dbox-dependent manner by
neuronal APCCDH1 is the helix-loop-helix protein Id2 (inhibitor of
DNA binding 2) (Lasorella et al.,
2006). Finally, APCCDH1 regulates synaptic function
through the Dbox-containing substrate Liprin-
in flies
(van Roessel et al., 2004). A
postsynaptic role for the APC/C in the restriction of glutamate receptor
abundance has also been described in worm
(Juo and Kaplan, 2004).
However, despite these recent findings, the number of identified APC/C
substrates in differentiated cells remains limited and the regulation of its
activity, beyond cell division, is still poorly described.
In the model plant Arabidopsis thaliana, single-copy genes encode
counterparts of all known vertebrate APC/C subunits, except for APC3/CDC27
(Capron et al., 2003a). Nine
APC/C activators have also been identified: six CDC20 and three CCS52/CDH1
isoforms. Several reports support a role for plant APC/C in the regulation of
the cell cycle. Thus, mitotic cyclins are degraded in a Dbox-dependent manner
(Genschik et al., 1998) and
their degradation is required for the reorganization of mitotic microtubules
to the phragmoplast and for proper cytokinesis
(Weingartner et al., 2004).
Furthermore, Arabidopsis apc2
(Capron et al., 2003b),
apc6/NOMEGA (Kwee and Sundaresan,
2003) loss-of-function mutants are impaired in megagametogenesis
after the first mitotic division. In addition, in Medicago species,
CDH1-type activators are involved in endoreduplication, a modified cell cycle
during which DNA continues to be duplicated in the absence of mitosis
(Cebolla et al., 1999;
Vinardell et al., 2003).
Finally, the removal of CDC27B/HBT APC/C subunit in Arabidopsis root
or leaf sectors revealed defects in both cell division and endoreduplication
(Serralbo et al., 2006).
Here, we have used two different cyclin reporter constructs to probe APC/C E3 ligase activity during plant development and particularly in various differentiated cell types. Strikingly, we found that the APC/C remains active in most post-mitotic cells. Second, we engineered different APC/C hypomorphic Arabidopsis transgenic lines and found that a reduction in the activity of this E3 ligase leads to multiple developmental abnormalities. Among them, the cellular organization of the inflorescence stems revealed increased amounts of vascular tissues.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The fragments carrying the coding regions of tobacco
cyclins with native or mutated Dbox motifs were cloned into pBluescript II
SK+ (Stratagene) vector in frame with the β-Glucuronidase
(GUS)-coding sequence. Subsequently, the BamHI-SacI
fragments were subcloned into the binary pBI121.1 vector (Clontech), replacing
the GUS reporter gene.
35S::APC10/APC6 constructs
Fragments of 323 bp (nucleotides 191 to 514) from the APC10-coding sequence
and of 456 bp (nucleotides 1046 to 1502) from the APC6-coding sequence were
cloned into the pB2GW7 [described by Karimi et al.
(Karimi et al., 2002)] to
generate the 35S::APC10 and the 35S::APC6 co-suppression constructs,
respectively.
RNAi constructs
The vector pFGC5941 (Kerschen et al.,
2004) was used to generate the RNAi constructs. The same APC10 and
APC6 fragments as indicated above were cloned into the PFGC5941 vector. One
sequence was cloned at the XhoI and NcoI sites. and the
other was cloned in the other orientation at the XmaI and
XbaI sites. These constructs generate hairpin structured RNA that
will trigger RNAi silencing of APC10 and APC6, respectively.
Transgenic plants
Plant transformation was performed by the floral dip method
(Clough and Bent, 1998).
Wassilewskija (WS) ecotype was used to generate transgenic plants expressing
cyclin-GUS fusion. We isolated a minimum of 17 T1 lines for each construct and
selected at least five T3 lines on the basis of their segregation ratio for
kanamycin resistance.
Columbia (Col0) ecotype was used to generate APC10 and APC6 co-suppressing lines (APC10S and APC6S), and RNAi lines for APC10 and APC6. We isolated a minimum of 15 T1 lines for each construct for further analysis.
BY2 cell culture, transformation and synchronization
The tobacco BY2 suspension culture (Nicotiana tabacum L. cv.
Bright Yellow 2) was maintained according to Nagata et al.
(Nagata et al., 1992).
Transgenic BY2 cells generated by Agrobacterium-mediated transformation were
synchronized and analyzed as described by Genschik et al.
(Genschik et al., 1998).
GUS assays
Histochemical localization of GUS activity was performed as described by
Jefferson et al. (Jefferson et al.,
1987). Several independent T3 lines, at least three per
construction, were assayed for GUS activity at various developmental stages,
using in vitro grown plantlets. The expression patterns were qualitatively the
same among these lines. Thereafter, the pictures shown in this study are
typical examples of the different patterns observed.
Three-week-old in vitro grown plantlets were used for quantitative GUS assays performed with the GUS-Light kit (Tropix) in a microplate luminometer (TR717 Tropix, Applied Biosystems) according to the manufacturer's instructions. GUS activity is expressed as RLU per 5 µg proteins after 30 minutes of enzymatic reaction.
For the transgenic BY2 cells, fluorimetric GUS assay were performed
according to Jefferson et al. (Jefferson
et al., 1987). GUS activity is expressed as pmoles of MU produced
per minute per milligram of protein.
RNA and DNA gel blotting and RT-PCR analysis
Total RNAs from plantlets were isolated as described by Verwoerd et al.
(Verwoerd et al., 1989) and
blotted as indicated in Criqui et al.
(Criqui et al., 2002). The
genomic DNA extraction was performed using the Plant DNAZOL Reagent
(Invitrogen).
For RT-PCR on Arabidopsis leaves, RNA was extracted from 1, 2, 3
and 5 mm long leaves of a ProCycB1;1::NterCycB1;1-GUS line using the RNeasy
plant mini kit (Qiagen). Total RNA (3 µg) was reverse transcribed with High
Capacity cDNA Reverse Transcription kit (Applied Biosystems). The PCR reaction
was carried out with 1 µl template and 20 pmoles of specific primer
(details can be provided on request). For small RNA analysis, total RNA was
extracted from flower buds from different RNAi APC6 lines with Tri-Reagent
(Sigma). RNA gel blot analysis of low molecular weight RNA was conducted on 15
µg of total RNA as described by Akbergenov et al.
(Akbergenov et al., 2006).
Flow cytometry
The method is described elsewhere
(Cebolla et al., 1999).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) to monitor cell division activity and harvested
Arabidopsis leaves at different developmental stages, ranging from
very young leaves with high cell division activity to fully expanded leaves,
in which cell division activity is scarce and most cells are differentiated
(Fig. 1A). This analysis
includes selected subunits of the APC/C
(Capron et al., 2003a) and,
most importantly, different APC/C activators (CDC20 and CCS52/CDH1), as well
as two specific ubiquitin-conjugating enzymes (UBC19 and UBC20). In contrast
to AtCYCB1;1, all APC/C subunits tested (APC3/CDC27A, APC4, APC6 and APC10)
were expressed similarly at the different leaf-stages
(Fig. 1B). In particular, their
transcript levels remained high in fully expanded leaves (lane 5). However,
both classes of APC/C activators were expressed differentially. Under our
experimental conditions, we could detect in leaves the transcripts of only two
of the six CDC20-related subunits (CDC20-1 and CDC20-2). Both showed an
expression profile similar to AtCYCB1;1, suggesting that they are cell cycle
regulated. In contrast to the CDC20-types of activator, CCS52/CDH1-related
subunits, as well as the E2 UBC19 and to a lesser extent UBC20, remained
expressed in older leaves, suggesting their involvement in the function of
APC/C in post-mitotic cells. These data are consistent with the recent finding
that some APC/C subunits are expressed in organs with overall low cell
division rate, such as siliques (Eloy et
al., 2006).
Proteolysis of the chimeric cyclin-GUS proteins throughout the cell cycle
As a next step, we wished to visualize directly the APC/C activity in
various tissues and during the process of plant organ development and growth.
We have previously reported that a fusion protein between the N-terminal
domain of either Nicta;CYCA3;1 or Nicta;CYCB1;1 tobacco mitotic cyclins and
the chloramphenicol acetyl transferase (CAT) reporter protein is degraded in a
cell cycle-dependent manner in synchronized tobacco BY2 cells
(Genschik et al., 1998). Both
cyclin N-terminal domains carry the well-characterized Dbox motif, which is
one of the degrons recognized by the APC/C
(Klotzbücher et al.,
1996). To visualize the accumulation of these APC/C substrates in
planta by histochemical analysis, we replaced the CAT gene by the
GUS reporter gene (Fig.
2A). Constructs CycA-GUS and CycB-GUS carry a native Dbox motif in
the cyclin N-terminal domains, whereas the two highly conserved amino acids
are mutated from RxxLxx(L/I)xN to
GxxVxx(L/V)xN in CycA
Box-GUS and
CycBBox-GUS constructs (Genschik et
al., 1998). We first investigated the stability of these chimeric
proteins during the cell cycle.
The constructs were introduced into tobacco BY2 cells by
Agrobacterium-mediated transformation, as this cell suspension still
represents one of the best and most homogenous systems to synchronize plant
cells. Each synchronization experiment (as illustrated in
Fig. 2B-F) was repeated at
least twice by using different transgenic lines per construct. As expected,
the GUS protein alone, expressed under the control of the constitutive CaMV
35S promoter, did not exhibit a significant difference in the pattern of its
accumulation (Fig. 2B).
However, both the CycA-GUS and CycB-GUS chimeric protein levels oscillated
during the cell cycle (Fig.
2C,D) in a similar manner as previously described for their CAT
variants (Genschik et al.,
1998). Thus, the CycB-GUS chimeric protein accumulates during G2
and is actively degraded early in mitosis, always before the mitotic index
reaches its maximum (Fig. 2D).
By contrast, the accumulation pattern of the CycA-GUS protein is broader
(Fig. 2C,E). The fusion protein
has already accumulated during S-phase and is also degraded later in mitosis,
as its level declined always after the mitotic index pick. The differential
accumulation patterns of A- and B-type cyclin reporters indicates that the
plant APC/C destroys sequentially these cyclins during the cell cycle, similar
to the situation in animal cells (Sigrist
et al., 1995). For both constructs 35S::CycADbox-GUS and
35S::CycBDbox-GUS, mutations inside the Dbox motif abolished the cell
cycle-specific oscillations of the fusion proteins
(Fig. 2F; and data not shown).
From these experiments, we can conclude that these chimeric proteins can be
used as tools to reflect the APC/C activity in planta, at least in the cell
types in which the 35S promoter is expressed.
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The histochemical staining at different developmental stages of plants
expressing the CycB-GUS chimeric protein shows a patchy distribution of
stained cells, which is restricted to regions of cell division, such as in
young leaves (Fig. 3A). As
leaves expand, a longitudinal gradient in the frequency of GUS-expressing
cells developed. GUS activity is confined to the base of the developing leaf
(Fig. 3D) and thus follows the
basiplastic patterns of cell cycling described by Donnelly et al.
(Donnelly et al., 1999). In
cotyledon (Fig. 3A) and older
leaves (Fig. 3G), the GUS
staining is maintained in a group of cells associated with the vascular system
(primary, secondary and tertiary veins) and can also be visualized at the
hydathodes (Fig. 3F). However,
GUS staining was never observed in any other cell types of fully
differentiated leaves, including branched trichomes
(Fig. 3H). In the root, GUS
staining is detected with a patchy pattern at the level of the root meristem,
but not the elongation zone (Fig.
3K). CycB-GUS reporter protein accumulation was also observed in
cells in the stele of the primary root
(Fig. 3J). Conversely, plants
expressing the CycBDbox-GUS protein variant show a uniform distribution
of the GUS staining in the different plant tissues
(Fig. 3B,E,I,L), which is
similar to the 35S::GUS control plants (not shown). To further demonstrate
that the absence of GUS staining in non-cycling plant cells is due to the
active degradation of the fusion protein, we used MG132, an inhibitor of the
26S proteasome. Indeed, we observed the appearance of GUS staining in 14
hours-MG132 treated CycB-GUS plants (Fig.
3C). From these experiments, we conclude that CycB-GUS reporter
protein is actively degraded in non-dividing cells in a Dbox-dependent manner,
indicating that the APC/C is active in most post-mitotic plant cells.
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Dbox-GUS protein exhibit a GUS signal uniformly distributed in all
cells (Fig. 4B,E,I). Overall, we conclude that the APC/C is maintained active in many different post-mitotic cell types. To obtain more insight into the role of the APC/C during plant development, we generated transgenic lines with reduced APC/C activity.
Production of APC/C knockdown mutant
Because APC/C activity is essential in plants
(Capron et al., 2003b;
Kwee and Sundaresan, 2003;
Perez-Perez et al., 2008), we
decided to generate hypomorphic mutant lines using RNAi. Two subunits of the
complex, APC6 and APC10 were chosen because both are encoded by single copy
genes and are expressed in post-mitotic cells
(Fig. 1B). We believe that this
method is particularly appropriate to characterize the function of APC/C in
differentiated cells, because it has been shown that RNAi is less efficient in
dividing cells in meristems or in Agrobacterium-induced tumors
(Voinnet et al., 1998;
Dunoyer et al., 2006), and
thus should permit the recovery of viable plants. To do so, we followed two
strategies: first, we produced co-suppression lines in which a fragment of
each gene was overexpressed under the control of the strong 35S promoter (see
Fig. S2A in the supplementary material). Several independent lines were
selected and the expression of the corresponding endogenous gene was tested by
RT-PCR. In some of these lines, we observed a significant reduction in the
expression of APC10 and APC6 (see Fig. S2B in the supplementary material).
Second, the same cDNA fragments of APC10 and APC6 were cloned into the
pFGC5941 vector, on both sides of the chalcone synthase intron into opposite
orientation (see Fig. S2A in the supplementary material). This vector
generates hairpin structured RNA that triggers RNAi silencing of APC10 and
APC6, respectively. Expression level of the endogenous genes and accumulation
of small RNA were tested in the selected lines (see Fig. S2C in the
supplementary material). A significant reduction in the expression of the
endogenous APC6 and APC10 mRNAs was observed in the RNAi lines, which
correlates with a high accumulation of siRNAs (see Fig. S2C in the
supplementary material).
If silencing of APC10 and APC6 has an effect on APC/C activity in planta, we could expect to see a higher accumulation of the artificial cyclin-GUS substrates in those mutant lines. To address this issue, we introgressed the 35S::CycB-GUS construct into two of these mutant lines: APC6S-4 and APC10S-6. Indeed, by histochemical analyses we observed a stronger accumulation of the CycB-GUS protein in the APC/C hypomorphic mutant backgrounds compared with wild type (see Fig. S3 in the supplementary material). By contrast, histochemical analyses of the Dbox-mutated version of the 35S::CycB-GUS showed no difference between wild-type and hypomorphic mutant backgrounds (see Fig. S3 in the supplementary material). This result indicates that the APC/C activity is reduced in the APC10 and APC6 hypomorphic mutants and that these lines are appropriate for further characterization of the role of APC/C in plant development.
APC/C hypomorphic mutants display endoreduplication defect in rosette leaves
The phenotype of the APC/C hypomorphic lines was followed during plant
development. We could not detect major morphological difference at the young
seedling stage between wild-type and the hypomorphic lines
(Fig. 5A, upper panel). In
contrast to the hobbit mutant (Willemsen
et al., 1998), we could not observe any defect during the root
growth (see Fig. S4A in the supplementary material). At the rosette stage, we
also did not detect major differences between the wild type and APC10S and
APC6S lines (Fig. 5A, bottom
panel). However, several RNAi APC/C lines exhibited smaller and denser rosette
and curled leaves (Fig. 5A,C).
Moreover, even without major morphological differences, we noticed that in
hypomorphic lines the cotyledon vein patterning is altered
(Fig. 5B). In
Arabidopsis cotyledon, the vein pattern is simple, containing a
single primary vein that extends though the center of the cotyledon and
additional four secondary veins that form four closed loops. In the
hypomorphic mutants, the primary vein shows no defect, but in most cases,
secondary veins formed only two or three loops
(Fig. 5B).
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Endoreduplication is a post-mitotic mechanism during which several rounds
of DNA replication occur without cell division. The regulation of this
mechanism is not well understood; however, CCS52, a CDH1-type activator of
APC/C complex, has been previously involved in endoreduplication in plant
cells (Cebolla et al., 1999;
Vinardell et al., 2003).
Therefore, we analyzed the DNA content in leaf cells of the APC hypomorphic
mutants. In the strongest APC/C knockdown lines (RNAi-APC6 and RNAi-APC10), we
observed an important reduction in the 8C and 16C DNA contents compared with
the wild-type plants and conversely an increase of cells with 4C and 2C DNA
contents (Fig. 5E), which
correlates with reduced leaf cell size
(Fig. 5D). Interestingly, in
the co-suppression lines (APC6S and APC10S), we could already detect a
similar, although weaker, phenotype on the ploidy level
(Fig. 5E). This result supports
a function of the APC/C in the mechanism of plant endoreduplication, as
earlier suggested (Cebolla et al.,
1999).
APC hypomorphic mutants are impaired in inflorescence architecture and vascular tissue organization
Strikingly, at later developmental stages, the mutant lines developed
dramatic morphological aberrations during shoot elongation and inflorescence.
These plants showed shorter stems and a severe shortening of the internodes
causing the formation of a `broomhead-like' cluster of siliques at the top of
stems (Fig. 6A). This phenotype
is more or less severe according to the lines, but was observed for both the
co-suppression and the RNAi lines. In the case of a `strong broomhead'
phenotype, stem elongation is extremely reduced and the inflorescences appear
at the level of the plant rosette (Fig.
6A, part e). Apart from this inflorescence phenotype, the flowers
and the siliques are normal, as is the fertility of the hypomorphic lines.
To investigate in more detail the cellular organization in the inflorescence stem of the APC/C hypomorphic mutants, we performed histological analyses. Longitudinal and transverse sections of different organs were colored with Toluidine Blue (Fig. 6B; see Fig. S4B in the supplementary material). This dye stains specifically the lignified cells like xylem and secondary vascular tissues. First, the sections of 8-day-old seedling hypocotyls reveals that the APC/C hypomorphic lines present all differentiated cell types, with the epidermal, cortical and endodermal layers and the xylem and phloem in the inner stele tissue (see Fig. S4B, parts a,b in the supplementary material). This correlates with the lack of morphological abnormalities of these lines at this developmental stage. Next, we made sections of stems of 5-week-old plants, which show a `broomhead' phenotype. In the upper stem, the vascular system of wild-type Arabidopsis is organized in discrete collateral bundles with the phloem towards the outside and the xylem towards the inside of the bundle and separated by interfascicular regions (Fig. 6B, parts a,g). By contrast, the hypomorphic mutants exhibit an increased amount of vascular tissue, most notably cambium, xylem and lignified sclerenchyma, organized in a continuous ring (Fig. 6B, parts b,h). No interfascicular fibers are distinguishable in hypomorphic mutant stem sections. In addition to the overproliferation of the cambium, we could observe enlarged lignified cells (see arrows in Fig. 6B, parts d,h), which have disorganized cell division planes (Fig. 6B, part d). Moreover, cells in the cortex and epidermis are larger in the hypomorphic mutant stems compared with wild type (Fig. 6B, parts g,h).
The secondary growth shown in the sections of the base of the stem (Fig. 6B, part e) is characterized by the presence of vascular cambium, which originates from procambium within the vascular bundles and from parenchyma cells in the interfascicular region. As in the upper part, the hypomorphic mutants exhibit an increased amount of vascular tissues (Fig. 6 B, part f). In addition to the curled leaf phenotype (see Fig. S4B, part d in the supplementary material), there is also a disorganized vascular tissue in the primary vein of the strong hypomorphic mutant (see Fig. S4B, part f in the supplementary material) compared with wild type (see Fig. S4B, part e in the supplementary material). Within this vein, vascular tissues are generally arranged with the xylem on the upper (adaxial) side of the leaf and the phloem on the abaxial side (see Fig. S4B, part e in the supplementary material). In the APC/C hypomorphic lines, some cells on the abaxial side are differentiated into xylem (see Fig. S4B, part f in the supplementary material).
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). Its promoter drives the expression in the protoxylem and
metaxylem of roots, shoots and leaves
(Yamaguchi et al., 2008) (see
Fig. S5A,C,E). When we introgressed the PVND7::GUS reporter within
the APC/C hypomorphic mutant background, we did not observe any difference in
the GUS expression patterns (see Fig. S5B,D,F,G,H in the supplementary
material), suggesting that cell differentiation into xylem occurs normally in
these lines. |
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To visualize the spatial and temporal APC/C activity in various plant
organs, we expressed two types of cyclin-GUS reporter proteins under the
control of the constitutive 35S CaMV promoter. The B-type cyclin reporter
lines showed GUS stained cells essentially in tissues with high rates of cell
division activity, such as in meristems, young leaves and cells in the
procambium. This correlates well with G2/M stabilization of the Dbox
containing reporter protein in cycling cells
(Colon-Carmona et al., 1999;
Donnelly et al., 1999), in
which B-type cyclins and other regulators need to accumulate. Conversely, no
GUS staining was associated with many differentiated cells, including
mesophyll and pavement cells, guard cells and trichomes. Thus, as in mammalian
neurons and hepatocytes (Gieffers et al.,
1999; Wirth et al.,
2004), differentiated non-dividing plant cells keep an active
APC/C, despite the fact that no mitotic cyclins and most probably no other
mitotic regulators are expressed. This implies that the APC/C recognizes other
substrates containing a Dbox motif in post-mitotic cells.
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) and
thus the N terminus of cyclin A alone may just be less efficiently degraded in
some differentiated cell types. Overall, the findings that APC/C and CDH1-related subunits are expressed in mature plant leaves and that reporter proteins caring a Dbox degron are actively degraded in differentiated post-mitotic cells suggests that the activity of APC/C is required outside of the plant cell cycle.
The APC/C in Arabidopsis is required for endoreduplication
Loss-of-function mutants analyses for most APC/C core subunits leads to
lethality in yeast and multicellular organisms. Mice APC2 knockouts
are embryonic lethal (Wirth et al.,
2004), whereas the loss of function of this gene in
Drosophila and Arabidopsis leads to late larval lethality
(Kashevsky et al., 2002) and
female gametophyte arrest (Capron et al.,
2003b), respectively. Thus, to address the function of APC/C in
developing plants and in differentiated cell types, we used RNAi strategies to
target two of its subunits: APC6 and APC10. It is noteworthy that RNAi, in
contrast to the action of miRNAs, is less efficient in dividing cells (e.g.
meristems) or in Agrobacterium-induced tumors
(Voinnet et al., 1998;
Dunoyer et al., 2006).
Endoreduplication is an alternative cell cycle in which at least two
successive rounds of DNA replication occur without intervening mitosis. This
phenomenon is observed in some animal cell types, but is much more frequent in
plant cells (Edgar and Orr-Weaver,
2001; Kondorosi and Kondorosi,
2004). Interestingly, endoreduplication operates during
development and in differentiated cells, such as in plant trichomes. A role
for the APC/C in endoreduplication was already suspected as the function of
CDH1-type activators of APC/C is required for this process in both fly and
plant (Sigrist and Lehner,
1997; Cebolla et al.,
1999). Hence, very recent work in Drosophila elucidated
the role of APC/C in the process of endoreduplication
(Zielke et al., 2008;
Narbonne-Reveau et al., 2008).
Our finding that the downregulation of APC/C activity in Arabidopsis
leads to a significant reduction in endocycles in leaves together with the
report that the loss-of-function of Arabidopsis HBT/CDC27B leads to a
similar effect in root cells (Serralbo et
al., 2006) indicates that this pathway is functionally conserved
throughout evolution. Nevertheless, in fly the APC/C regulates
endoreduplication by mediating Geminin oscillation
(Zielke et al., 2008), but an
obvious ortholog of Geminin has not yet been identified in plants
(Caro and Gutierrez, 2007);
thus, the mechanism how APC/C regulates endoreduplication in plants remains
still unclear. Interestingly, two A-type cyclins, both containing a Dbox
degron, have been involved in endoreduplication in Arabidopsis
(Yu et al., 2003;
Imai et al., 2006).
Strikingly, ectopic expression of a Dbox-deficient, and thus non-degradable,
cyclin CYCA2;3 significantly restrained endocycles in various plant organs
(Imai et al., 2006),
suggesting this cyclin as a good candidate substrate of the APC/C in the
regulation of this process.
The APC/C restricts cell proliferation in the vasculature
At adult stage, Arabidopsis APC/C knockdown lines exhibited a
severe phenotype regarding stem elongation. In some lines, stem elongation was
that much reduced that all inflorescences appeared to emerge directly from the
plant rosette. This phenotype is associated with overproliferation of vascular
cells at the expanses of the interfascicular tissue, which can even lead to a
continuous ring-like pattern of xylem and phloem cells. Although plant and
animal developmental pathways are under the control of undoubtedly different
mechanisms and signals, it is intriguing that the loss of APC/C function in
vertebrates results in unscheduled proliferation of some differentiated cell
types. Thus, the conditional inactivation of APC2 subunit in mouse hepatocytes
led those cells to re-enter the cell cycle even without any proliferative
stimulus (Wirth et al., 2004).
In zebrafish, the loss of APC/C activity also resulted in improper re-entry
into the cell cycle of quiescent cells
(Wehman et al., 2007). The
mechanism by which the APC/C restrains cell division in plant vascular tissue
is still unclear. Either the APC/C restrains cell cycle re-entry of G0 cells
or it acts as a `safeguard' to avoid unscheduled ongoing cell proliferation,
which may result in abnormal differentiation. Whether APC/C function in
vascular development is post-mitotic or still cell cycle based, this E3 ligase
may act by suppressing the accumulation of extracellular or intracellular
mitogens. Indeed active degradation of cyclin B1 by the APC/CCDH1
in post-mitotic rat neurons prevents them from cell cycle re-entering and
subsequently cell death (Almeida et al.,
2005). However, ectopic expression of a functional and
non-destructible plant cyclin B1 leads to abnormal mitosis and many
developmental abnormalities but does not induce vascular tissue
overproliferation (Weingartner et al.,
2004), indicating that cyclin B1 accumulation cannot explain this
phenotype.
Several Arabidopsis mutants have been described that are affected
both in the growth of inflorescence stems and ectopic vascular proliferation
and/or differentiation. Among them, mutants impaired in auxin response or
transport, have been reported, as auxin plays a central role in vascular
patterning and differentiation (reviewed by
Berleth et al., 2000;
Rolland-Lagan, 2008).
Overproliferation of vascular tissues occurs in plants in which the transport
of auxin is chemically inhibited or by mutation of the efflux carrier PIN1
(Mattsson et al., 1999). Local
auxin action probably induces genes that are required for vascular patterning.
Thus, ATHB-8, a gene encoding a class III homeodomain-leucine zipper
(HD-Zip III) transcription factor exclusively expressed in procambium tissue,
is activated by auxin (Baima et al.,
1995) and acts as a positive regulator of cambial cell
proliferation and differentiation (Baima et
al., 2001). AtHB15 is another member of the HD-Zip III gene
family, strictly expressed in vascular bundles, that alters the vascular
system by accelerated vascular cell differentiation from cambial cells when
the transcript level is drastically reduced
(Kim et al., 2005).
Interestingly, it has been reported that the 26S proteasome is involved in
the vascular patterning through the regulatory subunit RPN9, a subunit of the
19S RP lid (Jin et al., 2006).
RPN9-silenced tobacco plants display extra leaf vein formation with increased
xylem and decreased phloem. These authors stipulated that RPN9 functions at
least in part through regulation of auxin transport. It is noteworthy that
mutations in the HOBBIT/CDC27B gene were previously reported to
affect both auxin responsiveness and AXR3/IAA17 protein abundance
(Blilou et al., 2002).
Therefore, to investigate whether APC/C regulates auxin transport or signaling
or even auxin downstream regulators during vascular patterning are important
issues to address in the future.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/136/9/1475/DC1
|
ACKNOWLEDGMENTS |
|---|
|
Footnotes |
|---|
* Present address: ZMBP-Developmental Genetics, Universität
Tübingen, Germany 
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REFERENCES |
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