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First published online 3 July 2008
doi: 10.1242/dev.019349
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1 Umeå Plant Science Centre, Department of Plant Physiology, Umeå
University, 90187 Umeå, Sweden.
2 Instituto de Biología Molecular y Celular de Plantas (CSIC-UPV),
Universidad Politécnica de Valencia, Avda de los Naranjos s/n, 46022
Valencia, Spain.
Author for correspondence (e-mail:
mblazquez{at}ibmcp.upv.es)
Accepted 26 May 2008
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: ACL5, Arabidopsis, Cell death, Secondary cell wall, Tracheary element, Xylem specification
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Plant hormones are involved in the control of all the different stages of
xylem development. Physiological and pharmacological studies have demonstrated
the important role of auxins and cytokinins in controlling the activity of the
vascular cambium and the initiation of xylem development, while
brassinosteroids, ethylene and gibberellins are important in the modulation of
the cambial activity and the control of xylem differentiation (reviewed by
Ye, 2002
). Mutations in the
various components of hormone synthesis, transport or signal transduction in
Arabidopsis have largely confirmed the action of the various hormones
and demonstrated the involvement of additional compounds, such as sterols
(reviewed by Fukuda, 2004).
Mutations affecting hormone transport and/or signaling provide evidence for
the role of auxins in the initiation of the vascular meristem and the
maintenance of vascular continuity
(Gälweiler et al., 1998;
Hardtke and Berleth, 1998;
Hobbie et al., 2000), the role
of cytokinins in phloem specification
(Mähönen et al.,
2000) and the inhibition of protoxylem differentiation
(Mähönen et al.,
2006), and the role of brassinosteroids in promoting xylem
differentiation (Caño-Delgado et
al., 2004). However, Arabidopsis mutants have
demonstrated that there are as yet unknown signals that regulate xylem
development (Koizumi et al.,
2000; Parker et al.,
2003). In addition, the signals controlling the maturation of
xylem elements remain largely unknown.
Polyamines (PAs) are low molecular weight cationic molecules, the synthesis
of which is initiated by decarboxylation of arginine or ornithine to produce
putrescine, and sequential addition of two aminopropyl groups to putrescine
through the activity of the aminopropyltransferases spermidine synthase and
spermine synthase, to produce the triamine spermidine and the tetraamine
spermine, respectively (Ikeguchi et al.,
2006). Arabidopsis has two putative spermidine synthases
(SPDS1 and SPDS2) and two putative spermine synthases (SPMS and ACL5)
(Imai et al., 2004b;
Panicot et al., 2002).
Bacterially produced ACL5 was recently related to synthesis of thermospermine,
which is an isomer of spermine (Knott et
al., 2007), but this remains to be validated in planta. PAs have
been shown to be involved in a variety of processes, such as cell
proliferation and defense against both abiotic and biotic stresses, but they
are also associated with the normal development of plants
(Kumar et al., 1997;
Walden et al., 1997). It has
also been proposed that they participate in the control of vascular
development based on their effect on cell division, interaction with other
hormones and H2O2 produced during PA catabolism that
could potentially affect processes such as vascular cambial activity, cell
differentiation and cell death (Bais and
Ravishankar, 2002;
Møller and McPherson,
1998; Sebela et al.,
2001). Indirect support for this suggestion was provided by
spermidine- and spermine-deficient transgenic plants, which exhibited a
stunted phenotype (Kumar et al.,
1996). In addition, an Arabidopsis mutant in the
polyamine biosynthesis-related S-adenosylmethionine decarboxylase, with
slightly reduced spermidine and spermine levels, was found to be stunted and
to exhibit severely altered vascular development
(Ge et al., 2006). However, a
more direct involvement was demonstrated only recently, with the
identification of ACAULIS 5 (ACL5) as a putative spermine
synthase (Hanzawa et al.,
2000). The Arabidopsis acaulis mutants were isolated on
the basis of their severely impaired internodal elongation after the
transition from the vegetative to the reproductive stage
(Akamatsu et al., 1999), and
acl5 was found to display overproliferation of xylem elements
(Hanzawa et al., 1997).
Furthermore, the thickvein mutant, harboring another loss-of-function
allele of ACL5, was shown to display thicker veins and an increased
number of vascular cells in the inflorescence stems
(Clay and Nelson, 2005).
Interestingly, changes to vascular development seem rather specific to the
acl5 mutant, as the lack of spermidine synthesis in
Arabidopsis is embryo lethal
(Imai et al., 2004b), whereas
mutations in SPMS, which encodes the major spermine synthase, do not
affect plant development (Imai et al.,
2004a). Recently, a mutation that allows higher production of a
bHLH transcription factor, SAC51, was shown to suppress all the defects
associated with the loss of ACL5 function
(Imai et al., 2006), but as
the suppressor mutant of bHLH was dominant it is not entirely clear whether
its function is directly related to PA signaling. Therefore, the underlying
mechanism for the action of PAs in plant growth and development remains
unclear.
ACL5 is specifically expressed in the procambial and/or the
provascular tissues during primary growth of the root
(Birnbaum et al., 2003;
Clay and Nelson, 2005), but it
does not seem to have any major function at this stage
(Clay and Nelson, 2005). To
elucidate the function of ACL5 during vascular development, we therefore
focused in the current study on the vascular tissues of the hypocotyl, which
display extensive secondary growth during prolonged growth period. We
demonstrate here that, in the hypocotyl as well as in the inflorescence stem,
ACL5 is expressed not just broadly with respect to vasculature, as
shown earlier (Clay and Nelson,
2005), but specifically in the xylem vessel elements at a strictly
defined developmental stage, suggesting direct involvement of ACL5 in xylem
vessel differentiation. Furthermore, we show that the acl5 mutant
displays severe overall inhibition of the secondary growth of the vascular
tissues, dramatic alteration in the morphology of the vessel elements and
complete lack of xylem fibers. Finally, we propose a mechanistic model for the
function of ACL5 in xylem specification, based on experiments carried out in
transgenic plants expressing a DT-A toxin gene under the control of
the ACL5 promoter and in the Zinnia elegans tracheary
element differentiation system.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Construction of transgenic ProACL5:DT-A and ProACL5:GUS plants
2.48 kb of the ACL5 (At5g19530) sequence, upstream from the start codon,
was amplified from Col-0 genomic DNA using the primers
5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTCCACCATCGAATGGTATGC-3' and
5'-GGGGACCACTTTGTACAAGAAAGCTGGGTATCCAAGTTGAGGAGAAGAT-3', which
include recognition sequences for the Gateway recombination system
(Invitrogen). Amplification conditions followed the manufacturer's
instructions, and the promoter fragment was sequenced after the first cloning
step in pDONR201.
To create ProACL5:DT-A plants, the 3'UTR of the
ACL5 gene was amplified from Col-0 genomic DNA with the primers
5'-CACAGTCGACAGACGAACCGGTTTCAGTTTC-3' and
5'-CACAGAATTCAGATTTGGTGTGGAGAAATAAG-3', which include
restriction sites for SalI and EcoRI, respectively
(underlined). This was subsequently cloned into pBluescript SK II to produce
pACL-3'UTR/SK. The sequence of the diphtheria toxin A chain (DT-A) was
amplified from pEW3 (Nilsson et al.,
1998) with primers
5'-GAGTCGACATGGATCCTGATGATGTTGTTG-3' and
5'-CCACGTCCAGACGTCGAC-3', including restriction sites
for SalI, and was cloned into pACL-3'UTR/SK. Orientation and
sequence fidelity were checked by sequencing. The DTA-ACL5 3'UTR
cassette was transferred, as a KpnI/SacI restriction
fragment, to the binary vector pMDC205
(Curtis and Grossniklaus,
2003), the GFP-coding sequence of which was simultaneously removed
to create the construct DTA-utr/pMDC205. Finally, the 2.48 kb ACL5 upstream
sequence was recombined into DTA-utr/pMDC205 using the Gateway LR reaction
(Invitrogen), and the resulting vector was transformed into Col-0 plants by
the method of Clough and Bent (Clough and
Bent, 1998). Transformants were selected on MS medium containing
20 g/l sucrose and 50 µg/ml hygromycin. Homozygous and heterozygous plants
were identified by PCR and segregation analysis.
To create the ProACL5:GUS plants, the 2.48 kb ACL5
promoter fragment was cloned into pK2GWFS7.0
(Karimi et al., 2002). This
vector was transformed into Col-0 plants using the method described by Clough
and Bent (Clough and Bent,
1998). Transformants were selected on MS medium containing 20 g/l
sucrose and 50 µg/ml kanamycin. A non-segregating line was crossed to each
of the acl5 mutant and a heterozygous
ProACL5:DT-A line 4. The resultant progenies were screened
for homozygosity of either the acl5 mutation or the
ProACL5:DT-A transgene in F2 by the seedling phenotype and
for the ProACL5:GUS transgene in F3 by antibiotic
resistance.
Microscopic analyses
Histochemical β-glucuronidase (GUS) activity assays were performed for
whole seedlings grown in vitro or tissue pieces that were excised from plants
grown in soil for indicates times. The chromogenic substrate
5-bromo-4-chloro-3-indoxyl-beta-D-glucuronide cyclohexylammonium (Gold
Biotechnology) was used, according to the manufacturer's instructions, to
detect GUS activity. The GUS-stained whole seedlings were examined directly by
microscopy. The excised tissue pieces were mounted after GUS staining in LR
white resin (TAAB Laboratories) containing 10% PEG400 and sectioned at 25
µm. If needed, the sections were counterstained with 0.05% Ruthenium
Red.
Localization of the ACL5 mRNA was determined, using 8 µm
sections embedded in paraplast, by in situ hybridization, as described by
Jackson (Jackson, 1991).
Briefly, antisense and sense riboprobes were generated from the complete cDNA
of ACL5 isolated from the PRL1 gene library
(Newman et al., 1994), using
SP6 and T7 RNA polymerases, respectively, and then hydrolyzed by carbonate
hydrolysis into 100-200 bp fragments. Probes were labeled with digoxigenin and
immunodetected with an alkaline phosphatase-conjugated antidigoxigenin
antibody. Alkaline phosphatase was detected using the BCIP-NBT procedure.
Photographs were taken under the bright field of a Nikon Eclipse
microscope.
For anatomical characterization, hypocotyls were collected from plants that were grown in soil for indicates times, fixed in FAA (5% formaldehyde, 10% acetic acid, 50% ethanol) and embedded in LR white resin (TAAB Laboratories). Transverse and longitudinal sections were taken and stained with 0.05% Toluidine Blue (Merck).
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Light microscopy images were taken using a Zeiss Axioplan II microscope equipped with Zeiss AxioCam CCD camera (Zeiss, Oberkochen, Germany). Xylem vessel elements were measured using the microscope images and Zeiss Axiovision 3.1 software.
Electron microscopy images were taken of hypocotyls embedded in Spurr resin
(Sigma) according to Rensing (Rensing,
2002), and examined in a Hitachi H-7000 transmission electron
microscope (Hitachi, Tokyo, Japan).
Confocal images of vessels were taken using a Leica TCS SP2 microscope (Leica Microsystems, Wetzlar, Germany) with an excitation wavelength of 568 nm (helium-neon laser) and an emission wavelength of 585 nm. Projections of the confocal data were exported using TCS software.
Zinnia elegans xylogenic cell cultures and pharmacological treatments
The first pair of leaves from 14-day-old seedlings of Zinnia
elegans cv Envy (Hem Zaden BV, Venhuizen, Holland) were used to isolate
mesophyll cells for xylogenic cell suspension cultures according to the method
of Fukuda and Komamine (Fukuda and
Komamine, 1980). Cells were cultured in an induction medium
containing 0.1 mg/l 
-naphthaleneacetic acid and 0.2 mg/l benzyladenine
(Sigma-Aldrich).
For the pharmacological treatments, 1 ml of the differentiating cell culture was treated, in 12-well plates, with various amounts of spermine (Sigma-Aldrich), ranging from 0 to 200 µM final concentration, and monitored for 7 days. Quantification of the tracheary element (TE) differentiation efficiency, as well as the width, length and type of TEs, were recorded using microscope images for 50 cells from each replicate cell culture.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Clay and Nelson, 2005). We
detected with high-resolution analyses using in situ hybridization and
resin-embedded sections of ProACL5:GUS plants that, in all
parts of the plant, ACL5 was expressed in the vascular tissues
(Fig. 1). More specifically,
ACL5 expression was confined to xylem vessel elements in the vascular
bundles of the inflorescence stems (Fig.
1C,E,F), in the junction between the silique and the pedicel
(Fig. 1A) and in the hypocotyl
(Fig. 1H). Expression was
apparent immediately after expansion of the vessel elements, but before the
onset of the secondary cell wall deposition. Protoxylem cells did not express
ACL5 but sometimes parenchymatic cells next to the protoxylem
elements showed expression in the inflorescence stem
(Fig. 1F) and the roots (data
not shown). In line with the earlier reports, we also found expression of
ProACL5:GUS in the procambial and/or provascular tissues
of young hypocotyls (Fig. 1G).
To assess the function of ACL5 during vascular development, we chose to focus
on the hypocotyls, where large numbers of the ACL5-expressing vessels
are continuously being formed.
Loss of ACL5 function alters morphology of xylem vessel elements
Consistent with the expression profile of ACL5, its loss of
function caused severe defects in the vasculature of the hypocotyl.
Seven-day-old acl5 seedlings looked normal and the diameter of the
stele was indistinguishable from that of the wild type
(Fig. 2A,B) although slight
changes, such as asymmetry of the stele and of the whole hypocotyl, could be
discerned. As with the overproliferation of xylem vessels in the inflorescence
stems and leaf veins reported previously
(Clay and Nelson, 2005;
Hanzawa et al., 1997), the
hypocotyls of 13-day-old acl5 seedlings had more xylem vessels than
the wild type (Fig. 2E,F).
However, during prolonged growth of the plants, acl5 did not display
further secondary growth, and the hypocotyls of 35-day-old acl5
seedlings were significantly thinner than those of the wild type
(Fig. 2G,H). The lack of
secondary growth was accompanied by complete lack of xylem fibers in the
acl5 hypocotyls (Fig.
2H). In addition to the weak secondary growth of the hypocotyl,
defects were visible in the secondary cell wall patterning of the
acl5 mutant. Instead of having clearly defined spiral-type protoxylem
and pitted-type metaxylem vessels, as in the wild type
(Fig. 2C), acl5 seemed
to form spiral-type vessels that were slightly reticulated, i.e. that had a
few interconnecting strands between the spiral whorls
(Fig. 2D).
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To get a more in depth insight into the morphological changes of the xylem
elements, hypocotyls of wild-type and acl5 plants were macerated in
an alkaline medium, and the size and secondary cell wall architecture of the
individual xylem elements that remained intact after the procedure were
examined. Hypocotyls were collected from two-month-old plants, which normally
at this age exhibit extensive secondary growth. Marked differences were
observed in the morphology of vessel elements that were classified as annular,
spiral, reticulate or pitted according to their secondary cell wall pattern
(Esau, 1977). The vessel
elements of acl5 were mainly spiral, while only a very small portion
of the vessel elements were of this type in the wild-type
(Fig. 4A,
Fig. 2J). Most strikingly, the
pitted elements, that were dominant in the wild type during secondary growth,
were completely missing in acl5
(Fig. 4A,
Fig. 2I). The length and the
width of the vessel elements were also reduced in acl5
(Fig. 4B,C). In addition, we
were able to verify the absence of xylem fibers in the macerates of all
acl5 plants examined (see Fig.
7E). Thus, our microscopic analyses showed that the absence of
ACL5 function has a profound effect on xylem development. In particular, the
pattern of xylem maturation was altered in a dramatic way, which raises the
issue of whether it is causally related to the observed alterations in xylem
specification.
Vessel cell death is activated in acl5 before the onset of secondary cell wall formation
To further examine the function ACL5 in xylem specification and maturation,
acl5 was crossed to two different marker lines that are indicative of
vascular development. ProDR5:GUS is a well-established
marker for endogenous auxin levels
(Ulmasov et al., 1997),
normally showing highest expression in the root tips and no expression in the
hypocotyls of young Arabidopsis seedlings
(Fig. 5A). However, when
expressed in the acl5 background, ProDR5:GUS
showed weak activity in the developing xylem vessel elements of the hypocotyls
(Fig. 5B). As auxin is
effective in stimulation of cambial activity, increase in the expression of an
auxin marker is in line with the observed increase in the cambial activity and
vessel differentiation during secondary growth of the young acl5
seedlings.
ProXCP2:GUS is a marker for cell death in the xylem
elements of Arabidopsis seedlings
(Funk et al., 2002). The
XCP2 promoter is isolated from a gene encoding a xylem-specific
cysteine protease that is believed to function as an effector protease during
autolysis of the cell contents (Zhao et
al., 2000). In the wild-type background,
ProXCP2:GUS was expressed in the maturing xylem vessels of
the hypocotyls, but not in the immature vessel elements without secondary cell
wall thickenings (Fig. 5C). A
slightly different pattern was observed in the acl5 background;
ProXCP2:GUS was expressed not only in the maturing vessels
but also at an earlier developmental stage in the immature vessel elements
(Fig. 5D). Therefore, the
results are in accordance with the results of the electron microscopy
analysis, suggesting premature onset of the cell death program in relation to
the formation of the secondary cell walls in the vessel elements of
acl5.
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).
Concentrations of exogenous spermine were physiologically relevant as the
endogenous levels of spermine were at a maximum 
50 µM in the
Zinnia cells (data not shown).
Exogenous spermine delayed the time of TE appearance and reduced the rate
of TE differentiation in a dose-dependent manner
(Fig. 6A). Spermine also
altered TE type by stimulating differentiation of the more elaborate,
metaxylem-type TEs (characterized by their reticulated or pitted secondary
cell walls). While control TE cultures contained about 25% spiral and 75%
reticulated cells, the addition of 50 µM spermine caused the appearance of
pitted cells amounting to about 10% of the total
(Fig. 6B). This trend was
enhanced with higher concentrations of spermine: at 100 µM spermine, the
proportion of reticulated and pitted cells was roughly equivalent
(Fig. 6B). The same was true
for 200 µM spermine (Fig.
6B,E), although very few TEs differentiated under these conditions
(Fig. 6A). Concentrations above
200 µM were lethal for the cell culture. The length and the width of the
TEs were also strongly affected, with a dose-dependent increase of over
threefold for the width and twofold for the length, compared with the control
values (Fig. 6C). In summary,
these results show that spermine: (1) delays the time of TE appearance, (2)
increases TE size and (3) favors pitted-type TE differentiation. It was
recently reported that, instead of spermine synthase, ACL5 might
encode an enzyme for synthesis of thermospermine, which is a more rare
tetraamine (Knott et al.,
2007). Our conclusions on the function of ACL5 in vessel
specification, which is analogous to what we observed here for spermine,
indicate that if thermospermine is the product of ACL5 activity it has at
least to some extent, if not completely, equivalent role to spermine in the
xylem vessels.
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). The general phenotype of ProACL5:DT-A plants resembled that of the acl5 mutant, with the severity depending on the gene dose (Fig. 7). Heterozygous plants were more slender and slightly smaller than wild-type plants, while homozygous plants had a greatly reduced rosette leaf size and stem length, resembling an extreme acl5 phenotype (Fig. 7A). It was evident that the expression of DT-A from the ACL5 promoter delayed the onset of xylem differentiation by a few days in in vitro grown seedlings, but 6 days after germination a broad cambium was already present and numerous vessels were being produced in the hypocotyl (Fig. 7L). This pattern was similar to the one observed in acl5, even though minor differences in the general anatomy of these two genotypes could be discerned (Fig, 7L,P). Further progression of the secondary growth was also similar to acl5: transverse sections of 2-month-old hypocotyls revealed that the secondary growth was severely reduced in the homozygous ProACL5:DT-A hypocotyls compared with the wild type (Fig. 7B-D). Comparison of the xylem cell types present in hypocotyls of two independent transgenic lines, after maceration, confirmed the similarity between the ProACL5:DT-A plants and the acl5 mutant in terms of xylem specification. The reticulate-type vessel elements were predominant in the homozygous ProACL5:DT-A lines, the pitted-type vessels were almost completely missing (Fig. 7F, Fig. 8A) and the vessel elements were significantly shorter and thinner than in the wild-type (Fig. 8B). Mature xylem fibers were not encountered in the macerates of the ProACL5:DT-A or acl5 hypocotyls (Fig. 7E,F), whereas this was the predominant type of xylem element produced in the wild-type hypocotyl at this stage (Fig. 7G). In conclusion, similarity in xylem specification and cell morphology of the ProACL5:DT-A plants to that observed in acl5 supports the proposed role of ACL5 in preventing premature death of the vessel elements.
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; Hanzawa et al.,
2000). Together, our results using the
ProACL5:GUS marker support: (1) the production of DT-A
toxin in the xylem vessel elements of the ProACL5:DT-A
plants at a stage which allows further maturation of the cells and (2) that
the toxin-induced premature death of the vessels is causally related to the
altered xylem specification of ProACL5:DT-A plants.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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That polyamines protect against premature cell death and senescence is not
surprising. A large number of studies demonstrate the role of polyamines in
protection from apoptotic cell death in animals, even though the opposite
function has also frequently been reported
(Seiler and Raul, 2005). In
plants, it has been suggested that polyamines retard senescence by maintaining
membrane stability and by reducing the quantity and effects of free radicals
(Bais and Ravishankar, 2002).
In addition, putrescine, spermidine and in particular spermine have been shown
to block tonoplast cation channels
(Brüggemann et al., 1998);
this is believed to block ion leakage from the vacuoles and contribute to the
regulation of osmotic potential in the cell and provide protection against
salt stress (Yamaguchi et al.,
2006; Yamaguchi et al.,
2007). On the basis of our results, we cannot predict the exact
molecular mechanism of the ACL5-mediated protection against cell death, but
the fact that alterations in ion fluxes, especially across the tonoplast, are
known to be central in the control of xylem cell death
(Kuriyama, 1999) makes it
tempting to speculate that ACL5 may function through regulation of tonoplast
channels in the xylem elements. An analogous mechanism was proposed for
polyamines in the regulation of mitochondrial integrity, which is central to
apoptotic cell death in animals (Tassani
et al., 1995). In this context, it is interesting to note that
spermine has been shown to inhibit mitochondrial membrane permeability in oats
(Curtis and Wolpert,
2002).
Premature vessel cell death in acl5 alters xylem specification
The premature vessel death in acl5 and in the transgenic
ProACL5:DT-A plants is accompanied by smaller sized
vessels and the formation of the simpler spiral- and reticulate-type secondary
cell walls instead of the more elaborate pitted-type secondary cell walls that
usually dominate (Figs 4 and
8). Consistent with these
results, an increase in the duration of differentiation in the Zinnia
xylogenic cultures induced the formation of large tracheary elements and a
shift towards the more elaborate type of secondary cell walls
(Fig. 6). Our results therefore
support the importance of the duration of vessel differentiation in
determining vessel specification and especially the formation of the
pitted-type vessels. It is generally believed that longer duration of
secondary cell wall formation allows increased deposition of the secondary
cell wall material (Barnett,
1981). It is therefore possible that ACL5 controls xylem
specification by extending the secondary cell wall deposition phase of the
vessels to allow formation of the most elaborate types of vessels that also
require the most extensive secondary cell wall deposition. However, it is also
possible that, instead of the duration of vessel maturation, ACL5 mediates an
increase in the duration of vessel expansion, resulting in an increase in the
size of the vessel elements, which in turn determines the complexity of the
secondary cell wall patterning, as suggested by Roberts and Haigler
(Roberts and Haigle,
1994).
The molecular control of xylem cell specification is poorly understood. The
NAC family transcription factors VND7 and VND6 have been suggested to control
the differentiation of the protoxylem and metaxylem elements, respectively
(Kubo et al., 2005). We found
increased expression of VND6 and especially VND7 in
acl5, suggesting that defects in xylem specification are not due to
lack of expression of either of these genes (data not shown).
Galactoglucomannans
(Benová-Kákosová et
al., 2006) and an arabinogalactan protein
(Dahiya et al., 2006) have
been related to control of protoxylem versus metaxylem type vessel elements,
but no information exists about their mode of function. Recently, reduced
cytokinin signaling was shown to result in high abundance of protoxylem
vessels in Arabidopsis roots
(Mähönen et al.,
2006). However, the predominance of protoxylem-type vessels in
acl5 does not seem to be due to impaired cytokinin signaling as
acl5 roots were actually shown to display increased cytokinin
sensitivity (Clay and Nelson,
2005).
Although earlier studies were correct in concluding that acl5
mutants exhibit enhanced vascular development with respect to the number of
vessels in the inflorescence stems and leaves
(Clay and Nelson, 2005;
Hanzawa et al., 1997), this
proliferation does not lead to enhanced overall size of the vasculature but
rather to a dramatic decrease in the width of the vasculature and the whole
stem in both the inflorescence stem (data not shown) and in the hypocotyl
primarily because of the complete lack of fibers
(Fig. 2). There could be
several reasons for the lack of fibers, but the occasional presence of
(immature) fibers in ProACL5:DT-A plants suggests that
fiber development is somehow dependent on the correct specification of
vessels, and that differentiation of the pitted-type vessels (also
occasionally observed in the ProACL5:DT-A plants) is
required for fiber differentiation. There are, to our knowledge, no other
mutants that have the interfascicular fibers (see
Hanzawa et al., 1997) but that
completely lack the xylary fibers, and ACL5 therefore seems to control fiber
development through a completely novel mechanism.
Our results, when considered together, suggest that ACL5 is required for correct xylem specification through regulation of the lifetime of the xylem elements. The shorter lifetime of the xylem vessels in the acl5 mutant results in the development of only the simple type of xylem vessels and the complete lack of xylem fibers.
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ACKNOWLEDGMENTS |
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Footnotes |
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Present address: Departamento de Biología Celular y Genética,
Universidad de Alcalá, Campus Universitario, 28870 Alcalá de
Henares, Spain
Present address: John Innes Centre, Norwich Research Park, Colney Lane,
Norwich NR4 7UH, UK
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Akamatsu, T., Hanzawa, Y., Ohtake, Y., Takahashi, T., Nishitani,
K. and Komeda, Y. (1999). Expression of endoxyloglucan
transferase genes in acaulis mutants of Arabidopsis. Plant
Physiol. 121,715
-721.
Bais, H. P. and Ravishankar, G. A. (2002). Role
of polyamines in the ontogeny of plants and their biotechnological
applications. Plant Cell Tissue Organ Cult.
69, 1-34.[CrossRef]
Barnett, J. R. (1981). Secondary xylem cell
development. In Xylem Cell Development (ed. J. R.
Barnett), pp. 47-95. Kent: Castle House
Publications.
Benová-Kákosová, A., Digonnet, C., Goubet,
F., Ranocha, P., Jauneau, A., Pesquet, E., Barbier, O., Zhang, Z., Capek, P.,
Dupree, P. et al. (2006). Galactoglucomannans increase cell
population density and alter the protoxylem/metaxylem tracheary element ratio
in xylogenic cultures of Zinnia. Plant Physiol.
142,696
-709.
Birnbaum, K., Shasha, D. E., Wang, J. Y., Jung, J. W., Lambert,
G. M., Galbraith, D. W. and Benfey, P. N. (2003). A gene
expression map of the Arabidopsis root. Science
302,1956
-1960.
Brüggemann, L. I., Pottosin, I. I. and Schönknecht,
G. (1998). Cytoplasmic polyamines block the fast-activating
vacuolar cation channel. Plant J.
16,101
-105.[CrossRef]
Caño-Delgado, A., Yin, Y., Yu, C., Vafeados, D.,
Mora-Garcia, S., Cheng, J. C., Nam, K. H., Li, J. and Chory, J.
(2004). BRL1 and BRL3 are novel brassinosteroid receptors that
function in vascular differentiation in Arabidopsis.
Development 131,5341
-5351.
Clay, N. K. and Nelson, T. (2005). Arabidopsis
thickvein mutation affects vein thickness and organ vascularization,
and resides in a provascular cell-specific spermine synthase involved in vein
definition and in polar auxin transport. Plant
Physiol. 138,767
-777.
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.[CrossRef][Medline]
Curtis, M. D. and Grossniklaus, U. (2003). A
Gateway cloning vector set for high-throughput functional analysis of genes in
planta. Plant Physiol.
133,462
-469.
Curtis, M. J. and Wolpert, T. J. (2002). The
oat mitochondrial permeability transition and its implication in victorin
binding and induced cell death. Plant J.
29,295
-312.[CrossRef][Medline]
Dahiya, P., Findlay, K., Roberts, K. and McCann, M. C.
(2006). A fasciclin-domain containing gene, ZeFLA11, is
expressed exclusively in xylem elements that have reticulate wall thickenings
in the stem vascular system of Zinnia elegans cv Envy.
Planta 223,1281
-1291.[CrossRef][Medline]
Esau, K. (1977). Anatomy of Seed
Plants, 2nd edn. New York: John Wiley & Sons.
Fukuda, H. (2004). Signals that control plant
vascular cell differentiation. Nat. Rev. Mol. Cell
Biol. 5,379
-391.[CrossRef][Medline]
Fukuda, H. and Komamine, A. (1980).
Establishment of an experimental system for the study of tracheary element
differentiation from single cells isolated from the mesophyll of Zinnia
elegans. Plant Physiol.
65, 57-60.
Funk, V., Kositsup, B., Zhao, C. and Beers, E. P.
(2002). The Arabidopsis xylem peptidase XCP1 is a tracheary
element vacuolar protein that may be a papain ortholog. Plant
Physiol. 128,84
-94.
Gälweiler, L., Guan, C., Müller, A., Wisman, E.,
Mendgen, K., Yephremov, A. and Palme, K. (1998). Regulation
of polar auxin transport by AtPIN1 in Arabidopsis vascular tissue.
Science 282,2226
-2230.
Ge, C., Cui, X., Wang, Y., Hu, Y., Fu, Z., Zhang, D., Cheng, Z.
and Li, J. (2006). BUD2, encoding an S-adenosylmethionine
decarboxylase, is required for Arabidopsis growth and development.
Cell Res. 16,446
-456.[CrossRef][Medline]
Hanzawa, Y., Takahashi, T. and Komeda, Y.
(1997). ACL5: an Arabidopsis gene required for
internodal elongation after flowering. Plant J.
12,863
-874.[CrossRef][Medline]
Hanzawa, Y., Takahashi, T., Michael, A. J., Burtin, D., Long,
D., Pineiro, M., Coupland, G. and Komeda, Y. (2000).
ACAULIS5, an Arabidopsis gene required for stem elongation,
encodes a spermine synthase. EMBO J.
19,4248
-4256.[CrossRef][Medline]
Hardtke, C. S. and Berleth, T. (1998). The
Arabidopsis gene MONOPTEROS encodes a transcription factor
mediating embryo axis formation and vascular development. EMBO
J. 17,1405
-1411.[CrossRef][Medline]
Hobbie, L., McGovern, M., Hurwitz, L. R., Pierro, A., Liu, N.
Y., Bandyopadhyay, A. and Estelle, M. (2000). The
axr6 mutants of Arabidopsis thaliana define a gene involved
in auxin response and early development. Development
127, 23-32.[Abstract]
Ikeguchi, Y., Bewley, M. C. and Pegg, A. E.
(2006). Aminopropyltransferases: function, structure and
genetics. J. Biochem.
139, 1-9.
Imai, A., Akiyama, T., Kato, T., Sato, S., Tabata, S., Yamamoto,
K. T. and Takahashi, T. (2004a). Spermine is not essential
for survival of Arabidopsis. FEBS Lett.
556,148
-152.[CrossRef][Medline]
Imai, A., Matsuyama, T., Hanzawa, Y., Akiyama, T., Tamaoki, M.,
Saji, H., Shirano, Y., Kato, T., Hayashi, H., Shibata, D. et al.
(2004b). Spermidine synthase genes are essential for survival of
Arabidopsis. Plant Physiol.
135,1565
-1573.
Imai, A., Hanzawa, Y., Komura, M., Yamamoto, K. T., Komeda, Y.
and Takahashi, T. (2006). The dwarf phenotype of the
Arabidopsis acl5 mutant is suppressed by a mutation in an upstream
ORF of a bHLH gene. Development
133,3575
-3585.
Jackson, D. (1991). In-situ hybridization in
plants. In Molecular Plant Pathology: A Practical
Approach (eds D. J. Bowles, S. J. Gurr and M. McPherson), pp.163
-174. Oxford University Press, Oxford,
UK.
Karimi, M., Inzé, D. and Depicker, A.
(2002). GATEWAYTM vectors for
Agrobacterium-mediated plant transformation. Trends Plant
Sci. 7,193
-195.[CrossRef][Medline]
Knott, J. M., Römer, P. and Sumper, M.
(2007). Putative spermine synthases from Thalassiosira
pseudonana and Arabidopsis thaliana synthesize thermospermine
rather than spermine. FEBS Lett.
581,3081
-3086.[CrossRef][Medline]
Koizumi, K., Sugiyama, M. and Fukuda, H.
(2000). A series of novel mutants of Arabidopsis
thaliana that are defective in the formation of continuous vascular
network: calling the auxin signal flow canalization hypothesis into question.
Development 127,3197
-3204.[Abstract]
Kubo, M., Udagawa, M., Nishikubo, N., Horiguchi, G., Yamaguchi,
M., Ito, J., Mimura, T., Fukuda, H. and Demura, T. (2005).
Transcription switches for protoxylem and metaxylem vessel formation.
Genes Dev. 19,1855
-1860.
Kumar, A., Taylor, M. A., Mad Arif, S. A. and Davies, H. V.
(1996). Potato plants expressing antisense and sense
S-adenosylmethionine decarboxylase (SAMDC) transgenes show altered levels of
polyamines and ethylene: antisense plants display abnormal phenotypes.
Plant J. 9,147
-158.[CrossRef]
Kumar, A., Taylor, M., Altabella, T. and Tiburcio, A. F.
(1997). Recent advances in polyamine research. Trends
Plant Sci. 2,124
-130.[CrossRef]
Kuriyama, H. (1999). Loss of tonoplast
integrity programmed in tracheary element differentiation. Plant
Physiol. 121,763
-774.
Mähönen, A. P., Bonke, M., Kauppinen, L., Riikonen,
M., Benfey, P. N. and Helariutta, Y. (2000). A novel
two-component hybrid molecule regulates vascular morphogenesis of the
Arabidopsis root. Genes Dev.
14,2938
-2943.
Mähönen, A. P., Bishopp, A., Higuchi, M., Nieminen, K.
M., Kinoshita, K., Törmäkangas, K., Ikeda, Y., Oka, A., Kakimoto, T.
and Helariutta, Y. (2006). Cytokinin signaling and its
inhibitor AHP6 regulate cell fate during vascular development.
Science 311,94
-98.
Møller, S. G. and McPherson, M. J.
(1998). Developmental expression and biochemical analysis of the
Arabidopsis atao1 gene encoding an
H2O2-generating diamine oxidase. Plant
J. 13,781
-791.[CrossRef][Medline]
Newman, T., de Bruijn, F. J., Green, P., Keegstra, K., Kende,
H., McIntosh, L., Ohlrogge, J., Raikhel, N., Somerville, S., Thomashow, M. et
al. (1994). Genes galore: a summary of methods for accessing
results from large-scale partial sequencing of anonymous Arabidopsis
cDNA clones. Plant Physiol.
106,1241
-1255.[Abstract]
Nilsson, O., Wu, E., Wolf, D. S. and Weigel, D.
(1998). Genetic ablation of flowers in transgenic
Arabidopsis. Plant J.
15,799
-804.[CrossRef][Medline]
Panicot, M., Minguet, E. G., Ferrando, A., Alcázar, R.,
Blázquez, M. A., Carbonell, J., Altabella, T., Koncz, C. and Tiburcio,
A. F. (2002). A polyamine metabolon involving aminopropyl
transferase complexes in Arabidopsis. Plant Cell
14,2539
-2551.
Parker, G., Schofield, R., Sundberg, B. and Turner, S.
(2003). Isolation of COV1, a gene involved in the
regulation of vascular patterning in the stem of Arabidopsis.
Development 130,2139
-2148.
Rensing, K. H. (2002). Chemical and
cryo-fixation for transmission electron microscopy of gymnosperm cambial
cells. In Wood Formation in Trees: Cell and Molecular Biology
Techniques (ed. N. J. Chaffey), pp.65
-81. London, UK: Taylor & Francis
Books.
Roberts, A. W. and Haigler, C. H. (1994). Cell
expansion and tracheary element differentiation are regulated by extracellular
pH in mesophyll cultures of Zinnia elegans L. Plant
Physiol. 105,699
-706.[Abstract]
Sebela, M., Radová, A., Angelini, R., Tavladoraki, P.,
Frébort, I. and Pec, P. (2001). FAD-containing
polyamine oxidases: a timely challenge for researchers in biochemistry and
physiology of plants. Plant Sci.
160,197
-207.[Medline]
Seiler, N. and Raul, F. (2005). Polyamines and
apoptosis. J. Cell. Mol. Med.
9, 623-642.[Medline]
Tassani, V., Biban, C., Toninello, A. and Siliprandi, D.
(1995). Inhibition of mitochondrial permeability transition by
polyamines and magnesium: Importance of the number and distribution of
electric charges. Biochem. Biophys. Res. Commun.
207,661
-667.[CrossRef][Medline]
Ulmasov, T., Murfett, J., Hagen, G. and Guilfoyle, T. J.
(1997). Aux/IAA proteins repress expression of reporter genes
containing natural and highly active synthetic auxin response elements.
Plant Cell 9,1963
-1971.[Abstract]
Walden, R., Cordeiro, A. and Tiburcio, A. F.
(1997). Polyamines: small molecules triggering pathways in plant
growth and development. Plant Physiol.
113,1009
-1013.[CrossRef][Medline]
Yamaguchi, K., Takahashi, Y., Berberich, T., Imai, A., Miyazaki,
A., Takahashi, T., Michael, A. and Kusano, T. (2006). The
polyamine spermine protects against high salt stress in Arabidopsis
thaliana. FEBS Lett.
580,6783
-6788.[CrossRef][Medline]
Yamaguchi, K., Takahashi, Y., Berberich, T., Imai, A.,
Takahashi, T., Michael, A. and Kusano, T. (2007). A
protective role for the polyamine spermine against drought stress in
Arabidopsis. Biochem. Biophys. Res. Commun.
352,486
-490.[CrossRef][Medline]
Ye, Z.-H. (2002). Vascular tissue
differentiation and pattern formation in plants. Annu. Rev. Plant
Biol. 53,183
-202.[CrossRef][Medline]
Zhao, C., Johnson, B. J., Kositsup, B. and Beers, E. P.
(2000). Exploiting secondary growth in Arabidopsis. Construction
of xylem and bark cDNA libraries and cloning of three xylem endopeptidases.
Plant Physiol. 123,1185
-1196.
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