RESEARCH ARTICLE 2573
Development 135, 2573-2582 (2008) doi:10.1242/dev.019349
ACAULIS5 controls Arabidopsis xylem specification through
the prevention of premature cell death
Luis Muñiz1,*,†, Eugenio G. Minguet2,*, Sunil Kumar Singh1,*, Edouard Pesquet1,‡, Francisco Vera-Sirera2,
Charleen L. Moreau-Courtois1, Juan Carbonell2, Miguel A. Blázquez2,§ and Hannele Tuominen1
Cell size and secondary cell wall patterning are crucial for the proper functioning of xylem vessel elements in the vascular tissues of
plants. Through detailed anatomical characterization of Arabidopsis thaliana hypocotyls, we observed that mutations in the
putative spermine biosynthetic gene ACL5 severely affected xylem specification: the xylem vessel elements of the acl5 mutant were
small and mainly of the spiral type, and the normally predominant pitted vessels as well as the xylem fibers were completely
missing. The cell-specific expression of ACL5 in the early developing vessel elements, as detected by in situ hybridization and
reporter gene analyses, suggested that the observed xylem vessel defects were caused directly by the acl5 mutation. Exogenous
spermine prolonged xylem element differentiation and stimulated cell expansion and cell wall elaboration in xylogenic cell cultures
of Zinnia elegans, suggesting that ACL5 prevents premature death of the developing vessel elements to allow complete expansion
and secondary cell wall patterning. This was further supported by our observations that the vessel elements of acl5 seemed to
initiate the cell death program too early and that the xylem defects associated with acl5 could be largely phenocopied by induction
of premature, diphtheria toxin-mediated cell death in the ACL5-expressing vessel elements. We therefore provide, for the first time,
mechanistic evidence for the function of ACL5 in xylem specification through its action on the duration of xylem element
differentiation.
INTRODUCTION
Organ differentiation in eukaryotes typically requires the proper
coordination of several different developmental processes. For
example, the shift from vegetative to reproductive development in
Arabidopsis thaliana involves a significant increase in the internode
length of the inflorescence stem, which has to be coordinated with
building of a functional vascular system, including the elongated
structures of xylem. During the active elongation phase of the
inflorescence stem, primary ‘protoxylem’ vessel elements
differentiate and develop spiral or annular secondary cell wall
thickenings that allow longitudinal expansion of the cells and,
therefore, elongation of the stem. When internode elongation ceases,
primary ‘metaxylem’ vessel elements with the more elaborate type
of reticulate or pitted secondary cell wall thickenings differentiate.
The primary vascular development is followed by the formation of
vascular cambium and secondary growth, which, in addition to
vessel differentiation, involves the formation of xylem fibers.
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
1
Umeå Plant Science Centre, Department of Plant Physiology, Umeå University,
90187 Umeå, Sweden. 2Instituto de Biología Molecular y Celular de Plantas
(CSIC-UPV), Universidad Politécnica de Valencia, Avda de los Naranjos s/n,
46022 Valencia, Spain.
*These authors contributed equally to this work
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
§
Author for correspondence (e-mail: mblazquez@ibmcp.upv.es)
†
Accepted 26 May 2008
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
DEVELOPMENT
KEY WORDS: ACL5, Arabidopsis, Cell death, Secondary cell wall, Tracheary element, Xylem specification
2574 RESEARCH ARTICLE
MATERIALS AND METHODS
Plant material and growth conditions
The analyses of the Arabidopsis thaliana acl5 mutant were conducted using
the mutant allele acl5-4 (in Ler background) or the SALK_028736 line,
when comparisons were made with transgenic ProACL5:DT-A, ProDR5:GUS,
or ProXCP2:GUS lines, which were all in the Arabidopsis Col-0 background.
Soil-grown plants were kept in a walk-in climate chamber (Kryo Service,
Helsinki, Finland) under long-day conditions (18 hours light/6 hours
darkness, 70% humidity, 21°C/18°C). The plants grown in vitro were kept,
without selection, on MS plates (Duchefa) containing 10 g/l sucrose, in
growth rooms under long-day conditions (18 hours light/6 hours darkness,
21°C/18°C).
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⬘-GAGTCGACATGGATCCTGATGATGTTGTTG3⬘ 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 DTAutr/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-4chloro-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.
DEVELOPMENT
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 Sadenosylmethionine 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-offunction 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.
Development 135 (15)
ACAULIS 5 delays xylem vessel death
RESEARCH ARTICLE 2575
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).
For analysis of the individual xylem elements, hypocotyls were
macerated from soil-grown 2-month-old plants that were stimulated for
extensive secondary growth by continuous decapitation of the inflorescence
stems. The tissues were incubated at 95°C for 4 hours in 3% H2O2/50%
acetic acid, gently washed twice with distilled water and neutralized by the
addition of solid Na2CO3 to the last wash. Samples were then disaggregated
mechanically and stored at room temperature. The cell suspensions were
stained with 0.01% CelluFluor (Polysciences) and examined under UV
illumination with a light microscope. For each genotype, three plants were
analyzed and 50-300 vessel elements per plant were scored for length, width
and the pattern of secondary cell wall thickenings.
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
RESULTS
Cell-specific expression of ACL5 in the xylem
vessel elements
The expression of ACL5 was earlier localized into the procambial
and/or provascular tissues of Arabidopsis plants (Birnbaum et al.,
2003; 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.
Fig. 1. ACL5 is specifically expressed in the xylem vessel
elements. (A-E) In situ hybridization of ACL5. Sections were taken
from the junction between the silique and the pedicel (A,B) and the
basal part of the inflorescence stem (C-E), and analyzed using antisense
(A,C,E) or sense (B,D) probes for the ACL5 gene. (A,B) Longitudinal
sections; (C-E) an area of the stem with the vascular bundles in the
transverse plane. The sections hybridized with the sense probe showed
sometimes dark coloration of the vascular tissues (B) but not the
positive purple precipitate derived from the chromogenic substrate
(A,C,E). (F-H) Histochemical β-glucuronidase (GUS) staining of
transgenic ProACL5:GUS seedlings. Transverse sections were taken from
the inflorescence stem (one vascular bundle shown in F), the hypocotyl
of a 4-day-old seedling (G) and the hypocotyl of a 1.5-month-old
seedling (H). do, developing ovary; pc, procambium; ph, phloem; sx,
secondary xylem; v, vessel element; vc, vascular cambium; xp, xylem
parenchyma. Asterisks indicate the protoxylem poles (G). Scale bars: 20
μm in E-G; 50 μm in C,D,H; 100 μm in A,B.
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. Sevenday-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
DEVELOPMENT
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 α-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 (SigmaAldrich), 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.
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).
Fig. 2. Xylem development is severely distorted in the acl5
mutant. (A-H) General anatomy of acl5 and wild-type hypocotyls was
examined by light microscopy in transverse sections (A,B,E-H) and in
longitudinal sections (C,D) from seedlings grown for seven (A-D), 13
(E,F) or 35 days (G,H). (I) A confocal image of a representative vessel
element from wild type. (J) A confocal image of a representative vessel
element from acl5. mx, metaxylem; px, protoxylem; sx, secondary
xylem; vc, vascular cambium. Scale bars: 50 μm in A-F,H; 100 μm in G.
Development 135 (15)
In addition, electron micrographs of 3-week-old hypocotyls
showed the presence of numerous vessels with altered patterns of
secondary cell wall deposition and the lack of xylem fibers in acl5
(Fig. 3E). Interestingly, the pattern of vessel maturation seemed to
differ as well. Normal maturation of vessel elements involves
extensive deposition of secondary cell wall material, which is
terminated by the collapse of the central vacuole and the release of
the vacuolar contents into the cytoplasm, leading into rapid cell
death and concomitant autolysis of the vessel elements. Maturation
can proceed after the death of the vessel elements, even though
majority of the secondary cell wall material is normally deposited
before the collapse of the vacuole. In the wild type, it was always
possible to distinguish a few maturing vessel elements with an intact
central vacuole and ongoing secondary cell wall deposition (Fig. 3BD). However, we could not find any vessel elements in acl5
hypocotyls that had distinguishable secondary cell walls and that
still had intact central vacuole (Fig. 3F-H). Therefore, the maturing
vessel elements observed in acl5 must have surpassed the vacuolar
collapse, and it seems that the vacuole collapses in acl5 too early in
relation to the progress of the cell wall deposition.
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 wellestablished 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
DEVELOPMENT
2576 RESEARCH ARTICLE
ACAULIS 5 delays xylem vessel death
RESEARCH ARTICLE 2577
Fig. 3. The vacuole collapses early during vessel maturation in acl5. (A-D) Xylem anatomy and cell morphology of the wild-type. (E-H) Xylem
anatomy and cell morphology of acl5. An overview of the xylem tissues (A,E) reveals the absence of fiber differentiation in acl5. Individual vessel
elements are shown during early maturation (B,F), moderate maturation (C,G) and late maturation (D,H). All panels represent electron microscopy
images of transverse sections from the hypocotyls of 3-week-old plants. The central vacuole is absent in maturing (i.e. secondary cell wall
depositing) vessel elements of acl5, even at the earliest stage of maturation (F). f, fiber; cv, central vacuole; n, nucleus; v, a vessel element that is
either living or undergoing cell death. The arrows indicate presence of secondary cell walls. The asterisks indicate dead, autolyzed vessel elements.
Scale bars: 10 μm in A,E; 2 μm in B-D,F-H.
Fig. 4. Xylem vessel elements of the acl5 mutant are small and
simple in structure. Two-month-old hypocotyls of acl5 and wild-type
plants were macerated and their vessel element morphology was
investigated using light microscopy. (A) Proportion of the different types
of vessel elements. (B) Length of the vessel elements. (C) Width of the
vessel elements. More than 200 cells were examined from three plants
of each genotype. Data were compared (B,C) using a Welch corrected
t-test (***P<0.005; acl5 versus wild-type), and presented as
average±s.e.m.
Spermine prolongs differentiation and increases
the size of xylem vessels in Zinnia elegans cell
cultures
To investigate the function of polyamines in xylem maturation and
cell wall patterning in a simplified system, we examined the effect
of spermine in the in vitro Zinnia elegans xylogenic system. This
system allows direct transdifferentiation of freshly isolated
mesophyll cells of Zinnia into structures similar to xylem vessels,
commonly called tracheary elements (TEs), within a time frame of
72 to 96 hours and in a semi-synchronous manner (Fukuda and
Komamine, 1980). 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
DEVELOPMENT
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.
2578 RESEARCH ARTICLE
Development 135 (15)
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.
Fig. 6. Exogenous spermine modifies tracheary element
differentiation in Zinnia elegans xylogenic cell cultures.
(A) Tracheary element (TE) differentiation efficiency, expressed as the
number of TEs as a percentage of all cells, in response to 50-200 μM
spermine 84 and 168 hours after the initiation of the cell culture.
(B) Proportion of the different types of TEs at 168 hours in response to
50-200 μM spermine. (C) The size of the TEs (±s.e.m.) after 168 hours
in response to 50-200 μM spermine. Statistics are presented (C) for
each treatment compared with the previous treatment, using a KruskalWallis test (***P<0.005). (D) Typical TEs after 168 hours without the
addition of spermine. (E) Typical TEs 168 hours after the addition of
200 μM spermine. Scale bar: 50 μm in D,E.
Premature vessel death mimics the effect of loss
of ACL5 function
The evidence presented above is compatible with a model in which
ACL5 prevents premature cell death in developing xylem vessel
elements in order to allow proper xylem specification and especially
formation of the elaborate type of vessels. One prediction based on
this model is that the induction of premature death of the ACL5expressing subset of cells should render a phenotype similar, in
terms of xylem differentiation, to the one caused by the loss of
ACL5 function. To test this, we constructed transgenic Arabidopsis
plants expressing the diphtheria toxin A chain (DT-A) under the
control of the ACL5 promoter. DT-A has been previously used in
plants as a tool to ablate specific cell types by directing the
production of this cell-autonomous toxin to those target cells
(Nilsson et al., 1998).
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
DEVELOPMENT
Fig. 5. Expression of an auxin and a cell death marker is altered
in acl5. (A,B) Histochemical GUS staining of 7-day-old ProDR5:GUS (A)
and acl5 ProDR5:GUS (B) seedlings grown in vitro. ProDR5:GUS expression
is present in the developing vessel elements of acl5 hypocotyl but not in
the wild type. (C,D) Histochemical GUS staining in 7-day-old
ProXCP2:GUS (C) and in acl5 ProXCP2:GUS (D) seedlings grown in vitro.
ProXCP2:GUS activity in the immature vessel elements (arrowheads) of
acl5 is indicative of an early onset of the vessel cell death program.
Scale bar: 50 μm.
ACAULIS 5 delays xylem vessel death
RESEARCH ARTICLE 2579
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.
To confirm that the observed alterations in vessel morphology of
the ProACL5:DT-A lines were caused directly by DT-A toxin
production and not, for example, by induction of ectopic non-DT-Aexpressing xylem elements resulting from early ablation of the
xylem elements, we analyzed expression of the ProACL5:GUS
marker in the homozygous ProACL5:DT-A line 4. As expression of
both GUS and DT-A are driven by the ACL5 promoter, it can be
assumed that GUS activity reveals the sites of toxin production in
the ProACL5:DT-A ProACL5:GUS plants. Whole-mount staining of
hypocotyls revealed that GUS activity was limited to the vasculature
of these plants (Fig. 7K). Higher resolution images allowed
definition of this activity into developing xylem with highest activity
in the incipient vessel elements (Fig. 7L,M) and sometimes even in
the maturing vessel elements (Fig. 7N). The expression domain of
ProACL5:GUS was broader both in ProACL5:DT-A (Fig. 7K-N) and
acl5 seedlings (Fig. 7O-Q) compared with the wild type (Fig. 7HJ), which is most probably related to the initial increase in the
cambial activity and overproliferation of vessel elements in the
young ProACL5:DT-A and acl5 seedlings. A similar increase in the
expression of ACL5 in the acl5 background was earlier suggested to
DEVELOPMENT
Fig. 7. Expression of ProACL5:DT-A alters plant growth and xylem
development. (A) The general phenotype of 1-month-old wild-type,
ProACL5:DT-A heterozygous line 4, ProACL5:DT-A homozygous line 4 and
acl5 seedlings. (B-D) Resin-embedded transverse sections of 2-monthold acl5 (B), ProACL5:DT-A homozygous line 4 (C) and wild-type (D)
hypocotyls stained with Toluidine Blue. (E-G) Appearance of xylem
elements after maceration of the hypocotyls of 2-month-old acl5 (E),
ProACL5:DT-A homozygous line 4 (F) and wild type (G). Asterisks indicate
the presence of xylem fibers in the wild-type (G). (H-Q) Expression of
ProACL5:GUS in wild-type (H-J), ProACL5:DT-A homozygous line 4 (K-N)
and acl5 seedlings (O-Q). Histochemical GUS staining is shown for
hypocotyls of whole mounts (H,J,K,M,N,O,Q) and transverse sections of
resin-embedded hypocotyls (I,L,P). Xylem differentiation was delayed in
ProACL5:DT-A seedlings, and comparisons were therefore made between
3-day-old wild-type and acl5 and 6-day-old ProACL5:DT-A in vitro grown
seedlings. The arrows indicate expression of ProACL5:GUS and therefore
DT-A toxin production in the incipient vessel elements (with first signs
of secondary cell wall deposition in cell corners) of the ProACL5:DT-A
seedlings (L). sx, secondary xylem; vc, vascular cambium. Scale bars: 20
μm in I,J,L,M,N,P,Q; 50 μm in E,F,G,H,K,O; 100 μm in B,C; 200 μm in D.
Fig. 8. ProACL5:DT-A expressing plants show acl5-like xylem
specification. (A) Proportion of the different types of the vessel
elements in the hypocotyls of wild-type, acl5 and ProACL5:DT-A
homozygous lines 4 and 5. (B) The length and width of individual xylem
vessel elements in the hypocotyls of the different genotypes. More than
200 cells were scored from macerated hypocotyls of three 2-month-old
plants for each genotype. Data are presented as average±s.e.m.
***P<0.005, as compared with the wild type using a Kruskal-Wallis
test.
indicate negative-feedback regulation of ACL5 expression (Clay and
Nelson, 2005; 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 toxininduced premature death of the vessels is causally related to the
altered xylem specification of ProACL5:DT-A plants.
DISCUSSION
ACL5 prevents premature death of the xylem
vessel elements
Our work establishes that ACL5 has a prominent role in the correct
specification of xylem cells. This is supported by at least three
observations: (1) the xylem-specific expression of ACL5 (Fig. 1);
(2) the predominance of the spiral-type vessels and the lack of pitted
vessels as well as fibers in the acl5 mutant (Figs 2-4); and (3) the
misregulation of vascular-related markers in the acl5 mutant (Fig.
5). The earliest xylem defects of acl5 colocalise with the ACL5expressing subset of cells, and we therefore conclude that xylem
specification is the primary function of ACL5. Activity of ACL5 as
a polyamine biosynthetic enzyme indicates a completely novel role
for polyamines in control of xylem specification by preventing
premature death of the xylem vessel elements. This conclusion is
substantiated by our observations of the early onset of the cell death
program in the vessel elements of acl5 (Fig. 3, Fig. 5D) and by the
fact that the xylem vessel defect of acl5 could be phenocopied by
the induction of premature DT-A toxin-mediated cell death in the
ACL5-expressing vessels (Figs 7 and 8).
Development 135 (15)
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,
DEVELOPMENT
2580 RESEARCH ARTICLE
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.
We thank Detlef Weigel for providing access to the pEW3 plasmid, Eric Beers
for the gift of the ProXCP2:GUS seeds, Karin Ljung for assistance with the auxinrelated experiments and Alan Marchant for valuable comments on the
manuscript. The work was supported by the Swedish Research Council
Formas, the Kempe foundation and the Swedish Foundation for Strategic
Research. M.A.B. is an EMBO Young Investigator. E.G.M. has been funded by
Fellowships from the Spanish Ministry of Education and the Spanish National
Research Council I3P Program.
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