Download Nitric oxide is essential for vesicle formation and

Journal of Experimental Botany, Vol. 63, No. 13,
2, pp.
2012
pp.695–709,
4875–4885,
2012
doi:10.1093/jxb/err313
doi:10.1093/jxb/ers166 Advance
AdvanceAccess
Accesspublication
publication 412November,
July, 20122011
This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)
RESEARCH PAPER
Nitric
oxide isoceanica
essentialcadmium
for vesicle
formation
and trafficking
In
Posidonia
induces
changes
in DNA
in Arabidopsis
root
hair growth
methylation
and
chromatin
patterning
1
2,
M. C. Lombardo
and L.
LamattinaLeonardo
*
Maria
Greco, Adriana
Chiappetta,
Bruno and Maria Beatrice Bitonti*
1
Departamento
de Biología
e Instituto
de Investigaciones
Biológicas,
Universidad Nacional
de MarBucci,
del Plata,
Consejo
Nacional
de
Department
of Ecology,
University
of Calabria,
Laboratory of
Plant Cyto-physiology,
Ponte Pietro
I-87036
Arcavacata
di Rende,
Investigaciones
Cosenza,
Italy Científicas y Técnicas, CC 1245, 7600 Mar del Plata, Argentina
2
Instituto de Investigaciones Biológicas, Universidad Nacional de Mar del Plata, Consejo Nacional de Investigaciones Científicas y
* To whom correspondence should be addressed. E-mail: [email protected]
Técnicas, CC 1245, 7600 Mar del Plata, Argentina
* To whom
should
be2011;
addressed.
E-mail:
[email protected]
Received
29correspondence
May 2011; Revised
8 July
Accepted
18 August
2011
Received 8 December 2011; Revised 13 March 2012; Accepted 8 May 2012
Abstract
In
mammals, cadmium is widely considered as a non-genotoxic carcinogen acting through a methylation-dependent
Abstract
epigenetic mechanism. Here, the effects of Cd treatment on the DNA methylation patten are examined together with
Theeffect
functions
of nitric oxide
(NO) in processes
associated
withDNA
rootmethylation
hair growthlevel
in Arabidopsis
were
analysed.
its
on chromatin
reconfiguration
in Posidonia
oceanica.
and pattern
were
analysedNO
in
is
located
at
high
concentrations
in
the
root
hair
cell
files
at
any
stage
of
development.
NO
is
detected
inside
of Cd,
the
actively growing organs, under short- (6 h) and long- (2 d or 4 d) term and low (10 mM) and high (50 mM) doses of
vacuole ina immature
actively growing
root hairs and,
later, NO is localized
in the
cytoplasm
when they become
mature.
through
Methylation-Sensitive
Amplification
Polymorphism
technique
and
an immunocytological
approach,
Experiments
performed
by
depleting
NO
in
Arabidopsis
root
hairs
indicate
that
NO
is
required
for
endocytosis,
vesicle
respectively. The expression of one member of the CHROMOMETHYLASE (CMT) family, a DNA methyltransferase,
formation,
and
trafficking
and
it
is
not
involved
in
nucleus
migration,
vacuolar
development,
and
transvacuolar
strands.
was also assessed by qRT-PCR. Nuclear chromatin ultrastructure was investigated by transmission electron
The Arabidopsis
G’4,3 mutant
(double
mutant
nia1/nia2) is severely
impaired
in NO production
and generates
microscopy.
Cd treatment
induced
a DNA
hypermethylation,
as well
as an up-regulation
of CMT,
indicating smaller
that de
root
hairs
than
the
wild
type
(WT).
Root
hairs
from
the
Arabidopsis
G’4,3
mutant
show
altered
vesicular
trafficking
novo methylation did indeed occur. Moreover, a high dose of Cd led to a progressive heterochromatinization
of
and are reminiscent
NO-depleted
root
hairs
from
the Arabidopsis
WT. Interestingly,
normal
formation
interphase
nuclei andofapoptotic
figures
were
also
observed
after long-term
treatment. The
datavesicle
demonstrate
thatand
Cd
traffickingthe
as DNA
well as
root hair growth
is restored
exogenous of
NOa application
in the Arabidopsis
G’4,3changes
mutant.are
All
perturbs
methylation
status through
theby
involvement
specific methyltransferase.
Such
together,
results
firmly support
the essential
played byaNO
in the
Arabidopsis
root-hair-growing chromatin.
process.
linked
to these
nuclear
chromatin
reconfiguration
likely role
to establish
new
balance
of expressed/repressed
Overall, the data show an epigenetic basis to the mechanism underlying Cd toxicity in plants.
Key words: Actin, Arabidopsis G’4,3 mutant, endocytosis, nitric oxide, root hair, tip growing, vesiculation.
Key words: 5-Methylcytosine-antibody, cadmium-stress condition, chromatin reconfiguration, CHROMOMETHYLASE,
DNA-methylation, Methylation- Sensitive Amplification Polymorphism (MSAP), Posidonia oceanica (L.) Delile.
Introduction
Root hairs are specialized root epidermal cells of higher plants
Introduction
whose functions are water absorption and anchorage. The charIn
the Mediterranean
ecosystem,
thewith
endemic
acteristic
polarized growthcoastal
of root hairs
is shared
a few
seagrass
Posidonia
oceanica
(L.)
Delile
plays
a
relevant
role
other cells such as pollen tubes, moss protonemata, and funby
ensuring
primary
production,
water
oxygenation
gal hyphae. All of these cells show polar cell expansion and
and
provides
some(Reiss
animals,
besides1979;
counteracting
have beenniches
widely for
studied
and Herth,
Heath and
coastal
erosion
its widespread meadows (Ott, 1980;
Geitmann,
2000;through
Hepler et al., 2001).
Piazzi
et
al.,
1999;
Alcoverro
et al., that
2001).
There
also
Tip growth depends on several processes
include
Ca2+isinflux,
considerable
evidence
that
P.
oceanica
plants
are
able
to
MAPK cascades, microtubular and actin arrangements, vacuolar
absorb
and
accumulate
metals
from
sediments
(Sanchiz
development, nucleus migration, and vesicular trafficking, among
et
al., 1990;
Pergent-Martini,
1998;et al.,
Maserti
etChytilova
al., 2005)et al.,
thus
others
(Galway
et al., 1997; Baluška
2000;
influencing
metal
bioavailability
in
the
marine
ecosystem.
2000; Hepler et al., 2001; Ketelaar et al., 2002; Takahashi et al.,
For
reason,
this seagrass
is widely
considered to be
2003;this
Šamaj
et al., 2004a,
b, 2006; Jones
and Smirnoff, 2006).
a metal
bioindicator
species
(Maserti
et
al.,
1988;arePergent
At the time of root hair initiation, the trichoblasts
extenet
al.,
1995;
Lafabrie
et
al.,
2007).
Cd
is
of most
sively vacuolated, the nucleus migrates from one
a position
in
widespread
metals intoward
both the
terrestrial
andemerging
marine
the middle ofheavy
the trichoblast
site of the
environments.
hair and, eventually, it enters the growing hair (Klahre and
Chua, 1999).
Although
not have
essential
growth,
in terrestrial
Several studies
shownfor
thatplant
polarized
plant cells
display a
plants,
Cd
is
readily
absorbed
by
roots
and
translocated
into
2+
tip-focused gradient of cytosolic Ca (Yokota et al., 2000; Chen
aerial
organs
while,
in
acquatic
plants,
it
is
directly
taken
up
2+
et al., 2008). External Ca is required for normal root-hair elonby
leaves.
In
plants,
Cd
absorption
induces
complex
changes
2+
gation. The Ca influx may be maintained by the continual fusion
at
the2+-channel-containing
genetic, biochemical
and at
physiological
levelsthewhich
of Ca
vesicles
the tip, providing
cytoultimately
account
for
its
toxicity
(Valle
and
Ulmer,
plasmic region with a random fine F-actin organization. The1972;
actin
Sanitz
di Toppi
and Gabrielli,
1999;vesicular
Benavides
et al., 2005;
cytoskeleton
organization
and polar
trafficking
are
Weber
et al., 2006;
Liutip-growth
et al., 2008).
The most
obvious
critical components
of the
mechanism
in pollen
tubes
symptom
of Cd
toxicity
is aet al.,
reduction
plant
growth
due to
(Hepler et al.,
2001;
Šamaj
2004b)inand
root
hairs (Šamaj
an
inhibition
of
photosynthesis,
respiration,
and
nitrogen
et al., 2006). In the root hair, the growing tip can be reached
metabolism,
as wellstreaming
as a reduction
water and
andorganelles
mineral
because cytoplasmic
transportsinvesicles
uptake
(Ouzonidou
et
al.,
1997;
Perfus-Barbeoch
et
al.,
from the root hair base to the tip and then back. This type of2000;
cytoShukla
al., 2003;isSobkowiak
and Deckert,streaming
2003). (Hepler
plasmic et
streaming
called reverse-fountain
At the
genetic
in both
animals
and plants,
Cd
et al.,
2001)
wherelevel,
the main
direction
of organelle
transport
can induce chromosomal aberrations, abnormalities in
© 2011
The Author
[2012]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.
ª
The Author(s).
For Permissions,
please article
e-mail:distributed
[email protected]
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is an Open Access
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4876 | Lombardo and Lamattina
reverses 180 degrees before the cell tip is reached (Sieberer and
Emons, 2000). When cytoplasm is transported back by F-actin, it
forms a cytoplasmic strand that runs through the vacuole and is
called the transvacuolar strand (Tominaga et al., 2000).
In growing root hairs, new plasma membrane (PM) and cell wall
components are incorporated continuously into the growing tips
by polarized exocytosis (Galway et al., 1997). The growing process is driven by the co-ordinated trafficking of secretory vesicles
(Ovečka et al., 2005); thus, the tip presents a remarkable vesiclerich cytoplasm (Miller et al., 1999). Root hairs severely affected
in growth lacks this tip-localized cytoplasm and has a vacuole
that extends into the root-hair tip (Miller et al., 1999; Jones and
Smirnoff, 2006). Although there is considerable exocytosis at the
tip of root hairs, there must also be active endocytosis to retrieve
the excess membrane that is secreted (Picton and Steer, 1983;
Derksen et al., 1995). The growing root hair is filled with a large
central vacuole that enlarges as the cell expands (Galway et al.,
1997). The RHD3 (ROOT HAIR DEFECTIVE3) gene encodes
a putative GTP-binding protein required for appropriate cell
enlargement in Arabidopsis (Wang et al., 1997). The Arabidopsis
thaliana rhd3 mutant shows the importance of the vacuole in the
development of root hairs because it produces short and wavy root
hairs with an average volume less than one-third of the wild-type
hairs, indicating abnormal cell expansion (Galway et al., 1997).
Root hair growth is accompanied by the migration of the
nucleus into the shank (Jones and Smirnoff, 2006), at a certain
distance of the tip (Miller et al., 1997). In mature root hairs, the
movement of the nucleus is bidirectional (Chytilova et al., 2000;
Ketelaar et al., 2002). Chytilova et al. (2000) demonstrated that
rapid and, in some cases, long-distance intracellular movement
of nuclei within actively growing cells occurs. This fact has been
widely observed, and the advantage for a cell to keep the nucleus
in the vicinity of sites with highly required protein production
like growing PM and new wall deposition is obvious (Ketelaar
et al., 2002). Despite these observations, multiple root hairs
showed that the requirement of nuclei migration is not absolute
(Jones and Smirnoff, 2006).
The actin cytoskeleton is responsible for different processes
during root hair tip growth (Miller et al., 1999; Baluška et al.,
2000), and microtubules are required to maintain the direction
of the tip growth but not the growing process per se (Bibikova
et al., 1999). Pharmacological experiments provide evidence that
the maintenance of the nucleus in the vicinity of the growing
root hair tip is a process that involves the sub-apical fine F-actin
cytoskeleton, but microtubules are not implicated (Ketelaar et al.,
2002). In root hair cells, actin is involved in vesicle trafficking; it
mediates endocytosis, directs the vesicles into endosomal compartments, and brings secretory vesicles back to the PM (Geldner
et al., 2001; Baluška et al., 2002; Kasprowicz et al., 2009). Many
reports show that the actin cytoskeleton and vesicular trafficking
are strongly associated processes in root hairs (Voigt et al., 2005;
Šamaj et al., 2006; Galleta and Cooper, 2009).
Nitric oxide (NO) is a bioactive molecule that is involved in
numerous plant physiological processes such as the regulation
of defence-related gene expression and programmed cell death,
seed germination, stomatal closure, and root development,
among others (Lamattina et al., 2003; Delledonne, 2005; Corpas
et al., 2007; Besson-Bard et al., 2008; Wawer et al., 2010).
Several works have demonstrated the involvement of NO in
root development. It was shown that NO mediates the auxin
response and cell wall biosynthesis in lateral root formation
(Correa-Aragunde et al., 2004, 2008). NO also induces adventitious root development (Pagnussat et al., 2004) and modulates
dynamic actin cytoskeleton and vesicle trafficking in root apices
(Kasprowicz et al., 2009). NO was also shown to be implicated
in Arabidopsis root hair formation in both the initiation and elongation processes (Lombardo et al., 2006).
NO was also reported to be responsible for the S-nitrosylation
of dynamin in animal cells (Wang et al., 2006). Dynamin is a
GTPase that regulates endocytic vesicle budding from the PM,
a central process in tip growth. Furthermore, it has also been
shown that NO modulates the organization of actin filaments in
tip growing cells like pollen tubes (Wang et al., 2009). In this
work, it has been demonstrated that NO is required for some
dynamin- and actin-regulated processes in root hair growth such
as vesicle formation and release and fine F-actin formation in the
cytoplasmic region and the exocytic route.
Materials and methods
Plant material and treatments
Arabidopsis thaliana ecotype Col-0 and G’4,3 (double mutant nia1
and nia2, chlorate resistant due to very low levels of nitrate reductase
activity, about 0.5% of the level in Columbia; Wilkinson and Crawford,
1991) seeds were obtained from the Arabidopsis Biological Resource
Center (Ohio State University, Columbus).
Seeds were surface-sterilized by immersion in 70% (v/v) ethanol
for 5 min and 20% (v/v) bleach for 20 min, followed by three rinses
in sterile water. Seeds were washed with sterilizing solution 30%
(v/v) bleach with 20% (v/v) Triton X-100. Seeds were sown in Petri
dishes with ATS medium: 0.6% (w/v) agar, 1% (w/v) sucrose, and
mineral nutrients according to Wilson et al. (1990), kept for 24 h
at 4 °C and then incubated in a chamber at 25 °C with a photoperiod of 14 h for 5 d (Col-0) and 8 d (mutant). Then, Col-0 seedlings
were transferred to fresh medium with 500 µM of the NO scavenger
2-(4-carboxydiphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, (cPTIO), 200 µM of the NO donor S-nitroso N-acetyl penicillamine (SNAP) or nothing for 3 d. To see the reversion of the G’4,3
mutant phenotype, 8-d-old seedlings were transferred to fresh medium
supplemented with 200 µM SNAP for 2 d. Roots were stained with
Toluidine Blue O (TBO) and observed by light microscopy (LM).
Pictures were taken with an Olympus SP-350 camera attached to the
microscope.
NO detection
For the detection of NO, Arabidopsis seedlings were incubated in
10 µM of the cell-permeable fluorescent probe 3-amino, 4-aminomethyl-2,7-difluorofluorescein diacetate (DAF-FM DA, excitation at
490 nm, emission at 525 nm; Calbiochem, San Diego, CA, USA) in
20 mM HEPES-NaOH (pH 7.5) for 1 h. Thereafter, roots were washed
three times with fresh buffer and examined with a Nikon Eclipse 200
(Tokyo, Japan) Fluorescence Microscope (FM) and a Nikon C1Confocal Laser Scanning Microscope (CLSM). Pictures were captured with a
Nikon 900 camera. Fluorescence was quantified with ImageJ program
and expressed in arbitrary units (AU).
Vacuolar development and nucleus migration
CPTIO-treated and -untreated seedlings were processed in accordance
with LM or Transmission Electron Microscopy (TEM) protocols. Acetic
Orcein (4%, w/v) treatment for 4 min was used to observe the nucleus
Nitric oxide is essential for Arabidopsis root hair growth | 4877
position in LM and photographs were recorded with an Olympus SP/350
digital camera attached to the microscope. For the vacuolar position,
samples were analysed by the Electron Microscopy Laboratory, Scientific Technologic Center (CCT-BB, Bahía Blanca, Argentina). Samples
were from 0.5 cm above the root apices for each treatment. Explants were
fixed in 2.5% (v/v) glutaraldehyde and processed according to Reynolds
(1963): 80 kv, uranyl acetate for 2 min and lead for 40 s; longitudinal
sections of 800 Å were mounted in a copper grid with a formvar film for
observation. A JEOL 100 CXII (Tokio, Japan, 1983) TEM was used to
observe and record the samples. A grid of small squares of 0.02 µm2 over
pictures of 4.08 µm2 was used to assess the area covered with ER.
Vesicle formation and vesicular trafficking
FM4 64 endocytic tracer (excitation at 488 nm, emission at 760 nm)
was used at a final concentration of 50 µM for studying vesicular trafficking. Seedlings were stained for either 5 s or 5 min according to the
experiment. Root hair movies and pictures of samples were shot and
recorded with a Nikon C1 CLSM. Fluorescence intensity was quantified
with EZ-C1 3.9 Free Viewer software and expressed in AU. Unit areas
of 0.25 µm2 were used to analyse changes in the fluorescent intensity.
Cytoplasmic area and transvacuolar strands
Root hair movies and pictures from Differential Interference Contrast
(DIC) Microscopy were obtained with a Nikon C1 CLSM. The size of
the cytoplasmic area was measured with EZ-C1 3.9 Free Viewer software. Transvacuolar strands were monitored through movies.
Results
The NO levels are higher in trichoblast than in
atrichoblast
In a previous work, it has been demonstrated that NO is implicated
in the events leading to root hair formation in Arabidopsis thaliana (Lombardo et al., 2006). Arabidopsis roots were loaded with
the fluorescent probe DAF-FM DA and observed. The detection
of fluorescence indicates that NO is not homogeneously distributed in Arabidopsis roots. NO is located at higher concentrations in
trichoblasts (Fig. 1A). Figure 1B shows that root hair cell files have
nearly 50% more NO-dependent fluorescence than atrichoblasts.
The subcellular localization of NO in growing root hairs was analysed by Confocal Laser Scanning Microscope (CLSM) in 8-d-old
seedlings loaded with DAF-FM DA. In growing, immature root
hairs, NO is inside the vacuole whereas, in mature root hairs, NO
is localized outside the vacuole (Fig. 2). In mature root hairs, green
fluorescence appeared as a punctate pattern in the cytoplasm.
NO depletion induces abnormalities in root hair growth
Since previous data showed that treatments with the NO scavenger cPTIO prevented the normal elongation of root hairs in a
dose-dependent manner (Lombardo et al., 2006), the phenotype of
root hairs grown in the presence of 500 µM cPTIO was analysed.
Figure 3A shows DIC images of the different root hair forms
found in Arabidopsis roots depleted in NO. Most root hairs appear
sinuous and, in less proportion, thickness and ramified, dichotomously branched, and/or double. Figure 3B shows the percentage
of the main root hair phenotypes formed when NO is depleted.
Microscopic analysis also showed that, in some trichoblasts, root
hair bulges were present but they did not elongate (not shown).
Fig. 1. NO concentration is higher in Arabidopsis trichoblasts.
Roots from 8-d-old Arabidopsis Col-0 seedlings were loaded with
10 µM DAF-FM DA probe and NO localization was detected by
fluorescence microscopy. (A) Representative picture of fluorescent
trichoblasts. (B) Roots were treated or not with 200 µM SNAP to
show the presence of the fluorescent probe in trichoblasts and
atrichoblasts. The bright green fluorescence corresponding to NO
in trichoblast files, trichomes, and atrichoblasts is quantified in the
lower pannel and expressed as arbitrary units (AU). The pictures are
representative of at least three independent experiments. Arrows
indicate trichoblasts (A) and trichoblasts files (B). Bar: 100 µm.
Vacuolar development and nucleus migration are not
affected by NO depletion
Vacuolar development and nucleus migration are events involved
in tip-growing processes and were analysed in NO-depleted
root hairs. To know if NO is implicated in vacuolar development, root hair ultrastructure was observed with Transmission
4878 | Lombardo and Lamattina
Fig. 2. NO changes its subcellular location during the growth of root hairs. Arabidopsis Col-0 roots were loaded with 10 µM DAFFM DA probe to detect intracellular NO localization. The images of root hairs in different stages of maturity were taken by CLSM. V,
vacuole; N, nucleus; DIC, Differential Interference Contrast; Fl, Fluorescence. Bars: 20 µm. The inset in the mature root hair is shown to
distinguish V clearly. The pictures are representative of at least three independent experiments.
Electron Microscopy (TEM) in cPTIO-treated root hairs.
Figure 4A shows the presence of completely enlarged vacuoles in both cPTIO-treated and untreated root hairs. Untreated
root hairs show the normal size of mitochondria and rough
endoplasmic reticulum (ER) with parallel cisterns, whereas
cPTIO-treatment results in root hair cells with proportionally
more abundant ER and an irregular disposition of cisterns,
some of them being concentric (Fig. 4B). CPTIO-treated and
untreated Arabidopsis roots were stained with the nuclear dye
Acetic Orcein to reveal the position of nuclei. Figure 5 shows
that root hairs had nuclei in the trichome vacuolar zone in both
control and cPTIO-treated Arabidopsis roots. No nuclei were
found outside the vacuolar zone in 100% of both control and
cPTIO-treated root hairs indicating that NO is not essential for
nucleus migration.
manner to the tonoplast. Movies of two minutes in length
were recorded and analysed. Figure 6 shows the dynamics of
vesicle formation with five pictures taken from each movie.
Fluorescence intensity was quantified with EZ-C1 3.9 Free
Viewer software and expressed as arbitrary units (AU). Control
root hairs show the permanent formation of vesicles as numerous red dots along the fluorescent line (Fig. 6A) and marks of
high intensity in the same points (Fig. 6C). By contrast, cPTIOtreated root hairs present no changes in the fluorescent base line
corresponding to the PM (Fig. 6B, C). In some cases, a few
permanent red dots are detected in cPTIO-treated root hairs
(Fig. 6B), indicating that the process of vesicle endocytosis is
strongly prevented (Fig. 6C). A slight decrease in basal fluorescence can be seen after 2 min that might be due to bleaching
processes (Fig. 6A, B).
Vesicle formation is thoroughly affected by NO depletion
The Arabidopsis mutant G’4,3 is defective in nitrate
reductase activity and develops abnormal root
hairs growth
To observe the dynamic of endocytosis, control and cPTIOtreated root hairs were stained with the endocytosis-marker FM4
64 (amphiphilic styryl dye). The dye infiltrates the outer lipid
bilayer of the PM and then moves in a temperature-dependent
The root growing process was studied in the nia1/nia2
Arabidopsis mutant G’4,3. The NO content was analysed in the
Nitric oxide is essential for Arabidopsis root hair growth | 4879
Fig. 3. NO depletion induces abnormalities in root hair
morphology. Arabidopsis Col-0 seedlings were treated with
500 µM of the specific NO scavenger cPTIO. CLSM DIC images
(A) show different forms of root hairs found in cPTIO-treated roots:
sinuous (a), thickness and ramified (b), dichotomously branched
(c), and double hair (d). Bar: 20 µm. (B) Percentages of root hair
phenotypes found in cPTIO-treated Arabidopsis roots.
G’4,3 mutant and compared with Col-0. Eigth-day-old seedlings
were treated with the NO-sensitive fluorophore DAF-FM DA.
Figure 7A and B indicate that the Arabidopsis G’4,3 mutant has
a lower NO concentration than trichoblasts and trichomes from
Col-0, suggesting the involvement of nitrate reductase in NO formation in root hairs. To verify that there is no preferential uptake
of DAF FM by the G’4,3 mutant and/or differential distribution
of the dye in subcellular compartments; seedlings were grown in
a medium supplemented with the NO donor SNAP to observe if
NO-derived fluorescence is ubiquitously located along the root
hair. The results indicate that, in roots hairs treated with 200 µM
SNAP, DAF-FM DA fluorescence observed in the vacuole and
cytoplasm of both the G’4,3 mutant and the Arabidopsis WT (see
Supplementary Fig. S1 at JXB online), is indistinguishable.
Roots were then stained with TBO and observed with LM (Fig.
7C). The G’4,3 root hair length (225.5 ± 69 µm) is, on average,
46.8% lower than Col-0 (480 ± 92 µm). When G’4,3 seedlings
are treated with the NO donor SNAP, the length of the root hair is
restored to the values observed in WT root hairs (432.6 ± 52 µm).
NO participates in the dynamic of endocytic route
Root hairs from G’4,3 loaded with the endocytosis-maker FM4
64 for 5 s show basal fluorescence without red dots in the PM
indicating that vesicle formation is dramatically impaired
(Fig. 8A). Interestingly, the SNAP treatment results in increased
vesicle formation in the G’4,3 mutant, indicating that exogenous
NO application can rescue the mutant phenotype.
Arabidopsis Col-0 seedlings treated or untreated with
500 µµM cPTIO, and Arabidopsis mutant G’4,3 were stained
with FM4 64 for 5 min (long labelling). Long labelling allows
Fig. 4. NO depletion does not affect vacuolar development but
does ER organization. Eigth-day-old Arabidopsis Col-0 seedlings
were treated with 500 µM of the NO scavenger cPTIO (cPTIO) or
were untreated (Control). Root hairs from the growing root hair
region (immature) were observed with TEM (A). On the top (Control
and cPTIO), the insets show a transverse section of Arabidopsis
roots observed with light licroscopy. The photomicrographs show
the organization of the subcellular structures; ec1, epidermal
cell 1; ec2, epidermal cell 2; ER, endoplasmic reticulum; m,
mitochondrion; N, nucleus; n, nucleolus; T, trichome; V, vacuole.
Bars: 10 µm and 0.5 µm (details). (B) The proportion of ER in the
cytoplasm of control and cPTIO-treated root hairs was calculated
and expressed as a percentage.
fluorescence to be followed along the whole endocytic route.
Figure 8B shows that Arabidopsis Col-0 root hairs have uniformly stained cytoplasm. However, the overexposure to FM4
64 results in the agglomeration of vesicles in cPTIO-treated
Col-0 and G’4,3 root hairs, reminiscent of Brefeldin A-formed
vesicles (Fig. 8B) (Ovečka et al., 2005; Kasprowicz et al., 2009).
The agglomerations are caused by alterations of regular vesicle
trafficking.
4880 | Lombardo and Lamattina
Fig. 5. NO depletion does not alter nucleus migration in root
hairs. Eigth-day-old Arabidopsis Col-0 seedlings treated with
500 µM of cPTIO (cPTIO) or untreated (Control) were stained with
the nuclear dye Acetic Orcein and examined by light microscopy
to reveal the position of the nuclei (arrows). I, Immature;
M, mature. Bars: 50 µm.
Cytoplasmic region but not transvacuolar strands is
affected by NO depletion
The effect of NO in the cytoplasmic region was studied.
Movies from the root hairs of cPTIO-treated and untreated
Arabidopsis Col-0 and G’4,3 mutant (see Supplementary
Fig. S2B, A, and C, respectively, at JXB online) were analysed and indicate that the transvacuolar strands are unaltered
when NO is depleted. Figure 9A shows a reduced cytoplasmic
region in both the cPTIO-treated Col-0 and the G’4,3 mutant
compared with the WT. Figure 9B shows that this area was
reduced by 67% in the cPTIO-treated Col-0 and G’4,3 mutant
compared with Col-0.
Discussion
Like all tip-growing cells, root hairs carry out the typical growth
processes characterized by cell wall softening, vacuolar enlargement, vesicle trafficking, nucleus migration in concert with
cytoplasmic streaming in a reverse fountain. In this work, it is
reported that, in Arabidopsis root hairs, some of those processes
rely on an adequate NO concentration.
Vacuolar genesis and nucleus migration seems to proceed
independently of the NO concentration in root hairs. By contrast,
an altered morphology was detected in NO-depleted root hairs.
From bulge to the mature root hair, NO depletion results in the
Fig. 6. NO is required for vesicle formation during root hair
growth. Root hairs from 8-d-old Arabidopsis Col-0 seedlings
treated with 500 µM cPTIO (cPTIO) or untreated (Control) were
loaded with the endocytosis-marker FM4 64 for 5 s. Films were
shot with CLSM. Five pictures from the 2-min movies show the
dynamics of vesicle formation. (A) Control root hair. (B) cPTIOtreated root hair. (C) Vesicle formation was quantified using EZ-C1
3.90 Free Viewer software. Changes in fluorescence intensity
were recorded in a 0.25 µm2 area of the plasma membrane and
quantified as arbitrary units (AU). Arrows denote places in which
vesicle formation are detected. Bar: 5 µm.
vacuole occupying part of the tip region, a reduced cytoplasmic
area, and very limited vesicle trafficking.
Preliminary studies based on pharmacological approaches
showed that microtubules are not involved in positioning the
nucleus but sub-apical fine F-actin between the nucleus and the
Nitric oxide is essential for Arabidopsis root hair growth | 4881
Fig. 7. The Arabidopsis G’4,3 mutant defective in nitrate reductase shows low NO concentration and altered root hair growth. The
Arabidopsis Col-0 and G’4,3 mutant were loaded with DAF-FM DA (A, B). (A) Immature trichomes from Col-0 and G’4,3 observed with
FM, the fluorescence intensity was quantified with ImageJ and expressed in arbitrary units (AU) (right side). (B) Fluorescence of mature
trichomes was registered with CLSM and the intensity was quantified with ImageJ and expressed in AU (right side). (C) G’4,3 root
seedlings were treated with the 200 µM of the NO donor SNAP to analyse the reversion of the mutant phenotype. Root seedlings were
observed with light microscopy. The length of the trichome is represented on the right side (n = 150). Bars: A, B, 20 µm; C, 100 µm.
Figure represents the results of at least three independent experiments.
root hair apex is required for maintaining the nuclear position
with respect to the growing apex (Ketelaar et al., 2002). Villin
is required for nucleate actin polymerization and cross-linking
filaments (Hampton et al., 2008). Villin-like actin-binding proteins are expressed ubiquitously in Arabidopsis (Klahre et al.,
2000). It was shown that the injection of an antibody against
plant villin leads to actin filament unbundling and movement of
the nucleus closer to the apex. Thus, the bundled actin at the
tip side of the nucleus prevents the nucleus from approaching
the apex (Ketelaar et al., 2002). It is shown here that NO is not
required to upset nucleus migration in Arabidopsis root hairs
and, thereby, it is speculated that villin-regulated processes are
not NO-dependent during root hair tip growth.
A large centrally positioned vacuole filled the basal regions
of the Arabidopsis WT growing root hairs, with smaller vacuoles or finger-like extensions of the larger vacuole between the
subapical and basal regions. In Arabidopsis, the RHD3 (Root
Hair Defective 3) gene encodes a putative GTP-binding protein
required for appropriate cell enlargement. The development of
the root hairs in the atrhd3 mutant is similar to the wild-type
regarding the position and migration of nuclei, and as the wild
type, rhd3 hairs emerge at the apical ends of the trichoblasts but
4882 | Lombardo and Lamattina
Fig. 8. NO is necessary for the dynamics of the endocytic route
and vesicle formation in root hairs. (A) Root hairs from 8-d-old
Arabidopsis Col-0, and G’4,3 mutant seedlings grown in the
presence or absence of 200 µM SNAP were loaded with the
endocytosis marker FM4 64 for 5 s and observed with CLSM.
The arrows denote places in which vesicle formation are detected.
(B) The roots from 8-d-old Arabidopsis Col-0 seedlings treated with
500 µM cPTIO (Col-0+cPTIO) or untreated (Col-0), and Arabidopsis
G’4,3 mutant (G’4,3) were loaded with the endocytosis-marker
FM4 64 for 5 min. Overexposure of the root hair to the FM4 64
probe provoked fully fluorescent root hairs due to its internalization.
Arrows indicate the agglomeration of vesicles. Bar: 10 µm.
display a striking reduction in vacuole size and a corresponding
increase in the relative proportion of cytoplasm throughout hair
development. Furthermore, the hairs from the mutant rdh3 differ
in vesicle distribution during hair growth (Galway et al., 1997).
The phenotype of root hairs in the atrhd3 mutant is, in some way,
reminiscent of the cPTIO-treated WT root hairs. However, it is
shown here that NO seems not to be involved in vacuolar genesis
and this suggests that the phenotype generated by NO depletion
does not exactly fit with the rhd3 phenotype.
In this work, it is demonstrated that vesicle formation and
release, as well as the exocytic route are severely affected by NO
depletion in Arabidopsis root hairs. Previous work has shown
that different actin-binding proteins (ABPs) are involved in
these processes, for example, the Arp2/3 complex, profilin, and
endocytic proteins such as clathrin and dynamin (Galleta and
Cooper, 2009). It has been also reported that Rab GTPases play a
role in the trafficking of vesicles during tip growth (Preuss et al.,
2004, 2006; de Graaf et al., 2005). The endocytic marker FM4 64
shows that vesicle formation is not dynamic in the PM of cPTIOtreated Arabidopsis root hairs, indicating that vesicle release
is affected. In animal cells, NO regulates endocytosis through
S-nitrosylation of dynamin (Wang et al., 2006). It would be interesting to study if the same type of NO-mediated regulation of
root hair endocytosis occurs through dynamin S-nitrosylation.
Brefeldin A (BFA) is an inhibitor of vesicular trafficking that
prevents vesicle formation in the exocytosis pathway (Robineau
et al., 2000). Ovečka et al. (2005) used BFA to observe the fate
of endocytic vesicles. They showed active endocytosis at the
tip of growing root hairs and further trafficking of the endocytosed membranes through different putative endosomal compartments either back to the PM or towards the tonoplast. Ovečka
et al. (2005) concluded that BFA does not affect the endocytic
route but does inhibit exocytosis. It is demonstrated here that
over-exposure to the FM4 64 probe reveals FM-positive vesicle
agglomeration in root hairs from seedlings grown in the absence
of low concentrations of NO. The NO depletion provoked BFAlike bodies in Arabidopsis root hairs indicating that the exocytic
route is probably affected. ABPs are responsible for new F-actin
strand formation or branching, active processes in both endocytosis and exocytosis. It is suggested that specific ABPs involved
in exocytosis, as those required in vesicle cover or transport,
should be affected by NO depletion.
Our results indicate that transvacuolar strands are not affected
by NO depletion. Villin is a calcium-controlled actin-modulating
protein (Hesterberg and Weber, 1983). At low calcium concentrations, villin is an F-actin nucleating and cross-linking protein
(Hampton et al., 2008). Villin is responsible of F-actin binding
which produces transvacuolar strands and permit root hairs to
generate the characteristic reverse streaming (Tominaga et al.,
2000). A high Ca2+ concentration in the root hair tip is sufficient
to inhibit villin (Yokota et al., 2000; Hepler et al., 2001) and to
induce actin cytoskeleton disorganization at the apex. It is suggested that NO depletion keeps the Ca2+ concentration and gradient compatible with the occurrence of active villin and preserves
transvacuolar strands at a low level.
In this work, it is shown that NO is present in trichoblasts
and trichomes; it is localized inside the vacuole in immature
root hairs and in the cytosol when the root hair become mature.
Granstedt and Huffaker (1982) reported the vacuole as a major
nitrate/nitrite storage pool. Nitrate and nitrite are NO precursors
at lower pHs like those found in vacuoles (Stöhr and Ullrich,
2002). Thus, it can be speculated that vacuoles modify nitrate/
nitrite accumulation and/or pH regulation for controlling NO
generation during the root-hair-growing process. In addition,
nitrate and nitrite are also substrates for NR activity leading to NO
generation (Yamasaki et al., 1999). Interestingly, Stöhr (2007)
has proposed the relevance of a nitrite:NO reductase (NI-NOR)
activity that is bound to root-specific plasma membrane, and that
the main pathway of NO formation in roots is nitrite-dependent.
NO could also be associated with the regulation of the oscillating
growth of the root hair as described by Monshausen et al. (2007).
Overall, NO production and its intracellular localization seem to
Nitric oxide is essential for Arabidopsis root hair growth | 4883
Fig. 9. NO depletion affects cytoplasmic tip region of root hairs. Root hairs from the growing region of 8-d-old Arabidopsis Col-0
seedlings treated with 500 µM cPTIO (Col-0+cPTIO) or untreated (Col-0), and the Arabidopsis G’4,3 mutant (G’4,3) were observed
with CLSM. (A) DIC images showing transvacuolar currents (arrows) and the cytoplasmic area (arrowheads). Bars: 10 µm. (B) Length of
cytoplasmic area.
be tightly linked to the root hair growing process and arrest when
attaining its mature size.
Supplementary data
Supplementary data can be found at JXB online.
Supplementary Fig. S1. The fluorescent probe DAF FM DA is
homogenously distributed in root hairs treated with 200 µM of
the NO donor SNAP.
Supplementary Fig. S2. Transvacuolar strands are not affected
by NO depletion.
Acknowledgements
We thank Dr Beatriz G Galati for her kind help with the observations of TEM photographs and Dr Laura de la Canal for providing us with the fluorescent probe FM4 64. This work was
supported by the Universidad Nacional de Mar del Plata, Consejo
Nacional de Investigaciones Científicas y Técnicas (CONICET)
and Agencia Nacional de Promoción Científica y Tecnológica
(FONCyT).
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