1 Instituto Gulbenkian de Ciência, PT-2780-156 Oeiras, Portugal
2 University of Missouri-Rolla, Department of Biological Sciences, 105 Schrenk
Hall, 1870 Miner Circle, Rolla, MO 65409, USA
3 Centro de Biotecnologia Vegetal, Faculdade de Ciências, Universidad de
Lisboa, Campo Grande, Ed.C2. PT-1749-016 Lisboa, Portugal
* Author for correspondence (e-mail: jose.feijo{at}fc.ul.pt)
Accepted 26 February 2004
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SUMMARY |
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Key words: Pollen, NO, cGMP, Peroxisome, Guidance, Arabidopsis
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Introduction |
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However, given the biological relevance of fertilization, it is plausible
that evolution has created functional redundancy or co-functionality for
different molecules. In fact, theoretical arguments have been raised that a
single chemical gradient could hardly be responsible for guidance in most
species, which led Lush et al. (Lush et
al., 1998) to propose mechanical/structural stringencies as
co-operative mechanisms in the guidance of pollen tubes. Classical experiments
show that directionality of growth along the pistil/ovary can in principle
occur in more than one direction, restricting the guidance cue necessity to
just a few crucial steps along the pollen tube path and overruling positive
single molecule chemotropism as the sole mechanism of guidance (reviewed by
Heslop-Harrison, 1987
;
Mascarenhas, 1993
; Lord and
Russel, 2002).
In a effort to bridge the gap between in vitro and in vivo experiments of
pollen tube growth manipulation, our attention was drawn to NO as a possible
communication molecule in this system on the basis of a number of well-known
characteristics derived from studies in animals (reviewed by
Ignarro, 2000;
Stamler, 1994
): (1) NO
diffuses freely across cell membranes; (2) it is known to act as an intra and
inter-cellular messenger in a number of regulation mechanisms; (3) it is known
to act as positional cue diffusing from point sources; and (because it is a
gas) (4) it acts on minimal thresholds over considerable distances. In plants,
NO has been proposed as a regulator of growth and developmental processes
(Lamattina et al., 2003
), as
exemplified in roots, where NO mediates the response to indole acetic acid
during adventitious root formation
(Pagnussat et al., 2003
), in
senescence by downregulating ethylene emission
(Leshem et al., 1998
) and
through the stimulation of seed germination
(Beligni and Lamattina, 2000
).
NO also promotes adaptive responses against drought stress operating
downstream from ABA (Mata and Lamattina,
2001
), and it has been implicated in the establishment of legume
Rhyzobium symbiosis
(Hérouart et al.,
2002
). In plant disease resistance, NO plays a role by enhancing
the induction of hypersensitive response
(Delledonne et al., 1998
;
Durner et al., 1998
;
Huang et al., 2002
).
We present data to indicate that NO can function as a pollen tube growth modulator by inducing growth re-orientation, the crucial cellular response to pollen navigation on the pistil. Pollen tubes respond to threshold concentrations of NO by sharp re-orientation, and this reaction is totally abrogated by adding the NO scavenger CPTIO to the medium. Furthermore, we provide data to indicate that this response is mediated through a cGMP pathway, and that NO is primarily synthesized in peroxisomes. On the basis of these data, we propose an NO-based regulatory growth mechanism that could account for the basic curvature needed for ovule targeting by pollen tubes.
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Materials and methods |
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NO imaging
The presence of NO in pollen tubes was assayed and visualized with 10 µM
4,5-diaminofluorescein diacetate (DAF-2DA, Molecular Probes). Pollen tubes
longer than 200 µm were grown in a glass coverslip coated with 0.01% PLL
(poly-l-lysine hydrobromide, Mr 331; Sigma), incubated for
5 minutes and perfused to wash excess fluorophore. Imaging was carried out
using confocal (488 nm) or two-photon excitation (890 nm) on a BioRad
MRC1024MP with a Coherent Mira/Verdi Ti-Sa laser, using a Nikon PlanFluo NA1.3
lens. Emission was collected with a 522DF35 filter. Images were processed with
Metamorph (MM; Universal Imaging Corporation, v. 6.1). Kymographs were
produced by averaging pixel intensity along a linescan of the whole pollen
tube at each time point. Pollen tube length is represented on the horizontal
axis of the kymograph and time on the vertical axis. The tip boundary was
aligned on the right side of the kymograph by applying a custom-made journal
under MM.
NO flux measurements
Carbon fiber microelectrodes were built and operated as previously
described for NO flux measurements (Cahill
and Wightman, 1995; Friedman
et al., 1996
; Porterfield et
al., 2001
). Electrodes were polarized for NO detection at +9.0 V
(versus Ag/AgCl half cell connected to the solution by a 0.5% agarose/3 M KCl
bridge) and calibrated by dilution of a standard 2 mM NO solution
(Gevantman, 1995
). To
characterize the NO gradients created by SNAP, an artificial NO source (aNOs)
was immersed in medium and allowed to reach equilibrium. The self-referencing
polarographic NO vibrating-electrode was stepped linearly from the aNOs tip at
10 µm intervals and NO fluxes measured at each point. The diffusion of NO
is described by Fick's Law (J=D
C/
r), where J is expressed
as pmol cm2 s1, D is the diffusion
coefficient for NO (2.6x105 cm2
s1),
C is the concentration difference between two
electrode positions and
r is the excursion path of the electrode (10
µm). The conversion of the electrode signal to a concentration differential
followed a previously established protocol
(Porterfield and Smith, 2000
).
Data acquisition, processing and control of electrode movements were
accomplished using a 3D stepper micropositioner and amplifier
(www.applicableelectronics.com)
controlled by ASET software
(www.ScienceWares.com)
(Shipley and Feijó,
1999
).
Subcellular characterization
Pollen tubes loaded with DAF2-DA were co-incubated with specific probes for
mitochondria (Rhodamine 1,2,3; 10 µM, Molecular Probes), acidic organelles
(LysotrackerRed, 100 µM, Molecular Probes) and Golgi (Bodipy-TR, 1 µM,
Molecular Probes) and observed by confocal microscopy. Peroxisomes were imaged
by transient expression of an ECFP-Peroxi construct (6931-1, Clontech). This
vector contains a fusion between ECFP and the peroxisomal targeting signal 1
(PTS1), which was extracted and cloned into a construct containing the LAT52
pollen-specific promoter (Twell et al.,
1990) in a pBluescript SKII vector
(Chen et al., 2002
). Tungsten
particles (1.1 µm, BioRad, Hercules, CA) were coated with this construct
and bombarded into Lilium pollen using the biolistic PDS-1000/He
system (BioRad). After bombardment, pollen was germinated in coverslip-bottom
Petri dishes coated with 0.01% poly-l-lysisine hydrobromide,
Mr 331 and imaged 10 hour after germination with a Leica
Confocal microscope TC-SP2/AOBS (excitation at 458 nm to a spectral gate
ranging from 469 to 500 nm).
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Results |
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cGMP mediates pollen tube re-orientation response
The re-orientation of the growth axis in the presence of an external
gradient lead us to further investigate the existence of downstream messengers
of NO. In animal cells, NO effects can be mediated by cGMP-independent
signaling pathways (Ignarro,
2000), and it is well established that this second messenger
conveys NO signaling in a number of physiological conditions. cGMP levels are
modulated by NO in animal cells, and equilibrium concentrations of cGMP are
dependent on NO-activated guanylate cyclases (GC) and breakdown activity of
phospodiesterases (PDEs). Although these enzymes have not been well
characterized in plants, we tested the effects of a number of described
effectors of its activity: IBMX, a general inhibitor of the PDE family, and
sildenafil citrate (SC; VIAGRATM), a drug that inhibits cGMP degrading
phosphodiesterases (PDE5 and PDE6) in mammals
(Corbin and Francis, 1999
). The
use of IBMX at different concentrations promoted the occurrence of diverse tip
abnormal or subnormal morphologies, pointing to the occurrence of pleiotropic
effects (data not shown). Although the drug clearly disrupted growth
regulation, the pleiotropic responses made it difficult to isolate or test its
specificity to the cGMP hypothesis.
SC, however, has recently been shown to delay flower senescence
(Leshem, 2000). If the
re-orientation response of pollen tubes to exogenous NO involves cGMP and if
SC inhibited cGMP degradation in plants, the drug should sensitize pollen
tubes to NO and/or prolong the re-orientation response. To test this
possibility we exposed pollen tubes to suboptimal doses of SNAP. The criterion
to validate this experiment was the detection of pollen tubes insensitive to a
less concentrated NO source. Thus, pollen tubes that were previously shown not
to re-orient its growth axis on a lower SNAP concentration were submitted to a
SC final concentration of 339 µM. Under these conditions, eight out of nine
pollen tubes (n=9) showed re-orientation after addition of SC, and
some even re-oriented to previously never observed angles of 180°
(Fig. 1D). We further confirmed
this result by measuring an increase of the average re-orientation angle to
120° ±12 (n=9). This angle is 25% steeper than the control
but, more significantly, is obtained with much lower concentrations of NO.
Dose-response curves of SC also showed a dose-dependent stimulatory effect of
30% on the growth rate of pollen tube at 50 µM. This finding suggests
that the effect of exogenous NO on pollen tube growth is mediated by cGMP, and
that this second messenger is in the signaling cascade that affects the growth
regulation mechanism.
Nitric oxide is produced in peroxisomes
Once a re-orientation response takes place, we asked whether this means
that the extracellular NO challenge induces this response by modulating the
intracellular NO levels. To address this question, we searched to see if NO
was endogenously produced in pollen tube. Live cells were loaded with the NO
sensitive fluorophore, DAF2-DA (4,5-diaminofluorescein diacetate) and imaged
by confocal or two-photon microscopy (Fig.
2). This probe was previously shown to be NO-specific in plant
tissues (Foissner et al.,
2000). Fluorescence was found throughout the cytosol, although in
the region subjacent to the tip it was very low
(Fig. 2, 1'). A very
strong signal was found in round organelles of about 2 µm diameter
(Fig. 2, 1'). The
spatiotemporal dynamics of intracellular NO (iNO) are shown in the
form of kymographs in which we averaged an active representative region inside
each pollen tube at each time-point as a color-coded line, and plotted these
lines as a function of time (YY' axis) and pollen tube length (XX'
axis) (Fig. 2). For the sake of
clarity, the pollen tube tips were right-side aligned, and therefore the slope
on the left side of the kymograph reflects the pollen tube growth rate. In
non-challenged pollen tubes, no significant variation over time is seen. NO
levels are very low in the tip and highest in the subapical domain (control;
Fig. 2). To validate the
specificity of the observed signal, the NO scavenger CPTIO
[2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide] was
applied in continuous perfusion. The fluorescence signal was always reduced in
tubes that were perfused with CPTIO. Typically, cytosolic NO was almost
completely eliminated, confirming the presence of NO inside pollen tubes and
the specificity of the probe. Even though the cytosolic NO signal decayed, the
round organelles still showed a clear signal even after 44 minutes, suggesting
that they continued to generate NO at a high rate
(Fig. 2, 44' and lower
kymograph). These brightly fluorescent organelles visible after DAF2-DA
exposure have a diameter of about 2 µm (2.17±0.17). We performed
double-labeling experiments using different organelle-specific probes and
DAF-2DA probe to determine their identity. Double labeling with DAF2-DA
(green) and selective, color complementing dyes (red) for Golgi and ER
(Bodipy-TR, Fig. 3A),
mitochondria (rhodamine 123, Fig.
3B) and acidic organelles (Lysotracker,
Fig. 3C) showed no
co-localization. As peroxisomes and plastids are still within this size range,
we transiently transformed pollen tubes with a peroxisomal targeting signal
(PxTS) fused to ECFP under the control of the pollen-specific promoter LAT52.
LAT52 is a very late-acting promoter in monocots, and thus we imaged pollen
tube 10 hours after germination. The double labeling with ECFP and DAF2-DA
co-localized to the same organelles (Fig.
3D) and morphometric analysis (diameter and circularity index)
indicated that the double-stained organelles did not include plastids. These
results identify peroxisomes as the NO-producing organelles.
|
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We also monitored the NO levels after perfusion with CPTIO (Fig. 4E-G). Challenge with the NO-source provoked a slight increase in NO but immediately after, it decreased progressively to levels bellow the initial basal concentration. This level then remained constant (inserted kymograph). Under these conditions, no re-orientation took place, and growth rate slowly recovered. Together these results show a direct relationship between iNO and the regulation of cell growth and re-orientation.
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Discussion |
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We also show that NO is synthesized in the peroxisomes, a hypothesis
previously proposed in plants (Barroso et
al., 1999; del Rio et al.,
2002
).
Our findings indicate that NO is a negative regulator of growth, at least
in pollen tubes. We show that endogenously generated NO is low or absent at
the tip of pollen tube, but is present at higher levels behind the tip, where
the presumed NO-generating peroxisomes are located. We suggest that this
pattern of NO production is permissive for pollen tube growth at the tip and
may, in the absence of an exogenous NO source, act as a positive feedback
reinforcement of elongation in a straight growth axis. The ubiquitous presence
of NO in the medium, by addition of SNAP, prevents germination and inhibits
pollen tube growth rate. However, pointed application of an NO donor near the
pollen tube tip results in transient growth arrest, which is followed by
re-directed growth. This effect is mediated by NO that diffuses into the tip.
Growth arrest is then associated with changes in cell polarity and peroxisome
redistribution, as shown by the extension of the streaming lanes into the tip.
We assume that it is the localization of peroxisomes and thus the site of
endogenous NO production, which eventually determines the direction into which
pollen tube growth resumes. Peroxisomes are the plant cell oxidative
organelles and there are reports that highlight the relevance of this
organelles in the production of signal molecules, i.e. NO and reactive oxygen
species. These organelles have a rich enzymatic machinery and are reported to
participate in developmental processes such as photomorphogenesis in
Arabidopsis (Hu et al.,
2002; Barroso et al.,
1999
; del Rio et al.,
2002
).
Although our data show that endogenous NO production is correlated with the
regulation of pollen tube growth, the in vivo confirmation of this is made
difficult by various experimental obstacles. Real-time imaging of pollen tube
guidance in vivo would imply the possibility of optical sectioning closed
flowers, which implies demanding technical conditions (two-photon excitation
and water-immersion, long working distance objectives) far from optimized for
this specific application. Excitation-derived photo-damage of cells or, more
dramatically, any sort of ovary dissection or injury of the ovary is not an
option because it will generate stress-induced bursts of NO production
(Lamattina et al., 2003). A
possibility for overcoming these obstacles will be either the use of pollen
tubes expressing highly fluorescent reporter genes to closely monitor the
pollen tube-pistil interaction, or, otherwise, the use of floral mutants with
open ovaries and exposed, yet functional, transmitting tissue and ovules.
Another problem is related to the high reactivity of NO, with an half life
depending on the redox status of the surrounding environment, namely when ROS
are present (Ignarro, 2000;
Thomas et al., 2001
). This
makes it difficult to gauge the amount of NO being produced in vivo, so no
invasive techniques for NO can easily quantify a putative signal from the
female tissue. A self-referencing NO selective electrode could be used, but
again tissue accessibility is a limiting factor.
Several difficulties arise when interpreting chemical cues identified in
different plant species: it can be argued that general mechanisms do not
assure species specificity to avoid widespread cross-fertilization
(Johnson and Preuss, 2002).
One possible explanation could be related to different threshold sensitivities
operating for a given molecule from species to species. Otherwise a different
species could use similar mechanism, but with derivatised molecules within a
single chemical family, which would be transduced into different effects.
Given the diversity of molecules shown to have guidance effects on pollen
tubes, and predicting that more will be uncovered through successive genetic
screens, it is likely that chemical signaling between the pollen tube and
pistil could convey specificity by using universal molecules in various
combinations.
This finding indicates the need for further cues from the pistil. Whether or not NO takes part in communication cannot be deduced from in vitro studies, but the striking re-orientation response warrants for further investigation. In the context of our data, a feasible NO-guidance mechanism would be possible if there were specialized female tissues acting as NO `hot spots', for example, at the base of the funiculus, where a sharp change in pollen tube growth direction is required, or near the embryo sac after fertilization, in order to prevent secondary pollen tube from penetrating the micropyle. The indication that nos1, the only bona fide NO-producing mutant so far described, shows fertility deficiencies is a positive indication that NO may be involved in pollen tube guidance.
In past research, pollen tube guidance could not be fully explained by the
actions of positive guidance cues. In addition, it remains debatable that
tracking down a molecule will overcome questions related to pollen tube path
length and thickness (Lush et al.,
1998). Yet, a gaseous molecule may overcome these barriers easily.
In proposing NO, a diffusible gas, as a candidate for pollen tube guidance, we
may address a controversial aspect of pollen tube guidance. In
Arabidopsis, Hülskamp et al.
(Hülskamp et al., 1995
)
propose that each ovule guides the pollen tube by chemotatic gradients with
100 µm range of action at the junction of the ovule with the placenta.
However, wild-type Arabidopsis pollen tubes make a sharp turn to
enter the mycropyle in 10 µm of this area
(Shimizu and Okada, 2000
).
Surface localized diffusion of chemotatic signals effective through 50 cells
diameters would require signal molecules of less than one kDa
(Ray et al., 1997
;
Crick, 1970
). The ability of NO
to function as a messenger across cell layers and to trigger cellular
processes is nowadays well established in animals
(Ignarro, 2000
). The negative
chemotropism described here for NO is reminiscent of the effects of
semaphorins on axon guidance in animals: these proteins function as
chemorepellents, which prompt axons to make right angle turns within an
environment that contains both attractants and repellents
(Tessier-Lavigne and Goodman,
1996
). Similarly, NO acts as negative effector on the retinal
patterning of the optical lobe in Drosophila, where NO prevents
further extension of axons beyond their target neurons
(Gibbs and Truman, 1998
). NO
function as a guidance cue implies that (1) it is be able to form a
concentration gradient, (2) it produces a specific response, (3) it remains
stable for a given period of time, and (4) it varies in effectiveness with
distance to the target (Palanivelu and
Preuss, 2000
). Our in vitro data support these criteria. We were
able to detect an artificially generated external NO gradient to which the
pollen tubes respond in a specific way (re-orientation growth axis), the
gradient is maintained in time as a single pollen tube can undergo consecutive
re-orientation responses with the same NO source. In addition, the response
can be prevented if the gradient is perturbed or annihilated by an NO
scavenger.
The events downstream of NO seem to be, at least in part, mediated by cGMP.
Support for this assertion comes from our finding that, among other tested
chemicals, sildenafil citrate, a drug that inhibits cGMP-selective PDEs of
mammals, facilitated the redirected growth of pollen tubes in response to low
doses of NO donors that were themselves ineffective. Previous studies with
cyclic nucleotide analogues also suggest that cGMP and cAMP are involved in
pollen tube growth control (Moutinho et
al., 2001; Elias et al.,
2001
). A likely target downstream of cGMP is a family of cyclic
nucleotide-gated channels (CNGs) (Leng et
al., 1999
), also represented in the pollen transcriptome
(Becker et al., 2003
). Directly
or coupled with other transporters, CNGs may regulate the flux of ions such as
Ca2+, H+ and Cl that are known to be involved in pollen
tube growth control (Feijó et al.,
2001
; Becker et al.,
2003
; Feijó et al.,
1999
; Zonia et al.,
2002
). Cyclic nucleotide balance, modulation of Ca2+
channels and the control of nerve growth bi-directional axon guidance have
recently been linked (Nishyama et al., 2003). These findings encourage further
efforts to characterize the various components of the NO signal pathway in
plants and to endue in genetic and biochemical approaches.
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ACKNOWLEDGMENTS |
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Footnotes |
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