Molecular Plant • Volume 1 • Number 4 • Pages 703–714 • July 2008 RESEARCH ARTICLE

Targeting of Pollen Tubes to Ovules Is Dependenton Nitric Oxide (NO) Signaling

Ana Margarida Pradoa, Renato Colacxoa, Nuno Morenoa, Ana Catarina Silvaa and Jose A. Feijoa,b,1

a Instituto Gulbenkian de Ciencia, Centro de Biologia do Desenvolvimento, PT-2780–156 Oeiras, Portugalb Universidade de Lisboa, Faculdade de Ciencias, Dept Biologia Vegetal, Campo Grande, Ed.C2. PT-1749–016 Lisboa, Portugal

ABSTRACT The guidance signals that drive pollen tube navigation inside the pistil and micropyle targeting are still, to

a great extent, unknown. Previous studies in vitro showed that nitric oxide (NO) works as a negative chemotropic cue for

pollen tube growth in lily (Lilium longiflorum). Furthermore, Arabidopsis thaliana Atnos1 mutant plants, which show de-

fective NO production, have reduced fertility. Here, we focus in the role of NO in the process of pollen–pistil communi-

cation, using Arabidopsis in-vivo and lily semi-vivo assays. Cross-pollination between wild-type and Atnos1 plants shows

that the mutation affects the pistil tissues in a way that is compatible with abnormal pollen tube guidance. Moreover, DAF-

2DA staining for NO in kanadi floral mutants showed the presence of NO in an asymmetric restricted area around the

micropyle. The pollen–pistil interaction transcriptome indicates a time-course-specific modulation of transcripts of AtNOS1

and two Nitrate Reductases (nr1 and nr2), which collectively are thought to trigger a putative NO signaling pathway. Semi-

vivo assays with isolated ovules and lily pollen further showed that NO is necessary for micropyle targeting to occur. This

evidence is supported by CPTIO treatment with subsequent formation of balloon tips in pollen tubes facing ovules. Ac-

tivation of calcium influx in pollen tubes partially rescued normal pollen tube morphology, suggesting that this pathway is

also dependent on Ca21 signaling. A role of NO in modulating Ca21 signaling was further substantiated by direct imaging

the cytosolic free Ca21 concentration during NO-induced re-orientation, where two peaks of Ca21 occur—one during the

slowdown/stop response, the second during re-orientation and growth resumption. Taken together, these results provide

evidence for the participation of NO signaling events during pollen–pistil interaction. Of special relevance, NO seems to

directly affect the targeting of pollen tubes to the ovule’s micropyle by modulating the action of its diffusible factors.

Key words: pollen tubes; NO; fertilization; guidance; calcium signaling.

INTRODUCTION

Despite recent advances, the way by which pollen tubes find

their way into the micropyle and, generally speaking, the mech-

anismsbywhichpollentubeguidance is achievedwithinthepis-

til are still, to a great extent, unknown (Boavida et al., 2005;

Marton and Dresselhaus, 2008; Higashyiama and Hamamura,

2008). Several mutagenesis studies point to the existence of

genes associated with long and short-range chemotropic cues

that drive pollen tube–pistil communication (Palanivelu and

Preuss, 2006). But simple molecules of physiological relevance

have also been systematically associated with guidance, namely

nitric oxide (NO), which, in previous studies, we found to be

produced in pollen, and to act as a negative chemotropic agent

to pollen tube growth (Prado et al., 2004; Feijo et al., 2004).

Of relevance, NO is the only chemotropic agent so far described

capableofpromotingturninganglesof90�andmore,neededto

achievesomestepsofthefertilizationprocess.Theimplicationof

NO in fertilization has recently gained support also in Senecio

squalidus and Arabidopsis thaliana (McInnis et al., 2006).

Various mechanistic cues for guidance have been proposed

over the years, namely the ones involving the intimate contact

with the components of the extracellular matrix of the trans-

mitting tract (Cheung et al., 1995; Wheeller et al., 2001). In lily

(Lilium longiflorum), for instance, the stylar pectin and stylar

cysteine-rich adhesin are binding partners promoting in-vivo

adhesion between pollen tube and style (Jauh et al., 1997),

and a chemocyanin was identified as a new chemotropic mol-

ecule in the stigma (Kim et al., 2003). In Nicotiana, the pistil TTS

proteins are incorporated in the pollen tube wall and tip, but

also show an apparent gradient of glycosilation, reaching from

the stigma to the ovary (Wang et al., 1993; Cheung et al., 1995;

1 To whom correspondence should be addressed. E-mail jose.feijo@fc.ul.pt.

ª The Author 2008. Published by the Molecular Plant Shanghai Editorial

Office in association with Oxford University Press on behalf of CSPP and

IPPE, SIBS, CAS.

doi: 10.1093/mp/ssn034, Advance Access publication 27 June 2008

Received 5 March 2008; accepted 23 April 2008

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Wu et al., 1995). On the other hand, the role of female sporo-

phytic tissues and embryo sac in long-range guidance mecha-

nisms are highlighted in the analysis of Arabidopsis ovule

mutant studies. One such example is the evidence for an

ovule-derived guidance cue with long-range activity in com-

parison with mutant ovules incapable of proper guidance

(Hulskamp et al., 1995). The participation of ovule sporophytic

cells for successful pollen tube and pistil communication was re-

cently shown (Ray et al., 1997). Another example is the impaired

funicular guidance in the Arabidopsis mutant inner-no-outer

(ino), in which ovules are deprived of the outer integument

and cannot be fertilized despite the functional embryo sac

(Baker et al., 1997; Sawnson et al., 2004). In another screen

to detect defective ovule targeting, the pollen pistil interac-

tion 2 (pop2) mutant was characterized as having pollen

tubes with random growth throughout the ovary (Whilhemi

and Preuss, 1996). pop2 was later identified to encode

a transaminase that degrades c-amino butyric acid (GABA),

thus hypothetically forming a gradient in the pistil to pro-

mote growth, suggestive of a putative guidance mechanism

for Arabidopsis pollen tubes in vivo (Palanivelu et al., 2003;

Swanson et al., 2004). Finally, in thegametophyticmagatama1

and 3 (maa) mutants, which show delayed female gametophytic

development, pollen tubes are directed to the ovule but show

impaired short-range micropyle targeting. In these gameto-

phytic mutants, sometimes two pollen tubes seem to be simulta-

neously attracted by the ovule (Shimizu and Okada, 2000;

reviewed by Franklin-Tong, 2002).

Introduction of Torenia as an experimental model turned

out to be a significant breakthrough in the field (review in

Higashiyama and Hamamura, 2008). Of relevance, laser abla-

tion of synergids indicates that these cells secrete a guidance

cue that directs pollen tube targeting to the ovules over

100–200 lm (Higashiyama et al., 2003). Moreover, semi-vivo

inter-specific crosses between closely related species of the

genera Torenia show that the attraction of the pollen tube

to the embryo sac was impaired when the elongating cell ar-

rived close to the embryo sac, indicating that species preferen-

tiality to the guidance cue may serve as a reproductive barrier

in the final step of directional control of the pollen tube

(Higashiyama et al., 2006). The ZmE1 protein was also recently

identified as a maize-specific micropyle guidance protein, se-

creted by the egg apparatus and shown to have a direct role on

close-range pollen tube guidance (Marton et al., 2005).

The NO hypothesis was indirectly brought into board again

when Boisson-Bernier et al. (2008) showed that a peroxin loss-

of-function mutation (amc, abstinence by mutual consent) in

Arabidopsis affects peroxisomal protein import in both pollen

and ovules but, more importantly, does not affect pollen tube

growth or guidance, but prevents pollen tube reception from

happening, producing a self-sterile mutant. When both the

pollen tube and the ovule carry the amc mutation (amc/+), pol-

len reception is substituted by continued growth of the pollen

tube, which cancoil or branch. Theamc/– ovulecannevertheless

attract several pollen tubes. This paper shows for the first time

that functional peroxisomes must be present in either the male

or the female gametophyte for pollen tube guidance to work.

Interestingly, we had previously found that peroxisomes seem

to be the main source of NO in pollen tubes (Prado et al., 2004).

Several difficulties arise when interpreting chemotropic

cues identified in different plant species, even when compared

to similar systems in other kingdoms (Marton and Dresselhaus,

2008). 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. Given the diversity

of molecules shown to have guidance effects on pollen tubes,

and predicting that more will be uncovered through successive

genetic and physiological screens, it seems conceivable that

chemical signaling between the pollen tube and pistil could

convey specificity by using specific transduction events result-

ing from various combinations of universal molecules and

specifically secreted ones (Prado et al., 2004).

In our past research, we have shown that NO acts as a neg-

ative chemotropic cue in pollen tube guidance (Prado et al.,

2004). Simultaneously, the Atnos1 mutant, which was proven

to be defective in NO production, and was initially thought to

encode for a distinct nitric oxide synthase, was shown to have

reduced fertility (Guo et al., 2003). Both these forms of evi-

dence give ground to argue for a function of NO on in-vivo

guidance. Despite much scepticism based on the absence of

demonstrable genetic mechanisms for its production and

transduction, NO roles in plant biology have been the focus

of numerous approaches. In fact, a growing body of evidence

demonstrates that NO is an important signal in a variety of pro-

cesses, from fundamental development to plant–pathogen

interactions (Lamattina et al., 2003; Mur et al., 2006; Besson-

Bard et al., 2008).

Here, we further investigated the presence of NO in theAra-

bidopsis pistils resorting to Atnos1 and kanadi mutants, the

latter showing ectopic expression of ovules in open ovaries

(Kerstetter et al., 2001). We have also addressed the putative

action of a NO signaling cascade in lily pollen tube targeting.

Our findings suggest the involvement of Ca2+ as a downstream

messenger from NO for targeting to take place—a conclusion

also supported by demonstration of a Ca2+-specific response

during NO-induced pollen tube re-directioning. In this study,

we further provide evidence that NO is involved in pollen

tubes guidance and targeting to the micropyle.

RESULTS

Ovule Targeting Relies on Nitric Oxide Signaling

In previous studies, we showed that NO is able to re-direct

the growth axis of Lilium longiflorum pollen tubes (Prado et al.,

2004), therefore suggesting a role as a guiding cue in pollen–pistil

interaction. Here, we further investigate this relationship.

For this purpose, we started by characterizing in detail the

Atnos1 plants in what regards its phenotype during the

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pollen–pistil communication process. The involvement of NO

in Arabidopsis pollen tube–pistil communication was sug-

gested in the original publication by Guo et al. (2003), who

described the Atnos1 mutant plants as showing reduced fertil-

ity. The authors have isolated a homozygous mutant line with

DNA insertion in the first exon of this gene and confirmed the

mutation by polymerase chain reaction genotyping and se-

quencing. The fact that the phenotype could be rescued by

exogenous applications of an NO source (e.g. SNAP) and

had a phenotype consistent with the pharmacology implicated

in NO signaling led the authors to consider that the gene could

be the long sought NO synthase (hence the name At NOS1).

While the mutant is still generally accepted as being NO defec-

tive, the gene was later shown probably not to be a true NO

synthase, but some downstream regulation protein (Zemojtel

et al., 2006). Therefore, we decided to investigate whether the

low levels of NO in the AtNOS1 mutant could be responsible

for any defects on in-vivo guidance. To evaluate how guidance

or targeting to the ovules was affected in Atnos1 plants, we

proceeded with Atnos1 self- and cross-pollinations with wild

type. We have used aniline-blue staining of the callose pollen

tube cell wall to allow the detection of any abnormal pollen

tube morphology and the visualization of the pollen tube path

through the pistil tissues (Figure 1).

Contrary to wild type (Figure 1A), Atnos1 self-pollination

revealed clear variations and abnormalities to normal tip

growth morphology (Figure 1C and 1D). In contrast, the

cross-pollination between Atnos1 pollen and wild-type pistil

did not show defective pollen tube targeting to the ovules,

or abnormal growth (Figure 1B). This observation did not hold

for the reciprocal crosses. When wild-type pollen tubes grew in

Atnos1 pistil, we could again detect the presence of abnormal

tip morphology (Figure 1E). Of relevance, most of these defor-

mations seem to occur close to the ovules (Figure 1D) or even

the micropyle (Figure 1C and 1E). These data suggest that

pollen tube guidance in Atnos1 mutants is partially affected

at some stage of the pollen tube path along the pistil and that

the mutation affects predominantly the pistil.

As a result of these abnormal growth patterns, and in accor-

dance with the previous data reported by Guo et al. (2003), we

estimated an average 40% reduction in the number.of seeds

on ATNOS1 selfing when compared to wild type, or $ Wt

X #Atnos1. In order to address the question of whether this

phenotype could be directly attributed to the participation

of NO in the success of fertilization, we then tried to pheno-

copy the mutation by treating plants with the NO scavenger,

PTIO. Wild-type Arabidopsis plants were watered with various

pharmacologically active concentrations of PTIO, and seed set

production was scored. Table 1 shows these results, and they

show clearly that, in all concentrations tested, there was inhi-

bition of the seed set. This result is particularly relevant for the

lower concentrations, where no other measurable effect could

be detected or any visible developmental parameters of the

plants seem altered. Of the total of seeds scored (n = 1547),

PTIO caused an average decrease in the seed set close to

50%. Only in concentrations over 1 mM did PTIO start having

other deleterious effects on the development of the plant. The

fact that this treatment phenocopied the characteristic Atnos1

lower seed set phenotype suggests that, in fact, NO depriva-

tion may be responsible for the lower seed set by perturbing

the normal guidance mechanisms.

If NO is needed for guidance, and the AtNOS1 is mainly af-

fecting the female part, then NO should be produced in some

part of the pistil in a structural context compatible with the

phenotype observed. From our observations, the foremost

candidates to produce NO in a regulated fashion should be

the ovules, as we have noticed that pollen tubes show disrup-

ted polarized growth mostly at the micropyle entrance (see

Figure 1C–1E). To test this hypothesis, we decided to apply

the NO-specific dye DAF-2DA, which was previously used with

success in pollen tubes (Prado et al., 2004) and pistils (McInnis

et al., 2006). However, the fact that we were specifically inter-

ested in ovules posed a significant methodological problem, as

it would imply the dissection of the ovaries in normal wild-type

plants. NO being immediately released upon stress conditions,

any such surgery is prone to generate artefacts by lesion, which

would be impossible to control. We circumvented this situa-

tion by resorting to the Arabidopsis mutant kanadi (Kerstetter

et al., 2001). In this mutant, the ovary does not finish its regular

development of closure, the ovules thus remaining exposed

and accessible to image without any manipulation. Further-

more, despite this gross anatomical deficiency, kanadi flowers

are functional and fertile, and therefore the physiological

guidance mechanisms are operating as well (Kerstetter

et al., 2001). In accordance with our prediction, but with re-

markable specificity, the probe fluorescent signal was detected

at the edges of the micropyle (Figure 2A and 2B) in a restricted

and well defined asymmetric set of cells (arrows). This obser-

vation is suggestive of a confinement of pollen tube path and

penetration into the ovule through the zone delimited by the

unlabeled cells, since NO functions as a negative chemotropic

cue in pollen tube growth (Prado et al., 2004).

Finally, we addressed the issue of whether the genes neces-

sary for an NO response were present in the pistil tissue. As

mentioned before, this is a controversial question, as no un-

equivocal NO synthase or transducing mechanism is known

in plants. Yet, on our best judgement of the available litera-

ture, we decided to focus on AtNOS1 (Locus At3g47450),

decidedly involved in NO accumulation, and the Nitrate Reduc-

tase nr1 (Locus At1g77760) and nr2 (Locus At1g37130), be-

cause of their capacity to generate NO from nitrite, which

contributes to the NO-dependent stomatal closure (Bright

et al., 2006) and the fact that they are generally accepted as

the best candidates for an arginine-dependent NO synthesis

(Besson-Bard et al., 2008). In order to understand the possible

involvement of NO signaling pathway triggered in pollen pistil

communication, we analyzed the data gathered by Boavida

et al. (submitted) in which the gene responses during

pollen–pistil interaction were analyzed by transcriptomics at

various time points after fertilization. mRNA levels of these

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genes at different hours after pollination (HAP) as well as in

several tissues is depicted in Figure 2C. The fluctuations ob-

served in transcript levels were statistically analyzed using

Dchip software, which allowed the comparison of expression

levels and the calculation of the lower confidence bound of

the fold change. The lower confidence bound criterion indi-

cates 90% confidence that the fold change is a value between

the lower confidence bound and a variable upper confidence

bound (Pina et al., 2005). The Atnos1 transcript variations

noted in Figure 2C at 0.5, 3, and 8 HAP and unpollinated pistil

were statistically analyzed and did not show any significant

changes. In contrast, however, the transcriptomic data for

nr1 and nr2 show a different picture. Only nr2 shows both

a present call in pollen as well as in pistil transcriptome (Becker

Table 1. Average Seed Set (Avg. 6 SE) of Wild-Type PlantsWatered with Different Dosages of PTIO, a NO Scavenger.

Control 200 lM 500 lM 1 lM

19.0 (6 1.6 SE) 7.4 (6 0.9) 9.4 (6 1.3) 8.8 (6 1.7)

The wild-type seed set was lower in all concentrations, which indicatesa phenocopy of Atnos1 mutant seed set.

Figure 1. Detection of Abnormal Pollen Tube Guidance in Self- and Cross-Pollinations between Atnos1 and Wt Plants.

Fluorescence microscopy images of Wt and Atnos1 pistils in self and cross-pollinations after 6 h. Staining of callose walls with aniline-blue.(A) Wt self-pollinated pistil, arrow pointing to Wt pollen tube targeting to the micropyle. Scale bar: 40 lm.(B) Cross-pollination between $ Wt X #Atnos1, arrow pointing to Atnos1 pollen tubes at the micropyle entrance. Scale bar: 80 lm.(C, D) Atnos1 self-pollinated pistil. (C) Arrow pointing to abnormal pollen tube tip with comb-like shape; (D) arrow pointing to pollen tubethat shows a swollen tip near the micropyle (mp) entrance (white dashed line).(E) Cross-pollination between $ Atnos1 X #Wt arrow pointing to swollen tip of a pollen tube growing on top of an ovule wall. Scale bars:24 lm.

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et al., 2003; Pina et al., 2005; Boavida et al., submitted). The

expression levels of the three transcripts at different HAP were

compared with each other and with unpollinated pistil. Signif-

icant changes were detected above the 1.2 cut-off when com-

paring nr1 and nr2 at 8 HAP versus 0.5 HAP and 8 HAP versus

3 HAP. The comparison between the levels of expression of nr1

at 8 HAP versus 0.5 HAP also showed a negative 1.55-fold

change, while nr2 showed a 1.23-fold change. These variations

revealed that the nr1 transcript is being down-regulated at

8 HAP, while nr2 is up-regulated at 8 HAP. These differences

may be correlated with post-fertilization events, since these

time points were chosen to illustrate the hydration/germina-

tion and the final targeting points, respectively. Moreover,

the comparison between the levels of expression of nr1 and

nr2 at 8 HAP versus 3 HAP showed a negative 1.73 and 1.65

positive fold change, respectively, again showing the down-

regulation of nr1 and up-regulation of nr2 likely associated

with post-fertilization events. Taken together, these results

suggest a need for constant basal NO production through

the AtNOS1-dependent pathway, and a temporal regulated

production by the nitrate reductase pathway.

NO-Mediated Pollen Tube Re-Direction Is Ca2+ and Ovule

Factor-Dependent

The above results suggest a NO dependency of the pollen pistil

interaction, whose physiology may not be trivial, since it would

be dependent on a constant presence of NO, but a regulated

boost at certain temporal stages or structural locations. To

move forward in the understanding of NO as an in-vivo cue

for tube guidance, we have devised a semi-vivo assay, using lily

as an experimental model. A semi-vivo system for Arabidopsis

has also been described (Palanivelu and Preuss, 2006) but, in

our hands, the lily system, besides allowing direct physiological

extrapolation from previous results (Prado et al., 2004), also

worked in a more reliable and robust way. Lily ovules were iso-

lated and placed in an agarose medium adapted from Rosen

(1961), who also studied pollen tube chemotropism in lily. All

ovules were linearly placed so that the micropyle entrance

faced a row of pollen grains, and targeting events were scored.

We defined positive targeting when pollen tubes grow in the

region of the micropyle and/or along the walls of the ovules

and/or entry in the micropyle. Figure 3 shows images of lily

pollen tubes targeting to ovules. In Figure 3A, we can observe

the entry of a pollen tube in the micropyle making a right an-

gle to the point of entrance. The images from aniline-blue

staining also showed some pollen tubes coiled in the micropyle

region and along the side and base of the micropyle. We

scored a total of 270 ovules, of which 19.63% exhibited target-

ing performance.

To test whether NO is involved in lily targeting to ovules, we

added carboxy-PTIO, a non-cell-permeable NO scavenger, to

the medium. This scavenger allows not only the elimination

Figure 2. Identification of Putative NO Production Spots in Restricted Areas of the Micropyle of Arabidopsis Floral Mutants.

(A, B) Fluorescence microscopy images of Arabidopsis floral mutant kannadi. (A) and (B) show exposed ovules labeled with DAF-2DA at themicropyle entrance area. The DAF-2DA signal probes for NO presence. Labeling is observed in both images, being restricted to a cell layer inthe immediate vicinity of the micropyle entrance area. Dashed white line shows contour of ovules.(C) Graph shows the variation of levels of expression of Atnos1 (Arabidopsis thaliana nitric oxide synthase 1), nr1, and nr2 (nitrate reductase1 and 2) transcripts in different tissues namely the pistil at different hours after pollination (HAP).

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of external NO in the preparation, but also a decrease in the

intracellular levels of NO in lily pollen tubes, as previously

shown (Prado et al., 2004). Surprisingly, an unusual behavior

was observed as a result of this assay: pollen tubes growing

from the row of pollen in the direction of the ovules (the

top half of the semi-vivo preparation) showed balloon-like

tips, whereas the inferior half exhibited normal polarized pol-

len tube growth. Figures 3D and 3E show, respectively, pollen

tube tip behavior in the superior and inferior halves of

the semi-vivo preparation (arrows pointing at dilated, de-

polarized tips). Figure 3F illustrates the division in superior

and inferior halves of the preparation as we describe it in

the presence of the NO scavenger (see table 2 for quantifica-

tion). The loss of polarized growth by pollen tubes is long

known to result from perturbation of the intracellular Ca2+

gradient at the tip. Disruption of the Ca2+ gradient results

in the cessation of pollen tube growth, which can be accom-

panied by alterations in pollen tube tip morphology (Pierson

et al., 1994; Holdaway-Clarke and Hepler, 2003). Nevertheless,

the molecular entity responsible for the Ca2+ influx at the

Figure 3. Lilium Pollen Tube Targeting to the Ovules.

(A–C) Lilium longiflorum pollen tubes targeting the micropyle of Lilium isolated ovules in semi-vivo preparations. (B) and (C) pollen tube cellwall stained with aniline-blue. (B) The pollen tube curls near the micropyle region. (C) Pollen tube making a curve along the base of themicropyle region. Scale bars: 75 lm.(D, E) Targeting abrogation and pollen tube growth, respectively. (D) and (E) show the superior and inferior halves of the semi-vivo prep-aration where isolated ovules and pollen tubes are under the presence of CPTIO, a NO scavenger, in the medium. (D) Abrogation of target-ing by pollen tube showing a balloon tip in the proximity of the micropyle region. (E) Pollen tubes growing in the inferior half of the semi-vivo preparation, which is deprived of ovules.(F) Schematic diagram of the semi-vivo preparation to illustrate that the line of pollen divides the preparation into two halves (frontierbetween halves is represented by a green line). The top half presents isolated ovules (represented in green) that are aligned with themicropyle facing the row of pollen. Growing pollen tubes are represented by black lines and dots. Black dots represent the balloon pollentube tips.(G) Graph showing pollen tube tip morphological differences and targeting events (% 6 SE) in semi-vivo preparations treated with CPTIO,D-Ser and CPTIO + D-Ser. In all treatments with D-Ser, a decrease in the exploded and balloon tips as well as the promotion of targeting wereobserved.

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pollen tube tip remains elusive. In plant cells, the mechanism

through which Ca2+ entry is possible is still a matter of research

and debate, with few (if any) truly multidisciplinary conclusive

studies. Of relevance, Qi et al. (2006) described an Arabidopsis

glutamate receptor (GR) that mediates Ca2+ entry with a broad

agonist activity contemplating Glu, Gly, Ser, Ala, Asn, and Cys

(Qi et al., 2006). D-Ser has also been shown to produce a rise in

Ca2+ influx in tobacco pollen tubes (Michard and Feijo, in prep-

aration). In our study, the observation of loss of polarized

growth with concomitant formation of a balloon tip led us

to consider the following hypothesis: an ovule-derived signal

might trigger a NO-dependent pathway that directs the cor-

rect guidance of the pollen tube to the ovule through

a Ca2+-mediated process. Therefore, we hypothesized that if

we can interfere with the Ca2+ response in the presence of

CPTIO, we may recover the pollen tube targeting response.

We thus sought to test whether Ca2+ signaling is involved in

the NO targeting pathway, by adding D-Ser (10 mM) to the me-

dium (Kartvelishvily et al., 2006; Qi et al., 2006). If Ca2+ signal-

ing is implied in the process of NO pollen tube guidance, then

the balloon tip percentage should lower and targeting events

should rise in the presence of D-Ser. Figure 3G shows this to be

the case, with a partial but significant (p , 0.05 ANOVA, n =

52) rescue of pollen tube polarized growth and targeting to

ovules, and significant decrease in tubes with exploded tips.

Balloon tips also show a slight decrease, although this one

proved to be non-significant. These results suggested that

in-vivo involvement of NO in pollen tube growth and targeting

is likely mediated by a downstream Ca2+ signal.

That being the case, we would expect cytosolic Ca2+ to be

directly affected by the NO above some threshold concentra-

tion. Our previously developed bio-assay (Prado et al., 2004)

provided the ideal experiment to test this hypothesis, if mod-

ified to conditions in which pollen would be loaded with

a cytosolic Ca2+ indicator. Figure 4 shows such an experiment.

Pollen tubes were pressure-injected with the cytosolic Ca2+ in-

dicator Oregon-green BAPTA, on its dextranated form

(10 KDa), and followed by confocal imaging during a typical

NO challenging experiment as described in Prado et al.

(2004). This is basically achieved by filling a large-tip

(;50 lm) micropipette with SNAP-containing agarose, which

enables a slow and steady release of NO as directly demon-

strated (Prado et al., 2004). Under these conditions, pollen

tubes systematically show a reaction of slowdown/arrest,

and further re-orientation in a new growth axis (Figure 4B

and 4C). We focused on the typical tip-focused Ca2+ gradient

(Figure 4A) and quantified the normalized fluorescence of the

first 15 lm from the tip. As depicted in the graph of Figure 4D,

the Ca2+ response seems to anticipate the forthcoming cellular

response, and follows the previously measured intracellular

NO levels (Prado et al., 2004). In this specific experiment, a first

peak occurs at about 3 min, precisely the point at which pollen

tubes were predicted to reach the physiological active concen-

tration thresholds of NO (see Prado et al., 2004 for quantifica-

tion), and consequently slow down, sometimes up to growth

arrest. In this specific case, the tube did not arrest completely,

but a second peak of Ca2+ followed at about 6 min after chal-

lenge, precisely when the pollen started to change direction,

and resume growth in the new axis. Afterwards (.7 min), the

pollen tube resumed growth at normal growth rate and the

Ca2+ level returned to normal basal levels.

DISCUSSION

Our data provide new evidence for an in-vivo role of NO in

pollen tube guidance and targeting to the micropyle. The sub-

stantiation for the involvement of mostly unknown cues in

pollen tube guidance is largely based on observations that

in-vitro grown pollen does not generally exhibit any inherent

directionality and that its growth rate is usually significantly

slower than in vivo (Wheeler et al., 2001). The Nicotiana-

glycosylated TTS proteins provide one of the best examples

of pollen tube elongation. Nevertheless, TTS proteins show

a path in the style but do not dictate pollen tube targeting

(Wu et al., 1995). In Arabidopsis, the GABA gradient from

stigma to micropyle acts like a positive chemotropic cue for

pollen tube growth, but, nevertheless, it does not show any

effect in vitro, indicating that other intervening molecules

are needed in pollen tube growth to contextualize the com-

munication pattern (Palanivelu et al., 2003). In the particular

case of the Atnos1 mutant plants, where NO production is im-

paired and seed set is affected, we have a clear indication that

NO is a new candidate molecule for in-vivo pollen tube guid-

ance. We have gathered data from Atnos1 self-pollination and

cross-pollinations between Atnos1 mutant and wild-type

plants that suggest the action of NO in pollen tube guidance

in vivo. The observation that both wild-type and mutant pol-

len fail to fertilize the ovules in Atnos1 pistils, while mutant

pollen can fertilize wild-type ovules, indicates a possible NO

ovule signal function that orchestrates the final re-orientation

of pollen tube penetration into the micropyle (see Figure 1).

This result may explain the reduced seed set observed by

Guo et al. (2003) and it indicates that the mutation affects

the female tissues. Further support for this hypothesis stems

from the fact that we could successfully phenocopy the repro-

ductive phenotype of the mutant by treatment of wild-type

plants with PTIO, a permeable NO scavenger. This treatment

resulted in an overall decrease of more than half of the seed

set in comparison with the control—a value close to the 40%

Table 2. Distribution (% 6 SE) of Normal and Balloon Tipsbetween the Top and Inferior Halves of the Semi-VivoPreparation.

Top half Inferior half

Normal tip Balloon tip Normal tip Balloon tip

43.2% (6 18.4) 56.8 % (6 9.4) 97.7% (6 1.5) 2.3% (6 0.6)

The table shows a higher percentage of balloon tips in the top halfversus the inferior half with a higher percentage of normal tips in thepresence of CPTIO.

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decrease observed in the AtNOS1 mutant plant. These results

indicate that NO presence is necessary for the success of sexual

plant reproduction.

The cellular mechanism, underlying how the pollen tube–

pistil NO-mediated communication operates, remains very

hard to pin down. Nevertheless, it is evident that functional

peroxisomes are a necessary condition for Arabidopsis pollen

tube fertilization (Boisson-Dernier et al., 2008). The misalloca-

tion of a peroxisomal protein in the amc mutant may impair

the peroxisome from producing NO or ROS, and, in the ab-

sence of these signaling molecules, communication is pre-

vented between female and male gametophytes and

fertilization is abrogated.

This is an attractive working hypothesis in our work as well.

Nevertheless, so far, we have not gathered practical evidence

for the NO extracellular diffusion from pollen tubes, or from

the micropyle cells—a task which implies, for the moment, in-

surmountable experimental problems, which we are currently

trying to address.

New evidence for a role of NO during pollen–pistil interac-

tion was recently shown by McInnis et al. (2006). The work by

these authors suggests that possibly NO functions as an exter-

nal signaling molecule triggering the reduction of ROS/H2O2 in

stigmatic papillae. The experimental proof supporting this hy-

pothesis is the decrease in ROS/ H2O2 in papillae when sub-

jected to SNP that releases NO. The distinction between the

role of a NO–ROS/H2O2 in species recognition or discrimination

between pollen and micro-organisms in the stigma are still not

apparent.

To further understand our results, we analyzed the tran-

scriptomic data gathered by Boavida et al. (submitted) on pol-

len–pistil interactions regarding Atnos1, nr1 and nr2 genes.

We have chosen these genes since they code for different

enzymes already known for their ability to produce or modu-

late the production of NO (Guo et al., 2003; Bright et al., 2006).

Both NR1 and NR2 in Arabidopsis have the capacity to gener-

ate NO from nitrite, and both can contribute to NO-dependent

stomatal closure (Bright et al., 2006; Boisson-Demier et al.,

2008).

The possible contribution of the different transcripts at dis-

tinct stages after pollination as well as in several tissues, as

depicted in the graph of Figure 2, shows significant changes

detected above the 1.2 cut-off that arose between nr1 and

nr2 at 8 HAP versus 0.5 HAP and 8 HAP versus 3 HAP, with

no significant changes found for any comparison involving

Atnos1. These data reveal the down-regulation ofnr1 transcript

Figure 4. Cytosolic Free Calcium (c[Ca2+]) Dynamics during NO Challenging.

Lilium pollen tubes were microinjected with Oregon-Green BAPTA-Dextran (10 KDa) and imaged in a confocal microscope to visualize thetypical tip-focused gradient (A). Tubes growing straight with rates . 8 lm min�1 (B) were challenged with a SNP-filled pipette source asdescribed in Prado et al. (2004) until the typical re-orientation response takes place (C). The plot in (D) shows the alteration in the nor-malized fluorescence level of the first 15 lm from the apex of a pollen tube during a typical experiment (dots = experimental values;line = moving average, n = 2). As the tube enters the diffusion gradient from the pipette, the levels of c[Ca2+] start to raise (1.5 minonwards) until reaching a first peak. This peak coincides with a slowdown, sometimes full halt, of growth. The levels of c[Ca2+] then some-what decrease, and start to raise again to a peak that corresponds to the turning point, and growth restart (4–6 min). After that, the pollentubes resume growth at normal rate in the new growth axis (.7 min), and the levels of c[Ca2+] return to basal levels (here normalized to;100–110 arbitrary units of fluorescence on a 8-bit scale).

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versus the up-regulation ofnr2both at 8 HAP. This suggests that

the differences observed between the expression levels of these

transcripts may be correlated with post-fertilization events. We

cannot judge from these results if both nr’s will contribute to

any NO signaling pathway. Nevertheless, the participation of

these transcripts in the activation of NO pollen–pistil communi-

cation processes should be taken into consideration in further

studies. Genetic data fromA. thaliana show that NR is the major

source of NO in guard cells in response to ABA-mediated H2O2

synthesis (Bright et al., 2006). In sunflowers, the regulation of

NO production is controlled by nitrate reductase in vivo and

in vitro (Rockel et al., 2002). High NO emission rates correlate

with NR activation in the dark and accumulation of nitrite levels

in anoxia (Rockel et al., 2002; Kaiser et al., 2002; for a review, see

Crawford and Guo, 2005). The role of Atnos1 remains unclear,

since no significant changes in transcript levels were detected

and the specific activity of the protein is low (5 nmol min�1

mg�1) (Crawford and Guo, 2005). Nevertheless, micropyle tar-

geting is somehow affected in this mutant.

To better address the putative contribution of NO in pollen

tube micropyle targeting, we resorted to the use of the Kanadi

mutants. Kanadi expression is required for some aspects of ad-

axial–abaxial polarity in the Landsberg erecta background. It

shows floral defects such as production of ectopic ovules, for-

mation of projections of carpel and style or stigmatic tissue

from the base of the pistil (Kerstetter et al., 2001). The detec-

tion of DAF2-DA signal in the micropyle area of the exposed

ovules prompts us to propose the production of NO in this

structure. Interestingly, the signal is localized in a restricted

number of cells that border the micropyle opening. The obser-

vation of a putative restricted site of NO production at the bor-

der of the micropyle is compatible with targeting by growing

pollen tubes to the micropyle being confined by NO negative

tropism and re-orienting to the micropyle locus through the

area deprived of the putative NO signal.

Nevertheless, it is important to note that NO imaging with

DAF-2DA allows the detection of NO at potential sites of

in-vivo NO production—a quality not shared by other methods

of NO detection. Nevertheless, there is some controversy in the

field on the precise nature of the NO signal, as DAF-2DA reacts

more readily with more oxidized forms of NO as well as it can

react to changes in ROS and ascorbic acid (Planchet and Kaiser,

2006). In our work, though we cannot readily exclude the par-

ticipation of other chemical species that may contribute to the

DAF-2DA signal, the use of this probe in plant biology has

revealed amazing results such as, for instance, the detection

of asymmetric intracellular NO during gravitropic bending

of soybean roots (Hu et al., 2005).

In soybean roots, gravitropic bending is mediated by NO, in

a mechanism triggered by auxin with concomitant asymmetric

NO accumulation in the root (Hu et al., 2005). This finding illus-

trates that NO can function at a specific cell row, in this case for

gravitropic bending to be achieved.

Palanivelu and Preuss (2006) have defined three steps reg-

ulatingA. thaliana pollen tube guidance: (1) contact-mediated

competence conferred by the stigma and style, (2) diffusible

ovule-derived attractants, and (3) repellents from recently

targeted ovules. Our new data may add to the overall frame

of A. thaliana pollen tube guidance as defined by Palanivelu

and Preuss (2006), where a negative tropic response from a pol-

len tube may actually result in micropyle targeting.

That the real mechanism may, however, involve a more

sophisticated NO concentration dependency then the above

conclusion veiculates. In Lilium ovule, targeting events are cor-

related with a NO signaling pathway as shown by CPTIO affect-

ing tube targeting and growth parameters. The intriguing

result on this experiment stems from the fact that the pollen

tube population was divided into two groups, the first one lo-

cated in the top half facing the ovules exhibited a predomi-

nance of balloon tips and cessation of targeting versus the

second group in the inferior half of the preparation, in the ab-

sence of ovules, which retained polarized growth. Besides

proving the need of NO for targeting, the different behavior

of the tubes exposed to ovules can only be explained by

assuming that the ovules produce some sort of diffusible mol-

ecule that interacts or needs a basal level of NO to properly

work. This is consistent with our earlier hypothesis of combi-

natorial stimuli as the basis of the specificity in the usage of

ubiquitous signaling molecules (Prado et al., 2004). The loss

of polarized growth by pollen tubes is already well described

in the literature to be correlated with the loss of the tip-

focused Ca2+ gradient (Pierson et al., 1994; Holdaway-Clarke

and Hepler, 2003; Michard et al., 2008). Therefore, we consider

that besides the NO signal, together with an ovule-derived

factor, we also equate the necessity of a Ca2+ gradient at

the tip of the pollen tube as an element required for the sig-

naling pathway that drives pollen tube growth and targeting.

Cross-talk between NO and Ca2+ signaling has been recently

highlighted as essential in a number of systems (Besson-Bard

et al., 2008). And, in fact, with the experiment depicted in

Figure 4, we provide the first direct evidence that cytoplasmic

NO does in fact anticipate the cytosolic Ca2+ and the cellular

response of re-directioning. These are technically demanding

experiments, involving multiple micro-manipulators and

sophisticated imaging, and therefore this result should be

the object of further scrutiny, and better pharmacology, but

the fact remains that in all experiments, the Ca2+ reaction to

NO was observed.

How does NO, an ovule-derived signal, and a tip Ca2+ gra-

dient fit together to build up a NO-mediated pathway that

leads to lily pollen tube targeting? In the semi-vivo prepara-

tion, pollen tubes are growing in the presence of ovules

and submitted to a theoretically predicted ovule-derived fac-

tor (Lush, 1998). This ovule-derived factor is expected to trig-

ger a signaling cascade in the pollen tube that will redirect its

growth and promote targeting to the ovule. We predict that

the signaling cascade triggered by the ovule-derived factor

will be orchestrated by NO. The NO level at the tip of the

pollen tube may rise upon detection of the ovule-derived sig-

nal, and likely activate tip Ca2+ channels. In animal cells, it is

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well established that NO regulates the activity of Ca2+ chan-

nels, namely the activity of the NMDA receptor in synapses

(Boehning and Snyder, 2003). Likewise, a possible physiologi-

cal sequence for pollen tube targeting would then be: (1)

ovule-derived signal release, (2) increase of NO at the tip,

(3) activation of putative Ca2+ channel at the pollen tube

tip, (4) re-orientation of the pollen tube mediated by the

Ca2+ gradient, and (5) re-directioning of the growth axis.

The data presented in Figure 4 directly support steps 3 and

4, while step 2 had been shown by our previous work (Prado

et al., 2004). Considering this hypothesis, the addition of CPTIO

to the medium will block this cascade of events at step (2). In

agreement, we previously observed decrease of tip intracellu-

lar NO levels in lily pollen tubes after CPTIO treatment, fol-

lowed by subsequent blockage of pollen tube re-orientation

(Prado et al., 2004). To further evaluate the intervention of

a Ca2+ signaling pathway directing pollen tube targeting,

we added to the semi-vivo preparation medium 10 mM

D-Ser, alone and simultaneously with CPTIO, to assess whether

D-Ser can promote the recovery of normal pollen tube tip mor-

phology and targeting to ovules. The partial rescue of normal

tip morphology and targeting obtained further re-enforces

the hypothesis that step (3) is likely to occur. While we just of-

fer physiological evidence for this mechanism, one should bear

in mind that a possible molecular mechanism is also offered by

the fact that Frietsch et al. (2007) have recently found a striking

pollen phenotype in pollen for a mutation on a putative cyclic

nucleotide gated channel (CNGC), which, in parallel, was

shown to affect Ca2+ accumulation when overexpressed in bac-

teria. While confirmation of its role as a bona fide Ca2+

channel in pollen tubes as well is still required, the possibility

that NO may regulate such channels through a cGMP pathway,

as suggested in our previous experiments (Prado et al., 2004),

remains an exciting possibility to link our physiological data,

and establish a more defined and testable role for NO during

pollen–pistil interaction.

The direct and indirect evidences gathered in this study are

compatible with the involvement of NO in pollen tube guid-

ance in Arabidopsis and lily. All approaches used on both

species seem to point out a NO participation in ovule

targeting—the very final step prior to fertilization.

METHODS

Plant Material and Pollination Conditions

Wild-type Arabidopsis thaliana Columbia ecotype (Col-0),

Atnos1 mutant, and Kanadi mutant plants were used in this

study. Seeds were surface sterilized with sodium hypochlorite,

washed with sterile water and then spread on Petri dishes con-

taining MS medium (Duchefa, Haarlem, The Netherlands) so-

lidified with 0.8% (w/v) phytagar (Duchefa). Atnos1 mutant

seeds were selected in MS medium with kanamycin (50 lg

ml�1). Seeds were then cold-treated for 3 d at 4�C to ensure

uniform germination. Plates were transferred to short-day

conditions (8 h of light at 21–23�C); when full vegetative

growth was achieved, plants were transferred to long-day con-

ditions to promote flowering (16 h light).

The Atnos1 mutation was found at the T-DNA mutant col-

lection of the SALK Institute Genome Analysis Laboratory

(http://signal.salk.edu) and seeds were obtained from Notting-

ham Arabidopsis Stock Center (NASC, Nottingham, UK). The

Kanadi mutant plants in Landersberg erecta genetic back-

ground were kindly supplied by Dr John Bowman (School of

Biological Sciences, Monash University). Controlled pollina-

tions were performed first by emasculating closed flower buds

and only the pistils showing a turgid stigmatic papilla were

used for crosses in the next day.

Imaging

Detection of Arabidopsis pollen tube trajectory inside pistil tis-

sues in wild-type and mutant plants was done with 0.1% (w/v)

aniline-blue staining of pollen tube callose wall, 6 h after pol-

lination pistils were stained and squashed between slide and

cover slip. Monitoring of NO production in unpollinated

kanadi ovules was performed with 10 lM 4,5-diaminofluores-

cein diacetate (DAF-2DA, Molecular Probes) incubated for 4 h

prior to visualization. Preparations were observed using Leica

DMRA2 microscope (Leica Microsystems, Heerburg, Germany)

equipped with UV (340–380 nm) excitation filter, and blue

(440–480 nm) excitation filter.

Semi-Vivo and Watering Assays

Pistils of lily were collected from flowers during anthesis and

the ovaries were isolated. Under a stereoscope, the ovary was

cut in half longitudinally with a scalpel. From the ovary loculus,

exposed ovules were isolated one by one with a precision nee-

dle. Ovules were then collect in a row into a slide covered with

medium and stored in a humid chamber. The pollen grains

were then placed in a row on the slide bellow the ovules.

Lilium medium composition (Rosen, 1961): 10% sucrose, 1%

agarose (low meting point, Sigma), 0.16 mM yeast extract.

Variations to this medium were done by adding 200 lM

2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-

oxide, K+-salt (CPTIO, Calbiochem) in the presence or absence

of 10 mM D-Ser (Sigma) concentration. Wild-type Arabidopsis

Col-0 plants were water treated with 200 and 500 lM as well as

1 mM 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide

(PTIO, Sigma). Once siliques were formed, seed number was

scored.

Genechip Statistical Analysis

The pollen–pistil interaction transcriptome data analyzed

were gathered by Boavida et al. (submitted). The transcripts

Atnos1 (Locus At3g47450), nr1 (Locus At1g77760), and nr2

(Locus At1g37130) expression levels at different HAP were

compared with each other and with unpollinated pistil. The

Dchip software was used to statistically analyze and compare

the expression levels of the three chosen transcripts through

the calculation of the lower confidence bound of the fold

change. The lower confidence bound criterion indicates

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90% confidence that the fold change is a value flanked by the

lower confidence bound and a variable upper confidence

bound (Pina et al., 2005).

Cytosolic Ca2+ Imaging and NO Challenge

Pollen grains where mixed in a 1% agarose thin layer and kept

at 4�C for 5 min for agarose solidification. Purpose-built ger-

mination chambers were filled with regular medium and

germination followed as previously described. For injection,

needles where pulled with a Sutter P-97 using borosilicate

glass 1 mm diameter and micro-injection was preformed with

a Eppendorf Cell Tram injector filled with water. Pollen tubes

where microinjected with 0.5 mM of Oregon-Green Bapta-

Dextran (10 KDa; estimated dilution ; 1/1000 inside the cell).

Images where made in a Zeiss LSM510, using the 488 laser line

for excitation, and a BP filter 500–550 for emission. Image

Analysis was made with ImageJ v. 1.39. NO gradient formation

was performed with SNAP-filled pipettes, as previously de-

scribed (Prado et al., 2004; tip diameter ; 50 um).

FUNDING

This work was supported by FCT (POCTI/34772/BCI/2000; POCTI/BIA-

BCM/60046/2004; PPCDT/BIA-BCM/61270/2004).

ACKNOWLEDGMENTS

Kanadi mutant plant seeds were kindly supplied by Dr John

Bowman (School of Biological Sciences, Monash University). We

kindly acknowledge Steven Neill (University of the West of Eng-

land, Bristol) and Joao Laranjinha (Center for Neuroscience and Cell

Biology, university of Coimbra) for fruitful discussions, and Leonor

Boavida and Jorg Becker for help with the microarray data. A.M.P

acknowledges an FCT PhD fellowship (SFRH/BD/6278/2001).

No conflict of interest declared.

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