Molecular Plant Advance Access originally published online on June 27, 2008
Molecular Plant 2008 1(4):703-714; doi:10.1093/mp/ssn034
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Targeting of Pollen Tubes to Ovules Is Dependent on Nitric Oxide (NO) Signaling
a Instituto Gulbenkian de Ciência, Centro de Biologia do Desenvolvimento, PT-2780–156 Oeiras, Portugal
b Universidade de Lisboa, Faculdade de Ciências, Dept Biologia Vegetal, Campo Grande, Ed.C2. PT-1749–016 Lisboa, Portugal
1 To whom correspondence should be addressed. E-mail jose.feijo{at}fc.ul.pt.
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Abstract |
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 TOP Abstract INTRODUCTIONRESULTSDISCUSSIONMETHODSFUNDING |
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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 defective NO production, have reduced fertility. Here, we focus in the role of NO in the process of pollen–pistil communication, 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. Activation of calcium influx in pollen tubes partially rescued normal pollen tube morphology, suggesting that this pathway is also dependent on Ca2+ signaling. A role of NO in modulating Ca2+ signaling was further substantiated by direct imaging the cytosolic free Ca2+ concentration during NO-induced re-orientation, where two peaks of Ca2+ 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
Received for publication March 5, 2008. Accepted for publication April 23, 2008.
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INTRODUCTION |
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSFUNDING |
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Despite recent advances, the way by which pollen tubes find their way into the micropyle and, generally speaking, the mechanisms by which pollen tube guidance is achieved within the pistil 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; Feijó et al., 2004). Of relevance, NO is the only chemotropic agent so far described capable of promoting turning angles of 90° and more, needed to achieve some steps of the fertilization process. The implication of 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 transmitting 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 molecule 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; Wu et al., 1995). On the other hand, the role of female sporophytic tissues and embryo sac in long-range guidance mechanisms 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 comparison with mutant ovules incapable of proper guidance (Hülskamp et al., 1995). The participation of ovule sporophytic cells for successful pollen tube and pistil communication was recently 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 interaction 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 
-amino butyric acid (GABA), thus hypothetically forming a gradient in the pistil to promote growth, suggestive of a putative guidance mechanism for Arabidopsis pollen tubes in vivo (Palanivelu et al., 2003; Swanson et al., 2004). Finally, in the gametophytic magatama 1 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 gametophytic mutants, sometimes two pollen tubes seem to be simultaneously 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 ablation of synergids indicates that these cells secrete a guidance cue that directs pollen tube targeting to the ovules over 100–200 µm (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 arrived close to the embryo sac, indicating that species preferentiality 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, secreted by the egg apparatus and shown to have a direct role on close-range pollen tube guidance (Márton 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/+), pollen reception is substituted by continued growth of the pollen tube, which can coil or branch. The amc/– ovule can nevertheless 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 resulting 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 negative 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 evidence 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 processes, 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 the Arabidopsis 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.
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RESULTS |
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSFUNDING |
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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 pollen–pistil communication process. The involvement of NO in Arabidopsis pollen tube–pistil communication was suggested in the original publication by Guo et al. (2003), who described the Atnos1 mutant plants as showing reduced fertility. 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 sequencing. 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 defective, 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).
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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 deformations 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 accordance 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 phenocopy 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 inhibition 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 deprivation may be responsible for the lower seed set by perturbing the normal guidance mechanisms.
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If NO is needed for guidance, and the AtNOS1 is mainly affecting 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 disrupted 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 interested 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 situation 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. Furthermore, 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 remarkable 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 observation 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).
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Finally, we addressed the issue of whether the genes necessary for an NO response were present in the pistil tissue. As mentioned before, this is a controversial question, as no unequivocal NO synthase or transducing mechanism is known in plants. Yet, on our best judgement of the available literature, we decided to focus on AtNOS1 (Locus At3g47450), decidedly involved in NO accumulation, and the Nitrate Reductase nr1 (Locus At1g77760) and nr2 (Locus At1g37130), because 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 genes at different hours after pollination (HAP) as well as in several tissues is depicted in Figure 2C. The fluctuations observed 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 indicates 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 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. Significant changes were detected above the 1.2 cut-off when comparing 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/germination 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 isolated 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 angle 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 targeting performance.
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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 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 pollen 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 quantification). 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 accompanied 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 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 Feijó, in preparation). 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 correct 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 medium (Kartvelishvily et al., 2006; Qi et al., 2006). If Ca2+ signaling 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 concentration. Our previously developed bio-assay (Prado et al., 2004) provided the ideal experiment to test this hypothesis, if modified 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+ indicator 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 µm) micropipette with SNAP-containing agarose, which enables a slow and steady release of NO as directly demonstrated (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 µm 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 concentration thresholds of NO (see Prado et al., 2004 for quantification), 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 challenge, 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.
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DISCUSSION |
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSFUNDING |
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Our data provide new evidence for an in-vivo role of NO in pollen tube guidance and targeting to the micropyle. The substantiation 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 communication pattern (Palanivelu et al., 2003). In the particular case of the Atnos1 mutant plants, where NO production is impaired and seed set is affected, we have a clear indication that NO is a new candidate molecule for in-vivo pollen tube guidance. 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 pollen 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 reproductive 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% 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 misallocation of a peroxisomal protein in the amc mutant may impair the peroxisome from producing NO or ROS, and, in the absence of these signaling molecules, communication is prevented 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, insurmountable experimental problems, which we are currently trying to address.
New evidence for a role of NO during pollen–pistil interaction was recently shown by McInnis et al. (2006). The work by these authors suggests that possibly NO functions as an external signaling molecule triggering the reduction of ROS/H2O2 in stigmatic papillae. The experimental proof supporting this hypothesis is the decrease in ROS/ H2O2 in papillae when subjected 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 transcriptomic data gathered by Boavida et al. (submitted) on pollen–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 modulate the production of NO (Guo et al., 2003; Bright et al., 2006). Both NR1 and NR2 in Arabidopsis have the capacity to generate 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 distinct 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 of nr1 transcript versus the up-regulation of nr2 both 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 communication processes should be taken into consideration in further studies. Genetic data from A. 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 targeting 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 adaxial–abaxial polarity in the Landsberg erecta background. It shows floral defects such as production of ectopic ovules, formation of projections of carpel and style or stigmatic tissue from the base of the pistil (Kerstetter et al., 2001). The detection 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 observation of a putative restricted site of NO production at the border 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 participation 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 illustrates 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 regulating A. 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 pollen 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 correlated with a NO signaling pathway as shown by CPTIO affecting 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 located in the top half facing the ovules exhibited a predominance of balloon tips and cessation of targeting versus the second group in the inferior half of the preparation, in the absence 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 molecule that interacts or needs a basal level of NO to properly work. This is consistent with our earlier hypothesis of combinatorial 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 signaling 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+ gradient fit together to build up a NO-mediated pathway that leads to lily pollen tube targeting? In the semi-vivo preparation, pollen tubes are growing in the presence of ovules and submitted to a theoretically predicted ovule-derived factor (Lush, 1998). This ovule-derived factor is expected to trigger 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 signal, and likely activate tip Ca2+ channels. In animal cells, it is well established that NO regulates the activity of Ca2+ channels, namely the activity of the NMDA receptor in synapses (Boehning and Snyder, 2003). Likewise, a possible physiological 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 intracellular NO levels in lily pollen tubes after CPTIO treatment, followed 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 morphology 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 offer 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 bacteria. 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 guidance 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.
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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 containing MS medium (Duchefa, Haarlem, The Netherlands) solidified with 0.8% (w/v) phytagar (Duchefa). Atnos1 mutant seeds were selected in MS medium with kanamycin (50 µg 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 conditions to promote flowering (16 h light).
The Atnos1 mutation was found at the T-DNA mutant collection of the SALK Institute Genome Analysis Laboratory (http://signal.salk.edu) and seeds were obtained from Nottingham Arabidopsis Stock Center (NASC, Nottingham, UK). The Kanadi mutant plants in Landersberg erecta genetic background were kindly supplied by Dr John Bowman (School of Biological Sciences, Monash University). Controlled pollinations 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 tissues in wild-type and mutant plants was done with 0.1% (w/v) aniline-blue staining of pollen tube callose wall, 6 h after pollination pistils were stained and squashed between slide and cover slip. Monitoring of NO production in unpollinated kanadi ovules was performed with 10 µM 4,5-diaminofluorescein 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 needle. 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 µM 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 µM 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 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 germination 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 described (Prado et al., 2004; tip diameter 50 um).
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This work was supported by FCT (POCTI/34772/BCI/2000; POCTI/BIA-BCM/60046/2004; PPCDT/BIA-BCM/61270/2004).
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Acknowledgements |
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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 England, Bristol) and João Laranjinha (Center for Neuroscience and Cell Biology, university of Coimbra) for fruitful discussions, and Leonor Boavida and Jörg 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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