Role of peroxynitrite in programmed cell death induced in self-incompatible pollen

Abstract

Reactive oxygen species and NO are involved in the signaling pathway of programmed cell death (PCD). Information concerning the role of these molecules in self-incompatible pollination is scarce especially in non-model species studied in vivo. We recently reported that in the olive tree, compatible and self-incompatible pollen have different levels of reactive oxygen and nitrogen species and that PCD is induced in self-incompatible pollen. Levels of O₂^.- and NO are higher in pollen after self-incompatible pollination than after compatible pollination. The presence of these reactive species was concomitant with the presence of peroxynitrite. Similar results were obtained on pollen-germination experiments both in vivo and in vitro. These data, together with observations made after treating pollinated flowers with scavengers, suggest that peroxynitrite plays a role in PCD induced after self-incompatible pollination and we propose here a model to describe the way in which it might work.

The ability of plants to eliminate self- or genetically-related pollen from close relatives is vital to the avoidance of self-fertilization. Among the strategies that a plant can use to limit inbreeding, self-incompatibility (SI) is one of the most widespread and more thoroughly studied.^1,2 Different SI systems have been identified involving specific pollen and pistil molecules the interaction of which determines the specificity of pollination.^2-6

Although pollen and pistil determinants of model species have been characterized, there are other factors which, while not contributing directly to the determination of S-specificity, do play important roles in pollen rejection. Among these are molecules involved in programmed cell death (PCD), which plays an important role in the rejection of self-incompatible pollen in both sporophytic and gametophytic SI.^7,8

In the olive (Olea europaea L.) the SI type has not been established but in previous experiments using in vivo pollination we demonstrated that PCD was triggered in self-incompatible pollen after its germination on the stigma.⁹

Reactive oxygen and nitrogen species (ROS and RNS) are highly reactive molecules that play important roles in cell signaling and have recently been identified in the regulation of PCD during self-incompatible responses in Papaver.¹⁰ Within this context, we have assessed the involvement of ROS and NO in PCD caused by SI in our paper, “Peroxynitrite mediates programmed cell death in both papillar cells and in self-incompatible pollen in the olive (Olea europaea L.).”¹¹ The production of hydrogen peroxide, superoxide anion and nitric oxide was studied using specific fluorescent probes (DCF-DA, DHE and DAF2-DA respectively) during different stages before and after free and controlled pollination using confocal laser microscopy. There was a reduction on H₂O₂ signal in pistils after pollination whilst fluorescence corresponding to O₂^.- and NO was increased. These results were confirmed by in vitro germination experiments, in which an H₂O₂ signal was detectable in those pollen tubes germinated in the absence of pistils, or germinated in the presence of pistils of a self-compatible cultivar (Arbequina), but it was not detectable when the pollen grains were germinated in the presence of pistils of the same cultivar (Picual, self-incompatible). The reduction of H₂O₂ suggests no direct role of this reactive species in triggering PCD in response to self-incompatibility in the olive in contrast to that occurring in the context of plant-pathogen interactions during hypersensitive response (HR)¹² and in tobacco culture cells treated with ROS/NO donors,¹³ then suggesting a different pathway for PCD in SI response. For this reason, we investigated other possible triggers of PCD. Both O₂^.- and NO were present in pollen tubes germinating on the stigma, which was confirmed by in vitro experiments, these reactive species being detectable only when the pollen grains were germinated in the presence of the self-incompatible pistil.

Since O₂^.- and NO were the two reactive species present in pollen grains and pollen tubes, we studied their potential enzymatic sources. When pollinated pistils were incubated with diphenyliodonium (DPI) and sodium azide, O₂^.--dependent fluorescence decreased in pollen grains and pollen tubes, suggesting that both NADPH oxidase and peroxidase activities may be responsible for O₂^.- production. In addition, RT-PCR experiments showed an increase in the expression of NADPH oxidase in pollen grains germinated in vitro. Incubation of pollinated pistils with NG-nitro-l-arginine methyl ester (L-NAME), a nitric oxide synthase (NOS) inhibitor, resulted in a decrease in NO-dependent fluorescence in pollen grains germinated on the stigma, suggesting that a NOS-like activity could be responsible for NO production although we cannot exclude other NO sources such as nitrate reductase.

To evaluate the function of O₂^.- and NO in PCD during free pollination, we treated pollinated flowers with scavengers of these molecules (2,2,6,6-tetramethylpiperidinooxy (TMP) and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO). Samples were collected when the fertilization stage was reached and analyzed by trypan blue staining. We have shown previously that this staining correspond to PCD in our system.⁹ In both scavenger treatments, signs of cell death in pollen grains were reduced, indicating that O₂^.- and NO are involved in PCD induction. The coexistence of O₂^.- and NO after pollination led us to study the possible production of peroxynitrite (ONOO^-), which has been described as a key factor in PCD in animals.¹⁴ External high concentrations of ONOO^- seems not to be essential to induce PCD in Arabidopsis plants¹² probably due to the ability of plants to detoxify ONOO^- under physiological conditions.¹⁵ However, an increase in nitrated proteins and an inhibition of the plant antioxidant system involved in ONOO^- detoxification has been shown in vivo during the progression of the HR-dependent PCD,^15,16 thus we decided to analyze peroxynitrite function during PCD dependent of SI. In the olive, peroxynitrite was measured directly by a specific fluorescent probe (HKGreen-2)¹⁷ and also by immunodetection of tyrosine nitration.¹⁸ An increase in peroxynitrite-dependent fluorescence was detected in incompatible pollination compared with compatible pollination and, a reduction in the number of pollen grains undergoing PCD during SI was shown in samples treated with the specific ONOO^- scavenger, epicatechin. This result suggests that peroxynitrite is a key signal for PCD triggered by self-incompatibility. Additionally, nitration and concomitant PCD were shown in serial sections of different pollinated pistils were it was found that pollen containing nitrotyrosine signal were positive to TUNEL reaction while pollen not showing this signal were negative to TUNEL reaction.¹¹

On the basis of these data we postulate a model in which the balance ROS/NO can regulate a cross-talk between stigma and pollen and thus trigger compatible or incompatible responses, although the mechanisms involved in discriminating both responses have not been established so far (Fig. 1). A decrease in H₂O₂ can be detected in self-incompatible pollen, compared with compatible pollen; probably as result of an increase in catalase and ascorbate peroxidase activity as it has been shown in lily stigmas after self-incompatible pollination.¹⁹ At the same time, O₂^.- and NO increase in self-incompatible pollen, leading to the increase of ONOO^-, which results in a rise in the number of nitrated proteins probably involved in the induction of PCD, although the specific role of protein nitration in plant PCD has not been established so far. Recently a relationship between disturbances in the actin cytoskeleton and PCD in conditions of self-incompatibility has been reported.¹⁰ In this scenario, PCD triggered by ONOO^- could be due to actin modification by nitration, giving rise to disturbances in actin polymerization, as has been observed in animal cells.²⁰ It has been also suggested that RNases play an important role in PCD induced by self-incompatibilty⁸ and, although no information on the regulation of RNases by nitration has been demonstrated so far in plants, in seminal vesicles RNase VS1 is activated 130–200% by nitration.²¹ Further studies are required to unravel the role of protein nitration in self-incompatibility.

Figure 1. Role of peroxynitrite in programmed cell death induced in self-incompatible pollen. A decrease in H₂O₂ can be seen in self-incompatible pollen, compared with compatible pollen; at the same time, there is an increase in O₂^.- and NO in self-incompatible pollen, leading to the rise of ONOO^-. This fact give rise an increase in the number of nitrated proteins, which can trigger PCD in pollen tubes. Actin and RNases could be targets of the nitration promoting PCD in self-incompatible pollen. A cross-talk between ROS/NO from pistils and pollen could also influence incompatible response in the pollen, although the mechanisms involved have not been established so far (question mark). Grey shading and dashed arrows represent results from different or unconfirmed studies. Cat, catalase; APX, ascorbate peroxidase; NR, nitrate reductase; TMP, 2,2,6,6-tetramethylpiperidinooxy; 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO).

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.