Signaling on the Stigma

Abstract

Reactive oxygen species (ROS) and reactive nitrogen species, particularly NO, are key components of diverse signaling networks in animals and plants. We have recently shown that epidermal cells of stigmas from a range of different angiosperms accumulate relatively large amounts of ROS, principally H₂O₂, whereas pollen produces NO. Importantly, ROS/H₂O₂ levels appeared reduced in stigma cells supporting developing pollen grains compared to cells without pollen grains attached. To explore a possible link between pollen NO production and reduced levels of stigmatic ROS/H₂O₂, we supplied stigmas with NO and observed an overall reduction in levels of stigmatic ROS/H₂O₂. These new and unexpected data suggest a potential new signaling role for ROS/H₂O₂ and NO in pollen-stigma recognition processes.

ROS Accumulates in Angiosperm Stigmas

Our interest in the function of stigmatic peroxidases, which are an ubiquitous feature of angiosperm stigmas when they are mature and most receptive to pollination^1–4 led us to screen stigmas from a range of angiosperms for the presence of ROS as potential substrates for stigmatic peroxidases. Using confocal microscopy and the ROS-sensitive fluorescent probe dichlorodihydrofluorecein diacetate (DCFH₂-DA) we detected high levels of ROS accumulation in stigmatic epidermal cells (papillae) of Senecio squalidus (Asteraceae) and Arabidopsis thaliana.⁵ Treating these stigmas with sodium pyruvate (a scavenger of H₂O₂) prior to DCFH₂-DA staining resulted in reduced fluorescence, indicating that H₂O₂ is the major stigmatic ROS. To facilitate more rapid ROS/H₂O₂ detection in larger stigmas from other species we used the dye TMB (3,3′,5,5′-tetramethylbenzidine-HCl), which is also more specific to H₂O₂ than DCFH₂-DA,^6,7 and light microscopy. Stigmas of 20 species from 20 different angiosperm families stained positive for ROS/H₂O₂ suggesting that accumulation of ROS/H₂O₂ is probably a general feature of angiosperm stigmas.⁵ A next important step is therefore to determine the enzymatic source of ROS/H₂O₂ in stigmas—is one or more NADPH oxidase the source, or are other ROS-generating enzymes⁸ involved?

No is Produced by Pollen

In contrast to ROS, levels of NO accumulation in stigmas of S. squalidus and A. thaliana were low when assayed with the NO-specific probe diaminofluorescein diacetate (DAF2-DA) and confocal microscopy. However, far higher levels of NO were observed in the pollen of both S. squalidus and A. thaliana,⁵ NO being consistently present in pollen, both before and during germination (particularly in the emerging pollen tube). We are currently screening pollens from other angiosperm species (Fig. 1) to determine whether NO production is a general feature of angiosperm pollen. Plants produce NO in a range of different ways, both enzymatic and nonenzymatic,⁹ so it will be important to determine the principal source of NO in pollen. The main contenders are nitrate reductase and nitric oxide synthase (NOS)-like enzymes.⁹ However, it was suggested recently that NOS1 from A. thaliana has no NOS activity,^10–12 so the search for potential NO-generating enzymes in pollen needs to be investigated in a robust manner.

Figure 1.

Measurement of NO in pollen of Lilium longiflorum as assayed with DAF2-DA and confocal microscopy. cPTIO is a cell permeable scavenger of NO. Mean pixel intensity of fluorescence was calculated from twenty independent pollen grains according to McInnis et al.⁵

Potential Signaling Crosstalk Between Pollen No and Stigmatic ROS/H₂O₂

When levels of ROS/H₂O₂ were investigated in stigmas of S. squalidus and A. thaliana after pollination, levels of DCFH₂-DA fluorescence were consistently lower in stigmatic papillae in contact with developing pollen grains when compared to papillae that were not in contact with a pollen grain. To explore the possibility of pollen NO affecting levels of stigmatic ROS/H₂O₂ we exposed stigmas of S. squalidus and A. thaliana to sodium nitroprusside (SNP), which generates NO in the presence of light. Subsequent assays of ROS/H₂O₂ with DCFH₂-DA revealed dramatically reduced levels (up to 80% in S. squalidus) of ROS/H₂O₂ in NO-treated stigmas. This exciting new finding raises the possibility of a biologically relevant interaction between NO (from pollen) and ROS/H₂O₂ (in stigmatic papillae) in vivo as part of the signaling exchange that constitutes the pollen-pistil interaction.¹³ This molecular ‘courtship’ between the male pollen grain/pollen tube and the female cells of the pistil determines whether fertilization will occur and effectively acts as a maternal ‘screen’ for potential mates. In order for a pollen grain to germinate, produce a pollen tube that then finds its way through the female tissues of the pistil to locate the egg-containing ovules ahead of fertilization, the male pollen and female pistil cells must exchange a continuous stream of signals to reinforce the ‘compatibility’ of the two mating partners.^13–15 In recent years a diverse array of signals has been identified involved in all stages of the pollen-pistil interaction from pollen adhesion and germination to pollen tube growth and guidance.^16–20 Our observation of a possible in vivo interaction between NO from pollen and ROS/H₂O₂ in stigmatic papillae, spotlights a potential new dimension in signaling between pollen and stigma. One potential role for a NO/ROS interaction could be in species recognition, for which almost nothing is known (but see Swanson et al. and ref. 19 for recent review). Another possibility is that the interaction might be part of a more basic recognition system that discriminates between pollen and microbes. It is well known that stigmas, despite often producing sugary or lipidic secretions,²¹ are highly resistant to attack by microbes when receptive to pollen, so it is conceivable that high levels of stigmatic ROS/H₂O₂ may be sufficiently toxic to make stigmas resistant to attack by pathogens just as has been suggested for the high levels of ROS in nectar.^22,23 If such levels of stigmatic ROS/H₂O₂ are toxic to microbes why are they not toxic to pollen? The fact that levels of ROS/H₂O₂ appear reduced in stigmatic papillae in contact with pollen grains suggests that pollen grains are able to ‘switch’ off the ROS/H₂O₂ pathogen barrier, thereby allowing the emergent pollen tube to gain access to the stigma. Could pollen NO be the signal needed to release this barrier? In support of this hypothesis we have shown that external NO can reduce levels of stigmatic ROS/H₂O₂, so the next step is to establish a direct relationship between reduced levels of stigmatic ROS and release of pollen NO. Conversely, we also need to determine whether pollen NO production is altered by interaction with the stigma and if so, is this an effect of stigmatic ROS/H₂O₂? Our initial findings of potential signaling ‘cross-talk’ between pollen NO and stigmatic ROS/H₂O₂ thus raise many exciting and challenging questions which offer a new direction for studies of pollen-pistil interactions. Our findings also predict an intriguing parallel with sperm-egg interactions in animals, where NO and ROS are known to interact in processes such as sperm capacitation.^24,25

Addendum to: McInnis SM, Desikan R, Hancock JT, Hiscock SJ. Production of Reactive Oxygen Species and Reactive Nitrogen Species by Angiosperm Stigmas and Pollen: Potential Signalling Crosstalk. New Phytol. 2006;172:221–228. doi: 10.1111/j.1469-8137.2006.01875.x.