The plant stigma exudate

Abstract

During sexual reproduction, pollen performance is greatly influenced by the female tissues. The stigma exudate, i.e., the extracellular secretion that covers the stigma outermost surface, has been usually regarded as a reservoir of water, secondary metabolites, cell wall precursors and compounds that serve as energy supply for rapid pollen tube growth. In an attempt to identify the proteins present in the stigma secretome, we performed a large-scale analysis in two species (Lilium longiflorum and Olea europaea) following a proteomic-based approach. The resulting data strongly suggest that the stigma exudate is not a mere storage site but also a biochemically active environment with a markedly catabolic nature. Thus, this secretion may modulate early pollen tube growth and contribute to the senescence of stigma after pollination. In addition, a putative cross-talk between genetic programs that regulate stress/defense and pollination responses in the stigma is also suggested. The stigma exudate might also functionally diverge between species on the basis on their ecology and the biochemical, morphological and anatomical features of their stigmas. Unexpectedly, we identified in both exudates some intracellular proteins, suggesting that a mechanism other than the canonical ER-Golgi exocytic pathway may exist in the stigma and contribute to exudate secretion.

During sexual reproduction, pollen performance is greatly influenced by the biochemical, morphological and anatomical features of the female counterpart (i.e., the pistil). In some plants, the stigma outermost surface is coated by a secretion that is released by specialized cells called papillae. This extracellular fluid, the so-called stigma exudate, mostly contains water, sugars, lipids and proteins,¹ but other compounds such as phenols, amino acids, ROS/RNS and Ca²⁺ ions are also present in measurable amounts.^2-5 The picture of the stigma secretome remains fragmentary and only a few proteins have been identified and characterized so far.^6-8 In a recent publication, we performed a large-scale protein profiling study of the stigma exudate in two plant species, namely Eastern lily (Lilium longiflorum) and olive (Olea europaea), that substantially differ in their taxonomical position, pistil morphology and anatomy, ecology and cultivation purposes.⁹ Following a proteomic-based strategy, we identified 51 and 57 unique proteins in lily and olive, respectively, most of which are first described in this secretion. The resulting data shows that the complexity of the stigma exudate, in terms of protein diversity, is higher than initially expected.¹⁰ Under a functional perspective, this secretion has often been regarded as a mere source of water for pollen hydration, and storage compounds for sustaining pollen tube growth.¹¹ On the contrary, our data show that exudate proteins are involved in at least 80 different biological processes and 97 molecular functions, suggesting that this secretion is indeed a biochemically active site.

First, proteomic analysis revealed a catabolic nature of the stigma exudate, which is very rich in hydrolytic enzymes such as O-glycosylases, esterases/lipases and proteases. This biochemical machinery may contribute to degrade secreted polymers to smaller units, which would be easier to uptake by elongating pollen tube tips. Pollen tube growth in the stigma might be regulated through the selective action of specific sets of cell wall-degrading enzymes (e.g., pectinases). Thus, key features of pollen tube growth such as cell wall mechanical stability and extensibility may be modulated by these enzymes. Other proteins identified in the stigma exudate such as chemocyanin, SCA, FLA and phospholipase C among others, are typically implicated in cell to cell interaction and signaling. These proteins might mediate reactions important for pollen-stigma interaction events including pollen adhesion and recognition or, alternatively, generate signals to guide pollen tubes toward the style.

The presence of several PRP families (e.g., β-glucanases) and other defense-related proteins in the stigmatic exudate suggests a putative cross-talk between genetic programs regulating stress/defense and pollination responses in the stigma. For instance, some extracellular chitinases promote interactions between plants and compatible symbiotic mycorrhizal fungi by degrading fungal elicitors.¹² The mycorrhizal hyphae growth in the root has a parallelism with that of the compatible pollen tube in the pistil. Therefore, chitinases may participate in promoting compatible pollen tube growth by degrading or generating signal molecules. In that respect, it is interesting to note that extracellular chitinases are able to use N-acetylglucosamine and glucosamine-containing AGPs as substrates.¹³ Alternatively, in partnership with other secreted proteins identified in the stigma exudate (e.g., S1-type nucleases), chitinases may take part in the PCD of the stigma after either compatible pollination or incompatible pollen rejection.¹⁴ Temperature stress also affects several aspects of plant reproduction such as stigmatic receptivity, pollen performance and self-incompatibility relationships.¹⁵ At molecular level, plants tackle heat stress through the action of a set of molecular chaperones known as HSPs, which are emerging extracellular mediators in cell signaling in response to stress.¹⁶ Therefore, it is also plausible to hypothesize that the HSPs secreted by the stigma may modulate temperature-dependent reproductive events in this tissue.

It is interesting to point out that the lily and olive stigma exudates only share about 22–25% of the identified proteins. This fact might reflect some functional divergences of this secretion between these two species. Such differences can be mostly explained on the basis of their diverse ecology, pistil anatomy and morphology, and chemical composition of the exudate. For instance, O-glycosylases were very abundant in L. longiflorum but scarce in O. europaea, which is consistent with the marked polysaccharidic and lipidic nature, respectively, of their stigmatic exudates. Direct contact with pollinators could make the lily stigma exudate very susceptible to fungi infection. This fact may also explain the profusion of chitinase isoforms and classes in this species in contrast to olive, a wind-pollinated species, where these enzymes were not detected. The presence of numerous HSPs in the olive stigma exudate suggests that temperature fluctuations at the time of pollination might be more critical for the reproduction process in this species. However, despite these qualitative differences observed, we should be cautious since protein identification may be biased by the fact that both species are not fully annotated in databases. Thus, it is very likely that the stigma secretomes of these plants are still incomplete. Moreover, some of the proteins identified in the olive may be rather associated to the cell wall/apoplast due to the method of exudate collection in this species, which involved brushing of the stigma surface. Hence, it would be necessary to complete the stigma secretome maps of these and other plant species, and to carry out additional validation experiments to prove the existence of such functional divergences.

Based on in silico prediction analyses, about 30% of matched proteins in lily and up to 65% in olive lack a peptide signal. Although signal peptide-predicting programs are subjected to fail and most of proteins were actually identified in other plant species, the extracellular localization of some proteins in the stigma exudate was puzzling. Recently, a novel mechanism of secretion that involves the release of nanovesicles, termed “pollensomes,” has been described in the olive and other pollen tubes.¹⁷ The presence of exosome-like vesicles has been also reported in apoplastic fluids of sunflower seeds.¹⁸ Exosomes contain a number of proteins, many of which are typically intracellular.^17,19 Interestingly, the olive stigma exudate shows a manifest vesicular aspect at anthesis, and contains numerous vesicle-like particles in the size range (40–100 nm) expected for exosomes.^1,9 Therefore, a similar mechanism of secretion may also exist in the plant stigma in addition to the canonical ER-Golgi exocytic pathway, although this possibility needs to be further investigated.

Glossary

Abbreviations:

AGP

arabinogalactan protein

FLA

fasciclin-like AGP

HSP

heat shock protein

PCD

programmed cell death

PRP

pathogenesis-related protein

RNS

reactive nitrogen species

ROS

reactive oxygen species

SCA

stigma/stylar cysteine-rich adhesin