Signaling at Physical Barriers during Pollen–Pistil Interactions

In angiosperms, double fertilization requires pollen tubes to transport non-motile sperm to distant egg cells housed in a specialized female structure known as the pistil, mediating the ultimate fusion between male and female gametes. During this journey, the pollen tube encounters numerous physical barriers that must be mechanically circumvented, including the penetration of the stigmatic papillae, style, transmitting tract, and synergid cells as well as the ultimate fusion of sperm cells to the egg or central cell. Additionally, the pollen tube must maintain structural integrity in these compact environments, while responding to positional guidance cues that lead the pollen tube to its destination. Here, we discuss the nature of these physical barriers as well as efforts to genetically and cellularly identify the factors that allow pollen tubes to successfully, specifically, and quickly circumnavigate them.

Morphological, biochemical and molecular hallmarks of programmed cell death in stigmatic papillae of Brassica oleracea L.

The aim of this study is to determine the programmed cell death hallmarks in the stigmatic papillae of Brassica oleracea L. The flower development was divided in two main stages; pre-anthesis and post-anthesis. Programmed cell death hallmarks were examined in parallel to these stages. At pre-anthesis, the stigmatic papillae were ovoid and their dense cytoplasm were rich in insoluble polysaccharide and protein. At post-anthesis, vacuolization and enlargement were quite evident in papillae. Besides, the protein content decreased, but reactive oxygen species increased in comparison to the pre-anthesis stage. Although no significant change in superoxide dismutase activity was detected, catalase activity decreased and hydrogen peroxide content increased at post-anthesis. DAPI stained nuclei appeared rounded and smooth appearance at pre-anthesis, however, some invaginations and fragmentation in nuclei were observed at post-anthesis. Although, TUNEL staining was negative at pre-anthesis, while TUNEL positive reaction was significant in the nuclei of papillae at post-anthesis. In comparison to the pre-anthesis, the number of fragmented nuclei monitored by DAPI and TUNEL staining increased at post-anthesis.

Autophagy is required for self-incompatible pollen rejection in two transgenic Arabidopsis thaliana accessions

Successful reproduction in the Brassicaceae is mediated by a complex series of interactions between the pollen and the pistil, and some species have an additional layer of regulation with the self-incompatibility trait. While the initial activation of the self-incompatibility pathway by the pollen S-locus protein11/S-locus cysteine-rich peptide and the stigma S Receptor Kinase is well characterized, the downstream mechanisms causing self-pollen rejection are still not fully understood. In previous studies, we had detected the presence of autophagic bodies with self-incompatible pollinations in Arabidopsis lyrata and transgenic A. thaliana lines, but it was not known if autophagy was essential for self-pollen rejection. Here, we investigated the requirement of autophagy in this response by crossing mutations in the essential AUTOPHAGY7 (ATG7) gene into two different transgenic self-incompatible A. thaliana lines in the Col-0 and C24 accessions. By using these previously characterized transgenic lines that express A. lyrata and A. halleri self-incompatibility genes, we demonstrated that disrupting autophagy can weaken their self-incompatible responses in the stigma. When the atg7 mutation was present, an increased number of self-incompatible pollen were found to hydrate and form pollen tubes that successfully fertilized the self-incompatible pistils. Additionally, we confirmed the presence of GFP-ATG8a labelled autophagosomes in the stigmatic papillae following self-incompatible pollinations. Together, these findings support the requirement of autophagy in the self-incompatibility response and add to the growing understanding of the cellular events that take place in the stigma to reject self-pollen.One Sentence SummaryIn self-incompatible transgenic Arabidopsis thaliana lines, autophagy is an integral part of the cellular responses in the stigma to efficiently block fertilization by self-incompatible pollen.

Elongation of stigmatic papillae induced by heat stress is associated with disturbance of pollen attachment in Arabidopsis thaliana

ABSTRUCTHeat stress can seriously impact on yield production and quality of crops. Many studies uncovered the molecular mechanisms that regulate heat stress responses in plants. Nevertheless, effects of heat stress on the morphology of plants were still not extensively studied. In this study, we observed the detailed morphological changes of reproductive organs in Arabidopsis thaliana caused by heat stress. Larger area of stigma, and shorter length of anthers, filaments and petals were observed in plants subjected to heat stress compared to those under controlled conditions. Scanning electron microscopy (SEM) observation showed that length of stigmatic papillae without pollens seemed to be longer than that with pollens. In addition, classification of stigmas based on pollen attachment patterns together with artificial pollination assay revealed that pollen attachment onto stigma was clearly decreased by heat stress, and indicated that heat induced elongation of stigmatic papillae might be associated with disturbance of pollen attachment onto stigma. Furthermore, histochemical staining experiments revealed that crosstalk between Ca2+ and NO derived from pollens and O2− derived from stigma might be associated with morphological alteration of stigma.

Attachment-based mechanisms underlying capture and release of pollen grains

Successful insect pollination can be achieved by a sequence of numerous attachment and detachment events at various biological surfaces. However, the quantitative measurements of pollen adhesion on biological surfaces have been poorly studied so far. We performed atomic force microscopy adhesion measurements of pollen on two most important floral parts for Asteraceae in a course of pollination: the stigma and style of Hypochaeris radicata plant . The results indicated distinct adhesive properties of them—the pollen adhesion on stigmatic surfaces drastically increased over prolonged contact time, while the pollen adhesion increase on stylar surfaces was rather restrained. Based on the observation with cryo-scanning electron microscopy, we explained the experimental results by the presence of morphological features in form of flexible stigmatic papillae that may play a crucial role in enhancing both capillary attraction and van der Waals forces. The distinct adhesive properties seemingly originate from the unique adhesive tasks that each of the floral parts requires to achieve successful pollination. The insights into the adhesive interaction between pollen and the floral parts, obtained in the present study, may lead to better understanding of pollination mechanisms, which are strongly related to our food production. Additionally, the novel pollen adhesive mechanisms learned from the stigma of the studied Asteraceae plant can inspire biomimetic designs of spontaneous gripping systems.

Identification of a role for an E6-like 1 gene in early pollen-stigma interactions in Arabidopsis thaliana

In Arabidopsis, successful pollen-stigma interactions are dependent on rapid recognition of compatible pollen by the stigmatic papillae located on the surface of the pistil and the subsequent regulation of pollen hydration and germination, and followed by the growth of pollen tubes through the stigma surface. Here we have described the function of a novel gene, E6-like 1 (E6L1), that was identified through the analysis of transcriptome datasets, as one of highest-expressed genes in the stigma, and furthermore, its expression was largely restricted to the stigma and trichomes. The first E6 gene was initially identified as a highly-expressed gene during cotton fiber development, and related E6-like predicted proteins are found throughout the Angiosperms. To date, no orthologous genes have been assigned a biological function. Both the Arabidopsis E6L1 and cotton E6 proteins are predicted to be secreted, and this was confirmed using an E6L1:RFP fusion construct. To further investigate E6L1’s function, one T-DNA and two independent CRISPR-generated mutants were analyzed for compatible pollen-stigma interactions, and pollen hydration, pollen adhesion and seed set were mildly impaired for the e6l1 mutants. This work identifies E6L1 as a novel stigmatic factor that plays a role during the early post-pollination stages in Arabidopsis.Key MessageWe describe a function for a novel Arabidopsis gene, E6-like 1 (E6L1), that was identified as a highly-expressed gene in the stigma and plays a role in early post-pollination stages.

Structure of floral nectaries and comparison of reproductive and vestigial organs in the staminate and pistillate flowers of dioecious Silene latifolia (Caryophyllaceae)

Silene latifolia Poiret of Eurasia has established in North America, prompting this structural study of its mature unisexual buds and flowers. Floral nectaries, anther and stigma changes, and vestigial reproductive structures were studied using light and scanning electron microscopy. In staminate flowers, anthers dehisced before anthesis and >90% of their pollen was liberated within 36 h. Accumulated in the tubular calyx, nectar descended an anthophore from the stomatal-bearing nectary at the stamen bases. Nectary tissue surrounded the pistillode, a central filamentous organ lacking ovules but tipped by hairs resembling stigmatic papillae. In pistillate flowers, nectar flowed into an inflated calyx. The annular nectary had 10 regularly spaced, stomatal-lined craters and was continuous with the adaxial surfaces of the infertile antisepalous and epipetalous staminodes. Key elements of entomophilous pollination leading to successful sexual reproduction of this invasive species include secondary nectar presentation from disparate floral nectaries that, for pistillate flowers, also incorporate the staminodes; rapid pollen release from anthers; and elongation of papillae by tip growth that enhances each stigma’s receptive surface. Context is also provided for future studies of floral nectary development in this model dioecious species.