## 4 Conclusions  
In this study, the effect of GO interaction with the pollen\(-\)stigma system was verified on the entire reproduction process of the model plant, _C. pepo_, which is also an economically important vegetable. The stigmatic surface integrity was not compromised by GO; still, pollen adhesion and germination over the stigma decreased, fruit development was altered, and seed production was completely suppressed. The similar effect of GO and PGO and the comparable effect of further 2D-NMs (i.e., FLG and muscovite mica) support the hypothesis that the physical superposition of planar nanoparticles between pollen and stigma compromises the reproduction process by lowering the pollen load and affecting pollen-stigma signaling. However, the necrotic effect of GO on fruits also suggests a chemical interaction of this material (and its potential contaminants) with the plant tissues,  
Figure 4: Photographs of 5-week-old fruits of _Cucurbita pepo_ L., entire (left column) or dissected (right column), originated from stigmas coated with 0 (CTRL) (**a**,**b**) or 1 mg of graphene oxide (GO) (**c**,**d**) and purified GO (PGO) (**e**,**f**), and pollinated after three hours (for more details, see Sections 2.2 and 2.5). GO flakes/nanoparticles, seeds and dead tissues are indicated with arrows, arrowheads and asterisks, respectively. Bars = 2 cm.  
similar to other reactive substances. Our samples were exposed to high amounts of GO which were unlikely to occur in the environment in a plant whose flowers remain open for a few hours only. Nonetheless, this allowed us to highlight possible effects due to GO depositions on stigmas caused by very localized and abundant releases of these materials, such as sprays of GO-based fungicides or pesticides. Future research should test quantities more similar to what is expected to be found in the environment and with techniques mimicking atmospheric dry depositions. Special attention should be focused on anemophilous plants, whose flowers remain exposed to airborne particulate for days or even weeks.  
The following are available online at [[https://www.mdpi.com/article/10.3390/app11136150/s1](https://www.mdpi.com/article/10.3390/app11136150/s1)]([https://www.mdpi.com/article/10.3390/app11136150/s1](https://www.mdpi.com/article/10.3390/app11136150/s1)), Table S1: GLM results of pollen adhesion % in in vivo germination, Table S2: GLM results of fresh mass, length, maximum circumference of fruits and number of seeds produced per fruit.  
Conceptualization, D.Z., F.C.C. and M.T.; methodology, D.Z., F.C.C. and M.T.; validation, D.Z. and F.C.C.; formal analysis, D.Z.; investigation, D.Z. and F.C.C.; resources, D.Z. and F.C.C.; writing--original draft preparation, D.Z. and F.C.C.; writing--review and editing, D.Z., F.C.C. and M.T.; visualization, D.Z., F.C.C. and M.T.; supervision, F.C.C. and M.T.; project administration, F.C.C. and M.T.; funding acquisition, M.T. All authors have read and agreed to the published version of the manuscript.  
This work was supported by the Graphene Flagship Core 2 and Core 3 grant agreement (no. 785219 and 881603, respectively), the University of Trieste DOTTAMBIENTEVITA34-18.  
Not applicable.  
The data presented in this study are available on request from the corresponding author.  
The authors would like to thank [PERSON] (Fondi) for the advice and discussion on plants growth, [PERSON] and [PERSON] (University of Trieste), for the help with the ESEM analysis.  
The authors declare no conflict of interest.