Generation of nitric oxide by olive (Olea europaea L.) pollen during in vitro germination and assessment of the S-nitroso- and nitro-proteomes by computational predictive methods

- Positive correlation between NO production and pollen viability is demonstrated.
- Olive pollen emits NO/GSNO to the culture medium in a time dependent manner.
- Exogenous NO inhibits pollen germination and tube growth.
- Disturbed pollen tube morphology occurs after treatment with either NO donors or NO scavenger.
- Pollen predictive S-nitroso- and nitro-proteomes are highly complex and functional.
- Such NO-PTM proteins can be detected and identified by using specific antibodies.

Nitric oxide is recognized as a signaling molecule involved in a broad range of physiological processes in plants including sexual reproduction. NO has been detected in the pollen grain at high levels and regulates pollen tube growth. Previous studies demonstrated that NO as well as ROS are produced in the olive reproductive tissues in a stage- and tissue-specific manner. The aim of this study was to assess the production of NO throughout the germination of olive (Olea europaea L.) pollen in vitro. The NO fluorescent probe DAF-2DA was used to image NO production in situ, which was correlated to pollen viability. Moreover, by means of a fluorimetric assay we showed that growing pollen tubes release NO. GSNO -a mobile reservoir of NO, formed by the S-nitrosylation of NO with reduced glutathione (GSH) - was for the first time detected and quantified at different stages of pollen tube growth using a LC-ES/MS analysis. Exogenous NO donors inhibited both pollen germination and pollen tube growth and these effects were partially reverted by the specific NO-scavenger c-PTIO. However, little is known about how NO affects the germination process. With the aim of elucidating the putative relevance of protein S-nitrosylation and Tyr-nitration as important post-translational modifications in the development and physiology of the olive pollen, a de novo assembled and annotated reproductive transcriptome from olive was challenged in silico for the putative capability of transcripts to become potentially modified by S-nitrosylation/Tyr-nitration according to well-established criteria. Numerous gene products with these characteristics were identified, and a broad discussion as regards to their potential role in plant reproduction was built after their functional classification. Moreover, the importance of both S-nitrosylation/Tyr-nitrations was experimentally assessed and validated by using Western blotting, immunoprecipitation and proteomic approaches.