In vivo cross-linking combined with mass spectrometry analysis reveals receptor-like kinases and Ca2+ signalling proteins as putative interaction partners of pollen plasma membrane H+ ATPases

During fertilisation in plants, pollen grains germinate and generate a pollen tube which grows through the style tissue to the egg apparatus delivering the two sperm cells for fertilisation. For this process, adaption to specific environmental conditions and communication between male and female organs are essential, requiring the sensing of internal and external signals which are translated into tube growth. The plasma membrane (PM) H+ ATPase energises the pollen plasma membrane for nutrient, ion and water uptake, but additionally, its activity directly affects the germination frequency and drives the elongation of pollen tubes. A combination of in vivo cross-linking with para-formaldehyde, immunoaffinity purification of cross-linked PM H+ ATPase complexes and subsequent mass spectrometry analysis revealed putative interaction partners of the PM H+ ATPase of lily pollen, which are possibly involved in the perception and transduction of intra- and extracellular signals. Major interactions partners included (i) membrane-localised receptor-like kinases (RLKs) with the leucine-rich repeat RLKs (LRR-RLKs) forming the largest group, (ii) interacting protein kinases, phosphatases, WD-40 domain proteins and 14-3-3 proteins that may transduce intracellular, phosphorylation-dependent signals and (iii) specific cytosolic Ca2+ signatures may be decoded by interacting Ca2+ sensor proteins, calmodulin and calmodulin-like proteins, and Ca2+-dependent protein kinases, which were all identified as interaction partners of the PM H+ ATPase in lily pollen. These identified interaction partners suggest new putative regulation mechanisms of the PM H+ ATPase in general and new insights in regulating pollen tube growth rates in particular. Furthermore, the optimised experimental strategy can be applied to other non-model organisms to identify membrane protein interactions.