Combined Proteomic and Cytological Analysis of Ca²⁺-Calmodulin Regulation in Picea meyeri Pollen Tube Growth

Abstract

Ca²⁺-calmodulin (Ca²⁺-CaM) is a critical molecule that mediates cellular functions by interacting with various metabolic and signaling pathways. However, the protein expression patterns and accompanying serial cytological responses in Ca²⁺-CaM signaling deficiency remain enigmatic. Here, we provide a global analysis of the cytological responses and significant alterations in protein expression profiles after trifluoperazine treatment in Picea meyeri, which abrogates Ca²⁺-CaM signaling. Ninety-three differentially displayed proteins were identified by comparative proteomics at different development stages and were assigned to different functional categories closely related to tip growth machinery. The inhibition of Ca²⁺-CaM signaling rapidly induced an increase in extracellular Ca²⁺ influx, resulting in dramatically increased cytosolic Ca²⁺ concentrations and ultrastructural abnormalities in organelles as the primary responses. Secondary and tertiary alterations included actin filament depolymerization, disrupted patterns of endocytosis and exocytosis, and cell wall remodeling, ultimately resulting in perturbed pollen tube extension. In parallel with these cytological events, time-course experiments revealed that most differentially expressed proteins showed time-dependent quantitative changes (i.e. some signaling proteins and proteins involved in organelle functions and energy production changed first, followed by alterations in proteins related to cytoskeletal organization, secretory pathways, and polysaccharide synthesis). Taken together, Ca²⁺-CaM dysfunction induced serial cytological responses and temporal changes in protein expression profiles, indicating the pivotal role of Ca²⁺-CaM in the regulation of tip growth machinery.