Vacuolar CBL-CIPK12 Ca2+-Sensor-Kinase Complexes Are Required for Polarized Pollen Tube Growth

Abstract

Polarized tip growth is a fundamental process of specialized eukaryotic cells like neuronal axons, fungal hyphae, and plant root hairs and pollen tubes. In pollen tubes, a tip-focused oscillating Ca2+ gradient governs ions fluxes, vesicle transport, and cytoskeleton dynamics to ensure proper polarized cell growth [1, 2]. While a crucial role of vacuolar Ca2+ signaling is established for cellular movements like guard cell dynamics [3-5], its contribution to polarized growth remains to be defined. Here we identified the two closely related tonoplast-localized Ca2+-sensor proteins CBL2 and CBL3 as crucial regulators of vacuolar dynamics and polarized pollen tube growth. Overexpression of CBL2 or CBL3 in Arabidopsis and tobacco pollen tubes affected vacuolar morphology, pollen germination, and tube growth, but did not alter actin organization, PI(4,5)P-2 distribution, or tip-focused Ca2+ oscillations. Similarly, loss of function of each single Ca2+ sensor and cbl2/cbl3 double mutants exhibited impaired pollen tube growth in vitro and in vivo. Both Ca2+ sensors interacted with the kinase CIPK12, which translocated from the cytoplasm to the vacuolar membrane upon this interaction. Also, overexpression of CIPK12 induced severe vacuolar phenotypes, and loss of function of CIPK12 lead to impairment of polar growth. Remarkably, co-expression of CBL2 or CBL3 with CIPK12 resulted in a phosphorylation-dependent, massively enhanced vacuolar inflation and further disruption of polar growth. Together, these findings identify an essential role of the vacuole and vacuolar Ca2+ signaling for polarized tip growth. We propose that a faithfully balanced activity of Ca2+-activated CBL2/3-CIPK12 complexes fulfills fundamental functions to enable the fast growth of pollen tubes in higher plants.