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 Ca(2+) gradient governs ions fluxes, vesicle transport, and cytoskeleton dynamics to ensure proper polarized cell growth [1, 2]. While a crucial role of vacuolar Ca(2+) 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 Ca(2+)-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)P2 distribution, or tip-focused Ca(2+) oscillations. Similarly, loss of function of each single Ca(2+) sensor and cbl2/cbl3 double mutants exhibited impaired pollen tube growth in vitro and in vivo. Both Ca(2+) 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 Ca(2+) signaling for polarized tip growth. We propose that a faithfully balanced activity of Ca(2+)-activated CBL2/3-CIPK12 complexes fulfills fundamental functions to enable the fast growth of pollen tubes in higher plants.

Advances and current challenges in calcium signaling

“…Mechanical forces most likely trigger Ca 2+ signals during pollen tube bursting, and FERONIA is also involved in Ca 2+ responses to mechanical forces in roots (Shih et al, 2014). Interestingly, it was recently reported that Ca 2+ signaling processes at the vacuolar membrane are crucial for pollen tube growth and plant fertility, pointing to a so far not appreciated additional role of Ca 2+ in these processes (Steinhorst et al, 2015). The first example for multiparameter analyses was the simultaneous visualization of Ca 2+ and ABA (Waadt et al, 2017).…”

Signaling with Ions: The Keystone for Apical Cell Growth and Morphogenesis in Pollen Tubes

“…They display an even distribution throughout the cytosol while being weakly expressed in other tissues (Zhou et al, 2015a). Different AtCBL/AtCIPK pairs have been implicated in the activation of various kinds of channels (for review, see Steinhorst and Kudla, 2013), and in PTs, AtCBL2 and AtCBL3 were described to interact with AtCIPK12 in the tonoplast, with phenotypic consequences at the level of growth and PT morphogenesis, implicating the vacuole in Ca 2+ homeostasis and signaling (Steinhorst et al, 2015). A possible integration of these pathways is offered in Figure 4.…”

N‐terminal S‐acylation facilitates tonoplast targeting of the calcium sensor CBL6

“…More recent work has uncovered that four of the ten CBLs are localized at the tonoplast membrane [17], and reverse genetic analyses of the calcium sensors CBL2 and CBL3 mutants have uncovered their function in regulating cellular ion homeostasis and their essential role for Arabidopsis pollen, seed and early embryonal development [18][19][20]. Subsequent studies of CIPK quadruple mutants identified CIPK3/ 9/23/26 as the CBL2/3 interacting kinases functioning in regulating cellular magnesium homeostasis [21,22].…”