Vacuolar CBL-CIPK12 Ca(2+)-sensor-kinase complexes are required for polarized pollen tube growth.
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.
Institut für Biologie und Biotechnologie der Pflanzen, Westfälische Wilhelms-Universität, 48149 Münster, Germany.
Leonie
L
Steinhorst
Institut für Biologie und Biotechnologie der Pflanzen, Westfälische Wilhelms-Universität, 48149 Münster, Germany.
Anette
A
Mähs
Albrecht-von-Haller-Institut für Pflanzenwissenschaften, Abteilung Biochemie der Pflanze, Georg-August-Universität Göttingen, 37077 Göttingen, Germany.
Till
T
Ischebeck
Institut für Biologie und Biotechnologie der Pflanzen, Westfälische Wilhelms-Universität, 48149 Münster, Germany.
Chunxia
C
Zhang
Institut für Biologie und Biotechnologie der Pflanzen, Westfälische Wilhelms-Universität, 48149 Münster, Germany.
Xinxin
X
Zhang
Institut für Biologie und Biotechnologie der Pflanzen, Westfälische Wilhelms-Universität, 48149 Münster, Germany.
Sibylle
S
Arendt
Institut für Biologie und Biotechnologie der Pflanzen, Westfälische Wilhelms-Universität, 48149 Münster, Germany.
Stefanie
S
Schültke
Institut für Biochemie und Biotechnologie, Abteilung Zelluläre Biochemie, Martin-Luther-Universität Halle-Wittenberg, 06120 Halle (Saale), Germany.
Ingo
I
Heilmann
Institut für Biologie und Biotechnologie der Pflanzen, Westfälische Wilhelms-Universität, 48149 Münster, Germany. Electronic address: jkudla@uni-muenster.de.
Jörg
J
Kudla
D008841:Actin Cytoskeleton / Q000378:metabolism; D017360:Arabidopsis / Q000502:physiology*; D029681:Arabidopsis Proteins / Q000378:metabolism*; D020013:Calcium Signaling; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D010641:Phenotype; D053205:Pollen Tube / Q000254:growth & development*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D014617:Vacuoles / Q000201:enzymology
D029681:Arabidopsis Proteins; D002135:Calcium-Binding Proteins; D017346:Protein Serine-Threonine Kinases; C417306:CBL protein, Arabidopsis; C480758:At5g55990 protein, Arabidopsis; C484079:CBL3 protein, Arabidopsis; C000605949:CIPK12 protein, Arabidopsis