Pollen Tube Growth Regulation by Free Anions Depends on the Interaction between the Anion Channel SLAH3 and Calcium-Dependent Protein Kinases CPK2 and CPK20

Abstract

(J.A.F.).Apical growth in pollen tubes (PTs) is associated with the presence of tip-focused ion gradients and fluxes, implying polar localization or regulation of the underlying transporters. The molecular identity and regulation of anion transporters in PTs is unknown. Here we report a negative gradient of cytosolic anion concentration focused on the tip, in negative correlation with the cytosolic Ca 2+ concentration. We hypothesized that a possible link between these two ions is based on the presence of Ca 2+ -dependent protein kinases (CPKs). We characterized anion channels and CPK transcripts in PTs and analyzed their localization. Yellow fluorescent protein (YFP) tagging of a homolog of SLOW ANION CHANNEL-ASSOCIATED1 (SLAH3:YFP) was widespread along PTs, but, in accordance with the anion efflux, CPK2/CPK20/CPK17/CPK34:YFP fluorescence was strictly localized at the tip plasma membrane. Expression of SLAH3 with either CPK2 or CPK20 (but not CPK17/CPK34) in Xenopus laevis oocytes elicited S-type anion channel currents. Interaction of SLAH3 with CPK2/CPK20 (but not CPK17/CPK34) was confirmed by Förster-resonance energy transfer fluorescence lifetime microscopy in Arabidopsis thaliana mesophyll protoplasts and bimolecular fluorescence complementation in living PTs. Compared with wild-type PTs, slah3-1 and slah3-2 as well as cpk2-1 cpk20-2 PTs had reduced anion currents. Double mutant cpk2-1 cpk20-2 and slah3-1 PTs had reduced extracellular anion fluxes at the tip. Our studies provide evidence for a Ca 2+ -dependent CPK2/CPK20 regulation of the anion channel SLAH3 to regulate PT growth.

Arabidopsis CALCIUM-DEPENDENT PROTEIN KINASE8 and CATALASE3 Function in Abscisic Acid-Mediated Signaling and H2O2 Homeostasis in Stomatal Guard Cells under Drought Stress

“…Furthermore, CPK12 serves as a negative ABA-signaling regulator in seed germination and postgermination growth, which is different from the function of its homologs, CPK4 and CPK11 (Zhao et al, 2011). In addition to abiotic stress, some Arabidopsis CDPKs have been reported to be involved in the plant innate immune response (CPKs 4, 5, 6, and 11 [Boudsocq et al, 2010], CPK1 [Coca and San Segundo, 2010], and CPK5 [Dubiella et al, 2013]), herbivoryinduced signaling network (CPK3 and CPK13; Kanchiswamy et al, 2010), regulation of pollen tube growth (CPK17 and CPK34 [Myers et al, 2009], CPK11 and CPK24 , CPK2 and CPK20 [Gutermuth et al, 2013], and CPK32 [Zhou et al, 2014]), and stem elongation and vascular development (CPK28; Matschi et al, 2013).…”

Genome-wide analysis and expression profiling of calcium-dependent protein kinases in potato (Solanum tuberosum)

“…Twelve members of the Arabidopsis CDPK gene family are predominantly expressed in pollen (Myers et al 2009). For AtCPK2/AtCPK20 and AtCPK17/AtCPK34, a role in pollen tube growth has been demonstrated experimentally in double mutant lines (Gutermuth et al 2013). StCPK13, expressed exclusively in potato flowers, is very similar to AtCPK2/AtCPK20, while StCPK18, highly expressed in potato flowers, is closely related to AtCPK17/AtCPK34.…”

“…StCPK13, expressed exclusively in potato flowers, is very similar to AtCPK2/AtCPK20, while StCPK18, highly expressed in potato flowers, is closely related to AtCPK17/AtCPK34. AtCPK2/CPK20 and AtCPK17/AtCPK34 are involved in the regulation of pollen tube growth, and AtCPK2/CPK20 influence the anion channel SLAH3 (Gutermuth et al 2013). Moreover, PiCDPK1 from petunia (Petunia inflata), similar to AtCPK17/AtCPK34, has also been implicated in pollen tube growth polarity .…”

Investigation of the regulation mechanism of Arabidopsis thaliana anion channel SLAH2

“…SLAH3 has an overlapping function with SLAC1 in guard cells for stomatal closure, but it exhibits a stronger selectivity for nitrate over chloride compared with SLAC1 (Geiger et al, 2011). On the other hand, SLAH3 is also highly expressed in roots, mesophyll cells, and pollen tubes (Geiger et al, 2009;Gutermuth et al, 2013;Zheng et al, 2015). SLAH3 is regulated by CPK2 and CPK20 in a Ca 2+ -dependent manner and is involved in the regulation of pollen tube growth (Gutermuth et al, 2013).…”

“…On the other hand, SLAH3 is also highly expressed in roots, mesophyll cells, and pollen tubes (Geiger et al, 2009;Gutermuth et al, 2013;Zheng et al, 2015). SLAH3 is regulated by CPK2 and CPK20 in a Ca 2+ -dependent manner and is involved in the regulation of pollen tube growth (Gutermuth et al, 2013). SLAH3 also plays a role in nitrate-dependent alleviation of ammonium toxicity in Arabidopsis roots (Zheng et al, 2015).…”