An interconnection between tip-focused Ca2+ and anion homeostasis controls pollen tube growth

Abstract

Plant reproduction is the basis for economically relevant food production. It relies on pollen tube (PTs) growth into the female flower organs for successful fertilization. The high cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) at the PT tip is sensed by Ca²⁺-dependent protein kinases (CPKs) that in turn activate R- and S-type anion channels to control polar growth. Lanthanum, a blocker for plant Ca²⁺-permeable channels was used here to demonstrate a strict dependency for anion channel activation through high PT tip [Ca²⁺]_cyt. We visualized this relationship by live-cell anion imaging and concurrent triggering of Ca²⁺-elevations with the two-electrode voltage-clamp (TEVC) technique. The anion efflux provoked by a TEVC-triggered [Ca²⁺]_cyt increase was abolished by Lanthanum and was followed by an overall rise in the cytosolic anion concentration. An interrelation between Ca²⁺ and anion homeostasis occurred also on the transcript level of CPKs and anion channels. qRT-PCR analysis demonstrated a co-regulation of anion channels and CPKs in media with different Cl^- and NO₃^- compositions. Our data provides strong evidence for the importance of a Ca²⁺-dependent anion channel regulation and point to a synchronized adjustment of CPK and anion channel transcript levels to fine-tune anion efflux at the PT tip.

Keywords: ALMT12; CPK; Ca2+-permeable channel; Pollen tube growth; R-type anion channel; S-type anion channel; SLAH3; anion channel; calcium gradient; calcium-dependent protein kinase; live-cell imaging.

Figure 1.

Pollen tube tip anion release depends on apical Ca²⁺-channel activity. The TEVC technique was used simultaneously with live-cell anion imaging with a FRET-based Cl^–-Sensor during repetitive −200 mV induced [Ca²⁺]_cyt increases in PTs. A brightfield and false color-coded FRET-ratio image of a PT expressing the Cl^–-Sensor is shown (left). One kymograph of a representative cytosolic anion concentration ([anion]_cyt) time-lapse imaging series (out of n = 9) with 1 Hz imaging intervals is displayed. Five repetitive −200 mV (hyperpolarization) voltage pulses followed by five +60 mV (depolarization) voltage pulses were applied with an interval of 15 sec, followed by a continuous series of −200 mV pulses with an interval of 20 sec as indicated by the upper bars. Application of 100 µM LaCl₃ as pointed out by the bar abolished the repetitive decrease in anion-ratio signal at the tip. The presence of Lanthanum did not influence the −200 mV-induced rhythmic decrease of the ratio signal at the electrode insertion site indicating a Ca²⁺-leak there. Note that the presence of LaCl₃ increased the overall anion concentration, a cause of blocking Ca²⁺-permeable channels and thus Ca²⁺-signaling for anion release.

Figure 2.

Interrelation between anion channel and CPK expression levels in different pollen tube growth conditions. A quantification of PT transcripts of all (A) R- and (B) S-type anion channels as well as (C) selected CPKs was performed by qRT-PCR on PT cDNA grown in three distinct media containing different anion concentrations (each n = 6). The semi-solid growth medium consisted of (in mM): 1.6 H₃BO₃, 1 MES/pH 5.8 with TRIS and either 20 Ca(NO₃⁻)₂ (white bar), 20 CaCl₂ (black bar) or 0.5 Ca(NO₃⁻)₂ + 0.5 CaCl₂ + 19 Ca²⁺-Gluconate (patterned bar). Asterisks *, ** and *** indicate unpaired t test values of p < 0.05, p < 0.005 and p < 0.0005, respectively.