Exogenous γ-aminobutyric acid (GABA) affects pollen tube growth via modulating putative Ca2+-permeable membrane channels and is coupled to negative regulation on glutamate decarboxylase.

γ-Aminobutyric acid (GABA) is implicated in pollen tube growth, but the molecular and cellular mechanisms that it mediates are largely unknown. Here, it is shown that exogenous GABA modulates putative Ca(2+)-permeable channels on the plasma membranes of tobacco pollen grains and pollen tubes. Whole-cell voltage-clamp experiments and non-invasive micromeasurement technology (NMT) revealed that the influx of Ca(2+) increases in pollen tubes in response to exogenous GABA. It is also demonstrated that glutamate decarboxylase (GAD), the rate-limiting enzyme of GABA biosynthesis, is involved in feedback controls of Ca(2+)-permeable channels to fluctuate intracellular GABA levels and thus modulate pollen tube growth. The findings suggest that GAD activity linked with Ca(2+)-permeable channels relays an extracellular GABA signal and integrates multiple signal pathways to modulate tobacco pollen tube growth. Thus, the data explain how GABA mediates the communication between the style and the growing pollen tubes.

Reciprocal regulation of Ca²+-activated outward K+ channels of Pyrus pyrifolia pollen by heme and carbon monoxide.

• The regulation of plant potassium (K+) channels has been extensively studied in various systems. However, the mechanism of their regulation in the pollen tube is unclear. • In this study, the effects of heme and carbon monoxide (CO) on the outward K+ (K+(out)) channel in pear (Pyrus pyrifolia) pollen tube protoplasts were characterized using a patch-clamp technique. • Heme (1 µM) decreased the probability of K+(out) channel opening without affecting the unitary conductance, but this inhibition disappeared when heme was co-applied with 10 µM intracellular free Ca²+. Conversely, exposure to heme in the presence of NADPH increased channel activity. However, with tin protoporphyrin IX treatment, which inhibits hemeoxygenase activity, the inhibition of the K+(out) channel by heme occurred even in the presence of NADPH. CO, a product of heme catabolism by hemeoxygenase, activates the K+(out) channel in pollen tube protoplasts in a dose-dependent manner. The current induced by CO was inhibited by the K+ channel inhibitor tetraethylammonium. • These data indicate a role of heme and CO in reciprocal regulation of the K+(out) channel in pear pollen tubes.

Identification of hyperpolarization-activated calcium channels in apical pollen tubes of Pyrus pyrifolia.

The pollen tube has been widely used to study the mechanisms underlying polarized tip growth in plants. A steep tip-to-base gradient of free cytosolic calcium ([Ca(2+)](cyt)) is essential for pollen-tube growth. Local Ca(2+) influx mediated by Ca(2+)-permeable channels plays a key role in maintaining this [Ca(2+)](cyt) gradient. Here, we developed a protocol for successful isolation of spheroplasts from pollen tubes of Pyrus pyrifolia and identified a hyperpolarization-activated cation channel using the patch-clamp technique. We showed that the cation channel conductance displayed a strong selectivity for divalent cations, with a relative permeability sequence of barium (Ba(2+)) approximately Ca(2+) > magnesium (Mg(2+)) > strontium (Sr(2+)) > manganese (Mn(2+)). This channel conductance was selective for Ca(2+) over chlorine (Cl(-)) (relative permeability P(Ca)/P(Cl) = 14 in 10 mm extracellular Ca(2+)). We also showed that the channel was inhibited by the Ca(2+) channel blockers lanthanum (La(3+)) and gadolinium (Gd(3+)). Furthermore, channel activity depended on extracellular pH and pollen viability. We propose that the Ca(2+)-permeable channel is likely to play a role in mediating Ca(2+) influx into the growing pollen tubes to maintain the [Ca(2+)](cyt) gradient.