The involvement of calcium and calcium-activated calmodulin (Ca2-CaM) in heat shock (HS) signal transduction in wheat (Triticum aestivum) was investigated. Using Fluo-3/acetoxymethyl esters and laser scanning confocal microscopy, it was found that the increase of intracellular free calcium ion concentration started within 1 min after a 37℃ HS. The levels of CaM mRNA and protein increased during HS at 37℃ in the presence of Ca2. The expression of hsp26 and hsp70 genes was up-regulated by the addition of CaCl2 and down-regulated by the calcium ion chelator EGTA, the calcium ion channel blockers LaCl3 and verapamil, or the CaM antagonists N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide and chlorpromazine. Treatment with Ca2+ also increased, and with EGTA, verapamil, chlorpromazine, or trifluoperazine decreased, synthesis of HS proteins. The temporal expression of the CaM1-2 gene and the hsp26 and hsp70 genes demonstrated that up-regulation of the CaM1-2 gene occurred at 10 min after HS at 37℃, whereas that of hsp26 and hsp70 appeared at 20 min after HS. A 5-min HS induced expression of hsp26 after a period of recovery at 22℃ after HS at 37℃. Taken together, these results indicate that Ca2-CaM is directly involved in the HS signal transduction pathway. A working hypothesis about the relationship between upstream and downstream of HS signal transduction is presented.

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 ([Ca2+]cyt) is essential for pollen-tube growth. Local Ca2+ influx mediated by Ca2+-permeable channels plays a key role in maintaining this [Ca2+]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 (Ba2+)≈Ca2+>magnesium (Mg2+)> strontium (Sr2+)>manganese (Mn2+). This channel conductance was selective for Ca2+ over chlorine (Cl-) (relative permeability PCa/PCl=14 in 10 mM extracellular Ca2+). We also showed that the channel was inhibited by the Ca2+ channel blockers lanthanum (La3+) and gadolinium (Gd3+). Furthermore, channel activity depended on extracellular pH and pollen viability. We propose that the Ca2+-permeable channel is likely to play a role in mediating Ca2+ influx into the growing pollen tubes to maintain the [Ca2+]cyt gradient.

The role of heterotrimeric G proteins in pollen germination and tube growth was investigated using Arabidopsis thaliana plants in which the gene (GPA) encoding the G-protein α subunit (G α) was null or overexpressed. Pollen germination, free cytosolic calcium concentration ([Ca2+ ] cyt) and Ca2+ channel activity in the plasma membrane (PM) of pollen cells were investigated. Results showed that, compared with pollen grains of the wild type (ecotype Wassilewskija, ws), in vitro germinated pollen of G α null mutants (gpa1-1 and gpa1-2) had lower germination percentages and shorter pollen tubes, while pollen from G α overexpression lines (wG α and cG α) had higher germination percentages and longer pollen tubes. Compared with ws pollen cells, [Ca2+] cyt was lower in gpa1-1 and gpa1-2 and higher in wG α and cG α. In whole-cell patch clamp recordings, a hyperpolarization-activated Ca2+-permeable conductance was identified in the PM of pollen protoplasts. The conductance was suppressed by trivalent cations but insensitive to organic blockers; its permeability to divalent cations was Ba2+>Ca2+>Mg2+>Sr2+>Mn2+. The activity of the Ca2+-permeable channel conductance was down-regulated in pollen protoplasts of gpa1-1 and gpa1-2, and up-regulated in wG α and cG α. The results suggest that G α may participate in pollen germination through modulation of the hyperpolarization-activated Ca2+ channel in the PM of pollen cells.