Fast loading ester fluorescent Ca2+ and pH indicators into pollen of Pyrus pyrifolia

Abstract

Loading of Ca(2+)-sensitive fluorescent probes into plant cells is an essential step to measure activities of free Ca(2+) ions in cytoplasm with a fluorescent imaging technique. Fluo-3 is one of the most suitable Ca(2+) indicators for CLSM. We loaded pollen with fluo-3/AM at three different temperatures. Fluo-3/AM was successfully loaded into pollen at both low (4°C) and high (37°C) temperatures. However, high loading temperature was best suited for pollen, because germination rate of pollen and growth of pollen tubes were relatively little impaired and loading time was shortened. Moreover, Ca(2+) distribution increased in the three apertures of pollen after hydration and showed a Ca(2+) gradient, similar to the tip of growing pollen tubes. The same protocol can be used with the AM-forms of other fluorescent dyes for effective labeling. When loading BCECF-AM into pollen at high temperature, the pollen did not show a pH gradient after hydration. Ca(2+) activities and fluxes had the same periodicity as pollen germination, but pH did not show the same phase and mostly lagged behind. However, the clear zone was alkaline when pollen tube growth was slowed or stopped and turned acidic when growth recovered. It is likely that apical pH(i) regulated pollen tube growth.

Investigation and Prevention of Cork Spot Disorder in ‘Akizuki’ Pear (Pyrus pyrifolia Nakai)

“…Observation of Ca 2+ localization. The localization of free Ca 2+ was observed by fluorescence imaging as previously described by Qu et al (2012), with some modifications. Thin slices of flesh were collected from the same region under the peel tissue of normal and cork spot fruit by using a razor blade.…”

Arabidopsis thaliana CML25 mediates the Ca2+ regulation of K+ transmembrane trafficking during pollen germination and tube elongation

“…The fluorescent calcium indicator Fluo-3/AM was loaded into pollen grains and pollen tubes and subsequent measurement of [Ca 2+ ]cyt was performed using a published protocol (Qu et al 2012). In brief, pollen grains were hydrated at 28°C for 30 min, incubated with Fluo-3/AM at 37°C for 30 min, washed three times with the culture medium and then cultivated in the dark for 30 min at 20°C.…”

The Role of Ethylene and Calcium in Programmed Cell Death of Cold-Stored Cucumber Fruit

“…The histological sections with about 100 μm thickness comprising of epidermis and hypodermis were free-hand sliced. The following process was done with reference to Qu et al (2012) and there were some improvements. The slices were washed twice by 25 M 4-(2-hydroxyethyl)-1-piperazineethane-sulfonic acid (HEPES, ≥99.0%, Amresco) buffer solution (pH 7.0), then incubated at 4C for 2 h and followed by 1 h incubation at 37C in the dark.…”