title: 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.
authors.affiliation: College of Horticulture, Nanjing Agricultural University, No.1. WeiGang, Nanjing, Jiangsu, China. haiyongqu@126.com
authors.forename: Haiyong
authors.initials: H
authors.lastname: Qu
authors.forename: Xueting
authors.initials: X
authors.lastname: Jiang
authors.forename: Zebin
authors.initials: Z
authors.lastname: Shi
authors.forename: Lianmei
authors.initials: L
authors.lastname: Liu
authors.forename: Shaoling
authors.initials: S
authors.lastname: Zhang
mesh_terms: D000814:Aniline Compounds / Q000378:metabolism*; D017360:Arabidopsis / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000378:metabolism; D004533:Egtazic Acid / Q000494:pharmacology; D005452:Fluoresceins / Q000378:metabolism*; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000378:metabolism*; D018525:Germination / Q000187:drug effects; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D007202:Indicators and Reagents; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism; D018613:Microscopy, Confocal; D018517:Plant Roots / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D011058:Pollen / Q000166:cytology / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D053205:Pollen Tube / Q000166:cytology / Q000187:drug effects / Q000254:growth & development; D031989:Pyrus / Q000166:cytology / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D015203:Reproducibility of Results; D013045:Species Specificity; D013696:Temperature; D013997:Time Factors; D014966:Xanthenes / Q000378:metabolism*
chemical_list: D000814:Aniline Compounds; D002135:Calcium-Binding Proteins; D005452:Fluoresceins; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D014966:Xanthenes; C495639:yellow cameleon; C057433:2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester; C059715:Fluo-3; D004533:Egtazic Acid; D002118:Calcium