title: 21-kDa polypeptide, a low-molecular-weight cyclophilin, is released from pollen of higher plants into the extracellular medium in vitro.
abstract: Calcium ions play a key role in the elongation and orientation of pollen tubes. We found that significant amounts of 21-kDa polypeptide were specifically released into the extracellular medium when pollen grains of lily, Lilium longiflorum Thunb., were incubated in the presence of EGTA or at low concentrations of Ca2+. This phenomenon was also dependent on pH and on the concentrations of MgCl2 in the medium; the release of 21-kDa polypeptide from pollen was suppressed by increasing the MgCl2 concentration and by lowering pH. Germination of pollen grains was inhibited in the medium into which the 21-kDa polypeptide had been released. This inhibition was irreversible; germination did not occur on transfer of the pollen grains into basal culture medium. Immuno-electron microscopy using an antibody against 21-kDa polypeptide showed that this polypeptide was present in the cytoplasm, vegetative nucleus and generative cell. When the pollen was treated with a medium containing EGTA, the density of 21-kDa polypeptide in the cytoplasm significantly decreased, but its density in vegetative nuclei and the generative cell did not, suggesting that only cytoplasmic 21-kDa polypeptide was released into the extracellular medium. The 21-kDa polypeptide was also present in the pollen of other higher-plant species, such as Tradescantia virginiana L., Nicotiana tabacum L. (angiosperms), and Cryptomeria japonica D. Don. (gymnosperm), and was also released into the medium in the presence of EGTA. In the case of C. japonica, however, it was released from pollen at alkaline pH above 8.5. The expression of 21-kDa polypeptide was not pollen-specific, because 21-kDa components immunoreactive with the anti-21-kDa polypeptide serum also existed in vegetative organs and cells of lily or tobacco. However, the 21-kDa polypeptide was not released into the extracellular medium from cultured tobacco BY-2 cells, even in the presence of EGTA. Amino acid sequences of two peptide fragments derived from 21-kDa polypeptide matched well those of low-molecular-weight cyclophilin (CyP). The antiserum against 21-kDa polypeptide recognized the CyP A from calf thymus and that in A431 carcinoma cells. The 21-kDa polypeptide fraction purified from lily pollen possessed peptidyl-prolyl cis- trans isomerase activity, which was suppressed by cyclosporin A (CsA), an inhibitor of enzyme activities of CyPs. From these results, we concluded that the 21-kDa polypeptide is a low-molecular-weight CyP. The present study showed that CyP in the pollen of higher plants is released into the extracellular matrix under unfavorable conditions.
authors.affiliation: Department of Life Science, Graduate School of Science, Himeji Institute of Technology, Harima Science Park City, 678-1297 Hyogo, Japan. yokota@sci.himeji-tech.ac.jp
authors.forename: Etsuo
authors.initials: E
authors.lastname: Yokota
authors.forename: Takahiro
authors.initials: T
authors.lastname: Ohmori
authors.forename: Shoshi
authors.initials: S
authors.lastname: Muto
authors.forename: Teruo
authors.initials: T
authors.lastname: Shimmen
mesh_terms: D000595:Amino Acid Sequence; D003470:Culture Media; D021983:Cyclophilins / Q000302:isolation & purification / Q000378:metabolism* / Q000502:physiology*; D018525:Germination; D006863:Hydrogen-Ion Concentration; D027762:Lilium / Q000201:enzymology; D008970:Molecular Weight; D011058:Pollen / Q000201:enzymology*
chemical_list: D003470:Culture Media; D021983:Cyclophilins