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Regulation of callose synthase activity in situ in alamethicin-permeabilized Arabidopsis and tobacco suspension cells

Aidemark, Mari LU ; Andersson, Carl-Johan; Rasmusson, Allan LU and Widell, Susanne LU (2009) In BMC Plant Biology 9.
Abstract
Background: The cell wall component callose is mainly synthesized at certain developmental stages and after wounding or pathogen attack. Callose synthases are membrane-bound enzymes that have been relatively well characterized in vitro using isolated membrane fractions or purified enzyme. However, little is known about their functional properties in situ, under conditions when the cell wall is intact. To allow in situ investigations of the regulation of callose synthesis, cell suspensions of Arabidopsis thaliana (Col-0), and tobacco (BY-2), were permeabilized with the channel-forming peptide alamethicin. Results: Nucleic acid-binding dyes and marker enzymes demonstrated alamethicin permeabilization of plasma membrane, mitochondria and... (More)
Background: The cell wall component callose is mainly synthesized at certain developmental stages and after wounding or pathogen attack. Callose synthases are membrane-bound enzymes that have been relatively well characterized in vitro using isolated membrane fractions or purified enzyme. However, little is known about their functional properties in situ, under conditions when the cell wall is intact. To allow in situ investigations of the regulation of callose synthesis, cell suspensions of Arabidopsis thaliana (Col-0), and tobacco (BY-2), were permeabilized with the channel-forming peptide alamethicin. Results: Nucleic acid-binding dyes and marker enzymes demonstrated alamethicin permeabilization of plasma membrane, mitochondria and plastids, also allowing callose synthase measurements. In the presence of alamethicin, Ca2+ addition was required for callose synthase activity, and the activity was further stimulated by Mg2+ Cells pretreated with oryzalin to destabilize the microtubules prior to alamethicin permeabilization showed significantly lower callose synthase activity as compared to non-treated cells. As judged by aniline blue staining, the callose formed was deposited both at the cell walls joining adjacent cells and at discrete punctate locations earlier described as half plasmodesmata on the outer walls. This pattern was unaffected by oryzalin pretreatment, showing a quantitative rather than a qualitative effect of polymerized tubulin on callose synthase activity. No callose was deposited unless alamethicin, Ca2+ and UDP-glucose were present. Tubulin and callose synthase were furthermore part of the same plasma membrane protein complex, as judged by two-dimensional blue native SDS-PAGE. Conclusion: Alamethicin permeabilization allowed determination of callose synthase regulation and tubulin interaction in the natural crowded cellular environment and under conditions where contacts between the cell wall, the plasma membrane and cytoskeletal macromolecules remained. The results also suggest that alamethicin permeabilization induces a defense response mimicking the natural physical separation of cells (for example when intercellulars are formed), during which plasmodesmata are transiently left open. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
BMC Plant Biology
volume
9
publisher
BioMed Central
external identifiers
  • wos:000265062400001
  • scopus:64749106707
ISSN
1471-2229
DOI
10.1186/1471-2229-9-27
language
English
LU publication?
yes
id
58cfd4b0-6518-4d7f-a1f5-994184748610 (old id 1400725)
date added to LUP
2009-06-12 10:29:53
date last changed
2017-08-06 04:20:17
@article{58cfd4b0-6518-4d7f-a1f5-994184748610,
  abstract     = {Background: The cell wall component callose is mainly synthesized at certain developmental stages and after wounding or pathogen attack. Callose synthases are membrane-bound enzymes that have been relatively well characterized in vitro using isolated membrane fractions or purified enzyme. However, little is known about their functional properties in situ, under conditions when the cell wall is intact. To allow in situ investigations of the regulation of callose synthesis, cell suspensions of Arabidopsis thaliana (Col-0), and tobacco (BY-2), were permeabilized with the channel-forming peptide alamethicin. Results: Nucleic acid-binding dyes and marker enzymes demonstrated alamethicin permeabilization of plasma membrane, mitochondria and plastids, also allowing callose synthase measurements. In the presence of alamethicin, Ca2+ addition was required for callose synthase activity, and the activity was further stimulated by Mg2+ Cells pretreated with oryzalin to destabilize the microtubules prior to alamethicin permeabilization showed significantly lower callose synthase activity as compared to non-treated cells. As judged by aniline blue staining, the callose formed was deposited both at the cell walls joining adjacent cells and at discrete punctate locations earlier described as half plasmodesmata on the outer walls. This pattern was unaffected by oryzalin pretreatment, showing a quantitative rather than a qualitative effect of polymerized tubulin on callose synthase activity. No callose was deposited unless alamethicin, Ca2+ and UDP-glucose were present. Tubulin and callose synthase were furthermore part of the same plasma membrane protein complex, as judged by two-dimensional blue native SDS-PAGE. Conclusion: Alamethicin permeabilization allowed determination of callose synthase regulation and tubulin interaction in the natural crowded cellular environment and under conditions where contacts between the cell wall, the plasma membrane and cytoskeletal macromolecules remained. The results also suggest that alamethicin permeabilization induces a defense response mimicking the natural physical separation of cells (for example when intercellulars are formed), during which plasmodesmata are transiently left open.},
  author       = {Aidemark, Mari and Andersson, Carl-Johan and Rasmusson, Allan and Widell, Susanne},
  issn         = {1471-2229},
  language     = {eng},
  publisher    = {BioMed Central},
  series       = {BMC Plant Biology},
  title        = {Regulation of callose synthase activity in situ in alamethicin-permeabilized Arabidopsis and tobacco suspension cells},
  url          = {http://dx.doi.org/10.1186/1471-2229-9-27},
  volume       = {9},
  year         = {2009},
}