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

Aidemark M, Andersson CJ, Rasmusson AG, Widell S - BMC Plant Biol. (2009)

Bottom Line: 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.This pattern was unaffected by oryzalin pretreatment, showing a quantitative rather than a qualitative effect of polymerized tubulin on callose synthase activity.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell and Organism Biology, Lund University, Lund, Sweden. mari.Aidemark@cob.lu.se

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.

Show MeSH

Related in: MedlinePlus

Two-dimensional blue native/SDS-PAGE and immunoblotting of solubilized BY-2 plasma membranes. Native, n-octyl-β-D-glucoside-soluble, plasma membrane protein complexes were separated in a first dimension using Blue native PAGE. After denaturation, the complexes were thereafter separated into their subunits in a second dimension using SDS-PAGE. After separation, callose synthase (180 kDa), sucrose synthase (90 kDa) and β-tubulin (50 kDa) were detected by immunoblotting in separate blots with the respectively specific antibodies. The figure is a composite of these separate blots. Native molecular masses for the first dimension are denoted in kDa below the blots. The upper line depicts the start and direction of the first dimension separation gel.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2667179&req=5

Figure 7: Two-dimensional blue native/SDS-PAGE and immunoblotting of solubilized BY-2 plasma membranes. Native, n-octyl-β-D-glucoside-soluble, plasma membrane protein complexes were separated in a first dimension using Blue native PAGE. After denaturation, the complexes were thereafter separated into their subunits in a second dimension using SDS-PAGE. After separation, callose synthase (180 kDa), sucrose synthase (90 kDa) and β-tubulin (50 kDa) were detected by immunoblotting in separate blots with the respectively specific antibodies. The figure is a composite of these separate blots. Native molecular masses for the first dimension are denoted in kDa below the blots. The upper line depicts the start and direction of the first dimension separation gel.

Mentions: The data presented above indicate an interaction between callose synthase and microtubules/tubulin that remained after alamethicin permeabilization. To further test this possible interaction, we used blue native SDS-PAGE to separate plasma membrane protein complexes isolated from untreated BY-2 cells, as was successfully done earlier with spinach leaf plasma membranes [25]. In BY-2 cells, callose synthase appeared in two different protein complexes with masses of approximately 1500 kDa and at 800 kDa, each comigrating with tubulin, that was more abundant at the same masses (Fig. 7). The comigration suggests that callose synthase and tubulin are part of the same complexes through a relatively strong physical interaction, sufficient for the binding to remain during isolation and gel analysis. A mass of around 800 kDa for the callose synthase complex was also found with spinach leaf plasma membranes [25]. Sucrose synthase, on the other hand, was not here associated with callose synthase but found in a separate complex, with a molecular mass between 400 and 500 kDa (Fig. 7), consistent with the enzyme being a tetramer in vivo [50].


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

Aidemark M, Andersson CJ, Rasmusson AG, Widell S - BMC Plant Biol. (2009)

Two-dimensional blue native/SDS-PAGE and immunoblotting of solubilized BY-2 plasma membranes. Native, n-octyl-β-D-glucoside-soluble, plasma membrane protein complexes were separated in a first dimension using Blue native PAGE. After denaturation, the complexes were thereafter separated into their subunits in a second dimension using SDS-PAGE. After separation, callose synthase (180 kDa), sucrose synthase (90 kDa) and β-tubulin (50 kDa) were detected by immunoblotting in separate blots with the respectively specific antibodies. The figure is a composite of these separate blots. Native molecular masses for the first dimension are denoted in kDa below the blots. The upper line depicts the start and direction of the first dimension separation gel.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2667179&req=5

Figure 7: Two-dimensional blue native/SDS-PAGE and immunoblotting of solubilized BY-2 plasma membranes. Native, n-octyl-β-D-glucoside-soluble, plasma membrane protein complexes were separated in a first dimension using Blue native PAGE. After denaturation, the complexes were thereafter separated into their subunits in a second dimension using SDS-PAGE. After separation, callose synthase (180 kDa), sucrose synthase (90 kDa) and β-tubulin (50 kDa) were detected by immunoblotting in separate blots with the respectively specific antibodies. The figure is a composite of these separate blots. Native molecular masses for the first dimension are denoted in kDa below the blots. The upper line depicts the start and direction of the first dimension separation gel.
Mentions: The data presented above indicate an interaction between callose synthase and microtubules/tubulin that remained after alamethicin permeabilization. To further test this possible interaction, we used blue native SDS-PAGE to separate plasma membrane protein complexes isolated from untreated BY-2 cells, as was successfully done earlier with spinach leaf plasma membranes [25]. In BY-2 cells, callose synthase appeared in two different protein complexes with masses of approximately 1500 kDa and at 800 kDa, each comigrating with tubulin, that was more abundant at the same masses (Fig. 7). The comigration suggests that callose synthase and tubulin are part of the same complexes through a relatively strong physical interaction, sufficient for the binding to remain during isolation and gel analysis. A mass of around 800 kDa for the callose synthase complex was also found with spinach leaf plasma membranes [25]. Sucrose synthase, on the other hand, was not here associated with callose synthase but found in a separate complex, with a molecular mass between 400 and 500 kDa (Fig. 7), consistent with the enzyme being a tetramer in vivo [50].

Bottom Line: 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.This pattern was unaffected by oryzalin pretreatment, showing a quantitative rather than a qualitative effect of polymerized tubulin on callose synthase activity.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell and Organism Biology, Lund University, Lund, Sweden. mari.Aidemark@cob.lu.se

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.

Show MeSH
Related in: MedlinePlus