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Localization of calcium and magnesium by secondary ion mass spectrometry in flax plantlets
32" Colloque SFME, Rouen-Mont St Aignan, juillet 1992
265
SENSITIVITY OF EPIDERMIS CELL WALLS OF FLAX TO ENDOPOLYGALACTURONASE ACCORDING TO T H E I R CALCIUM AND PECTIN CONTENTS : INVESTIGATIONS BY TEM AND SIMS
LOCALIZATION OF CALCIUM AND MAGNESIUM BY SECONDARY ION MASS SPECTROMETRY IN FLAX PLANTLETS J A U N E A U Alain, L E F E B V R E Fabrice P A R I O T Christine, K R E Z E L Chantal, R I H O U E Y Christophe, RIPOLL Camille, D E M A R T Y Maurice, T H E L L I E R Michel
J A U N E A U Alain., M O R V A N Claudine, CABINFLAMAN Armelle, R I H O U E Y Christophe, R I P O L L Camille, D E M A R T Y Maurice.
Laboratoire des Processus Ioniques CeIlulaires, S.C.U.E.O.R., CNRS UA 203, Facult~ des Sciences de Rouen, BB 118 76134 Mont-Saint-Aignan
Laboratoire des Processus Ioniques Cellulaires, S.C.U.E.O.R., CNRS UA 203, Facultd des Sciences de Rouen, BP 118, 76134 Mont-Saint Aignan
In the epidermis cells, the SIMS images show that calcium is more concentrated in the tangential walls and in the intercellular junctions than it. is in the radial walls. Using substractive methods and T.E.M., it was shown that i) pectins linked by calcium bridges, were mainly located in the outer part of the wall close to the cuticle) and in the intracellular junction 1) ii) pectins were removed by b-elimination from the inner part of the epidermis walls. After an incubation of 30 min, an endoPGH had caused a massive damage in the inner part of the wall compared to the outer part and to the intercellular junctions. It may seem paradoxical that the enzyme damages the part of the wall rich in acidic pectins much slower than the part with methylated pectins. It was inferred that e n d o P G H is sensitive to inhibition by Ca + ÷, in situ, as it was observed in vitro (90 % inhibition occuring with 5 mM Ca + *).
The mapping of the alkaline earth cations, Ca > and Mg 2÷ in the cotyledons and hypocotyls of three days old flax plantlets (Linum usitatissfmum) was performed using secondary ion analytical microscopy (SIMS). We have compared the relative abilities of the conventional glm~.raldedhyde method and of the pyroantimonate precipitation method (1) for the retention of the alkaline earth cations during preparation of the plant samples. The tissue retention of the cations ~s much better when using the precipitation method than the conventional method. Even if we can't exclude that some ion diffusion occured during sample preparation, it is nevertheless possible to observe reliable differences in the tissue distribution of Ca z+ and Mg 2+. (1) MENTRE P., ESCAIG F. (1988). J. Histochem. Cytochem., 36:49-54.
(1) JAUNEAU A., MORVAN C., LEFEBVRE F., DEMARTY M., RIPOLL C., THELLIER M. (1992). J. histochem. Cytochem. Sous presse.
ENDOPOLYGALACTURONASE-GOLD COMPLEX TO LOCATE BY TEM THE PECTIC ACIDS IN THE CELL WALL OF FLAX HYPOCOTYL : PARAMETERS INFLUENCING THE LABELLING EFFICIENCY
VISUALIZATION OF CALCIUM DISTRIBUTION AND PECTIC ACIDS PA'Iq'ERNS IN THE CELL WALLS OF RIPENING CHERRY TOMATOES ROY St~,phane', J A U N E A U Alain", L E F E B V R E Fabrice", VIAN Brigitte', RIPOLL Camille"
CABIN-FLAMAN Armelle, J A U N E A U Alain, MORVAN Claudine, D E M A R T Y Maurice
"Laboratoire des Biomembranes, ENS, 46 me d'OTm, 75230 Paris Cedex 05
Laboratoire des Processus ioniques Celhdaires, S.C.U.E.O.R., CNRS UA 203, Facultd des Sciences de Rouen BP 118, 76134 Mont-Saint-Aignan
"'Laboratoire des Processus loniques Cellulaires, S.C.U.E.O.R., CNRS UA 203, Facult~ des Sciences de Rouen, BP 118, 76134 Mont-Saint-Aignan
An endopolygalacturonase (E.C. 3.2.1.15) was tagged with gold particle. This enzyme • previously characterized has a low molecular weight (close to 35 kD) and an isoelectric point close to 4.5. The greatest labelling efficiency (1) was obtained when one protein molecule was adsorbed, at pH 5.9 , onto one gold particle with a 9.6 nm diameter. This probe might be used after two centrifugations at 30, 000 rpm for one hour to remove free enzymes in the preparation. Then it could be applied on section, at a concentration such as the optical density was close to 1.0 at 522-523 rim, at room temperature and for three hours. Osmication of tim vegetal sample might be irnperatively omitted. Used in conjonction with appropriate controls, showing the specificity of tiffs probe, endopolygalacturonase-gold labelling provided reformation about distribution of pectic acids in flax hypocotyl. (1/PARK K. (19891 Rcann. Micrnse. ,qunnl.. 3: 15-25.
Pectic acids were visualized using JIM5 monoclonal antibodies with immuno-fluorescence technique or IGSS (immunogold silver staining) technique(l). Calcium was located using SIMS (Secondary ion mass spectrometry). In the mature-green tomato, pectic acids are referentially localized in the junctions as seems also to e the calcium. In the ripe tomato, the extension of the separation spaces seems to be limited by pectic acids areas particularly rich in calcium. During the ripening, the scanning ion images show an increase of the calcium content m the cell walls, and preferentially in the intercellular junctions. This work shows that the combination of immunocytochemistry methods and SIMS imaging could provide new informations about the role of calcium during the fruit ripening.
~
(1) KNOX J.P., LINSTEAD P.J., KING J., C O O P E R C., R O B E R T S K. (1990). P[anta 181:512-521. 9a
265
SENSITIVITY OF EPIDERMIS CELL WALLS OF FLAX TO ENDOPOLYGALACTURONASE ACCORDING TO T H E I R CALCIUM AND PECTIN CONTENTS : INVESTIGATIONS BY TEM AND SIMS
LOCALIZATION OF CALCIUM AND MAGNESIUM BY SECONDARY ION MASS SPECTROMETRY IN FLAX PLANTLETS J A U N E A U Alain, L E F E B V R E Fabrice P A R I O T Christine, K R E Z E L Chantal, R I H O U E Y Christophe, RIPOLL Camille, D E M A R T Y Maurice, T H E L L I E R Michel
J A U N E A U Alain., M O R V A N Claudine, CABINFLAMAN Armelle, R I H O U E Y Christophe, R I P O L L Camille, D E M A R T Y Maurice.
Laboratoire des Processus Ioniques CeIlulaires, S.C.U.E.O.R., CNRS UA 203, Facult~ des Sciences de Rouen, BB 118 76134 Mont-Saint-Aignan
Laboratoire des Processus Ioniques Cellulaires, S.C.U.E.O.R., CNRS UA 203, Facultd des Sciences de Rouen, BP 118, 76134 Mont-Saint Aignan
In the epidermis cells, the SIMS images show that calcium is more concentrated in the tangential walls and in the intercellular junctions than it. is in the radial walls. Using substractive methods and T.E.M., it was shown that i) pectins linked by calcium bridges, were mainly located in the outer part of the wall close to the cuticle) and in the intracellular junction 1) ii) pectins were removed by b-elimination from the inner part of the epidermis walls. After an incubation of 30 min, an endoPGH had caused a massive damage in the inner part of the wall compared to the outer part and to the intercellular junctions. It may seem paradoxical that the enzyme damages the part of the wall rich in acidic pectins much slower than the part with methylated pectins. It was inferred that e n d o P G H is sensitive to inhibition by Ca + ÷, in situ, as it was observed in vitro (90 % inhibition occuring with 5 mM Ca + *).
The mapping of the alkaline earth cations, Ca > and Mg 2÷ in the cotyledons and hypocotyls of three days old flax plantlets (Linum usitatissfmum) was performed using secondary ion analytical microscopy (SIMS). We have compared the relative abilities of the conventional glm~.raldedhyde method and of the pyroantimonate precipitation method (1) for the retention of the alkaline earth cations during preparation of the plant samples. The tissue retention of the cations ~s much better when using the precipitation method than the conventional method. Even if we can't exclude that some ion diffusion occured during sample preparation, it is nevertheless possible to observe reliable differences in the tissue distribution of Ca z+ and Mg 2+. (1) MENTRE P., ESCAIG F. (1988). J. Histochem. Cytochem., 36:49-54.
(1) JAUNEAU A., MORVAN C., LEFEBVRE F., DEMARTY M., RIPOLL C., THELLIER M. (1992). J. histochem. Cytochem. Sous presse.
ENDOPOLYGALACTURONASE-GOLD COMPLEX TO LOCATE BY TEM THE PECTIC ACIDS IN THE CELL WALL OF FLAX HYPOCOTYL : PARAMETERS INFLUENCING THE LABELLING EFFICIENCY
VISUALIZATION OF CALCIUM DISTRIBUTION AND PECTIC ACIDS PA'Iq'ERNS IN THE CELL WALLS OF RIPENING CHERRY TOMATOES ROY St~,phane', J A U N E A U Alain", L E F E B V R E Fabrice", VIAN Brigitte', RIPOLL Camille"
CABIN-FLAMAN Armelle, J A U N E A U Alain, MORVAN Claudine, D E M A R T Y Maurice
"Laboratoire des Biomembranes, ENS, 46 me d'OTm, 75230 Paris Cedex 05
Laboratoire des Processus ioniques Celhdaires, S.C.U.E.O.R., CNRS UA 203, Facultd des Sciences de Rouen BP 118, 76134 Mont-Saint-Aignan
"'Laboratoire des Processus loniques Cellulaires, S.C.U.E.O.R., CNRS UA 203, Facult~ des Sciences de Rouen, BP 118, 76134 Mont-Saint-Aignan
An endopolygalacturonase (E.C. 3.2.1.15) was tagged with gold particle. This enzyme • previously characterized has a low molecular weight (close to 35 kD) and an isoelectric point close to 4.5. The greatest labelling efficiency (1) was obtained when one protein molecule was adsorbed, at pH 5.9 , onto one gold particle with a 9.6 nm diameter. This probe might be used after two centrifugations at 30, 000 rpm for one hour to remove free enzymes in the preparation. Then it could be applied on section, at a concentration such as the optical density was close to 1.0 at 522-523 rim, at room temperature and for three hours. Osmication of tim vegetal sample might be irnperatively omitted. Used in conjonction with appropriate controls, showing the specificity of tiffs probe, endopolygalacturonase-gold labelling provided reformation about distribution of pectic acids in flax hypocotyl. (1/PARK K. (19891 Rcann. Micrnse. ,qunnl.. 3: 15-25.
Pectic acids were visualized using JIM5 monoclonal antibodies with immuno-fluorescence technique or IGSS (immunogold silver staining) technique(l). Calcium was located using SIMS (Secondary ion mass spectrometry). In the mature-green tomato, pectic acids are referentially localized in the junctions as seems also to e the calcium. In the ripe tomato, the extension of the separation spaces seems to be limited by pectic acids areas particularly rich in calcium. During the ripening, the scanning ion images show an increase of the calcium content m the cell walls, and preferentially in the intercellular junctions. This work shows that the combination of immunocytochemistry methods and SIMS imaging could provide new informations about the role of calcium during the fruit ripening.
~
(1) KNOX J.P., LINSTEAD P.J., KING J., C O O P E R C., R O B E R T S K. (1990). P[anta 181:512-521. 9a