Protoplasma (1997) 198:9-19 PROTOPLASMA 9 Springer-Verlag 1997 Printed in Austria Micro-heterogeneity of pectins and calcium distribution in the epidermal and cortical parenchyma cell walls of flax hypocotyl Alain Jauneau*, Michael Quentin, and Azeddine Driouich Laboratoire Parois et Polym~res Paridtaux, Service Co6peration Universit6 Entreprise Organismes Rdgionaux, CNRS, URA 203, Facult6 des Sciences, Universit6 de Rouen, Mont Saint-Aignan Received December 10, 1996 Accepted February 26, 1997 Summary. Pectic polysaccharides are major components of the plant cell wall matrix and are known to perform many important functions for the plant. In the course of our studies on the putative role of pec- tic polysaccharides in the control of cell elongation, we have exam- ined the distribution of polygalacturonans in the epidermal and corti- cal parenchyma cell walls of flax seedling hypocotyls. Pectic components have been detected with (1) the nickel (Ni2+) staining method to visualize polygalacturonates, (2) monoclonal antibodies specific to low (JIM5) and highly methylesterified (JIM7) pectins and (3) a combination of subtractive treatment and PATAg (periodic acid-thiocarbohydrazide-silver proteinate) staining. In parallel, cal- cium (Ca2+) distribution has been imaged using SIMS microscopy (secondary ion mass spectrometry) on cryo-prepared samples and TEM (transmission electron microscopy) after precipitation of calci- um with potassium pyroantimonate. Our results show that, at the tis- sular level, polygalacturonans are mainly located in the epidermal cell walls, as revealed by the Ni 2+ staining and immunofluorescence microscopy with JIM5 and JIM7 antibodies. In parallel, Ca 2§ distri- bution points to a higher content of this cation in the epidermal walls compared to cortical parenchyma walls. At the ultrastructural level, immunogold labeling with JIM5 and JIM7 antibodies shows a differ- ential distribution of pectic polysaccharides within cell walls of both tissues. The acidic polygalacturonans (recognized by JIM5) held through calcium bridges are mainly found in the outer part of the external wall of epidermal cells. In contrast, the labeling of methyl- esterified pectins with JIM7 is slightly higher in the inner part than in the outer part of the wall. In the cortical parenchyma cells, acidic pectins are restricted to the cell junctions and the wall areas in contact with the air-spaces, whereas methylesterified pectins are evenly distributed all over the wall. In addition, the pyroantimonate precipitation method reveals a clear difference in the Ca2+ distribu- tion in the epidermal wall, suggesting that this cation is more tightly *Correspondence and reprints: Laboratoire Parois et Polym6res Pari6taux, SCUEOR, Facult6 des Sciences, Universit6 de Rouen, F- 76821 Mont Saint-Aignan Cedex, France. bound to acidic pectins in the outer part than in the inner part of that wall. Our findings show that the distribution of pectic polysaccha- rides and the nature of their linkages differ not only between tissues, but also within a single wall of a given cell in flax hypocotyls. The differential distribution of pectins and Ca 2+ in the external epidermal wall suggests a specific control of the demethylation of pectins and a central role for Ca 2+ in this regulation. Keywords: Calcium; Demethylation; Epidermal wall; Polygalactur- onate; Polygalacturonic acid-Ca2+ conformation. Abbreviations: Cdta diamino-l,2-cyclohexane tetra-acetic acid; PATAg periodic acid-thiocarbohydrazide-silver proteinate; PGA polygalacturonic acid; PME pectin methylesterase; RG I rhamno- galacturonan I; SIMS secondary ion mass spectrometry; TEM trans- mission electron microscopy. Introduction The two major constituents of pectins in dicotyledo- nous plants are polygalacturonic acid (PGA), which contains homogalacturonans composed of ~-(1-4)- linked galacturonic acids, and rhamnogalacturonan I (RG I), which is composed of a highly branched poly- saccharide containing a backbone of ~-(1-2) linked L-rhamnose and ct-(1-4) linked D-galacturonic acid (Carpita and Gibeaut 1993). Pectins are known to be synthesized with a high degree of methyl-esterifica- tion and secreted into the wall, where some of the methylester groups are cleaved by pectin methyl- esterases (Zhang and Staehelin 1992, Goldberg et al. 1996). The unesterified homogalacturonans can be cross-linked by Ca 2+ ions causing them to form a gel- like matrix (Jarvis 1984). The first function of pectins