Mg2+ Ions Stimulate both Helical Protoplast-Callose-Fiber Formation and Protoplast Division in a Mangrove Tree, Sonneratia caseolaris: Analysis of Sub-fibril Structures of PCF by Atomic Force Microscopy

Abstract

The effects of four salts, NaCl, KCl, MgCl2 and CaCl2, on protoplast division and protoplast-callose-fiber (PCF) formation were examined using suspension cultured cells of a mangrove tree, Sonneratia caseolaris. Basal medium was hormone-free Murashige & Skoog’s medium containing 0.8 M sorbitol and 3% sucrose. Addition of 50-100 mM of Mg2+ ions highly stimulated both cell division and PCF formation, while addition of Ca2+ ions was inhibitory. Addition of Na+ ions at 10-25 mM and K+ ions at 50-100 mM stimulated protoplast division but not PCF formation. Helical PCF rapidly elongated from a specific site of cell division plate after 3 days of culture. The β-1,3-glucan (callose) component of the protoplast-fibers was stained with Aniline Blue fluorescent dye and was re-certified enzymatically using laminarinase. After selecting the PCF using a micromanipulator, we analyzed the fibril- and sub-fibril- structures using laser confocal scanning microscopy (LCSM), and atomic force microscopy (AFM). We discuss the uniqueness of the PCF of S. caseolaris as compared to PCFs of several plants.

Effects of Salts on Helical Protoplast-Callose-Fiber Formation and Cell Division in Leaf Protoplast Culture of Arabidopsis thaliana: Ultrastructure of PCF Using Transmission Electron Microscopy

“…They were similar to the PCFs of Larix and Betula, which elongated from a specific site on spherically enlarged protoplast without cell division (Sasamoto et al, 2003;Fukumoto et al, 2005). Observation of PCF after less than a week of culture revealed speedy PCF formation, which was similar to that in Bruguiera sexangula (Kurita-Tashiro et al, 2020) and in Sonneratia caseolaris (Oyanagi et al, 2012a,b;2024), compared with those after 2 weeks in Larix, one to two months in Betula (Sasamoto et al, 2003), and in Avicennia mangrove protoplasts (Kurita-Tashiro et al, 2020). Figure 4a shows an elongated helical PCF in the medium containing an additional 25 mM KCl, and Figure 4b and c show PCFs in the medium containing an additional 10 and 50 mM CaCl 2 , respectively.…”

“…Figure 5 shows the effects of four salts on the number of Arabidopsis PCF per well cultured for 40 days. The PCFs were counted under a fluorescent microscope after Aniline Blue staining, as in Sonneratia caseolaris PCFs (Oyanagi et al, 2024). There were fewer PCFs after 27 days of culture under an inverted microscope without staining, though the tendency of the salts effect was similar (data not shown).…”

“…In suspension cultured cells of another mangrove tree, Sonneratia caseolaris, Mg 2+ ion, but not Ca 2+ ion, stimulated both protoplast growth (Sasamoto & Hasegawa, 2021) and rapid PCF formation in sucrose medium (Oyanagi et al, 2012a,b;2024).…”

“…The callose component of PCFs was determined by using specific degrading enzymes for callose and cellulose in Larix (Fukumoto et al, 2005), mangrove trees, Bruguiera sexangula (Kurita-Tashiro et al, 2020), and Sonneratia caseolaris (Oyanagi et al, 2012a,b;2024). Observation of specific blue-green fluorescence after Aniline Blue staining of helical PCFs under a fluorescence inverted microscope can be used for a criterion of PCFs (Sasamoto et al, 2003).…”

“…In another mangrove, Sonneratia caseolaris, only Mg 2+ ion strongly stimulated both protoplast growth and PCF formation. PCF elongated from early divided protoplasts with a division plate (Oyanagi et al, 2012a,b;2024).…”