New Factors for Protoplast-Callose-Fiber Formation in Salt-Tolerant Mangrove Plants, Avicennia alba and Bruguiera sexangula and Analysis of Fiber Substructures

Abstract

Elongated and spiral β-1,3-glucan (callose) fibers were obtained by new factors from protoplasts cultured in liquid medium from suspension cultured cells of two salt-tolerant mangrove species; Avicennia alba and Bruguiera sexangula. Differences in salt factor for protoplast-fiber formation were compared with those of the callose fibers developed from protoplasts of non-mangrove tree plants, Larix leptolepis and Betula platyphylla, which high concentrations of divalent cations, Mg2+ (50 mM) or Ca2+ (100 mM), were stimulatory. In the halophilic A. alba protoplasts, whose cell division was stimulated by up to 400 mM NaCl, addition of Mg2+, Ca2+, K+ ions inhibited protoplast-fiber formation. In B. sexangula, protoplast-fibers were rapidly and efficiently formed only by another new factor, electric cell fusion treatment of protoplasts. Spiral fibers developed from mangrove protoplasts were detected under an inverted microscope, and their specific blue-green color for callose after staining with Aniline Blue dye was detected under a fluorescence microscope. Enzymatic certification of callose was further performed with laminarinase, specific for callose, in comparison with cellulase CBH1, specific for cellulose. Differences in sub-structures, fibrils and sub-fibrils of two mangrove protoplast-fibers were analyzed using laser confocal scanning microscopy, atomic force microscopy and image J analysis. Tube-like fine structure was observed using transmission electron microscopy in single protoplast-fiber of B. sexangula selected with a micromanipulator.

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

“…In protoplast culture of suspension cells obtained from the Na + salt-tolerant mangrove tree, Bruguiera sexangula, Ca 2+ and Mg 2+ ions stimulated protoplast growth at low concentrations, but inhibited it at high concentrations (Fukumoto et al, 2004). Although no stimulation of PCF formation was observed with high concentrations of cationic ions, electric treatment for cell fusion caused rapid (within one week) PCF formation (Kurita-Tashiro et al, 2020).…”

“…In suspension cultured cells of the Na + halophilic mangrove tree, Avicennia alba, Na + and Mg 2+ ions stimulated protoplast growth (Hasegawa et al, 2013), and Na + ion, at only low concentrations, stimulated slow PCF formation (> 0.5 mm long with 20 μm diameter), though Ca 2+ ion inhibited both protoplast growth and PCF formation (Kurita-Tashiro et al, 2020).…”

“…The hollow fibril sub-structure of Betula PCF was observed by TEM using the antibody of callose (Seyama et al, 2008). Sub-fibrils and tubes-like structures of Bruguiera sexangula PCF were observed by single cell TEM (Kurita-Tashiro et al, 2020).…”

“…Since the findings of Larix and Betula PCFs, the stimulation factors for such helical PCF formation have been investigated in protoplast cultures of both tree species (Fukumoto, 2006;Fukumoto et al, 2005;Kurita-Tashiro et al, 2020;Oyanagi et al, 2012aOyanagi et al, ,b, 2024Sasamoto et al, 2020b;and Seyama et al, 2008 though the culture conditions were wrongly described), and herbaceous plant species (Fukumoto, 2006;Sasamoto et al, 2020a;Tagawa & Kondo, 2018).…”

“…Fine sub-structures (e.g., fibril and sub-fibril) of helical PCF were investigated using TEM, atomic force microscopy (AFM) and laser confocal scanning microscopy (LCSM), and single cell TEM which was performed to clarify the cellular elongation site, in Larix (Fukumoto, 2006;Fukumoto et al, 2005;Oyanagi et al, 2014) and mangrove trees, e.g., Avicennia alba, Bruguiera sexangula (Kurita-Tashiro et al, 2020), and Sonneratia caseolaris (Oyanagi et al, 2012a(Oyanagi et al, ,b, 2024.…”