Cellular Calcium Distribution Modulates the Growth of Callus and Protoplasts of Halophyte Mangrove Plant, Avicennia Alba - an X-ray Microanalysis

Abstract

Two cultured cell lines were developed from cotyledons of a halophyte mangrove, Avicennia alba. In the high-Ca callus line, which was sub-cultured in a modified amino acid medium containing 3 mM CaCl 2 , growth of calluses and their protoplasts were both inhibited by low concentrations of CaCl 2 in the culture medium. Removal of Ca 2+ from the culture medium stimulated callus growth and the calluses could be sub-cultured without CaCl 2 (low-Ca callus line). The intra-(cytoplasmic matrix and vacuole) and extra-(cell wall) cellular concentrations of elements, i.e., [Ca] [S] were investigated using quantitative X-ray microanalysis of cryosections of calluses from both cell lines. [Ca] was high in the cytoplasmic matrix and cell wall of the high-Ca line. [Ca] was lowered in the low-Ca line in all cell compartments, though still detected. Ca-containing electron-dense precipitates were accumulated in the middle lamella of cell walls in resin-embedded sections of the high-Ca line. CaCl 2 in the medium stimulated protoplast growth only in the low-Ca line. These results suggested that a low cellular [Ca] is needed for protoplasts growth of A. alba. The importance of cellular [Ca] for the growth of halophilic mangrove plant cells was discussed.

High Allelopathic Activity of Carotenoid-accumulating Callus of a Halophilic Mangrove Plant, Avicennia alba: Protoplast Co-culture Method with Digital Image Analysis

“…Protoplasts of suspension-cultured cells from cotyledons of Sonneratia alba and Avicennia alba, which are distributed in the seaward-side area, were found to be halophilic to NaCl . The cellular distribution of salts elements, Na, K, Mg, Ca, Cl, P and S, in suspension-cultured cells of S. alba (Hayatsu et al, 2014) and A. alba (Hayatsu et al, 2017) were investigated using quantitative X-ray microanalysis of cryosections to study the cellular mechanism(s) of strong salt tolerance or halophilism.…”

“…Ultrathin sections of ~70 nm thickness were cut using ultramicrotome (Ultracut-N; Reichert-Jung, Vienna, Austria), and stained with uranyl acetate and lead citrate. The sections were examined with a transmission electron microscope (H-7650, Hitachi Ltd., or JEM-2100EX, JEOL, Akishima, Tokyo, Japan) operated at an accelerating voltage of 80 kV or 100 kV as described previously (Aoki et al, 2011;Hayatsu et al, 2014Hayatsu et al, , 2016Hayatsu et al, , 2017.…”

“…Electron dense crystalloids and particle structures were found in the plastid of yellow A. alba callus using transmission electron microscopy (section 3.6). Such structures in the plastid were not observed in non-yellow A. alba suspension cells originating from cotyledons (Hayatsu et al, 2017). During fruit ripening, many electron dense particle structures, plastoglobuli, and more developed plate-like crystalloids in chromoplasts were found in carotenoid-containing plant tissues (Aoki et al, 2011;Hayatsu et al, 2016;Suzuki, 2017).…”

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

“…Samples for TEM observation were prepared as previously described (Hayatsu et al, 2012(Hayatsu et al, , 2014(Hayatsu et al, , 2017. Samples were fixed in 2% glutaraldehyde with PB (pH7.2) at 4º C for more than 24 hrs and fixed in 1% osmium tetroxide with PB (pH7.2) at 4º C for 12 hrs.…”

In vitro Bioassay of Allelopathic Activities of a Mangrove Tree, Kandelia obovata, and Fast-growing Trees, Betula platyphylla and Populus alba, Using Protoplast Co-culture Method

“…In addition to stimulation by NaCl, different responses to other sea salt ions (K + , Mg 2+ , Ca 2+ ) added to MS basal medium were examined. Comparison of the responses of rice cells and lettuce protoplasts revealed specific Ca 2+ -related cellular mechanisms of halophilism (Hayatsu et al, 2014(Hayatsu et al, , 2017.…”