Biochemical and structural characterization of an endoplasmic reticulum-localized late embryogenesis abundant (LEA) protein from the liverwort Marchantia polymorpha.

Late embryogenesis abundant (LEA) proteins, which accumulate to high levels in seeds during late maturation, are associated with desiccation tolerance. A member of the LEA protein family was found in cultured cells of the liverwort Marchantia polymorpha; preculture treatment of these cells with 0.5M sucrose medium led to their acquisition of desiccation tolerance. We characterized this preculture-induced LEA protein, designated as MpLEA1. MpLEA1 is predominantly hydrophilic with a few hydrophobic residues that may represent its putative signal peptide. The protein also contains a putative endoplasmic reticulum (ER) retention sequence, HEEL, at the C-terminus. Microscopic observations indicated that GFP-fused MpLEA1 was mainly localized in the ER. The recombinant protein MpLEA1 is intrinsically disordered in solution. On drying, MpLEA1 shifted predominantly toward α-helices from random coils. Such changes in conformation are a typical feature of the group 3 LEA proteins. Recombinant MpLEA1 prevented the aggregation of α-casein during desiccation-rehydration events, suggesting that MpLEA1 exerts anti-aggregation activity against desiccation-sensitive proteins by functioning as a "molecular shield". Moreover, the anti-aggregation activity of MpLEA1 was ten times greater than that of BSA or insect LEA proteins, which are known to prevent aggregation on drying. Here, we show that an ER-localized LEA protein, MpLEA1, possesses biochemical and structural features specific to group 3 LEA proteins.

Development of desiccation tolerance and vitrification by preculture treatment in suspension-cultured cells of the liverwort Marchantia polymorpha.

Some cultured plant cells are able to acquire tolerance to various stresses when they are cultured under suitably controlled conditions. Induction of a high level of desiccation tolerance in suspension-cultured cells of the liverwort Marchantia polymorpha was examined for studying the mechanisms of desiccation tolerance and vitrification at the cellular level. Desiccation tolerance level of cells was very low and the survival rate was less than 10% after exposure to drying below 0.1 g H(2)O g(-1) dry weight (DW). Preculture treatment in 0.5 M sucrose medium was the most effective method for inducing a high level of desiccation tolerance in cells and the survival rate was 87% even after being desiccated to below 0.1 g H(2)O g(-1) DW. Preculture treatment caused alteration of cell structures and accumulation of a large amount of sucrose and newly synthesized proteins in cells. Abundant sucrose and preculture-induced proteins were necessary for full development of desiccation tolerance in the cells. When water content decreased to below 0.1 g H(2)O g(-1) DW, desiccation-tolerant cells that had been precultured were vitrified above 0 degrees C and maintained stable viability. We have succeeded in the induction of desiccation tolerance that allows formation of intracellular glass with cell viability at ambient temperatures by controlling culture conditions, and our results suggest that suspension-cultured cells of M. polymorpha are useful for studying cellular mechanisms for the development of desiccation tolerance and the stabilization of vitrified cells.

Induction of multinucleation by beta-glucosyl Yariv reagent in regenerated cells from Marchantia polymorpha protoplasts and involvement of arabinogalactan proteins in cell plate formation.

Arabinogalactan proteins (AGPs) are abundant plant cell surface proteoglycans widely distributed in plant species. Since high concentrations of beta-glucosyl Yariv reagent (betaglcY), which binds selectively to AGPs, inhibited cell division of protoplast-regenerated cells of the liverwort Marchantia polymorpha L. (Shibaya and Sugawara in Physiol Plant 130:271-279, 2007), we investigated the mechanism underlying the inability of the cells to divide normally by staining nuclei, cell walls and beta-1,3-glucan. Microscopic observation showed that the diameter of regenerated cells cultured with betaglcY was about 2.8-fold larger than that of cells cultured without betaglcY. The cells cultured with betaglcY were remarkably multinucleated. These results indicated that betaglcY did not inhibit mitosis but induced multinucleation. In the regenerated cells cultured with low concentrations of betaglcY (5 and 1 microg ml(-1)), the cell plate was stained strongly by betaglcY, suggesting abundant AGPs in the forming cell plate. In these cell plates, beta-1,3-glucan was barely detectable or not detected. In multinucleated cells, cell plate-like fragments, which could not reach the cell wall, were frequently observed and they were also stained strongly by betaglcY. Our results indicated that AGPs might have an important role in cell plate formation, and perturbation of AGPs with betaglcY might result in remarkable multinucleation in protoplast-regenerated cells of M. polymorpha.

Ultrastructural implications of pretreatment for successful cryopreservation of Oncidium protocorm-like body.

By applying pre-treatment with high concentrations of sucrose and glycerol prior to desiccation and subsequent freezing in liquid nitrogen, successful cryopreservation with high recovery rate was achieved in Oncidium PLB (protocorm-like body). Cellular and subcellular changes after each step in various cryopreservation regimes were examined to elucidate the mechanism of the beneficial effect of the pretreatment which confers resistance to desiccation and freezing.