Modulation of calcium, callose synthesis, membrane permeability and pectin methyl-esterase activity affect cell wall composition and embryo yield during Brassica napus microspore embryogenesis.

Background and aims: Microspore embryogenesis is a convenient inducible system to study the changes associated to the developmental reprogramming of cells. In this work, Brassica napus microspore cultures were used to study the role in the embryogenic switch of callose and pectin cell wall composition, which depends on Ca2+ levels.

Methods: We used different chemicals to modulate Ca2+, callose and pectin methyl-esterification, including Ca(NO3)2, InsP3, 2-deoxy-D-glucose, benzyl alcohol, chitosan, epigallocatechin gallate and pectin methyl-esterase. Ca2+distribution, callose and cellulose were imaged with FluoForte, aniline blue and Pontamine Fast Scarlett stainings, respectively, and observed with confocal microscopy.

Key results: Inhibition of callose synthesis with 2-deoxy-D-glucose evidenced that callose is essential for induction of microspore embryogenesis. A moderate increase of Ca2+ levels with Ca(NO3)2 or InsP3 promoted increased callose synthesis and deposition in the cell wall. However, the use of benzyl alcohol and chitosan to permeabilize the plasma membrane and allow for Ca2+ influx was not positive, as this prevented embryo development by inducing callus formation. Benzyl alcohol did not affect callose and cellulose deposition, but chitosan induced the formation of callose plugs, similar to those formed in response to pathogen attack. Inhibition of pectin methyl-esterase (PME) activity with epigallocatechin gallate during the first 3 days of culture produced ~70% more embryos, but prolonged exposures were negative. Instead, increased PME activity during the first three days was not positive, but when applied for 7 days, embryos increased ~60%.

Conclusions: Together, these results confirm the relevant role of calcium and callose during the first stages of microspore induction, and suggest that the levels of pectin methyl-esterification in the cell wall are dynamic, and different cell wall compositions are required during the different stages of microspore embryogenesis.