Analyses of Anandamide-Mediated Growth Inhibition in Physcomitrella Patens

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In higher plants, a class of bioactive fatty acid ethanolamides or N-acylethanolamines (NAEs) mediate growth, development, cellular organization and response to stress, in an abscisic acid (ABA)-dependent or independent manner. Unlike in higher plants, Physcomitrella patens, a bryophyte contains anandamide or NAE 20:4, a mammalian endocannabinoid ligand that mediates a multitude of physiological functions including development and stress. Unique lipids in mosses are considered vital for their resilience to environmental stresses; such lipids might enable them to recognize stress at the cellular level, and respond with membrane reorganization and altered growth. Since the identification of anandamide in moss, we have shown that, like abscisic acid (ABA), it inhibits gametophyte growth in a dose-dependent manner and reduced chlorophyll content. It is hypothesized that moss gametophores undergo morphological and cellular changes during anandamide-mediated growth inhibition. To test this, gametophyte growth and morphological changes in phyllodes, under different concentrations of NAE 20:4, were digitally captured using Canon EOS 70D, and analyzed using ImageJ software. NAE 20:4 but not its free fatty acid, arachidonic acid, not only inhibited growth of both shoots and rhizoids in a dose-dependent manner but also showed remarkable cellular changes. Phyllodes and protonemal cells of NAE 20:4 treated plants were further examined under stereo and compound light microscopes. Long- and short-term treatment with anandamide resulted in reduced chloroplast number, cytoplasmic shrinkage and plasmolysis in phyllodes and protonemal cells. A 100 micromolar NAE 20:4 treatment resulted in complete loss of green pigmentation in phyllodes. Effects of anandamide on cytoskeletal organization will be studied using Physcomitrella plants expressing GFP-talin and tubulin, via confocal microscopy. Together, these data will provide insights into anandamide-mediated cellular responses during growth inhibition.


Johnson City, TN

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