Evidence for Deposition of Cellulose Prior to Dark-starvation Treatment During Spherulation in Physarum microplasmodia

Abstract

Physarum plasmodium living as a slimy mass of protoplast in the dark is fragmented into small multinucleated microplasmodia (mPL) in a liquid medium. When mPL were exposed to several unfavorable environments, they transformed into “spherules” with the cell wall structure. We established a synchronous spherule-induction system for mPL consisting of 3 steps: Step 1, subculture in the 1% medium (1% sucrose and 1% soytone peptone) for 84 h at 25°C; Step 2, preculture in the 2% medium (2% sucrose and 2% soytone peptone) for 72 h at 25°C; and Step 3, dark-starvation treatment in the spherulation medium for 96 h at 25°C. Approximately 100% spherulation was observed within 48 h in Step 3. By fluorescence microscopy, we confirmed for the first time that cellulose was the major component of the cell wall in the Physarum spherule. From the results of experiments using the synchronous spherule-induction system and those of experiments on the inhibitory effect of 2,6-dichlorobenzonitrile (DCB) on cellulose synthesis in mPL in the preculture period, we presumed that the nourished medium in the preculture period was essential for mPL prior to spherulation to attain 100% of spherule. Our in vivo labeling experiments revealed that mPL of multinucleated protoplasts produced cellulose in the preculture period by de novo synthesis. We conclude that the Physarum plasmodia always supply themselves with materials to adapt to unfavorable environmental conditions such as starvation as rapidly as they can, even when they are under excellent conditions.