The Effects of the Phospholipase D-Antagonist 1-Butanol on Seedling Development and Microtubule Organisation in Arabidopsis

Abstract

;The organisation of plant microtubules into distinct arrays during the cell cycle requires interactions with partner proteins. Having recently identified a 90-kDa phospholipase D (PLD) that associates with microtubules and the plasma membrane [Gardiner et al. (2001) Plant Cell 13: 2143], we exposed seeds and young seedlings of Arabidopsis to 1-butanol, a specific inhibitor of PLD-dependent production of the signalling molecule phosphatidic acid (PA). When added to agar growth media, 0.2% 1-butanol strongly inhibited the emergence of the radicle and cotyledons, while 0.4% 1-butanol effectively blocked germination. When normal seedlings were transferred onto media containing 0.2% and 0.4% 1-butanol, the inhibitor retarded root growth by about 40% and 90%, respectively, by reducing cell elongation. Inhibited plants showed significant swelling in the root elongation zone, bulbous or branched root hairs, and modified cotyledon morphology. Confocal immunofluorescence microscopy of root tips revealed that 1-butanol disrupted the organisation of interphase cortical microtubules. Butanol isomers that do not inhibit PLD-dependent PA production, 2-and 3-butanol, had no effect on seed germination, seedling growth, or microtubule organisation. We propose that production of PA by PLD may be required for normal microtubule organisation and hence normal growth in Arabidopsis.

Enhanced induction of microspore embryogenesis after n-butanol treatment in wheat (Triticum aestivum L.) anther culture

“…Among the three butanol isomers applied, only n-butanol had a clearly observable effect on anther culture response. This confirms that the effects of n-butanol were specific and could be attributable to the ability of n-butanol to disrupt cortical MT arrays (Dhonukshe et al 2003;Gardiner et al 2003;Hirase et al 2006). This is in accordance with the stimulation of microspore embryogenesis produced by antimicrotubule agents, as previously reported in anther and microspore culture of wheat (Barnabás et al 1991;Soriano et al 2007) and in other species (Herrera et al 2002;Zhou et al 2002).…”

“…In the present work, toxicity was not detected at the low concentrations (0.1-0.2%) of n-butanol applied, as previously reported by Dhonukshe et al 2003 andGardiner et al 2003. Both Pavon and Caramba showed similar response to n-butanol treatments.…”

“…Treatment of Arabidopsis seedlings with n-butanol also produced cortical MT disruption, and inhibited seed germination and seedling growth (Gardiner et al 2003). The effect of n-butanol on plant MTs, is thought to be mediated by its known ability to activate phospholipase D (PLD) (Munnik et al 1995).…”

“…The reported activities of these butanol isomers have been shown to be reversible and non-toxic (Dhonukshe et al 2003;Gardiner et al 2003). Therefore, in this work we applied n-butanol, sec-butanol and tert-butanol during the first hours of bread wheat anther culture, in order to evaluate if n-butanol could specifically stimulate microspore embryogenesis.…”

Generation of noncentrosomal microtubule arrays

“…Specific inhibition or activation of p90/PLD, an enzyme that associates with MTs and membranes (Gardiner et al, 2001;Marc et al, 1996), affects the organization of interphase cortical MTs, leading to either the disruption of cortical MTs or their uncoupling from the plasma membrane (Dhonukshe et al, 2003;Gardiner et al, 2003).…”