Abscisic acid signal transduction in guard cells is mediated by phospholipase D activity

Abstract

In guard cells, the plant hormone abscisic acid (ABA) inhibits stomatal opening and induces stomatal closure through the coordinated regulation of ion transport. Despite this central role of ABA in regulating stomatal function, the signal transduction events leading to altered ion fluxes remain incompletely understood. We report that the activity of the enzyme phospholipase D (PLD) transiently increased in guard cell protoplasts at 2.5 and 25 min after ABA application. Treatment of guard cell protoplasts with phosphatidic acid (PtdOH), one of the products of PLD activity, led to an inhibition of the activity of the inward K ؉ channel. PtdOH also induced stomatal closure and inhibited stomatal opening when added to epidermal peels. Application of 1-butanol (1-buOH), a selective inhibitor of PtdOH production by PLD, inhibited the increase in PtdOH production elicited by ABA. 1-BuOH treatment also partially prevented ABA-induced stomatal closure and ABA-induced inhibition of stomatal opening. This inhibitory effect of buOH was enhanced by simultaneous application of nicotinamide, an inhibitor of cADP ribose action. These results suggest that in the guard cell, ABA activates the enzyme PLD, which leads to the production of PtdOH. This PtdOH is then involved in triggering subsequent ABA responses of the cell via a pathway operating in parallel to cADP ribose-mediated events.

Inhibition of Phospholipase Dα byN-Acylethanolamines

“…Our results are consistent with these findings and they indicate that selective biochemical inhibition of PLD␣ by NAEs results in an interference with ABA-induced stomatal closure (Tables II-IV). In addition, our results continue to point to a general role for PLD␣ in ABA signaling as has been inferred from studies with a number of plants systems (Fan et al, 1997;Ryu et al, 1997;Ritchie and Gilroy, 1998;El Maarouf, 1999;Jacob et al, 1999;Frank et al, 2000). Membrane-permeable NAEs should provide a new biochemical tool for the dissection of PLD␣ physiological function(s) in plants.…”

“…Treatment of epidermal cell layers of tobacco and Commelina communis with NAE 12:0 abrogated the ABA-induced closure of stomatal guard cells, a process mediated by PLD␣ (Jacob et al, 1999;Sang et al, 2001). Together, our results suggest a novel lipid-mediator role for NAEs in higher plants as a potential endogenous inhibitor of PLD␣, and they suggest that products of PLD␤ or ␥ (NAEs) can regulate the activity of PLD␣ in plant cells.…”

“…2 and 3), two orders of magnitude lower than LPE and within the concentration range of NAEs measured in vivo in plant tissues (for summary, see Chapman, 2000). PLD has been implicated by others in the signal transduction pathway of guard cells in response to ABA (Jacob et al, 1999;Sang et al, 2001). Recent transgenic approaches with plants devoid of PLD␣ activity clearly demonstrated that abrogation of PLD␣ activity delayed the ABA-induced closure of stomata and resulted in profound physiological effects in plants exposed to drought stress (Sang et al, 2001).…”

“…The unregulated activity of PLD␣ in plant cells, then, potentially would lead to membrane damage and loss of cellular function, and cells likely have mechanisms in place to regulate PLD␣ activity. In addition, a signal transduction role for the PLD␣ isoform has been implicated from studies in several plant systems in which PLD␣ mediates, in part, the cellular responses to abscisic acid (ABA; Fan et al, 1997;Ritchie and Gilroy, 1998;Jacob et al, 1999;Frank et al, 2000;Sang et al, 2001).…”

“…The unregulated activity of PLD␣ in plant cells, then, potentially would lead to membrane damage and loss of cellular function, and cells likely have mechanisms in place to regulate PLD␣ activity. In addition, a signal transduction role for the PLD␣ isoform has been implicated from studies in several plant systems in which PLD␣ mediates, in part, the cellular responses to abscisic acid (ABA; Fan et al, 1997;Ritchie and Gilroy, 1998;Jacob et al, 1999;Frank et al, 2000;Sang et al, 2001).Recent evidence in tobacco (Nicotiana tabacum) cells indicated that a novel class of lipids, N-acylethanolamines (NAEs), was released from the membrane phospholipid, N-acylphosphatidylethanolamine (NAPE), in a signal-mediated fashion Tripathy et al, 1999). A PLD-type activity was identified that hydrolyzed NAPE to NAE in vitro , and a subsequent biochemical survey of PLD catalytic properties of recombinant isoforms (Pappan et al, 1998) suggested that the activity in tobacco microsomes likely was attributed to the ␤ or ␥ isoforms.…”