Membrane lipids play fundamental structural and regulatory roles in cell metabolism and signaling. Here, we report that phosphatidic acid (PA), a product of phospholipase D (PLD), regulates MAP65-1, a microtubule-associated protein, in response to salt stress. Knockout of the PLDa1 gene resulted in greater NaCl-induced disorganization of microtubules, which could not be recovered during or after removal of the stress. Salt affected the association of MAP65-1 with microtubules, leading to microtubule disorganization in plda1cells, which was alleviated by exogenous PA. PA bound to MAP65-1, increasing its activity in enhancing microtubule polymerization and bundling. Overexpression of MAP65-1 improved salt tolerance of Arabidopsis thaliana cells. Mutations of eight amino acids in MAP65-1 led to the loss of its binding to PA, microtubule-bundling activity, and promotion of salt tolerance. The plda1 map65-1 double mutant showed greater sensitivity to salt stress than did either single mutant. These results suggest that PLDa1-derived PA binds to MAP65-1, thus mediating microtubule stabilization and salt tolerance. The identification of MAP65-1 as a target of PA reveals a functional connection between membrane lipids and the cytoskeleton in environmental stress signaling.

The regulation of hypocotyl elongation is important for plant growth. Microtubules play a crucial role during hypocotyl cell elongation. However, the molecular mechanism underlying this process is not well understood. In this study, we describe a novel Arabidopsis thaliana microtubule-destabilizing protein 25 (MDP25) as a negative regulator of hypocotyl cell elongation. We found that MDP25 directly bound to and destabilized microtubules to enhance microtubule depolymerization in vitro. The seedlings of mdp25 mutant Arabidopsis lines had longer etiolated hypocotyls. In addition, MDP25 overexpression resulted in significant overall shortening of hypocotyl cells, which exhibited destabilized cortical microtubules and abnormal cortical microtubule orientation, suggesting that MDP25 plays a crucial role in the negative regulation of hypocotyl cell elongation. Although MDP25 localized to the plasma membrane under normal conditions, increased calcium levels in cells caused MDP25 to partially dissociate from the plasma membrane and move into the cytosol. Cellular MDP25 bound to and destabilized cortical microtubules, resulting in their reorientation, and subsequently inhibited hypocotyl cell elongation. Our results suggest that MDP25 exerts its function on cortical microtubules by responding to cytoplasmic calcium levels to mediate hypocotyl cell elongation.

The organization and dynamics of microtubules are regulated by microtubule-associated proteins, or MAPs. In Arabidopsis (Arabidopsis thaliana), nine genes encode proteins of the evolutionarily conserved MAP65 family. We proposed that different MAP65s might have distinct roles in the interaction with microtubules. In this study, two AtMAP65 proteins, AtMAP65-1 and AtMAP65-6, were chosen to test this hypothesis in vitro. Although both fusion proteins were able to cosediment with microtubules in vitro, different properties on tubulin polymerization and microtubule bundling were observed. AtMAP65-1 was able to promote tubulin polymerization, enhance microtubule nucleation, and decrease the critical concentration for tubulin polymerization. It also induced the formation of large microtubule bundles by forming cross-bridges between microtubules evenly along the whole length of microtubules. In the presence of AtMAP65-1, microtubule bundles were more resistant to cold and dilution treatments. AtMAP65-6, however, demonstrated no activity in promoting tubulin polymerization and stabilizing preformed microtubules. AtMAP65-6 induced microtubules to form a mesh-like network with individual microtubules. Cross-bridge-like interactions were only found at regional sites between microtubules. The microtubule network induced by AtMAP65-6 was more resistant to high concentration of NaCl than the bundles induced by AtMAP65-1. Purified monospecific anti-AtMAP65-6 antibodies revealed that AtMAP65-6 was associated with mitochondria in Arabidopsis cells. It was concluded that these two MAP65 proteins were targeted to distinct sites, thus performing distinct functions in Arabidopsis cells.

Microfilament dynamics play a critical role in regulating stomatal movement; however, the molecular mechanism underlying this process is not well understood. We report here the identification and characterization of STOMATAL CLOSURE-RELATED ACTIN BINDING PROTEIN1 (SCAB1), an Arabidopsis thaliana actin binding protein. Plants lacking SCAB1 were hypersensitive to drought stress and exhibited reduced abscisic acid-, H 2 O 2 -, and CaCl 2 -regulated stomatal movement. In vitro and in vivo analyses revealed that SCAB1 binds, stabilizes, and bundles actin filaments. SCAB1 shares sequence similarity only with plant proteins and contains a previously undiscovered actin binding domain. During stomatal closure, actin filaments switched from a radial orientation in open stomata to a longitudinal orientation in closed stomata. This switch took longer in scab1 plants than in wild-type plants and was correlated with the delay in stomatal closure seen in scab1 mutants in response to drought stress. Our results suggest that SCAB1 is required for the precise regulation of actin filament reorganization during stomatal closure.