A 90-kD Phospholipase D from Tobacco Binds to Microtubules and the Plasma Membrane

Abstract

The organization of microtubule arrays in the plant cell cortex involves interactions with the plasma membrane, presumably through protein bridges. We have used immunochemistry and monoclonal antibody 6G5 against a candidate bridge protein, a 90-kD tubulin binding protein (p90) from tobacco BY-2 membranes, to characterize the protein and isolate the corresponding gene. Screening an Arabidopsis cDNA expression library with the antibody 6G5 produced a partial clone encoding phospholipase D (PLD), and a full-length gene was obtained by sequencing a corresponding expressed sequence tag clone. The predicted protein of 857 amino acids contains the active sites of a phospholipidmetabolizing enzyme and a Ca 2 ؉ -dependent lipid binding domain and is identical to Arabidopsis PLD ␦ . Two amino acid sequences obtained by Edman degradation of the tobacco p90 are identical to corresponding segments of a PLD sequence from tobacco. Moreover, immunoprecipitation using the antibody 6G5 and tobacco BY-2 protein extracts gave significant PLD activity, and PLD activity of tobacco BY-2 membrane proteins was enriched 6.7-fold by tubulin-affinity chromatography. In a cosedimentation assay, p90 bound and decorated microtubules. In immunofluorescence microscopy of intact tobacco BY-2 cells or lysed protoplasts, p90 colocalized with cortical microtubules, and taxol-induced microtubule bundling was accompanied by corresponding reorganization of p90. Labeling of p90 remained along the plasma membrane when microtubules were depolymerized, although detergent extraction abolished the labeling. Therefore, p90 is a specialized PLD that associates with membranes and microtubules, possibly conveying hormonal and environmental signals to the microtubule cytoskeleton. INTRODUCTIONMicrotubules (MTs), a major component of the plant cytoskeleton, are involved in important cellular functions during growth and development (Lloyd, 1991; Cyr, 1994; Cyr and Palevitz, 1995; Kost et al., 1999;Nick, 1999). Before mitosis, MTs form a preprophase band, which predicts the plane of future cell division. Subsequently, they rearrange into the mitotic spindle and eventually the phragmoplast during cytokinesis. During interphase, they form parallel ordered arrays on the internal face of the plasma membrane. The parallel organization of newly deposited cellulose microfibrils in the cell wall mirrors the orientation of the interphase MT array (Williamson, 1991). Conversely, the MT network also is known to respond to changes in cell wall organization (Fisher and Cyr, 1998). Through their interaction with cellulose microfibril deposition, MTs play an important role in directional cell expansion (Gertel and Green, 1977; Giddings and Staehelin, 1991).Interphase MT arrays in the cell cortex respond to diverse developmental and extracellular stimuli. Cortical MT arrays typically change their orientation from transverse to longitudinal to the growth axis as cells mature (Laskowski, 1990; Cyr and Palevitz, 1995; Wymer and Lloyd, 1996; Granger and Cyr, 2001). Wounding cause...

The role of phospholipase D in plant stress responses

“…A future study of PLDδ localization promises interesting results; this PLD isoform is reportedly localized on the plasma-membrane and bound to microtubules (Gardiner et al, 2001;Wang and Wang, 2001). Furthermore, it is known to be activated in response to dehydration (Katagiri et al, 2001), H 2 O 2 , cold , salinity stress and wounding (Chapters 2 and 3).…”

“…This hypothesis makes use of the twostep transphosphatidylation reaction, and states that the phosphatidyl-PLD intermediate could serve as a transient membrane anchor. The hypothesis was based on the finding that a PLD was found to bind microtubules and the plasma membrane in tobacco cell-suspension cultures (Gardiner et al, 2001). Separate groups have presented experimental data that support this hypothesis.…”

“…Functions for PLD have been suggested in membrane degradation (Ryu and Wang, 1995), vesicular trafficking (Roth et al, 1999), membrane tethering (Gardiner et al, 2001;Dhonukshe et al, 2003) and intracellular signaling Wang, 2002;Laxalt and Munnik, 2002). Plants possess a vast variety of PLDs, whereas mammalians have just two PLDs and only one has been found in yeast (Elias et al, 2002 The Arabidopsis genome contains 12 PLDs that can be subdivided into several classes depending on their sequence similarity, substrate specificity and enzymatic requirements.…”

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).…”

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

“…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). This enzyme is tightly associated with plasma membrane and with the MT cytoskeleton (Gardiner et al 2001), and its stimulation has been correlated with microtubular rearrangements (Dhonukshe et al 2003). PLD hydrolyses membrane phospholipids into the second messenger phosphatidic acid (PA) and free head groups.…”