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.
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...
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