Supplemental Data

Files in this Data Supplement:

• Supplemental Figure 1 - Amino Acid Sequence Alignment of Pet PLC1 and two Arabidopsis PLCs, At PLC2 and At PLC4. The sequences were aligned using ClustalW 1.82. Among the nine PLCs of Arabidopsis, At PLC2 is most similar to Pet PLC1 and At PLC4 is expressed in pollen. Pet PLC1, like other plant PLCs, contains a C2 Ca²⁺/phospholipid-binding domain, the conserved X and Y catalytic domains, and a region homologous to the second loop of the Ca²⁺-binding EF hand of PLC δ, the so-called “EF loop” (Otterhag et al., 2001). Pet PLC1 lacks the PH domain involved in membrane targeting and processive catalysis in nearly all animal PLCs. Amino acids shown in red are the starting amino acids for the X+Y+C2 truncations used in this study on Pet PLC1and by Otterhag et al., (2001) on At PLC2. The catalytic His¹²⁶, which was mutated to Ala¹²⁶ to produce Pet PLC1-H126A, is shown in light blue.
• Supplemental Movie 1 - Time Lapse of a Growing Pollen Tube Expressing GFP:Pet PLC1. Note pollen tube shows phases of rapid growth interspersed by periods of slow growth/arrest when GFP:Pet PLC1 accumulates to the very apex of the tube. The duration of the Movie is 26 min. Scale bar represents 10 μm.
• Supplemental Movie 2 - Time Lapse of a Growing Pollen Tube Expressing GFP:Pet PLC1 and stained with 5 μM FM 4-64. Note the apical plasma membrane and the ‘vesicle cone’ (arrow) are both labeled. The duration of the Movie is 9 min 30 s. Scale bar represents 10 μm.
• Supplemental Movie 3 - Time Lapse of a Non-Growing Pollen Tube Expressing GFP:Pet PLC1 and stained with 5 μµM FM 4-64. Note the entire plasma membrane is labeled by both probes,with no evidence of a vesicle rich cone of staining in the apical cytoplasm seen in the growing tube. The duration of the Movie is 3 min 30 s. Scale bar represents 10 μm. Scale bar represents 10 µm.
• Supplemental Movie 4 - Fluorescence Recovery After Photobleaching of a Pollen Tube Expressing GFP:Pet PLC1.Photobleaching of the apical; cytoplasm was applied as indicated and was limited to the region denoted by the dashed circle. The duration of the Movie is 3 min 40 s. Scale bar represents 10 μm.
• Supplemental Movie 5 - Fluorescence Recovery After Photobleaching of a Pollen Tube Expressing GFP:Pet PLC1. Photobleaching of the plasma membrane was applied when indicated and was limited to the region of the lateral plasma membrane denoted by the dashed box. The duration of the Movie is 6 min 40 s. Scale bar represents 10 μm.
• Supplemental Movie 6 - Golgi Dynamics in Wild-Type Growing Pollen Tubes. Golgi motility was visualized by expressing the Golgi marker, NAG:CFP, made from a fusion of the first 79 amino acids of an A. thaliana N-acetylglucosaminyl transferase I to CFP. Note the linear tracking of the Golgi in the shank of the tube and exclusion from the very apex. The duration of the Movie is 125 s.
• Supplemental Movie 7 - Golgi Dynamics in Pollen Tubes Expressing Pet PLC1- H126A. Golgi motility was visualized by expressing the Golgi marker, NAG:CFP, made from a fusion of the first 79 amino acids of an A. thaliana N-acetylglucosaminyl transferase I to CFP. Note the linear tracking of the Golgi in the shank of the tube seen in wild-type pollen is maintained but Golgi now traverse the very apex of the bulging tip. The duration of the Movie is 125 s.
• Supplemental Movie 8 - Time Lapse of a Growing Pollen Tube Expressing GFP:PH to visualize PtdInsP₂ localization. The pollen tube has also been stained with 5 μM FM 4-64. Note gradient of GFP:PH fluorescence to the apex of the growing tube. The duration of the Movie is 18 min 30 s. Scale bar represents 10 μm.
• Supplemental Movie 9 - Time Lapse of a Non-Growing Pollen Tube Expressing GFP:PH to visualize PtdInsP₂ localization. The pollen tube has also been labeled with 5 μM FM 4-64. Note the gradient of GFP:PH fluorescence towards the apex seen in a growing tube is no longer evident. The duration of the Movie is 7 min 2 s. Scale bar represents 10 μm.