Plasmid mini kit

Pollen Tube Growth Assay Flowering plants were used for collecting mature pollen grains used for bombardment-mediated transformation as described below. For transient expression of CNGCs and CaM2 in pollen tubes, the coding regions of CNGCs and CaM2 were PCR-amplified with specific primers and cloned into pGEM-T Easy vector (Promega, USA). After sequencing, the cDNAs were cloned into pBI221 vector (CLONTECH Laboratories, Mountain View, CA) in which CaMV 35S::GUS was replaced with the LAT52 promoter driving GFP (Twell et al., 1990) . All resulting constructs were purified using plasmid mini kits (QIAGEN, Germany). Mature pollen grains were collected from N. benthamiana plants right before each experiment, and were transformed with construct DNA using a particle bombardment procedure described previously (Fu et al., 2001) . Briefly, 0.5 mg of gold particles was coated with 0.8 mg of plasmid DNA. Co-bombardments were achieved by coating particles with 1 mg of each plasmid construct. The transformed pollen grains were incubated at 28 C for 3 hours. For quantitative analysis of growth phenotype, images of transformed pollen tubes were captured using a cooled CCD camera (model DP70) attached on an Olympus microscope (model BX51). Pollen tube length and maximum tip width were measured through Zeiss LSM image browser (version 3.2).

Plasmids used for transfection (p35S::CNGC18-YFP, p35S::CNGC8-YFP, p35S::CNGC18-YFP N , p35S::CNGC8-YFP C ) were transformed into E. coli and purified with plasmid mini kits (QIAGEN, Germany). Arabidopsis protoplasts were isolated and transfected as previously described (Yoo et al., 2007) . Briefly, 10-15 Arabidopsis leaves were cut into 0.5 mm wide strips that were placed into 15 mL enzyme solution for vacuum infiltration. The protoplasts in the enzyme solution were collected by passing a nylon mesh and centrifuging at 150 g for 3 min. Protoplast pellets were resuspended in 5 mL cold W5 solution and used for transfection by mixing 10 mL plasmid DNA (about 1 mg/mL) and 110 ml 40% PEG for every aliquot of 100 mL protoplasts. The mixture was incubated for 30 minutes at room temperature, followed by addition of 500 mL W5 solution and centrifugation at 300 g for about 2 min. and the protoplast pellet was resuspend in 1mL W5 solution and incubated overnight. YFP fluorescence was visualized with a confocal scanning microscope (Zeiss LSM510 META, Germany).

Plasmid EX-TO223-M13 containing flag tagged HS3ST1 enzyme sequence was purchased from GeneCopoeia. Bacterial transformation of E.coli DH5α (Invitrogen) was performed using the heat shock method. Bacterial cells lysis and plasmid purification were performed using Plasmid Mini Kits (Qiagen). To generate an empty plasmid (plasmid without HS3ST1 sequence), 3 μg of plasmid were digested with 30U of BsrGI (Bsp 1407I, ThermoFisher). The digested plasmid was extracted using QIAquick Gel Extraction Kit Plasmid extraction. The final plasmid was ligated 3h at room temperature using T4 DNA ligase. Cells transfections were performed using Effectene Reagent (Qiagen) in combination with an enhancer and a DNA-condensation buffer. The following day, fresh media containing 200 µg/mL of selective antibiotic G418 was added to the transfected cells. Colonies were isolated using cloning cylinders.