RNA Extraction and RT-qPCR

A fixing solution (4% [v/v] formaldehyde, 10% [w/v] Suc, 50 mM of 2,2'-piperazine-1,4-diylbisethanesulfonic acid, 5 mM of MgSO₄, and 0.5 mM of CaCl₂ at pH 7.0) was added to liquid pollen germination medium, and pollen tubes were collected by centrifugation at 12,000g for RNA isolation. Total RNA was extracted from pollen tubes using the modified CTAB method as described in Gasic et al. (2004). After digestion with DNase I (Thermo Fisher Scientific), 2 μg of total RNA was reverse-transcribed to cDNA using the iScript cDNA Synthesis Kit (Bio-Rad). RT-qPCR analysis was performed with iQ SYBR Green Supermix in an iCycler iQ5 system (Bio-Rad) following the manufacturer’s instructions. Each reaction was replicated three times per biological replicate, with ACTIN as an internal reference gene. The relative expression of each gene was calculated using the 2^−ΔCT method (Udvardi et al., 2008). Sequences of the primers for RT-qPCR used in this study are listed in the Supplemental Table.