Figure 4

Antisense knockdown of candidate genes with putative role in polarized pollen-tube tip expansion. All samples were observed with differential interference contrast (DIC, left panel) and fluorescence microscopy (right panel). The NtpSF3-Lim2a:GFP construct, an actin-cytoskeleton marker [36], was used to monitor possible defects in cytoskeleton organization. (a) Wild-type control and cytofectin-treated pollen tubes. (b) Quantitative analysis of mean pollen-tube length (± SEM) from NTP303 and eIF5A control. Lower panel; semi-quantitative RT-PCR analysis was used to determine transcript levels in each treatment. 1: wild-type control 2: cytofectin-treated pollen tubes; 3: sense-transfected pollen tubes; 4: antisense-transfected pollen tubes. The same numbers key is used for panels (d), (f) and (h). (c, d) Aberrant pollen-tube growth following PD40 knockdown with significant reduction in pollen-tube length (p < 0.001), however with no evident defect in cytoskeleton biogenesis or organization. (e, f) Severe pollen-tube growth defects and occasional abnormal tip morphology (inset, yellow arrows) following CSD1 knockdown. Pollen tubes were significantly shorter (p < 0.001), with 'hook-like' morphology generated immediately after the initiation of germination. (g, h) Less severe but significant (p < 0.01) reduction in NtRFC1-depleted pollen-tube growth. Moderate reduction in transcripts levels were also observed in sense-treated pollen tubes, although this was not reflected by alterations in the pollen-tube phenotype. Scale bar = 20 μM.