Phosphatidyl inositol 4,5-bisphosphate (PI 4,5-P2) accumulates in a Rac/Rop-dependent manner in the pollen tube tip plasma membrane, where it may control actin organization and membrane traffic. PI 4,5-P2 is hydrolyzed by phospholipase C (PLC) activity to the signaling molecules inositol 1,4,5-trisphosphate and diacyl glycerol (DAG). To investigate PLC activity during tip growth, we cloned Nt PLC3, specifically expressed in tobacco (Nicotiana tabacum) pollen tubes. Recombinant Nt PLC3 displayed Ca2+-dependent PI 4,5-P2–hydrolyzing activity sensitive to U-73122 and to mutations in the active site. Nt PLC3 overexpression, but not that of inactive mutants, inhibited pollen tube growth. Yellow fluorescent protein (YFP) fused to Nt PLC3, or to its EF and C2 domains, accumulated laterally at the pollen tube tip plasma membrane in a pattern complementary to the distribution of PI 4,5-P2. The DAG marker Cys1:YFP displayed a similar intracellular localization as PI 4,5-P2. Blocking endocytic membrane recycling affected the intracellular distribution of DAG but not of PI 4,5-P2. U-73122 at low micromolar concentrations inhibited and partially depolarized pollen tube growth, caused PI 4,5-P2 spreading at the apex, and abolished DAG membrane accumulation. We show that Nt PLC3 is targeted by its EF and C2 domains to the plasma membrane laterally at the pollen tube tip and that it maintains, together with endocytic membrane recycling, an apical domain enriched in PI 4,5-P2 and DAG required for polar cell growth.

Our results show that scent is an important flower trait that defines plant-pollinator interactions at the level of individual plants. The genetic basis underlying such a major phenotypic difference appears to be relatively simple and may enable rapid loss or gain of scent through hybridization.

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The underlying mechanisms are thought to be highly conserved in evolution (2, 4). RNAi in animals is initiated by doublestranded RNAs (dsRNAs) similar in sequence to the transcribed region of target genes. These dsRNAs undergo endonucleolytic cleavage to generate 21-to 23-nt-long small interfering RNAs (siRNAs), which then promote RNA degradation (5-7) Remarkably, the silent state in transgenic plants and in C. elegans can spread from cell to cell and even systemically throughout the organism, implying the existence of mobile silencing signals (2, 8). Little is known about the chemical nature of these signals, but it seems likely that the sequence-specific component is an RNA (8-11). The finding that siRNA and dsRNA accumulate in silent tissues, together with studies of informative stable transformants and PTGS induced by RNA viruses, supports the view that dsRNAs and siRNAs have key roles in plant PTGS (12-14). Nevertheless, direct evidence that these or other RNAs can induce systemic PTGS or comprise silencing signals in plants is lacking.In the present study, we used a positive marker system and real-time monitoring of green fluorescent protein (GFP) expression to show that double-stranded siRNAs, large sense, antisense, and double-stranded RNAs delivered biolistically into plant cells trigger PTGS capable of spreading locally and systemically. The introduced siRNAs trigger the production of siRNAs derived from sequences both 3Ј and 5Ј of the inducing siRNAs. Our findings support the hypothesis that siRNAs themselves or intermediates induced by siRNAs could comprise silencing signals and are generated in a self-amplifying fashion. Materials and Methods