In vitro inhibition of incompatible pollen tubes in Nicotiana alata involves the uncoupling of the F-actin cytoskeleton and the endomembrane trafficking system
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In the S-RNase-based self-incompatibility system, subcellular events occurring in the apical region of incompatible pollen tubes during the pollen rejection process are poorly understood. F-actin dynamics and endomembrane trafficking are crucial for polar growth, which is temporally and spatially controlled in the tip region of pollen tubes. Thus, we developed a simple in vitro assay to study the changes in the F-actin cytoskeleton and the endomembrane system at the apical region of incompatible pollen tubes in Nicotiana alata. Growth but not germination of pollen tubes of S c10 -, S 70 -, and S 75 -haplotypes was selectively inhibited by style extracts carrying the same haplotypes. Pollen F-actin cytoskeleton and endomembrane system, visualized by fluorescent markers, were normal during the initial 60 min of pollen culture in the presence of compatible and incompatible style extracts. Additional culture resulted in complete growth arrest and critical alterations in the integrity of the F-actin cytoskeleton and the endomembrane system of incompatible pollen tubes. The F-actin ring and the V-shaped zone disappeared from the apical region, while distorted F-actin cables and progressive formation of membrane aggregates evolved in the subapical region and the shank. The vacuolar network of incompatible pollen tubes invaded the tip region, but vacuolar membrane integrity remained mostly unaffected. The polar growth machinery of incompatible pollen tubes was uncoupled, as evidenced by the severe disruption of colocalization between the F-actin cytoskeleton and the endomembrane compartments. A model of pollen rejection integrating the main subcellular events occurring in incompatible pollen is discussed.
KeywordsEndomembrane system F-actin cytoskeleton Nicotiana alata Polar growth Self-incompatibility
Actin binding proteins
We thank Dr Carlos Mas and Dr Cecilia Sampedro for their assistance in digital image processing and Gabriela Diaz Cortez for editorial and language assistance. The vPPase antibody was kindly provided by Dr Thomas Phillips (University of Missouri). This work was supported by funds from the following Argentina state agencies: Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT; PICT 32933); Consejo Nacional de Investigaciones Científicas y Tecnológicas (CONICET; PIP 11220090100265); and Secretaría de Ciencia y Técnica-Universidad Nacional de Córdoba (SECyT-UNC; 05/C466) to AG. AG is a member of CONICET. JAR has a CONICET postdoctoral scholarship. HJR is a SECyT-Universidad Nacional de Córdoba scholarship holder.
Conflict of interest
The authors declare that they have no conflicts of interest.
Elongation rate and vesicle dynamics of a pollen tube started to be tracked 50 min after challenging with a compatible style extract. (MPG 8824 kb)
Elongation rate and vesicle dynamics of a pollen tube started to be tracked 50 min after challenging with an incompatible style extract. (MPG 6998 kb)
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