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Protoplasma

, Volume 252, Issue 1, pp 63–75 | Cite as

In vitro inhibition of incompatible pollen tubes in Nicotiana alata involves the uncoupling of the F-actin cytoskeleton and the endomembrane trafficking system

  • Juan A. Roldán
  • Hernán J. Rojas
  • Ariel GoldraijEmail author
Original Article

Abstract

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.

Keywords

Endomembrane system F-actin cytoskeleton Nicotiana alata Polar growth Self-incompatibility 

Abbreviations

ABPs

Actin binding proteins

GM

Growth medium

SI

Self-incompatibility

vPPase

Vacuolar pyrophosphatase

Notes

Acknowledgments

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.

Supplementary material

709_2014_658_Fig9_ESM.gif (32 kb)
Fig. S1

Pollen of S c10 -, S 70 -, and, S 75 - haplotypes were cultured in GM. Percentages of germinated (white) and non-germinated (grey) pollen grains are shown. The values are the average ± SD of three or more independent experiments. (GIF 32 kb)

709_2014_658_MOESM1_ESM.eps (790 kb)
High Resolution Image (EPS 790 kb)
709_2014_658_Fig10_ESM.gif (86 kb)
Fig. S2

In vitro growth of Nicotiana alata pollen tubes challenged with compatible and incompatible style extract. Pollen tubes were cultured for 6 h in the presence of compatible (white) and incompatible (gray) style extracts (a-c). The pollen S c10 - and the style extract S c10 S c10 in a were prepared from different S c10 S c10 individuals. The values are the average ± SD of three or more independent experiments. Data were statistically analyzed using one-way ANOVA followed by Tukey test. ***P<0,001. (GIF 85 kb)

709_2014_658_MOESM2_ESM.eps (1.9 mb)
High Resolution Image (EPS 1899 kb)
709_2014_658_Fig11_ESM.gif (85 kb)
Fig. S3

Patterns of F-actin cytoskeleton disorganization after 180 m of culture with incompatible style extract. Images are confocal projections of F-actin stained with fluorescent phalloidin. a F-actin cables distorted and slightly fragmented. b F-actin cables severely fragmented. c F-actin foci. Patterns a, b, and c represented 88%, 9%, and 3%, respectively, of the total pollen tubes analyzed. Bars 10 μm. (GIF 85 kb)

709_2014_658_MOESM3_ESM.tif (2 mb)
High Resolution Image (TIFF 2071 kb)
video online resource 1

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)

video online resource 2

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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Copyright information

© Springer-Verlag Wien 2014

Authors and Affiliations

  • Juan A. Roldán
    • 1
  • Hernán J. Rojas
    • 1
  • Ariel Goldraij
    • 1
    Email author
  1. 1.Centro de Investigaciones en Química Biológica de Córdoba (CIQUIBIC–CONICET), Departamento de Química Biológica, Facultad de Ciencias QuímicasUniversidad Nacional de CórdobaCórdobaArgentina

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