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Immunolocalization and histochemical evidence for the association of two different Arabidopsis annexins with secretion during early seedling growth and development

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Abstract

Annexins are a multigene, multifunctional family of calcium-dependent, membrane-binding proteins found in animal and plant cells. In plants, annexins have been localized in the cytoplasm and at the cell periphery of highly secretory cell types, and in the tip region of polarly growing cells. Consequently, one proposed function for annexins in plant cells is participation in the Golgi-mediated secretion of new wall materials. In Arabidopsis, there are eight different annexin cDNAs, which share between 30% and 81% deduced amino acid sequence identity. We have used two monospecific Arabidopsis anti-annexin antibodies, raised against divergent 31-mer peptides from AnnAt1 and AnnAt2 and a previously characterized pea anti-annexin p35 antibody, for Western blot and immunolocalization studies in Arabidopsis. Western blot analyses of various Arabidopsis protein fractions showed that the two Arabidopsis antibodies are able to specifically recognize annexins in both soluble and membrane fractions. Immunofluorescence results with the three annexin antibodies show staining of secretory cells, especially at the cell periphery in developing sieve tubes, outer root cap cells, and in root hairs, consistent with previous results. In developmentally different stages some staining was also seen near the apical meristem, in some leaf cells, and in phloem-associated cells. Autoradiography following 3H-galactose incorporation was used to more clearly correlate active secretion of wall materials with the localization patterns of a specific individual annexin protein in the same cells at the same developmental stage. The results obtained in this study provide further support for the hypothesis that these two Arabidopsis annexins function in Golgi-mediated secretion during early seedling growth and development.

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Fig. 1a–e
Fig. 2a–f
Fig. 3a–i
Fig. 4a–g

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Abbreviations

AGP :

Arabinogalactan protein

SDS-PAGE :

Sodium dodecylsulphate polyacrylamide gel electrophoresis

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Acknowledgements

This work is dedicated in loving memory to Marianne Dauwalder. She was a best friend and great colleague during the past 20 years and she is dearly missed. Her commitment to always doing the hard work needed to get things right kept our research grounded and took our science together to a higher level. I thank her for teaching me much about science and life. This work was supported by NASA grants (NAGW 1519 and NAG2 1586) to S.J. Roux.

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  1. School of Biological Sciences, Section of Molecular Cell and Developmental Biology, University of Texas, Austin, TX 78712, USA

    Gregory B. Clark, Dongwoo Lee, Marianne Dauwalder & Stanley J. Roux

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  1. Gregory B. Clark
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  2. Dongwoo Lee
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  3. Marianne Dauwalder
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  4. Stanley J. Roux
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Correspondence to Stanley J. Roux.

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Clark, G.B., Lee, D., Dauwalder, M. et al. Immunolocalization and histochemical evidence for the association of two different Arabidopsis annexins with secretion during early seedling growth and development. Planta 220, 621–631 (2005). https://doi.org/10.1007/s00425-004-1374-7

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