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Planta

, Volume 227, Issue 5, pp 1047–1061 | Cite as

Heterologous expression of IAP1, a seed protein from bean modified by indole-3-acetic acid, in Arabidopsis thaliana and Medicago truncatula

  • Alexander Walz
  • Claudia Seidel
  • Gordana Rusak
  • Seijin Park
  • Jerry D. CohenEmail author
  • Jutta Ludwig-Müller
Original Article

Abstract

The seed protein IAP1 from bean (PvIAP1; Phaseolus vulgaris L.) that is modified by the phytohormone indole-3-acetic acid (IAA) was heterologously expressed in the two reference plant species Arabidopsis thaliana and Medicago truncatula. For the transformation of Medicago we devised a novel protocol using seedling infiltration. When PvIAP1 was overexpressed under the control of the constitutive 35SCaMV promoter in Arabidopsis, the plants showed signs of earlier bolting and enhanced branching. Expression of a fusion protein of PvIAP1 with both a green fluorescence protein (GFP) as reporter and 6× histidine (His) tag under the control of the native bean IAP1 promoter resulted in the accumulation of the protein in both plant species exclusively in seeds as shown by immunoblotting and by fluorescence microscopy. During seed development, PvIAP1 was first expressed in the vascular bundle of Arabidopsis, whereas in later stages GFP fluorescence was visible essentially in all tissues of the seed. Fluorescence decreased rapidly after imbibition in the seeds for both Arabidopsis and Medicago, although the fluorescence persisted longer in Arabidopsis. GFP fluorescence was distributed evenly between an organelle fraction, the microsomal membrane fraction, and the cytosol. This was also confirmed by immunoblot analysis. Clusters of higher GFP fluorescence were observed by confocal microscopy. Although PvIAP1 protein accumulated in seeds of both Arabidopsis and Medicago, neither species post-translationally modified the protein with an indoleacyl moiety as shown by quantitative GC–MS analysis after alkaline hydrolysis. These results indicate an apparent specificity for IAA attachment in different plant species.

Keywords

Arabidopsis thaliana Auxin conjugates Indole-3-acetic acid Medicago truncatula Plant transformation Protein modification 

Abbreviations

CaMV

Cauliflower mosaic virus

GC–MS

Gas chromatography–mass spectrometry

GFP

Green fluorescent protein

IAA

Indole-3-acetic acid

IAP

IAA attached to protein

LEA

Late embryogenesis abundant

Notes

Acknowledgments

This work was supported by a grant from the Deutsche Forschungsgemeinschaft within SPP 1067 to J.L.-M. (Lu500/8), a grant from the German Ministry for Science and Education (WTZ grant HRV 01/025) to J.L.-M and G.R., grants from the National Science Foundation (IBN 01-11530 and DBI 06-06666) to J.D.C. and support from the Minnesota Agricultural Experiment Station and the Gordon and Margaret Bailey Endowment for Environmental Horticulture. We thank Dr. David Mok for the gift of the genomic library from bean and Dr. Cesar V. Mujer for critical reading of the manuscript. The help with Confocal Laser Scanning Microscopy by Dr. Hella Hartmann, BIOTEC, Technische Universität Dresden is gratefully acknowledged.

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Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  • Alexander Walz
    • 1
    • 2
  • Claudia Seidel
    • 1
  • Gordana Rusak
    • 3
  • Seijin Park
    • 4
  • Jerry D. Cohen
    • 4
    Email author
  • Jutta Ludwig-Müller
    • 1
  1. 1.Institut für BotanikTechnische Universität DresdenDresdenGermany
  2. 2.Vital Probes, Inc.MayfieldUSA
  3. 3.Department of Biology, Faculty of ScienceMarulicev trg 20/IIZagrebCroatia
  4. 4.Department of Horticultural Science and the Microbial and Plant Genomics InstituteUniversity of MinnesotaSaint PaulUSA

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