Dynamic trafficking of wheat γ-gliadin and of its structural domains in tobacco cells, studied with fluorescent protein fusions

Mathilde Francin-Allami, Amélie Saumonneau, Laurence Lavenant, Axelle Bouder, Imogen Sparkes, Chris Hawes, Yves Popineau

Research output: Contribution to journalArticle (Academic Journal)peer-review

15 Citations (Scopus)

Abstract

Prolamins, the main storage proteins of wheat seeds, are synthesized and retained in the endoplasmic reticulum (ER) of the endosperm cells, where they accumulate in protein bodies (PBs) and are then exported to the storage vacuole. The mechanisms leading to these events are unresolved. To investigate this unconventional trafficking pathway, wheat γ-gliadin and its isolated repeated N-terminal and cysteine-rich C-terminal domains were fused to fluorescent proteins and expressed in tobacco leaf epidermal cells. The results indicated that γ-gliadin and both isolated domains were able to be retained and accumulated as protein body-like structures (PBLS) in the ER, suggesting that tandem repeats are not the only sequence involved in γ-gliadin ER retention and PBLS formation. The high actin-dependent mobility of γ-gliadin PBLS is also reported, and it is demonstrated that most of them do not co-localize with Golgi body or pre-vacuolar compartment markers. Both γ-gliadin domains are found in the same PBLS when co-expressed, which is most probably due to their ability to interact with each other, as indicated by the yeast two-hybrid and FRET-FLIM experiments. Moreover, when stably expressed in BY-2 cells, green fluorescent protein (GFP) fusions to γ-gliadin and its isolated domains were retained in the ER for several days before being exported to the vacuole in a Golgi-dependent manner, and degraded, leading to the release of the GFP 'core'. Taken together, the results show that tobacco cells are a convenient model to study the atypical wheat prolamin trafficking with fluorescent protein fusions.

Original languageEnglish
Pages (from-to)4507-20
Number of pages14
JournalJournal of Experimental Botany
Volume62
Issue number13
DOIs
Publication statusPublished - Aug 2011

Keywords

  • Actins
  • Brefeldin A
  • Endoplasmic Reticulum
  • Fluorescence
  • Gliadin
  • Green Fluorescent Proteins
  • Immunoblotting
  • Plant Epidermis
  • Plants, Genetically Modified
  • Protein Structure, Tertiary
  • Protein Transport
  • Recombinant Fusion Proteins
  • Subcellular Fractions
  • Tobacco
  • Transformation, Genetic
  • Triticum
  • Vacuoles
  • Journal Article
  • Research Support, Non-U.S. Gov't

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