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

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.

Biochemical and Functional Properties of Wheat Gliadins: A Review

“…Extensive and intensive research is being carried out on gluten proteins to determine their properties and structure. Based on their solubility in aqueous alcohol solutions, prolamins of wheat have been divided into two classes, the gliadins and glutenins (Francin-Allami et al, 2011). Together, gliadins and glutenins represent 80-85% of the total proteins of wheat flour (Veraverbeke and Delcour, 2002) and impart the unique properties-extensibility and elasticity to the wheat dough.…”

Protein accumulation and rumen stability of wheat γ‐gliadin fusion proteins in tobacco and alfalfa

“…Here, we have demonstrated that both a γ‐gliadin‐δ‐zein protein and a γ‐gliadin‐GFP fusion protein can accumulate to very high levels in transgenic tobacco. Previous studies demonstrated that when ectopically expressed in yeast or in tobacco BY‐2 cells, γ‐gliadin and γ‐gliadin fused to other proteins were transported to central vacuoles and degraded, probably due to lack of an ER retention signal (Francin‐Allami et al ., ; Rosenberg et al ., ). Napier et al .…”

Identification of the region of rice 13 kDa prolamin essential for the formation of ER-derived protein bodies using a heterologous expression system

“…[5][6][7][8]11,19) In addition, rice prolamin has been shown to form ER-derived PBs in plant tissues such as rice leaves, roots, 12) and calli.…”

“…The N-terminal domains of γ-zein and γ-gliadin are important for ER retention or the formation of ERderived PBs in heterologous tissue, and these domains are highly hydrophobic. 6,11) In addition to cereal prolamin, hydrophobic proteins such as hydrophobin (fungi) and elastin (mammalian) also formed PBs in a heterologous expression system. 22,23) These data suggest that the hydrophobic properties of the polypeptide promote aggregation in ER, resulting in the formation of ER-derived PBs.…”