The dynamic behavior of storage organelles in developing cereal seeds and its impact on the production of recombinant proteins

Abstract

Cereal endosperm is a highly differentiated tissue containing specialized organelles for the accumulation of storage proteins, which are ultimately deposited either within protein bodies derived from the endoplasmic reticulum, or in protein storage vacuoles (PSVs). During seed maturation endosperm cells undergo a rapid sequence of developmental changes, including extensive reorganization and rearrangement of the endomembrane system and protein transport via several developmentally regulated trafficking routes. Storage organelles have been characterized in great detail by the histochemical analysis of fixed immature tissue samples. More recently, in vivo imaging and the use of tonoplast markers and fluorescent organelle tracers have provided further insight into the dynamic morphology of PSVs in different cell layers of the developing endosperm. This is relevant for biotechnological applications in the area of molecular farming because seed storage organelles in different cereal crops offer alternative subcellular destinations for the deposition of recombinant proteins that can reduce proteolytic degradation, allow control over glycan structures and increase the efficacy of oral delivery. We discuss how the specialized architecture and developmental changes of the endomembrane system in endosperm cells may influence the subcellular fate and post-translational modification of recombinant glycoproteins in different cereal species.

Keywords: cereal biotechnology; endosperm; molecular farming; recombinant glycoproteins; storage organelles.

FIGURE 1

Comparison of endosperm cells from the subaleurone layer at mid-maturation stage in wheat (A), barley (B), rice (C), and maize (D). Semithin sections (1 μm in thickness), stained with toluidine blue for light microscopy (Arcalis et al., 2004). (A) Wheat endosperm cells show a predominant, large central PSV. Note the huge prolamin aggregates stained in light blue (*) and the scarce globulin (triticin) bodies stained in dark blue at the periphery of the prolamins. Several smaller vacuolar compartments containing prolamins can also be observed (arrows), and well developed endoplasmic reticulum (ER) and abundant spindle like starch grains (s). (B) Barley endosperm cells show abundant protein deposits forming a multiphasic protein body (see the different shades of blue), habitually within a PSV. Note the abundant ER, the spheroidal starch grains (s) and an apoptotic nucleus (n). (C) Rice endosperm cells are the smallest depicted in this figure. Rice endosperm accumulates mainly glutelins in protein storage vacuoles (arrows). Note the abundant PSVs containing a blue stained inclusion, normally close to the tonoplast and the spherical, densely packed prolamin bodies or PB-I (arrowheads). Starch (s). (D) Maize endosperm stores mainly prolamins in ER-derived protein bodies (zein bodies). Zein bodies (arrowheads) reach a diameter of 1 μm, are very abundant and appear evenly spread within the cytoplasm. Several storage vacuoles can be also observed (arrows) either completely filled or with peripherical protein deposits. Starch grains (s) are spheroidal and similar in size to the PSVs. Scale bar equals 10 μm.

FIGURE 2

TIP3-GFP-labeled PSVs contain protein bodies and are involved in fusion and rupture in barley endosperm. (A) TIP3-GFP-labeled PSVs comprise large protein bodies stained with ER-Tracker^TM red (asterisks). Note the three-dimensional surface rendering (and the magnified inset) of 16 sections with a step size of 0.5 μm. (B) Live cell imaging of TIP3-GFP-labeled PSVs show fusion (a′) and collapse (b′) processes in the subaleurone at 10 DAP. Note the presence of TIP3-GFP-labeled vesicles (a′, arrowheads) after PSV fusion (a′, arrows). Images were acquired every 6 s. Scale equals 5 μm. Figure partially reproduced from Ibl et al. (2014).

FIGURE 3

Schematic overview of the spatiotemporally regulated dynamics of storage organelles in cereal seeds during development and germination. The spherical PSVs in the aleurone remain constant throughout seed maturation. Note the presence of zein-rich protein inclusions (asterisk) and intravacuolar membranes (indicated as short black lines). Upon germination, GA₃-dependent vacuolation results in the formation of one large central vacuole and secondary lytic vacuoles. The dramatic change from nutrient-storing compartments to lytic organelles is indicated by different shadings. In the subaleurone, large PSVs turn into smaller, spherical structures enclosing protein bodies (gray), followed by vacuolar fusion resulting in the formation of larger, composite protein bodies. The surrounding membranes gradually lose their integrity (indicated by broken lines). In the central starchy endosperm, the large PSVs containing protein bodies (gray) are shrinking. At later developmental stages, the protein bodies are tightly enclosed by PSV membranes, which degenerate during development, leaving protein bodies with no continuous PSV membrane or no membrane at all.