The changing fate of a secretory glycoprotein in developing maize endosperm

Abstract

Zeins are the major storage proteins in maize (Zea mays) endosperm, and their accumulation in zein bodies derived from the endoplasmic reticulum is well characterized. In contrast, relatively little is known about post-Golgi compartments or the trafficking of vacuolar proteins in maize endosperm, specifically the presence of globulins in structures resembling protein storage vacuoles that appear in early to mid-stage seed development. We investigated this pathway by expressing and analyzing a recombinant reporter glycoprotein during endosperm maturation, using a combination of microscopy and sensitive glycopeptide analysis. Specific N-glycan acceptor sites on the protein were followed through the stages of grain development, revealing a shift from predominantly paucimannosidic vacuolar glycoforms to predominantly trimmed glycan structures lacking fucose. This was accompanied by a change in the main subcellular localization of the protein from large protein storage vacuole-like post-Golgi organelles to the endoplasmic reticulum and zein bodies. The endogenous storage proteins corn alpha-globulin and corn legumin-1 showed a similar spatiotemporal profile both in transgenic plants expressing the reporter glycoprotein and in wild-type plants. This indicates that the shift of the intracellular trafficking route, as observed with our reporter glycoprotein, may be a common strategy in maize seed development.

Figure 1.

Accumulation of phytase and seed storage proteins during seed maturation. Total soluble protein was extracted from transgenic seeds at various developmental stages (10–30 DAP) at a fixed w/v ratio, and 10 μL were loaded per lane. Immunoblots were incubated with antisera against phytase (A), γ zein (B), CAG (C), and CL-1 (D).

Figure 2.

N-glycan structures attached to recombinant phytase glycopeptides TYNYS LGADD LTPFG EQELV NSGIK (A) and YSALIEEIQQNATTFDGK (B), derived from developing maize endosperm at 20, 30, and 40 DAP. The vacuolar-type glycans MMXF and MMX were major N-glycan structures in phytase derived from young seeds (20 DAP), whereas small structures consisting of only one or two core GlcNAc residues dominated in phytase isolated from more mature seeds (30 and 40 DAP). See http://www.proglycan.com for an explanation of N-glycan structure abbreviations.

Figure 3.

Development of maize seeds. Sections after embedding in Spurr's resin. A to C, Light microscopy, toluidine blue: stage 1 (A), stage 2 (B), and stage 3 (C). Aleurone (Al) and endosperm (En). Cells accumulate starch (s), some storage vacuole-like compartments are already seen (arrowheads), smaller refringent vacuolar compartments are also observed (B, arrow), zein bodies appear as small blue stained accretions, few nuclei (n) are still present. D and E, Electron microscopy, general nonspecific contrast: stage 2. Zein bodies (zb) and PSV. Note the globulin-like inclusion in the vacuole-like compartments (*). rER, Rough ER. Bars 20 μm (A–C), 0.5 μm (D and E). [See online article for color version of this figure.]

Figure 4.

Localization of recombinant phytase. Sections after embedding in LRWhite resin. A to C, Fluorescence microscopy. D to F, Electron microscopy. Comparison of the distribution pattern of recombinant phytase between endosperm cells in stage 1 (A), stage 2 (B), and stage 3 (C). Note the storage vacuole-like compartments in each of the developmental stages (arrows) and the nonlabeled spherical zein bodies evenly spread within the cytoplasm (B and C). No significant labeling in the apoplast (arrowheads) or the starch granules (s). D, Stage 1. Note the heavy labeling on a small PSV. E, Stage 3. Gold particles decorating the periphery of a zein body (zb). F, Stage 3. Double labeling, α-zein (15 nm), and phytase (10 nm). Presence of recombinant phytase on the ER and also on the periphery of the protein body. Note that α-zein and phytase do not colocalize. rER, Rough ER. Bars 20 μm (A–C), 0.5 μm (D–H). [See online article for color version of this figure.]

Figure 5.

Characterization of the storage compartments. Sections after embedding in LRWhite resin. A to D, Stage 2. F and G, Fluorescence microscopy. E, Light microscopy. Lugol's iodine staining. A, Localization of phytase. s, Starch. B, Localization of α-zein. C, Merged. Note that phytase accumulates in storage vacuole-like compartments (arrows), but there is no colocalization with α-zein. D, Localization of phytase. Strong labeling in PSV-like organelles (arrows). E, Lugol's iodine staining of the same cell, vacuole-like compartments do not stain (arrows). s, Purple-stained starch grains. F, Detection of post-Golgi glycan modifications with bee venom anti-serum. Significant labeling in the storage vacuoles (arrows). s, Starch. G, Control: no signal after competition with bromelain. Bars 20 μm.

Figure 6.

Deposition of maize globulins. Sections after embedding in LRWhite resin. Electron microscopy. A and B, Localization of CL-1. C and D, Localization of CAG. A and C, Stage 2. B and D, Stage 3. A and C, Significant labeling for CL-1 and CAG in the PSV. B to D, Note labeling for both CL-1 and CAG, now distributed around the periphery of the zein bodies (zb; arrows). Bars 0.25 μm.