Delivery of prolamins to the protein storage vacuole in maize aleurone cells

Abstract

Zeins, the prolamin storage proteins found in maize (Zea mays), accumulate in accretions called protein bodies inside the endoplasmic reticulum (ER) of starchy endosperm cells. We found that genes encoding zeins, α-globulin, and legumin-1 are transcribed not only in the starchy endosperm but also in aleurone cells. Unlike the starchy endosperm, aleurone cells accumulate these storage proteins inside protein storage vacuoles (PSVs) instead of the ER. Aleurone PSVs contain zein-rich protein inclusions, a matrix, and a large system of intravacuolar membranes. After being assembled in the ER, zeins are delivered to the aleurone PSVs in atypical prevacuolar compartments that seem to arise at least partially by autophagy and consist of multilayered membranes and engulfed cytoplasmic material. The zein-containing prevacuolar compartments are neither surrounded by a double membrane nor decorated by AUTOPHAGY RELATED8 protein, suggesting that they are not typical autophagosomes. The PSV matrix contains glycoproteins that are trafficked through a Golgi-multivesicular body (MVB) pathway. MVBs likely fuse with the multilayered, autophagic compartments before merging with the PSV. The presence of similar PSVs also containing prolamins and large systems of intravacuolar membranes in wheat (Triticum aestivum) and barley (Hordeum vulgare) starchy endosperm suggests that this trafficking mechanism may be common among cereals.

Figure 1.

Expression of Zeins and Other Storage Proteins in Aleurone and Starchy Endosperm Cells. (A) RT-PCR analysis of expression of genes encoding zeins, α-globulin, and legumin-1 in aleurone peels (A) and starchy endosperm samples (S) from 18- and 22-DAP kernels. Ubc9 (Ubiquitin-conjugating enzyme 9; Chung et al. 2009), Bt1 (starchy endosperm-preferred expression), and Vp1 (aleurone-preferred expression) were used as controls. PCR products were visualized by staining with ethidium bromide. (B) Expression profile of Bt1 and Vp1 in aleurone and starchy endosperm samples from 18-DAP kernels determined by real-time quantitative RT-PCR. The expression of both genes in the two cell types was first normalized to Ubc9 and then normalized to the transcript levels of that particular gene in the starchy endosperm (Vp1) or in the aleurone cells (Bt1). The results represent the average and the standard error of two biological samples analyzed by triplicate. (C) Immunoblot analysis of storage proteins in aleurone (A) and starchy endosperm (S) protein extracts. Bip was used as loading control.

Figure 2.

Localization of Storage Proteins in Aleurone PSVs. (A) Electron micrograph of an aleurone cell at 22 DAP containing lipid bodies (LB) and PSVs with large inclusions (asterisks). CW, cell wall. (B) to (H) Immunogold labeling of aleurone cells at 22 DAP ([B], [D], and [F]) and 18 DAP ([C], [E], [G], and [H]) with antibodies against maize storage proteins. Most of the signal was detected on PSV inclusions (arrowheads). G, Golgi; IM, intravacuolar membranes. Bars = 500 nm.

Figure 3.

Electron Tomographic Analysis of Aleurone and Starchy Endosperm Cells at 22 DAP. (A) Tomographic slice (4.3 nm thick) of a starchy endosperm cell containing multiple protein bodies (asterisks) within the ER. The corresponding tomographic reconstruction can be seen in Supplemental Movie 1 online. (A’) Tomographic model derived from the tomogram depicted in (A). (B) Tomographic slice (4.3 nm thick) of an aleurone cell. PSVs with large inclusions (asterisks) and lipid bodies (LB) are the predominant storage compartment in these cells. CW, cell wall. (B’) Tomographic model derived from the tomogram shown in (B). For simplicity, only PSVs, prevacuolar compartments (PrVC), and a Golgi stack (G) are depicted. This is a serial tomogram reconstructed from four serial 250-nm-thick serial sections. (C) and (C’) Tomographic slice and tomographic model of a PSV. Note the presence of a large inclusion (asterisk), intravacuolar membranes (IM), and a globoid (GL). The corresponding tomographic reconstruction can be seen in Supplemental Movie 2 online. Bars = 100 nm.

Figure 4.

Immunogold Detection of ER Proteins inside Aleurone PSVs at 18 DAP. (A) and (B) TIP3-4 labeling on PSV inclusions (A) and PSV intravacuolar membranes (B). (C) and (D) ACA2 detection on PSV inclusions (C) and PSV intravacuolar membranes (D). (E) FL1 labeling on PSV inclusions. (F) FL1 preimmune serum. (G) and (H) calnexin/calreticulin labeling on PSV inclusions (G) and ER cisternae (H). Note the complete lack of labeling on Golgi stacks (G). In all panels, arrowheads indicate the positions of gold particles. CW, cell wall; M, mitochondria P, plastid. Bars = 500 nm.

Figure 5.

Analysis of Zein-Containing Compartments, MVBs, and Putative Autophagosomes in Aleurone Cells. (A) and (A') Tomographic slice (A) and derived tomographic model (A') of a membrane-bound compartment containing an aggregate or inclusion (I) identified as zeins (see [B]) and two electron-dense globoids (arrows). (B) Immunodetection of 15-kD β-zein in an organelle similar to the one depicted in (A) and (A'). (C) to (F) Tomographic reconstructions of zein-containing prevacuolar compartments. Tomographic slice (C) and derived tomographic model (C') of an organelle consisting of several layers of concentric membranes, with aggregates or inclusions (I) identified as zeins (see [D]), and an electron-dense globoid (arrow). (D) Immunodetection of 22-kD α-zein in an organelle similar to the ones depicted in (C), (C'), (E), (E'), (F), and (G). (E) and (E’) Tomographic slice (E) and corresponding tomographic model (E’) of a prevacuolar compartment containing internal membranes (IM) and enclosed ribosomes (yellow arrows). For direct comparison, some ribosomes located in the cytoplasm are indicated by red arrows in (E). The corresponding tomographic reconstruction can be seen in Supplemental Movie 3 online. (F) Tomographic model of another zein-containing prevacuolar compartment. The corresponding three-dimensional rendition of this model can be seen in Supplemental Movie 4 online. (G) to (I) Electron micrographs of three different types of membrane-bound organelles that are likely involved in transport to PSVs in aleurone cells. Prevacuolar compartment containing an inclusion (I) and a globoid (arrow) (G); MVB (H); and autophagosome-like structure (I). For easy side-by-side comparison, all the images in (G) to (I) are depicted at the same magnification. Bars = 100 nm in (A) to (F) and 200 nm in (G) to (I).

Figure 6.

Electron Tomographic Analysis of PSVs in Aleurone Cells at 14 DAP. (A) to (B') Tomographic slices ([A] and [B]) and derived tomographic models ([A’] and [B’]) of aleurone cells showing the distribution of zein-rich inclusions (asterisks) and intravacuolar membranes (depicted in green) within the developing PSVs. (C) to (D') Tomographic slices ([C] and [D]) and corresponding tomographic models ([C’] and [D’]) of intravacuolar membranes and zein-rich inclusions in PSVs. Note that some intravacuolar membranes form completely closed or partially open (arrows in [C] and [D]) multivesicular, spherical structures. In some cases, intravacuolar membranes enclose, partially or completely, the zein-rich inclusions ([D] and [D’]). (E) Quantitative analysis of changes in intravacuolar surface area (expressed as a percentage of the total vacuolar membrane including the tonoplast), volume occupied by the zein-rich inclusions (expressed as a percentage of the total PSV volume), and PSV diameter between 14- and 22-DAP aleurone samples. Thirteen PSVs at 14 DAP and 12 PSVs at 22 DAP were considered in this analysis. The error bars denote the standard deviation. LB, lipid body; M, mitochondrion; N, nucleus; P, plastid. Bars = 500 nm in (A) and (B) and 200 nm in (C) and (D).

Figure 7.

Immunodetection of β1-2 Xylose on Aleurone Cells at 22 DAP. (A) β1-2 Xylose-positive labeling of PSV matrix. The PSV inclusion is indicated by an asterisk. (B) and (C) β1-2 Xylose gold labeling (arrowheads) of MVBs. (D) and (E) β1-2 Xylose gold labeling (arrowheads) of zein-containing prevacuolar compartments. Bars = 500 nm in (A) and 200 nm in (B) to (E).

Figure 8.

FL1 and ATG8 Distribution in Aleurone and Starchy Endosperm Cells. (A) to (C) Expression of FL1-mOrange in starchy endosperm cells of in vitro–cultured endosperms at different developmental stages: 6 DAP plus 4 DIC (A), 6 DAP plus 6 DIC (B), and 6 DAP plus 8 DIC (C). Note the change in the FL1-mOrange localization pattern, from a reticulated distribution in (A) to a punctuate pattern in (B) and (C). (D) and (E) Expression of FL1-mOrange in aleurone cells of in vitro cultured endosperms at 6 DAP plus 6 DIC (D) and 6 DAP plus 8 DIC (E). (F) Coexpression of FL1-mOrange and the ER marker GFP-KDEL in aleurone cells of in vitro–cultured endosperms at 6 DAP plus 8 DIC. Arrowheads indicate the localization of FL1-mOrange as discrete spherical structures in the ER. (G) FL1 accumulation in starchy endosperm (S) and aleurone (A) samples at 18 and 22 DAP revealed by immunoblots. Bip was used as loading control. (H) Profile of ATG8 proteins in starchy endosperm (S) and aleurone (A) samples at 18 and 22 DAP. Total protein extracts were subjected to SDS-PAGE in the presence of urea followed by immunodetection with antibodies against ATG8. Free (ATG8) and lipidated (ATG8-PE) forms were detected. Bip was used as loading control. (I) Profile of ATG8 proteins in starchy endosperm and aleurone samples treated with ConA for 0 or 16 h. Bip was used as loading control. (J) Coexpression of FL1-mOrange (white arrowheads) and ATG8a-YFP (blue arrowheads) in an aleurone cell of an in vitro–cultured endosperm at 6 DAP plus 8 DIC. Note the lack of colocalization of the two fluorescent signals. (K) and (L) Immunogold detection of ATG8 (arrow heads) on double-membrane, autophagosome-like structures in starchy endosperm cells. Bars = 500 nm in (K) and (L), 20 μm in (A) to (F), and 10 μm in (J).

Figure 9.

Model Depicting the Mechanisms of Accumulation and Transport of Zeins in Starchy Endosperm and Aleurone Cells. In starchy endosperm cells (left), zeins are synthesized and stored in the ER as protein bodies (asterisk). In aleurone cells (right), zeins first accumulate in the ER as protein aggregates (asterisk) and then are sequestered into complex prevacuolar compartments (PVC) that fuse with the PSV. The presence of internal membranes and ribosomes inside the prevacuolar compartments suggest that these compartments are originating, at least partially, by autophagy. The glycoproteins found in the PSV matrix traffic through a typical ER-Golgi-MVB pathway, although it is possible that the MVBs carrying these glycoproteins fuse with the autophagic PVCs before reaching the PSV.