The Changing Fate of a Secretory Glycoprotein in · The Changing Fate of a Secretory Glycoprotein in Developing Maize Endosperm1[C][W] Elsa Arcalis, Johannes Stadlmann, Sylvain Marcel2,

The Changing Fate of a Secretory Glycoprotein inDeveloping Maize Endosperm1[C][W]

Elsa Arcalis, Johannes Stadlmann, Sylvain Marcel2, Georgia Drakakaki3, Verena Winter, Julian Rodriguez,Rainer Fischer, Friedrich Altmann, and Eva Stoger*

Department of Applied Genetics and Cell Biology and Department of Chemistry, University of NaturalResources and Applied Life Sciences, 1190 Vienna, Austria (E.A., J.S., V.W., J.R., F.A., E.S.); and Institute forMolecular Biotechnology, Biology VII, Aachen University, 52074 Aachen, Germany (S.M., G.D., R.F., E.S.)

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

Storage proteins in cereal seeds accumulate in dif-ferent compartments of the endosperm cell, and theirabundance and distribution varies according to thespecies. While in most cereals prolamins are the moreabundant class of storage proteins, small-grain species(e.g. wheat [Triticum aestivum], oat [Avena sativa], andbarley [Hordeum vulgare]) may contain variable pro-portions of both prolamins and globulins, and theseare delivered to the protein storage vacuole (PSV) viaGolgi-dependent and Golgi-independent pathways(Wettstein, 1980; Levanony et al., 1992; Herman andSchmidt, 2004; Takahashi et al., 2005; Cameron-Millsand von Tosi et al., 2009). In rice (Oryza sativa), whereglobulins and prolamins accumulate in distinct stor-age compartments, most globulins (mainly glutelins)

accumulate in PSVs whereas prolamins aggregate intodense protein bodies within the rough endoplasmicreticulum (ER) and remain in ER-derived organelles(Okita and Rogers, 1996). Maize (Zea mays) storesmainly prolamins (zeins) comprised in three zeinsubfamilies (a, g, and d) that form ER-derived zeinbodies. Mature zein bodies consist of a central core ofa and d zeins, while g zeins are mainly found in theperiphery (Lending and Larkins, 1989). Small amountsof globulins also accumulate in maize endosperm, i.e.corn a-globulin (CAG) and corn legumin-1 (CL-1; Wooet al., 2001). Unlike legumin homologs in other plantspecies including cereals, CL-1 lacks the canonicalasparaginyl endopeptidase cleavage sequence (Wooet al., 2001), so it is not cleaved into a and b chains(Yamagata et al., 2003). CAG has been observed insmall, PSV-like compartments within the maize endo-sperm cell (Woo et al., 2001) and a similar fate has beenpredicted for CL-1 (Yamagata et al., 2003). The iden-tification and localization of globulins in maize in-dicates the presence of storage vacuoles in maizeendosperm, but it does not address the questionwhether the size and number of these organelles issignificant in maize, whether they change morpholog-ically during seed maturation, and how proteins reachthis destination.

Proteins may reach the PSV by different routes, andin some species storage protein trafficking appears toundergo changes during seed development. For ex-ample, in the context of 2S and 11S storage proteintrafficking in pumpkin (Cucurbita pepo) and castor

1 This work was supported by the Alexander von HumboldtFoundation and the European Union project PharmaPlanta.

2 Present address: Department of Soil and Crop Sciences, TexasA&M University, 370 Olsen Blvd., 2474 TAMU, College Station, TX77843.

3 Present address: Department of Plant Sciences, University ofCalifornia, Davis, CA 95616.

* Corresponding author; e-mail [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policydescribed in the Instructions for Authors (www.plantphysiol.org) is:Eva Stoger ([email protected]).

[C] Some figures in this article are displayed in color online but inblack and white in the print edition.

[W] The online version of this article contains Web-only data.www.plantphysiol.org/cgi/doi/10.1104/pp.109.152363

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bean (Ricinus communis) it has been proposed that seeddevelopmental stages may be important in determin-ing the transport routes to the PSV (Vitale and Hinz,2005). A seed-development-mediated change in thetrafficking route of wheat prolamins has been sug-gested earlier as well (Shy et al., 2001; Tosi et al., 2009).One approach to study such change in traffickingroutes along seed maturation is to scrutinize theglycosylation pattern of proteins destined to the PSV,taking advantage of the fact that the intracellulartrafficking route of a glycoprotein determines its finalN-glycan structures (Lerouge et al., 1998).

The first stage of N-glycosylation (which takes placein the ER) involves the cotranslational addition of aprecursor oligosaccharide (Glc3Man9GlcNAc2) that ismodified by various glycosidases and glycosyltrans-ferases to form the final glycan structure as the proteinmigrates through the endomembrane system (Lis andSharon, 1993; Lerouge et al., 1998). ER-resident glyco-proteins contain high-Man-type N-glycans whereasproteins passing though the Golgi apparatus containcomplex-type N-glycans that include a(1-3)-Fuc and/or b(1-2)-Xyl residues (Lerouge et al., 1998). Whilesecreted glycoproteins contain terminal GlcNAc resi-dues in addition to the core Fuc and Xyl, these termi-nal residues are trimmed off by enzymes either enroute to the vacuole or within the vacuole (Lerougeet al., 1998). Thus the structure of N-glycans is a usefulindicator for the intracellular pathway of a protein(Vitale and Hinz, 2005).

Unfortunately, most seed storage proteins, particu-larly those in cereals, are not glycosylated. However,information on N-glycan structures can be obtainedfrom recombinant glycoproteins. For example, aKDEL-tagged antibody, which was located primarilyin ER-derived zein bodies, was predominantly madeup of molecules with single GlcNAc residues lackingFuc (Rademacher et al., 2008). In contrast, recombinanthuman lactoferrin isolated from maize seeds wasreported to contain pauci-Man-type N-glycans withb(1,2)-Xyl and a(1,3)-linked core Fuc (Samyn-Petitet al., 2001). Interestingly, this glycan pattern suggestsa vacuolar location of this recombinant protein, andprovides a second strong evidence for the presence ofPSVs in maize, although the actual subcellular local-ization of lactoferrin in maize endosperm cells has notbeen confirmed.

In previous studies we have shown that recombi-nant glycoproteins can help to clarify questions aboutthe intracellular trafficking of proteins in cereal en-dosperm, and we found that a recombinant fungalphytase, although secreted from leaf cells, is mainlylocalized in the PSVs of wheat and rice endosperm(Arcalis et al., 2004; Drakakaki et al., 2006). In thisstudy we used recombinant phytase to facilitate thevisualization and characterization of the PSVs inmaize, and we followed the intracellular fate of re-combinant phytase in developing endosperm using acombination of microscopy and N-glycan analysis,revealing that the trafficking of the protein does

indeed change as the seed matures. This behavior ismirrored by the two endogenous (aglycosylated) glob-ulins, CAG and CL-1, indicating that the diversion ofstorage proteins may be a common strategy in seeddevelopment.

RESULTS

Comparative Accumulation of Recombinant Phytase andEndogenous Storage Proteins

Transgenic maize plants expressing a fungal phy-tase under the control of the endosperm-specific riceglutelin-1 promoter (Drakakaki et al., 2005) were usedas a model to study the trafficking of a recombinantglycoprotein carrying anN-terminal sequence for entryinto the endomembrane system. As expected from thepreviously established activity of the promoter in thisbackground, only small amounts of recombinant phy-tase were produced at 10 d after pollination (DAP), butlarger amounts were detected between 15 and 20 DAP.Recombinant phytase extracted from maize seeds mi-grated as a blurred band with an apparent molecularmass of 60 to 65 kD, as observed earlier with glycosy-lated phytase from rice seeds (Drakakaki et al., 2006).Phytase remained at high levels in further develop-mental stages, close to maturity (30 DAP), appearingonly slightly diluted by the progressive accumulationof starch toward the end of seed maturation (Fig. 1A).

Very similarly, in both transgenic and wild-typeplants, the relative amount of three endogenous stor-age proteins (g-zein, CAG, and CL-1) accumulatedprogressively during grain development and peakedat approximately 25 DAP before slightly declining atlater stages (Fig. 1, B–D) due to dilution by the pro-gressive accumulation of starch. Thus, the accumula-tion rate of the recombinant phytase closely resemblesthat of endogenous storage proteins, demonstrating ahigh stability of the protein.

The N-Glycan Structure of Phytase Changes during

Endosperm Maturation

Tomonitor changes in the glycosylation pattern of phy-tase during development we analyzed the N-glycanprofile of the protein isolated from maize endospermat different stages of maturity. Since it was not possibleto obtain sufficient amounts of the protein from seedsyounger than 20 DAP, we compared the N-glycanstructures of phytase isolated from maize endospermat midmaturation stage (20 DAP), seeds close to ma-turity (30 DAP), and mature, dry seeds. Protein sam-ples were separated by SDS-PAGE, and the phytaseband was excised and digested with trypsin. Glyco-peptide analysis allowed us to follow up specificglycosylation sites through different developmentalstages. The results for the glycopeptides TYNYSLGADD LTPFG EQELV NSGIK and YSALIEEIQQ-NATTFDGK are shown in Figure 2 and SupplementalFigures S1 and S2. The absence of unglycosylated

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peptides at all stages confirms the efficiency ofN-glycosylation. Figure 2 shows that the glycan patternsof different peptides are not identical, most likely dueto different accessibility of the individual glycosylationsites, but the major changes in abundance of vacuolarglycoforms and trimmed GlcNAc structures duringdevelopment are very similar for both glycopeptides.At 20 DAP, we identified large amounts of glycopeptidespecies containing the N-glycans Man3XylFucGlcNAc2(Fig. 2, A and B) and Man3XylGlcNAc2 (Fig. 2B). Thesestructures, also known as MMXF and MMX (Altmann,2007), are typically found on vacuolar glycoproteins(Lerouge et al., 1998). Minor peaks corresponded to thepeptide containing trimmed glycans, some of whichcontained Fuc on the core GlcNAc, representing deg-radation products of Golgi-modified complex glycans.At 30 DAP the relative amount of the formerly pre-dominant glycan MMXF had decreased dramatically,and the predominant glycopeptide species had a mo-lecular mass of 2,934.4 amu or 2,231 amu, respectively,indicating the presence of trimmed structures consist-ing of core GlcNAcs alone. Single GlcNAcs have beenfound previously on a KDEL-tagged recombinant pro-tein frommaize (Rademacher et al., 2008). After 30 DAPthe N-glycan profile did not change significantly asshown with phytase isolated from fully mature seeds(40 DAP; Fig. 2).

Ultrastructural Changes in Maize Seedsduring Development

Changes in the N-glycan profile of phytase duringdevelopment suggested that the subcellular localiza-tion of the protein might also change as seed matured.We therefore investigated the subcellular organizationof maize endosperm cells during development tomonitor any changes in the different protein storagecompartments. Based on the ultrastructural character-istics of the cells in the different stages studied, wedefined three representative developmental stages, inwhich major changes in subcellular organization wereobserved: stage 1 (young seeds, around 10 DAP), stage2 (midmaturation stage, seeds around 20 DAP), andstage 3 (seeds close to maturity, around 30 DAP).

Stage 1

In young seeds, there is no clear difference betweenthe aleurone and the subaleurone layers. Thus, there isan outermost layer of cubical aleurone cells, with ahigh content in aleurone grains. Layers 2 to 4 could beconsidered as transition cells, as there is a progressiveloss of aleurone grains, accompanied by a gain instarch grains of increasing size. Storage protein syn-thesis is in progress and zein bodies can be found fromlayer 5 onwards. Several PSV-like organelles, approx-imately spherical and similar in size to starch grains(approximately 2 mm), can be identified in the samelayers (Fig. 3A).

Stage 2

The aleurone layer presents its characteristic appear-ance of polyhedral cells with thick cell walls, clearlydistinct from the rest of the endosperm. The starchcontent of the endosperm has increased significantlyand also the number and size of the protein bodies(Fig. 3B). Although already observed in younger cells(Fig. 3A), vacuolar storage compartments becomemoreprominent at this stage and they are more abundantand have reached a diameter of approximately 4 to5 mm (Fig. 3, B, D, and E). Some small vacuole-likecompartments, similar in size to the zein bodies can alsobe observedwithin the endosperm cells (Fig. 3B). In theelectronmicroscope, vacuolar storage compartments inOsO4-fixed samples appear to contain a globulin-likeinclusion, which is highly electron dense (Fig. 3, Dand E). The electron-dense inclusion in the PSV-likestructures consists of CAG (Woo et al., 2001) andprobably CL-1 (Yamagata et al., 2003).

Stage 3

Endosperm cells are packed with starch and pro-teins, and the number of visible or identifiable PSVshas apparently decreased (Fig. 3C). The average size ofthe zein bodies has increased above 1 mm and that ofthe starch grains up to approximately 8 mm. In laterdevelopmental stages zein bodies and starch grainsenlarge even further, but no significant morphological

Figure 1. Accumulation of phytase and seed storageproteins during seed maturation. Total soluble pro-tein was extracted from transgenic seeds at variousdevelopmental stages (10–30 DAP) at a fixed w/vratio, and 10 mL were loaded per lane. Immunoblotswere incubated with antisera against phytase (A), gzein (B), CAG (C), and CL-1 (D).

Post-Golgi Protein Accumulation in Maize Endosperm

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changes can be observed. The mature endosperm cellsare tightly packed with enlarged zein bodies that fillthe spaces between the starch grains (data not shown).

The Subcellular Localization of Phytase Changes duringSeed Development

We determined the localization of recombinant phy-tase in sections of endosperm cells at the differentdevelopmental stages described above. The ultrastruc-tural changes observed during development werereflected in the deposition of the recombinant proteinas the distribution of phytase changed along matura-tion (Fig. 4, A–C).

Stage 1

Young endosperm cells showed strong labelingin PSV-like structures. Consistent with the ultrastruc-tural observations, labeled putative PSVs in these cellswere small (approximately 2 mm). In some PSV-like

structures the signal was equally distributed, othersshowed labeled phytase deposits at the periphery only(Fig. 4, A and D). There was no significant labelingwithin the spherical zein bodies, but some signal wasvisible in the cytoplasm, probably reflecting the ongo-ing trafficking of recombinant phytase through theendomembrane system. No significant labeling wasdetected in the starch granules or in the apoplast(Fig. 4A).

Stage 2

Labeling was still concentrated in the putative PSVsin middevelopment endosperm cells (Fig. 4B). Thislocalization is in agreement with the main N-glycanstructure attached to phytase at this stage (MMXF),which is typically found on vacuolar glycoproteins(Lerouge et al., 1998). The labeled PSV-like structureshad increased considerably both in size (approxi-mately 5 mm) and in number, as described in theprevious section for this developmental stage (Fig. 4B).

Figure 2. N-glycan structures attached torecombinant phytase glycopeptides TYNYSLGADD LTPFG EQELV NSGIK (A) and YSA-LIEEIQQNATTFDGK (B), derived from devel-oping maize endosperm at 20, 30, and 40DAP. The vacuolar-type glycans MMXF andMMX were major N-glycan structures in phy-tase derived from young seeds (20 DAP),whereas small structures consisting of onlyone or two core GlcNAc residues dominatedin phytase isolated from more mature seeds(30 and 40 DAP). See http://www.proglycan.com for an explanation of N-glycan structureabbreviations.

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As in stage 1, recombinant phytase was detectedalmost exclusively in PSV-like organelles, to the ex-clusion of the clearly distinct zein bodies (Fig. 5, A–C).

Stage 3

A striking change in the localization of recombinantphytase was observed in older endosperm cells, con-sistent with the shift from MMXF to GlcNAc glycansthat was also observed in seeds close to maturation.Now only a few vacuolar compartments could beclearly identified, and these residual PSVs were stilllabeled (Fig. 4C), but a strong signal was also observedaround the periphery of the zein bodies (Fig. 4, Eand F). Moreover, significant labeling in the ER wasalso observed (Fig. 4F).To investigate if the reduced Golgi-mediated trans-

port that was observed for recombinant phytaseduring late seed development is due to a reducedavailability of Golgi organelles we used a markerantibody to visualize and fluorescently label Golgistacks in vibratome sections of developing endosperm(Horsley et al., 1993). Using confocal microscopy wedetermined the number of Golgi compartments percell (Supplemental Fig. S3) and observed a 4-foldincrease between stage 1 and stage 3.

Proteins in the PSV Contain Golgi-Modified N-Glycans

By developmental stage 2, the PSVs are similar insize to starch grains, but can be distinguished becausethey do not stain with Lugol’s iodine (Fig. 5, D and E).The glycan composition of glycoproteins within thePSVs was confirmed by the strong labeling observedusing an antiserum specific for fucosylated N-glycans(Fig. 5F). The labeling could be outcompeted by theaddition of bromelain, an unrelated plant glycoproteinwith complex paucimanosidic N-glycans, thus con-firming the specificity of the signal (Fig. 5, F and G).This confirms that the putative PSVs contain glyco-proteins that have undergone Golgi-specific modifica-tions. PSVs reacting with the antiserum specific forfucosylated N-glycans are also present in wild-typeseeds (data not shown), indicating that endogenousglycoproteins accumulate in a similar fashion.

Endogenous Maize Globulins Show Similar Changes in

Subcellular Localization during Seed Development

To determine whether the developmental profile ofphytase was mirrored by endogenous storage globu-lins, we carried out localization experiments as de-scribed above, this time using CL-1 and CAG.At stage 2

Figure 3. Development of maizeseeds. Sections after embedding inSpurr’s resin. A to C, Light microscopy,toluidine blue: stage 1 (A), stage 2(B), and stage 3 (C). Aleurone (Al) andendosperm (En). Cells accumulatestarch (s), some storage vacuole-likecompartments are already seen (arrow-heads), smaller refringent vacuolarcompartments are also observed (B,arrow), zein bodies appear as smallblue stained accretions, few nuclei (n)are still present. D and E, Electronmicroscopy, general nonspecific con-trast: stage 2. Zein bodies (zb) and PSV.Note the globulin-like inclusion in thevacuole-like compartments (*). rER,Rough ER. Bars 20 mm (A–C), 0.5 mm(D and E). [See online article for colorversion of this figure.]





the distribution of both CAG and CL-1 is similar to thatof phytase in this developmental stage, and both glob-ulins could be found within the PSVs (Fig. 6, A and C).At stage 3 the shift in the localization of phytase is alsoobserved for CL-1 and CAG, which are now found atthe periphery of the protein bodies, forming a ringaround the zein inclusion (Fig. 6, B and C).

DISCUSSION

Previous studies have suggested that the intracellu-lar trafficking and distribution of storage proteins maychange during seed development (Levanony et al.,1992; Shy et al., 2001; Vitale and Hinz, 2005). To in-vestigate this phenomenon in maize endosperm, wetracked the recombinant model glycoprotein phytaseand two endogenous storage globulins (CAG andCL-1) through the development of the maize seedusing a combination of microscopy and N-glycananalysis. In all three cases, we observed a majordevelopmental switch from post-Golgi PSV-like or-ganelles to ER-derived zein bodies, confirming thatseeds alter protein trafficking and accumulation aspart of the developmental program.

PSV-like structures appear to be the major storagesites in young endosperm cells for all three proteins weinvestigated. Storage vacuoles have been the focus ofnumerous recent studies looking at structure, classifi-cation, and protein trafficking but most of these stud-ies have concerned dicotyledonous plants (Wenzelet al., 2005; Hinz et al., 2007; Craddock et al., 2008).In cereals, studies of storage protein accumulationhave focused mainly on the prolamins, and this is

particularly the case in maize where the vast majorityof storage protein is made up of prolamins (zeins) thataccumulate in ER-derived zein bodies. In contrast,only small amounts of globulins are stored in maizeendosperm, and only two previous studies haveaddressed their spatiotemporal expression and local-ization (Woo et al., 2001; Yamagata et al., 2003).

We have confirmed that both CAG and CL-1 accu-mulate in PSV-like compartments in early endospermdevelopment. Woo et al. (2001) reported that theputative PSVs increased in size in maturing endo-sperm cells, eventually swelling to three times the sizeof zein bodies. Labeling with an antibody against CAGwas less dense in the larger compartments, leading theauthors to assume that CAG was being diluted andwas therefore more difficult to detect. Since phytase isan easily detectable reporter protein, the labeling ofphytase greatly facilitated the visualization of evenlarge PSV-like organelles. Thus, we were able to showthat PSVs at developmental stage 2 varied greatly insize, although they were on average much larger thanthose at earlier stages, and that they contained glycanstructures with trans-Golgi-specific modifications,clearly identifying them as post-Golgi compartments.Since both CAG and CL-1 lack N-glycan chains, thefact that vacuolar N-glycans were also detected in thePSVs of stage-matched wild-type seeds indicates thatthese organelles must contain endogenous glyco-proteins that remain to be identified. Indeed, theanalysis of total soluble proteins fromwild-type maizeendosperm revealed the presence of glycoproteinswith complex paucimannosidic N-glycans (data notshown), and those are probably residents of the PSV-like organelles.

Figure 4. Localization of recombinantphytase. Sections after embedding inLRWhite resin. A to C, Fluorescencemicroscopy. D to F, Electron micros-copy. Comparison of the distributionpattern of recombinant phytase be-tween endosperm cells in stage 1 (A),stage 2 (B), and stage 3 (C). Note thestorage vacuole-like compartments ineach of the developmental stages (ar-rows) and the nonlabeled sphericalzein bodies evenly spread within thecytoplasm (B and C). No significantlabeling in the apoplast (arrowheads)or the starch granules (s). D, Stage 1.Note the heavy labeling on a smallPSV. E, Stage 3. Gold particles deco-rating the periphery of a zein body (zb).F, Stage 3. Double labeling, a-zein (15nm), and phytase (10 nm). Presence ofrecombinant phytase on the ER andalso on the periphery of the proteinbody. Note that a-zein and phytase donot colocalize. rER, Rough ER. Bars 20mm (A–C), 0.5 mm (D–H). [See onlinearticle for color version of this figure.]

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In immature cereal endosperm cells PSVs appear tobe a preferred accumulation site for recombinant pro-teins bearing an N-terminal signal peptide for entryinto the endomembrane system, even though suchproteins are normally secreted from vegetative tis-sues. This phenomenon has been reported for phy-tase expressed in rice and wheat (Arcalis et al., 2004;Drakakaki et al., 2006), and for a secretory antibodyand lysozyme expressed in rice (Yang et al., 2003;Nicholson et al., 2005).We found that in maize the number of identifiable

PSVs decrease relative to the other organelles later inendosperm development, concomitant with a pro-gressive change in the subcellular localization ofrecombinant phytase and both endogenous globulins.At this stage, a very clear label became visible aroundthe periphery of the zein bodies in transgenic plantsexpressing phytase. This raises the question as towhether or not an interaction takes place betweenphytase and g zein, which is also located in the outerpart of the mature zein bodies (Lending and Larkins,1989). However, phytase is localized exclusively

around the periphery of the protein bodies, whereasdeposits of g zein are sometimes also found in theinner areas, suggesting that the proteins do not colo-calize (Fig. 4E).

It was conceivable that the developmental change inthe subcellular localization of recombinant phytaseand the two native storage proteins could perhaps berelated to the induction of an unfolded proteinresponse, triggered by the recombinant protein.However, in our transgenic plants, the levels of thechaperone BiP were not significantly increased ascompared to wild-type levels (data not shown). Inaddition, although an influence of BiP cannot be ruledout, the fact that CAG and CL-1 accumulated in thesame compartments with the same developmentalprofile in wild-type plants and transgenics suggeststhat its role is not specific in transgenic plants.

Both of the endogenous globulins lack N-glycanacceptor sites so although their final destination can bedetermined it is more difficult to follow their pathwaythrough the cell. In contrast, the N-glycan acceptorsites on phytase allow the route taken by this protein

Figure 5. Characterization of the storage compart-ments. Sections after embedding in LRWhite resin.A to D, Stage 2. F and G, Fluorescence microscopy.E, Light microscopy. Lugol’s iodine staining. A, Lo-calization of phytase. s, Starch. B, Localization ofa-zein. C, Merged. Note that phytase accumulates instorage vacuole-like compartments (arrows), butthere is no colocalization with a-zein. D, Localiza-tion 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 com-petition with bromelain. Bars 20 mm.





to be determined by examining the N-glycan struc-tures. The developmental switch from PSV to zeinbody as preferred accumulation site for phytase ismirrored by a corresponding change in N-glycanstructures. Initially, the glycan residues were indicativeof vacuolar localization, consistent with immunode-tection in the PSV-like organelles. Later in develop-ment and simultaneous with the changing subcellularlocalization, the relative abundance of glycoforms withcomplexmodifications including b(1,2)-Xyl and a(1,3)-linked core Fuc decreased while small glycans con-sisting only of one or two GlcNAc residues withoutFuc became dominant. There is some evidence for thetime-dependent trafficking of storage proteins inwheat, where the amount of transcripts for Golgi-associated proteins declines with seed maturation.Therefore, the role of Golgi vesicles in the traffickingof prolamins in wheat appears mainly restricted to theearly stages of seed development (Parker, 1982; Galiliet al., 1993; Shy et al., 2001). In maize we observed thatthe number of Golgi organelles per cell increases 4-foldbetween stage 1 and 3. Although the volume of thecells and the synthesis of storage proteins increasemarkedly at the same time, this result does not indicatea striking decline of Golgi activity during maize en-dosperm development. Nevertheless, we observed atime-dependent protein trafficking in maize resultingin two pools of phytase, an early pool that reflects

trafficking through the ER and Golgi vesicles to thePSV, and a later pool accumulating in the ER and inER-derived compartments. This is consistent with thechanging N-glycan profiles of recombinant phytaseand with the occurrence of trimmed glycoforms. Wehave previously observed the same N-glycan struc-tures attached to a KDEL-tagged recombinant anti-body, which accumulated in ER-derived zein bodies(Rademacher et al., 2008). Interestingly, recombinanthuman lactoferrin isolated from mature maize seedswas reported to contain almost exclusively (98%)pauci-Man-type N-glycans with b(1,2)-Xyl and a(1,3)-linked core Fuc (Samyn-Petit et al., 2001). How-ever, it is important to note that in this study, as in mostprevious reports, glycans were enzymatically releasedfrom the protein prior to analysis, and this methodwould not have identified small glycan structures,such as single or double GlcNAcs. Thus, it is possiblethat the described vacuolar glycan pattern character-izes only one part of the lactoferrin, whereas a secondpart with single GlcNac structures would not havebeen noticed. In this study we also identified GlcNAcswith core Fuc residues on a small proportion ofrecombinant phytase molecules isolated from endo-sperm cells, apparently representing the result ofglycan degradation in post-Golgi compartments. Thetrimmed glycan structures clearly indicate thepresence of various glycanase activities in maizeendosperm. Endoglycanase (ENGase and PNGase)activities in cereal seeds have been reported previ-ously, but were thought to be mainly derived from theembryo (Chang et al., 2000; Vuylsteker et al., 2000).

In conclusion, the use of a model glycoprotein (inaddition to two endogenous globulins) allowed us tovisualize and characterize the vacuolar compartmentsin maize endosperm, and follow the developmentalchanges in protein trafficking and distribution. Incontrast to earlier reports, we have shown that thevacuolar compartments are significant storage com-partments in developing maize endosperm, particu-larly at early developmental stages. A combination ofmicroscopy and N-glycan analysis provided evidencethat protein trafficking in maize is time dependent, aspreviously suggested in wheat. Further experimentsare under way to determine the fate of the PSVs in laterdevelopmental stages and the implications of ourfindings on the production of recombinant pharma-ceutical proteins in cereal grains.

MATERIALS AND METHODS

Transgenic Plants

The transgenic maize (Zea mays) plants have been described previously

(Drakakaki et al., 2005) and contained the construct pLPL-phyA (including the

Aspergillus niger phyA gene preceded by the N-terminal signal peptide from

the murine immunoglobulin k chain) under the control of the rice (Oryza

sativa) glutelin-1 promoter. Fourth generation plants (T4) homozygous for the

transgene were maintained in a controlled growth room at 28�C/20�C day/

night temperature with a 16-h photoperiod and 60% to 90% relative humidity.

Nontransgenic control plants were regenerated from the same batch of callus

Figure 6. Deposition of maize globulins. Sections after embedding inLRWhite resin. Electron microscopy. A and B, Localization of CL-1. Cand D, Localization of CAG. A and C, Stage 2. B and D, Stage 3. A andC, Significant labeling for CL-1 and CAG in the PSV. B to D, Notelabeling for both CL-1 and CAG, now distributed around the peripheryof the zein bodies (zb; arrows). Bars 0.25 mm.

Arcalis et al.




material as used for transformation (Drakakaki et al., 2005), and propagated

and grown under the same conditions as the transgenic lines. Developing ears

were harvested at 10, 15, 20, 25, 30, and 40 DAP.

Immunoblot Analysis

Total soluble protein was extracted from seeds as described (Arcalis et al.,

2004) using phosphate-buffered saline (PBS) containing 10 mM ascorbic acid,

500 mM NaCl, and 5% b-mercaptoethanol. After centrifugation at 17,500g, 4�C,the supernatants were boiled for 10 min and separated by 12% (w/v) SDS-

PAGE under reducing conditions. Immunoblot analysis was carried out

according to standard protocols. For each time point, five kernels from the

same ear were pooled for each sample.

Protein Extraction and Liquid Chromatography-MassSpectrometry Analysis

Proteins were extracted from ground seeds with PBS containing 10 mM

ascorbic acid, 500 mM NaCl, and 5% b-mercaptoethanol to minimize enzy-

matic activities post extraction. Phytase was isolated from the extracts by

sequential ammonium sulfate fractionation as described by Arcalis et al.

(2004) and concentrated by ultrafiltration before separation by 12% (w/v)

SDS-PAGE under reducing conditions. The bands corresponding to phytase

were excised, destained, carbamidomethylated, digested with trypsin, and

extracted from gel pieces as described (Kolarich and Altmann, 2000; Kolarich

et al., 2006). Peptide fractionation and analysis using a Q-TOF Ultima Global

(Waters Micromass) mass spectrometer was performed as described previ-

ously (Kolarich and Altmann, 2000; Van Droogenbroeck et al., 2007).

The mass spectrometry data from the tryptic peptides were compared to

datasets generated by in silico tryptic digestion of A. niger phytase using the

PeptideMass program (http://www.expasy.org/tools/ peptide-mass.html).

Tryptic glycopeptide datasets were generated by the addition of glycan mass

increments to the masses of the potential glycopeptides.

Light and Electron Microscopy

A minimum of 10 phyA-containing grains from three different ears were

examined for each developmental stage. Seeds were bisected longitudinally

and the embryo was removed. One half of the endosperm was processed for

recombinant protein analysis by immunoblot. Thin slices were cut from the

remaining half with a razor blade under phosphate buffer (0.1 M, pH 7.4).

Tissue pieces were fixed in 2% (w/v) paraformaldehyde and 2.5% (v/v)

glutaraldehyde in phosphate buffer (0.1 M, pH 7.4) for ultrastructural analysis

or in 4% (w/v) paraformaldehyde and 0.5% (v/v) glutaraldehyde in phos-

phate buffer (0.1 M, pH 7.4) for immunolocalization studies and then

processed as described previously (Arcalis et al., 2004). For light microscopy,

1-mm sections were stained in toluidine blue. Starch was stained by incubating

1-mm sections with Lugol’s iodine solution for 5 min. For electron microscopy,

sections showing silver interference colors were stained in 2% (w/v) aqueous

uranyl acetate. The sections were observed using a Philips EM-400 transmis-

sion electron microscope.

Sections mounted either on glass slides for fluorescence microscopy or on

gold grids for electron microscopy were preincubated in 5% (w/v) bovine

serum albumin (BSA; fraction V) in phosphate buffer (0.1 M, pH 7.4) and then

incubatedwith the appropriate dilution of polyclonal rabbit anti-phytase, anti-

CL-1, and anti-CAG or monoclonal rat anti-a-zein. Sections were then treated

with the secondary antibody diluted in phosphate buffer (0.1 M, pH 7.4). This

was a goat anti-rabbit IgG Alexa Fluor 594 or goat anti-rat IgG Alexa Fluor 488

for fluorescence microscopy and a goat anti-rabbit IgG 10-nm gold and goat

anti-rat IgG labeled with 15-nm gold particles for electron microscopy.

Immunofluorescence Analysis and Confocal Microscopy

Seeds of wild-type maize (10 and 25 DAP) were fixed in 2% (w/v)

paraformaldehyde for 12 to 72 h. After washing with 0.1 M phosphate buffer

(pH 7.4), 60 to 100 mm vibratome sections were prepared and placed on glass

slides coated with 0.1% (w/v) poly-Lys (Sigma), respectively. Sections were

dehydrated by an ethanol series and equilibrated in 0.1 M phosphate buffer.

The cell wall was digested with 2% (w/v) cellulase (Onozuka R-10 from

Trichoderma viride) in phosphate buffer (0.1 M, pH 7.4) for 1 h at room

temperature. Following a treatment with 0.5% Triton X-100 in 0.1 M phosphate

buffer for 1 h at room temperature, nonspecific binding sites were blocked

with 3% (w/v) BSA (fraction V) in PBS for 10 min. Sections were incubated

with monoclonal antibody JIM84 diluted 1:50 in phosphate buffer (0.1 M,

pH 7.4) overnight at 4�C (Horsley et al., 1993). Antibody binding was

visualized by Cy3 conjugated goat anti-rabbit IgM, m-chain-specific antibody

(Jackson Immunoresearch). The sections were mounted in 50% glycerol in

phosphate buffer (0.1 M, pH 7.4) and observed in the confocal laser scanning

microscope (TCS-SP2, Leica) via the Leica confocal software (version 2.61) and

long-distance 403, 633 water-immersion objectives. Excitation wavelengths

were 488 nm (argon laser) for Cy3. Emission was detected between 551 and

616 nm. Z sections were collected and overlay pictures were generated using

ImageJ. The Golgi compartments were counted per cell, excluding overlap

areas between two adjoining cells. A minimum of five cells were counted for

each sample.

Bromelain Competition

Polyclonal rabbit anti-bee venom serum, which recognizes core-linked a

(1-3)-Fuc (Prenner et al., 1992), was diluted (1:100) in phosphate buffer (0.1 M,

pH 7.4) with or without 10% (w/v) bromelain (preheated to destroy enzy-

matic activity) and incubated at room temperature for 30 min. Sections were

preincubated in 5% (w/v) BSA (fraction V) and then incubated with the anti-

bee venom antiserum, which binds to core Fuc glycan residues. Samples were

then treated with the secondary antibody (goat anti-rabbit Alexa Fluor 594)

diluted in phosphate buffer (0.1 M, pH 7.4).

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure S1. Deconvoluted liquid chromatography-mass

spectrometry spectrum of the glycopeptide TYNYS LGADD LTPFG

EQELV NSGIK.

Supplemental Figure S2. Deconvoluted liquid chromatography-mass

spectrometry spectra of the glycopeptide YSALIEEIQQNATTFDGK.

Supplemental Figure S3. Golgi bodies visualized in endosperm cells at

different stages.

ACKNOWLEDGMENTS

The authors would like to thank Dr. Richard M. Twyman for critical

assessment and help with manuscript preparation and Dr. Rudolf Jung for

helpful discussions and for providing antibodies against corn legumin, CAG,

and a-zein. We would also like to thank Dr. A. Ohmann for providing

antibodies against phytase, and the Pathology Department University Hospi-

tal of the Rheinisch-Westfalische Technische Hochschule Aachen for allowing

us to use their microscopy facilities.

Received December 23, 2009; accepted April 10, 2010; published April 13,

2010.

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Arcalis et al.




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