Protein Storage Vacuoles Originate from RemodeledPreexisting Vacuoles in Arabidopsis thaliana1[OPEN]

Mistianne Feeney,a,2 Maike Kittelmann,b,2 Rima Menassa,c Chris Hawes,b and Lorenzo Frigerioa,3

aSchool of Life Sciences, University of Warwick, Coventry CV4 7AL, United KingdombPlant Cell Biology, Biological and Medical Sciences, Oxford Brookes University, Oxford OX3 0BP,United KingdomcLondon Research and Development Centre, Agriculture and Agri-Food Canada, London, Ontario,Canada N5V 4T3

ORCID IDs: 0000-0002-0057-7753 (M.F.); 0000-0003-3536-0332 (R.M.); 0000-0003-4856-7690 (C.H.); 0000-0003-4100-6022 (L.F.).

Protein storage vacuoles (PSV) are the main repository of protein in dicotyledonous seeds, but little is known about the origins ofthese transient organelles. PSV are hypothesized to either arise de novo or originate from the preexisting embryonic vacuole (EV)during seed maturation. Here, we tested these hypotheses by studying PSV formation in Arabidopsis (Arabidopsis thaliana)embryos at different stages of seed maturation and recapitulated this process in Arabidopsis leaves reprogrammed to anembryogenic fate by inducing expression of the LEAFY COTYLEDON2 transcription factor. Confocal and immunoelectronmicroscopy indicated that both storage proteins and tonoplast proteins typical of PSV were delivered to the preexisting EVin embryos or to the lytic vacuole in reprogrammed leaf cells. In addition, sectioning through embryos at several developmentalstages using serial block face scanning electron microscopy revealed the 3D architecture of forming PSV. Our results indicate thatthe preexisting EV is reprogrammed to become a PSV in Arabidopsis.

During seed development, protein reserves andminerals are stored in specialized vacuoles called pro-tein storage vacuoles (PSV). PSV are functionally dif-ferent from lytic vacuoles (LV), which are present inmost vegetative plant tissues and function as lysosome-like, degradative organelles (Marty, 1999; De, 2000).

PSV exist in both monocots and dicots but are themain site of storage protein accumulation in dicotyle-donous species. In spite of the global importance of PSVas primary repositories for storage proteins, very little isknown about their origins. It is debated whether theyarise de novo during seed maturation or whether theyderive from the vacuoles present in embryo cells,henceforth named embryonic vacuoles (EV), which

undergo a functional reprogramming during seedmaturation. Whatever the intracellular origin of PSV, aspecific PSV developmental program must exist, as thesimple overexpression of seed storage proteins in non-seed organs is not sufficient to either induce PSV for-mation or change the function of the existing LV (Baggaet al., 1992; Frigerio et al., 1998, 2000). PSV have beenshown to arise by a de novo mechanism in cotyledonsof developing pea (Pisum sativum), and a similarmechanism may operate in Medicago truncatula em-bryos (Hoh et al., 1995; Frigerio et al., 2008). However,there have been few studies addressing the early stagesof PSV formation in Arabidopsis (Arabidopsis thaliana;Mansfield and Briarty, 1992).

In Arabidopsis cells, an EV is present during earlyembryogenesis (Rojo et al., 2001; D’Ippólito et al., 2017).During thematuration phase of seed development, PSVarise to accumulate storage reserves and ultimatelybecome the only detectable vacuole (Mansfield andBriarty, 1992; Otegui et al., 2006). PSV persist in em-bryonic cells until seed germination, when reserves aremobilized to provide nutrients for the growing seed-ling. The PSV-to-LV transition during germinationhas been studied in tobacco (Nicotiana tabacum) roottip cells: LV were shown to arise by a reprogrammingand fusion of PSV (Zheng and Staehelin, 2011). PSVtonoplast markers also are replaced by LV markers inboth tobacco and Arabidopsis seedling cells (Gattolinet al., 2011; Zheng and Staehelin, 2011). It is not yetclear whether similar reprogramming mechanismsapply to PSV formation during seed maturation inArabidopsis.

1 This work was funded by the Leverhulme Trust (grant RPG-327to L.F.) and the Biotechnology and Biological Sciences ResearchCouncil (grants BB/J017582/1 to L.F. and BB/M000168/1 to C.H.).

2 These authors contributed equally to the article.3 Address correspondence to l.frigerio@warwick.ac.uk.The author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Lorenzo Frigerio (l.frigerio@warwick.ac.uk).

M.F. generated the materials, performed all confocal microscopyand immunogold labeling, and contributed to preparation and imag-ing of EM samples; M.K. prepared and imaged EM and SBF-SEMsamples and prepared all reconstructions; R.M. contributed towardgeneration of the materials; C.H. analyzed the data; L.F. conceivedthe project and analyzed the data; all authors contributed to the writingof the article.

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The study of PSV formation is challenging, as PSV aretransient organelles and the EV-to-PSV transition oc-curs relatively quickly (Mansfield and Briarty, 1992).Genetic approaches have not pinpointed the actualformation of PSV. Mutants of the ESCRT componentFREE1/FYVE1 (Gao et al., 2015; Kolb et al., 2015), theSNARE proteins VTI11 and VTI12 (Sanmartin et al.,2007; Zheng et al., 2014), the VAMP727 SNARE com-plex (Ebine et al., 2008), the d-subunit of the AP-3adaptor complex (Zwiewka et al., 2011), and the shootmeristem identity protein TFL1 (Sohn et al., 2007), forexample, all show disruptions in protein traffickingto PSV rather than in PSV formation. In addition,vacuoleless mutants are embryo lethal (Rojo et al., 2001;D’Ippólito et al., 2017). Finally, there is a shortage ofboth EV- and PSV-specific markers, which makes itdifficult to identify the PSV unequivocally and to dis-tinguish it from the EV or LV, particularly during de-velopmental transitions such as embryo formation orseed germination, respectively.

Here, we used a combination of experimentalapproaches to address the origins of PSV.We producedtransgenic Arabidopsis lines expressing fluorescentprotein-taggedmarkers for the tonoplast and the lumenof PSV as well as tonoplast markers for preexistingvacuoles (i.e. EV in embryos and LV in leaves). In ad-dition, we visualized PSV using both fluorescentpH-sensitive stains and inherent PSV autofluorescence.Altogether, these markers were used to observe PSVformation in two experimental systems: embryo cellsfrom maturing seeds and leaf cells reprogrammedtoward a seed developmental program by over-expressing LEAFY COTYLEDON2 (LEC2), a masterregulator of seed maturation (Stone et al., 2001; Feeneyet al., 2013a). These experimental systems were used toexplore the two conceptually simplest scenarios of PSVformation (Fig. 1). If PSV arise de novo, it is likely that,as the seed matures, the newly synthesized PSV tono-plast markers will label new compartments, which areseparate from the EV. These separate compartmentswill contain storage proteins (Fig. 1A). If, however, PSVderive from the reprogramming of existing EV, then thePSV membrane markers will label the preexisting ton-oplast and will colocalize with the EV tonoplastmarkers, while storage proteins will appear in the EVlumen (Fig. 1B). We complemented these approacheswith immunoelectron microscopy and serial block facescanning electron microscopy (SBF-SEM) to studyvacuoles in developing embryos in 3D. Our findingsindicate that Arabidopsis PSV arise by the remodelingof preexisting vacuoles rather than by the de novo bi-ogenesis of PSV.

RESULTS

Timing of PSV Formation

To capture the time during development when PSV,here defined as vacuolar structures containing storage

proteins, are formed, we dissected Arabidopsis em-bryos from developing siliques and assigned them todifferent stages of maturation (Fig. 2A). The stages wedefined here reflect the overall morphology of the em-bryos after dissection from the ovule: heart for heart-shaped embryos, torpedo for elongated but not yetcurved embryos, walking stick for all embryos withcotyledons bent less than 90° relative to the radicle, andbent cotyledon for all further developed green embryos(Fig. 2A).We further divided thewalking stick and bentcotyledon stages into early, mid, and late substages.Mature embryos were dissected from dry seeds afterimbibing them in water for approximately 3 h.

For an initial analysis of the embryo ultrastructure,embryos were fixed for transmission electron micros-copy (TEM). The EV is clearly visible as a transparentstructure in torpedo and walking stick embryos (Fig.2B). However, at the early-bent cotyledon stage,electron-opaque material begins to appear within theEV, and this material gradually fills the vacuolar lumenin mid- and late-bent cotyledon embryos (Fig. 2B). Inagreement with Mansfield and Briarty (1992), we esti-mate that PSV formation from an empty EV to a fullPSV takes approximately 5 d. Therefore, within thistime frame, we focused on the transition between late-walking stick and bent cotyledon stages, whichencompasses the onset of storage protein deposition.For consistency, we chose to study cotyledon cells forboth electron microscopy (EM) and live cell imaging.

During the most critical time of PSV formation, theoverall morphology of the bent cotyledon embryosdoes not change significantly (Fig. 2). Therefore, we relyon PSV characteristics to evaluate the stage of PSVformation. EM shows the gradual accumulation ofelectron-opaque material in the vacuole and indicates

Figure 1. Hypotheses for PSV formation tested in thiswork. A, PSV formde novo, briefly coexisting with the EV to eventually become thedominant structure in mature seeds. During the transition, PSV-specifictonoplast markers (green outline) appear alongside the EV tonoplastmarkers (red outline). B, PSV arise through reprogramming of the EVby the accumulation of seed storage proteins in the EV lumen (greenpattern fill). PSVand EV tonoplast markers coexist (yellowoutline) whilethe EV is converted to a PSV.

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the stage of PSV formation. Likewise, confocal micros-copy of fluorescent protein-tagged tonoplast markersreveals vacuolemorphologies that characterize formingPSV. With the large numbers of embryos imaged byconfocal microscopy, we noted that particular eventsoccurring during the vacuole transition often could notbe pinned down to a specific developmental substage,even among bent cotyledon embryos isolated from thesame plant. This variability has been observed previ-ously (Mansfield and Briarty, 1992). Consequently, forconfocal analysis, we often avoided assigning an eventto a particular substage of bent cotyledon embryos.

PSV Tonoplast Markers Colocalize with TonoplastMarkers Labeling the Preexisting EV inDeveloping Embryos

We used the temporal expression patterns of a panelof known vacuolar markers to identify, and distinguishbetween, the EV and PSV. As a first step to determinewhether PSV are formed de novo or by a reprogram-ming of the preexisting vacuole, we used confocal mi-croscopy to localize two PSV-specific aquaporins,Tonoplast Intrinsic Protein3;1 (TIP3;1) and TIP3;2(Gattolin et al., 2011). The expression of both TIP3 iso-forms is restricted to seeds and begins in bent cotyledonstage embryos, which makes them suitable PSVmarkers (Johnson et al., 1990; Jauh et al., 1999; Hunteret al., 2007; Gattolin et al., 2011). To visualize the EV inlate-walking stick to early-bent cotyledon embryos, weimaged the 35S-driven expression of known tonoplastproteins: TPK1-GFP (Voelker et al., 2006; Maîtrejeanet al., 2011), VHA-a3-mRFP (Brux et al., 2008), andTIP1;1-RFP (Gattolin et al., 2009). These constitutivelyexpressed markers are present on the EV tonoplastbefore the TIP3 PSV markers are expressed (Fig. 3,A–D); thus, we believe that this is a viable approach tostudy the EV-to-PSV transition.

We imaged embryos in early- and late-bent cotyle-don stages expressing either TIP3;1-YFP or TIP3;2-mCherry, under the control of their native promoters,in combination with 35S:TPK1-GFP (Maîtrejean et al.,2011; Fig. 3). Constitutively expressed TPK1-GFP isvisible in all bent cotyledon embryos and labels theEV membrane before TIP3;1-YFP or TIP3;2-mCherrymarkers are observed (Fig. 3, A–D). Once PSVmarkers begin to appear, they label the samemembraneas TPK1-GFP, as indicated by the colocalization of bothmarkers (Fig. 3, E–L). It should be noted that, duringembryo development, chlorophyll autofluorescencefrom plastids often is detectable in the GFP/YFPemission channel. To distinguish our GFP- and YFP-labeled makers from plastids, a separate channel forchlorophyll autofluorescence is shown (Fig. 3, C, G, andK). At no stage were we able to visualize, at least at theresolution and sensitivity of the confocal microscope,TIP3;1-YFP- or TIP3;2-mCherry-labeled structures thatwere distinct from the EV tonoplast.

Seed Storage Proteins Accumulate in the EV Lumen inDeveloping Embryos

If the PSV tonoplast markers localize to the EVmembrane, we then hypothesized that seed storageproteins also would accumulate in the lumen of thevacuole labeled by both EV and PSV tonoplast markers.Therefore, we studied an Arabidopsis line coexpressingTIP3;2-mCherry and the seed storage protein 2S1 al-bumin fused to GFP (2S1-GFP), both driven by theirendogenous promoters. When first detectable in early-bent cotyledon embryos, 2S1-GFP is located both inpunctate structures in the cytosol and within the lumenof the existing vacuoles (Fig. 4, A–D). At the same time,the TIP3;2-mCherry signal labels the tonoplast but alsothe endoplasmic reticulum (ER) and the plasma mem-brane, as observed previously (Gattolin et al., 2011).

Figure 2. Stages assigned to Arabi-dopsis embryo development and anoverview of PSV formation in a rep-resentative cell from selected stages.A, Images of heart, torpedo, walkingstick, and bent cotyledon embryosdissected from Arabidopsis ovules.The mature embryo was dissectedfrom a dry seed. Bar = 100 mm. B,PSV arise during the maturationphase of embryonic development.Images shows single micrographsfrom SBF-SEM stacks of embryos atdifferent developmental stages. De-posits of electron-opaque material(arrows) are first observed in the vac-uole lumen and along the tonoplast ofthe EV in early-bent cotyledon em-bryos. The deposits accumulate and

eventually fill the vacuolar lumen in mature seed. Asterisks indicate oil bodies. Bar at left for high-magnification images = 1 mm; bar at right for insetoverview images = 100 mm.

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We reasoned that the 2S1-GFP punctate structurescould be prevacuolar compartments/multivesicularbodies (PVC/MVB) on account of their size and dis-tribution, as described previously (Miao et al., 2008).Staining with FM4-64 can be used to study membranetrafficking events in plant cells. The dye inserts into theouter leaflet of the plasma membrane and is thought toenter the secretory pathway by endocytosis (Bolte et al.,2004). As FM4-64 is endocytosed, it is transported to

the trans-Golgi network/early endosome, where it issorted to the MVB (Tse et al., 2004; Dettmer et al., 2006;Ebine et al., 2008; Viotti et al., 2010) along with newlysynthesized proteins, such as 2S albumins (Otegui et al.,2006; Miao et al., 2008). MVB then fuse with the vacuoleto release their contents (Otegui et al., 2006; Ebine et al.,2008; Scheuring et al., 2011). Long-term staining ofembryos with FM4-64 revealed the localization of 2S1-GFP-labeled punctate structures to subdomains of

Figure 4. 2S1 albumin seed storageproteins accumulate initially in punc-tate cytosolic structures and ultimatelyas deposits inside the lumina of formingPSV in bent cotyledon embryos. 2S1-GFP (green) labels small punctatestructures (arrows) that accumulate inthe EV/PSV lumen (asterisk). The PSVtonoplast marker TIP3;2-mCherry (red)is localized to the ER, tonoplast, andplasma membrane (open arrowheads).Chlorophyll is shown in blue. A to D,2S1-GFP signal is first observed assmall punctate structures in the cyto-plasm and accumulates in PSV luminawhose tonoplasts are labeled withTIP3;2-mCherry. E to H, In late-bentcotyledon embryos, the 2S1-GFP signalis observed only in vacuole lumina.Subregions of more intense 2S1-GFPfluorescence are visible in PSV lumina(closed arrowheads). Bars = 5 mm.

Figure 3. PSV tonoplast markersappear on the preexisting EV tono-plast in bent cotyledon embryos.Embryos constitutively expressingthe EV tonoplast marker 35S:TPK1-GFP (green) and the PSV tonoplastmarkers (red) TIP3;1:TIP3;1-YFP (topand middle rows) or TIP3;2:TIP3;2-mCherry (bottom row) are shown.Chlorophyll autofluorescence isshown in blue. A to D, In early-bentcotyledon embryos, the EV is labeledwith TPK1-GFP before PSV markersare expressed. E to L, In late-bentcotyledon embryos, PSV tonoplastmarkers colocalize with the EV ton-oplast marker. Bars = 5 mm.

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larger structures labeled by FM4-64 (Supplemental Fig.S1A). Using the high-resolution Zeiss Airyscan detec-tor, the FM4-64-stained structures that associated withthe punctate 2S1-GFP signals appeared brighter thanother cellular structures (Supplemental Fig. S1A). Ad-ditionally, 2S1-GFP puncta no longer associating withthe FM4-64-stained structures were observed to accu-mulate inside vacuolar lumina (Supplemental Fig. S1B),implying that the 2S1-GFP cargo was delivered to thevacuole. At no point were the 2S1-GFP puncta seen tofuse or accumulate into a separate compartmentother than EV lumina. Taken together, these observa-tions suggest that the 2S1-GFP puncta that associatewith larger FM4-64-labeled structures are likely to bePVC/MVB.In late-bent cotyledon embryos of the Arabidopsis

line coexpressing TIP3;2-mCherry and 2S1-GFP, theTIP3;2-mCherry signal becomes more visible on thetonoplast while 2S1-GFP no longer labels punctatestructures and is observed only within the lumina ofvacuoles (Fig. 4, E–H). Within the vacuoles, subregionsof more intense 2S1-GFP signal are observed (Fig. 4,E–H, arrowheads). We also were able to observe thecombined arrival of a PSV tonoplast marker and lu-minal marker to the EV in a triple transgenic line wherethe EV was labeled by 35S:TIP1;1-RFP (SupplementalFig. S2). In agreement with our previous results (Fig.3, E–L), EV (TIP1;1-RFP) and PSV (YFP-TIP3;1) tono-plast markers were located on the same membrane(Supplemental Fig. S2). At the same time, the PSV lu-minal marker (2S1-GFP) localized to the lumen of

vacuoles labeled with both PSV and EVmarkers. Takentogether, our fluorescent protein-tagged PSV markersconsistently associate with the vacuole labeled by ourEV markers, suggesting that PSV originate from thepreexisting EV.

PSV Autofluorescence and pH-Sensitive Fluorescent DyesAre Detected within the EV

Due to the limited choice of markers available to labelEV and PSV, we took advantage of the PSV’s affinity forstaining with pH-sensitive fluorescent dyes (Hara-Nishimura et al., 1987; Otegui et al., 2006; Gattolinet al., 2011) and their inherent luminal autofluorescence(Fuji et al., 2007; Hunter et al., 2007; Feeney et al., 2013b)to map the formation of PSV. Bent cotyledon em-bryos were stained with two acidotropic dyes:Neutral Red (NR) and 29,79-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein, acetoxymethyl ester (BCECF-AM). At neutral pH, these stains pass freely throughmembranes in their unprotonated forms, but proton-ation in acidic compartments reduces their permeabil-ity and leads to their accumulation (Dubrovsky et al.,2006; Scheuring et al., 2015).

Both stains accumulated and fluoresced within vac-uoles or subregions of the vacuoles whose tonoplastswere labeled with constitutively expressed TPK1-GFPand VHA-a3-RFP (Fig. 5, A–H). PSV autofluorescencewas associated with the staining patterns (Fig. 5, C, G,and K). To investigate whether the staining patterns are

Figure 5. Forming PSV are identifiedby the acidotropic stains NR andBCECF-AM and PSV luminal auto-fluorescence in bent cotyledon em-bryos. A to H, NR and BCECF-AMstain vacuoles labeled with the ton-oplast markers TPK1-GFP (A) andVHA-a3-RFP (E), respectively. Vacu-ole lumen autofluorescence (blue)colocalizes with the stains (D andH). I to L, Embryos accumulating 2S1albumin-GFP were stained with NR.The 2S1-GFP signal fills vacuole lu-mina, and areas of more intense GFPfluorescence are observed (arrow-heads in I). NR stains distinct subre-gions of the vacuole lumina (J). TheseNR-stained subregions colocalizewith PSV lumen autofluorescence(K) and with areas of intense 2S1-GFP fluorescence (L). Bars = 10 mm(A–H) and 5 mm (I–L).

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associated with the accumulation pattern of seed stor-age proteins, bent cotyledon embryos expressing 2S1-GFP were stained with NR. We observed the 2S1-GFPsignal to disperse within the entire vacuole lumen (Fig. 5,I–L), as also shown in Figure 4. However, bright areasof NR stain and PSV autofluorescence were both ob-served to colocalize with areas of more intense 2S1-GFPfluorescence in subregions of the vacuoles (Fig. 5, I–L).These intensely fluorescent 2S1-GFP subregions alsowere seen without NR staining, as shown in Figure 4.

To further investigate the pattern of staining and theoccurrence of PSV autofluorescence, bent cotyledonembryos expressing the PSV-specific tonoplast markerYFP-TIP3;1 under the control of its native promoterwere stained with NR. As PSV form from the large,round EV (Supplemental Fig. S3A) and remodel to as-sume the characteristic mature PSV morphology(Supplemental Fig. S3, B and C), NR staining becomesmore prominent. The stain initially accumulates indiscrete zones within the vacuolar lumen (SupplementalFig. S3A) to then eventually stain the entire lumen inlate-bent cotyledon embryos (Supplemental Fig.S3C). At no point are the stains observed to accu-mulate in structures other than vacuoles labeled byEV (Fig. 5, A–H) or PSV (Supplemental Fig. S3) ton-oplast markers. A similar pattern is observed for PSVautofluorescence in forming PSV (Supplemental Fig.S3, D–F). Taken together, these data indicate that theaccumulation of both acidotropic stains and the in-herent PSV autofluorescence associate with the EVreprogramming to become the PSV.

PSV Tonoplast Markers and Seed Storage Proteins Label aTransitioning EV

Our light microscopy observations were corrobo-rated by immunoelectron microscopy to identifywhere PSV marker proteins begin to accumulate. Forembryos prepared for immunogold labeling, the EV ischaracterized by a large, translucent lumen in torpedo,walking stick, and early-bent cotyledon stage embryos(Fig. 6, A, C, and E). However, unlike routine EM (Fig.2B), specimens for immunogold labeling were notfixed with osmium. Oil bodies (which, due to the lackof osmium in this preparation, also appear translu-cent) were differentiated from the EV by their ratheruniform size, round shape, and lack of flocculent lu-minal material.

Antibodies against TIP3;1 as well as 2S albumin and12S globulin seed storage proteins showed no signifi-cant gold labeling in embryos from heart to walkingstick stages (Supplemental Fig. S4). In early-bent coty-ledon embryos, anti-TIP3;1 antiserum labeled the ton-oplast of the large, translucent EV (Fig. 6A), and anti-2Sand anti-12S were detected along the inner periphery ofthe tonoplast and on electron-opaque material withinthe lumen of the vacuole (Fig. 6, C and E). In late-bentcotyledon embryos, the lumen of the maturing PSVis completely filled with electron-opaque material

(Fig. 2B) and shows a high density of gold labeling withanti-TIP3;1, anti-12S, and anti-2S antibodies (Fig. 6, B,D, and F). To allow a quicker visualization of the dis-tribution of the gold particles, we highlighted them ontransparent versions of the micrographs (SupplementalFig. S5). These results support our confocal microscopyobservations that suggest that TIP3 isoforms and seedstorage proteins appear at the tonoplast and in the lu-men of the EV, respectively (Fig. 4; SupplementalFig. S5).

In addition, the electron-opaque material accumu-lating along the periphery and dispersed within theEV/PSV lumen was labeled by anti-complex glycanantiserum (Laurière et al., 1989), confirming that at least

Figure 6. Immunogold labeling reveals the localization of PSV tono-plast aquaporin TIP3;1 and the 2S albumin and 12S globulin seedstorage proteins to the EV in bent cotyledon embryo cells. A and B, Anti-TIP3;1 antibody labels the tonoplast of transitioning vacuoles. C and D,Anti-12S globulin antibody labels electron-opaque material accumu-lating along the luminal side of the tonoplast (C) and the entire PSVlumen in late-bent cotyledon embryos (D). E and F, Anti-2S antibodylabels electron-opaquematerial accumulating along the luminal side ofthe tonoplast (E) as well as electron-opaque material in the vacuolelumen (F). OB, Oil bodies. Bar = 500 nm.

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a proportion of PSV glycoproteins underwentN-glycanprocessing in the Golgi (Supplemental Fig. S6).

PSV Form through Remodeling of the LV in Leaf CellsReprogrammed by LEC2

Our data so far indicate that PSV are formed in em-bryos by the repurposing of the preexisting vacuole. Inorder to test whether this functional transition could berecapitulated in a system where the only vacuole pre-sent is the LV, we investigated the formation of PSV inleaves reprogrammed to follow a seed developmentalpathway by the overexpression of LEC2 (Feeney et al.,2013b). LEC2 is a key transcriptional regulator of seed

development and, when overexpressed in vegetativetissues, causes cells to change their developmentalpathway and acquire characteristics of maturationphase embryos. We showed previously that, in thissystem, leaf LV are replaced by PSV (Feeney et al.,2013a). However, the details of the LV-to-PSV transi-tion were not explored. Therefore, with a similar strat-egy to that used for embryos as described above, weimaged leaf cells of Arabidopsis 35S:LEC2-GR lineswhere the LV was labeled with a constitutivelyexpressed tonoplast marker, TPK1-GFP, and observedthe localization of the PSV marker TIP3;1-YFP, ex-pressed under the control of its native, seed-specificpromoter (Fig. 7, A–P). After 14 d with dexametha-sone (DEX) treatment to induce LEC2 overexpression

Figure 7. PSV form through remod-eling of the LV in Arabidopsis leafcells reprogrammed by LEC2. A to P,Representative images of transition-ing vacuoles in LEC2-induced leafcells at 14 d (A–D), 17 d (E–H), and20 d (I–P) with DEX. The TIP3;1-YFPPSV tonoplast marker (red) accumu-lates on the preexisting LV (E, I,and M) in Arabidopsis lines constitu-tively expressing the tonoplast markerTPK1-GFP (green). Chlorophyll auto-fluorescence is shown in blue. Aster-isks show asynchronous remodelingof the tonoplast in neighboring cells.Bars = 10 mm. Q to T, Electron mi-croscopy of leaf cells 14 d after LEC2induction with DEX. Electron-opaquePSV material (black) accumulatesalong the luminal side of the tonoplastand disperses in the vacuole lumen.Bars = 500 nm (Q–S) and 2 mm (T).

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(Fig. 7, A–D), the PSVmarker is not yet detectable whilethe TPK1-GFP marker labels the LV tonoplast. Between15 and 20 d with DEX, the PSV marker becomes de-tectable and is observed to colocalize to the LV markeron the same tonoplast (Fig. 7, E–P), indicating that theLV is reprogrammed to become a PSV. During theLV-to-PSV transition, leaf LV morphology changesfrom a large central compartment that mirrors the cell’sshape (Fig. 7, A–D) to smaller vacuoles that are moreround in shape, resembling PSV (Fig. 7, M–P). In ad-dition, the images show an asynchronous remodelingof tonoplast in neighboring cells; LV highlighted solelywith TPK1-GFP lack tonoplast folds (Fig. 7, E–H, as-terisks). However, adjacent vacuoles accumulatingboth TPK1-GFP and TIP3;1-YFP possess brightly fluo-rescent tonoplast folds, as reported by Feeney et al.(2013a, 2013b).

In a similar manner to developing embryos, LEC2-induced leaf cells accumulate PSV material in theLV/PSV (compare Fig. 7, Q–T, with Figs. 2B and 6).TEM of leaf cells from 35S:LEC2-GR plants grown in thepresence of DEX for 14 d provides a representation ofthe LV-to-PSV transition (Fig. 7, Q–T). Electron-opaquematerial accumulates along the inner periphery of theLV tonoplast and is observed to disperse within thevacuole lumen. These electron-opaque deposits wereshown previously to contain 12S globulin and 2S al-bumin seed storage proteins by immunogold labeling(Feeney et al., 2013a).

The LV-to-PSV transition occurs over approximately5 d in LEC2-induced leaf cells, as shown in Figure 7.To visualize the highly dynamic nature of the vacuoleremodeling occurring over this time course, opticalsections were taken through leaf cell vacuoles of LEC2-induced plants harboring 35S:TPK1-GFP. Maximum

intensity projections of representative images areshown in Figure 8. At approximately 14 d on DEX, LVappear normally shaped (Fig. 8A) but possess moretransvacuolar strands than control leaf cells withoutDEX (Fig. 8F). However, as the LV transitions to a PSV,the tonoplast undergoes extensive remodeling (Fig. 8,B–D). At approximately 20 d on DEX, vacuoles aremore round in shape and resemble PSV (Fig. 8E). A timeseries taken from a representative cell during thistransition stage reveals fast tonoplast remodeling,compared with minimal remodeling in noninducedcells (Supplemental Fig. S7; Supplemental Movies S1and S2).

PSV Formation Involves Extensive Remodeling of the EV

Embryo cells contain one large EV at the torpedostage. However, inmature embryo cells, there appear tobe multiple, smaller PSV present (Fig. 2B). To betterunderstand the EV-to-PSV transition, we used SBF-SEM to obtain 3D data sets of embryos at different de-velopmental stages. By serially imaging the block facescut at 100-nm intervals through whole cells with high-resolution SEM,wewere able to acquire image stacks ofentire cells and reconstruct the entire vacuolar systemin developing embryos. For comparison, we chose cellsin the cotyledon tip of embryos from torpedo, early-,mid-, and late-bent cotyledon stages and from a matureseed (Supplemental Fig. S8). We reconstructed the trans-lucent lumen of the EV, electron-opaque PSV materialcomposed of seed storage proteins (according to our an-tibody labeling; Fig. 6), the nucleus, and the cell mem-brane (Fig. 9). For the original data sets and animatedreconstructions, see Supplemental Movies S3 to S10.

A B C

D E F

Figure 8. The tonoplast undergoesextensive remodeling during the LV-to-PSV transition in LEC2-inducedleaf cells. Representative imagesshow the progression of tonoplastremodeling during the LV-to-PSVtransition at 14 d (A), 17 d (B and C),and 20 d (D and E) with DEX or at14 d without DEX (F). Arabidopsis35S:LEC2-GR lines harboring 35S:TPK1-GFP (green) were imaged.Images are maximum intensity pro-jections of Z-stacks taken throughleaf epidermal cells. Bars = 10 mm.

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During seed maturation, storage proteins (Fig. 9,green) increasingly accumulate along the periphery ofthe EV lumen (Fig. 9, blue) and take up an increasingproportion of the vacuolar volume (Supplemental Fig.S9). At the same time, the large EV divides into smallervacuoles, which are eventually filled with storage pro-tein. In the mature seed, the EV has been replaced bymultiple PSV. Electron-opaque PSV lumina possessedsmall, translucent, and partly crystalline inclusions(Fig. 2B; Supplemental Fig. S9). These inclusions oftenare identified as globoids (Jiang et al., 2001). To main-tain consistency in our reconstructions, these translu-cent inclusions were considered to be EV (Fig. 9;Supplemental Fig. S9), although we are unable tospeculate on their ontology. Additionally, as the em-bryo matures, the nucleus changes position from the

cortex to the center of the cell (Fig. 9, yellow). Thesesnapshots of PSV formation during embryo devel-opment, captured in 3D using SBF-SEM, providefurther evidence that the PSV originates from thepreexisting EV.

DISCUSSION

Our data provide a dynamic picture of PSV forma-tion during seed maturation and indicate that, inArabidopsis embryo cells, PSV do not arise de novobut result from the repurposing of the preexisting EV.While it has been reported previously that matureseeds only appear to contain PSV (Otegui et al., 2006;Hunter et al., 2007), the events leading to the formation

Figure 9. Remodeling of the EV to PSV during embryo maturation. The lumen of the EV (blue), electron-opaque PSV luminalmaterial (green), nucleus (yellow), and plasma membrane (gray) were rendered from SBF-SEM image stacks in cotyledon cells ofembryos in the torpedo, early-, mid-, and late-bent cotyledon, or mature embryo stages. As electron-opaque material initiallyaccumulates along the periphery of the EV lumen, the vacuole separates into several smaller vacuoles, which are eventually filledwith electron-opaque PSV luminal material aside from small translucent areas. The nucleus changes position from the cell cortexto the center of the cell. Bar = 5 mm.

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of PSV in Arabidopsis have not been documentedpreviously.

To establish where PSV originate in Arabidopsisembryo cells, we used lines expressing fluorescentlylabeled vacuolar markers, acidotropic stains, PSVautofluorescence, high-resolution Zeiss Airyscan con-focal laser scanning microscopy detection, SBF-SEM,and immunoelectron microscopy. According to ourdefinition of PSV as vacuolar structures that containseed storage proteins, our data indicate that PSV for-mation begins between the late-walking stick and early-bent cotyledon stages (Fig. 2B).

PSV formation involves an accumulation of electron-opaque material in the lumen of the preexisting vacu-ole. The elemental composition of the electron-opaquematerial accumulating in PSV has been characterizedby means of energy-dispersive x-ray spectroscopyanalysis by Zheng and Staehelin (2011) andOtegui et al.(2002). The principal components of the spectrum werepotassium, calcium, magnesium, as well as phospho-rus, which was characterized as phytate (myoinositol-hexakisphosphate). Our immunogold labeling showsthat seed storage proteins (Fig. 6; Supplemental Fig. S5)and glycoproteins that have trafficked through theGolgi (Supplemental Fig. S6) also are found within theelectron-opaque material. Similarly, the seed storageprotein vicilin and barley (Hordeum vulgare) lectin weredetected in electron-opaque material in transitioningpea and barley vacuoles (Hoh et al., 1995; Olbrich et al.,2007) as well as 12S globulins and 2S albumins in LVtransitioning to PSV in leaf cells reprogrammed byLEC2 overexpression (Feeney et al., 2013a). Zheng andStaehelin (2011) distinguished between vacuoles un-dergoing PSV-to-LV transitions in tobacco root meri-stematic cells by the presence of electron-opaque PSVmaterial using TEM. We similarly observed the ap-pearance of electron-opaque deposits during the EV-to-PSV and LV-to-PSV transitions in embryos and leavesreprogrammed by LEC2 overexpression, respectively.

Nascent vacuolar proteins traffic from the ER to theirultimate destination in the EV/PSV. Throughout thevacuolar transition, we observed trafficking of themarker proteins. As PSV form, tonoplast markers,particularly the TIP isoforms, are observed in the ER enroute to the tonoplast (Fig. 4; Supplemental Fig. S2).Similarly, the seed storage protein 2S1 albumin-GFP isobserved as punctate structures at its earliest time ofdetection (Fig. 4, A–D; Supplemental Fig. S1). Seedstorage proteins are sorted at the Golgi apparatus intodense vesicles that fuse with other small vesicles car-rying proteolytic enzymes to give rise to PVC/MVB(Otegui et al., 2006). These organelles function as pre-vacuolar compartments in the secretory pathway andultimately fuse with the vacuole (Ebine et al., 2008;Scheuring et al., 2011). Therefore, the punctate 2S1-GFPstructures that we observe during early PSV formationare likely to be PVC/MVB, as described previously(Ebine et al., 2008; Miao et al., 2008).

Mature PSV can be identified by the accumulation ofacidotropic fluorescent stains and PSV autofluorescence

in their lumina (Fuji et al., 2007;Hunter et al., 2007; Feeneyet al., 2013b), although we do not yet know what iscontributing to PSV autofluorescence. We show herethat NR, BCECF-AM, and PSV autofluorescence can beused to identify forming PSV (Fig. 5; Supplemental Fig.S3). Within early PSV lumina, the colocalization of NR,PSV autofluorescence, and 2S1-GFP fluorescence intodistinct, brighter areas suggest the existence of subre-gions in the matrix of the transitioning vacuole duringseed filling (Fig. 5, I–L). A similar observation wasmade in vacuoles of developing pumpkin (Cucurbitamaxima) cotyledons stained with NR (Hara-Nishimuraet al., 1987). In our study, these distinct areas were firstobserved to accumulate in the EV/PSV lumen as small,highly fluorescent regions that increased in volume,eventually filling the PSV lumen (Supplemental Fig.S3). Looking at cross sections of our 3D SBF-SEM datasets, we can sometimes observe a similar accumulationof electron-opaque material in one area of the EV, po-tentially representing such a subregion (SupplementalFig. S9, early-bent cotyledon). It will be interesting toexplore the nature of these EV/PSV subregions andhow they relate to the biogenesis of the PSV as a com-pound organelle (Jiang et al., 2000, 2001).

Once we understood that PSV originate from EV indeveloping embryos, we documented how a single EV,occupying nearly the entire cell volume, transitions tobecome a collection of numerous, smaller-sized PSVwithin the mature embryo cell. SBF-SEM allowed usto image the entire embryo to visualize the 3D organi-zation of cells and tissues and, likewise, the spatialorganization of vacuolar structures in those cells. Re-constructions from SBF-SEM data of cotyledon cellsfrom one embryo each of torpedo, early-, mid-, and late-bent cotyledon stages and a mature embryo (Fig. 9;Supplemental Fig. S9) provide snapshots of how EVremodel to form PSV. In mature seeds, PSV exist as agroup of separate, individual entities that do not appearto form an interconnected network, as suggested pre-viously by light microscopy data (Hegedus et al., 2015).

The biogenesis of PSV has been a long-standing topicof debate. In cotyledon cells of developing pea em-bryos, an accumulation of electron-opaque materialwas observed within a tube-like, membrane-boundedstructure surrounding the EV during embryo devel-opment. PSV and EV membranes were labeled withanti-TIP3;1 and anti-TIP1;1 antibodies, respectively(Hoh et al., 1995). Altogether, these results supported ade novo mechanism for PSV development. Therefore, ifnascent PSV arise separately from the preexisting vac-uole, we may anticipate their respective tonoplasts topossess unique TIP isoform markers and, more im-portantly, seed storage proteins should accumulate inthe lumen of the PSV and not within the lumen of thepreexisting vacuole. Others have reported TIP1;1-labeled internal membranes in PSV lumina (Gillespieet al., 2005; Bolte et al., 2011). In Arabidopsis embryos,however, we were unable to detect TIP1;1 accumula-tion on the EV tonoplast (data not shown), and our EVand PSV tonoplast markers appear to colocalize on the

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same EV membrane (Fig. 3). This is in agreement withprevious results in Arabidopsis embryo cells (Oteguiet al., 2006; Gattolin et al., 2011) and in PSV forming inleaf cells reprogrammed by LEC2 (Feeney et al., 2013a).Moreover, we consistently observe seed storage pro-teins accumulating in the lumen of the vacuole labeledby both EV and PSV tonoplast and luminal markers.Therefore, we reason that, in Arabidopsis, PSV arise bya remodeling of the preexisting vacuole.In Arabidopsis, the presence of adjacent tonoplasts

within embryo cells was observed in single sections(Frigerio et al., 2008), potentially suggesting the pres-ence of separate vacuoles. Our study, which coversseveral developmental time points during PSV bio-genesis across whole cellular volumes, shows that ex-tensive membrane remodeling occurs during seedmaturation. Therefore, it is possible that the multiplemembranes observed at a fixed point may reflect ton-oplast remodeling rather than the presence of a separatestructure. Upon close inspection of our TEM data, wedid not observe a membrane surrounding the storageprotein aggregates forming in bent cotyledon embryosor transitioning vacuoles of LEC2-induced leaf cells(Supplemental Fig. S10). In both cases, the tonoplastappears as a clearly defined interface surroundingthe outer periphery of the protein aggregates. However,the inner periphery of these aggregates, which facesthe vacuole lumen, appears fuzzy and poorly defined,which does not suggest that it is membrane bounded.In addition, immunolabeling of transitioning LEC2-induced leaf vacuoles with an anti-TIP3;1 antiserumshows that the PSV membrane does not enclose theseed proteins but is localized solely to the tonoplast ofthe preexisting LV (Feeney et al., 2013a; SupplementalFig. S10). Therefore, our results do not support thehypothesis that the PSV arise by a de novo structure butmore strongly support a remodeling mechanism forPSV biogenesis.This work provides a foundation to further explore

the mechanism of PSV biogenesis. During the EV-to-PSV transition, it will be interesting to understand howthe EV lumen environment changes to accommodatethe influx of seed storage proteins. The overexpressionof seed storage proteins targeted to the leaf LV resultsin degradation of the proteins (Bagga et al., 1992;Frigerio et al., 1998, 2000); however, reprogrammingof the LV to a PSV alters the luminal environmentto accommodate incoming storage proteins (Feeneyet al., 2013a). While it is established that storage pro-teins are delivered to the nascent PSV by the fusion ofMVB (Otegui et al., 2006), additional processes mustbe at play.That PSV can arise from both the EV and LV raises

some general questions about the relationship betweenthe EV and LV. In lower plants, there is no separationbetween embryo morphogenesis and postembryonicdevelopment (West and Harada, 1993). During the ev-olution of seed plants, the maturation phase was inte-grated into embryogenesis to enable plants to interrupttheir life cycle (Vicente-Carbajosa and Carbonero,

2005). A network of transcriptional regulators, such asLEC2, are responsible for regulating the activities oc-curring during this developmental phase (Braybrookand Harada, 2008; Baud et al., 2016), including theformation of PSV (Feeney et al., 2013a, 2013b). Alto-gether, this suggests that the evolution of seeds hascommandeered the existing cellular structures and that,perhaps, the EV and LV are the same vacuole but ac-commodate a different complement of proteins.

In conclusion, our analysis of the early stages of PSVformation in Arabidopsis suggests that, rather than by ade novo process, PSV arise by functional reprogram-ming of the EV. While we do not attempt here to elu-cidate how this change happens, our findings pave theway to understanding the processes underpinning sucha developmental change.

MATERIALS AND METHODS

Plant Materials

Arabidopsis (Arabidopsis thaliana) lines used in this study for confocalmicroscopic analysis were Columbia-0 (Col-0) or lines harboring 35S:TPK1-GFP (Voelker et al., 2006; Maîtrejean et al., 2011), 35S:VHA-a3-mRFP (Bruxet al., 2008), 35S:TIP1;1-RFP, TIP3;1:TIP3;1-YFP/TIP1;1:TIP1;1-RFP, TIP3;1:YFP-TIP3;1, and TIP3;2:TIP3;2-mCherry (Gattolin et al., 2009, 2011), and 35S:LEC2-GR (Stone et al., 2008). The 2S1 albumin marker line 2S1:2S1-GFPwas modifiedfrom Miao et al. (2008). The 2S1-GFP fusion from vector pBI221 (Miao et al.,2008) was PCR amplified with BamHI and EcoRI restriction sites, and the pro-duct was ligated into binary vector pCaMterX (Harris and Gleddie, 2001). The2S1 promoter region was cloned from Col-0 genomic DNA by amplifying914 bp upstream of the 2S1 coding sequence (At4g27140) with the addition ofClaI and BamHI restriction sites. The 35S promoter was excised from pCaMterXby cuttingwithClaI and BamHI andwas replacedwith the 2S1 promoter region.For experiments, crossesweremade for combinations of the abovemarker lines.For EM studies, Col-0 plants orWassilewskija-0 plants harboring the 35S:LEC2-GR construct (Stone et al., 2008) were used.

Tissue Culture Conditions and LEC2 Induction

Sterilized seeds were transferred to germination medium consisting ofMurashige and Skoog (MS) salts (Sigma-Aldrich) supplemented with full-strength MS vitamins and 0.4 mg L21 thiamine-HCl, 100 mg L21 myoinositol,30 g L21 Suc, and 0.75% (w/v) agar, pH 5.8. Seeds were stratified for 3 to 4 d at4°C in the dark and transferred to a growth chamber for germination. Thegrowth chamber was set at 24°C day/22°C night with 70 mmol m22 s21 illu-minance and with a 16-h-light/8-h-dark photoperiod.

To induce LEC2 overexpression, stratified seeds were allowed to germinateand grow for 7 d on germination medium. Seedlings were then transferred toinduction medium composed of MS germination medium supplemented with30 mM DEX (Sigma-Aldrich). DEX was solubilized in dimethyl sulfoxide.Seedlings were incubated on DEX for up to 21 d before imaging.

Greenhouse Growth Conditions and Embryo Isolationfrom Siliques

Seedlings germinated in tissue culture were transferred to soil and grown at20°C day/18°C night with a minimum of 178 mmol m22 s21 illuminance andwith a 16-h-light/8-h-dark photoperiod for approximately 6 weeks. For con-focal imaging, one flower stalk was selected for each line. Siliques (eight to 12)were harvested from the mid to lower region of the flower stalk, closest to therosette. Using a stereomicroscope, ovules were dissected from siliques andtransferred to a 1.5-mL tube. A pestle was used to gently squash the ovules torelease embryos. The plant material was then transferred to a petri dish filledwith water. Embryos were gently separated from the debris and were classifiedand sorted according to their developmental stage as described in “Results”and shown in Figure 2A.

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Fluorescence and Confocal Laser Scanning Microscopyand Staining

Seeds and seedlings were screened for fluorescence using a Leica MZ FLIIIfluorescence stereomicroscope. To observe GFP and YFP fluorescence, a stan-dard GFP filter (excitation, BP480/40 nm; emission, LP510 nm) was used. Toobserve RFP fluorescence, an RFP filter (excitation, 546/10 nm; emission,LP590 nm) was used.

For confocal microscopy, embryo and leaf sampleswere observed directly orstained before examination with a Zeiss 880 confocal microscope. All imagingwas performed on embryo cotyledon cells. To stain vacuole lumina, embryoswere stained for 5minwith 17.5mMNR (Sigma-Aldrich) or for 30minwith 10mM

BCECF-AM (Molecular Probes). Embryos were washed three times with waterbefore imaging. For long-term staining with FM4-64, embryos were incubatedfor 1 h in 8 mM FM4-64, washed three times with water, and incubated for afurther 2.5 to 3 h in water before imaging. Confocal imaging was performedusing a 633 (1.4 numerical aperture) oil-immersion lens. A 405-nm laser linewas used to visualize PSV autofluorescence, and 440- to 490-nm emission wascollected. A 488-nm laser line was used to excite GFP and BCECF-AM, andemissions were collected as 505 to 540 nm for GFP and 500 to 560 nm forBCECF-AM. A 514-nm laser line was used to excite YFP and FM4-64, andemissions were collected as 525 to 585 nm for YFP and 615 to 645 nm for FM4-64. A 561-nm laser line was used to excite RFP and NR, and emissions werecollected as 565 to 640 nm for RFP and 560 to 615 nm forNR. A 630-nm laser linewas used to visualize chlorophyll autofluorescence, and 645- to 720-nm emis-sions were collected. Sequential detection of combinations of the above fluo-rophores was performed by combining their settings in the frame-scanningmode. Z-stacks were recorded using optimal scan parameters. For Airyscandetection, samples were imaged using a 1003 (1.46 numerical aperture) oil-immersion lens. Image processing was performedwith Zeiss Zen Lite software.Temporal color coding of time series data was performed using FIJI software.

Immunogold Labeling and TEM

Embryos were isolated from siliques as described above and fixed in2.5% (v/v) glutaraldehyde and 4% (w/v) paraformaldehyde in 0.1 M sodiumphosphate buffer, pH 7.4, at 4°C as described previously (Feeney et al., 2013a).Embryos were dehydrated in a graded ethanol series and then infiltrated inincreasing concentrations of LR White resin. Infiltration in pure LRWhite resinwas carried out for 3 d. In the last 24 h of infiltration, 0.5% (w/v) benzoinmethylether was added as a catalyst for polymerization. Embryos were embedded inflat embedding dishes sealed with Melinex film (Agar Scientific) and poly-merized under UV light at 220°C for 24 h followed by 0°C for 24 h.

Specimens were cut into 70-nm-thick sections and collected on nickel meshgrids for immunogold labeling experimentsusingaPowerTomeultramicrotome(RMC). Sections were blocked with goat normal serum (Aurion) for 30 minfollowed by 2 hwith primary antibodies dilutedwith dilution buffer (0.2% [v/v]BSA-c [Aurion], 0.05% [v/v] Tween 20, and 1% [w/v] BSA in PBS, pH 7.4).Primary antibodies were rabbit anti-TIP3;1 (1:10; Jauh et al., 1999), rabbit anti-12S globulin (1:500; Shimada et al., 2003), rabbit anti-2S albumin (1:500;Scarafoni et al., 2001), and rabbit anti-complex glycan (1:500; Laurière et al.,1989). Specimens were incubated for 1 h with secondary antibodies diluted 1:10with dilution buffer. All secondary antibodies were IgGs produced in goats andconjugated to 15-nm gold particles (Aurion). All specimens were stained for30 min with 4% (w/v) uranyl acetate (UA) and 20 min with lead citrate(Reynolds, 1963) followed by 3 min with 4% (w/v) UA. Specimens were ex-amined with an Hitachi H-7650 transmission electron microscope operating at100 kV.

Leaves were collected from LEC2-induced plants andwere fixed, infiltrated,and embedded in LR Gold resin according to Feeney et al. (2013a). Specimenswere cut into 60-nm-thick sections and stained for 10 min with 5% (w/v) UAand for 1 min with lead citrate (Feeney et al., 2013a) and were examined with aCM-10 transmission electron microscope (Philips) operating at 80 kV.

SBF-SEM

Embryos were isolated as described above and fixed in 1% (v/v) glutaral-dehyde and 1% (w/v) paraformaldehydewith 2% (w/v) Suc and 2mMCaCl2 in0.1 M sodium cacodylate (NaCac) buffer, pH 6.9, for 60 min at room tempera-ture. Embryos were washed in NaCac buffer and then incubated in 1% (w/v)tannic acid in 0.1 M NaCac buffer for 60 min. Embryos were washed with water3 3 10 min, stained in 1% (w/v) aqueous osmium tetroxide for 2 h at room

temperature, washed in deionized water, and dehydrated in a graded ethanolseries. Embryos were then infiltrated in gradually higher concentrations ofSpurr resin. Infiltration with pure resin was carried out for 3 d, with resinreplaced every 24 h. Embryos were embedded in flat dishes, and the resin waspolymerized at 70°C for 12 h. Embryosweremounted onto SBF-SEM stubswithconductive adhesive resin as described previously (Kittelmann et al., 2016).Trimmed blocks were gold sputter coated for 30 s (;20 nm thick). Serialoverview images of the entire embryo as well as high-magnification and high-resolution images of the cotyledon tips were collected with a Zeiss MerlinCompact SEM device fitted with a Gatan 3View system. Microscope settingswere as follows: 4 kV, 50 Pa at variable pressure mode, and 30-mm aperture.3View settings were as follows: 100-nm sections, pixel size of ;0.004 mm, andpixel dwell times of 3 ms for high-magnification images and 2 ms for overviewimages.

3D Reconstruction

The IMODsoftware packagewas used for stack formation, image alignment,trimming, andGaussianfiltering. Rendering of structures of interestwasdone inAmira Software (FEI) using the magic wand tool to semiautomatically segmentthe EV and PSV and the brush to manually segment the plasma membrane andnucleus. Labels were modeled using surface generation, and the number oftriangles was reduced for visualization. Movies were generated using the An-imation tool in Amira.

Accession Numbers

Accession numbers are as follows: AtTPK1, AT5G55630; AtVHA-a3,AT4G39080; AtTIP3;1, AT1G73190; AtTIP3;2, AT1G17810; AtTIP1;1,AT2G36830; At2S1, AT4G27140; and AtLEC2, AT1G28300.

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. Punctate cytosolic 2S1-GFP-labeled structuresassociate with FM4-64-stained structures and appear to accumulate invacuole lumina.

Supplemental Figure S2. Fluorescently labeled PSV tonoplast and lumenmarkers associate with the preexisting EV in bent cotyledon embryos.

Supplemental Figure S3. NR staining pattern and appearance of PSVautofluorescence in EV/PSV lumina of bent cotyledon embryos.

Supplemental Figure S4. Immunogold labeling of the 2S albumin seedstorage proteins in torpedo and early- to mid-walking stick embryosshow no significant signal.

Supplemental Figure S5. Highlighted gold particles indicating immuno-gold labeling with antibodies against TIP3;1, 12S globulins, and 2S al-bumins in the cells shown in Figure 6.

Supplemental Figure S6. Anti-complex glycan antibody labeling in early-and late-bent cotyledon embryos.

Supplemental Figure S7. Vacuoles exhibit highly dynamic remodelingduring the LV-to-PSV transition in LEC2-induced leaf epidermal cells.

Supplemental Figure S8. Regions of interest of selected embryos at differ-ent developmental stages imaged with SBF-SEM.

Supplemental Figure S9. Cross-section view through vacuoles in cotyle-don cells of embryos in the torpedo, early-, mid-, and late-bent cotyle-don, and mature embryo stages.

Supplemental Figure S10. Storage proteins accumulate within the lumenof the preexisting vacuole.

Supplemental Movie S1. Arabidopsis 35S:LEC2-GR line expressing 35S:TPK1-GFP after 18 d with DEX.

Supplemental Movie S2. Arabidopsis 35S:LEC2-GR line expressing 35S:TPK1-GFP after 14 d without DEX.

Supplemental Movie S3. 3D reconstruction of one cell in the cotyledon of atorpedo stage embryo.

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Supplemental Movie S4. Orthoslice moving through the image stack of atorpedo stage embryo cotyledon.

Supplemental Movie S5. 3D reconstruction of one cell in the cotyledon ofan early-bent cotyledon stage embryo.

Supplemental Movie S6. Orthoslice moving through the image stack of anearly-bent cotyledon stage embryo cotyledon.

Supplemental Movie S7. 3D reconstruction of one cell in the cotyledon of alate-bent cotyledon stage embryo.

Supplemental Movie S8. Orthoslice moving through the image stack of alate-bent cotyledon stage embryo cotyledon.

Supplemental Movie S9. 3D reconstruction of one cell in the cotyledon of amature seed stage embryo.

Supplemental Movie S10. Orthoslice moving through the image stack of amature stage embryo cotyledon.

ACKNOWLEDGMENTS

We thank J. Richardson and I. Hands-Portman for technical support. We aregrateful to J. Harada for donating 35S:LEC2-GR seeds, J. Rogers and T. Okita fordonating the anti-TIP1;1 and anti-TIP3;1 antibodies, I. Hara-Nishimura for do-nating the anti-12S antibody, A. Scarafoni for donating the anti-2S antibody, L.Jiang for the 35S:2S1-GFP construct, A. Vitale for the gift of the 35S:TPK1-GFPseeds and the anti-complex glycan antibody, and K. Schumacher for donating35S:VHA-a3-RFP seeds.

Received January 8, 2018; accepted March 9, 2018; published March 19, 2018.

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