organization of xylan production in the golgi during secondary … · organization of xylan...

Organization of Xylan Production in the Golgi DuringSecondary Cell Wall Biosynthesis1[OPEN]

Miranda J. Meents,a,b Sanya Motani,a Shawn D. Mansfield,b and A. Lacey Samuelsa,2,3

aDepartment of Botany, University of British Columbia, Vancouver V6T 1Z4 British ColumbiabDepartment of Wood Science, University of British Columbia, Vancouver V6T 1Z4 British Columbia

ORCID IDs: 0000-0003-3706-1766 (M.J.M.); 0000-0002-0175-554X (S.D.M.); 0000-0002-0606-8933 (A.L.S.).

Secondary cell wall (SCW) production during xylem development requires massive up-regulation of hemicellulose (e.g.glucuronoxylan) biosynthesis in the Golgi. Although mutant studies have revealed much of the xylan biosynthetic machinery,the precise arrangement of these proteins and their products in the Golgi apparatus is largely unknown. We used a fluorescentlytagged xylan backbone biosynthetic protein (IRREGULAR XYLEM9; IRX9) as a marker of xylan production in the Golgi ofdeveloping protoxylem tracheary elements in Arabidopsis (Arabidopsis thaliana). Both live-cell confocal and transmission electronmicroscopy (TEM) revealed SCW deposition is accompanied by a significant proliferation of Golgi stacks. Furthermore, althoughGolgi stacks were randomly distributed, the organization of the cytoplasm ensured their close proximity to developing SCWs.Quantitative immuno-TEM revealed IRX9 is present in a specific subdomain of the Golgi stack and was most abundant in the ringof the inner margins of medial cisternae where fenestrations are abundant. Conversely, the xylan product accumulated in swollentrans cisternal margins and the Trans-Golgi network (TGN). The irx9 mutant lacked this expansion for both the cisternal marginsand the TGN, whereas Golgi stack proliferation was unaffected. Golgi in irx9 also displayed dramatic changes in theirstructure, with increases in cisternal fenestration and tubulation. Our data support a new model where xylan biosynthesisand packaging into secretory vesicles are localized in distinct structural and functional domains of the Golgi. Rather thanpolysaccharide biosynthesis occurring in the center of the cisternae, IRX9 and the xylan product are arranged in successiveconcentric rings in Golgi cisternae.

The successful colonization of land by plants hasbeen ascribed in part to the development of secondarycell walls (SCWs), which reinforce water-conductingvessel elements and supportive fibers. Branched poly-saccharides, termed hemicelluloses, are essential chem-ical components of SCWs that can account for asmuch as30% to 50% of thewall byweight (Scheller andUlvskov,2010). In eudicots, the major SCW hemicellulose is axylan characterized by a Xyl backbone decorated withside chains of GlcA andmethyl and acetyl substitutions(Ebringerová, 2005). Xylan is synthesized in the Golgi

by a suite of glycosyltransferases working in concertwith other transferases as well as substrate biosyn-thetic proteins and transporters. Among these proteins,IRREGULAR XYLEM 9 (IRX9) is a type-II transmem-brane protein proposed to form part of a xylan back-bone biosynthetic complex (Rennie and Scheller, 2014).Loss of IRX9 function results in reduced growth, col-lapsed xylem vessels, and reduced xylan content andxylosyltransferase activity, collectively supporting arole for IRX9 in SCW xylan biosynthesis (Lee et al.,2007; Lee et al., 2010; Wu et al., 2010). The proposedxylan backbone biosynthetic complex includes not onlyIRX9, but also IRX9-LIKE (IRX9L; Lee et al., 2010; Wuet al., 2010), as well as IRX10/IRX10L (Brown et al.,2009; Wu et al., 2009), and IRX14/IRX14L (Kepplerand Showalter, 2010). The xylan backbone precursor,UDP-Xyl, must be transported into the Golgi by UDP-Xyl Transporters (UXTs), primarily UXT1 (Ebert et al.,2015). In addition, the xylan backbone is decoratedwith GlcA by xylan glucuronosyltransferases of theGUX family (Mortimer et al., 2010; Rennie et al., 2012),whereas methylation of the GlcA residues is catalyzedby glucuronoxylan methyltransferases (Lee et al., 2012;Urbanowicz et al., 2012). Xylan is acetylated primarilyby the protein ESK1TBL29 (Xiong et al., 2013; Yuan et al.,2013; Urbanowicz et al., 2014), although other TrichomeBirefringence-Like (TBL) proteins also acetylate xylanat different positions and with various affinities (Yuanet al., 2016a, 2016b; Zhong et al., 2017a). Despite theidentification of each of these proteins, little is known

1This work was supported by the Gouvernement of Canada j Nat-ural Sciences and Engineering Research Council of Canada (Conseilde recherches en sciences naturelles et en génie du Canada) (discov-ery grants to S.D.M. and A.L.S.); Canada postgraduate scholarships(to M.J.M.); and the Faculty of Graduate Studies, University of BritishColumbia (four-year fellowship to M.J.M.). The authors declare noconflicts of interest.

2Author for contact: [email protected] author.The 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: A. Lacey Samuels ([email protected]).

M.J.M. and A.L.S. wrote the article with contributions from all theauthors; M.J.M. performed most of the experiments and analyzed thedata; S.M. provided technical assistance to M.J.M.; A.L.S. and S.D.M.supervised the experiments; A.L.S. conceived the original research.

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about how they are organized in the Golgi and howthey work together to synthesize the xylan that is ulti-mately deposited in the SCW.

The Golgi apparatus is a central component of theendomembrane system involved in protein processingand trafficking in addition to synthesis of cell wallpolysaccharides like xylan. During primary cell wall(PCW) production, increased demand for Golgi pro-ducts is met, in part, by a proliferation in the number ofGolgi stacks making up the Golgi apparatus (Garcia-Herdugo et al., 1988; Seguí-Simarro and Staehelin,2006; Young et al., 2008; Toyooka et al., 2014). Whetherthe onset of xylan synthesis during SCW developmentresults in similar increases in Golgi number is unknown.Furthermore, during SCW synthesis, secretion of xylanfrom the Golgi must be targeted to specific plasmamembrane domains to produce the patterned SCWscharacteristic of cells like protoxylem tracheary elements(Chou et al., 2018). This targeting may be facilitated by aclose association between Golgi and the forming SCWs,as suggested by live-cell imaging of Golgi containingfluorescently tagged SCW CELLULOSE SYNTHASES(CESAs; Schneider et al., 2017). However, it is unclearif the proximity of Golgi to SCWs observed in these ex-periments is due to preferential targeting or randomchance.

Each individual Golgi body is composed of a stack offlattened membrane-bound cisternae arranged in apolar fashion from cis to trans. Proteins enter the Golgiat the cis-most cisterna via the endoplasmic reticulum(ER; Brandizzi and Barlowe, 2013), and Golgi productstypically exit at the trans cisterna and associated TransGolgi Network (TGN; Kang et al., 2011; Uemura, 2016;Wang et al., 2017). Golgi-resident proteins that carryout polysaccharide synthesis and modification aremaintained in Golgi cisternae, whereas their productstransit through the Golgi stack. The cisternal matura-tion model posits that cisternae change over time fromcis to medial and then trans cisternae (reviewed byGlick and Luini, 2011). This model is supported by ex-periments in yeast (Saccharomyces cerevisiae) showingthe population of resident proteins in a cisterna changesas cis-type residents are replaced by more trans-typeresidents via retrograde trafficking from later to ear-lier cisternae (Losev et al., 2006; Matsuura-Tokita et al.,2006). In mammalian cells, COAT PROTEIN I (COPI)-coated vesicles are important for this intra-Golgi ret-rograde transport of Golgi resident proteins, and COPItubules can mediate transport in both the anterogradeand retrograde directions (Yang et al., 2011; Park et al.,2015). Although COPI-coated tubules have not yet beenreported in plant Golgi, intra-Golgi COPI vesicles havebeen identified (Donohoe et al., 2007), and they havebeen hypothesized to play a role in glycosyltransferaserecycling (Donohoe et al., 2007; Kang et al., 2011). Thisis supported by studies showing that disruption ofCOPI vesicle machinery using the inhibitor brefeldin Aor mutations leads to mislocalization of a heterolo-gously expressed Golgi resident protein (Nebenführet al., 2002; Naramoto et al., 2014). In the context of

xylan biosynthesis, cisternal maturation implies xylanmoves through the Golgi with the maturation of thecisternae, whereas the xylan biosynthetic enzymes areactively recycled to previous cisternae to maintain theirposition in the Golgi. Because all reports of xylan bio-synthetic proteins demonstrate strict Golgi localization,these proteins must also be sequestered from the xylanproduct no later than the trans most cisternae, wherethis final flattened cisternamatures into TGN/secretoryvesicle clusters (Kang et al., 2011; Toyooka et al., 2014),although the mechanism by which this occurs has notbeen resolved. In addition to the cis-trans polarity ofGolgi stacks, each cisterna also varies in structure andcomposition between the flat center of the cisternae andtheir swollen outer margins with associated vesiclesand fenestrations (Mogelsvang et al., 2003; Mogelsvanget al., 2004; Koga and Ushiki, 2006; Kang and Staehelin,2008; Kang et al., 2011; Donohoe et al., 2013). Freeze-fracture images through the center of Golgi cisternae inroot cells reveals arrays of integral membrane proteinsproposed to be glycosyltransferases producing cell wallpolysaccharides (Staehelin et al., 1990). However, thehypothesis that the flattened center of the Golgi is thesite of cell wall biosynthesis has not been directly tested.The goal of this study was to examine both the cis-to-trans and the center-to-margins distribution of a xylanbiosynthetic protein (IRX9) at its native expression levelduring SCW synthesis.

Direct localization of Golgi-resident proteins likeglycosyltransferases is difficult because their abun-dance is often too low to detect (Fukuda et al., 1996).The few studies localizing glycosyltransferases withinthe Golgi used overexpression of genes in heterologoussystems (Chevalier et al., 2010), which may not reflectthe localization found with native expression levels.The production of large amounts of glucuronoxylanduring SCW biosynthesis therefore provides an ex-cellent opportunity to study the cell wall productionmachinery in the Golgi. SCW biosynthesis can be ex-perimentally triggered in an Arabidopsis (Arabidopsisthaliana) model system where an inducible version ofthe master transcription factor VASCULAR RELATEDNAC-DOMAIN7 (VND7) initiates transdifferentiationof cells throughout the plant into protoxylem trachearyelements with thick helical SCWs (Kubo et al., 2005;Yamaguchi et al., 2010a). Furthermore, during SCWsynthesis, IRX9 gene expression naturally increasesover 4-fold, and production of large amounts of glu-curonoxylan in the Golgi is triggered (Yamaguchi et al.,2010b; Yamaguchi et al., 2010a).

To locate IRX9-containing Golgi, we generated afunctional, fluorescently tagged version of IRX9 drivenby its native promoter, and introduced this into theVND7-induction system. We then used a combinationof confocal and transmission electron microscopy(TEM) to show that the number of Golgi stacks in-creases significantly with the onset of SCW production.Within individual Golgi stacks, IRX9 and its polysac-charide product were localized to concentric circleswithin medial and trans Golgi cisternae, respectively.

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The greatest amount of IRX9 was in the fenestratedregion of the cisternae, whereas the xylan productcorrelated with swollen cisternal edges and the TGN.The localization of a required SCW biosynthetic pro-tein, and its product, in successive concentric rings inthe Golgi cisternae generates new hypotheses about themechanisms driving Golgi protein recycling and xylanproduct packaging in the maturing, dynamic, andstructurally complex Golgi cisternae.

RESULTS

Live-Cell Imaging of Functional Fluorescently TaggedIRX9 Driven by the Native Promoter

To test if a fluorescently tagged IRX9 is still func-tional, a C-terminal fusion of IRX9-GFP was introducedinto the irx9-2 T-DNA insertional mutant line (Lee et al.,2010; Wu et al., 2010). The dwarf growth phenotype ofthe mutant was restored to wild-type levels when irx9was transformed with proIRX9:IRX9-GFP (SupplementalFig. S1A). Wild-type vascular bundles have large, opentracheary elements in toluidine blue-stained stem cross-sections (Supplemental Fig. S1B), which are frequentlycollapsed in irx9 (Supplemental Fig. S1C). Restoration ofopen vessel elements was apparent in the complementedlines (Supplemental Fig. S1D). Complementation of themutant phenotypes indicates the GFP-tagged IRX9 wasfunctional.In live-cell imaging of seedling roots from plants

containing proIRX9:IRX9-GFP, GFP signal was detectedin the paired cell files of the developing root protoxylemtracheary elements (Supplemental Fig. S1E). Althoughthe native promoter drove expression in the appropri-ate cell type, imaging the tagged proteins in these cellsdemonstrated the typical low resolution associatedwith imaging native tracheary elements deep in an or-gan. Furthermore, the rapid programmed cell death ofprotoxylem tracheary elements meant only a few cellsper root could be imaged. To overcome these inherentlimitations, the VND7-VP16-GR experimental systemwas adopted.Plants carrying the proIRX9:IRX9-GFP in both the

wild-type and irx9-2 backgrounds were transformedwith a proUBQ10-driven version of VND7-VP16-GR.Upon induction of SCW synthesis in this complementedline, IRX9-GFP localized to numerous punctae whichstreamed through the cytoplasm, consistent with Golgilocalization (Supplemental Fig. S1F). Similar resultswere obtained when proIRX9:IRX9-GFPwas introducedinto a 35S:VND7-VP16-GR line in a wild-type back-ground in which a very large number of cells undergotransdifferentiation. To control for the effect of IRX9copy number, the subcellular localization of IRX9-GFPwas compared with the irx9-complemented line irx9/proIRX9:IRX9-GFP/proUBQ10:VND7-VP16-GR, and noobvious differences in IRX9-GFP localization wereapparent (Supplemental Fig. S1G). This suggests thepresence of wild-type IRX9 did not alter the localization

of IRX9-GFP. Introduction of a functional IRX9-GFPinto a systemwith inducible transdifferentiation of cellsinto protoxylem tracheary elements permitted investi-gation of IRX9-GFP in the Golgi apparatus as a wholeand within each Golgi stack.

The Number of Golgi Stacks Increases with Onset ofSCW Deposition

To test the hypothesis that Golgi stack proliferationcoincides with the surge of glucuronoxylan biosynthesisduring SCW production, it was necessary to compareGolgi numbers at well-defined xylem developmentalstages. As timing of transdifferentiation between cellsand seedlings can be variable, the timing of xylemdifferentiation in each cell was characterized usingbrightfield and confocal microscopy of hypocotylsfrom 4- to 7-d-old seedlings, 17 to 30 h after inductionof VND7. The presence of IRX9-GFP signal and theprominence of SCW bands in brightfield and afterpropidium iodide stainingwere used to define the stageof SCW deposition. Cells in the early-SCW stage (Fig. 1,A–E) had no visible SCW in brightfield (Fig. 1A), al-though faint SCW bands were sometimes seen withpropidium iodide staining (Fig. 1, B and C), and IRX9-GFP expression was detectable (Fig. 1, D and E). Themid-SCW stage was characterized by the first appear-ance of SCW in brightfield, increasing propidium io-dide stain (Fig. 1, F–H), and strong IRX9-GFP signal inpunctae (Fig. 1, I and J). By the late-SCW stage the SCWwas conspicuous in brightfield, stained intensely withpropidium iodide, and IRX9-GFP signal in punctae wasprominent (Fig. 1, K–O). According to these definitions,the early-SCW stage was most abundant 17–20 h fol-lowing VND7 induction, whereas the mid-SCW stagewas more prevalent at 20–26 h, and the late-SCW stageat 26–30 h (Fig. 1P). This characterization of early-, mid-, and late-SCW stages during transdifferentiation ofprotoxylem tracheary elements identified 17–30 h as theperiod of maximum SCWdeposition, and this was thenused to guide subsequent experiments.Within each Golgi stack, the IRX9-GFP signal dis-

played a ring-shaped localization (Fig. 1Q). To verifythe ring-shaped fluorescent bodies observed in confocalmicroscopy of IRX9-GFP represent Golgi bodies, a well-characterized Golgi marker, proUBQ10:MANI-mCherry(Nebenführ et al., 1999) was introduced, and colocaliza-tionwith IRX9-GFPwas assessed. In every cell expressingIRX9-GFP and MANI-mCherry, both markers were pre-sent in all structures (Fig. 1, Q–S). When the Mander’scolocalization coefficient (Manders et al., 1993) wasaveraged for 26 cells from 5 seedlings, 73.3% of IRX9-GFP signal overlapped with MANI-mCherry signal,confirming the Golgi localization of IRX9-GFP. Inter-estingly, the MANI-mCherry signal was distributedthroughout the center of Golgi stacks (Fig. 1R) whereIRX9-GFP signal was absent (Fig. 1S) resulting in only39.6% of MANI-mCherry signal overlapping withIRX9-GFP, and a Pearson correlation coefficient of 0.322.

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Despite thesedifferences in distributionofMANI-mCherryand IRX9-GFP within individual Golgi bodies, they werealways found together, suggesting all stacks in the Golgiapparatus were participating in xylan production.

Confocal imaging of Golgi bodies does not providethe context of the surrounding unlabeled cytoplasm;therefore, the same stages of SCW deposition were

examined in developing tracheary elements using TEM(Fig. 2, A–C). The time following VND7 induction wasselected based on the predominant stage of SCW de-velopment according to the live cell imaging (Fig. 1P) toenrich for cells at the early-SCW (17–20 h) or late-SCW(22–30 h) stages. Seedlings were high-pressure-frozen/freeze-substituted and embedded in Spurr’s resin for

Figure 1. IRX9-GFP localizes to theGolgi throughout SCW deposition. A toO, IRX9-GFP and SCW localization atthe early-SCW, mid-SCW, and late-SCW stages. SCW bands are visible inbrightfield (BF) in mid-SCW and late-SCW stages, and are sometimes alsoapparent when early-SCW cells arestained with propidium iodide (CellWall). IRX9-GFP is found in Golgi-likepunctae in all stages. Cell wall imageswere acquired using different sensitiv-ities. Images are representative singleoptical sections through the cell cortex.Arrowheads 5 SCW bands. Scalebar 5 10 mm. P, The stage of SCW de-velopment varies among populations oftransdifferentiating cells. The percent ofIRX9-GFP expressing cells assignedto the early, mid, or late stages of SCW-deposition at each time point followingtreatment with DEX. Included per in-duction time point were 60–204 cells,across 4 replicate experiments. Q to S,IRX9-GFP and MANI-mCherry coloc-alize in the Golgi apparatus. Spinningdisk confocal colocalization of thexylan biosynthetic IRX9-GFP (Q) andthe Golgi-marker MANI-mCherry (R).The co-occurrence of the proteins inevery Golgi stack is apparent in themerged image (S). Distinct distributionswithin theGolgi are visible as ring-shapedIRX9-GFP and solid MANI-mCherrysignals. Scale bar 5 5 mm.


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morphological characterization. Plants lacking theVND7-induction construct, and thus producing onlyPCWs, were imaged as a control. These PCW-producingcells were dominated by a large central vacuole (Fig. 2A).After SCW deposition was triggered, small SCW intru-sions (more electron-lucent than the PCW) were visibleat repeating intervals along the cell periphery at theearly-SCW stage (Fig. 2B). By the late-SCW stage, theSCWs protruded into the cell (Fig. 2C) and bundles ofmicrotubules were visible lining the plasma membraneat SCW-domains (Fig. 2C, arrowheads). To understandhowxylan production contributes to the cellular changesoccurringwith the onset of SCWsynthesis, irx9-2mutantlines transformed with proUBQ10:VND7-VP16-GRweresimilarly prepared for TEM imaging. This mutant pro-duces significantly less xylan than wild type (Brownet al., 2007; Lee et al., 2010; Wu et al., 2010). Trans-differentiating irx9-2 cells continued to produce pat-terned SCW thickenings of approximately the samewidth, but the SCW did not extend into the cell to thesame extent as thewild-type late-SCWcells (Fig. 2, C andD). Integrating this characterization of SCW depositionin TEM with the stages of development outlined usingconfocal microscopy provides a multiscale platform,which was used to further examine the Golgi duringSCW biosynthesis.Given the rapid deposition of SCWs over a 12-h pe-

riod (Fig. 1P), the abundance of Golgi stacks wasquantified to determine if this shift in demand for Golgiproducts correlated with an increase in the number ofGolgi stacks in the cell. The number of Golgi stacks wasquantified in a TEM survey of the cortical cytoplasmsurrounding the large central vacuole before SCW de-position (wild-type PCW), and after SCW depositionhad commenced (wild-type SCW; Fig. 2, A and C). Thenumber of mitochondria was also examined for com-parison purposes. The number of Golgi in cells pro-ducing SCW increased compared with cells producingonly PCW (Supplemental Fig. S2). However, cells pro-ducing SCWs had significantly thicker cortical cyto-plasm from tonoplast to plasmamembrane (Fig. 2E). Toisolate the change in organelle abundance from thisincrease in cortical cytoplasm, Golgi and mitochondrialabundance was calculated per cell perimeter (counts/micron), rather than per cytoplasmic area (counts/mi-cron squared; Supplemental Fig. S2). Golgi counts were

Figure 2. Golgi abundance increaseswith SCWdeposition in wild-typeand irx9 cells. A to D, TEM images of cell cortex in wild-type cellsbefore deposition (A;wild-type [WT] PCW), early in SCWdeposition (B;wild-type Early-SCW), late in SCW deposition (C; wild-type Late-SCW),and in irx9 during SCW deposition (D; irx9 SCW). Representative

images are from 2 to 3 replicate high-pressure freezing experiments.Mito, mitochondrion; arrowheads5microtubules lining SCW domain.Scale bar5 200 nm. E to G,Quantification of cellular features observedin TEM revealed increased thickness of cytoplasm (E), and Golgi (F) andmitochondrial abundance per cell perimeter (G) in cross-sections dur-ing SCW synthesis. Golgi abundance has been normalized by averagecisternal diameter in each type of cell. Means 6 95% CI. Differentletters (a to c) in (E to G) indicate statistically significant differences.Statistics5 one-way ANOVAs with Tukey HSD post hoc tests (P, 0.05);wild-type PCW and wild-type late-SCW (n 5 31 cells, 4 to 5 seedlings,4 high-pressure freezing experiments); irx9 SCW (n 5 17 cells, 7 seed-lings, 2 high-pressure freezing experiments).



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also adjusted for differences in Golgi width to correctfor overestimation of Golgi abundance in SCW-producingcells due to more frequent detection of larger Golgi inthin sections (Supplemental Fig. S2). With use of thismethod, the number of Golgi per cell perimeter wasfound to increase significantly in wild-type SCW cells(Fig. 2F). Interestingly, the number of mitochondria percell perimeter also increased significantly with SCWproduction (Fig. 2G). To test if xylan production con-tributes to the increased Golgi abundance during SCWdeposition, these analyses were repeated in the irx9mutant background. Golgi and mitochondrial abun-dances in irx9 SCWcells were not different than those inwild-type SCW cells (Fig. 2, F and G), suggesting Golgiand mitochondrial proliferation during SCW synthesisis independent of xylan biosynthesis. The increase inGolgi abundance was also quantified using confo-cal imaging of the Golgi marker MANI-mCherry andIRX9-GFP during PCW and SCW production, respec-tively (Fig. 3, A and B). Dividing cells into early-, mid-,and late-SCW stages revealed a significant, consistent,and gradual increase in Golgi abundance in the corticalcytoplasm over the course of SCW deposition (Fig. 3C).This significant increase in Golgi density with the onsetof SCW synthesis was also observed in plants in the irx9mutant background with IRX9-GFP complementation(Supplemental Fig. S3). Together, the TEM and confocalquantification of the Golgi indicate the absolute numberof Golgi stacks increased during SCW production, andthis was not influenced by loss of IRX9-dependentproduction of xylan in the Golgi.

Cytoplasmic Geometry Constrains Randomly PositionedGolgi Stacks to Regions near SCW Domains

In live-cell imaging of Golgi labeled with a YFP-tagged SCW CESA7, Golgi have been observed topause at SCW-domains (Watanabe et al., 2015), and

85% of Golgi were found ‘underneath’ SCW bands(Schneider et al., 2017). These results suggest Golgi maypreferentially associate with SCWs, perhaps to facili-tate targeting of secretion to these domains. Confocalimaging of IRX9-GFP in optical sections through thecell cortex (Fig. 4A) or through the cell center (Fig. 4B)illustrated how the large central vacuole restricts theGolgi to the cell periphery. Cross-sections through thecell using TEM (Fig. 4C) revealed a very thin layer ofcortical cytoplasm that accommodated mitochondria,chloroplasts, ER bodies, the nucleus, and other organ-elles, in addition to Golgi. To test the hypothesis thatGolgi preferentially associate with SCWs, the distancebetween Golgi stacks and the nearest SCW was mea-sured (Fig. 4C). These values were then compared withthe predicted results of a random distribution of Golgiusing random points in the available cytoplasm. Thedistance between mitochondria and the SCW was alsoanalyzed for comparison, as mitochondria are a similarsize to Golgi in these cells. The majority of Golgi andrandom points were found within 1 mm of a SCW(Fig. 4D). A similar percentage of Golgi (38.6%) andrandom points (41.7%) were found within 500 nm of aSCW, whereas a larger proportion of mitochondria(68.4%) were found within this distance (Fig. 4D). Asdistance from the SCW increased, the abundance of allthree dropped exponentially. The average distance ofGolgi stacks to the SCW was not significantly differentfrom that of random points in the cytoplasm (Fig. 4E).This is in contrast with the distribution ofmitochondria,which are found significantly nearer to SCWs than bothrandom points and Golgi stacks (Fig. 4E). This confirmsthis analysis is capable of detecting whether organellesare significantly closer to SCWs than random. As such,the random distribution of Golgi during SCW synthesisindicates most of the free cytoplasmic space is nearSCWs in these cells; therefore, Golgi are naturally moreabundant in these regions. Thus, although Golgi arefound in close proximity to SCWs, they do not appear to

Figure 3. IRX9-GFP-labeledGolgi increase in densityas SCW deposition progresses. A and B, Golgi in thecell cortex in a PCW-producing cell with the Golgimarker proUBQ10:MANI-mCherry (A) or a late-SCWcell expressing proIRX9:IRX9-GFP (B). Scale bar 5 5mm. C, Quantification of Golgi density in the cellcortex before SCW deposition (PCW) and at early,mid, and late stages of SCW deposition. Means 695% CI. Different letters (a to d) indicate statisticallysignificant differences. Statistics 5 one-way ANOVAand Tukey HSD post hoc test (P , 0.05). For eachstage n 5 223–342 cells from 31 to 78 plants in 4–6replicate confocal experiments.


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preferentially associate with the plasma membranelining domains of SCW secretion, unlike mitochondria.

Golgi Become Wider, and Cisternal Margins Expand,during SCW Synthesis

The onset of SCW synthesis during differentiation ofprotoxylem tracheary elements requires a shift in Golgifunction, from PCW production to SCW production(Meents et al., 2018; Watanabe et al., 2018). With theintense demands on the Golgi during rapid SCW syn-thesis, we hypothesized the diameter of Golgi and thesize of cisternal margins and TGN vesicles would in-crease. Golgi ultrastructure was characterized in TEMimages of cryo-fixed wild type (wild-type PCW) andwild type/35S:VND7-VP16-GR (wild-type early-SCWand wild-type late-SCW) seedlings. To assess thespecific impact of xylan biosynthesis on Golgi struc-ture, Golgi were also examined in xylan-deficient irx9mutant plants carrying the VND7 construct (irx9/proUBQ10:VND7-VP16-GR; irx9 SCW). VND7 activitywas induced for 22–28 h, except for the wild-typeearly-SCW plants, which were exposed for 17–20 h(Fig. 1P).With extensive sampling, the different planesof section of Golgi were identified (Supplemental Fig. S4),and cross-sections of Golgi cisternae were used for mea-surements of Golgi size and structure (SupplementalFig. S4, A–C). Clear qualitative differences were observed

in the structure of Golgi stacks (Fig. 5, A–H) and TGN(Fig. 5, I–L) during SCW biosynthesis. Golgi featureswere quantified to provide a more nuanced character-ization of the observed structural changes within indi-vidual Golgi stacks and across the different genotypes(Fig. 5M). Cisternae were categorized as cis, cis11, mid,trans-1, and trans, based on their position in the stackand their physical features (as described in the “Mate-rials and Methods”). Most Golgi contained 5 or 6 cis-ternae, and the number of cisternae did not changesignificantly with the onset of SCW synthesis or in theirx9 mutant (Supplemental Fig. S5B).Golgi cisterna have a flattened central region with

margins (i.e. the rounded profiles at the edge of eachcisterna) that increase in size from cis to trans. In othercell types, increases in cisternal margins have been at-tributed to accumulation of polysaccharide cargo, cul-minating in the production of secretory vesicles at theTGN (Zhang and Staehelin, 1992; Young et al., 2008;Wang et al., 2017). Analysis of margin size in this studyfound a similar trend with a significant increase in thesize of more trans cisternal margins, regardless of stageof SCW deposition or genotype (Supplemental Fig. S5).Furthermore, the onset of SCWproduction resulted in asignificant increase in margin size compared with wild-type PCW cells, with an ;18% increase in the marginsof cis-cisternae, up to a dramatic ;65% increase oftrans cisterna margins (Fig. 5N). The size of irx9 SCWGolgi margins was significantly smaller than Golgi in

Figure 4. Cytoplasmic geometry constrains randomlydistributed Golgi stacks to regions near SCWs. A andB, Optical sections through the cell cortex (A) andmore endoplasmic regions (B) in cells expressingIRX9-GFP (green) and with SCWs stained with pro-pidium iodide (magenta). The dark center of the cell in(B) is dominated by the vacuole, which is unlabeled.White boxes give an approximation of the size of thefield of view in (C). Scale bars5 5 mm. C, TEM cross-section of cortical cytoplasm showing the position ofGolgi stacks (green), mitochondria (Mito), and a ran-dom point (orange) relative to the SCW (magenta).Scale bar 5 500 nm. D, Histogram showing the dis-tance to the SCW in TEM images of random points,Golgi, and mitochondria using 500 nm bins. E, Av-erage distance to SCW from random points, Golgi, ormitochondria. Means6 95% CI. Different letters (a tob) indicate statistically significant differences. Statis-tics 5 one-way ANOVA with Tukey HSD post hoc(P , 0.05) for square-root transformed distances;n 5 168 (Random, Golgi) and 114 (Mitochondria)from 16 cells, 5 seedlings, and 3 high-pressurefreezing experiments.



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Figure 5. Cisternal structure changes with the onset of xylan biosynthesis and SCW deposition. A to D, Golgi cross-sections forwild-type (WT) PCW (A), wild-type Early-SCW (B), wild-type Late-SCW (C), and irx9 SCW (D) showing gaps in cisternae (ar-rowheads) and examples of size measurements of cisternal margins (brackets). Scale bars 5 200 nm. E to H, Sections throughtilted ‘Top View’ Golgi showing fenestrations (arrowheads) in the cisternae from wild-type PCW (E), wild-type Early-SCW (F),wild-type Late-SCW (G), and irx9 SCW cells (H). Scale bars5 200 nm. I to L, TGN from wild-type PCW (I), wild-type Early-SCW


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wild-type SCW-producing cells (Fig. 5N), consistentwith the hypothesis that cell wall polysaccharide ac-cumulation drives the expansion of Golgi margins.The TGN is composed of interconnected clusters of

tubules, secretory vesicle clusters, and clathrin-coatedvesicle buds (Young et al., 2008; Toyooka et al., 2009;Kang et al., 2011; Boutté et al., 2013). As the size of thesebuds can vary depending on the type of vesicle and itscontents, the size of these swollen membrane regions,termed ‘TGN vesicles’, was quantified. For all sampletypes, the size of TGN vesicles was not significantlydifferent than the size of the trans cisterna margins,supporting a model where the transmost cisterna ma-tures into the TGN (Supplemental Fig. S5C). A largeincrease in the size of TGN vesicles accompanied theonset of SCW production, shifting from ;60 nm inwild-type PCWTGNup to;100 nm inwild-type early-SCW and late-SCW cells (Fig. 5N). This changewas alsoapparent in the distribution of vesicle sizes at each stage(Fig. 5O). Reduced xylan biosynthesis in irx9 SCW cellsresulted in an intermediate TGN vesicle size with amean of;75 nm (Fig. 5N). The irx9 SCW TGN also hada more hybrid structure, with tubular regions like wild-type PCW TGN and regions with large vesicle budsmore like the wild-type SCW TGN (Fig. 5, I–L). Thesedifferent structures are reflected in a bimodal distribu-tion of irx9 TGNvesicle sizes (Fig. 5O). These data showthat although the packaging of xylan in secretory ves-icles contributes to increased TGN vesicle size duringSCW production, other SCW cargo likely contributeas well.In each Golgi stack, the length of each cisterna from

edge to edge was measured in Golgi cross-sections(Fig. 5, A–D). The largest cisternae in both wild-typePCW and wild-type SCW Golgi were the mid- andtrans-1 cisternae, and there was a significant (10% to20%) decrease in diameter from the trans-1 to transcisterna (Supplemental Fig. S5D). There was a sig-nificant (2-fold) change in cisternal diameter from thesmallest to largest cisternae for all sample types(Supplemental Fig. S5D). Averaging the diameter ofcis11 to trans-1 cisternae provided an estimate of Golgisize that was used to compare Golgi width in the dif-ferent samples. SCW production was accompanied by

an;50% increase inGolgiwidth inwild-type early-SCWand late-SCW cells (Fig. 5P). This increase in cisternaldiameter could have been driven by the accumulation ofxylan, in a similar fashion as the increase in margin size.However, this appears not to be the case, as a similarincrease in Golgi width was observed in irx9 SCW cellsas in the wild-type SCW cells (Fig. 5P). These data indi-cate that the onset of SCWproduction is accompanied byan increase in the overall width of Golgi cisternae;however, this increase appears to be independent ofxylan accumulation.

Golgi in irx9 Mutants Have Abundant Tubulesand Fenestrae

Another key feature of many Golgi cisternae arefenestrations, which often appear as gaps in cisternaledges in Golgi cross-sections (Staehelin and Kang, 2008;Kang et al., 2011; Donohoe et al., 2013). Despite theirubiquity, the function of Golgi fenestrations has notbeen resolved. The most fenestrated cisternae in theGolgi stacks were the mid and trans-1 cisternae, re-gardless of developmental stage or genotype (Fig. 5Q).Surprisingly, top-down views of irx9 SCW Golgishowed highly tubulated, web-like cisternae with largeand numerous fenestrations (Fig. 5H). To quantify thisincrease in fenestrations, the number of fenestrations inGolgi cisterna was determined by counting the numberof gaps in cisternal cross-sections (Fig. 5Q). As pre-dicted, the irx9 SCW Golgi had a significant 3-fold in-crease in the number of fenestrations compared with allother samples (Fig. 5R).Fine-scale quantification of cisternal architecture

confirmed that altering Golgi function by triggeringSCW synthesis changes Golgi structure with increasedSCW production correlating with increased cisternallength, margin width, and TGN vesicle size. Con-versely, decreased xylan production in the irx9 mutantled to a reduction in the size of the cisternalmargins andTGN vesicles, indicating xylan accumulation contrib-utes substantially to these aspects of Golgi structurebut not to Golgi diameter. Surprisingly, the irx9 Golgicisternae were also highly tubulated and displayed a

Figure 5. (Continued.)(J), wild-type Late-SCW (K), and irx9 SCW cells (L) showing the formation of large vesicles (arrowheads), small tubules (arrows),and examples of vesicle bud size measurements (brackets). Scale bars 5 200 nm. M, Diagram illustrating Golgi ultrastructuralfeatures quantified. The diameter (red arrow), margin, and TGN vesicle size (brackets) and number of gaps (blue arrowheads)were quantified for every cisterna in each stack. N, The diameter of cisternal margins and TGN vesicle buds in wild-type PCW,wild-type Early-SCW,wild-type Late-SCW, and irx9 SCWGolgi,measured as indicated in (M).Means695%CI. Statistics5 separateKruskal-Wallis and post hoc analysis (P , 0.05); n 5 35–55 Golgi and 327–640 TGN vesicles in 5–7 seedlings from 2 to 3 high-pressure freezing experiments. O, Distribution of TGN vesicle-bud sizes; n5 327–640 TGN vesicle buds in 5–7 seedlings from 2 to3 high-pressure freezing experiments. P, Golgi diameter inwild-type PCW,wild-type Early-SCW,wild-type Late-SCW, and irx9 SCWcells. Golgi diameter was calculated by averaging the diameter of cis11 to trans-1 cisternae. Means6 95%CI. Statistics5 one-wayANOVA and Tukey HSD post hoc analysis (P , 0.05); n 5 40–55 Golgi and in 5–7 seedlings from 2 to 3 high-pressure freezingexperiments. Q and R, Number of cisternal gaps in each cisterna (Q) and averaged across all cisternae (R) in each stack in wild-typePCW, wild-type Early-SCW, wild-type Late-SCW, and irx9 SCW Golgi. Means 6 95% CI. Statistics 5 separate Kruskal-Wallis andpost hoc analysis (P, 0.05);n5 35–44Golgi in 5–7 seedlings from2 to 3 high-pressure freezing experiments.Different letters (a to c)in (N and P to R) indicate statistically significant differences.



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significant increase in the number of fenestrations. Thepresence of IRX9 thus also promotes the formation ofsmooth cisternal sheets at the expense of fenestrationsand tubules.

IRX9-GFP and Xylan Localize to Different Regions ofGolgi Stacks

In live-cell imaging, IRX9-GFP is found in a ringaround the center of the Golgi stack (Fig. 1Q), but cur-rent models based on freeze-fracture data propose thesetypes of Golgi-localized biosynthetic proteins reside inthe cisternal centers (Staehelin et al., 1990). To reconcilethis difference, and test the existing model, the distri-butions of the Golgi-resident protein IRX9 and its pro-duct, xylan, were quantified across different domains ofGolgi cisternae. Immuno-TEM localization of IRX9-GFP and xylan, via anti-GFP and anti-xylan (CCRCM138) antibodies, respectively, was conducted on high-pressure frozen/freeze-substituted proIRX9:IRX9-GFP/35S:VND7-VP16-GR sampleswhere VND7 activitywasinduced for 22–26 h.

To map the distribution of IRX9 in Golgi and TGN,seedlings containing proIRX9:IRX9-GFPwere preparedfor immuno-TEM using anti-GFP antibodies. The anti-GFP antibody consistently labeled the Golgi in IRX9-GFP SCW cells (Fig. 6A), with very little nonspecificlabel in wild-type SCW cells from plants lacking GFP(Supplemental Fig. S6, A and B).When anti-GFP bindingwas quantified in IRX9-GFP lines, 46% of Golgi or TGNcontained one ormore gold particles,whereas only 7%ofGolgi were labeled in the plants not containing GFP(Fig. 6B). The average number of gold particles perGolgi/TGN increased 92% from 0.07 gold particle perGolgi in wild-type cells lacking IRX9-GFP to 0.83 inIRX9-GFP cells. The gold label in IRX9-GFP Golgi wassignificantly different than that of background label,according to a Χ2 test (P , 0.001). When mapped todistinct regions of the Golgi, IRX9-GFP label was foundin all cisternae with peak label in the midcisternae(33.9%), tapering to 8.6% in cis cisternae, and 12.4% intrans cisternae (Fig. 6D). A very small proportion of labelwas found in the TGN (2.2%).

To map the product of IRX9 during SCW biosyn-thesis, xylan distribution was quantified in Golgi andTGN using anti-xylan antibodies. Five antibodies weretested, including LM10 (McCartney et al., 2005; Kimand Daniel, 2012), CCRC-M138, CCRC-M147, CCRC-M149, and CCRC-M153 (Pattathil et al., 2010). AlthoughLM10 labeled the SCW as previously reported(McCartney et al., 2005; Kim and Daniel, 2012), theGolgi label in high-pressure frozen and freezesubstituted samples was too sparse for quantification.Of the other antibodies tested, CCRC-M138 had thestrongest signal and was therefore chosen for xylanquantification. The CCRC-M138 antibody recognizesunsubstituted xylopentaose units (Peralta et al., 2017)and was used previously in immunofluorescence ex-periments to label xylan in Arabidopsis SCWs (Pattathil

et al., 2010). CCRC-M138 binding was detected with asecondary antibody conjugated to colloidal gold andimaged with TEM. CCRC-M138 signal was found inGolgi, TGN, and SCWs in all cells undergoing SCWdeposition (Fig. 6C; Supplemental Fig. S6, C–E). In to-tal, 57% of Golgi and 57% of TGNwere labeledwith oneor more gold particles, averaging 1.17 gold particles perGolgi and 1.36 gold particles per TGN. The secondaryantibody was specific to CCRC-M138, as no gold la-beling was seen in the Golgi, TGN, or SCW in the ab-sence of the primary antibody (Supplemental Fig. S6,F–H). Furthermore, gold labeling was never observedin cells not producing SCWs (Supplemental Fig. S6I),suggesting CCRC-M138 is specific to an epitope presentduring SCW deposition, which is consistent with xylanspecificity. Mapping of xylan revealed 60% of the labelin the TGN and 40% in the Golgi, with label firstappearing in small quantities in the medial cisternae(2.3%) and becoming much more abundant in transcisternae (21.9%; Fig. 6D).

Lateral mapping of IRX9-GFP and xylan across thewidth of the cisternae was conducted by measuring thedistance from each gold particle to the edge and centerof the cisterna. Both GFP and xylan label was presentsignificantly closer to the margin of cisternae than thecenters, although the xylan label was significantlycloser to the margin than GFP (104 nm vs 157 nm, re-spectively; Fig. 6E). To better visualize the distributionof label across Golgi cisternae, every cisterna was di-vided into ten equal regions, giving 5 blocks for eachhalf cisterna from the cisternal center to the margin(Fig. 6, F and G). The number of gold particles fallinginto each block was then tallied and the distributionvisualized using a heat map (Fig. 6, F and G). As sug-gested by the previous analysis, IRX9-GFP and xylanlabel only overlapped slightly. IRX9-GFP label wasmost abundant in cis11, mid, and trans-1 cisternaeadjacent to the cisternal centers (Fig. 6F), whereas xylanlabel was found predominantly in the outermost mar-gins of trans-1 and trans cisternae (Fig. 6G). Combiningthese labeling patterns with the quantified architec-ture of Golgi stacks from the TEM (Fig. 5) allows scalingof the heat maps to account for the changing diameterof Golgi cisternae in wild-type SCW-producing cells(Supplemental Fig. S5D). Both IRX9-GFP and xylanlabel are low in the center of cisternae, and each is foundin spatially distinct regions of the Golgi (Fig. 6, H and I).The IRX9-GFP label appeared to be abundant in regionsof the Golgi that are highly fenestrated (Fig. 6H, andrefer to Fig. 5Q). Indeed, the amount of IRX9-GFP labelin each cisterna was strongly correlated with the aver-age number of cisternal fenestrations via Pearson’sr [r(3)5 0.99, P, 0.01], whichwas not the case for xylanlabel [r(3) 5 0.04, P . 0.05; Fig. 6J]. Conversely, xylanlabel highly correlatedwith the size of cisternal margins[r(3)5 0.91, P, 0.05; Fig. 6I, and refer to SupplementalFig. S5C], whereas IRX9-GFP label did not [r(3) 5 0.03,P . 0.05; Fig. 6K]. These results clearly demonstrateIRX9-GFP and its xylan product localize to distinctsubdomains of the Golgi in both the cis-to-trans and


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Figure 6. IRX9-GFP and xylan are spatially segregated in different regions of golgi cisternae. A, Anti-GFP labeling of Golgi inSCW-producing cells in a plant containing IRX9-GFP. Black circles outline gold particles. Scale bar5 200 nm. B, Anti-GFP signalabove background label. Distribution of gold label among Golgi in SCW-producing negative controls that lack IRX9-GFP(No GFP) and in similar cells in plants containing IRX9-GFP is shown; No GFP (n 5 20 gold particles, 277 Golgi, 19 cells,4 seedlings, 2 high-pressure freezing experiments); IRX9-GFP (n 5 410 gold particles, 493 Golgi, 39 cells, 4 seedlings, 2 high-pressure freezing experiments). C, Anti-xylan (CCRC M138) labeling of a Golgi in a SCW-producing cell in a plant containingIRX9-GFP. Black circle outlines a gold particle. Scale bar 5 200 nm. D, Percent of gold particles in Golgi cisternae from ciscisternae through to the TGN for anti-GFP (IRX9-GFP) or CCRC-M138 anti-xylan (Xylan). E, Average distance from gold label tothe cisterna center andmargin. All gold particles were pooled, regardless of cisterna labeled.Means6 95%CI. Different letters (ato d) indicate statistically significant differences. Statistics5 one-way ANOVAwith TukeyHSD post hoc (P, 0.05). F andG, Heatmap of gold distribution across the Golgi from cis to trans and in 10% blocks of cisternal diameter from the center to the edge ofeach cisterna.Distribution of IRX9-GFP (F) and xylan (G). H and I, Distributions of IRX9-GFP (H) and xylan (I) adjusted for changesin cisternal width from cis to trans using the average cisternal diameters of early-SCWGolgi. Blocks represent 10%of the cisternaldiameter. J and K, Correlation of number of fenestrations (J) or cisternalmargin size (K) in each cisternawith amount of GFP (green)or xylan (orange) label in each cisterna; Xylan (n5 136 gold particles, 17 cells); IRX9-GFP (n5 182 gold particles, 33 cells). Dataare from 4 seedlings and 2 high-pressure freezing experiments.



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centers-to-margins dimensions. These distributionsalso correlate with different structural features of theGolgi with xylan abundance correlating with the size ofthe cisternal margins and IRX9-GFP labeling with thenumber of fenestrations.

DISCUSSION

A Concentric Circles Model Defines Functional Domainsof Polysaccharide Production in Golgi Stacks

During SCW biosynthesis, the xylan biosyntheticprotein IRX9 and its xylan product were detected inconcentric circles in the Golgi cisternae. The live-cellimaging of Golgi ring-like structures, as well as quan-titative immuno-TEM mapping of IRX9 and xylan towell-defined Golgi structures, support this model ofhemicellulose production in the Golgi (Fig. 7). Impor-tantly, this concentric circles model emphasizes theenrichment of biosynthetic proteins and products in

specific functional Golgi domains with concomitantstructural features. In this model we propose (1) xylanbackbone synthesis occurs in a ring in the inner-marginof fenestrated, medial cisternae where IRX9-GFP is lo-calized and not in cisternal centers; (2) xylan accumu-lates in the outer-margins of trans-1 and trans cisternae,which become increasingly swollen as the cisternaemature; (3) IRX9 maintains its appropriate localizationin medial cisternae while IRX9 is depleted from transcisternae, suggesting active retrograde recycling; and(4) xylan is packaged into large secretory vesicle budsat the TGN for export to SCW domains. This model isconsistent with the cisternal maturation model ofGolgi processing (Glick and Luini, 2011) and theproposed formation of xylan biosynthetic complexescontaining IRX9 (Zeng et al., 2010; Jiang et al., 2016;Zeng et al., 2016).

The presence of xylan biosynthetic proteins in theinner margin of medial cisternae is consistent withthe ring-shaped distribution of IRX9-GFP in confocallive-cell imaging and was demonstrated quantitatively

Figure 7. Organization of xylan biosynthetic machinery and products in concentric circles defines functional regions of Golgicisternae. A, Proposed steps of xylan synthesis in Golgi cisternae and TGN in comparison with a schematic of the relativeabundance of IRX9 and xylan in each compartment. Backbone synthesis is proposed to predominate in mid and trans-1 cisternaewhere IRX9 is most abundant. IRX9 must be removed from cisternae during maturation of trans-1 into trans cisternae as it is muchdepleted in the trans cisternae. IRX9 is predicted to recycle via retrograde trafficking to cis11 or mid cisternae, where IRX9 firstappears in substantial amounts. After backbone synthesis, xylan processing, including addition of GlcA side chains, methylation,acetylation and deacetylation, likely occurs during maturation of trans-1 cisternae through to TGN. Final packaging of xylan intosecretory vesicles occurs at the TGN before secretion to the SCW. B, Top-down view of simplified cisternae showing the con-centric circle organization of xylan synthesis with an accumulation of xylan in outer cisternal margins, abundant IRX9 in inner-margins, and the absence of both IRX9 and xylan in cisternal centers. The size of each cisternae and the TGN is adjusted to reflecttheir size in a wild-type early-SCW Golgi. C, Distribution of IRX9 (blue) and xylan (magenta) abundance from Figure 6, super-imposed on the outline of a representative wild-type early-SCWGolgi cross-section from a TEMmicrograph. IRX9 is abundant inmore central regions of fenestrated mid and trans-1 cisternae, whereas xylan accumulates in the swollen outer margins of transcisternae and the TGN. The collapse of the flattened cisternal structure in the TGN is proposed to accompany the packaging ofxylan into secretory vesicles.


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using high-resolution immuno-TEM mapping. Al-though the distribution of IRX9-GFP in quantitativeimmuno-TEM may be skewed somewhat by the pres-ence of wild-type IRX9 in these plants, the pattern ob-served is consistent with the ring-shaped localization ofIRX9-GFP in the mutant irx9 background from confocalimaging, providing confidence the IRX9-GFP distribu-tion seen in Figure 6 is indicative of the true localizationof IRX9 in the Golgi. Other Golgi-localized proteinshave been found in similar localization patterns inconfocal imaging, including IRX9L (Zhang et al., 2016)and several COPI-related proteins (Ritzenthaler et al.,2002). Experiments in a variety of eukaryotes suggestoligomerization may influence protein distributionacross Golgi cisternae from the center to the margins. Inplants, the ring-shaped distribution of the PCW cellu-lose synthase CESA3 in the Golgi was lost in the stellomutant background, where CESA3 shifted toward amore central cisternal localization, coinciding with adecrease in the formation of CESA complexes (Zhanget al., 2016). In mammalian cells, induced aggregationof engineered Golgi-resident proteins caused them toshift toward cisternal centers, whereas disaggregationreturned them to the rims (Rizzo et al., 2013). Addi-tionally, localization of these proteins in the cisternalrims was required to maintain their position in appro-priate cisternae, suggesting aggregated Golgi-residentswere excluded from retrograde trafficking and thuscarried downstream with cisternal maturation. Fur-thermore, aggregation of membrane-associated cargoproteins led to aggregate accumulation in more centralregions of the cisternae, whereas aggregations of solu-ble cargo were detected in Golgi edges (Lavieu et al.,2013). The formation of a large xylan biosyntheticcomplex may similarly shift localization of these pro-teins toward the center of cisternae while allowing thesoluble cargo to accumulate in the outermost margins.The xylan biosynthetic complex may then dissociate ascisternae mature, allowing individual proteins to movetransiently to the margins where they become avail-able for retrograde trafficking to earlier cisterna wherethe complex reforms and the next round of xylan bi-osynthesis begins.Mapping of IRX9 to a ring in the inner-margin of

medial cisternae suggests synthesis of the xylan back-bone is occurring in these domains. Production of thexylan backbone for Arabidopsis SCWs requires threesets of proteins: IRX9/IRX9L (Lee et al., 2010; Wu et al.,2010), IRX10/IRX10L (Brown et al., 2009; Wu et al.,2009), and IRX14/IRX14L (Keppler and Showalter,2010). The other proteins involved in backbone synthe-sismay be similarly localized, as a number of experimentssuggest xylan is synthesized by a biosynthetic com-plex. Coimmunoprecipitation and bimolecular fluores-cence complementation studies during PCW productionof arabinoxylans in wheat (Triticum aestivum) and aspar-agus (Asparagus officianalis) have demonstrated in plantaheterodimerization of xylan biosynthetic proteins orthol-ogous to the Arabidopsis proteins (Zeng et al., 2010; Jianget al., 2016; Zeng et al., 2016). Eachproteinwas also shown

to homodimerize in planta, further increasing the sizeof the proposed xylan biosynthetic complex to at leastsix members (Zeng et al., 2016). Proteins involved indecorating the xylan backbone may also be incorpo-rated into the protein complex, as a PCW xylan bio-synthetic complex isolated from wheat also showedarabinosyltransferase and glucuronosyltransferase ac-tivity necessary for side chain addition (Zeng et al.,2010). Furthermore, because the UDP-Xyl substratefor xylan backbone synthesis is synthesized in the cy-tosol (Kuang et al., 2016; Zhong et al., 2017b), the Golgi-localized UDP-Xyl transporters, especially UXT1, arehypothesized to be associated with the xylan biosyn-thetic complex in the Golgi to ensure adequate substrateavailability (Ebert et al., 2015).Some degree of sequential processing in different

Golgi cisternae is likely occurring during xylan bio-synthesis. Following backbone production and addi-tion of side chains, methylation and acetylation arelikely occurring in different downstream regions of theGolgi. Analysis of xylans in various xylan biosyntheticmutants suggests the methylation rate of GlcA residuesis independent of the rate of xylan synthesis, which isconsistent with a model where methylation occurs fol-lowing backbone synthesis (Zhong et al., 2005; Peñaet al., 2007; Kuang et al., 2016). Xylans are also acety-lated in the Golgi (Urbanowicz et al., 2014) and thendeacetylated before deposition in the wall (Zhang et al.,2017). The resulting pattern of acetylation was essentialfor xylan interaction with cellulose (Grantham et al.,2017). Xylan acetylation and deacetylation almost cer-tainly occur in different regions of the Golgi. With thelocalization of xylan backbone synthesis to the medialGolgi, we can now formulate testable hypotheses aboutthe relative location of other steps in xylan biosynthesis.For example, the conserved reducing-end oligosaccha-ride (REO) found in many SCW xylans (Peña et al.,2016) has been hypothesized to be either a primer forconsecutive xylan synthesis or a terminator of synthesistransferred en bloc to the completed xylan backbone(York and O’Neill, 2008). If the REO is a primer for se-quential xylan biosynthesis, then we would expect theenzymes producing REOs to appear in cis-cisterna.However, if the REO is a xylan terminator the biosyn-thetic enzymesmay appear inmore-trans cisternae. Themapping of IRX9 therefore allows the medial Golgi tobe used as a benchmark to guide mapping of the vari-ous steps of xylan biosynthesis.Immuno-TEM labeling showed xylan was abundant

in themargins of later cisternae and in the TGN, and theswelling of the cisternal margins and large TGN vesi-cles was strongly correlated with the amount of xylanlabel in each cisterna. The increased accumulation ofpolysaccharide cargo in the cisternal periphery andTGN vesicles is also supported by the reduction inmargin size in irx9 Golgi. The decrease in margin sizeand TGN in irx9 is analogous to a similar decreaseobserved during Arabidopsis seed coat developmentin a mutant with substantially reduced pectin bio-synthesis (Young et al., 2008). There is a population of



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intermediate-sized TGN vesicle buds in irx9 mutantsthat are likely important for trafficking other cargo,including cellulose synthases. The ixr9 plants still pro-duce patterned SCWs containing cellulose (Petersenet al., 2012); therefore, the CESAs must still be traf-ficked to, and subsequently from, the SCW domains.

The interpretation of the anti-xylan antibody label-ing in this study depends on our understanding of theepitope the antibody recognizes, as well as potentialmasking by other cell wall components such as theGlcA substitutions on the xylan backbone. Becausethe CCRC-M138 anti-xylan antibody used recognizesunsubstituted xylopentaose (Peralta et al., 2017), andthe xylan backbone in Arabidopsis is substituted withGlcA at intervals greater than five Xyl residues(Bromley et al., 2013), CCRC-M138 labeling is likelyminimally affected byGlcA substitutions. However, wecannot exclude the possibility that the xylan labeldetected in our study reflects a more mature, partiallydeacetylated xylan. This could be investigated by ex-amining anti-xylan labeling in plants defective in xylanacetylation, such as the eskimo1 (esk1) mutant (Yuanet al., 2013; Urbanowicz et al., 2014). Immuno-TEMmapping using other anti-xylan antibodies (Ruprechtet al., 2017) may also shed light on the distribution ofvarious populations of xylans in the Golgi.

Abundant Golgi Fenestration and Tubulation in theAbsence of IRX9

The regions of the Golgi in which IRX9-GFP labelingwas strongest also contained the largest number offenestrations. The presence of fenestrations is welldocumented in plant (Kang et al., 2011; Donohoe et al.,2013), mammalian (Mogelsvang et al., 2004; Koga andUshiki, 2006), and yeast Golgi (Mogelsvang et al., 2003).Despite conservation of this Golgi feature across eu-karyotes, their function is unknown. There was a sur-prising and dramatic increase in fenestrations in theirx9 mutant. The mechanism by which IRX9 inhibitsfenestration formation is unclear. Several functionshave been proposed for fenestrations, including actingas ‘spot welds’ to control cisternal swelling, increasingmembrane curvature to facilitate the formation of ves-icle buds, and providing pathways for movement ofcytoplasm through cisternae to aid diffusion and/orGolgi movement (Ladinsky et al., 1999). This first hy-pothesis seems unlikely, as fenestration abundance in-creased in the irx9 mutant, despite decreased swellingof cisternal margins. Alternately, fenestrations couldbe important for increasing the membrane area ofspecific regions of the Golgi to accommodate abun-dant membrane-associated Golgi resident proteins.Regions with fenestrations are predicted to have highsurface area:volume ratios, in contrast with theswollen cisternal margins that must contain largevolumes of secretory cargo.

One model of fenestration formation requires ho-motypic fusion of tubules protruding from cisternal

margins and looping around to reconnect with theoriginating cisterna (Weidman et al., 1993). This modelis consistent with the increased tubulation and fenes-tration seen in irx9 Golgi. In irx9, the Golgi stacks arelacking the large quantities of xylan, which normallyaccumulate in the margins, but they are also lacking theGolgi resident protein IRX9. Furthermore, in transientexpression of an IRX9 homolog inNicotiana benthamiana,IRX9 did not localize to the Golgi unless also expressedwith IRX10 and IRX14 (Zeng et al., 2016). The absence ofIRX9 from the cell in this study may therefore similarlyprevent Golgi localization of other xylan biosyntheticproteins. To what extent can we then attribute thechanges in Golgi structure in irx9 to loss of Golgi pol-ysaccharide cargo versus Golgi residents? Given thatirx9 Golgi are of a similar size to wild-type Golgi, itis apparent that the additional fenestrations in irx9occur closer to the center of the Golgi cisternae whereIRX9-GFP is usually observed and xylan is absent. Theincreased abundance of fenestrations in more centralregions of irx9 cisternae is therefore more likely to be aresult of the lack of IRX9, rather than xylan, which is notabundant in these regions. Further experiments will berequired to elucidate the mechanism by which IRX9may inhibit Golgi fenestration. One possibility is that alarge xylan biosynthetic complex in the Golgi lumenphysically prevents spontaneous fusion of the mem-brane on either side of the lumen.

Cytoplasmic Geometry Constrains Golgi duringSCW Production

Confocal imaging of Golgi in the cell cortex allowssome characterization of the relationship between theGolgi and the SCW but gives little information aboutthe organization of other cellular components. TEMimaging takes into account the volume and organiza-tion of the cytoplasm and other organelles, allowing usto conclude the close proximity of Golgi to SCWs inconfocal imaging (Watanabe et al., 2015; Schneideret al., 2017) is due to a concentration of free cytoplas-mic space in this area. In any plant cell, the organizationof Golgi stacks at any given time is determined by thecontribution of both moving and stationary Golgi. This‘stop-and-go’ behavior has been well-characterized inseveral cell types, including Nicotiana tabacum BY2 cellcultures (Nebenführ et al., 1999), Nicotiana clevelandiipavement cells (Boevink et al., 1998), and Arabidopsistrichomes (Lu et al., 2005). In native root tracheary ele-ments, Golgi were similarly said to ‘pause’ in their rapidcytoplasmic streaming at SCWdomains (Wightman andTurner, 2008). This was also demonstrated in the VND7-induction system, where Golgi pausing near SCWs las-ted anywhere from 15 s to 3 min (Watanabe et al., 2015).However, Golgi paused at SCW domains may representa small proportion of the population of Golgi stacks,which may explain why the larger population of Golgibodies remains randomly distributed in the narrowcortical cytoplasm.


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Multitasking of the Golgi Apparatus duringSCW Synthesis

Deposition of SCWs requires abundant production ofxylan in the plant Golgi apparatus, as well as targetedsecretion of Golgi products to the plasma membranelining SCW domains. Accomplishing these tasks re-quires the concerted action of the population of Golgistacks streaming rapidly through the cytoplasm. In thisstudy, we demonstrate the onset of SCW production isaccompanied by significant proliferation in the numberof Golgi stacks. Increase in the abundance of Golgistacks also occurs during the onset of mucilage pro-duction in Arabidopsis seed epidermal cells (Younget al., 2008) and during mitosis (Garcia-Herdugo et al.,1988; Seguí-Simarro and Staehelin, 2006; Toyooka et al.,2014). The prevailing model for Golgi biogenesis inplants proposes Golgi stacks increase in diameter beforecisternal fission, creating two new stacks with the samenumber of cisterna as the ‘parent’ Golgi (Ito et al., 2014).Indeed, electron microscopy in various cell types havecaptured Golgi at what could be a ‘mid-division’ stage(Hirose and Komamine, 1989; Langhans et al., 2007;Staehelin and Kang, 2008). Our data are consistent withthis model, as Golgi proliferation during SCW synthesiswas accompanied by a significant increase in Golgi di-ameter. Similar increases in Golgi size have been ob-served in the transition from root meristem to columellacells (Staehelin et al., 1990).The observation that all Golgi stacks in the cell con-

tained both IRX9-GFP and the Golgi marker MANI-mCherry demonstrates the entire population of Golgistacks in the cell are likely working in concert to pro-duce xylan for SCWs. The Golgi marker MANI is in-volved in the early stages of glycoprotein processing inthe Golgi (Schoberer and Strasser, 2011), implying eachindividual Golgi stack is carrying out both glycoproteinprocessing and xylan biosynthesis simultaneously. Thisis consistent with previous work showing Golgi stackscan produce both pectin and xyloglucan concurrently(Zhang and Staehelin, 1992; Young et al., 2008) andperform polysaccharide biosynthesis at the same timeas glycoprotein processing (Moore et al., 1991). Theconservation of Golgi stack multitasking across tissuetypes and species strongly suggests multitasking is acommon feature of plant Golgi.

MATERIALS AND METHODS

Plant Materials and Growth Conditions

All Arabidopsis (Arabidopsis thaliana) seedswere sterilized using chlorine gasin a sealed container containing 100mL of bleach and 3mL of concentrated HClfor 3–6 h Seeds were then plated on Germination Media (13 Murashige andSkoog 1% [w/v] Suc, 13Gamborg’s Vitaminmix, 0.05% [w/v]MES, 0.8% [w/v]agar at pH 5.8) and then transferred to growth conditions. Plants were grownunder long-day conditions (16-h light/8-h dark) at 21°C in a vertical position. Theirx9-2 (irx9) mutant line (AT2G37090, SALK_057033) was obtained from theArabidopsis Biological Resource Center (The Ohio State University, Columbus;ABRC). The 35S:VND7-VP16-GR seeds and plasmid were kindly donatedby Taku Demura (Yamaguchi et al., 2010a). To induce VND7-VP16-GR activity,4- to 7-d-old seedlings were treated with 10 mL of 10 mM dexamethasone

(DEX; Sigma) for 17–36 h, depending on the experiment. The ArabidopsisColumbia-0 ecotype was used to generate all transgenic lines usingAgrobacteriumtumefaciens (strain GV3101) and the floral dip method. Primary transformantswere selected by plating on Germination Media plates containing 50 mg/mLkanamycin, 25 mg/mL hygromycin, or 25 mg/mL Basta.

Generation of Constructs

Constructs were primarily generated using Gateway cloning (Invitrogen),including PCR amplification with attB sequences, Gateway Clonase-mediatedinsertion into a donor vector, and then transfer to the destination vector. PCRwas conducted with Phusion High Fidelity DNA polymerase (New EnglandBiolabs) followed by two-step adapter PCR to incorporate the full attB se-quences for Gateway cloning. The native promoter used for IRX9 (AT2G37090)consisted of the 2045 bp intergenic sequences upstream of the start codon asin published promoter-GUS experiments (Wu et al., 2010). ProIRX9:IRX9was amplified from genomic DNA, cloned into pDONRzeo, and then intopMDC107 (Curtis and Grossniklaus, 2003) to incorporate an in-frameC-terminal GFP and stop codon, thereby generating proIRX9:IRX9-GFP.Seeds and plasmids containing a 35S-driven version of soybean (Glycinemax) MANI-mCherry were available, but signal was largely absent due tosilencing. To address this, the UBIQUITIN10 (UBQ10; AT4G05320) pro-moter was used to generate proUBQ10:MANI-mCherry by amplifyingMANI-mCherry from the published 35S:MANI-mCherry construct (Nelsonet al., 2007), cloning this into pDONRzeo, and then pUB-DEST to add theproUBQ10 promoter (Grefen et al., 2010). Alternate versions of the published35S:VND7-VP16-GR (Kanamycin resistant; KanR) construct (Yamaguchi et al.,2010a) were generated to reduce ongoing silencing problems believed to be ex-acerbated by the 35S promoter and to provide an alternate plant selectable-marker. VND7-VP16-GR was amplified from the existing plasmid and clonedinto pDONRzeo and then into pUB-DEST to generate proUBQ10:VND7-VP16-GR(Basta resistant; BasR) or pB2GW7 to generate pro35S:VND7-VP16-GR (BasR).

Complementation Testing

Proper function of proIRX9:IRX9-GFP was tested by transforming the irx9mutant and looking for complementation of its dwarf and collapsed xylemphenotypes (Lee et al., 2010; Wu et al., 2010). Restoration of wild-type heightwas measured in 40-d-old plants and averaged for 5 plants in each of the wildtype, mutant, and complemented lines. Complementation of the irregularxylem phenotype was observed in hand sections from the bottom 1 cm ofmature wild type, mutant, and complemented inflorescence stems stained with0.01% toluidine blue (Ted Pella) for 5 min and imaged on a Leica DMR mi-croscope equipped with a QICAM digital camera (QIMAGING). Images wereprepared in Image J.

Native Expression of proIRX9:IRX9-GFP

Live-cell imaging of irx9/proIRX9:IRX9-GFP in native roots was conductedon a Perkin-Elmer UltraView VoX spinning disk confocal mounted on a LeicaDMI6000 inverted microscope with a Hamamatsu 9100-02 CCD camera.Samples were mounted in water, and expression in developing tracheary ele-ments in the rootwas observed using a glycerol 203 objective (aperture 0.7), 488nm excitation filter, and 525 emission filter (GFP).

Subcellular Localization in the VND7-Induction System

Subcellular localization of IRX9-GFPwas assessed in the complemented irx9and in wild-type backgrounds. The complemented irx9/proIRX9:IRX9-GFPwastransformed with proUBQ10:VND7-VP16-GR (BasR), whereas the wild-typeline was generated by transforming 35S:VND7-VP16-GR (KanR) with proIR-X9:IRX9-GFP. The wild-type line was used in subsequent experiments, exceptwhere indicated otherwise. The developmental time-course of proIRX9:IRX9-GFP expression and subcellular localization was determined by imaging plantsat 2-h increments from 17 to 30 h following induction with DEX. Cells werecategorized as ‘early’ if a secondary cell wall was not visible in brightfieldimaging but IRX9-GFP was expressed. ‘Mid’-development coincided with theappearance of faint SCWs in brightfield, and ‘Late’ development was definedby obvious SCWs. The differences in deposition of the SCWduring these stageswas validated by separate experiments where imaging of IRX9-GFP and SCWsin brightfield coincidedwith staining of the cell wall with 10mg/mL propidium



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iodide for 5 min. Cells were observed on the Perkin-Elmer spinning disk set-up,described above, using a 633 objective (aperture 1.2).

Colocalization of IRX9-GFP and MANI-mCherry

Colocalization of IRX9-GFP andMANI-mCherry was conducted in F1 crossesof proIRX9:IRX9-GFP/35S:VND7-VP16-GR with proUBQ10:MANI-mCherry.Plants were imaged on a Perkin-Elmer UltraView VoX spinning disk confocalmounted on a Leica DMI DMi8 and a Hamamatsu 9100-02 CCD camera and animmersion oil 1003 objective (aperture 1.47) and the following excitation andemission filters: GFP (488 nm, 525 nm), RFP (561 nm, 595 nm). Images of 26 cellsfrom five seedlings were analyzed for quantification of colocalization via Pear-son’s Correlation Coefficient (PCC) and Mander’s overlap coefficients (Manderset al., 1993) inVolocity image analysis software (Improvision). All confocal imageswere acquired using Volocity and prepared in Image J.

Golgi Stack Abundance using Confocal Microscopy

ToquantifyGolgi density throughout SCWdeposition, plant cells expressingGolgi-localized proteins were imaged on a Perkin-Elmer UltraView VoXspinning disk confocal mounted on a Leica DMI DMi8 and a HamamatsuVOXC9100-23B camera with a glycerol 633 objective (aperture 1.2) and thefollowing excitation and emission filters: GFP (488 nm, 525 nm), RFP (561 nm,595 nm). Because IRX9 is not expressed during PCW biosynthesis in Arabi-dopsis, MANI-mCherry lines were used to count Golgi numbers before SCWdeposition. Golgi density during PCW synthesis was assessed using theproUBQ10:MANI-mCherry line and compared with Golgi density in SCW-producing cells using the proIRX9:IRX9-GFP lines described above. All plantswere treated with DEX for 17–30 h before imaging, and at least three plantswere imaged per time point for each experiment. For each cell analyzed, a singleoptical section through the cell cortex was selected and the number of Golgi-likepuncta was divided by the cell area in the section to calculate the Golgi density.Because stage of development varies from cell to cell in any one plant, the ap-pearance of the SCW in brightfield imagingwas used to assign the stage of SCWdeposition, according to the definitions laid out in the previous time-courseexperiments. Approximately 10 cells per plant were analyzed. All imageswere processed using Volocity image analysis software (Improvision) andImage J. PCW data were collected from 319 cells from 31 seedlings across sixreplicate experiments. Golgi density in the IRX9-GFP line in the wild-typebackground was measured for 795 cells from 78 seedlings across four repli-cate experiments. Data from the complemented line was collected from 150 cellsacross ten seedlings in two replicate experiments.

TEM Imaging

For TEM analysis, hypocotyls and petioles of 4-d-old seedlings were high-pressure frozen, freeze-substituted, sectioned, and prepared for TEM as pre-viously described (McFarlane et al., 2008). Briefly, dissected samples wereloaded into copper hats (Ted Pella) with 1-hexadecene as a cryoprotectant andfrozen using a Leica HPM-100 high-pressure freezer. Samples were transferredto freeze-substitutionmedia containing 2% (w/v) osmium tetroxide and 8% (v/v)dimethoxypropane in anhydrous acetone for morphological analysis, or 0.25%(v/v) glutaraldehyde, 0.1% (w/v) uranyl acetate, and 8% (v/v) dimethox-ypropane in acetone for immunolabeling. Freeze substitution was performed for5 d at280°C using an acetone/dry ice slush. Samples were transferred to220°Covernight, followed by 4°C for 2 h, and then transferred to room temperature.Sampleswere removed from the sample holders and rinsed in anhydrous acetoneseveral times before slowly infiltrating over the course of 4 d with increasingconcentrations of Spurr’s resin (Electron Microscopy Services) for analysis ofmorphology or LR White for immunolabeling (London Resin Company). Fol-lowing polymerization at 60°C, samples were sectioned using a Leica UltracutUCT Ultramicrotome (Leica Microsystems). Thin sections (70–80 nm thick) werepoststained using 2% uranyl acetate in 70% methanol and Reynolds lead citrateand viewed on a Hitachi H7600 PC-TEM at an accelerating voltage of 80 kV.Photographs were taken using an ATM Advantage HR digital CCD camera andimages analyzed in Image J.

TEM Ultrastructure Analysis

Golgi ultrastructure was quantified in TEM images of DEX-treated wildtype (wild-type PCW), wild type/35S:VND7-VP16-GR (wild-type SCW), and

irx9/proUB10:VND7-VP16-GR (irx9 SCW) seedlings. For wild type/35S:VND7-VP16-GR, Golgi were analyzed from both 16- to 18-h–induced (wild-typeEarly-SCW) and 22- to 30-h–induced (wild-type Late-SCW) plants. For eachof wild-type PCW, wild-type Early-SCW, wild-type Late-SCW, and irx9SCW, quantification data were collected from Golgi cross-sections in cellswith visible SCWs from replicate seedlings (8, 5, 8, 7, respectively) and ex-periments (3, 2, 3, 2, respectively).

The ‘cis’ cisternae were identified by lighter staining, visible lumen in thecisternal center, and smaller margins than cisternae on the opposite side of thestack (Staehelin et al., 1990; Samuels et al., 2002). The ‘trans’ cisterna was de-fined as the last structure on the trans face with a flattened, cisterna-like region.Cisternae adjacent to cis and trans were designated ‘cis11’ and ‘trans-1’, re-spectively, and the remaining intervening cisterna(e) classified as ‘mid’. Thenumber of fenestrations in each cisterna was estimated by counting cisternal‘gaps’ in Golgi cross-sections for each developmental stage or genotype. Thesegaps, defined as breaks in a cisterna or regions of lighter staining correspondingwith indentations in the cisternal membrane, were assumed to be fenestrationsbased on published Golgi tomography and transverse-sections of cisternae(Mollenhauer and Morré, 1998; Donohoe et al., 2013). Cisternal diameters weredetermined bymeasuring the length of each cisterna from one edge to the other.Golgi width was calculated by averaging cisternal diameter for cis11, mid andtrans-1 cisternae. Cis and trans cisternae were excluded as their structures wereassumed to be changing rapidly as the cisterna forms or collapses, respectively.Cisternal margins are known to become swollen in more-trans cisternae(Staehelin et al., 1990; Samuels et al., 2002). Margin size across cisternae wastherefore quantified by measuring the diameter of the margins of each cisterna.The diameter of round profiles on TGN was also quantified to estimate the sizeof budding or secretory vesicles. All measurements were conducted in ImageJ.

Golgi Abundance in TEM

For each cell quantified, the number of Golgi or mitochondria (termed‘counts’) were summed from nonoverlapping images of the cell cortex in wild-type PCW, wild-type Late-SCW, and irx9 SCW cells. For the purposes ofquantification of organelle abundance in TEM, Golgi counts included vesicleaggregations, as sections through cisternal margins could not be easily distin-guished from the TGN at lower magnifications. Counts/perimeter was thencalculated by dividing the number of counts in each cell by the length of the cellperimeter in the images from which the counts were obtained. The area of cy-toplasm in each image was then determined (excluding the vacuole, cell wall,plastids, nucleus, and ER bodies;1 mm2 or larger) and summed for all imagesin each cell. Counts/area was calculated by dividing the number of counts ineach cell by this summed area. Increases in Golgi diameter would increase theprobability of sectioning through individual Golgi in a cell, thereby artificiallyincreasing Golgi counts using this method. To account for this, the percent in-crease in Golgi width between wild-type PCW and wild-type Late-SCW or irx9SCW was divided from the respective Golgi/perimeter and Golgi/area aver-ages, thereby normalizing them to the width of wild-type PCWGolgi. Averagecytoplasm thickness was determined by dividing the measured perimeterlength from the cytoplasmic area. For all parameters quantified, values wereaveraged for 31 cells taken from four or five seedlings prepared in four replicatehigh-pressure freezing experiments (wild-type Pre-SCW and wild-type Late-SCW) or 17 cells from seven seedlings in two experiments (irx9 SCW).

Golgi Position in TEM

A subset of the images from the wild-type Late-SCW TEM organelle a-bundance quantifications were used to estimate the distance to the formingSCW from Golgi/TGN, mitochondria, or random positions in the cytoplasm. A500-nm grid was laid over each image, and Excel’s random number generatorused to select random points on the grid that a Golgi or mitochondrion could befound (i.e. excluding the vacuole, cell wall, nucleus, plastids, and large ERbodies). The shortest distance between each Golgi, mitochondrion, and randompoint and the nearest SCW was then measured. A total of 114 mitochondria,and 168 random points and Golgi, were quantified from sixteen cells, from fiveseedlings, in three replicate experiments. Values were square root transformedbefore statistical analysis to achieve a normal distribution.

TEM Immunogold Labeling and Mapping

Primary antibodies included the anti-xylan antibody CCRC M138 (Carbo-source) and a polyclonal anti-GFP (Torrey Pine Biolabs). Secondary antibodies


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included goat antimouse (CCRC M138) and goat antirabbit (anti-GFP) anti-bodies conjugated to 10-nm colloidal gold (Ted Pella). For immunogold la-beling, wild-type Late-SCW or IRX9-GFP Late-SCW (proIRX9:IRX9-GFP/35S:VND7-VP16-GR) samples were induced for 22–24 h before high-pressurefreezing, freeze-substitution, and embedding in LR-white, as describedabove. Formvar-coated nickel grids with 70-nm sections were blocked with5% (w/v) nonfat dry milk (NFDM) in 13 Tris-buffered saline/ 0.1% (v/v)Tween 20 (TBST) for 20 min. After removal of excess blocking solution byblotting, grids were transferred to the primary antibody, which was undi-luted (CCRCM138) or diluted 1:50 with 1% (w/v) NFDM in 13 TBST (TorreyPines anti-GFP) for 60 min. After being rinsed with 13 TBST, grids weretransferred to appropriate secondary antibodies diluted 1:100 in 1% (w/v)NFDM in 1x TBST for 60 min, rinsed again with 1 x TBST and then thoroughlywashed with distilled water. Grids were poststained for 5 min in uranyl ac-etate and 2 min in lead citrate before imaging as described above.

Ideal antibodies forGolgimappingwouldhave sufficient label in theGolgi ofthe cells making SCWs, but little or no label in negative controls. To assess thissignal-to-noise ratio, anti-xylan labeling in IRX9-GFP Late-SCW cells withSCWs was compared with cells in the same seedling not making SCWs andwith cells with SCWs but not exposed to the primary antibody. Anti-GFP an-tibodies were tested by comparing labeling in IRX9-GFP Late-SCW to simi-lar cells in the wild-type Late-SCW samples, as well as IRX9-GFP Late-SCWsamples not exposed to the primary antibody. Quantification of gold label inanti-xylan negative controls and anti-GFP no-primary-antibody controls wasunnecessary as they contained virtually no gold label. For comparison of anti-GFP label in IRX9-GFP for Late-SCW and wild-type Late-SCW, all Golgi andTGN in each cell examined were imaged and the number of gold particles ineachGolgi was counted. For this purpose, 493 Golgi/TGN in 39 cells (IRX9-GFPLate-SCW) and 277 Golgi/TGN in 19 cells (wild-type Late-SCW) were imagedfrom four seedlings in two replicate high-pressure freezing experiments. Thedifference between anti-GFP label and nonspecific binding was demonstratedby comparing the number of gold particles labeling Golgi in IRX9-GFP Late-SCW and wild-type Late-SCW.

Quantitative mapping in IRX9-GFP was completed using CCRC M138 forxylan labeling and Torrey Pines anti-GFP for IRX9-GFP labeling. Every goldparticle in a cell labeling the Golgi or TGN was quantified, and the position ofgold particles in the Golgi was assessed when plane-of-section and samplepreservation permitted. The results shown consist of data from 24 cells (xylanmapping) and 39 cells (GFP mapping) from four seedlings and two high-pressure freezing experiments. High-resolution mapping of gold label in ap-propriately oriented sections through Golgi stacks resulted in quantification of136 gold particles in 17 cells (xylan mapping) and 182 gold particles in 33 cells(GFP mapping).

The cisterna with gold label was identified according to the parametersoutlined in the ultrastructure characterization section above. The distance be-tween the gold particle and the cisternal edge, as well was the diameter of thecisterna, was measured. The distance to the cisternal center was calculated bydividing the cisternal width in two and subtracting the distance to the cisternaledge. To compare the cisternal position of xylan and GFP label, the averagedistance tocisternal centers andcisternal edgeswascalculated, irrespectiveof thecisterna in which the gold particle was found. To map the distribution of goldlabel across Golgi width, the distance to the cisternal edge was converted into apercent ofGolgiwidth for each goldparticle. Goldparticlesmapping to the samecisterna, and the same 10% block of cisternal width, were binned together forgenerationof the labelingheatmaps.Althoughmappingofgold label to theTGNwas largely independent of plane of section andpreservationquality, only;50%Golgi label could be accurately mapped to a specific region of individual cis-ternae. As such, the amount of TGN label could not be directly compared withthe Golgi-mapping frequencies. Instead, the proportion of TGN vs Golgi labelbefore mapping was used to scale down the amount of TGN label to coincidewith the amount of Golgi label that was actuallymapped. Heatmap colorsweregenerated in Excel (Microsoft) based on these final distributions. For easiercomparison of Golgi structure and the mapping distributions, the heat mapcolorswere thenmanually transferred to a realistic Golgi outline based onGolgiultrastructure imaging.

Statistical Analysis

Statistical analysiswas conductedusing SPSS 25 (IBM). Before comparison ofmeans, normality was assessed visually using histograms and Q-Q plots,supplemented with Kolmogorov-Smirnov and Shapiro-Wilkes tests. If the datawere approximately normally distributed, a t test for independent means was

used to compare two means or a one-way ANOVAwas used to compare morethan two means. If the ANOVA showed a significant difference, Tukey HSDpost hoc analysis was conducted to identify significant differences. If the datawere non-normal, data transformations were applied as indicated in the results,and normality retested as described above. If normality was achieved,the above parametric procedures were followed. If the data continued to benon-normal, nonparametric tests were performed with either the Mann-Whitney test (U) for two means or the Kruskal-Wallis test (H) for more thantwo means followed by post hoc analysis using Dunn’s procedure.

Accession Numbers

Sequence data from this article can be found using the following accessionnumbers: IRX9, AT2G37090; VND7, AT1G71930; UBQ10, AT4G05320; MANI,AT1G51590.

SUPPLEMENTAL DATA

The following supplemental materials are available.

Supplemental Figure S1. ProIRX9:IRX9-GFP complements the irx9 mu-tant phenotypes and is expressed in native root protoxylem trachearyelements.

Supplemental Figure S2. Comparing methods for quantifying the abun-dance of Golgi and mitochondria in WT PCW, WT Late-SCW and irx9SCW cells from TEM cross-sections.

Supplemental Figure S3. In the complemented irx9 background, IRX9-GFP-labeled Golgi punctae increase in density as SCW deposition progressescompared to MANI-mCherry-labeled Golgi in PCW-producing cells.

Supplemental Figure S4. Identifying cross-sections of Golgi stacks for ul-trastructure characterization.

Supplemental Figure S5. Additional quantification of Golgi structuralfeatures.

Supplemental Figure S6. Controls for immuno-TEM antibody labeling ofxylan and IRX9-GFP.

ACKNOWLEDGMENTS

The authors thank the staff of the UBC Bioimaging Facility for technicalsupport. Taku Demura from the Nara Institute of Science and Technologykindly donated the 35S:VND7-VP16-GR seeds and plasmid.

Received June 13, 2019; accepted August 2, 2019; published August 20, 2019.

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