Two Complementary Mechanisms Underpin Cell WallPatterning during Xylem Vessel Development OPEN

Rene Schneider,a,b Lu Tang,c Edwin R. Lampugnani,a Sarah Barkwill,d Rahul Lathe,b Yi Zhang,b

Heather E. McFarlane,a Edouard Pesquet,e,f Totte Niittyla,f Shawn D. Mansfield,d Yihua Zhou,c

and Staffan Perssona,b,1

a School of Biosciences, University of Melbourne, Parkville 3010, Melbourne, AustraliabMax-Planck Institute for Molecular Plant Physiology, Am Muehlenberg 1, 14476 Potsdam, Germanyc State Key Laboratory of Plant Genomics, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing100101, ChinadDepartment of Wood Science, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canadae Arrhenius Laboratories, Department of Ecology, Environment and Plant Sciences (DEEP), Stockholm University, 160 91 Stockholm,Swedenf Umeå Plant Science Centre, Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences,901 87 Umeå, Sweden

ORCID IDs: 0000-0003-3951-9742 (R.S.); 0000-0002-3666-7240 (E.R.L.); 0000-0002-1572-2837 (Y. Zhang); 0000-0002-6959-3284 (E.P.);0000-0001-8029-1503 (T.N.); 0000-0002-0175-554X (S.D.M.); 0000-0001-6644-610X (Y. Zhou); 0000-0002-6377-5132 (S.P.)

The evolution of the plant vasculature was essential for the emergence of terrestrial life. Xylem vessels are solute-transporting elements in the vasculature that possess secondary wall thickenings deposited in intricate patterns. Evenlydispersed microtubule (MT) bands support the formation of these wall thickenings, but how the MTs direct cell wall synthesisduring this process remains largely unknown. Cellulose is the major secondary wall constituent and is synthesized by plasmamembrane-localized cellulose synthases (CesAs) whose catalytic activity propels them through the membrane. We show thatthe protein CELLULOSE SYNTHASE INTERACTING1 (CSI1)/POM2 is necessary to align the secondary wall CesAs and MTsduring the initial phase of xylem vessel development in Arabidopsis thaliana and rice (Oryza sativa). Surprisingly, theseMT-driven patterns successively become imprinted and sufficient to sustain the continued progression of wall thickening inthe absence of MTs and CSI1/POM2 function. Hence, two complementary principles underpin wall patterning during xylemvessel development.

INTRODUCTION

The plant vasculature is one of the most important evolutionaryinnovations for terrestrial life, as it allowed plants to adapt and growto significant stature (Myburg et al., 2013). The xylem tissue pro-vides essential functions in the vasculature by distributing waterthroughout the plant and providing structural support to the plantbody. The xylem cells are encased by thickened cell walls thatreinforce them and therefore are essential for their function (Turneret al., 2007). The organization of the secondary cell walls differsbetween xylem vessel cell types and is typically described either asan annular/spiral pattern (called protoxylem) or a reticulate/pittedpattern (called metaxylem; Pesquet et al., 2011). Before thesethickenedsecondarywalls areassembled, the xylemcells, likeall plantcells, are encased by a flexible but strong primary cell wall (Somervilleet al., 2004). These walls largely comprise polysaccharides, ofwhich cellulose, an unbranched, linear b-1,4-linked glucan, forms

a significant constituent. Cellulose is synthesized at the plasmamembrane by large cellulose synthase (CesA) complexes (CSCs;Schneider et al., 2016). The CSCs are composed of a heterotrimericconfigurationof 18 to 24CesAswhereCesA1,CesA3, andCesA6-like(i.e., CesA2, 5, 6, and 9) CesAs produce primary wall cellulose inArabidopsis thaliana, and CesA4, CesA7, and CesA8 comprise theCSCs necessary to make secondary wall cellulose (Persson et al.,2007; Desprez et al., 2007; Taylor et al., 2003; Atanassov et al., 2009).The CSCs move along linear tracks at the plasma membrane

(Paredez et al., 2006), likely due to the catalytic activity of theCSCs.Nascent cellulosemicrofibrils becomeentrapped in thecellwall and further synthesis thereforeexertsa forceon theCSCs thatpropels them forward through the plasma membrane. Themovement of the CSCs is guided by cortical microtubules (MTs)during both primary and secondary wall cellulose synthesis(Paredez et al., 2006; Watanabe et al., 2015). The proteinCELLULOSE SYNTHASE INTERACTING1 (CSI1), also called POM-POM2 (POM2), is necessary for the MT-based guidance of theprimary wall CSCs, as lesions in the protein impaired coalignmentbetween tracksofprimarywallCSCsandcorticalMTs (Bringmannet al., 2012; Li et al., 2012). However, reports on the function ofCSI1/POM2 during secondary wall cellulose production differ. Guand Somerville (2010) reported no defects on secondary walls nordecreased cellulose content in csi1/pom2 mutant Arabidopsis

1 Address correspondence to staffan.persson@unimelb.edu.au.The author responsible for distribution of materials integral to the findingspresented in this article in accordance with the policy described in theInstructions for Authors (www.plantcell.org) is: Staffan Persson (staffan.persson@unimelb.edu.au).OPENArticles can be viewed without a subscription.www.plantcell.org/cgi/doi/10.1105/tpc.17.00309

The Plant Cell, Vol. 29: 2433–2449, October 2017, www.plantcell.org ã 2017 ASPB.

stems. By contrast, Derbyshire et al. (2015) showed that inductionof tracheary elements in Arabidopsis cell cultures was impaired incells with reduced CSI1/POM2 expression. The role of CSI1/POM2 in secondary wall cellulose production therefore remainsunclear.

Secondary walls are typically produced around cells that aresituated deep in tissues and that therefore are largely masked byother cells. This location makes it difficult to study secondary wallsynthesiswhere itnormallyoccurs. Instead,alternativesystemshavebeen developed for this purpose, including trans-differentiating cellcultures thatcanbe inducedbyhormonecocktails (Kuboetal., 2005;Demura et al., 2002; Pesquet et al., 2010) and inducible transcriptionfactor-based systems. The latter systems make use of the NAC-related transcription factors VASCULAR-RELATEDNAC-DOMAIN6(VND6) and VND7 that promote meta- and protoxylem-like cell wallstructures, respectively (Kubo et al., 2005; Yamaguchi et al., 2010;Oda et al., 2010). By selectively controlling the activity of the VNDswith an inducible promoter system, it is possible to induce andexplore secondary wall formation in cells that normally do not formthese structures. VND7-inducible Arabidopsis seedlings have beenused to evaluate the behavior of the secondary wall CSCs usinga fluorescently tagged CesA7 (Watanabe et al., 2015) and to assessthe coordination between transcripts and metabolites during thisprocess (Li et al., 2016a).

Here, we investigated how protoxylem vessel wall patterns arecontrolled by analyzing the coordination of MTs and cell wall de-position in Arabidopsis and rice (Oryza sativa). We found that CSI1/POM2 orchestrates cell wall synthesis along MTs during theinitial developmental phase of xylem vessel formation but thatsubsequent synthesis occurs via a CSI1/POM2 autonomousmechanism. Our results indicate that cell wall patterns aredirected by two complementary principles during xylem vesseldevelopment.

RESULTS

CSI1/POM2 Influences Xylem Vessel Wall Patterning

To evaluate if defects in CSI1/POM2 function alter cell wall pat-terning during xylem vessel formation in Arabidopsis, we exam-inedsecondarywall formation in threedifferent systemswhere thefunction of CSI1/POM2was impaired. First, we confirmed that thedownregulation of CSI1/POM2 caused aberrant secondary walldeposition in proto- and metaxylem trans-differentiating cellsuspension cultures (Derbyshire et al., 2015; SupplementalFigures 1Aand 1B). Using confocalmicroscopy,wequantified theoccurrenceof spiral, reticulate, andpitted secondarywall patternsand the percentage of calcofluor-stained irregular deposits in thesecondary walls of nontransgenic and CSI1 downregulated celllines (Supplemental Figures 1C and 1D). Although it was difficultto assess defects in cell wall patterning in these lines, down-regulation ofCSI1/POM2caused a significant increase in irregulardeposits along the secondarywalls (Supplemental Figures1Band1D).Thisdefectwas irrespectiveof thepatterningof thesecondarywalls (Supplemental Figure 1D).

Wenext investigated if the xylemofmature stemsof Arabidopsisplants showed structural defects when CSI1/POM2 function wasimpaired. We made longitudinal sections of the first internodes

allowing structural characterization of intact and transectedxylem vessels in the previously described csi1/pom2 mutantspom2-4 and csi1-1/pom2-8 (Bringmann et al., 2012) as well aswild-type plants (Figures 1A and 1B). We found that the sec-ondary wall bands were significantly more disordered in thepom2-4 and csi1-1/pom2-8 mutants, as evident from mea-suring the spread in orientation angles of neighboring wallbands (Figure 1C).We next used a VND7-inducible Arabidopsis line (Yamaguchi

et al., 2010) to studyprotoxylemvessel secondarywall patterning.Here, we observed xylem-related wall synthesis as indicated bywell-organized band patterns that were transversely and evenlydistributed around induced hypocotyl cells (Figure 1D). Wequantified the geometry of the bands and found that they werealigned tightly around an average angle of 0.66 3.8° (mean6 SD,132 cells from five seedlings) against the horizontal axis (Figures1D and 1F).To assess if the CSI1/POM2 function influenced the wall pat-

terns, we introgressed the pom2-4 mutant into the VND7-inducible line. The xylem vessel wall patternswere lesswell alignedin the pom2-4 background (Figures 1E and 1F). Here, the bandsdisplayed significantly wider and less uniform angles comparedwith control (22.4622.7°; 136 cells from five seedlings; Figures 1Eto1G). In addition, the band spacingwassubstantially altered in thepom2-4 mutant compared with the control VND7-inducible line(Figure 1H). These results indicate that while CSI1/POM2 is notessential for the formation of secondarywall bands, confirming thatxylem vessels are intact (Gu and Somerville, 2010), the proteininfluences the geometry and relative position of the deposition ofthe bands.To investigate if the defects in secondary wall patterning were

associated with changes in cell wall architecture and ultra-structure, we measured the microfibril angle (MFA), cell wallcrystallinity, degree of cellulose polymerization, and cellulosecontent in pom2-4mutant stems and compared the results withwild-type stems (Figures 1I to 1L). We found that the celluloseshoweddifferences inbothMFAsandcrystallinity (Figures1I and1J), corroborating defects in cellulose synthesis. We also founda slight increase in glucose content, most likely due to an in-crease in amorphous cellulose due to the decreased levelsof crystalline cellulose (Figure 1L). These data indicate thatCSI1/POM2 influence the quality of secondary wall cellulosesynthesis.

CSI1/POM2 Mimics the Behavior of, and Can Interact with,the Secondary Wall CesA Proteins

To investigate how the CSI1/POM2 behaves during the transitionfrom primary to secondary wall synthesis, we crossed plantsexpressing a functional, native promoter-driven triple (3x) YFPtranslational fusionwith CSI1/POM2 (Worden et al., 2015) into theVND7-inducible Arabidopsis line. The 3xYFP-CSI1/POM2 can beseen as fluorescent foci that track together with the CSCs at thecell cortex along linear trajectories during primary wall synthesis(Wordenet al., 2015). After inductionof VND7,weobservedaclearchange in the cellular distribution of the 3xYFP-CSI1/POM2. Al-though the 3xYFP-CSI1/POM2 foci maintained linear movement,the pattern of movement changed following induction. The foci

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Figure 1. Defects in CSI1/POM2 Cause Aberrant Xylem Vessel Patterns.

(A) and (B) Scanning electron micrographs of longitudinal sections of mature wild-type stems. Exposed (A) and transected (B) xylem vessels of wild-typeplants and pom2-4 and pom2-8 (csi1-1) mutants. Bar = 10 mm.(C)Band-to-bandorientations inpom2-4 (16 cells in 6 seedlings) andpom2-8 (44 cells in 6 seedlings) comparedwithwild-type xylem (27cells in 6 seedlings)obtained from the images in (A) and (B).(D)and (E)S4Bstainingof cellulose inVND7-inducedhypocotyls.Dotted lines indicatehighlyorderedbands in thesecondarywallsofwild-typecells (D)andirregular bands in pom2-4 mutant cells (E). Bar = 5 mm.(F) Distribution of the average band orientations (yellow lines in [A] and [B]).(G) The spread of band orientations within individual cells of induced pom2-4 cells (602 bands in 115 cells in 5 seedlings) compared with wild-type cells(824 bands in 132 cells in 5 seedlings).(H) Secondary wall band spacing in the pom2-4 mutant compared with wild-type cells.(I) MFA (relative to growth axis) in the pom2-4 mutant compared with the wild type.(J) Cell wall crystallinity in the pom2-4 mutant compared with the wild type.(K) Degree of polymerization (DP) in the pom2-4 mutant compared with the wild type.(L)Cellulosecontent (% fractionofdryweight) in thepom2-4mutant comparedwith thewild type.Allmeasurements ([I] to [L])weredoneongroundstemsof10-week-old, fully senesced plants grown in 16-h-light/8-h-dark conditions. Data aremeans6 SD. Statistical significancewas tested byWelch’s unpaired ttest: *P < 0.05, **P < 0.005, and ***P < 0.0005.

CSI1/POM2 Steers Xylem Vessel Wall Patterning 2435

were initially evenly distributed across the plasma membrane;however, thispatternchanged in favorofdenseand regularly spacedbanded patterns (Supplemental Movie 1).

Cortical MTs change their distribution during the progressionof xylem vessel production and form distinct banded or helicalarrays (Watanabe et al., 2015), similar to what we observed forthe 3xYFP-CSI1/POM2 foci. To see if the 3xYFP-CSI1/POM2 andMT re-distributions co-occurred during xylem vessel development,wecrossedamCherry-TUA5-expressingplantwith the3xYFP-CSI1/POM2 VND7-inducible plant and analyzed the progeny. We foundthatthechangesinthe3xYFP-CSI1/POM2patternsco-occurredwiththe rearrangement of theMT array during the transition from primaryto secondary wall synthesis (Figures 2A to 2E), indicating that theCSI1/POM2 likely trackswithbothprimaryandsecondarywallCSCs.

TheprimarywallCSCstypically trackwithaspeedof;250nm/min(Paredez et al., 2006), and CSI1/POM2 proteins track together withthese CSCs (Bringmann et al., 2012). However, the secondary wallCSCstracksignificantly faster than theprimarywallCSCs (Watanabeet al., 2015), and if the CSI1/POM2 proteins are associated with thesecondary wall CSCs, one would anticipate an increase in speed ofthe CSI1/POM2s over time after VND7 induction. To test this, wemeasured thespeedof the3xYFP-CSI1/POM2at early,mid, and latetime points after VND7 induction. We selected these time pointsbasedon theMT reorganization status after induction (SupplementalFigure 2), and they roughly coincide with time points used byWatanabe et al. (2015). We found that the CSI1/POM2 proteinsmovedwith a speedof;2666 35 nm/min (1015 foci in 12 cells from3 seedlings) in DMSO-treated cells and at 2936 98nm/min (905 fociin 17 cells in 15 seedlings) in cells in the early stagesof the secondarywall synthesis, i.e., where MTs still exhibited a primary wall-likepattern (Figures 2F to 2H). However, during the middle stages ofsecondary wall synthesis, i.e., where MTs formed diffuse bands, weobserved a significant increase in CSI1/POM2 speed (4226 72 nm/min;1391 foci in18cells from15seedlings).Oncesecondarycellwallsynthesis had progressed to late stages, we found that the speed oftheCSI1/POM2proteinsdeclined (211675nm/min; 234 foci in7cellsfrom15seedlings), possibly related to the initiationofprogrammedcelldeath. Notably, the secondary wall CSCs underwent a very similartransition in speedsduring early,mid, and late secondarywall stages(Watanabeetal., 2015).Thesedata indicate that theCSI1/POM2maytrackwith thesecondarywallCesAproteins, similar towhat hasbeenshown for the primary wall CesAs (Gu et al., 2010). To test whetherCSI1/POM2 can interact with secondary wall CesAs, we performedbimolecular fluorescence complementation (BiFC) assays betweenthe three secondary wall Arabidopsis CesAs and CSI1/POM2. Wefound that the proteins can interact when transiently expressed intobacco (Nicotiana tabacum) epidermal leaf cells (SupplementalFigure 3). Hence, the CSI1/POM2 proteins behave similarly to thesecondary wall CesAs and can interact with them.

The CSI1/POM2s Track with the Secondary Wall CesAs andAre Rapidly Recruited to Their Sites of Action

The CSI1/POM2 and secondary wall CesAs behave similarly andcan interact, suggesting that the proteins also track togetherduring xylem vessel development. To test this, we generatedplants expressing a mCherry-tagged CSI1/POM2 fusion proteinunder control of theCSI1/POM2promoter and introgressed these

plants with VND7-inducible lines expressing the YFP-CesA7construct. The two fluorescent proteins showed similar behaviorand closely colocalized throughout the different stages of VND7induction (Figures 3A to3D). Theseobservations are supportedbyclose inspections of kymographs from movies of the fluorescentproteins, where the tracking of the proteins coincided (Figure 3B).To assess the recruitment of CSI1/POM2 and CesA7 to their

sitesduring secondarywall synthesis,weperformed fluorescencerecovery after photobleaching (FRAP) experiments. We firstused the VND7-lines expressing mCherry-CSI1/POM2 andYFP-CesA7; however, the mCherry signal proved too weak toaccuratelyassessfluorescence recovery. Instead,weusedVND7-induced lines expressing either 3xYFP-CSI1/POM2 or YFP-CesA7 and counted the number of insertion events over time,which permitted measurement of the average insertion, or de-livery, times (Supplemental Figures 4A to 4D). The 3xYFP-CSI1/POM2 signal rapidly repopulated the bleached area after FRAP(Supplemental Figures 4A and 4D; recovery time 32 6 13 s,46 bands in 7 cells in 3 seedlings), whereas the recovery of theYFP-CesA7 was significantly slower (106 6 68 s, 32 bands in6cells in 4 seedlings).Wecalculated the ratio of the recoveryof thetwo fluorescently labeled proteins to be 3.3 6 2.5. As the sec-ondary wall CSCs contain CesA4, CesA7, and CesA8, possibly inequal stoichiometry (Gonneau et al., 2014; Hill et al., 2014), it islikely that eachCesA in the secondarywall CSC is associatedwithone CSI1/POM2 protein. However, it is important to note twothings: First, the pom2-4 and irx3-4 mutations were not homo-zygous in the 3xYFP-CSI1/POM2 and the YFP-CesA7 lines, re-spectively. While one might assume that each CSC will containboth labeled and unlabeled CesA7, and thus that each CSC istracked in our image analysis, it is possible that we underestimatethe numbers of CSI1/POM2s associatedwith eachCSC. Second,the analyses were done on seedlings with either the 3xYFP-CSI1/POM2 or YFP-CesA7, which alone may introduce experimentaldifferences. While we therefore favor a ratio between the CSI1/POM2 and CesA as 1:1 at a given secondary wall CSC, furtherexperiments are needed to firmly corroborate this hypothesis.

Optical Flow Analyses Support Global Bidirectionality, butLocal Unidirectionality, of the CSI1/POM2

To assess, in more detail, the migratory patterns of CSI1/POM2during xylemvessel development,weanalyzed thebehavior of the3xYFP-CSI1/POM2 using optical flow analyses (SupplementalFigures 5A to 5C). This analysis can examine the patterns ofapparentmotion andsize of fluorescent objects.We false-coloredthe motion of fluorescent objects based on direction, i.e.,movement to the left or right were colored purple and green, re-spectively (Figure 4A). We detected clear bidirectional movementof the 3xYFP-CSI1/POM2 objects in both DMSO and VND7-induced cells, i.e., the YFP-CSI1/POM2 trajectories clearly over-lapped along kymograph sections (Figure 4B), and the averageoptical flow images contained a significant number of white pixels(Figures 4Band4C). However, domains of apparent unidirectionalmovement were significantly larger in the cells undergoing xylemdifferentiation (Figure 4D). These data indicate that the flow of theCSI1/POM2, and therefore most likely also the CSCs, is prefer-entially bidirectional onacellular scale, butunidirectional ona local

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Figure 2. CSI1/POM2 Colocalizes with Cortical Microtubules and Behaves Similarly to Secondary Wall CesAs during Xylem Vessel Development.

(A) Time-averaged images of MTs and 3xYFP-CSI1 in DMSO-treated control cells (upper panel) and VND7-induced cells in mid stage (lower panel; seeSupplemental Figure 3 for definition of stages). Bar = 5 mm.(B) and (C) Fluorescence intensity plots of CSI1 (green) and MTs (lilac) in DMSO-treated (B) and VND7-induced (C) cells along the dashed lines in (A).(D) and (E) Measurement of the Pearson’s (D) and Mander’s (E) correlation coefficient comparing CSI1 colocalization with MTs during secondary walldevelopmental programcomparedwithDMSO-treated cells. The arrow scheme in (E) indicates intensity overlap ofMTswithCSI1 (lilac) andCSI1withMTs(green).(F) Single and time-averaged images of 3xYFP-CSI1 in DMSO-treated (left) and VND7-induced (right) cells. Bar = 5 mm.

CSI1/POM2 Steers Xylem Vessel Wall Patterning 2437

scale.Hence, in regionswhereoneCSI1/POM2migrated inadefineddirection, the majority of the associated CSI1/POM2s were likely tofollowthesamedirection (Figures4Band4C).Tosee if themovementof the 3xYFP-CSI1/POM2 foci depended on whether they were partof uni- or bidirectional domains, wemeasured the speeds of the focifrom the different domains. We found that the CSI1/POM2migratedwith similar speeds independent of being part of a bi- or unidirec-tionally moving domain (Figure 4E). These data indicate that thesecondarywall cellulose is preferentially produced in onedirection atany given subregion of the cell wall bands.

We observed many 3xYFP-CSI1/POM2 foci that first moved inone direction but suddenly stopped and changed direction (whitearrows in Figure 4B). Such events were detected only in xylemvessel-differentiating cells and not in the DMSO-treated cells.These observations suggest that CSI1/POM2 can be transferredbetween CSCs moving in opposite directions during secondarywall synthesis, perhaps to support tight associations between thesecondary wall CSCs and underlying MTs.

Mutations in CSI1/POM2 Cause Misalignments ofSecondary Wall CesAs and Microtubules

Lesions in CSI1/POM2 caused defects in the alignment of primarywall CesA trajectories and cortical MTs (Bringmann et al., 2012; Liet al., 2012). To investigate whether defects in CSI1/POM2 also

affected the alignment of the secondarywall CesAs and theMTs,wegenerated YFP-CesA7 mCh-TUA5 dual-labeled VND7-induciblelines in wild-type or pom2-4mutant backgrounds. The YFP-CesA7trajectories coaligned with cortical MTs during all stages of xylemvessel development in the wild-type background (SupplementalFigure 6). However, we observed clear defects in the alignment ofCesA7trajectoriesandMTs inpom2-4mutantcells (Figures5Ato5F;Supplemental Movie 2). Notably, substantial misalignment was ob-served only during early stages of secondary wall synthesis (Figure5D). TheseobservationswerecorroboratedbyquantificationofYFP-CesA7 and mCh-TUA5 colocalization, revealing a significant re-duction in CesA7 overlap with MTs during the early developmentalstage, but not during subsequent stages, in pom2-4 compared withthe wild type (Figures 5E and 5F).To further assesswhether defects inCSI1/POM2 influenced the

behavior of the secondary wall CSC, wemeasured insertion ratesand speeds of YFP-CesA7 in either VND7-induced wild-type orpom2-4mutant backgrounds.We found that the insertion timesofYFP-CesA7 in pom2-4 were not significantly different from thewild type (Supplemental Figures 4Cand4D; 84627s, 39bands in6 cells in 5 seedlings). By contrast, we observed changes in thedistribution of YFP-CesA7 speeds during the progression ofsecondary cell wall synthesis (Figure 5G). In the pom2-4 mutantbackground, YFP-CesA7 moved with higher speeds during earlystages of secondary wall synthesis, but showed significantly

Figure 2. (continued).

(G) Kymographs along the dotted lines in (F) comparing 3xYFP-CSI1 velocity in DMSO-treated and VND7-induced cells.(H) 3xYFP-CSI1migration speedsduring secondarywall developmental comparedwithDMSO-treated cells. Statistical significancewas testedbyWelch’sunpaired t test relative to DMSO-treated controls: *P < 0.05, **P < 0.005, and ***P < 0.0005.

Figure 3. CSI1/POM2 Comigrates with CesA7 and Recovers More Quickly Than CesA7 after Photobleaching during Xylem Vessel Formation.

(A) Average projections of mCherry-CSI1/POM2 and YFP-CesA7 in noninduced cells (DMSO) and early, mid, and late stages of secondary wall synthesis.Bar = 5 mm.(B)Kymographsalong thedotted lines in (A)showing themigrationofCSI1/POM2withYFP-CesA7 inearly,mid,and latestagesof secondarywall synthesis.Trajectories are indicated by arrowheads. Bar = 5 mm.(C)and (D)CSI1/POM2colocalizationwithCesA7 throughout secondarywall synthesis as shownbyPearson’s (C)andMander’scoefficients (D). Thearrowscheme in (D) indicates intensity overlapofCSI1withCesA7 (red) andCesA7withCSI1 (cyan). Statistical significancewas testedbyWelch’sunpaired t test:*P < 0.05, **P < 0.005, and ***P < 0.0005.

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slower speeds than the wild type during mid stages. In addition,during late secondary wall synthesis stages, the YFP-CesA7speeds appeared less tightly controlled in the pom2-4 mutantcompared with the wild type. These findings indicate that CSI1/POM2 is involved in regulating thespeedofsecondarywallCesAs,possibly by maintaining the CesAs in close vicinity of the MTs.

Xylem Vessel Cell Wall Patterns Can Be Maintained in theAbsence of Microtubules

CSI1/POM2 is regarded as the component that guides CSCsalongcorticalMTsduringprimarywall synthesis (Bringmannet al.,2012; Li et al., 2012). The observation that the CSI1/POM2 is notessential for alignment of the CSCs and MTs during the mid andlate stages of xylem vessel development indicated that thesestages do not depend onMT-based guidance tomaintain cell wallpatterning. To test this conclusion,wefirst established timepointswhen the MT array was reorganized during VND7-induced xylemvessel formation. In our hands, diffuse bands of MTs were notestablished until around 16 h after the VND7 induction (Figure 6A),and the bands became progressively more condensed during thesubsequent 8h. Toassess the influenceofMTsoncellwall patternmaintenance, we treated VND7-induced seedlings with theMT-depolymerizing drug oryzalin (Morejohn et al., 1987; 20 mM) atdifferent time points after induction, and then investigated theensuingwall patterns48hafter VND7 induction.Seedlings treatedwith oryzalin 8 h after induction lacked cell wall bands entirely. Bycontrast, cell wall bands were evident in seedlings treated withoryzalin 16 and 24 h after VND7 induction (Figure 6B, third andfourth image from left). These wall bands were not as well definedand evenly spaced as the control seedlings (DMSO-treated;Figure6B, left image).However,whencomparing thewall patternswith the typical MT array organization after 16 and 24 h VND7induction (Figure 6A, third and fourth images), the 16 and 24 hwallbands showed very similar distributions (Figures 6A and 6B). Inaddition, the wall bands in the oryzalin-treated seedlings (treated16 and 24 h, and imaged at 48 h, after VND7 induction) weresubstantiallymorepronouncedcomparedwith thewall patterns inseedlings at 24 h after VND7 induction (Figure 6B). These dataindicate that the xylem vessel wall patterns become reinforceddespite removal of the MT array.

Secondary Wall CesAs Remain Preferentially Delivered toSites of Secondary Cell Wall Bands in Absenceof Microtubules

To determine the behavior of the CSCs and the CSI1/POM2 in theabsence of MTs during the VND7 induction, we used the dual-labeled YFP-CesA7 mCherry-TUA5 and 3xYFP-CSI1/POM2mCherry-TUA5 lines. We treated the seedlings with oryzalin for4 h after 24-h VND7 induction, confirming effective MT de-polymerization, and assessed the behavior of the YFP-taggedproteins. While some YFP-CesA7 puncta clearly were not asso-ciated with distinct bands, many were, despite complete de-polymerization of MTs (Figure 6C). These observations wereconfirmedwith fluorescence intensity values along transects fromtime average images (Figures 6C and 6D). Similar observationsweremadeusing the3xYFP-CSI1/POM2 (Supplemental Figure7),indicating that the protoxylem vessels need MTs to establish thewall patterns but that the patterns can be maintained in the ab-sence of MTs.To investigate the dynamic behavior of the secondary wall

CSCs inmoredetail,wefirst lookedat thebehaviorofYFP-CesA7-containing Golgi bodies. We observed that the Golgi moved er-ratically at the cell cortex and that they preferentially associatedwith regions that coincidedwithMTbands (SupplementalMovie 3).

Figure4. CSI1/POM2Moves inLocalDirectionalPatchesalongMTBandsduring Xylem Vessel Development.

(A) Single, time-averaged, and time-averaged optical flow images of3xYFP-CSI1/POM2 time-lapse series in DMSO-treated cells (upper pan-els) and in mid stages of secondary wall synthesis (lower panels). Inset:Color code for movements to the left and right. Overlapping bidirectionalmovements are displayed in white. Bar = 5 mm.(B) Kymographs drawn along the dotted lines in (A) reveal predominantlybidirectional movement of individual CSI1/POM2s in DMSO-treated cells(left column) but local unidirectional movement of large patches of CSI1/POM2 toward the right (green, middle column) or the left (purple, rightcolumn) in inducedcells. IndividualCSI1/POM2 foci thatmovedagainst thedominant direction of the patch were able to change direction (arrow-heads). Bar = 2 mm.(C) Fraction of pixels in the time-averaged optical flow images that rep-resent bidirectional (white pixels in [A]) and local unidirectional (green andpurple pixels in [A]) movement for DMSO-treated and induced cells.(D) Size of bi- and unidirectionally moving CSI1/POM2 patches in DMSO-treated and induced cells.(E) Box plot of CSI1/POM2 speedsmoving in groupswith each other (parallel,left) or against the group (anti-parallel, right). Statistical significancewas testedby Welch’s unpaired t test: *P < 0.05, **P < 0.005, and ***P < 0.0005.

CSI1/POM2 Steers Xylem Vessel Wall Patterning 2439

Figure 5. Defects in CSI1/POM2 Cause Misalignment of YFP-CesA7 Trajectories and Cortical Microtubules during Early Stages of Xylem VesselDevelopment.

(A) Time-averaged images of YFP-CesA7 mCh-TUA5 in VND7-induced wild-type (upper panel) and pom2-4 mutant (lower panel) seedlings during earlystages of secondary wall synthesis. The YFP-CesA7 trajectories (dashed lines) fail to align with the MTs in the pom2-4 mutant (lower panel). Bar = 5 mm.(B)Enlarged regions of yellow, dashed rectangles in (A) for wild type (upper panel) and the pom2-4mutant (lower panel). Prominent YFP-CesA7 trajectories(dashed lines) arehighlighted in theMTandYFP-CesA7 images.Schematic illustrationsofMTs (lilac lines) andCesA7 trajectories (green lines) in thewild typeand pom2-4 reveal clear misalignment as indicated by the white arrow heads (right column). Bar = 5 mm.(C) Kymographs along the yellow, dashed lines in (B) confirming linear movement of YFP-CesA7 along the selected lines. Bar = 2 mm.(D)MisalignmentbetweenYFP-CesA7 trajectories andMTsduring early,mid, and late stagesof secondarywall synthesis. Note: Pronouncedmisalignmentis only present during early stages of secondary wall synthesis.(E) and (F) Quantification of colocalization of YFP-CesA7 and MTs in VND7-inducible wild-type and the pom2-4 mutant background using Mander’scoefficient describing the intensity overlap of MTs with YFP-CesA7 (upper panel) and YFP-CesA7 with MTs (lower panel).(G)YFP-CesA7 speeds during early,mid, and late stages of secondarywall synthesis inwild-type andpom2-4mutant background, respectively. Statisticalsignificance was tested by Welch’s unpaired t test: *P < 0.05, **P < 0.005, and ***P < 0.0005.

2440 The Plant Cell

Figure 6. Secondary Wall Patterning Can Be Maintained also in the Absence of MTs.

(A) Representative images of mCherry-TUA5-labeled MT arrays at 0, 8, 16, and 24 h after VND7 induction. Bar = 5 mm.

CSI1/POM2 Steers Xylem Vessel Wall Patterning 2441

We next investigated the behavior of the Golgi in cells whereMTshad been depolymerized by oryzalin treatment. Golgi followed verysimilar patterns of movement, i.e., they preferentially populatedregions where MT bands had been before the oryzalin treatment(Figures 6E to 6G; Supplemental Movie 4). To confirm these ob-servations,weanalyzed thenumberofGolgi localized towall bandsversus gaps using TrackMate.We found that 85%6 16% (mean6SD, n = 7 bands in 3 cells) of the Golgi were localized beneath wallbands, with the rest localized to gaps, in the presence ofMTs. Afteroryzalin treatment 75%6 18%of theGolgi were localized beneathwall bands. This difference was not significant (P = 0.39, Welch’sunpaired t test), indicating that Golgi movement is independent ofMTbands.Golgipositions typicallycorrespondtositesofdeliveryofCesAs (Crowell et al., 2009; Sampathkumar et al., 2013). To see ifCesAs were inserted to the plasmamembranemainly above areaswith Golgi movement, we applied FRAP and assessed how theYFP-CesA7 signal repopulated the bleached area. Indeed, theCesAswerepreferentially insertedat regionswhere thebulkofGolgiwas evident and, thus, in proximity of the wall bands both inpresence and absence of MTs (Figures 6E to 6G). To investigate ifthe delivered CesAs moved in any direction after delivery, or if theyfollowed the tracks of previous CesAs, we analyzed the CesAbehavior at the plasma membrane. While we found that the YFP-CesA7 foci moved slower in the absence of MTs compared withcells with MTs (Figures 6H and 6I), the majority of CesAs movedparallel to thecellwallbands. Inall, 88%64%and93%68%of thenewly inserted CesAsmoved parallel to cell wall bands before andafter oryzalin treatment, respectively (means6 SD, 101and35CesAsin 3 cells; Figure 6F). Given theGolgi behavior, our data indicate thatthe cellular regions where cell wall bands are made are different intheir molecular composition compared with interband regions.

Defects in CSI1/POM2 Affect Patterning of Cell Walls in RiceXylem Vessels

The importance of CSI1/POM2 in cellulose synthesis has beensupported by data from only Arabidopsis. To see if the protein isalso important for secondarywall synthesis in other plant species,we investigated the role of CSI1/POM2 in xylem vessel wall

formation in rice. CSI1/POM2 was in part discovered based oncoexpression of the corresponding gene with the primary wallCesAgenes inArabidopsis (Guet al., 2010).We thereforeexploredwhat rice CSI1/POM2 homolog displayed the closest coex-pression with the rice primary and secondary wall CesAs usingFamNet (Ruprecht et al., 2016). We found that the most likelycandidate for this functionwasOs06g11990, whichwe referred toas CSI-like 1 (CSIL1; Supplemental Figure 8A). These data werecorroborated by phylogenetic analyses, which revealed that thericeCSIL1wasclosely related to theArabidopsisCSI1/POM2,andthrough expression analyses that showed ubiquitous expressionof the gene and very low expression of the other CSIL genes(Supplemental Figures 8B to 8DandSupplemental Data Set 1). Toassess whether this protein affects rice growth, we generatedRNAi-mediated suppression constructs to downregulate CSIL1.Several independent homozygous T3 progeny of the trans-formants had substantially decreased CSI1L transcript abun-dance, as estimated by quantitative RT-PCR (Figure 7A), andshowed stunted growth with reduced cellulose content (Figures7B and 7C). Notably, when estimating the secondary cell wallthickness, we found that theCSIL1 RNAi plants had considerablythinner walls compared with control plants (Figures 7D and 7E).While these effects were more pronounced than what we ob-served in Arabidopsis, they clearly support a function of CSIL1 insecondary wall synthesis in rice. In addition, when we assessedthe xylemvesselwall patterns,we found that the spacingbetweenthe bands was significantly changed (Figures 7F and 7G). Thesechanges were in close agreement with the phenotypes we ob-served in theArabidopsis pom2-4 andpom2-8mutants (Figure 1).To assess if the CSIL1 can also interact with the rice secondary

wall CesAs, we performed split-luciferase assays of the ricesecondary wall CesAs, i.e., CesA4, CesA7, and CesA9, and theCSIL1. All the rice secondary wall CesAs could interact with theCSIL1 (Figure 7J; Supplemental Figure 8E), corroboratinga function of CSIL1 in rice secondary wall cellulose production. Inaddition, transient coinfiltration of mRFP-CSIL1 and GFP-CesA4intoNicotiana benthamiana leaves revealed tight colocalization ofthe proteins at plasmamembrane focal planes (Figures 7Hand7I).The patterns of colocalization were reminiscent of cortical MTs,

Figure 6. (continued).

(B)Representative images of cellulose-stained secondarywalls of VND7-induced seedlings 48h after induction.We appliedDMSOor 20mMoryzalin 8, 16,and 24h after induction until the final imaging at 48 h after induction to the seedlings. As comparison, a representative imageof the secondarywall 24 h afterinduction is shown on the right. Bar = 5 mm.(C) Single and time-averaged images of dual-labeled YFP-CesA7 (cyan) andmCherry-TUA5 (magenta) after being treatedwith DMSO (left panel) or 20mMoryzalin (right panel) for 4 h. Arrowheads indicate YFP-CesA7 trajectories still localized to distinct bands despite complete depolymerization of the corticalMT array. Bar = 5 mm.(D) Intensity crosssectionalongdotted line in (C). YFP-CesA7fluorescenceaccumulatesatdistinctbands (indicatedbydashed lines)wheremCherry-TUA5fluorescence is absent.(E) Single images of YFP-CesA7 and mCherry-Tubulin before, during, and after photobleaching a rectangular region (dotted box) in absence (left) andpresence (right) of oryzalin (40 mM for 2 h). Bar = 5 mm.(F) Trans-illumination image in absence (left) and presence (right) of oryzalin showing overlap of secondary wall thickenings and YFP-CesA7 bands(arrowheads).(G) Fluorescence recovery of secondary wall bands after photobleaching of YFP-CesA7. Bright objects (Golgi, purple circles) were tracked and foundaccumulating at and moving quickly between the previously bleached bands (see colored tracks coding for the vertical position of the Golgi). Bar = 5 mm.(H)Kymographs along the dotted lines in (E) show the insertion of individual CesAs to the secondary cell wall band (left) also in absence ofMTs (right). Bar =5 mm.(I) Velocity of CesA7 inserted into bands in the presence or absence of MTs. Statistical significance was tested byWelch’s unpaired t test: *P < 0.05, **P <0.005, and ***P < 0.0005.

2442 The Plant Cell

supportinga related functionof the riceCSIL1and theArabidopsisCSI1/POM2. Hence, we conclude that CSI1/POM2 also con-tributes to xylem vessel patterns in rice.

DISCUSSION

Cell wall patterning has been attributed to MT-based guidance ofCSCs (Oda and Fukuda, 2013; Schneider et al., 2016). While theguiding principles have been largely resolved for primary cell wallcellulose synthesis, the corresponding mechanisms for secondary

wall deposition have remained ill defined. We show that secondarywall patterningdependsonMT-basedcellwall deposition.However,once thewall patterns are established, they can also be sustained inthe absence of MTs, as hypothesized in Zinnia elegans cell sus-pensions (Roberts et al., 2004). The reorganization of the MT arraytherefore represents a critical initial establishment phase for thexylemvessel bands to form,whereas thepatternscanbemaintainedin the absence of MTs during the subsequent phases.Several MT-associated proteins have been implicated in the

MT reorganization during xylem vessel development, including

Figure 7. Rice CSIL1 Can Interact with Secondary Wall CESA4 and Affects Secondary Wall Cellulose Synthesis.

(A) RT-qPCR analysis of CSIL1 expression in rice wild-type and CSIL1 RNAi plants. The relative expression level was estimated by normalizing theexpression of CSIL1 with that of TP1 (control). Mean 6 SD (n = 3).(B) Four-month-old wild-type and two lines of CSIL1 RNAi transgenic plants. Bar = 12 cm.(C)Quantification of the cellulose content in the internodes of wild-type and RNAi plants. Mean6 SD (n = 4; biological replicates by using alcohol-insolubleresidues from eight plants).(D) Scanning electron micrographs of the sclerenchyma cells in cross sections of matured wild-type and CSIL1 RNAi internodes. Bar = 4 mm.(E) Quantification of the thickness of sclerenchyma cell walls shown in (D). Mean 6 SD (n = 40 cells from five plants).(F) Scanning electron micrographs of tracheary elements in wild-type and the CSIL1 RNAi internodes. Bar = 10 mm.(G) Quantification of the band space between wall spirals shown in (F). Mean 6 SD (n = 218 bands from five plants).(H) GFP-CESA4 and mRFP-CSIL1 are colocalized at foci at the plasma membrane of N. benthamiana epidermal cells. Bar = 10 mm.(I) Intensity plot of GFP-CESA4 and mRFP-CSIL1 from transects in ([H], right panel).(J) Split-luciferase complementation assay showing interactions between CESA4 and CSIL1. Bar = 1 cm. Statistical significance was tested by Welch’sunpaired t test: *P < 0.05, **P < 0.005, and ***P < 0.0005.

CSI1/POM2 Steers Xylem Vessel Wall Patterning 2443

MAP65-1, AIR9, MAP70-1, and MAP70-5 (Pesquet et al., 2010;Derbyshire et al., 2015). These proteins contribute to the MT-bundling/stabilization and are important to achieve the MT arrayrearrangements during secondary wall synthesis. In addition,several small GTPases, MICROTUBULE DEPLETION DOMAIN1,aKINESIN13A,anda recentlydescribedmemberof the IQD family(IQD13) are involved in depleting MTs from the areas betweenthickenings (Oda and Fukuda, 2012; Sugiyama et al., 2017).Nevertheless, the mechanism for how the MTs guide cellulosesynthesis during this important developmental process has re-mained elusive. Our results indicate that CSI1/POM2 is necessaryfor MT-based CSC guidance during the initial phase of xylemvessel development but that it is not needed during the sub-sequent stages. Time-course experiments using oryzalin cor-roborate that cell wall patterns, and the tracking of CSCs alongdefined bands, can be maintained also in the absence of MTs.These data indicate that other mechanisms, perhaps cell wall-mediated CSC guidance, may play significant roles during thesestages. It is plausible that xylansand/or othercellwall constituentsthat are deposited along the MT bands may serve this function inthe absence of CSI1/POM2. Computational modeling and NMRexperiments suggest a tight interplay between xylans and cellu-losemicrofibrils (Busse-Wicher et al., 2014; Simmons et al., 2016).Such interactions could influence the direction of the CSCs andtherefore cause them to successively align along the secondarywall bands. Apart from potential cell wall polymers directingcellulose synthesis, the observation that Golgi movement andCesA delivery are different at regions that underlie cell wall bandsindicates that other cellular features may also influence the cellwall band progression. We speculate that the membrane envi-ronment is different in these regions compared with membraneregions that lie between the bands and that these differencesinfluence themovement of theGolgi bodies and, thus, the deliveryof CesAs. Another possibility is that the movement of the Golgi isrestricted due to physical constraints. The secondary wall bandslead to deformation of the plasma membrane, i.e., the membraneis slightly indented below the bands. These deformations couldmake it difficult for Golgi to pass through these regions andperhaps could trap Golgi beneath the bands. While there is muchleft to explore about this process, we find it unlikely that themaintenance of the band patterning is solely due to the cell wallpolymers directing cellulose synthesis.

The primary wall CSCs typically track with a uniform speed andbidirectionality along cortical MTs (Paredez et al., 2006). Thespeed of the primary wall CSCs is reliant on CSI1/POM2 function,as CSC speeds were significantly reduced in csi1/pom2mutants(Gu et al., 2010). During the secondary wall synthesis, we also seeclear alterations in CesA7 speeds in pom2-4; however, thesechanges appear to be largely due to a wider spread of speedsrather than a uniform reduction and are tied to particular de-velopmental stages of xylem vessel development. The increasedvariance in theCSCspeedswasprimarily observedduring themidstagesofsecondarywall synthesis (Figure5G).Whileweobservedmajormisalignment between theMTs andCesA7 trajectories onlyduring the early stages, it is possible that the lack of direct en-gagement of the CSCs with the MTs causes difficulties in main-taining the speeds. It is worth highlighting that although weobservedclear secondarywall bands in thecsi1/pom2 lines, these

bands were less well ordered compared with the wild type. Wespeculate that CSI1/POM2 proteins provide a feedback functionto the formationofMTbandsand that thismaybecompromised inthecsi1/pom2 lines,which in turnmayaffect thefinal cellwall bandpatterns. This is in line with observations during primary wallsynthesiswhereMTorganization isperturbedwhenCSI1/POM2 ismutated (Bringmannet al., 2012; Landrein et al., 2013). If theCSCsare indeed also guided by cell wall components and membraneenvironment during this development stage, as discussed above,it is plausible that such guidance is not optimal and that it canmanifest in changes in the quality and quantity of cellulose that isproduced. These data are in agreement with our recorded changesin the MFA, cellulose microfibril crystallinity, and amorphous cel-lulose in the pom2-4 mutant.The CSI1/POM2 speeds changed during the different stages of

xylem vessel development. For example, the speeds significantlyincreased during the mid stages of transition (Figure 2H). Thesedata are very similar to what has independently been reported forthe secondary wall CesA7 (Watanabe et al., 2015), but contrastthose of Li et al. (2016b). Assuming that the speed of the CSCsrepresents catalytic activity, these findings support a scenario inwhichan increase in speedof tracking andCesAabundance leadsto a major boost in cellulose synthesis, which is compatible withthe rapid development and subsequent death of the xylem ves-sels. Li et al. (2016b) also studied secondarywall CesAbehavior inthe VND7-inducible system and concluded that the CSCsmovedunidirectionally as “swarms” (referred to as “directionally coherentmovement”; Li et al., 2016b) during xylem vessel development.Our data support this report, but it is important to note that theunidirectional movement apparent in CSI1/POM2 was observedbothduringprimarywall synthesis (DMSOtreatment; Figure4) andxylemvessel development and appears to depend on local versusglobal cell wall synthesis.In summary, CSI1/POM2 directs xylem vessel patterning by co-

ordinating the secondary wall CSCs and MTs during the transitionfrom primary to secondary wall synthesis. However, the bandingpatterns can largely be maintained in absence of CSI1/POM2 andMTs during later stages of development.We therefore conclude thatthe wall patterning during proto-xylem development is initiated andmore importantly sustained by two complementary mechanisms.

METHODS

More detailed descriptions of some procedures are provided in SupplementalMethods.

Plant Material

We used the previously described Arabidopsis thaliana lines pom2-4 andpom2-8/csi1-1 (SALK_136239; Bringmann et al., 2012). To generatemultiple marker lines in the VND7 background, we crossed seeds of pom2-4and native promoter-driven triple (3x)YFP-CSI1/POM2 (Worden et al., 2015)into the VND7-inducible Arabidopsis line proCaMV35s::VND7::VP16::GR(Yamaguchi et al., 2010). The F3 progeny was used for analysis. The pom2-4mutant was used as the main allele, as it produces seeds more readilythan some of the more severe csi1/pom2 lines. Note that the pom2-4was generated from a T-DNA population between Nossen and Columbia.We have backcrossed the pom2-4 extensively to Col-0 and we usedsegregating progeny from crosses of pom2-4 and different markers to

2444 The Plant Cell

assure the best possible genetic homogeneity between samples. Togenerate dual-labeled plants for MTs, we crossed 3xYFP-CSI1/POM2 inthe VND7background intomCherry-TUA5 (Gutierrez et al., 2009) and usedthe F2 progeny of that cross. To visualize secondary wall CesAs, wecrossed YFP-CesA7 in the irx3-4 background (Watanabe et al., 2015) intothe wild-type and pom2-4-mutated mCherry-TUA5-VND7 background.We used the F2 generation for experiments. We confirmed the homozy-gosity of the pom2-4 mutation by growing seedlings on solid (0.8% agar)half-strength Murashige and Skoog (MS) medium (pH 5.7) supplementedwith 5% sucrose allowing the identification of the obvious stunted rootphenotype and confirmed via PCR (Supplemental Table 1). We furtherchecked for the presence of YFP and mCherry markers using a fluores-cence stereomicroscope prior to further treatment.

For generation of CSIL1-RNAi plants, the targeted fragments wereamplified from the cDNA of rice (Oryza sativa) CSIL1 and inserted intoPKANNIBAL vectors (see below). The construct was transfected intoAgrobacterium tumefaciens EHA105 and introduced into the wild-typevariety Nipponbare.

Plant Growth Conditions and Treatments

Arabidopsisplantsweregerminatedandgrownessentially asdescribedbyLiu et al. (2016). More specifically, seeds of the VND7-inducible Arabi-dopsis linesdescribed abovewere surface-sterilizedbywashing for 10minin 1.25% sodium-hypochlorite solution supplemented with 0.05% Tween20. Sterilized seeds were washed excessively with sterile water. Seedswere plated on solid (0.8% agar) half-strength MS medium (pH 5.7) sup-plementedwith 1%sucrose for normal growth and5%sucrose forpom2-4genotyping purposes, respectively. Plates were stratified for at least 2 d ina dark cold room (4°C). Germination was triggered by exposing plates for8 h to light (100 mEm22 s21). Subsequently, the plates were wrapped withaluminumfoil andplacedvertically inagrowth roomair-conditioned to60%relative humidity and 21°C temperatures. Seedlings used for spinning discconfocal microscopy were grown for 3 d in the dark, transferred to 24-wellplates containing DMSO (control), or 10 to 100 mM dexamethasone (in-duction) to induce VND7. Subsequently, plates were wrapped with alu-minum foil and placed on a slowly rotating orbital shaker in the growthroom.

The plants used for determination of cell wall alterations were sterilizedfor 3 min in 70% ethanol followed by 10min in 10% bleach, then rinsed sixtimes in sterile distilled water, plated on solid (0.8% agar) half-strengthMSplates,andstratified for2d inadarkcold room (4°C)beforebeing incubatedat21°Cata16/8-h light/darkcycle.Ten-day-oldseedlingswere transferredtosoil andgrownunder thesameconditions for;9weeks throughmaturityto full senescence.

Arabidopsis cell suspension cultures were generated and trachearyelement formation induced, as previously described (Pesquet et al., 2010).

Riceplants, including thewild-typeplants andCSIL1-RNAiplants,weregrown in experimental fields at the Institute of Genetics and DevelopmentalBiology in Beijing and in Linshui, Hainan province during the natural growingseasons.

Nicotiana benthamiana plants were grown in soil in a glasshouse withcontinuous cool white fluorescent lights (100 mE m22 s21) and naturaldaylight at 20 to 26°C, as previously described (Lampugnani et al., 2016).

Live-Cell Imaging

Imaging was done essentially as described by Liu et al. (2016). Seedlingswere observed under the microscope between 10 and 30 h after VND7induction. Induced 3xYFP-CSI1/POM2 seedlings were imaged using theCSU-X1spinningdisk head (Yokogawa)mounted to an invertedNikonTi-Emicroscope equippedwith a 1003oil-immersion objective (PlanApoTIRF,NA 1.45). Fluorescence detection was achieved using an Evolve EM-CCDcamera (Photometrics Technology). Induced YFP-CesA7 seedlings were

imaged using the CSU-W1 spinning disk head (Yokogawa) mounted to aninverted Nikon Ti-E microscope equipped with a 1003 oil-immersionobjective (Apo TIRF, NA1.49). Fluorescencedetectionwas achieved usinga deep-cooled iXon Ultra 888 EM-CCD (Andor Technology). Both setupswere controlled via PC using MetaMorph (Molecular Devices). Photo-bleaching was achieved using either the iLas laser illumination system(Roper Scientific) or the Andor FRAPPA scanning instrument.

Seedlings weremounted on 1.5 grade glass cover slips and covered by1-mm-thick agarose pads made from water supplemented with 1%agarose. We imaged 3xYFP-CSI1/POM2, mCherry-TUA5, and YFP-CesA7 using time-lapse recordings with typical exposure times between200 and 400 ms, time intervals of 10 s, and total durations between 5 and10 min. Fluorescence recovery was recorded in intervals of 2 to 5 s for3xYFP-CSI1/POM2 and 10 s for YFP-CesA7.

For analysis of colocalization of rice CesA4-CSIL1, AgrobacteriumEHA105 harboring GFP-CesA4 andmRFP-CSIL1 were coinjected into thelower epidermis of 4-week-oldN. benthamiana leaves. After cultivation fortwo more days, the leaves were observed with oil immersed objective onthe spinning-disc confocal microscope (Perkin-Elmer UltarVIEW VoX). Toobtain theGFPandmRFP fluorescence images, the 488- and561-nm linesof laser were used for excitation, and emission was detected at 500 to540 nm and 600 to 640 nm, separately.

Scanning Electron Microscopy

For xylem defect analysis, the first internodes of 8-week-old wild-type,pom2-4, and pom2-8 (csi1-1) mutant plants were cut into longitudinalsections and immediately fixed in 2.5% glutaraldehyde in PBS buffer for30 min. Sections were washed three times in PBS and subsequently threetimes in water. Dehydration was achieved by washing the sections forminimum 1 h each in an ethanol series from 10% to 100% in 10% steps.After several washes with 100% ethanol, critical point drying was per-formed and the dried samples were gold-coated. Examination of thesamples was performed using an XL30 field-emission scanning electronmicroscope from Phillips.

The second internodes of mature wild-type and CSIL1-RNAi plantswere fixed in 4% paraformaldehyde (Sigma-Aldrich). To view the wallthickness of sclerenchyma cells, the internodes were transversely cut toexpose the epidermal sclerenchyma cells. To observe the secondarypattern of vessel cells, the internodes were longitudinally cut under thestereoscope. After critical-point drying, the samples were sprayed withgold particles and observed with a scanning electron microscope(S-3000N; Hitachi).

Cell Wall Staining

To label the secondary walls in VND7-induced wild-type and pom2-4mutants, DirectRed23 (Anderson et al., 2010; Sigma-Aldrich)was added toa final concentration of 0.06% to six-well plates containing 3-d-oldseedlings 24 h after induction. The samples were washed with ultrapurewater to reduce the amount of unbound dye. Subsequently, samples wereobserved under the spinning diskmicroscope by recording z-stacks usinga 561-nm laser and 610/40-nm emission filters.

Image Analyses

The velocity of CSI1 foci was measured using the open-source softwareFIESTA (Ruhnow et al., 2011). Briefly, the velocity of moving foci isdetermined by measuring their slope in kymograph projections. Wemeasured 1015 trajectories for noninduced cells and 905, 1391, and367 trajectories for induced cells in early, mid, and late stages of thesecondary wall program, respectively. Colocalization of CSI1 with MTsandCesA7wasmeasured using the JaCoPplug-in of Fiji. To increase thereliability of the colocalization measurements, we used a dual approach

CSI1/POM2 Steers Xylem Vessel Wall Patterning 2445

of measuring Pearson’s and Mander’s coefficients. Furthermore, weusedCostes randomization to validate the significanceof thedeterminedPearson’s coefficients. Costes-randomized image series always hada Pearson’s coefficient of at least a factor of 50 lower than the originalimage series.

To quantify the insertion rate of 3xYFP-CSI1 and YFP-CesA7 afterphotobleaching, we used the ThunderSTORM plug-in of Fiji to detect theappearanceof foci in thebleachedareas.Weanalyzed the recovery inareasslightly smaller than the bleached area to avoidmigrating complexes in theplasma membrane to be included in the recovery signal. We plotted thenumber of detected foci over time and analyzed the recovery usinga monoexponential growth model (reaction-limited case).

Themisalignment betweenCesA trajectories andMTswasmeasured indual-color average projections of the time series using Fiji. We measuredthe angle of short stretches of clearly visible CesA7 trajectories andcompared them to the angle of the underlying MTs. For each cell, at least10 trajectory-MT pairs were measured.

Orientation and spacing of secondary wall bands in VND7-inducedseedlingsweremeasuredusingFiji. Z-stacksweresmoothedandaverage-projected using inbuilt Fiji plug-ins. Subsequently, individual cells werecroppedand alignedwith the growth axis of the seedling. A total of 136 and132 VND7-induced cells were captured for wild-type and the pom2-4mutant, respectively. We quantified the average orientation of secondarywall bands and the variability of band orientations within each cell, termeddispersion, using theFiji plug-in “Directionality”with default settings. Bandspacingwasanalyzedusingacustom-madeMatlab (Mathworks) program.Briefly, the programdisplayed the intensity profile along the long axis of thecell and a graphical user interface subsequently allowed for the de-termination of band positions in a point-and-click manner.

Optical Flow Analysis

Theoptical flowwasanalyzedusing theFiji plug-inPIVanalyzer using4-by-4pixel averaging, interpolation, and a mask of 0.1. The image series werepreprocessed using subtract background (50 pixel sliding paraboloid) andfour-frame walking averaging. The resulting optical flow image series wasaverage projected to obtain images displaying the mean optical flow ofintensity. The direction of the optical flow was determined using Fiji bydecomposing themeanoptical flow images into hue (H), saturation (S), andbrightness (B) with the following thresholds: for movement to the right (Hbetween 34 and 94, S between 50 and 255, and B between 1 and 255), formovement to the left (Hbetween161and221,Sbetween50and255, andBbetween 1 and 255), for movement into both directions (H between 0 and255, S between 0 and 50, and B between 1 and 255).

Biochemical Analyses

The MFA of at least 18 Arabidopsis stems from VND7 and the pom2-4mutant in VND7 was measured using an x-ray diffraction technique(Ukrainetz et al., 2008). The bottom 3 cm of mature, senesced plant stemswas used for analysis. The 002 diffraction spectra of each stem werescreened for T-value distribution and symmetry on a Bruker D8 discoverx-ray diffraction unit equipped with an area array detector (GADDS). Wide-angle diffraction was used in the transmission mode, and measurementswere made with CuKa1 radiation (l = 1.54 Å). The x-ray source was fit witha 0.5-mm collimator and a GADDS detector collected the scatteredphotons. Thex-ray sourceand thedetectorwerebothset at a thetaangleof0°. The diffraction data were integrated using GADDS software and furtheranalyzed to estimate MFA values.

Cell wall crystallinity was determined on the same stems used formeasuring MFA, using the same x-ray unit and parameters as the MFAmeasurements, except the source thetawas set at 17°. Thediffractiondatawere integrated using GADDS software and the output data further

analyzed using a crystallinity calculation program based on the Vonkmethod (Vonk, 1973).

Cellulose Content

After x-raydatacollectionwascomplete, thesamestemswere thenpooledby genotype andground on aThomasWileyMiniMill to pass through a #60mesh (250 mm). The powdered sample was then dried for 24 h at 50°C and15 mg of tissue was weighed into each preweighed tube. At least threetechnical replicateswere done on each pooled genotype. First, the alcoholinsoluble residue (AIR) was prepared as described by Pattathil et al. (2012).The AIR was then subjected to a series of extractions in a proceduremodified from the AIR fractionation method also described by Pattathilet al. (2012). Themodifications involved completing the chlorite extractionfirst as well as the removal of both the 1 M and the postchlorite 4 M po-tassium hydroxide extractions. The resulting cellulose residue was thenpredried in a vacuum centrifuge and finished in a 50°C oven for 48 h beforethe final weights were measured.

Degree of Polymerization

The resulting cellulose was dissolved at 5 mg/mL in 9% lithium chloride(LiCl)/N,N-dimethylacetamide (DMAc) through a four-step solvent ex-change of nanopurewater, anhydrous ethanol, DMAc, and9%LiCl/DMAc.Once dissolved, the samples were diluted to 0.5 mg/mL cellulose in 0.9%LiCl/DMAc and each was separated on an Agilent 1100 SEC systemcontaining Waters Styragel HR4 and HR6 columns coupled to a WyattDawnHeleos II light scatteringdetector. Theaveragemolecularweightwascalculated from the output using Wyatt’s Astra 6 software before con-verting to degree of polymerization.

Expression and Phylogenetic Analyses

Rice CSI1 homologous genes were identified based on the annotation ofthe rice genome database (Rice Genome Annotation Project, http://rice.plantbiology.msu.edu/). The phylogenetic tree of CSI1 and its like proteinsin rice and Arabidopsis was generated using maximum likelihood with theMEGA5 softwarewith 1000 bootstrap replications (Supplemental Data Set1; Tamura et al., 2011). The spatio-temporal expression profiles of riceCSIL1 were from the expression data in RiceXPro database (http://ricexpro.dna.affrc.go.jp/).

To examine the expressionofCSIL1 in thewild-type and transgenic plants,total RNA was extracted from young internodes using the Plant RNA Puri-ficationReagent (Invitrogen), and cDNAwas synthesized fromRNAusingthe Reverse Transcription system kit (Takara). The expression level ofCSIL1 was examined by qPCR with a CFX96 real-time system (Bio-Rad)using riceHNRas internal control. Theprimers for theRNAi construct andqPCR analyses are listed in Supplemental Table 1.

Constructs

The CSIL1-RNAi construct was generated by amplifying CSIL1 from a ricecDNA library, and thecDNAwas inserted intoaPKANNIBALvector (Wesleyet al., 2001) usingBamHI andXbaI. The construct was transformed into thewild-type variety Nipponbare ecotype using Agrobacterium. The expres-sion level of CSIL1was quantified by qPCRwith aCFX96 real-time system(Bio-Rad). Coding sequences of Arabidopsis EH1, CSI1/POM2, CesA4,CesA7, andCesA8were amplified fromcDNAand cloned into pAMONandpSUR using the Gibson assembly method to generate N-terminal fusionsto VN155 (I152L) and VC155, respectively (Lee et al., 2014), for BiFCanalyses (see below). All primers are listed in Supplemental Table 1.

2446 The Plant Cell

Transgenic cell suspensions were produced by coculture with Agro-bacterium transformed with 35S:CSI1-RNAi construct (Derbyshire et al.,2015) as previously described byPesquet et al. (2010). Expression levels ofCSI1 were measured using RT-qPCR on five independent biological re-peats (primer sequences andmethod described in Derbyshire et al., 2015)and expressed as percentage of CSI1-to-UBIQUITIN gene ratio. Down-regulated lines showeda residualCSI1 expressionof 57%617% (mean6SD) compared with wild-type cells (100% 6 13%, P < 0.005, Welch’sunpaired t test). The expression of the different CSIs and primary andsecondary CesAs during the TE differentiation time course have beenchecked using macroarray data (GEOGSE73146; Derbyshire et al., 2015).

Interaction Analyses

Coding sequences of EH1, CSI1/POM2, CesA4, CesA7, and CesA8 wereamplified from cDNA using the primers defined in Supplemental Table 1and cloned into linearized BiFC vectors pURIL [GENE-V(I152L)N], pDOX(GENE-VC), pAMON [V(I152L)N-GENE], or pSUR (VC-GENE) using theGibson assembly method as previously described (Lampugnani et al.,2016). The pURIL and pDOX were linearized using KpnI and SfoI, whilepAMONand pSURwere linearized usingBamHI andSfoI. EH1was clonedinto pURIL and pDOX, whileCSI1/POM2,CesA4,CesA7, andCesA8werecloned into pAMON and pSUR to generate C-terminal and N-terminalfusions respectively. Constructs were introduced into Agrobacterium andcombinations of BiFC constructs, together with Agrobacterium strainscarrying 35S::CFP-N7 (Kaplan-Levyet al., 2014) andP19 ,were introducedinto N. benthamiana leaves by infiltration following the procedure de-scribed by Zhang et al. (2016). Leaves were examined for fluorescence 3 dpostinfiltrationonan invertedNikonTi-EmicroscopeequippedwithaCSU-W1 spinning disk head (Yokogawa). Detection occurred using a 1003 oil-immersion objective (Apo TIRF, NA 1.49) and an iXon Ultra 888 EM-CCD(Andor Technology). All BiFC combinations were imaged under the sameconditions. Specifically, a 445-nm laser line was used to excite CFP, whilea 515-nm laser line was used to excite YFP. Emissions were detected with470/40 and 535/30 band-pass filters. Z-stacks of images were collectedusing exposure times of 100 ms.

The split-luciferase complementation assay was performed as de-scribed (Chenetal., 2008). Inbrief, thecDNAofCSIL1,CESA4,CESA7, andCESA9were amplified (Supplemental Table 1) and inserted into the binaryvectors for expression fusedwith N- or C-terminal luciferase. The resultingconstructs were transfected into Agrobacterium strain C58 and infiltratedwith the leaves of 4-week-oldN. benthamiana. Interaction was determinedbased on the fluorescent signal intensity harvested by IndiGO software.

Accession Numbers

Sequence data from this article can be found in the Arabidopsis GenomeInitiative or GenBank/EMBL databases under the following accessionnumbers: CSI1/POM2, At2g22125; CesA4, At5g44030; CesA6, At5g64740;CesA7, At5g17420;CesA8, At4g18780; VND7,At1g71930; TUA5, At5g19780;EH1, At1g20760; OsCSIL1, Os06g11990; OsCesA4, Os01g54620; OsCesA7,Os10g32980; OsCesA9, Os09g25490; and GEO GSE73146.

Supplemental Data

Supplemental Figure 1. Defects in CSI1/POM2 Cause AberrantSecondary Wall Patterns.

Supplemental Figure 2. Representative Images of Primary WallSynthesis and Different Stages (Early, Mid, and Late) of Xylem VesselDevelopment.

Supplemental Figure 3. BiFC Assay Demonstrating Interactionsbetween CSI1/POM2 and Secondary Wall CesA4, CesA7, and

CesA8 Transiently Expressed in Epidermal Cells of N. benthamianaLeaves.

Supplemental Figure 4. CSI1/POM2 Recovers More Quickly afterPhotobleaching Than CesA7 during Xylem Vessel Formation.

Supplemental Figure 5. Schematic Workflow of Optical Flow Analyses.

Supplemental Figure 6. Secondary Wall CesA7 Tracks along Micro-tubules throughout All Stages of Xylem Vessel Development in Wild-Type Background, but Not in the pom2-4 Mutant.

Supplemental Figure 7. YFP-CSI1/POM2 Can Maintain Tracks alongBands in the Absence of MTs.

Supplemental Figure 8. Rice CSIL1 Is a Homolog of CSI1/POM2 andCan Interact with Secondary Wall Rice CesAs.

Supplemental Table 1. Primers used for BiFC constructs.

Supplemental Movie 1. Cellular Distribution of 3xYFP-CSI1/POM2 inNoninduced Cells and Early, Mid, and Late Stages of Secondary WallFormation.

Supplemental Movie 2. YFP-CesA7 Trajectories Are Not Aligned withCortical MTs in the pom2-4 Mutant during Early Stages of SecondaryWall Formation.

Supplemental Movie 3. YFP-CesA7 Quickly Recycles at MT Bandsafter Fluorescence Photobleaching.

Supplemental Movie 4. YFP-CesA7 Quickly Recycles to Sites ofSecondary Wall Formation also in the Absence of MTs.

Supplemental Data Set 1. Multiple Protein Sequence Alignment ofCSI Proteins in Arabidopsis and Rice.

Supplemental Data Set 2. ANOVA Tables.

Supplemental Methods.

ACKNOWLEDGMENTS

S.P. was funded by a R@MAP Professorship at University of Melbourne.Thisworkwas inpart supportedbyanARCDiscovery grant (DP150103495),a Future Fellowship grant (FT160100218), and the National Natural ScienceFoundation of China (Grant 31530051). S.D.M. acknowledges funding fromthe NSERC Discovery program. We thank Taku Demura for sharing theVND7-line. The P19 plasmid was a kind gift from David Baulcombe.

ReceivedApril 18, 2017; revisedAugust29, 2017; acceptedSeptember24,2017; published September 25, 2017.

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CSI1/POM2 Steers Xylem Vessel Wall Patterning 2449

DOI 10.1105/tpc.17.00309; originally published online September 25, 2017; 2017;29;2433-2449Plant Cell

McFarlane, Edouard Pesquet, Totte Niittyla, Shawn D. Mansfield, Yihua Zhou and Staffan PerssonRene Schneider, Lu Tang, Edwin R. Lampugnani, Sarah Barkwill, Rahul Lathe, Yi Zhang, Heather E.

DevelopmentTwo Complementary Mechanisms Underpin Cell Wall Patterning during Xylem Vessel

 This information is current as of December 13, 2020

 

Supplemental Data

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