Symplastic signaling instructs cell division, cell ... · Symplastic signaling instructs cell...

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Symplastic signaling instructs cell division, cell expansion, and cell polarity in the ground tissue of Arabidopsis thaliana roots Shuang Wu a,b,c,1 , Ruthsabel OLexy b , Meizhi Xu a,c , Yi Sang b,2 , Xu Chen a,c , Qiaozhi Yu a,c , and Kimberly L. Gallagher b,1 a College of Horticulture, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, China; b Department of Biology, 121 Carolyn Lynch Laboratories, University of Pennsylvania, Philadelphia, PA 19104; and c Haixia Institute of Science and Technology, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, China Edited by Sarah Hake, University of California, Berkeley, CA, and approved August 5, 2016 (received for review June 28, 2016) Cell-to-cell communication is essential for the development and patterning of multicellular organisms. In plants, plasmodesmata (PD) provide direct routes for intercellular signaling. However, the role that PD-mediated signaling plays in plant development has not been fully investigated. To gain a comprehensive view of the role that symplastic signaling plays in Arabidopsis thaliana, we have taken advantage of a synthetic allele of CALLOSE SYNTHASE3 (icals3m) that inducibly disrupts cell-to-cell communication specifi- cally at PD. Our results show that loss of symplastic signaling to and from the endodermis has very significant effects on the root, in- cluding an increase in the number of cell layers in the root and a misspecification of stele cells, as well as ground tissue. Surprisingly, loss of endodermal signaling also results in a loss of anisotropic elongation in all cells within the root, similar to what is seen in radially swollen mutants. Our results suggest that symplastic signals to and from the endodermis are critical in the coordinated growth and development of the root. symplastic signaling | plasmodesmata | cell division | cell expansion | cell polarity A n enduring question in developmental biology is how cellular patterning and specification of cell fate are achieved. How are the processes of cell division, cell expansion, and cell differ- entiation coordinated over multiple cell distances to produce a functional organ? In plants, where nearly all cells are connected to their neighbors by plasmodesmata (PD; intercellular bridges), coordination of cellular patterning with growth and development could occur symplastically (through PD). Indeed, numerous groups have shown that proteins and RNAs are able to move between PD and that this movement is correlated with predict- able changes in plant growth and development (reviewed in ref. 1). For example, in the patterning of root hairs, a mobile signal, CAPRICE (CPC) moves from the stele and from nonhair cells into the hair cells where it is sequestered by GLABRA3 to in- hibit the nonhair cell fate (24). Likewise, during flowering, the mobile signal FLOWERING LOCUS T (FT) moves from leaf tissue into the shoot apical meristem to induce floral develop- ment (57). Consistent with an essential role for PD in plant development, rather mild increases or decreases in PD-mediated signaling are associated with seedling and/or embryonic lethality (e.g., INCREASED SIZE EXCUSION LIMIT 1 and 2 and GFP ARRESTED TRAFFICKING 1) (812). Tissue-specific defects in PD-mediated protein movement are associated with defects in stomatal patterning and spacing and defects in root patterning and cellular specification (13, 14). One of the obstacles to understanding the function of PD in plant development has been a lack of tools for specifically modi- fying PD-mediated protein movement. There are no drugs that specifically inhibit PD-mediated protein trafficking, as there are for other processes like secretion and endocytosis. Recently, however, Vatén et al. designed a synthetic form of CALLOSE SYNTHASE 3 (icals3m) that can be used to inducibly block movement of proteins and RNA via PD (14). Induction of icals3m results in an increase in callose, which mechanically decreases the size of the PD ap- erture and blocks protein and RNA trafficking through PD. Pre- viously, we showed that stele-specific expression of the icals3m transgene in the roots of Arabidopsis thaliana blocked movement of the SHORT-ROOT (SHR) protein from the stele into the endodermis and transport of miRNA 165/66 from the endodermis into the stele, which resulted in defects in xylem patterning and the normal development of the ground tissue (14). Since then, the icals3m system has been used to show that changes in symplastic connectivity are required for generation and emergence of lateral roots and proper development of the shoot apical meristem (1518). A loss-of-function callose synthase system has been used to examine tropisms in the hypocotyl of A. thaliana (19). To address the role of PD in the coordination of tissue patterning and plant development, we use icals3m to specifically block PD-mediated movement to and from the endodermis in A. thaliana roots. By blocking trafficking to and from the endodermis, we restrict symplastic signals from the stele into outer cell layers and like- wise from the cortex or epidermis to the stele. We find that in the root meristem, symplastic signals are required for proper speci- fication of the endodermis, coordination of growth between the endodermis and the cortex, and maintenance of cell polarity in the ground tissue. Outside of the meristem, symplastic signals Significance Cell-to-cell communication is critical for the coordination of developmental processes between cells and tissues within an organism. Mechanical signals, secreted peptides, and hor- mones can all convey information between cells. In plants, neighboring cells can directly communicate with each other via plasmodesmata (PD). It has long been assumed that the exchange of proteins and RNAs via PD is important for the regulation of plant development. However, the tools for large-scale testing of this hypothesis have been lacking. Here we exploit a tool that effectively blocks movement of proteins and small RNAs between cells to show that PD-dependent cell- to-cell signaling is important for the coordination of cell divi- sions, cell polarity, and cell expansion between cell layers in the Arabidopsis root. Author contributions: S.W. and K.L.G. designed research; S.W., R.O., M.X., Y.S., Q.Y. and K.L.G. performed research; S.W., R.O., M.X., X.C., and K.L.G. analyzed data; and S.W. and K.L.G. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. 1 To whom correspondence may be addressed. Email: [email protected] or gallagkl@sas. upenn.edu. 2 Present address: Ministry of Education Key Laboratory of Cell Activities and Stress Adaptations, School of Life Sciences, Lanzhou University, Lanzhou 730000, China. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1610358113/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1610358113 PNAS | October 11, 2016 | vol. 113 | no. 41 | 1162111626 PLANT BIOLOGY Downloaded by guest on June 14, 2020

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Symplastic signaling instructs cell division, cellexpansion, and cell polarity in the groundtissue of Arabidopsis thaliana rootsShuang Wua,b,c,1, Ruthsabel O’Lexyb, Meizhi Xua,c, Yi Sangb,2, Xu Chena,c, Qiaozhi Yua,c, and Kimberly L. Gallagherb,1

aCollege of Horticulture, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, China; bDepartment of Biology, 121 Carolyn Lynch Laboratories,University of Pennsylvania, Philadelphia, PA 19104; and cHaixia Institute of Science and Technology, Fujian Agriculture and Forestry University, Fuzhou350002, Fujian, China

Edited by Sarah Hake, University of California, Berkeley, CA, and approved August 5, 2016 (received for review June 28, 2016)

Cell-to-cell communication is essential for the development andpatterning of multicellular organisms. In plants, plasmodesmata(PD) provide direct routes for intercellular signaling. However, therole that PD-mediated signaling plays in plant development has notbeen fully investigated. To gain a comprehensive view of the rolethat symplastic signaling plays in Arabidopsis thaliana, we havetaken advantage of a synthetic allele of CALLOSE SYNTHASE3(icals3m) that inducibly disrupts cell-to-cell communication specifi-cally at PD. Our results show that loss of symplastic signaling to andfrom the endodermis has very significant effects on the root, in-cluding an increase in the number of cell layers in the root and amisspecification of stele cells, as well as ground tissue. Surprisingly,loss of endodermal signaling also results in a loss of anisotropicelongation in all cells within the root, similar to what is seen inradially swollen mutants. Our results suggest that symplastic signalsto and from the endodermis are critical in the coordinated growthand development of the root.

symplastic signaling | plasmodesmata | cell division | cell expansion |cell polarity

An enduring question in developmental biology is how cellularpatterning and specification of cell fate are achieved. How

are the processes of cell division, cell expansion, and cell differ-entiation coordinated over multiple cell distances to produce afunctional organ? In plants, where nearly all cells are connected totheir neighbors by plasmodesmata (PD; intercellular bridges),coordination of cellular patterning with growth and developmentcould occur symplastically (through PD). Indeed, numerousgroups have shown that proteins and RNAs are able to movebetween PD and that this movement is correlated with predict-able changes in plant growth and development (reviewed in ref. 1).For example, in the patterning of root hairs, a mobile signal,CAPRICE (CPC) moves from the stele and from nonhair cellsinto the hair cells where it is sequestered by GLABRA3 to in-hibit the nonhair cell fate (2–4). Likewise, during flowering, themobile signal FLOWERING LOCUS T (FT) moves from leaftissue into the shoot apical meristem to induce floral develop-ment (5–7). Consistent with an essential role for PD in plantdevelopment, rather mild increases or decreases in PD-mediatedsignaling are associated with seedling and/or embryonic lethality(e.g., INCREASED SIZE EXCUSION LIMIT 1 and 2 and GFPARRESTED TRAFFICKING 1) (8–12). Tissue-specific defectsin PD-mediated protein movement are associated with defects instomatal patterning and spacing and defects in root patterningand cellular specification (13, 14).One of the obstacles to understanding the function of PD in

plant development has been a lack of tools for specifically modi-fying PD-mediated protein movement. There are no drugs thatspecifically inhibit PD-mediated protein trafficking, as there are forother processes like secretion and endocytosis. Recently, however,Vatén et al. designed a synthetic form of CALLOSE SYNTHASE 3(icals3m) that can be used to inducibly block movement of proteins

and RNA via PD (14). Induction of icals3m results in an increasein callose, which mechanically decreases the size of the PD ap-erture and blocks protein and RNA trafficking through PD. Pre-viously, we showed that stele-specific expression of the icals3mtransgene in the roots of Arabidopsis thaliana blocked movementof the SHORT-ROOT (SHR) protein from the stele into theendodermis and transport of miRNA 165/66 from the endodermisinto the stele, which resulted in defects in xylem patterning andthe normal development of the ground tissue (14). Since then, theicals3m system has been used to show that changes in symplasticconnectivity are required for generation and emergence of lateralroots and proper development of the shoot apical meristem (15–18). A loss-of-function callose synthase system has been used toexamine tropisms in the hypocotyl of A. thaliana (19). To addressthe role of PD in the coordination of tissue patterning and plantdevelopment, we use icals3m to specifically block PD-mediatedmovement to and from the endodermis in A. thaliana roots. Byblocking trafficking to and from the endodermis, we restrictsymplastic signals from the stele into outer cell layers and like-wise from the cortex or epidermis to the stele. We find that in theroot meristem, symplastic signals are required for proper speci-fication of the endodermis, coordination of growth between theendodermis and the cortex, and maintenance of cell polarity inthe ground tissue. Outside of the meristem, symplastic signals

Significance

Cell-to-cell communication is critical for the coordination ofdevelopmental processes between cells and tissues within anorganism. Mechanical signals, secreted peptides, and hor-mones can all convey information between cells. In plants,neighboring cells can directly communicate with each othervia plasmodesmata (PD). It has long been assumed that theexchange of proteins and RNAs via PD is important for theregulation of plant development. However, the tools forlarge-scale testing of this hypothesis have been lacking. Herewe exploit a tool that effectively blocks movement of proteinsand small RNAs between cells to show that PD-dependent cell-to-cell signaling is important for the coordination of cell divi-sions, cell polarity, and cell expansion between cell layers in theArabidopsis root.

Author contributions: S.W. and K.L.G. designed research; S.W., R.O., M.X., Y.S., Q.Y. andK.L.G. performed research; S.W., R.O., M.X., X.C., and K.L.G. analyzed data; and S.W. andK.L.G. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1To whom correspondence may be addressed. Email: [email protected] or [email protected].

2Present address: Ministry of Education Key Laboratory of Cell Activities and Stress Adaptations,School of Life Sciences, Lanzhou University, Lanzhou 730000, China.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1610358113/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1610358113 PNAS | October 11, 2016 | vol. 113 | no. 41 | 11621–11626

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from the endodermis are required for normal anisotropic cellexpansion in the root elongation zone.

Results and DiscussionTransient Induction of icals3m in the Endodermis Blocks both Targetedand Nontargeted Movement. Previously we showed that expressionof the icals3m transgene from the Cytokinin Response 1 (CRE1/akaWOODENLEG, WOL) promoter was sufficient to block move-ment of proteins out of the stele (14). To test the ability of icals3mto block movement into the endodermis, we expressed the icals3mtransgene from the Endodermis 7 (EN7) promoter. The EN7promoter is active in the endodermis, cortical endodermal initial(CEI) cells, and the cortical endodermal daughter (CED) cells,but absent from the quiescent center (QC) (20). As early as 3 hpostinduction with estradiol, we saw a clear accumulation of cal-lose in the endodermis of roots with the EN7:icals3m transgene(Fig. 1 A and B). Within 24 h of induction, the levels of callosewere much higher in the endodermis of induced roots comparedwith controls. To determine whether this increase in calloseblocked transport through PD, we introduced two markers: SHR:SHR-GFP and SUC2:GFP into the EN7:icals3m-expressing plants.SHR-GFP, which facilitates its own movement through PD, servesas a marker of targeted movement, whereas free GFP driven bythe SUC2 promoter serves as a marker for nontargeted movement(i.e., diffusion) (21, 22). Before induction with estradiol, SHR-GFP was present in the stele, endodermis, and QC cells (Fig. 1C).After induction of EN7:icals3m, SHR-GFP in the endodermisdiminished to background, but was still present in the QC, con-sistent with a lack of EN7 activity in QC cells (Fig. 1D and E). TheSUC2 promoter drives GFP expression in the phloem; from thephloem, GFP diffuses freely throughout the meristem (Fig. 1F)(14, 22). Induction of EN7:icals3m significantly limited movementof GFP into the endodermis and surprisingly all cells outside ofthe stele (Fig. 1 G and H). Because EN7 does not drive icals3mexpression in QC cells (20), this suggests that there is very little

shootward movement of GFP once it has trafficked through theQC into the columella (in the direction indicated by arrows in Fig.1G, Inset). Therefore, our results show that the expression oficals3m in the endodermis can effectively block both targeted andnontargeted movement into this cell layer. Additionally, basedupon the SUC2:GFP results, it appears that passive movementthrough the QC and then shootward into the lateral tissues (i.e.,lateral root cap, epidermis, and cortex) may be generally limited.

Symplastic Isolation of the Endodermis Causes Defects in Root Growthand Patterning and a Lack of Coordinated Cell Divisions Between theEndodermis and Cortex. To determine how a loss of PD-mediatedendodermal signaling affects root development, seedlings con-taining the EN7:icals3m transgene were transferred to estradiol-containing media or control media. The transferred seedlings werethen imaged at various time points, up to 60 h after transfer.During normal root development, cells expand anisotropically asthey exit the meristem (Fig. 2A). When SHR moves from thestele into the endodermis where it turns on miRNA165/166;miRNA165/66 in turn moves from the endodermis into the stele,where it patterns xylem in a concentration-dependent manner(23). Consistent with a loss of SHR or miRNA165/166 movement,the xylem was mispatterned in EN7:icals3m roots treated withestradiol (Fig. 2 A and B) (14, 23). In addition, the cells in theelongation zone were shorter and wider than controls, having anappearance similar to radially swollen mutants. These results in-dicate that endodermal signaling is required to maintain normaldevelopmental patterning and polarized cell elongation in multi-ple cell layers in the root.In the meristem, activation of EN7:icals3m resulted in the

expansion of endodermal cells and defects in radial patterning(Fig. 2 C–F). In wild-type roots, the endodermal cell lineageoccupies a single cell file (Fig. 2C). However, after inductionof EN7:icals3m, the endodermal lineage expanded into multiplecell files. Sixty hours after induction, nearly all cells within theendodermal cell lineage had divided, creating one to two

Fig. 1. Transient induction of icals3m in the endodermis blocks symplasticcommunication between cells. Aniline blue staining showing the accumulationof callose in the root tips of seedlings (A) before and (B) after induction of theEN7:icals3m transgene. Note the endodermal-specific accumulation of callosein B. (C–H) Confocal images of roots expressing the (C–E) SHR:SHR-GFP trans-gene and (F–H) SUC2:GFP (C and F) before and (D, E, G, and H) after inductionof the EN7:icals3m expression. Induction of the EN7:icals3m blocks movementof (D and E) SHR-GFP and (G and H) free GFP throughout the root meristem.Times after induction aremarked. E, endodermal lineage; D, epidermis; C, cortex.In all cropped half-root images (e.g., D–H) the epidermis is oriented to the leftside of the image. (Scale bars, 25 μm.)

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Fig. 2. Disruption of symplastic signaling in the endodermis alters endoder-mal cell expansion and radial patterning. (A and B) Confocal images of theelongation zone of roots (A) before and (B) after activation of the EN7:icals3mtransgene. White arrowheads in B point to areas of aberrant xylem develop-ment. (C–F) Confocal micrographs of the root meristem (C) before and (D–F)after induction with estradiol. (G and H) Two different roots 45 h after in-duction of the CRE1:icals3m transgene. Note that compared with D–F, theground tissue (G and H) is normally patterned following induction of CRE1:icals3m (quantified in Fig. S1). (I–M) Cross-sections through (I) uninduced EN7:icals3m roots, (J) roots expressing the SCR:SHR-nlsGFP transgene, and (K) in-duced EN7:icals3m roots; every other cortical cell is numbered. L and M aremagnified images of the same cells (yellow boxes) in I and K, respectively. E,endodermal lineage; C, cortex. Confocal images of roots of the EN7:icals3mgenotype are outlined in blue. Those expressing CRE1:icals3m are outlined ingreen. Times indicate duration of induction. (Scale bars, 25 μm.)

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additional cell layers (Fig. 2 D–F and Fig. S1A). We observed nodefects in the patterning of the cortex or the epidermis; theseremained as single cell layers, suggesting that the effect on radialpatterning is specific to the endodermis. To test whether thedefects observed require a bona fide endodermis, we blockedPD-mediated signaling in shr-2 roots, which lack a typical en-dodermis (24). To drive the expression of the icals3m transgenein the ground tissue of shr-2 roots, we tried both the EN7 pro-moter and the J0571:GAL4 enhancer trap system (https://www.arabidopsis.org/abrc/haseloff.jsp) (24). The EN7 promoter showedpoor expression in shr-2 roots (Fig. 3A). However, the levels ofJ0571:GAL4; UAS:erGFP in the ground tissue of shr-2 roots wassimilar to wild-type (Fig. 3B). J0571:GAL4 was therefore used toinducibly expressUAS:icals3m in shr-2 roots. shr-2 roots expressingthe J0571:GAL4; UAS:icals3m transgenes showed increased cellexpansion in the mutant cell layer, but no change in radial pat-terning (Fig. 3 B–E). Likewise wild-type roots expressing theJ0571:GAL4; UAS:icals3m transgenes showed expansion of boththe endodermis and cortex cell layers, but no change in the ori-entation of the cell division in the cortex (Fig. 3 F–J). By about45 h postinduction, the phenotypes of the wild-type roots expressingthe EN7:icals3m or the J0571:GAL4; UAS:icals3m transgenes weresimilar (Fig. 3I). These results suggest that the effect of the icals3mtransgene on cell division in the ground tissue is specific tothe endodermis.

Previously, Helariutta et al. showed that expression of SHRdirectly in the endodermis resulted in an increase in the numberof endodermal cell layers and the number of cortex cells (24).Likewise Nakajima and colleagues showed in argonaute 1 (ago1-101) roots that an increase in endodermal cell layers (25) is ac-companied by an increase in the number of cortex cells. In theSCR:SHR-GFP and the ago1-101 roots the cortex cell layer dividesanticlinally (in the longitudinal dimension) to produce additionalcortex cells (an increase from the normal eight cells per layer),which accommodates the expansion in endodermal cell layers (24,25). Shown here, the expression of SHR-nlsGFP (21) from theSCR promoter results in the same multilayered endodermalphenotype observed by Helariutta et al. (24) (Fig. 2J). Likewise,the number of cortex cells in the roots expressing SCR:SHR-nlsGFP increases (in the root shown from 8 to 18; Fig. 2J).Surprisingly in the estradiol-induced roots, there is an increasein the number of cell layers in the endodermis, but no com-pensatory change in cortex cell numbers (Fig. 2 K and M). Thecortex maintains the eight-cell number but the cells appearstretched, presumably to accommodate the increased root girth(Fig. 2 K and M). These cortex cells are alive; there is no pen-etration of propidium iodide into the cell (Fig. 2 D–F). Theseresults suggest that symplastic signals (as opposed to mechanicalsignals, for example) from the endodermis are required to in-duce compensatory changes in cell division in the cortex thatbalance the increase in cell number in the endodermis.

Fig. 3. Symplastic regulation of radial patterning isspecific to the endodermis. Confocal images of shr-2roots showing expression of EN7:H2B-GFP (A) and UAS:erGFP driven by the J0571 enhancer trap line (B–D).shr-2 roots expressing the J0571:GAL4; UAS:icals3mand UASerGFP transgenes (B) before and (C and D)after induction with estradiol. (D) Quantification ofground tissue width in shr-2 roots expressing theJ0571:GAL4; UAS:icals3m; UASerGFP transgenes.(E–I ) Wild-type roots expressing the J0571:GAL4;UAS:icals3m transgenes (F) before and (F–I) after in-duction with estradiol (times as indicated). (J–M) Wild-type roots expressing the E903:GAL4; UAS:icals3mtransgenes. The variable expression pattern of E903 isshown by the variability in erGFP expression: (J) only inthe epidermis; (K) epidermis and cortex; (L) cortex andendodermis; and (M) epidermis and cortex with weakexpression in endodermis. Note that only roots withsignificant expression in the endodermis (L, arrows)show changes in radial patterning in response to in-duction of the icals3m transgene. (N) Induction ofEN7:icals3m transgenes in pin2 roots causes defects inradial patterning. Arrows point to the periclinal celldivisions. E, endodermal lineage; M, mutant groundtissue layer in shr-2 mutants; C, cortex; D, epidermis.

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When the EN7:icals3m transgene is activated, symplastic sig-nals emanating from the cortex and epidermis into the endo-dermis are blocked. Likewise symplastic signals coming from thestele into the endodermis are also blocked. To determine whichcell layers are responsible for the phenotype seen in theEN7:icals3m roots, we expressed the icals3m transgene in the steleor in the cortex and epidermis. Induction of CRE1:icals3m resultsin the disruption of symplastic movement out of the stele (14).However, even after 60 h of induction of the CRE1:icals3m trans-gene, no significant changes in the orientation of cell divisions inthe endodermis (Fig. 2 G and H and Fig. S1) were observed. Theseresults suggest that a lack of stele to endodermis signaling alone isnot what drives the EN7:icals3m phenotype. To determine whethera loss of symplastic signals from the cortex or epidermis could in-duce periclinal cell divisions in the endodermis we made use of theE903 enhancer trap line (https://www.arabidopsis.org/abrc/poethig.jsp) to drive inducible expression of UAS:icals3m. The E903 linesshow variable expression of the UAS:erGFP marker in the epider-mis, cortex, and endodermis. In most roots, as marked by erGFP,expression is in the epidermis only (Fig. 3J) or epidermis and cortex(Fig. 3K). In these roots, activation of the UAS:icals3m has no ef-fect on the patterning of the endodermis. However, in roots whereexpression of UAS:erGFP is driven also in the endodermis (Fig.3L), activation of UAS:icals3m correlated with periclinal divi-sions in the endodermis. Weak expression in the endodermishad no effect on patterning (Fig. 3M). These results stronglysuggest that a loss of symplastic signaling from the cortex or epi-dermis into the endodermis is not sufficient to induce periclinalcell division in the endodermis. Instead the phenotype observedin the EN7:icals3m roots is the result of a loss of PD-mediatedsignals from both the stele and the cortex into the endodermisor signals between endodermal cells.

Signals from the Endodermis Are Not Required to Maintain the Activityof the CED Cells or for QC Maintenance or Function. Previous resultsby van den Berg et al. showed that isolation of CED cells fromtheir progeny in the cortex and endodermis (via laser ablation)resulted in a loss of periclinal cell divisions in the CED cells (26).This prompted the “top-down signaling hypothesis,” a specificmodel, which posited that top-down (hence the name) signalsderived from more mature tissues were transmitted into the CEDcells where they were required to maintain the formative periclinalcell divisions that produce the separate endodermis and cortex(26, 27). Because only complete ablation of all cortex and endo-dermal cells in direct contact with the CED affected periclinal celldivisions in the CED, the top-down signal was thought to besymplastic, juxtacrine, or mechanical. However, in our assays,neither activation of EN7:icals3m (no symplastic signals from theendodermis to the CED; Fig. 2 D–F) nor activation of J0571:GAL4; UAS:icals3m (no symplastic signals from the endodermisor cortex to the CED; Fig. 3 G–J) caused a loss of periclinal celldivision in the CED cells. This finding argues against symplasticconveyance of the top-down signal required to maintain theproper behavior of the CED stem cell populations, and instead forsome other form of signaling.The QC functions as an organizer of cellular patterning and

inhibits the differentiation of surrounding stem cells. The QC sitsat the apex of the cortical endodermal lineage and is in directcontact with the CEIs (28). To determine whether symplasticsignaling from the endodermis or the CEIs is required for main-tenance of the QC, we examined QC morphology and expressionof the QC marker, WOX5:erGFP after induction of EN7:icals3m(Fig. S2A) (29). Similar to what we saw in the cortex, the cells ofthe QC appeared stretched or compressed after induction of theEN7:icals3m transgene (Fig. S2 A–C). Despite the extensive de-fects in ground tissue patterning and changes in QC morphology,the expression ofWOX5 was well maintained after induction of EN7:icals3m (Fig. S2A). Consistent with this finding, the organization of

the columella was largely normal with a lack of starch in the colu-mella initials and starch accumulation in the columella (Fig. S2C)(29). These results suggest that symplastic signals from the en-dodermis are not required for QC function.Periclinal cell divisions of the CED cells requires the expression

of CYCD6;1 (30). Following asymmetric division of the CED cell,expression of CYCD6;1 is turned off and the cells in the newlyformed endodermis and cortex divide symmetrically in a series oftransit amplifying cell divisions. To determine whether expressionof CYCD6;1 is affected by a loss of endodermal signaling, theCYCD6;1:GFP-GUS marker was crossed into roots expressingEN7:icals3m. As expected, before estradiol induction, CYCD6;1expression was found predominately in the CEI and CED cells(Fig. S3A). After 20 h on estradiol, CYCD6;1 expression waspresent in the endodermal cell lineage (Fig. S3B). The occurrenceof elevated levels of CYCD6;1 appeared before the ectopic peri-clinal cell divisions in the endodermis. Longer treatment with es-tradiol correlated with a further expansion of CYCD6;1 expressionand increased numbers of periclinal cell divisions within the en-dodermis (Fig. S3 C and D). In no instances was CYCD6;1 ex-pression found in cortical or epidermal cells. These results indicatethat symplastic signals to the endodermis or between endodermalcells are required to inhibit inappropriate expression of CYCD6;1.The persistence of CYCD6;1 expression in the endodermis sug-gests that symplastic signals (perhaps from more mature cells inthe lineage) are required to promote the transition from a CEDcell fate to an endodermal precursor cell fate. Collectively, theseresults suggest that, whereas symplastic signals from the groundtissue are dispensable for asymmetric division of the CEDs and thefunction of the QC, signals from the endodermis may be importantin triggering a switch from CED cell fate to endodermal cell fate.

Symplastic Communication Is Essential for Specifying Cell Identity inthe Ground Tissue. Plant cells rely heavily on spatial context, ratherthan lineage to determine cell fate. To determine whether loss ofendodermal signaling affects cell fate in the radial dimension, weexamined markers of stele (SHR:erGFP; Fig. S4A), epidermal(WER:H2B-YFP; Fig. S4B), cortical (CO2:H2B-YFP; Fig. 4A), andendodermal (EN7:H2B-YFP and SCR:H2B-YFP; Fig. 4B) cell fate(20, 24, 31). We saw no significant changes in the expression ofSHR:erGFP (stele) or WER:H2B-YFP (epidermis) after induction(up to 60 h) of EN7:icals3m (Fig. S4 A and B). In contrast, theexpression of CO2:H2B-YFP, which is usually restricted to thecortex and CED cells, is turned on in the endodermis (Fig. 4A)(20). Expression of the CO2:H2B-YFP marker in the endodermispreceded large-scale changes in radial patterning. After division,most of the cell layers derived from the endodermal cell lineageexpressed the CO2:H2B-YFP marker. However, expression ofCO2:H2B-YFP in these cells was generally lower than in the cortexand not exclusive of EN7:H2B-YFP or SCR:H2B-YFP expression(Fig. 4B). Instead, most of the cells in the endodermal lineageexpressed the markers of both cortex and endodermis. AlthoughCO2:H2B-YFP became active in the endodermis, we did not seethe expression of endodermal markers in cortex, which is seen inthe shr-2 mutants (Fig. 3A). These results suggest that symplasticsignals to or between endodermal cells are required to maintainground tissue cell fates.

Auxin Signaling Is Affected by Symplastic Isolation of the Endodermis.The auxin efflux carrier PIN2, is a functional marker of cortical andepidermal cell identity in the root (Fig. 4C). Together with otherPIN family proteins, PIN2 facilitates the polarized flow of auxin viaasymmetric localization within the plasma membrane of corticaland epidermal cells. Whereas PIN2 localization is variable in theepidermis and cortex, the predominant localization is shootward inepidermal cells and rootward in the cortex (Fig. 4C and Fig. S5).PIN2 is not expressed in the endodermis (Fig. 4C). After 30 h onestradiol, and before ectopic periclinal cell divisions, PIN2:PIN2-GFP

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was expressed in the endodermis of EN7:icals3m roots (Fig. 4C andFig. S5). In the endodermis, PIN2 showed preferential (but not ex-clusive) localization to the rootward end of the cell (Fig. S5 B, E, andE′). At 30 h, we saw mild increases in the localization of PIN2 to thelongitudinal walls of the cortex and epidermis. In the cells created bythe periclinal division in the endodermis, PIN2 was also expressedand largely apolar (Fig. 4C and Fig. S5). This apolar localization ofPIN2 became more obvious with longer disruption (45 h) of sym-plastic communication in the endodermis (Fig. 4C). These resultsindicate that symplastic communication is required to maintainground tissue identity and for cell polarity.Because polar transport of auxin governs root gravitropism (32,

33), we examined whether the activation of EN7:icals3m affects rootgravitropic responses. Upon shifting EN7:icals3m roots to estradiol-containing medium, the roots became noticeably less gravitropic(Fig. 5 A and B). After 3 days on estradiol, some roots had grownin circles, whereas others grew in opposition to gravity. To testwhether this defect was caused by abnormal auxin distribution, weexaminedDR5:erGFP reporter in both wild-type (Fig. 5C) and EN7:icals3m (Fig. 5D) lines (34). In uninduced roots, grown vertically onplates, the fluorescence maximum of DR5:erGFP was in the distalregion of the root tip, with no signal in the epidermis or the lateral

root cap (Fig. 5C). In the induced roots, expression ofDR5:erGFP isaltered; the signal in the stele is increased with signals now in theepidermis and lateral root cap (Fig. 5D). IAA2:GUS showed asimilar trend (Fig. S6 A and B) (35). Treatment of wild-type rootswith auxin changes the pattern of DR5:erGFP signal so that thereare peaks in expression in the outermost cell layers of the rootmeristem similar to what we observed in the roots with activatedEN7:icals3m expression (Fig. 5D) (34). This result indicates thatsymplastic signals are necessary for proper gravity response throughthe control of PIN2-expressing cells.When wild-type roots are rotated by 90°, there is a redistribution

of auxin toward the lower edge of the root, as indicated by an in-creased level of DR5:erGFP signal (Fig. 5E). This precedes differ-ential expansion of the root to reorient growth downward.Following rotation of the EN7:icals3m roots, no changes in theauxin concentration were observed; both the upper and lower sidesof the root maintained high levels of auxin as indicated by the ex-pression of the DR5:erGFP reporter (Fig. 5F). These results suggestthat an inability to differentially localize auxin in the EN7:icals3mroots underlies their inability to respond to gravity. Interestingly,gravitropic defects caused by activation of EN7:icals3m can be re-covered. Treatment in estradiol for 2–3 d followed by recoveryin normal Murashige and Skoog (MS) medium restored rootgravitropism, suggesting that patterning can be recovered (Fig. 4B).

Fig. 4. A lack of endodermal signaling affects cell identity and PIN2 localiza-tion. Confocal images of roots expressing markers of the (A) cortex (CO2:H2B-GFP; grouped and outlined in blue) and (B) endodermis (SCR:H2B-GFP andEN7:H2B-GFP; grouped and outlined in yellow) before and after induction ofEN7:icals3m as labeled. After induction of the EN7:icals3m transgene, expres-sion of the (A) CO2:H2B-GFP marker expands into the endodermal cell file.(C) PIN2:PIN2-GFP expression (grouped and outlined in gray) in the epidermisand cortex in uninduced and induced roots (as labeled). Expression of PIN2-GFPis absent from the endodermis in roots before induction (0 h) of icals3m. PIN2expression turns on in the endodermis (arrows) after induction of icals3m, in-creasing in expression up to 45 h (the focal plane was chosen to highlight thePIN2-GFP in endodermis). E, endodermal lineage; C, cortex; D, epidermis. Alltimes are after induction. (Scale bars, 25 μm.)

Fig. 5. A loss of endodermal signaling results in changes in auxin distribu-tion and root gravitropism. (A) Seedlings were grown for 5 d on MS mediumbefore transfer for 3 d to estradiol containing medium. (B) Roots from Awere transferred back to MS medium and allowed to grow for 3 d duringwhich time normal growth was recovered; the white bars mark the positionof the root tip at the time of transfer to estradiol containing medium; theyellow bar marks the position of the root tip at the time of transfer tomedium without estradiol. (C–F) Expression of DR5:erGFP in vertically grownroots with the EN7:icals3m transgene. DR5:erGFP in (C) uninduced and (D)induced roots. (D) Arrows note the expression of DR5:erGFP in the lateralroot cap after a 2-d block to endodermal signaling. Vertically grown (E)uninduced and (F) induced EN7:icals3m roots expressing the DR5:erGFPmarker were gravistimulated (turned 90°). Yellow arrow points to the ex-pression of DR5:erGFP in the downward flank of the root meristem ofthe uninduced root following gravistimulation. (F) There is no change inDR5:erGFP expression in the induced root in response to gravistimulation.

Wu et al. PNAS | October 11, 2016 | vol. 113 | no. 41 | 11625

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Recent reports by Han et al. looking at hypocotyl tropism suggestthat PD can serve as conduits for auxin movement (19). They foundthat loss of GLUCAN SYNTHASE-LIKE 8 (GSL8) activity (viaRNAi) resulted in a significant reduction in hypocotyl accumulationof PD-localized callose and a loss of normal phototropic responses.They conclude that restricted movement of auxin through PDallows for the formation of an auxin gradient that regulatesphototropic responses. In the EN7:icals3m roots the loss of anormal gravitropic response is more likely the result of aber-rant PIN expression and localization rather than a restriction ofPD-mediated auxin flow. Han et al. show normal localization ofPIN3 in the hypocotyl of their gsl8 RNAi lines; however, be-cause PIN4 and PIN7, which play significant roles in the pho-totropic responses, are not examined, an effect on PIN proteinscannot be eliminated (19).To determine the extent to which ectopic expression of the

PIN2 protein in the endodermal cell file drives the pattern-ing defects after induction of EN7:icals3m, we expressed theEN7:icals3m transgene in pin2 (eir1) mutants (36). In these rootsthe frequency of periclinal cell divisions in the endodermallineage is very similar to wild type (79% divided in pin2 mutantsversus 88% divided in wild type at 45 h; Fig. S1A). The significantdifference in the response of the pin2mutants to induction of theicals3m transgene is a lack of radial swelling in the endodermisbefore periclinal cell division (Fig. 3O). Whereas root cells ex-pand in all directions following cell division, most expansion

occurs in the apical–basal axis; this allows for directional growth ofthe root. In wild-type plants, expression of EN7:icals3m resulted ina doubling of the width of endodermal cells compared with con-trols (uninduced EN7:icals3m versus EN7:icals3m after 1 d onestradiol; Student’s t test P values = 0.00038, n = 13–15); thisincrease in girth, preceded divisions in the endodermal cell file. Inthe pin2 mutants, periclinal cell divisions were induced in theendodermis without prior cell swelling (Fig. 3O). These resultsindicate that the cell expansion phenotype in the wild-type plantsexpressing EN7:icals3m is dependent upon an auxin response,whereas the ectopic periclinal divisions are not. In addition, theseresults uncouple the cell swelling phenotype from division, in-dicating that the periclinal cell divisions in the endodermis are nota response to an increase in cell girth. Collectively, our resultsshow that symplastic signaling is critical for the regulation of celldivisions, cell polarity, and cell expansion in the endodermis ofA. thaliana.

Materials and MethodsDetails on materials and methods, including the plant material and chemicals,plasmid construction, confocal imaging, and histology, as well as other analysesand tools used are provided in SI Materials and Methods.

ACKNOWLEDGMENTS. R.O. was supported by NIH Cell and Molecular BiologyTraining Grant GM-007229-37. S.W. and Y.S. were partially supported by Na-tional Science Foundation Grant 1243945 (to K.L.G.).

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