Novel Insights into the Organization of Laticifer Cells: A Cell ......From a panel of antibodies...

Novel Insights into the Organization of Laticifer Cells: ACell Comprising a Unified Whole System1

Lourdes Castelblanque, Begoña Balaguer, Cristina Martí, Juan José Rodríguez, Marianela Orozco, andPablo Vera*

Instituto de Biología Molecular y Celular de Plantas, Universidad Politécnica de Valencia-Consejo Superior deInvestigaciones Científicas, Ciudad Politécnica de la Innovación, 46022 Valencia, Spain

ORCID ID: 0000-0002-8918-8147 (C.M.).

Laticifer cells are specialized plant cells that synthesize and accumulate latex. Studies on laticifers have lagged behind in recentyears, and data regarding the functional role of laticifers and their fitness benefit still remain elusive. Laticifer differentiation andits impact on plant growth and development also remain to be investigated. Here, cellular, molecular, and genetic tools weredeveloped to examine the distribution, differentiation, ontogeny, and other characteristic features, as well as the potentialdevelopmental role of laticifer cells in the latex-bearing plant Euphorbia lathyris. The organization of the laticiferous systemwithin the E. lathyris plant body is reported, emerging as a single elongated and branched coenocytic cell, constituting the largestcell type existing in plants. We also report the ontogeny and organization of laticifer cells in the embryo and the identification ofa laticifer-associated gene expression pattern. Moreover, the identification of laticifer- and latex-deficient mutants (pil mutants)allowed for the identification of distinct loci regulating laticifer differentiation, growth, and metabolic activity. Additionally, pilmutants revealed that laticifer cells appear nonessential for plant growth and development, thus pointing toward theirimportance, instead, for specific ecophysiological adaptations of latex-bearing plants in natural environments.

In vascular plants, two prominent tubing systems,the tracheal-appearing xylem and the phloem, havebeen widely studied. Laticifer cells form an additionaltubing system based on living cell(s). They occurthroughout the Plantae, yet not as extensively as thexylem and phloem. Laticifers are specialized cells (orrow of cells) that synthesize and accumulate latex(Fahn, 1990). The latex produced is highly variable in itschemical composition, not necessarily of a dense milkyappearance but frequently white, and contains sus-pended colloids and carries a variety of dissolved sol-utes and macromolecules (Konno, 2011). According toKekwick (2002), latex is produced in approximately12,500 plant species, representing approximately 10%of all flowering plants (angiosperms), which belong to900 genera of approximately 20 plant families that growin a variety of ecological settings (Metcalfe, 1967;Lewinsohn, 1991; Agrawal and Konno, 2009). Thus,laticifers appear to be polyphyletic in origin. Moreover,their absence in primitive angiosperms suggests that

these cells developed more recently than most other celltypes. Despite their widespread presence in the plantkingdom, studies on laticifers have been lagging behindin recent years. Aspects that have been more deeplystudied are those related to the physiology and role oflaticifers in the production of latex in rubber tree (Heveabrasiliensis) or opium poppy (Papaver somniferum) as asource of rubber and opium, respectively. Also, the rel-evance of laticifers for insect defense and their involve-ment in the transport pathways of natural products hasbeen pointed out (Hagel et al., 2008). However, thereremains a paucity of information regarding the mecha-nisms of laticifer cell differentiation, the precise onto-genic origin, and the organization of the laticifer systemwithin the plant body. In fact, to find comprehensivereviews about laticifer cells it is necessary to visit theearly contributions provided by K. Esau (1965) and hercoetaneous authors (Fahn, 1990; Mahlberg, 1993).

Early anatomical and morphological observations oflaticifers were mostly provided by plant anatomists dur-ing the 19th century who focused on the large cosmopol-itan familyEuphorbiaceae (DeBary, 1884).As a result, twolaticifer cell types, nonarticulated and articulated, wereidentifiedwith distinct modes of cellular organization anddifferent ontogenic origins (Fahn, 1990). Nonarticulatedlaticifers are single elongated cells that develop and growintrusively between other cells via tip growth. This processrequires a partial disassembly of the cell wall componentsand adisruption of cellwall connectionswith surroundingmesophyll cells (Mahlberg, 1959, 1963). Nonarticulatedlaticifers develop from cells that are present in the em-bryo (i.e. laticifer initials; Mahlberg, 1961; Mahlberg and

1 This work was supported by the Spanish MINECO (BFU2015-68199-R to P.V.) and Generalitat Valenciana (Prometeo 2014/024 toP.V.).

* Address correspondence to [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Pablo Vera ([email protected]).

P.V., L.C., and C.M. designed the research; B.B., C.M., J.J.R., M.O.,and L.C. performed the experiments; P.V. wrote the article.

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1032 Plant Physiology�, October 2016, Vol. 172, pp. 1032–1044, www.plantphysiol.org � 2016 American Society of Plant Biologists. All Rights Reserved.

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Sabharwal, 1968). As the embryo grows into a matureplant, the laticifer initials elongate and undergo karyoki-nesis without forming cell plates. Thus, nonarticulatedlaticifers become large (e.g. tens of centimeters long), andconstitute the largest cell type described in plants to date.Articulated laticifers, as found in dandelion (Taraxacumspp.) or rubber tree, arise from a series of initials that de-rive from meristematic regions (i.e. apical meristem andcambium) that originate at different phases of plantgrowth. Within this region, adjacent cell walls undergopartial or complete perforation (due to the gradual re-moval of wall materials) to form a series of somewhatelongated cells that are connected through highly perfo-rated cell walls (Nessler and Mahlberg, 1979, 1981).No specific function has yet been ascribed to latici-

fers (Pickard, 2008). It has been observed that non-articulated laticifers do not contain chloroplast (Sacchettiet al., 1999), and they have no plasmodesmatal connec-tions with their neighbors. Thus, these cells presumablyobtain energy inputs from the apoplast. Laticifer load-ing has also been attributed to the symplastic transportof nutrients from phloem to parenchymal cells adjacentto the laticifers followed by entry into the apoplast,fromwhich nutrients are taken up by laticifers (Bouteauet al., 1991; St-Pierre et al., 1999; Santana et al., 2002)and converted into latex, the most obvious character-istic that distinguishes laticifer from other cell types.This latex is enriched in different isoprenoid moleculesfollowing species-specific patterns, and it oozes copi-ously whenever a laticifer is punctured. It is hypothe-sized that unpunctured laticifers are turgid as a resultof osmotic water uptake. From an ecological perspec-tive, laticifers have been touted as a defense againstinsect herbivory for more than a century (Dussourdand Eisner, 1987) where the pressurized flow of latexmay function as a form of physical defense, in additionto the potential antibiotic effects of the secondary me-tabolites stored in the latex (Agrawal and Konno, 2009;Huber et al., 2016).In this study, cellular, molecular, and genetic tools

were developed to examine the distribution, differen-tiation, and ontogeny of laticifer cells in the latex-bearing plant Euphorbia lathyris. We describe thedistribution of the nonarticulated laticifer networkwithin entire organs and approach the ontogeny oflaticifer initials in the embryo. Furthermore, throughthe identification of laticifer mutants, we show thatlaticifers and latex production appear not to be es-sential for plant growth and development, and insteadprobably have importance for the ecophysiologicaladaptation of plants in natural environments.

RESULTS

Organization of the Laticiferous System within E. lathyrisPlant Body

Knowledge of laticifer cells in latex-bearing plantshas been derived frommicroscopy studies of cryostat orparaffin sections of plant tissues and conventional

staining techniques. However, this approach does notallow for the three-dimensional distribution of laticifercells within a plant organ. Moreover, laticifer cells fre-quently adopt a sinuous elongation pattern of cellulargrowth, moving in and out of plane. Thus, tissue sec-tions only provide information for short distances alonga piece of plant tissue and not along a longitudinal axisof a complex tissue as a whole. To simplify the identi-fication of laticifer cells and the characterization ofthe three-dimensional relationships between them andtheir surrounding tissues in an intact seedling or plantorgan, a whole-mount histochemical staining proce-dure employing Sudan Black B stainingwas developed.Since this technique renders other tissues translucent ortransparent while staining the laticifer cells, it permitsmapping of entire laticifer supply patterns of organs.

Whole-mount staining of intact E. lathyris seedlingsrevealed the longitudinal growth of laticifer cells alongthe hypocotyl axis (Fig. 1, A and B). On average, 18 to21 laticifer cells were detected in the hypocotyl, andthese cells ran parallel to each other from the base of thehypocotyl toward the shoot apical meristem (SAM),reaching an average of at least 9 cm in length, which isthe length of the hypocotyl under our growing condi-tions. Therefore, laticifer cells constitute the largestplant cell type, if not the largest cell in nature. The la-ticifer cell asset of the hypocotyl was concentric to thecentral vascular cylinder and embedded among themesophyll cells of the cortex (Fig. 1C). SupplementalFigure S1 presents a scheme of laticifer organization inthe hypocotyl.

Laticifer cells were not identified in the roots of E.lathyris seedlings. Conversely, laticifer cells in the cot-yledonary region were observed to group together toform rows of longitudinal cells that were entangledalong the midrib of the petiole (Fig. 1D). This tier oflongitudinal- and vascular-associated laticifer cells ex-panded up to the tip of the cotyledon. From this centraldisposition along the midrib, laticifers were profuselybifurcated and elongated, and they encompassed theentire blade of the cotyledon (Fig. 1E). Consequently, acomplex labyrinth of laticifer cells formed, and noregular pattern of cellular distribution could be infer-red.

Along the stem, which is typically composed of24 xylem/phloem poles (Fig. 1, F and G), laticifer cellswere observed to elongate toward the apical meristemand paralleled to secondary vascular strands. Due tothe complexity of the tissues at this developmentalstage, the exact number of laticifers could not be accu-rately determined. However, laticifer cells were pre-dominant in the cortex and in an area proximal tothe vascular bundle (Fig. 1H; Supplemental Fig. S2).Scanning electron microscopy (SEM) across transversesections of the stem confirmed the distribution of la-ticifers observed in whole-mount preparations. Latici-fer cells proximal to the vascular bundle had a largerinternal diameter than those in the external cortex(Supplemental Fig. S3). A frequent and peculiar obser-vation was the angling of the laticifer cells toward a

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nearby secondary xylem pole (Fig. 1I) and their entryinto the lumen of hollowed tracheary elements to oc-cupy the empty cavity (Fig. 1, J and K). This phenom-enon represents to our knowledge a previously unknowncellular strategy by which a laticiferous system canenhance its invasive growth style.

In the leaves, laticifers appeared distributed paralleland along the midrib of all leaves (Fig. 1, L and M;Supplemental Fig. S4). Frequent bifurcations of thecentral laticifer cells occurred at right or almost rightangles, and these cells subsequently continued toelongate and bifurcate to expand along every direc-tion of the leaf blade. Thus, a myriad of laticifer cell-based crossroads appeared along the leaf lamina (Fig.1, N and O). The density of these cells progressively

decreased at the leaf margins, recalling a circulatorysystem like that of animals, exhibiting an unpredictablepattern of distribution with occasional aggregations oflaticifers assembled in parallel to one another and withY- and H-type bifurcations (Fig. 1, P and Q). However,these laticifers did not undergo anastomosis. Along theleaf blade, laticifers were distributed among the meso-phyll cells, primarily on the abaxial side of the leaflamina. Direct contact with the epidermal cell layer wasnot observed, although their proximity to the epidermisresulted in frequent dragging of the laticifer tubularstructureswith epidermalmanual peels (Fig. 1Q).Whole-mount staining of intact true leaves emerging fromthe apical meristem, which are undergoing leaf ex-pansion, provided a three-dimensional visualization

Figure 1. Distribution pattern of laticifer cells in E. lathyris intact plant structures as revealed bywhole-mount stainingwith SudanBlack B. A, Seedling, showing the first pair of true leaves, employed to identify laticifer cells as in B to E. B, A sector of the hy-pocotyl showing rows of laticifers running in parallel along the hypocotyl. C, Cross section of a hypocotyl region with laticifercells (marked by a white arrow) specifically stained for isoprenoids. D and E, Longitudinal sector of a whole-mount stainedcotyledon, along the lamina, in a region proximal to the node (D), where longitudinal laticifers cells concentrate along themidriband in a distal region from the node and midrib (E), where laticifers appear scattered. F and G, Cross section of the stem stainedwith toluidine blue (F) and phloroglucinol (G), where the lignified xylem poles become specifically stained in red. H and I,Close-up of a whole-mount preparation of the stem showing an ascending laticifer running parallel to one of the poles of thevascular cylinder (H) or curving toward the vasculature from the cortex in search of a xylem pole (I). J and K, Close-up of crosssections of the stem showing some laticifer cells occupying the internal cavity of the hollow cylinder of different tracheary el-ements. L, Sector of a blade from a whole-mount stained fully expanded leaf. M, Magnification of a sector showing the midrib ofthe leaf blade and the abundant presence of laticifer cells. N and O, Magnification of leaf blade sectors showing the differentdispositions and distribution patterns of laticifer cells. P and Q, Magnification of leaf blade laticifer cells showing characteristicY- and H-bifurcation patterns. R to U, Whole-mount preparation of an emerging leaf close to the apical meristem, with pre-dominant distribution of laticifer cells close to the midrib (R) and details of a sector close to the leaf tip at different magnifications(S–U). c, Cortex; l, laticifer; ph, phloem; pp, pith parenchyma; sv, secondary vasculature; x, xylem.

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of the interconnected tubular-like organization of thelaticiferous system, exhibiting both sinuous and wavegrowth patterns with frequent loops and curls that al-tered the direction of the elongating laticifer growth. Inaddition, repeated bifurcations occurred and pro-gressed toward the leaf margins as the leaf expanded(Fig. 1, R–U).

Characterization of Isolated Laticifer Cells

Nonarticulated laticifer cells have been documentedas coenocytic cells that undergo karyokinesis withoutforming a cell plate (Mahlberg and Sabharwal, 1966).To obtain a detailed characterization of E. lathyris la-ticifer cells, laticifer protoplasts, corresponding to largefragments of individual laticifer cells (up to 1 cm), wereisolated, and the distribution and spacing of nucleiwere studied upon 49,6-diamino-phenylindole (DAPI)staining (Fig. 2, A–D). The coenocytic nature of laticifercells was confirmed with nuclei fitting and occupyingthe internal lumen of the tubular laticifer cells. Nucleiappear flattened at the poles, adopting an oblate shapewith an averaged length of 10 mM and with a lineal andapparent constant spacing of 10 nuclei per mm of la-ticifer cell length (Fig. 2, E–G).When individual laticiferprotoplasts were derived from stable tetraploid plants(Supplemental Fig. S5), the size of the nuclei doubled(Fig. 2–H) and the nuclear spacing was retained alongthe coenocyte (Fig. 2E). Therefore, the lack of a cell wallseptum and consistent nuclei spacing, giving the ap-pearance of beads in a rosary, help define the longitu-dinal coenocytic organization of a laticifer cell.

Ontogeny and Organization of Laticifer Cells in the E.lathyris Embryo

Early anatomical studies of laticifer cells in em-bryos from different plant species were conducted bySchmalhausen (1877) and by Chauveaud (1891). Theseobservations, along with more recent studies byMahlberg (1961, 1993) and Mahlberg and Sabharwal(1968), have indicated that nonarticulated laticiferoussystems arise from a series of embryonal initials andtheir intrusively growing branches. Since whole-mountstaining in the embryo is impeded by the presence ofthe seed coat, an immunohistochemical approach usingfixed sections was applied to study laticifer organiza-tion in the embryo of E. lathyris and its ontogeny. In-trusive growth requires both disassembly and synthesisprocesses of different cell wall constituents. Conse-quently, differences in the wall composition of laticifercells were expected. From a panel of antibodies raisedagainst various cell wall components, we identified theLM6 antibody to specifically immunodecorate laticifercells in the mature embryo (Fig. 3, A and B). LM6 spe-cifically recognizes (1-5)-a-L-arabinan epitopes (Willatset al., 1998). When the LM6 antibody was used to studythe complexity of laticifer organization in the embryo, a

ring of interwoven laticifer cells, here designed as a“plexus,” was observed (Fig. 3D). These cells were lo-cated at the cotyledonary node, fromwhich branches oflaticifers extended upwards into the cotyledons parallelto the immature vascular strand (Fig. 3F). These latici-fers subsequently branched at right angles and ex-tended laterally into the swollen cotyledonary tissues(Fig. 3E). A frontal view of a mature embryo (Fig. 3G)showed the laticifer branches of the plexus thatascended into the cotyledons along the course of theimmature vascular strand from a different angle (Fig. 3,H and I). The SAM appeared to be devoid of laticifer

Figure 2. Isolation of coenocytic laticifer E. lathyris protoplasts anddistribution of nuclei. A to D, Fragments of different long individuallaticifer protoplast, either with bifurcations or lineal, viewed under theoptical microscope (A–C) or by fluorescence microscopy upon stainingwith DAPI (D). E, Nuclear density along the longitudinal laticifer pro-toplasts froma diploid (2x) and a tetraploid (4x) plant. F, Length of nucleifrom 2x and 4x plants. Bars represent mean 6 SD. For nuclear density,40 individual laticifer protoplasts of different lengthswere analyzed. Fornuclear length determination, 100 different nuclei from 10 differentlaticifer cells were measured. G andH,Magnification details of isolatedlaticifer protoplasts from 2x and 4x plants.



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strands (Fig. 3H). In addition, branches that extendedfrom the plexus downward into the radicle were muchless abundant than those extending toward the cotyle-dons (Fig. 3, C and G).

We subsequentlywonderedwhen andwhere laticiferinitials originate during embryo development. Sym-metric pairs of elongated laticifer initials in the earlystages of laticifer cellular differentiation (e.g. carryingeight to 12 nuclei) were detected at the heart stage ofembryo development, at the base of emerging cotyle-dons, and in the region where future procambial tissueeventually differentiate (Fig. 3J). These laticifer initialsappear to elongate bidirectionally, with the lower endpenetrating downward toward the root apex of theimmature embryo and the upper end penetrating to-ward the base of the developing cotyledons. Initially,each end of the laticifer intrusively thrusted its waybetween neighboring cells, with the initials decreasingin diameter until they terminated as a narrow tip withacuminate ends that wedged between two adjacentcells (Fig. 3K). The growing tips then followed the pathof the middle lamella and did not fuse or penetrate

adjacent cells. During their intrusive growth, the latic-ifers established contacts with new cells, while neigh-boring cells were forced apart from their original pointof contacts. Thus, intrusive growth appears establishedin the very early stages of laticifer differentiation and ismaintained throughout the life span of the laticifer. Tomonitor the abundance of the laticifer initials, bothtransversal and radial serial sectioning were performedalong the entire embryo at the heart stage of develop-ment. In this serial sectioning approach, up to 26 to28 initials were found to be present at this stage ofembryo development (Supplemental Fig. S6A). Fur-thermore, these laticifers were radially distributed andexhibited different degrees of differentiation, as if thedifferentiation process was not completely simulta-neous for all the initials. From that point, the laticiferinitials began to branch and elongate until they con-formed to the laticifer complexity that characterizes themature embryo as described above. The earliest laticiferinitials were detected when the embryo leaves theglobular stage (e.g. when no initials were detected;Supplemental Fig. S6B) and enters the early heart stage.

Figure 3. Organization and ontogeny of thelaticiferous system in the E. lathyris embryo. Aand B, Comparative immunohistochemicalstaining of E. lathyris embryo sections withLM6 (A) and a nonspecific (B) antibodyrevealed the specificity of LM6 to immunode-corate laticifer cells in the embryo. C, Sagittalsection along the longitudinal axis of the ma-ture embryo revealed with LM6 allowedidentifying laticifer cellular structures alongthe embryo axis. D, Magnification of the sectorshown in C showing the ring of interwovenlaticifer cells (plexus) in the cotyledonary nodeand the ascending row of laticifers parallel tothe immature vascular strand in the cotyledon.E and F, Details of branches of laticifer cellstructures extending laterally (E) in the cotyle-don tissues or extending upwards and parallelto the immature vascular strand (F). G–I,Frontal view of a mature embryo sectionimmunodecoratedwith LM6 (G;magnificationdetails (H and I) allowed identification of la-ticifer branches ascending into the cotyledons.Observed that the SAM appears devoid of la-ticifer strands. J, Detection of elongated latic-ifer initials in immature embryos (i.e. heartstage) as embedded in the seed. The laticifersappeared immunodecorated at the base of theemerging cotyledons. K, Detail of a laticiferinitial terminating as a narrow tip with acu-minate ends. L, Detection of earliest laticiferappearance detected using LM6 antibodies atlate globular stages of embryo development.Serial longitudinal sectioning (top) and cross-sectioning (bottom) of embryos revealed thepresence of a single pair of nonelongated ini-tials, marked with orange arrows and discern-ible at the time primordia of cotyledons start toform in the immature embryo.



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At this point, the embryo is approximately 180 mM inlength (Fig. 3L). At the time the primordia of the coty-ledons start to form in the embryo, we identified asingle pair of nonelongated and symmetrically posi-tioned laticifer initials that occupied a median positionrelative to the cotyledonary primordia. Both, serial lon-gitudinal sectioning (Fig. 3L, top) and cross-sectioning(Fig. 3L, bottom) of embryos confirmed the presence ofthese two initials. These observations may be taken asindicative that the laticiferous system is initiatedwith theearly differentiation of a single initial on either side of thebilateral axis of an immature embryo. Soon after, furthercellular differentiation events occur concurrent withthe embryo entering the heart stage of development.Supplemental Figure S7 summarizes this process ofdifferentiation and growth in the developing embryofor a single laticifer initial.

Identification of Laticifer-Associated GeneExpression Pattern

Oozing latex carries the cytosolic constituents of la-ticifers. Therefore, to identify genes related to laticifercell activity, we isolated latex from leaves, isolated itsRNA, and performed massive 454 sequencing. Wholeleaves were similarly processed and sequenced. Thenucleotide sequences obtained were compared withthe entries in databases to search for homologous se-quences. A subset of genes (Supplemental Table S1)was further selected for expression analysis by quanti-tative reverse transcription PCR (RT-qPCR). As shownin Supplemental Figure S8, mRNAs encoding con-served ACAT or HMGCoAR enzymes, key for theactivity of the cytosolic mevalonate pathway (MVA),were abundant in the latex as well as in the leaves,stem, and roots. In contrast, genes encoding DXS andDXR were found to be poorly expressed in laticifercells compared to other plant organs (SupplementalFig. S8). The latter are markers of the methylerythritolphosphate (MEP) pathway of the chloroplast. Both theMVA and MEP pathways contribute to the synthesisof the common five carbons in isopentenyl pyro-phosphate precursor required for isoprenoid biosyn-thesis in photosynthetic tissues (Vranová et al., 2013).Thus, isopentenyl pyrophosphate precursors in latic-ifer cells may be primarily provided by the cytosolicMVA pathway. This possibility is further supportedby the observation that laticifer cells are devoid ofchloroplasts (Mahlberg, 1993), which indeed containthe components of the MEP pathway (Vranová et al.,2013).Genes encoding cardinal enzymatic constituents of

the triterpenoid pathway, including SQS, SQE, CAS,and SMT1, are also expressed both in laticifers andin the different plant organs. Other genes highlyexpressed in laticifer cells, however, are expressed atmuch lower levels in samples representing whole plantorgans. These genesmay represent potential markers oflaticifer cells, such as PE and EG (Supplemental Fig. S8).

These later genes encode proteins involved in cell wallremodeling that might contribute to the disassembly ofthe cell walls of surrounding cells during the intrusivegrowth of laticifers. Other laticifer-specific markers in-cludedMLP, or EH, of unknown function, andDHDDS,involved in membrane metabolism. Conversely, twogenes were found strongly expressed in various plantorgans but strongly repressed in laticifer cells (i.e. PEI orPL). These latter proteins are hypothesized to provide acounterbalancing effect of the mesophyll cells to the cellwall remodeling enzymes secreted by laticifers. Asubset of these genes was subsequently employed asmarkers for the presence of laticifers.

Dynamics of Laticifer Cells in the Plant

To study laticifer metabolic activity, the latex com-ponents were analyzed by gas chromatography-massspectrometry (GC-MS). Four major isoprenoid specieswere found: cycloartenol (CYC), lanosterol (LAN),butyrospermol (BUT), and 24-methylene cycloartanol(24M), with distinctive retention times (Fig. 4, A and B).In whole-leaf extracts, all four latex-associated isopre-noids, along with b-sitosterol, which was absent in la-tex, where detected (Fig. 4C). Thus, the CYC, LAN,BUT, and 24M isoprenoid profile represents ametabolicfingerprint for E. lathyris laticifer cells. The relativecontent of each of these four isoprenoids varies betweenleaves and stems, with 24M accumulating at higherlevels in stems, apparently at the expense of the threeother isoprenoids (Fig. 4D). Thus, organ-specific ad-justments in the isoprenoid pathway may be made toallow common squalene intermediate to be trans-formed into any of the four major isoprenoids that ac-cumulate in laticifer cells (Supplemental Fig. S8). Acomparative analysis of isoprenoid content in stems,leaves, and roots was also conductedwith respect to thenet accumulation of isoprenoids in the whole plant. A3-fold increase in isoprenoid content was detected inthe leaves compared to the stems (Fig. 4E), while theywere barely detectable in roots. Taken together, theseresults reconcile with findings deduced from whole-mount staining (Fig. 1).

The abundance of laticifer cells was estimated using a“laticifer index” (LI) calculated based on the total length(in mm) of the tubular laticifer cells that occupied amicroscopic field area (in mm2) of the leaf lamina uponwhole-mount staining. Laticifer abundance was foundto positively correlate with the extent of leaf expansionat a fixed position in the stem (Fig. 4, F–H); leaf ex-pansion was arbitrarily numbered from 1 to 4 (Fig. 4, Fand G). Moreover, LI increase was observed in parallelwith greater isoprenoid accumulation as the leafexpands (Fig. 4I). RT-qPCR analysis expression ofSMT1, serving as a marker for isoprenoid biosynthesis,remained stable during leaf expansion (Fig. 4J). Con-versely, the expression of laticifer marker genes (e.g. PEand EH) declined in the late stages of leaf expansion,indicative that laticifers slow down their growth when



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leaf expansion decays. Thus, isoprenoid metabolismwithin laticifers remains fully active even in the absenceof laticifer growth.

Influence of the SAM on Laticifer Growth in the Cotyledon

Early reports proposed that a relationship betweenlaticifer growth and the meristematic activity in theapex might exist (Schaffstein, 1932). It was thus con-tended that the SAM was the source of a factor(s)influencing laticifer differentiation and growth. To de-termine the importance of the SAM on the laticiferoussystem, we searched for a mutant defective in shootapical meristem. To achieve this goal, we mutagenized9000 seeds of E. lathyris, which were allowed to growand selfed. M2 seeds were screened for mutants de-fective in SAM. One mutant was identified. When itwas assayed in homozygosis, it lacked shoot apicalgrowth due to the lack of a distinguishable SAM andthus did not develop true leaves (Fig. 5A). This mutantwas named shoot meristemless (sml).

In the early stages of seedling development, whole-mount staining of sml seedlings and wild-type seed-lings showed that the arrangement and growth of thelaticifer cells in the hypocotyl were similar, as it wasalso the case for the disposition of vascular strands (Fig.5B). Conversely, laticifer cell establishment was foundto be severely compromised in the cotyledons of the smlmutant, where laticifer structures were only sporadi-cally observed and showed no branching (Fig. 5B),while the development of the vasculature suffers novariation with respect to the parental line. RT-qPCRanalysis of the mRNAs extracted from cotyledonsshowed that expression of laticifer markers (e.g. EH, PE,and DHDDS) was severely repressed in the smlmutant(Fig. 5C), consistent with the diminution of laticifer cellsin this organ. In contrast, expression of triterpenoidbiosynthesis-related genes (e.g. HMGCoAR, SQS, orSMT1) remained nearly invariant in the sml mutant.Therefore, the smlmutant confirmed that SAM plays animportant role in the organization of the laticiferoussystem in cotyledons, reminiscent of a laticifer-specificchemotropic response.

Figure 4. Dynamics of E. lathyris laticifer dif-ferentiation and activity in the whole plant andduring leaf development. A, Major isoprenoidspresent in latex: CYC, cycloartenol; LAN, la-nosterol;BUT,butyrospermol;24M,24-methylene-cycloartanol. B and C, GC-MS analysis of latexextracts (B) and whole-leaf extracts (C). D, Rel-ative content of each of the four major latexisoprenoids in extracts derived from wholeplants, stems, and leaves. E, Comparative anal-ysis of triterpenoid content in stems, leaves, androots with respect to its net accumulation inthe whole developed plant. Bars representmean 6 SD, n = 9 independent plants. AnANOVA was conducted to assess significantdifferences in isoprenoid content, with a pri-ori P , 0.05 level of significance; the lettersabove the bars indicate different homoge-neous groups with statistically significantdifferences. F, Image of E. lathyris leaves at thefour stages (1–4) of leaf expansion. G, Whole-mount staining of leaves and close-up of asector of the leaf blade, showing the presenceof laticifer cells at the four stages of leaf ex-pansion shown in F. The insert cartoons serveto indicate the relative position of the selectedleaves in the stem. H, LI recording for eachstage of leaf expansion. I, Triterpenoid con-tent in leaves at different stages of leaf ex-pansion. Bars represent mean 6 SD, n =9 independent plants. J, Expression of the EH,PE, and SMT1 genes at different stages of leafexpansion. Relative expression was assayedby RT-qPCR on total RNA from leaves at theindicated stages. Data represent means 6 SD

(n = 3 biological replicates). Expression wasnormalized to the constitutive Histone H3gene, then to expression attained at stage 1 ofleaf expansion.



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Identification of E. lathyris Mutants Defective in LaticiferOrganization and Activity

We next approached the identification of mutantsdefective in laticifer differentiation and latex productionto unravel the importance of laticifer cells and latexproduction for plant growth and development. Wescreened for mutants devoid of latex production basedon the rationale that these mutants would reflect defectsin laticifer differentiation and growth or, alternatively,have defects in latex production. A total of 3000 M2plants were screened by pricking leaves and observingwhether latex oozed, and candidatemutantswere selfed.Five mutants were reconfirmed in the M3 and M4 gen-erations. These mutants, which showed reduced or nobleeding of latex upon severe injury, were coined poor inlatex (pil). Three of them (i.e. pil1, pil6, and pil10) pro-duced no latex in either the leaves or stems. The pil2 andpil3 mutants produced some latex, albeit much less co-piously than the parental plants (Fig. 6A). Backcrossingwith parental plants and segregation analysis of the F2progenies revealed that the mutants manifested asMendelian recessive genes (Supplemental Table S2),except for pil3, which behaved as dominant. As a com-plementation test, reciprocal crosses of the five pil mu-tants with each other and characterization of F1 plantsrevealed a wild-type phenotype in all cases, thereby in-dicating that none of the pil mutants were allelic.

Characterization of the pil1, pil6, and pil10 Mutants

The five PIL loci were found to affect distinct aspectsof laticifer activity and organization. In pil1, pil6, and

pil10 mutants, no identifiable laticifer cells were ob-served along the leaf lamina of full expanded leaves(Fig. 6B). Thus, a zero value LI was calculated for eachmutant (Fig. 6C). The lack of detectable laticifers wasalso consistent with the depletion of CYC, LAN, BUT,and 24M triterpenes in the crude leaf extracts obtainedfrom each of the three mutants (Fig. 6D; SupplementalFig. S9). Moreover, laticifer identity genes (e.g. EH, PE,and DHDDS) were severely repressed in pil1 and pil6mutants and much less acute in the pil10 mutant (Fig.6E). This posed a distinction of pil10with respect to pil1and pil6.

A detailed inspection of whole-mount preparationsof the intact primary meristematic leaves from each ofthe fivemutants revealed that the primary leaves frompil1 and pil6 exhibited only rudimentary versions oflaticifer cells (Fig. 6F), which were only proximal tothe central vein and were highly branched but did notelongate. The characteristic network of laticifersengulfing the entire leaf lamina in wild-type plantswas absent in these two mutants. Therefore, it washypothesized that the pil1 and pil16 mutants carrydefects in genes required for the elongation, but notfor branching, of laticifers. Remarkably, careful ob-servation of whole-mount preparations of primaryleaves in pil10 plants revealed the mutant indeedcontained laticifer cells, albeit they did not becomestained with the colorant; pil10 laticifers appearedtranslucent and could awkwardly be visualized uponilluminating the cleared whole-mount preparationswith intense bright light (Fig. 6G). Thus, while thepil10 plants maintained a normal laticifer network,they lacked production of latex. When longitudinal

Figure 5. Influence of the SAM on laticifer growthin the cotyledon of E. lathyris. A, Developingseedlings from wild type and the sml mutant.Observe the lack of true leaf formation in themutant. B, Close-up of the cotyledons and hypo-cotyls from wild type and sml mutant. Below isshown the presence and disposition of laticifers(top) and the vascular strands and venation pattern(bottom) in each genetic background and for bothorgans, and as revealed by whole-mount stainingwith Sudan Black B or upon acetone clarification,respectively, and visualization under the light mi-croscope. C, Expression of the EH, PE, DHDDS,HMGCoAR, SQS, and SMT1 genes in cotyledonsfromwild-type and sml plants. Relative expressionwas assayed by RT-qPCR on total RNA from leavesat the indicated stages. Data represent means6 SD

(n = 3 biological replicates). Expression was nor-malized to the constitutive Histone H3 gene, thento expression attained in wild-type plants.



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arrays of laticifer protoplasts were retrieved fromwild-type and pil10 fully expanded leaves (Fig. 6H),with pil1 plants serving as a negative control, DAPIstaining further showed that pil10 laticifer proto-plasts retained the characteristic coenocytic orga-nization observed in the wild type (Fig. 6H). Thus,laticifer differentiation, growth, and organization

can occur in the absence of latex and isoprenoidaccumulation.

In marked contrast with the adult mutant plants,immunohistochemical staining with an LM6 antibodyshowed that the growth and organization of laticifercells in these three mutant embryos were not severelycompromised. However, distribution and shape of the

Figure 6. Characterization of E. lathyris pilmutants. A, Comparative oozing of latex upon pricking of leaves fromwild-type, pil1,pil2, pil3, pil6, and pil10 plants. B, Whole-mount Sudan Black B staining of leaves and close-up of a sector of the leaf blade,showing the relative abundance of laticifer cells in the indicated genetic backgrounds. C, Comparison of LI as recorded in leaveslocated at the same position in plants of the indicated genetic backgrounds. D, Triterpenoid content in leaves from wild-typeplants and the pil mutants. Bars represent mean 6 SD, n = 9 independent plants. E, Expression of the EH, PE, DHDDS, and CASgenes in leaves from wild-type plants and pilmutants. Relative expression was assayed by RT-qPCR on total RNA from leaves ofthe indicated genotypes. Data represent means6 SD (n = 3 biological replicates). Expression was normalized to the constitutiveHistone H3 gene, then to expression attained in wild-type plants. F, Whole-mount preparation of emerging leaves close to theapical meristem showing defective laticifer elongation in pil1 and pil6 mutants. G, Presence of laticifer cells in leaves of pil10plants, with a density similar to that observed in wild-type plants, as revealed under the optical microscope upon Sudan Black Bstaining, intense clarification with ethanol, and illumination with white bright light. H, Long individual laticifer protoplastfragments released from wild-type plants and pil10 plants view under bright field in the optical microscope (left) or by fluores-cencemicroscopy upon stainingwith DAPI (right). An estimation of laticifer density (mmof longitudinal protoplast released to themedium per optical surface area recorded) from independent protoplast preparations (n = 4) from wild-type and pil10 leaves isshown at the right. The pil1mutant was used as a negative control revealing the lack of laticifer protoplast released to the mediumin this mutant.



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laticifer cells in pil1 and pil6 were partially distorted(Supplemental Fig. S10). These observations indicatethat the defective laticiferous system observed in plantais most likely due to a postembryonary cell elongationdefect, presumably in response to growth signals fromvegetative tissues. This would reconcile with a chem-iotropic model of laticifer growth in planta. Moreover,complementation of the mutant phenotypes, upon gen-erating chimeric E. lathyris plants obtained by graftingwhere the rootstock and scion were reciprocally inter-changed between wild-type plants and the pil1 and pil6plants, was not achieved (Supplemental Fig. S11), thusindicating cell autonomy of PIL1 and PIL6 genes duringlaticifer cell growth.With the exception of only a minor reduction in

height of pil1 plants, which, however, recovered at laterdevelopment stages, the pil plants grew and developedsimilarly to the wild-type plants (Supplemental Fig.S12). Therefore, laticifer cells and latex production arenot essential for plant development.

Characterization of the pil2 and pil3 Mutants

In the pil3 plants, laticifer cell differentiation andgrowth occurred, yet it was characterized by a markedreduction in the LI value (Fig. 6, B and C). Despite thisreduction, laticifer cells in the pil13 mutant elongatealong the leaf lamina to an extent similar to that achievedin wild-type plants. Expression of laticifer marker genes(Fig. 6E) and triterpene content (Fig. 6D; SupplementalFig. S9) also decline in pil3 plants, consistent with theobserved reduced LI value. Therefore, the PIL3 proteinmight be controlling a process related to branching oflaticifers but not to elongation, indicative that the twocellular processes distinctly regulate laticifer growth anddensity.The pil2 plants exhibited an inverse laticifer cellular

phenotype compared to that of the pil3 plants. Despitehaving reduced latex production, pil2 plants had en-hanced LI value (Fig. 6, B and C) and concurring in-creases in the expression of laticifer marker genes (Fig.6E). However, pil2 laticifers stained less intensivelywith Sudan Black B compared with wild-type ones.Consequently, we hypothesize that the higher com-plexity of the laticiferous system observed in pil2 plantsmight be due to the enhanced branching activity of thelaticifer cells. However, despite the enhanced LI valuein pil2, lower levels of triterpenoid accumulation wereobserved compared to the wild-type plants (Fig. 6D).Thus, laticifer growth via branching and certain aspectsof triterpenoid metabolism may be negatively inter-linked through the PIL2 locus.Taken together, these findings suggest that PIL2 and

PIL3 may function as negative and positive regulators,respectively, of laticifer growth via tip branching. In-stead, PIL1 and PIL6 appear to be pivotal for laticifergrowth by tip elongation, and PIL10 appears to becritical for coupling laticifer differentiation to latexproduction.

DISCUSSION

Terpenoids characterize the cytoplasmic content oflaticifers (Hagel et al., 2008), and these specializedelongated cells or vessel-like series of cells, first de-scribed by de Bary in 1884 (De Bary, 1884), representthe lengthiest eukaryotic cell identified to date. Hagelet al. (2008) have performed a clade diagram repre-senting the evolutionary relationship of the varioustaxa that possess laticifers that suggests that laticifershave arisen independently more than once duringevolution. This points toward the importance of latic-ifers for adaptation of plants to specific natural envi-ronments. Previously, knowledge of the presence oflaticifers was based on observations made on classicalmicroscopy methods for tissue sections. In the currentstudy, a whole-mount histochemical staining proce-dure allowed us to identify and map the entire laticifersupply within entire plant organs and seedlings.Consequently, the polarized growth of rows of latici-fers along the longitudinal axis of the hypocotyl andtoward the SAM could be observed. In contrast, anintricate and complex distribution pattern of laticifersalong the cotyledon and leaf lamina was observed: afew major laticifer cells run parallel to the primaryvasculature of the leaf and rendered, by repeated bi-furcations, lateral laticifers that subsequently elon-gated via intrusive growth. Subsequent repetitions ofthis dual process, branching and elongation, led to theformation of a laticifer network that encompassed theentire leaf organ. The pattern of this network of latici-fers resembled the distribution of blood vessels in thecirculatory system of animal organs. Establishment ofthis laticifer network in the leaf correlates with theexpansion and growth of this organ, indicative thatboth processes are coordinated. In fact, early studies onlaticifer organization support the hypothesis that la-ticifer growth andmeristematic activity are related andthat the shoot meristem is the source of a factor thatinfluences laticifer differentiation and growth (Schaffstein,1932; Mahlberg, 1993). The identification of the smlmutant, compromised in the maintaining and orga-nizing the meristematic activity, in a manner similarto that described for the Arabidopsis (Arabidopsisthaliana) STM gene (Endrizzi et al., 1996), revealedthat for a correct organization and growth of the laticif-erous system, SAM intactness is required and evokes alaticifer-specific chemotropic response for a signal syn-thesized in the meristem.

In the embryo, the pattern of laticifer cell distributionmirrors what will be later seen in an adult plant, withparticular enrichment in cotyledons and absence inroots, suggesting an early pre-establishment of the lat-iciferous system. Moreover, a plexus of laticifers in themature embryo, at a certain distance below the meri-stem, fromwhich tangential rows of laticifers emanatedto elongate and reach distal tissues of the embryo, ap-pears to function as a supply of laticifers for the estab-lishment of the laticifer network. These observationsare consistent with early interpretations by Mahlberg



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(1961) in his search for the organization of laticifer cellsin Nerium oleander embryos and thus indicates that thischaracteristic organization of laticifer organization isevolutionarily conserved.

Our immunohistochemical studies also revealed thatthe primary events of laticifer differentiation in E. lathy-ris, when the first single pair of laticifer initials wasidentified, occurred during the transition from theglobular to the early heart stage of embryo development.The initials were symmetrically positioned, according tothe bilateral axis, at a median position relative to thecotyledonary primordia. This first differentiation eventwas then followed by the rapid appearance of up to 26 to28 additional laticifer initials, which were distributedconcentric to the perimeter of the maturing embryo.Once the ring of differentiated laticifer initials was set,they started elongating via intrusive growth along thelongitudinal axis of the embryo. This elongation wasaccompanied by sequential karyokinesis events and didnot include the formation of cell plates. As a result, acharacteristic coenocytic-type appearance was observedfor the incipient laticifers. When elongation of the latic-ifer initials was initiated (e.g. when a row of six to eightnuclei was identified within the laticifer initials), the la-ticifers began branching at oblique angles and startedforming the complex laticifer network observed in themature embryos.

Our understanding of the role of laticifers in plantgrowth and development has remained very limited.Consequently, the identification the pil mutants pro-vided valuable insight onto how laticifer cells growand become organizedwithin the plant body. The PIL1and PIL6 loci appear to function as positive regulatorsof laticifer elongation, but not branching. Conversely,The PIL2 and PIL3 loci appear to function as nega-tive and positive regulators, respectively, of laticiferbranching without affecting cell elongation. There-fore, the concerted action of these PIL determinantson elongation and branching might be on the basisfor the final conformation of the laticifer network inthe plant. On the other hand, the identification of thePIL10 locus allowed us to conclude that laticifer dif-ferentiation and growth is independent of latex pro-duction. Therefore, PIL10 may represent a metabolicswitcher for latex formation in fully differentiatedlaticifer cells. Moreover, pil plants did not appear tohave any noticeable phenotypic effect on normalgrowth and development, this being indicative oflaticifer cells representing a specialized cellular ad-aptation to fulfill a specific role in latex-bearing plantsin their natural environments.

In sum, our results provide valuable insight into theparadigmatic mechanisms involving cellular differen-tiation, morphogenesis, and growth characteristics ofspecialized laticifer cells that have long been recog-nized yet have remained poorly understood. The eco-physiological tradeoffs and fitness effects of pilmutants in natural environments, as well as the iden-tification of the different PILs, is our next challenge forthe future.

MATERIALS AND METHODS

Plants Growth Conditions and Latex Isolation

Euphorbia lathyris plants were grown in a growth chamber (19°C–23°C, 85%relative humidity, 100 mE m22 s21

fluorescent illumination) on a 16 h:8 h light:dark photoperiod. For latex isolation, leaves were pricked at the central vein,and the latex oozing stored at 280°C.

Leaf Enzymatic Digestion, Laticifer Isolation, andDAPI Staining

Whole plants were vacuum infiltrated in ethanol-acetic acid-formaldehyde(50:5:10) for 20 min and fixed during 16 h at 4°C. Leaves were excised, washed,and immersed in an enzymatic solution containing 1% (w/v) Driselase (Sigma-Aldrich) in MP 0.6 medium (MSmineral solution [Murashige and Skoog, 1962],2.5 mM MES, 100 mM Suc, 400 mM mannitol, 100 mM Gly, 14 mM CaCl2, pH 5.7)during 16 h at 28°C in the dark. The digested tissue was stained with DAPI.

Generation of Tetraploid Plants

Hypocotyl explants were cultured in basal medium (MS solution, B5Gamborg vitamins [Gamborg et al., 1968], 2.5 mM MES, 87 mM Suc, pH 5.7)supplemented with 0.009 mM 6-benzylaminopurine and 0.0014 mM oryzalin.After 3 d, explants were transferred to fresh medium without oryzalin. After3 weeks, developed shoots were excised from the explant and transferred to aroot induction medium (basal medium supplemented with 0.068 mM indole-3-acetic acid). Ploidy level was evaluated by flow cytometry (Smulders et al.,1994) with the CyFlow Ploidy Analyzer (Partec). Over 5000 nuclei were mea-sured per sample. Tetraploid (2n = 4x), diploid (2n = 2x) or mixoploid (with 2xand 4x nuclei) plants could be identified.

Seed Mutagenesis

Seedsweremutagenizedbygamma ray at 300Gyat the InternationalAtomicEnergy Agency Laboratories in Seibersdorf (Vienna, Austria). The irradiationdosage was chosen based on the observation that a dose of 300 Gy resulted inapproximately 97% survival of M1 plants.

Genetic Analysis

Upon backcrossing with the parental line, segregation of phenotypes in the F2generationwas analyzedwith the x2 test for goodness offit. For complementationanalysis of the different pilmutants, each pilmutant was crossed with each of theother mutants and appearance of the pil phenotype in F1 plants recorded.

RNA Extraction, RT, and qPCR

RNAextractionandreverse transcriptionwasperformedasdescribed (Lópezet al., 2011). qPCRs were performed using an ABI PRISM 7000 sequence de-tection system and SYBR-Green (Perkin-Elmer Applied Biosystems). HistoneH3was chosen as the reference gene. Primers for amplicons covering each of thegenes studied are listed in Supplemental Table S3.

454 Sequencing

A 1 mg aliquot of mRNA was used as the template for first-strand cDNAsynthesis using a MINT-2 cDNA synthesis kit (Biocat). cDNA normalizationwas performed with a Trimmer-2 cDNA normalization kit (Biocat). cDNA wasdigested withGsuI (Fermentas) and purified using QIAquick columns (Qiagen)to eliminate oligo(dT). The cDNA quality was verified with an Agilent2100 Bioanalyzer (Agilent). A 1 mg aliquot of each cDNA or noncoding DNAsample was nebulized to produce fragments of a mean size of between 400 and800 bp. Preparation of cDNA fragment libraries and emulsion PCR conditionswere as described in the Roche GS FLX manual. Pyrosequencing was performedon a Roche Genome Sequencer FLX instrument (454LifeScience-Roche Diagnos-tics). The quality of the reads was assessed with PERL scripts developed atLifesequencing S.L. for trimming and validation of high-quality sequences.Adaptor sequences used for library preparation were entered in an adaptor-trimming database to the PERL Program. New SFF output files were generated



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with the sfftools (454 Life Science/Roche). Trimmed reads were assembled withNEWBLER version 2.3 (454 Life Science/Roche) with default parameters.

SEM

Tissue samples were processed for SEM as described (Dobón et al., 2015).Pictures were taken with a JSM-5410 scanning electron microscope (JEOL).

Isoprenoids Extraction and GC-MS Analysis

Isoprenoids were extractedwith heptane in a Soxhlet and saponified (Koopset al., 1991). Triterpenols were derivatized to trimethylsilylether derivativeswith Sylon HTP (Sigma-Aldrich) and analyzed by GC-MS on an Agilent 6890Ngas chromatograph attached to a low-resolution quadrupolar mass spectrom-eter Agilent 5973 with a HP-5MS UI (30 m, 0.25 mm inner diameter, 0.25 mm)column. Mass spectra were taken over the m/z 30 to 500 range with an ionizingvoltage of 70 eV. The individual compounds were identified by matching theacquired mass spectra with those stored in the reference libraries (NationalInstitute of Standards and Technology), and lanosterol and cycloartenol werecompared with samples of pure compounds (Sigma-Aldrich). A calibrationcurve was performed with lanosterol samples using 5-a-cholestan-3-one(Sigma-Aldrich) as internal standard.

Whole-Mount Staining and Laticifer Index

Entire plants were immersed in fixative (formaldehyde-acetic acid-ethanol,3.5:10:50) overnight at 4°C. Plant sectors were washed with 70% ethanol andstained with Sudan Black B (0.1% [w/v] in 70% ethanol [Jensen, 1962]) for 3 to4 h at room temperature, washed with 70% ethanol thenwith water, and placedin 2.5 M NaOH until the leaves were cleared (Ruzin, 1999). Tissues were ob-served under an EclipseE600 (Nikon) light microscope. LI was used to estimatethe profusion of laticifer cells in a tissue and was calculated by measuring, withthe ImageJ software, the total length (in mm) of the Sudan Black B-stained la-ticifer cells in a microscopic field area (in mm2).

Immunohistochemical Identification of Laticifersin Embryos

Embryoswerefixed overnight with 4%paraformaldehyde and embedded inparaffin. Samples were sectioned on a HM330 microtome at 8 mm and wereblocked and incubated with the monoclonal antibody LM6 [binding to a pecticpolysaccharide, anti-(1-5)-a-L-arabinan; PlantProbes] diluted 1:20 in PBS con-taining 0.1% (w/v) BSA and 0.05% (v/v) Tween 20. Control slides were treatedsimilarly with a nonspecific monoclonal diluted 1:20. An anti-rat IgG conju-gated with alkaline phosphatase (Sigma), diluted 1:2000, was used as a sec-ondary antibody, and sections were revealed with nitro-blue tetrazolium and5-bromo-4-chloro-3-indolyl-phosphate.

Accession Numbers

Nucleotide sequence data for the genes described in this article are availablefrom the GenBank database under the following accession numbers: ACAT(JQ434427), HMGCoAR (JQ694150), DXS (KT003670), DXR (JQ694151), SQS(JQ694152), SQE (JQ694153), CAS (JQ694154), SMT1 (JQ694155), EH (JQ694156),DHDDS (JQ694157), MLP (JQ694158), EG (JQ694159), PE (JQ694160), PEI(JQ694161), PL (JQ694162), and H3 (JQ966276).

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. Scheme summarizing the disposition and abun-dance of laticifer cells along the hypocotyl axis of E. lathyris seedlings.

Supplemental Figure S2. Scheme summarizing the disposition and abun-dance of laticifer cells along the stem and in leaves of an E. lathyris plant.

Supplemental Figure S3. SEM across the stem of a developing E. lathyrisplant.

Supplemental Figure S4. SEM across the lamina of a developing E. lathyrisleaf.

Supplemental Figure S5. Generation of E. lathyris tetraploid plants.

Supplemental Figure S6. Ontogeny and early distribution of the laticifer-ous system in the embryo of E. lathyris.

Supplemental Figure S7. Scheme summarizing the appearance and abun-dance of laticifer initials at different stages of embryo development inE. lathyris.

Supplemental Figure S8. RT-qPCR analysis of genes in latex and in intactplant organs in E. lathyris.

Supplemental Figure S9. Comparative triterpenoid GC-MS analysis of pa-rental E. lathyris plants and pil mutants.

Supplemental Figure S10. Organization of the laticiferous system in E.lathyris in mature embryos of pil mutants in comparison to wild-typeplants.

Supplemental Figure S11. Chimeric E. lathyris plants generated by graftingbetween wild-type and pil1 or pil6 plants.

Supplemental Figure S12. Appearance of pil mutants of E. lathyris grownunder glasshouse conditions.

Supplemental Table S1. Marker genes selected for transcriptomic analysisin E. lathyris.

Supplemental Table S2. Segregation analysis in pil mutants of E. lathyris.

Supplemental Table S3. Primer sequences used in this study for RT-qPCRanalyses in E. lathyris.

ACKNOWLEDGMENTS

We thank Javier Paz-Ares and Antonio Leyva for critical reading of themanuscript, Carlos Alonso for assistance during seed mutagenesis, VicenteRamírez for helpful discussions, andMaria Dolores Arocas andMarinaMolinerfor taking care of the plants.

Received June 16, 2016; accepted July 25, 2016; ; published July 28, 2016.

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Novel Insights into the Organization of Laticifer Cells: A Cell ......From a panel of antibodies...

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