A novel type of tube network within the stem bark of Olea europaea L

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A novel type of tube network within the stem bark of Olea europaea L
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  A novel type of tube network within the stem bark of   Olea europaea  L. George Kyriakis, Costas Fasseas   Agricultural University of Athens, Department of Agricultural Biotechnology, Electron Microscopy Laboratory, Iera Odos 75, 11855 Athens, Greece a r t i c l e i n f o  Article history: Received 2 October 2008Accepted 2 December 2008 Keywords:Olea europaea BarkTubeCortexAnatomyFluorescence a b s t r a c t The tube systems known to exist in tracheophytes arexylem, phloem and laticifers which are composedof living or non-living cells and secretory ducts/canals and aerenchyma that is of schizogenous orlysigenous srcin. Here, we describe a novel type of tube network of unknown function that ramifiesthrough the olive tree bark and is composed of groups of apparently empty, anastomosing tubulesinterrupted by perforated plates; side openings of the tubules connect them to the intercellular spacesof the cortex. &  2009 Elsevier GmbH. All rights reserved. Introduction The tissues most commonly found within the photosyntheticor storage cortex of the vascular plants (tracheophytes) arevarious parenchyma or fibre types such as collenchyma, scler-enchyma and gelatinous or g-fibres with mechanical functions,those involved in storing and transporting secondary metabolitessuch as secretorycavities, resin ducts/canals and laticifers and theair conducting tissue, the aerenchyma. These anatomical featuresare extensively dealt with in the classic contributions of, e.g., Esau(1977); Fahn (1990); Kozlowski and Pallardy (1996); Roth (1981). Theolive treeisoneoftheoldestcultivatedplants andhas beenfor centuries of great economic importance in the Mediterraneanbasin for its crop and wood production. It is an evergreensclerophyllous bush or tree, which grows up to 20m high. It isextremely long-lived, and may get an age of more than 1000 years,although this cannot be determined exactly due to its tree trunkmorphology that does not allow the application of dendrochrono-logical methods (Filippou et al., 2007; Kuniholm, 1995). Filippou et al. (2007), in their investigation of the photosyn-thetic characteristics of olive tree bark, report that young stemsarecoveredbypeltatehairs, similar tothose that cover the abaxialsurface of the leaves. Stomata are rare in the epidermis of youngshoots, and when secondary growth starts, lenticels develop. Thephotosynthetic cortex persists for more than 30 years when it isreplaced by the rhytidome. The present study examines theanatomy and ultrastructure of a not yet described tissue of unknown function that ramifies through the photosyntheticcortex of the  O. europaea  secondary stem. Materials and methods All samples were collected from the bark of well established,40–80-years-old olive trees ( O. europaea  L.), of ten differentvarieties growing in the olive grove of the Agricultural Universityof Athens and from stems, trunks and roots between 2 and 3weeks and 40 years old. Preliminary examination for relevantstructures with the light microscope (LM) was performed onstems of randomly selected species of trees or bushes growing inthe University campus, from a variety of families of Angiosperms.These were  Olea oleaster   Hoffmanns. & Link (Oleaceae, wild olive), Citrus limon  (L.) Burm.f. (Rutaceae, lemon tree),  Pistacia vera  L.(Anacardiaceae, pistachio nut tree),  Ficus carica  L. (Moraceae, figtree),  Ceratonia siliqua  L. (Leguminosae, carob tree),  Pyrus commu-nis  L. (Rosaceae, pear tree),  Platanus orientalis  L. (Platanaceae,Oriental plane),  Ligustrum vulgare  L. (Oleaceae, privet),  Jasminum sp. (Oleaceae, jasmine) and  Ailanthus altissima  Mill. (Simarouba-ceae, tree of Heaven). All specimens included all the bark tissuesoutside the xylem.Specimens for LM and transmission electron microscopy (TEM)were fixed in a mixture of 2.5% glutaraldehyde and 2% parafor-maldehyde in 0.1M cacodylate buffer at 4 1 C for 2–3h, post-fixedin 1% OsO 4  for 2h, washed in buffer, dehydrated in a series of ethanol followed by propylene oxide, embedded in Spurr epoxyresin and polymerised at 70 1 C for 36h. Semi- and ultra-thinsections were cut with a Reichert OMU-3 ultramicrotome withglass knives. Semi-thin sections for LM were stained with 0.5%toluidine blue in 1% borax in water, and ultra-thin sections for ARTICLE IN PRESS Contents lists available at ScienceDirectjournal homepage: www.elsevier.de/flora Flora 0367-2530/$-see front matter  &  2009 Elsevier GmbH. All rights reserved.doi:10.1016/j.flora.2008.12.004  Corresponding author. Tel./fax: +302105294331. E-mail address:  cfass@aua.gr (C. Fasseas).Flora 205 (2010) 90–93  ARTICLE IN PRESS TEM were stained with uranyl acetate and lead citrate. Specimenswere examined and photographed with a Jeol JEM 100-S TEM.For scanning electron microscopy (SEM), serial paradermalsections 40 m m thick were cut with a cryotome (Leica CM1850,Germany) at   10 1 C, embedded in Jung Tissue Freezing Medium(Leica Microsystems Nussloch GmbH, Germany), transferreddirectly into 3% glutaraldehyde in 0.1M phosphate buffer, pH 7.2at 4 1 C for 4h, dehydrated with acetone, critical point dried,mounted on stubs with self-adhesive double-sided carbon discs(Agar Scientific Ltd., UK) and sputter coated with gold. Observa-tions were made and digital photographs were taken with a Jeol JSM-6360 SEM at 20kV.For fluorescence microscopy (FM) and histochemistry, 20- m m-thick sections of fresh samples were cut with a cryotome, asdescribed above and examined directly with an Olympus BX40microscope equipped with a digital camera (DP71, Olympus, Japan). For FM, a BP 330–385 exciter filter and a BA 420 barrierfilter were used. For the tracing of the cortical tube network small3mm  5mm and 2mm deep pieces of bark (including outercork,cortex and phloem), were placed in small glass vials containingCalcofluor (Fluorescent brightener 28, Sigma) and a mild vacuumwas created with a tap pump until no more air bubbles escapedfrom the cut edges of the pieces of tissue (2–3s). The pieces of tissue were then rinsed thoroughly in distilled water, and 20- m m-thick paradermal sections were cut immediately with thecryotome, as described above, mounted on microscope slidesand observed under UV.Acidified phloroglucinol and Sudan IV ( Jensen,1962) wereusedfor the detection of lignin and lipids, respectively, on freshspecimens cut with the cryotome. Results During secondary growth of the olive tree stem, peltatetrichomes areshed and a typical peridermwith lenticels develops.At the same time, a sclerenchyma ring composed of groups of sclereids is formed which separates the photosynthetic from thestorage cortex (Fig. 1a). Within the photosynthetic and storagecortex of all stems with secondary growth, except for those thathave developed a rhytidome, a network of tubules develops as canbe seen in transverse (TS) and paradermal sections (Fig. 1a,b).This tissue forms a network of groups of elongated parallel cells,20–30 m m in diameter, irregularly polygonal in transverse section,that form anastomosing tubules (Figs. 1a,b, 2a). These cells have no intercellular spaces between them unlike the surroundingcortical cells (Fig. 1b). The network formed by this tissue wasfound to be evenly distributed within the cortex, irrespective of the orientation of the stem or branch.The elongated tubular cells can only be discerned in favourablycut paradermal sections and in such cases these were measured tobe up to 300 m m in length (Fig. 2b, dotted line). The transverseanticlinal walls of the tubular cells are reminiscent of theperforated plates of tracheary elements with 50–60 circularperforations of approximately 1 m m in diameter (Fig. 3d). Onmany occasions SEM images show openings on the periclinalwalls of the tubes that seem to establish a continuity of theirlumens with the intercellular spaces (Fig. 2c, arrow) and this isalso verified with TEM (Fig. 3, arrow). There, apparently lysis of the cell walls has occurred. LM, SEM and TEM have shown thatthese tubules are composed of empty, non-living cells as they lackany cytoplasm or any other substance except for some smallcrystalline structures, possibly calcium oxalate, which wereoccasionally observed near the perforated plates. These crystalswere similar in shape and size to those seen in the neighbouringliving cortical cells (Fig. 3). The cell walls of the tubular cellsappear much thinner than those of the surrounding cortical cells(Fig. 3). Fluorescence microscopy and histochemistry with SudanIV and acidified phloroglucinol showed the absence of lignin orany lipid substances from the cell walls of the tubular cells andperforated plates (results not shown), whereas staining withCalcofluor shows their cellulosic nature (Fig. 2).The influx of Calcofluor into the network was immediate as airbubbles stopped emerging only 2–3s after the application of mildvacuum. The result was staining, primarily, of the walls of thetubular cells indicating their cellulosic nature and in places someof the neighbouring cortical cells, presumably where the stain hadseeped through the openings of the tubules into the intercellularspaces (Fig. 2d, arrowheads).Of all tree species investigated only the cortex of those stemsbelonging to the  Olea  genus had developed this tissue irrespectiveof the age, branch position on the tree and orientation. No suchtissue was ever observed in the root cortex of the  Olea  trees or of other species investigated. Discussion The described tissue is, to the best of our knowledge, reportedhere for the first time and is different from the tissues expected tobe found within the photosynthetic or storage cortex of the stem Fig.1.  Light micrographs of semi-thin transverse sections from a 1-year-old stem.(a) The cortical tissues from the periderm to the storage parenchyma; the arrowsshow the locations where the network of tubules develops. (b) The cells indicatedwith asterisks are the transversely cut tubules. (cl – collenchyma; ph –photosynthetic parenchyma; pr – periderm; sc – slerenchyma; st – storageparenchyma). G. Kyriakis, C. Fasseas / Flora 205 (2010) 90–93  91  ARTICLE IN PRESS as these are described in plant anatomy books, e.g. Esau (1977)and Fahn (1990). The position of this tissue and its fibrillarstructure suggest a similarity to the extra-xylem gelatinous fibresthat have been observed in the cortex of   Gnetum gnemon (Carlquist, 1994; Tomlinson, 2001, 2003). However, in the case of  olive tree the development of this tissue does not seem to be theresult of a gravimorphic response as in the case of g-fibres in thetension wood and cortex of many angiosperms (Carlquist, 1994;Fisher and Tomlinson, 2003; Palhares et al., 2007; Tomlinson, 2001, 2003; Tomlinson and Fisher, 2005; Yoshida et al., 2003) or the cortical g-fibres that cause the coiling of the redvine( Brunnichia ovata  (Walt.) Shiners) tendrils (Meloche et al., 2007)as no difference in its distribution is observed between ortho-tropic and plagiotropic axes.The almost instant flooding of the tubule network withCalcofluor demonstrates the continuity of the tubule’s lumensthrough the perforated plates of the transverse anticlinal wallsand the lack of protoplasts. An occasional staining of the corticalcells in the vicinity of the tubules, verifies the existence of sideopenings tothe intercellular spaces as these areobservedwith theSEM and the TEM.The possibility of an artifactual removal of the g-layer or anyother cell contents is minimal as their empty lumen is verifiedusing a variety of microscopical methods, all coming to the sameresults. The thin cell walls, thinner than the surroundingparenchyma cells, of the tubules (or fibres) described here andtheir development after all other mechanical tissues havedeveloped, such as secondary xylem, sclerenchyma, collenchymaand periderm, suggest that their function is not mechanical.Furthermore, the side openings to the intercellular spaces,together with the thin cellulosic cell walls, rule out the possibilityof them transporting any substances, at least in liquid phase. Oneplausible hypothesis that requires further investigation would bethat this tissue might facilitate the diffusion of gases and vaporsbetween the cortical tissues and the external environmentthrough the lenticels. In this case this tissue could be classifiedas a special kind of aerenchyma even though its description fallsoutside the definition of this term which specifies that ‘‘aerench-yma is a parenchymatous tissue characterized by the presence of large intercellular spaces of schizogenous, lysigenous or rhex-igenous srcin, developing only in the submerged parts, roots orstems, of hydrophytes (mangrove plants) as a result of anaero-biosis’’ (Esau, 1977; Fahn, 1990; Kozlowski and Pallardy, 1996; Roth, 1981; Y   an˜ez-Espinoza et al., 2008).The network is found in the photosynthetic cortex of all  Olea stems with secondary growth that have not developed arhytidome. This tissue can easily be missed in transverse,longitudinal or paradermal sections, due to the difficulty insectioning such hard, structurally not uniform specimens. Thistissue has repeatedly been overlooked by us in the past (Filippouet al., 2007) and presumably by other investigators also who haveexamined olive tree bark (Kalahanis and Psaras, 2007). Thearrangement and the extent of it becomes evident only whenserial, exact paradermal sections of even thickness are cut and areexamined under the light microscope, whereas, SEM and TEM addmore information at the ultrastructural level. In our investigationof several tree species with persisting photosynthetic cortex, thistype of tissue was observed only in the  Olea  species examined.However, further examination of the photosynthetic bark of morespecies needs to be conducted. References Carlquist, S., 1994. Wood and bark anatomy of   Gnetum gnemon  L. Bot. J. Linn. Soc.116, 203–221.Esau, K., 1977. Plant Anatomy. Anatomy of the Seed Plants. Wiley, New York. Fig. 2.  a–c SEM micrographs of a paradermal section through the photosyntheticparenchyma. (a) The network of tubules (arrows). (b) The dotted line indicates asingle tubule with the two anticlinal walls having perforations (arrows). (c) Detailof the lower anticlinal wall of the tubule in (b) and side openings to theintercellular space system (arrows). (d) Fluorescence micrograph of a paradermalsection of fresh tissue treated with Calcofluor that has entered the tubules(arrows); the arrowheads indicate the sites where the dye has seeped into thesurrounding cortex. (ph – photosynthetic parenchyma; pp – perforated plate). Fig. 3.  TEM micrograph of several transversely cut tubules showing the muchthinner cell walls than those of the neighbouring cortical cells, a transversely cutperforated plate, an opening of a tubule to the intercellular space network (arrow)and some crystals near the perforated plate and in the protoplast of the corticalcells. (cr – crystals; is – intercellular space; pp – perforated plate;sg – starch grain;st – storage parenchyma cell). G. Kyriakis, C. Fasseas / Flora 205 (2010) 90–93 92
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