plants Article The Orchid Velamen: A Model System for Studying Patterned Article SecondaryArticle Cell Wall Development? The Orchid Velamen:Velamen: AA ModelModel SystemSystem forfor StudyingStudying PatternedPatterned 1,2 3 3 2,3,4,5, Nurul A. IdrisSecondary , Maketelana Aleamotu Cell ʻ a , WallDavid. W Development?McCurdyDevelopment? and David A. Collings * 1 Faculty of Science and Marine Environment, Universiti Malaysia Terengganu, Kuala Terengganu 21030, Nurul A. Idris 1,2Malaysia, ,Maketelana Maketelana; [email protected] Aleamotu Aleamotu ʻ a a3 3,, DavidDavid. W. W McCurdyMcCurdy 33 andand David David A. A. Collings Collings 2,3,4,52,3,4,5,,* * 2 School of Biological Science, University of Canterbury, Private Bag 4800, Christchurch 8140, New Zealand 3 School of Environmental and Life Sciences, The University of Newcastle, Callaghan, NSW 2308, Australia; [email protected] FacultyFaculty of of Sc(M.A.); Scienceience [email protected] and and Marine Marine Environment, Environment, Universiti Universiti (D.W.M.) Malaysia Malaysia Terengganu, Terengganu, Kuala Terengganu 21030, 4 School of Molecular Sciences,MalaysiaKuala TerengganuThe; [email protected] University 21030, of Western Malaysia; Australia, [email protected] Crawley, WA 6009, Australia 5 Harry Butler Institute,22 School SchoolMurdoch of of BiologicalUniversity, Biological Science, Murdoch Science, University, UniversityWA 6150, Australiaof of Canterbury, Canterbury, Private Private Bag Bag 4800 4800,, Christchurch Christchurch 8140, 8140, New New Zealand Zealand * Correspondence: [email protected] 33 SchoolSchool of of Environmental Environmental and and Life Life Sciences, Sciences, The The University University of of Newcastle, Newcastle, Callaghan, Callaghan, NSW NSW 2308, 2308, Australia Australia;; [email protected]@uon.edu.au (M.A.); (M.A.); [email protected] [email protected] (D.W.M.) (D.W.M.) Abstract: Understanding the mechanisms through which plants generate secondary cell walls is of 44 SchoolSchool of of Molecular Molecular Sciences, Sciences, The The University University of of Western Western Australia, Australia, Crawley Crawley,, WA WA 6009 6009,, Australia more than academic55 interest:HarryHarry Butler Butlerthe physi Institute, Institute,cal properties Murdoch Murdoch University,of University, plant-derived Murdoch Murdoch, materials, WA WA 6150,, 6150, including Australia Australia timber and textiles, all depend* Correspondence:Correspondence: upon secondary [email protected] [email protected] cellulose organization. Processes controlling cellulose in the secondary cell wall and their reliance on microtubules have been documented in recent dec- ades, but this understandingAbstract: is Understanding complicated, as secondarythe mechanisms walls normally through form which in the plants plant’s generate interior secondary cell wall wallss is of where live cell imaging is more difficult. We investigated secondary wall formation in the orchid more than academicacademic interest:interest: the the physical physical properties properties of of plant-derived plant-derived materials, materials including, including timber timber and velamen, a multicellular epidermal layer found around orchid roots that consists of dead cells with andtextiles, textiles all depend, all depend upon upon secondary secondary wall cellulosewall cellulose organization. organization. Processes Processes controlling controlling cellulose cellulose in the lignified secondary cell walls. The patterns of cell wall ridges that form within the velamen vary between different orchidinsecondary the secondaryspecies, cell but wall cellimmunolabelling and wall their and reliance their demonstrated reliance on microtubules on thatmicrotubules wall have deposition been have documented isbeen con- documented in recent in decades, recent de butc- Citation: Idris, N.A.; Aleamotu ʻ a, trolled by microtubules.ades,this understanding butAs thesethis understanding patterning is complicated, events is complicatedoccur as at secondarythe outer, as secondary surface walls of normally wallsthe root, normally form and as in form the plant’s in the plant’s interior interior where M.; McCurdy, D.W.; Collings, D.A. orchids are adaptablewherelive for cell tissuelive imaging cell culture imaging is and more genetic is difficult. more manipulation, difficult. We investigated We we investigatedconclude secondary that thesecondary wallorchid formation root wall formation in the orchid in the velamen, orchid The Orchid Velamen: A Model Sys- velamen may indeedvelamen,amulticellular be a suitable a multicellular model epidermal system epidermallayer for studying found layer the around found organization orchid around rootsof otherchid plant that roots consistscell wall. thatof consists dead cells of dead with cells lignified with tem for Studying Patterned Second- Notably, roots of thelignifiedsecondary commonly secondary cell grown walls. orchid cell The walls.Laelia patterns anceps The patterns ofappear cell wallideally of cell ridges suited wall that for ridges developing form that within form this the within velamen the velamen vary between vary ary Cell Wall Development? Plants Citation: Idris, N.A.;research. Aleamotu ʻ a, betweendifferent different orchid species, orchid butspecies, immunolabelling but immunolabelling demonstrated demonstrated that wall that deposition wall deposition is controlled is con- by 2021, 10, x. M.; McCurdy, D.W.; Collings, D.A. https://doi.org/10.3390/xxxxxCitation: Idris, N.A.; Aleamotu ʻ a, trolledmicrotubules. by microtubules. As these patterning As these patterning events occur events at the occur outer at surface the outer of thesurface root, of and the as root, orchids and are as The Orchid Velamen:Keywords: A Model lignin; lignification; microtubules; orchid velamen; secondary cell wall M.; McCurdy, D.W.; Collings, D.A. orchidsadaptable are for adaptable tissue culture for tissue and geneticculture manipulation,and genetic manipulation, we conclude thatwe conclude the orchid that root the velamen orchid mayroot System for Studying Patterned Academic Editors: ViktorThe Orchid Demko Velamen:, Ján A Model Sys- velamenindeed be may a suitable indeed model be a suitable system model for studying system the for organization studying the of organization the plant cell of wall. the plant Notably, cell rootswall. Secondary Cell Wall Development? Jásik, Alexander Luxtem and for Marek Studying Vac- Patterned Second- Notably, roots of the commonly grown orchid Laelia anceps appear ideally suited for developing this Plants 2021, 10, 1358. https:// of the commonly grown orchid Laelia anceps appear ideally suited for developing this research. ulík ary Cell Wall Development? Plants doi.org/10.3390/plants100713581. Introduction research. ʻ 2021, 10, x. Received: 6 June 2021 The primaryKeywords: componentlignin; of the lignification;plant cell wall microtubules; is cellulose, orchida β-1,4 velamen;polymer secondaryof D-glu- cell wall https://doi.org/10.3390/xxxxx Accepted: 24 June 2021Academic Editors: Viktorcose Demko, crystallized Keywords:into microfibrils lignin; that lignification; contain 18 m(toicrotubules; 24) separate orchid glucan velamen; chains [1,2secondary]. The cell wall Published: 2 July 2021Ján Jásik, Alexander Luxorganization and of this cellulose within the cell wall controls plant morphogenesis: for plant Academic Editors: Viktor Demko, Ján Marek Vaculík cells undergoing expansion with a thin, deformable primary cell wall, the transverse ori- Jásik, Alexander Lux and Marek Vac- Publisher’s Note: MDPI stays neu- entation of the cellulose1. Introduction microfibrils dictates the biophysical properties of the wall and tral with regard ulík to jurisdictional Received: 6 June 2021limits expansion to a direction perpendicular to the microfibrils [3]. In mature, nonex- claims in published maps and institu- 1. IntroductionThe primary component of the plant cell wall is cellulose, a β-1,4 polymer of D-glucose Accepted: 24 June 2021panding plant cells, cellulose is instead organized into oblique or cross-laminate patterns tional affiliations. crystallized into microfibrils that contain 18 (to 24) separate glucan chains [1,2]. The organi- Received: 6 June 2021that prevent further expansion.The primary A subset component of plant cells, of the however, plant cell develop wall ais secondary cellulose, cell a β-1,4 polymer of D-glu- Published: 2 July 2021 zation of this cellulose within the cell wall controls plant morphogenesis: for plant cells Accepted: 24 June 2021wall in which extracose cellulose crystallized is reinforced into microfibrils with the organic that containpolymer 18lignin (to 24)[4,5 ].separate In these glucan chains [1,2]. The Published: 2 July 2021secondary walls,organizationundergoing the organization expansion of thisof the cellulose cellulose with a thin,within again deformable remainsthe cell criticalwall primary controls for the cell strengthplant wall, morphogenesis: the transverse orientation for plant Publisher’s Note: MDPI stays neutral of the cell walls, andcellsof the thus undergoing cellulose of the overall microfibrils expansion plant tissue dictates with [3 a]. thin, theThese biophysical deformable secondary propertiescell primary walls are cell of of the wall, wall the and transverse limits expan- ori- Copyright: © 2021 bywith the regardauthors. to Li- jurisdictional claims in Publisher’s Note: MDPI stays neu- censee MDPI, Basel, Switzerland. economic importance:sion toplant a direction-based fibres perpendicular from the finest to cottons the microfibrils to the coarsest [3]. industrial In mature, nonexpanding plant published maps and institutional affil- entation of the cellulose microfibrils dictates the biophysical properties of the wall and This article is an opentral access with article regard tofibres jurisdictional are all derivedcells, from cellulose various is types instead of secondary organized cell into walls oblique and have or cross-laminateproperties de- patterns that prevent iations. limits expansion to a direction perpendicular to the microfibrils [3]. In mature, nonex- distributed under theclaims terms in and published con- mapspendent and institu- on cellulosepandingfurther organization expansion. plant cells, [6]. AS imilarly,cellulose subsetof the is plant propertiesinstead cells, organized however,of timber andinto develop paper oblique amade secondary or cross-laminate cell wall inpatterns which ditions of the Creativetional Commons affiliations. At- from wood pulp thatextradepend prevent cellulose on the further organization is reinforced expansion. of with the Acellulose the subset organic thatof plant polymer makes cells, up lignin thehowever, walls [4,5 ].of develop In these secondarya secondary walls, cell tribution (CC BY) license (http://crea- the xylem cells thatthe form organization the wood ofused the [7,8 cellulose]. again remains critical for the strength of the cell walls, and tivecommons.org/licenses/by/4.0/). wall in which extra cellulose is reinforced with the organic polymer lignin [4,5]. In these thus of the overall plant tissue [3]. These secondary cell walls are of economic importance: secondary walls, the organization of the cellulose again remains critical for the strength Copyright: © 2021 by the authors. plant-based fibres from the finest cottons to the coarsest industrial fibres are all derived Copyright: © 2021 by the authors. Li- of the cell walls, and thus of the overall plant tissue [3]. These secondary cell walls are of Licensee MDPI, Basel, Switzerland. from various types of secondary cell walls and have properties dependent on cellulose Plants 2021, 10, x.censee https://doi.org/10.3390/xxxxx MDPI, Basel, Switzerland. economic importance: plant-based fibres fromwww.mdpi.com/journal/ the finest cottonsplants to the coarsest industrial This article is an open access article organization [6]. Similarly, the properties of timber and paper made from wood pulp This article is an open access article fibres are all derived from various types of secondary cell walls and have properties de- distributed under the terms and depend on the organization of the cellulose that makes up the walls of the xylem cells that conditionsdistributed ofunder the Creativethe terms Commonsand con- pendent on cellulose organization [6]. Similarly, the properties of timber and paper made form the wood used [7,8]. Attributionditions of the (CC Creative BY) license Commons (https:// At- from wood pulp depend on the organization of the cellulose that makes up the walls of The deposition of cellulose within the plant cell wall is controlled, in part, by cortical creativecommons.org/licenses/by/tribution (CC BY) license (http://crea- the xylem cells that form the wood used [7,8]. microtubules, as demonstrated by live cell analyses that show cellulose synthase complexes 4.0/).tivecommons.org/licenses/by/4.0/).
Plants 2021, 10, x. https://doi.org/10.3390/xxxxx www.mdpi.com/journal/plants Plants 2021, 10, 1358. https://doi.org/10.3390/plants10071358 https://www.mdpi.com/journal/plants Plants 2021, 10, x FOR PEER REVIEW 2 of 16
Plants 2021, 10, 1358 2 of 16 The deposition of cellulose within the plant cell wall is controlled, in part, by cortical microtubules, as demonstrated by live cell analyses that show cellulose synthase com- plexes which synthesize new cellulose microfibrils moving along the microtubules in ex- which synthesize new cellulose microfibrils moving along the microtubules in expanding panding primary cells [9,10]. While similar studies have demonstrated a role for microtu- primary cells [9,10]. While similar studies have demonstrated a role for microtubules in bules in secondary cell wall development [11,12], these live cell imaging experiments have secondary cell wall development [11,12], these live cell imaging experiments have been been hampered by secondary cell wall development occurring in internal tissues, which hampered by secondary cell wall development occurring in internal tissues, which has has limited the clarity of the imaging conducted. Various systems have been developed limited the clarity of the imaging conducted. Various systems have been developed that that offer more tractable systems with which to study secondary cell wall formation. offer more tractable systems with which to study secondary cell wall formation. While While classic cell culture systems, such as Zinnia elegans mesophyll cells that can be in- classic cell culture systems, such as Zinnia elegans mesophyll cells that can be induced to ducedform xylem to form [13 xylem], are nontransformable,[13], are nontransformable,Arabidopsis Arabidopsis thaliana suspensionthaliana suspension cells expressing cells ex- pressingfluorescent fluorescent fusions can fusions be induced can be to induced undergo to secondaryundergo secondary cell wall development cell wall development [14]. More [14].recently, More inducible recently, systems inducible have systems been developed have been in developed which the in epidermal which the cells epidermal of A. thaliana cells ofroots A. thaliana are genetically roots are reprogrammed genetically reprogrammed to develop secondary to develop cell secondary walls and cell become walls and xylem be- comevessels xylem [15– 18vessels]. While [15–18]. these While experimental these experi systemsmental allow systems for theallow dissection for the dissection of the roles of theplayed roles by played microtubules by microtubules in secondary in secondary cell wall formation, cell wall formation, they remain they artificial, remain as artificial, the cells asare the in cells culture are and/orin culture are and/or triggered are totriggered develop to through develop nonstandard through nonstandard pathways. pathways. WeWe have previously investigatedinvestigated microtubules microtubules and and secondary secondary cell cell walls walls in in the the roots roots of ofthe the orchid orchidMiltoniopsis Miltoniopsis. These. These experiments experiments demonstrate demonstrate that microtubulesthat microtubules align withalign devel- with developingoping phi thickenings phi thickenings in the in root the cortex root cortex [19], these [19], thickeningsthese thickenings being being bands bands of secondary of sec- ondarycell wall cell that wall are that induced are induced in the in root the cortex root cortex of numerous of numerous species species including including orchids orchids [20], [20],while while preliminary preliminary observations observations demonstrated demonstrated that microtubulesthat microtubules are aligned are aligned with with develop- de- velopinging secondary secondary cell wall cell striations wall striations in the rootin the velamen root velamen [21]. In this[21]. study, In this we study, have expandedwe have expandedour observations our observations of microtubules of microtubules during velamen during development, velamen anddevelopment, we demonstrate and thatwe demonstratethe outer cell that layer the within outer thecell velamen layer within may the be avelamen useful system may be in a whichuseful tosystem investigate in which the torole investigate of the cytoskeleton the role of inthe secondary cytoskeleton cell wallin secondary formation. cell The wall velamen formation. is a layerThe velamen of dead iscells a layer with of secondary dead cells wall with striations secondary that wall surrounds striations the that cortex surrounds of orchid the cortex roots (Figure of orchid1). rootsIn terrestrial (Figure orchids,1). In terrestrial the velamen orchids, typically the velamen contains typically only a single contains cell layer only and a single coats cell the layerparenchymatic and coats the cortex. parenchymatic In epiphytic cortex. orchids In epiphytic that are prone orchids to dehydrationthat are prone because to dehydra- their tionaerial because roots typically their aerial only roots cling typically to the host only tree’s cling branches to the host surrounded tree’s branches by leaf surrounded litter rather bythan leaf actual litter soil, rather the than velamen actual is asoil, specialized, the velamen spongy, is a multilayered specialized, structurespongy, multilayered [22–25]. This structurevelamen is[22–25]. thought This to functionvelamen in iswater thought capture to function by the rootin water [24], andcapture in water by the retention root [24], in anddry conditionsin water retention [23,26], in with dry the conditions unusual nature[23,26], of with the the velamen unusual cell nature wall thought of the velamen to aid in cellthese wall roles. thought Classification to aid in these of the roles. different Classifi secondarycation of cellthe different patterns formedsecondary in thecell velamenpatterns formedhas defined in the 11 velamen different has velamen defined patterns 11 differen [24],t allowingvelamen taxonomicpatterns [24], keys allowing to utilise taxonomic velamen keyspatterning to utilise as avelamen defining patterning characteristic as a [defining27–29]. Incharacteristic several of these [27–29]. classes, In several velamen of these wall classes,thickenings velamen do not wall show thickenings banding patterns,do not show but banding some taxa patterns, show patterned but some wall taxa thickenings show pat- ternedin the velamenwall thickenings with helical in the or transversevelamen with ridges helical that or bear transverse superficial ridges similarities that bear to superfi- the wall cialthickenings similarities of primaryto the wall xylem. thickenings of primary xylem.
Figure 1. Velamen development in orchid roots. Orchid roots are co coveredvered by a thin layer of dead cells called the velamen which appears an off-white colour. Junctions between the newly formed velamen and the growing root are indicated by which appears an off-white colour. Junctions between the newly formed velamen and the growing root are indicated by arrows and are enlarged in the insets. (a) Miltoniopsis sp. (b) Laelia anceps. (c) Dendrobium sp. (d) Phalaenopsis sp. arrows and are enlarged in the insets. (a) Miltoniopsis sp. (b) Laelia anceps.(c) Dendrobium sp. (d) Phalaenopsis sp.
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We have previously demonstrated that the Miltoniopsis velamen exhibits the classic cymbidium-type velamen organization, with thin helical wall thickenings that show a webbed or mesh structure. Microtubules align along the length of these thickenings as they develop [21]. In this study, we wished to determine whether the orchid velamen might be developed as a model system in which live cell imaging can be used to investigate the role of the cytoskeleton in the formation of secondary cell walls. As a first step in this process, we surveyed cell wall organization in a range of commonly grown orchids and demonstrated that the outermost wall of the velamen shows striking patterns in several different orchids. We then investigated the role of microtubules in velamen development using conventional immunofluorescence microscopy. In all cases where microtubules were successfully imaged, they aligned along the developing cell wall ridges, but as these structures matured, the microtubules assumed a flanking position, a pattern also seen in developing vascular elements. Furthermore, as live cell imaging requires readily accessible outer epidermal and subepidermal layers, we placed extra emphasis on observing cell wall structures in the outermost cells of the velamen that would be most readily seen in such experiments. We demonstrate that in Laelia anceps, the outer face of the velamen has strongly patterned cell wall ridges and that strongly bundled bands of microtubules initially run parallel to these ridges before flanking the ridges on either side. Thus, the Laelia velamen may prove to be an interesting system in which to further study the cytoskeleton and cell wall development.
2. Results Although a range of different orchid species were initially investigated, we eventually limited imaging to four different species which show different classes of velamen thick- ening [24]. These included Miltoniopsis, which we had previously investigated, and three other species, Laelia anceps and commercial hybrids of Dendrobium and Phalaenopsis.
2.1. Velamen Structure in Miltoniopsis sp. Cross sections through the Miltoniopsis root stained for lignin with basic fuchsin showed multiple, lignin-reinforced secondary cell walls (Figure2). At the centre of the root, the stele was heavily lignified (Figure2a) and surrounded by a strongly lignified endodermis (en), although passage cells (*) through the endodermis remained unlignified. The outer cortex contained some lignified cells that had little cell wall patterning (Figure2 a, LC) but no phi thickenings were present. The velamen was separated from the root cortex by the exodermis (ex) in which the hexagonally shaped cells are strongly lignified, alternating with passage cells that had a thin, mostly unlignified wall (#). However, all exodermis cells had thickened walls separating the exodermis cells and velamen cells. The velamen surrounding the cortex showed a cymbidium-type velamen organiza- tion and consisted of six to eight layers of dead cells with larger, radially extended inner velamen cells and smaller outer velamen cells (Figure2a,b, V). Mature velamen cell walls were reinforced by long strands of cellulose forming a mesh-like appearance (Figure2 c), although this criss-crossing effect is due to adjacent cells having cellulose patterns run- ning in different orientations: in individual cells, the wall ridges were typically evenly spaced and roughly parallel, but their overall orientations showed no preferred direction (Figure2c, Supplementary Movie S1 in Supplementary Materials). Longitudinal sections demonstrated that the cell wall organization in the outer velamen layer was different, and instead of the highly regular patterns of cell wall ridges present in the inner velamen, these outer cells contained only a few striations that were more irregular (Figure2d, asterisk; Supplementary Movie S2 in Supplementary Materials). Plants 2021, 10, 1358 4 of 16 Plants 2021, 10, x FOR PEER REVIEW 4 of 16
FigureFigure 2. Velamen 2. Velamen organization organization in in MiltoniopsisMiltoniopsis sp.sp. roots. roots. Images Images (a,,cc,,dd)) areare maximummaximum projections projections of of confocal confocal optical optical series series showing basic fuchsin-stained lignin. (a,b) Cross section showing lignified cell walls and a concurrent transmitted light showing basic fuchsin-stained lignin. (a,b) Cross section showing lignified cell walls and a concurrent transmitted light image. The wide velamen (V) surrounds the lignified exodermis (ex) and the cortex which contains lignified cortical cells image. The wide velamen (V) surrounds the lignified exodermis (ex) and the cortex which contains lignified cortical cells (LC), while a lignified endodermis (en) surrounds the central stele. Passage cells through the endodermis (*) and velamen (LC), while a lignified endodermis (en) surrounds the central stele. Passage cells through the endodermis (*) and velamen (#) (#) walls are marked. (c) Higher resolution imaging of the criss-crossed cell wall thickenings in the velamen. (d) A longi- c d tudinalwalls section are marked. shows ( ) only Higher scattered resolution wall imaging thickenings of the criss-crossedin the outer cellwall wall of thickeningsthe outer velamen in the velamen. (asterisks). ( ) AA longitudinal region where thesection inner face shows of onlythese scattered cells was wall imaged, thickenings and which in the shows outer wall the criss-crossed of the outer velamen pattern, (asterisks). is indicated A regionby a double where arrowhead. the inner Seeface also of Supplementary these cells was imaged,Movies 1 and and which 2. Bar shows in (a) the= 200 criss-crossed µm for (a,b pattern,); bars in is ( indicatedc,d) = 100 by µm. a double arrowhead. See also Supplementary Movies 1 and 2. Bar in (a) = 200 µm for (a,b); bars in (c,d) = 100 µm. 2.2. Velamen Structure in Laelia anceps 2.2. Velamen Structure in Laelia anceps Basic fuchsin-stained cross sections of Laelia anceps roots revealed numerous strongly Basic fuchsin-stained cross sections of Laelia anceps roots revealed numerous strongly lignifiedlignified secondary secondary walls walls (Figure (Figure3 3).). The The most most dramatic dramatic structures structures were were the the near-complete near-complete corticalcortical rings rings of of phi phi thickenings thickenings that encircledencircled the the stele stele (Figure (Figure3a,b, 3a,b,Φ ).Φ While). While the the central central stelestele was was strongly strongly lignified, lignified, neither thethe endodermalendodermal cells cells nor nor the the exodermal exodermal cells cells of Laeliaof Laelia werewere lignified lignified (Figure (Figure 33a,a, enen andand ex),ex), although although the the outer outer exodermal exodermal wall wall that that joins joins to theto the velamenvelamen did did show show lignification. lignification. The The organization organization of of the the velamen velamen in inLaeliaLaelia anceps anceps waswas typ- icaltypical of the of epidendrum the epidendrum pattern, pattern, with with three three or or four four layers layers ofof longlong and and narrow narrow cells cells that that werewere reinforced reinforced by by thickened thickened bands ofof cellulosecellulose thatthat were were broadly broadly parallel parallel (Figure (Figure3c, 3c, SupplementarySupplementary Movie Movie S3 S3 in in Supplementary Materials)Materials) and and with with outer outer velamen velamen cells cells that that werewere smaller smaller and and not not elongated. elongated. Longitudinal sectionssections showed showed that that this this cell cell wall wall banding banding patternpattern continued continued through through to to the outer surfacesurface ofof thethe root root where where the the patterning patterning of of the the parallelparallel bands bands was strikingstriking and and showed showed evidence evidence of organizationof organization between between cells (cellsFigure (Figure3 d, 3d,Supplementary Supplementary Movie Movie S4 in S4 Supplementary in Supplementary Materials). Materials). Several Several locations locations where these where outer these velamen wall ridges ran at similar orientations in adjacent cells are highlighted in Figure3 d outer velamen wall ridges ran at similar orientations in adjacent cells are highlighted in with asterisks. Figure 3d with asterisks.
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FigureFigure 3. Velamen 3. Velamen organization organization in Laelia in Laelia anceps anceps roots.roots. Images Images (a (a,c,c,d,d)) are are maximum maximum projectionsprojections of of confocal confocal optical optical series series of berberineof berberine hemisulfate hemisulfate (a) and (a) basic and basic fuchsin fuchsin staining staining (c, (dc,).d ).(a (,ab,b) )Cross Cross section showingshowing lignified lignified cell cell walls, walls, along along with awith a concurrentconcurrent transmitted transmitted light light image. image. The The velamen velamen (V) (V) surrounds surrounds thethe lignifiedlignified exodermis exodermis (ex) (ex) and and the cortex.the cortex. Extensive Extensive phi phi thickeningsthickenings (ϕ) are (φ) present are present within within the the cortex. cortex. (c ()c )Higher Higher resolution resolution imageimage of of cell cell wall wall thickenings thickenings in the in highlythe highly elongated elongated inner innervelamen velamen cells. cells. (d) Longitudinal (d) Longitudinal section section showing showing wave-like wave-like wallwall thickenings thickenings in in the the outer outer surface surface of the of velamen. the velamen. See See also Supplementaryalso Supplementary Movies Movies S3 S3and and S4. S4. Bar Bar in in (a (a) )= = 200 200 µmµm for (aa,,bb);); bars bars in in (c (,dc,)d =) 100= 100µm. µm.
2.3.2.3. Velamen Velamen Structure Structure in in Dendrobium Dendrobium spp.spp. CellCell wall wall labelling labelling with with basic fuchsinfuchsin of of root root sections sections through through a Dendrobium a Dendrobiumhybrid hybrid showedshowed a wide a wide cortex, cortex, a avelamen velamen with with three three to to five five cell layers andand aa heavilyheavily lignified lignified en- dodermisendodermis with with unlignified unlignified passage passage cells cells (Fig (Figureure 4a,b).4a,b). The velamenvelamen showed showed helically helically ar- arranged wall thickenings characteristic of the dendrobium class (Figure4c, Supplementary ranged wall thickenings characteristic of the dendrobium class (Figure 4c, Supplementary Movie S5 in Supplementary Materials) which, in longitudinal sections, were widely spaced Movieand orientedS5 in Supplementary in a transverse pattern Materials) (Figure which,4d, Supplementary in longitudinal Movie sections, S6 in Supplemen- were widely spacedtary Materials).and oriented in a transverse pattern (Figure 4d, Supplementary Movie S6 in Sup- plementary Materials).
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FigureFigure 4. Velamen 4. Velamen organization organization in inDendrobiumDendrobium sp.sp. roots. roots. Images Images ((aa,,cc,d,d)) are are maximum maximum projections projections of confocal of confocal optical optical series series collectedcollected from from basic basic fuchsin fuchsin fluorescence. fluorescence. (a (,ab,)b A) A slightly slightly off-angle off-angle crosscross section section showing showing lignified lignified cell cell walls, walls, along along with awith a concurrentconcurrent transmitted transmitted light light image. image. The The velamen velamen (V) (V) surrounds surrounds thethe lignifiedlignified exodermis exodermis (ex) (ex) and and the cortex.the cortex. The partiallyThe partially lignifiedlignified endodermis endodermis (en) (en) with with multiple multiple passage passage cells cells (*) (*) surrounds surrounds the the central central stele. stele. (c )( Higherc) Higher resolution resolution image image of crossed of crossed cell wallcell wallthickenings thickenings in the in the outer outer velamen. velamen. ( (dd)) Longitudinal section section showing showing velamen velame cellsn withcells transversewith transverse wall thickenings wall thicken- ings onon the the velamen’s outer outer face. face. See See also also Supplementary Supplementary Movies Movies 5 and 5 6. and Bar 6. in Bar (a) = in 200 (a)µ =m 200 for µm (a,b );for bar (a in,b ();c )bar = 100 in µ(cm.;) =100 bar µm.; bar inin (d (d) )= =50 50 µm.µm.
2.4.2.4. Velamen Velamen Structure Structure in in Phalaenopsis Phalaenopsis sp.sp. TheThe commercial commercial PhalaenopsisPhalaenopsis hybridhybridimaged imaged in in these these experiments experiments had had a velamen a velamen layerlayer typical typical for for the the vanda vanda class class in in that that the the velamen velamen was was only only one one or or two two cell cell layers layers thick (Figurethick (5a,b,Figure Supplementary5 a,b, Supplementary Movie Movie S7 in S7 Su inpplementary Supplementary Materials). Materials). In In the the vanda vanda type, type, velamen cells are commonly smaller than the exodermis cells that are thickened in velamen cells are commonly smaller than the exodermis cells that are thickened in their their tangential and radial (partly thickened) walls. Longitudinal sections through the tangentialPhalaenopsis andvelamen radial showed (partly varying thickened) cell wall walls. patterns Longitudinal in the outer layer sections (Figure 5throughc). The the Phalaenopsisouter velamen velamen showed showed a considerable varying cell degree wallof patterns variability: in the while outer some layer cells (Figure showed 5c). The outerwidely velamen spaced showed bands of a near considerable transverse cellulosedegree ridgesof variability: (Figure5d), while most some cells contained cells showed widelyclosely spaced spaced, bands textured of near cell walls transverse in which cellulose the broad ridges thickening (Figure bands 5d), appeared most cells to weave contained closelyinto andspaced, out of textured the wall cell in a walls wickerwork-like in which the pattern broad (Figure thickening5e, Supplementary bands appeared Movie to S8 weave intoin and Supplementary out of the wall Materials). in a wickerwork-like pattern (Figure 5e, Supplementary Movie S8 in Supplementary Materials).
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FigureFigure 5. Velamen 5. Velamen organization organization in Phalaenopsis in Phalaenopsis sp. roots. sp. roots. Images Images (a,c– (ae,)c –aree) aremaximum maximum projections projections of of confocal confocal optical optical series collectedseries of collectedbasic fuchsin of basic labelling. fuchsin ( labelling.a,b) Cross (a section,b) Cross showing section showinglignified lignified velamen velamen (V) but (V)unlignified but unlignified exodermis exodermis (ex) walls, along(ex) with walls, a concurrent along with tr aansmitted concurrent light transmitted image. ( lightc) Higher image. resolution (c) Higher image resolution of cell image wall of thickenings cell wall thickenings in the velamen. in the (d) Longitudinalvelamen. sections (d) Longitudinal through sectionsthe outer through face of thethe outervelamen face showed of the velamen either showedirregular either cell wall irregular ridges cell (d wall) or more ridges commonly, (d) or narrowlymore spaced, commonly, broad narrowly swirls spaced, of secondary broad swirls walls of (e secondary). See also walls Supplementary (e). See also Supplementary Movies S7 and Movies S8. Bar S7 in and (a) S8. = 200 Bar inµm for (a,b); (bara) = in 200 (cµ) m=100 for µm.; (a,b); bars bar in in (c ()d =,e 100) = µ50m.; µm. bars in (d,e) = 50 µm.
2.5.2.5. Cytoskeletal Cytoskeletal Organi Organizationzation during during VelamenVelamen Development Development in Miltoniopsisin Miltoniopsis MicrotubuleMicrotubule immunolabelling immunolabelling waswas conductedconducted on on formaldehyde-fixed formaldehyde-fixed tissue tissue from from nearnear the the tip tip of of the the root root where where velamenvelamen development development was was occurring occurring (Figure (Figure1). The 1). two The two methodsmethods used used to toallow allow antibody antibody penetration penetration throughthrough the the cell cell wall wall were were sectioning sectioning with with a a vibratome and enzymatic digestion of root sections. While sectioning generally gave vibratome and enzymatic digestion of root sections. While sectioning generally gave more more consistent labelling patterns, enzymatic digestion of the walls proved superior when consistentviewing labelling the outer corticalpatterns, cells enzymatic and, in particular, digestion theof outerthe walls face ofproved the velamen. superior As when viewingpreviously the outer documented cortical [21 cells], tubulin and, immunolabellingin particular, the of outer Vibratome face sectionsof the velamen. of Miltoniopsis As previ- ouslyroots documented showed that [21], microtubules tubulin wereimmunolabelling strongly bundled of Vibratome early in the developmentsections of Miltoniopsis of the rootsvelamen showed when that the microtubules cell wall ridges were were strongly only just visiblebundled with early transmitted in the lightdevelopment (Figure6 a). of the velamenLater in when velamen the development,cell wall ridges as shown were byonly the strongjust visible development with transmitted of wall ridges light in the(Figure 6a).transmitted Later in velamen light image, development, bundled microtubules as shown by lay the parallel strong to development the cell wall ridges,of wall with ridges in thepaired transmitted microtubule light bundlesimage, bundled flankingeither microtubules side of the lay wall parallel ridges to (Figure the cell6b). wall Orthogonal ridges, with sections generated in ImageJ through optical stacks showed that these paired microtubule paired microtubule bundles flanking either side of the wall ridges (Figure 6b). Orthogonal bundles ran adjacent to the wall ridges and did not lie beneath them (Figure7, arrows). sections generated in ImageJ through optical stacks showed that these paired microtubule bundles ran adjacent to the wall ridges and did not lie beneath them (Figure 7, arrows).
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FigureFigure 6. 6.Microtubules Microtubules and and velamen velamen development development in inMiltoniopsis Miltoniopsis. Microtubule. Microtubule images images (left (left column) col- Figureareumn) maximum 6.are Microtubules maximum projections projections and of velamen confocal of confocal development optical seriesoptical in from series Miltoniopsis early from (a )early. and Microtubule late(a) and (b) velamenlate images (b) velamen development (left col- devel- umn)opment are andmaximum are shown projections alongside of confocalconcurrent optical transm seriesitted from light early images (a) and(right late column). (b) velamen Labelling devel- is and are shown alongside concurrent transmitted light images (right column). Labelling is of cells from opmentof cells andfrom are the shown inner alongsidevelamen in concurrent vibratome transm sections.itted When light cell images wall (rightridges column). first developed Labelling (a), is the inner velamen in vibratome sections. When cell wall ridges first developed (a), they were only ofthey cells were from only the innerweakly velamen visible inby vibratome transmitted sections. light but, When being cell unlignified, wall ridges were first notdeveloped yet autofluo- (a), theyweaklyrescent. were visible Microtubules only byweakly transmitted visibleformedlight by strongly transmitted but, being bundled, unlignified,light pa but,rallel being were arrays. unlignified, not Later yet autofluorescent. in development,were not yet Microtubules autofluo- when cell rescent.formedwall ridges Microtubules strongly were bundled, mo reformed pronounced parallel strongly arrays. (b bundled,), the Later immunolabelled pa inrallel development, arrays. microtubules Later when in celldevelopment, were wall often ridges whenpaired, were cell more run- wallpronouncedning ridges along were either (b), themo sidere immunolabelled pronounced of the cell wall (b), microtubulesridges the immunolabelled (arrows). were Bars often inmicrotubules ( paired,a,b) = 20 running µm. were alongoften paired, either side run- of ningthe cell along wall either ridges side (arrows). of the ce Barsll wall in (ridgesa,b) = 20(arrows).µm. Bars in (a,b) = 20 µm.
Figure 7. Split microtubule bands lie on either side but not underneath cell wall ridges. The microtubule splitting pattern in Miltoniopsis roots does not occur because of a change in focal plane of the microscope. Confocal optical sections were Figure 7. Split microtubule bands lie on either side but not undern underneatheath cell wall ridges. The microtubule splitting pattern incollected Miltoniopsis at 1 rootsµm intervals does not through occur because vibratome of a sectionschange inof focalinner plane velamen of the cells microscope. immunolabelled Confocal for optical (a) microtubules sections were (col- in Miltoniopsis roots does not occur because of a change in focal plane of the microscope. Confocal optical sections were collectedoured red at 1in µm the intervals overlay image)through and vibratome (b) stained sections with ofpontamine inner velamen for cellulose cells immunolabelled (coloured cyan for in (thea) microtubulesoverlay image). (col- (c) collected at 1 µm intervals through vibratome sections of inner velamen cells immunolabelled for (a) microtubules (coloured ouredOverlay red image. in the Microtubulesoverlay image) showed and (b distin) stainedct patterns with pontamine from the developing for cellulose cellulose (coloured wall cyan ridges. in Athe single overlay confocal image). optical (c) Overlayredsection in the image.is overlayshown Microtubules (panels image) XY and) showed with (b) stained YZ distin and with XZct patterns planes pontamine being from for computer-generated the cellulose developing (coloured cellulose orthog cyan wallonal in ridges. the reconstructions overlay A single image). confocal at the (c) Overlaylocations optical sectionimage.indicated Microtubulesis shown with the(panels dotted showed XY lines.) with distinct These YZ patternsand reconstructions XZ planes from the being developingshow computer-generated that late cellulose in velamen wall orthog ridges. development,onal A singlereconstructions microtubules confocal optical at the run locations section on either is indicatedshownside of (panels the with secondary XYthe) dotted with cellYZ lines. walland TheseridgeXZ planes (arrows)reconstructions being rather computer-generated thanshow directly that late unde in orthogonal velamenrneath them. development, reconstructions Bar in (c) microtubules = at20 theµm locations for all run images. indicatedon either sidewith of the the dotted secondary lines. Thesecell wall reconstructions ridge (arrows) show rather that than late directly in velamen unde development,rneath them. microtubulesBar in (c) = 20 runµm onfor either all images. side of the secondary cell wall ridge (arrows) rather than directly underneath them. Bar in (c) = 20 µm for all images.
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2.6. Microtubules and Velamen Development in Laelia 2.6.Immunolabelling Microtubules and Velamen experiments Development in the in developing Laelia Laelia root used enzyme-digested sectionsImmunolabelling to visualise microtubules experiments in in the the outermost developing cellsLaelia ofroot the usedvelamen, enzyme-digested where the most dramaticsections cell to visualisewall organization microtubules had in thebeen outermost observed cells (Figure of the 3d) velamen, and showed where the similar most pat- ternsdramatic of microtubule cell wall organization organization had to been those observed seen in (Figure Miltoniopsis3d) and. showedIn younger similar cells, patterns nearer the Miltoniopsis rootof tip, microtubule microtubule organization bundling to those preceded seen in the lignification. In younger of the cells,cell wall nearer ridges the root (Figure tip, microtubule bundling preceded the lignification of the cell wall ridges (Figure8a), 8a), whereas in older cells, between 1 and 2 mm further away from the root tip, lignifica- whereas in older cells, between 1 and 2 mm further away from the root tip, lignification tionof of the the ridges ridges had had begun, begun, and and the the microtubule microtubule bundles bundles which which had had been been whole whole showed showed evidenceevidence of ofsplitting splitting so so that that microtubules microtubules flanked flanked the the cell cell wall wall ridges ridges (Figure (Figure8c, arrows). 8c, arrows). WeWe have have attempted attempted microtubule microtubule immunolabelling immunolabelling in Dendrobium in Dendrobiumroots, as these roots, also as showed these also showedstriking striking patterns patterns in the outermost in the outermost velamen wall, velamen but to date,wall, labellingbut to date, experiments labelling have experi- mentsnot provenhave not to beproven successful. to be successful.
FigureFigure 8. Microtubules 8. Microtubules and and velamen velamen development development in in LaeliaLaelia. Labelling Labelling of of the the outermost outermost layer layer of theof the velamen velamen was achievedwas achieved usingusing cell wall cell wall digestion digestion enzymes. enzymes. Confocal Confocal maximum projections projections of microtubulesof microtubules (left-hand (left-hand column) column) and lignin and autofluo- lignin auto- fluorescencerescence (central (central column),column), along along with with concurrent concurrent transmitted transmitted light images light images (right-hand (right-hand column). (column).a,b) Early ( ina,b development,) Early in devel- opment,microtubules microtubules have have become become bundled, bundled, and while and cell while wall cell ridges wall were ridges visible were in transmittedvisible in tran light,smitted they had light, not they become had not becomelignified. lignified. Microtubules Microtubules ran parallel ran parallel to the wallto the ridges wall asridges single as bundles. single bundles. Confocal imagesConfocal of ligninimages and of microtubuleslignin and microtu- (a) bules were(a) were reoriented reoriented so that so the that outer the cell outer layers cell lie layers in the lie image in the plane, image while plane, the transmitted while the lighttransmitted image (b )light remains image unrotated. (b) remains unrotated.(b) Once (b) cellOnce wall cell ridges wall hadridges developed had developed lignification, lignification, the microtubule the microtubule bundles split bundles to run split on either to run flank on either of the ridgesflank of the ridges(arrows). (arrows). Bar Bar in (ina) ( =a 50) = µ50m µm for (fora,b); (a bar,b); in bar (c) in = 20 (c)µ =m. 20 µm.
2.7.2.7. Microtubules Microtubules and and Velamen Velamen Development in in Phalaenopsis Phalaenopsis CellCell wall wall digestion digestion of of root root sectionssections waswas also also used used to to observe observe the the outer outer face offace the of the velamen.velamen. While While strongly strongly bundled bundled microtubules were were seen seen early early in velamenin velamen development, development, againagain prior prior to to the the development development of significantsignificant cell cell wall wall lignification lignification (Figure (Figure9a), observing 9a), observing microtubules in older parts of the root proved to be problematic, presumably due to microtubules in older parts of the root proved to be problematic, presumably due to suboptimal enzyme digestion. However, in cases where labelling was observed, the suboptimalmicrotubule enzyme flanking digestion. patterns However, were sometimes in cases observed where (Figure labelling9b, arrows).was observed, Imaging the mi- crotubulefurther highlightedflanking patterns the variability were sometimes in the outer wallobserved of the (FigurePhalaenopsis 9b, velamenarrows). (compare Imaging fur- therlignin highlighted patterns inthe Figure variability9 to Figure in 5the). outer wall of the Phalaenopsis velamen (compare lignin patterns in Figure 9 to Figure 5).
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FigureFigure 9. Microtubules 9. Microtubules and and velamen velamen development development in in Phalaenopsis.. LabellingLabelling of of the the outermost outermost layer layer of the of velamenthe velamen was was achievedachieved using using cell wall cell walldigestion digestion enzymes. enzymes. Confocal Confocal maximu maximumm projections projections of ofmicrotubules microtubules (left-hand (left-hand column) column) and and lignin autofluorescencelignin autofluorescence (central column), (central column), along with along concurrent with concurrent transmitted transmitted light light images images (right-hand (right-hand column). column). ((aa)) Early Early in in de- velopment,development, microtubules microtubules have have become become bund bundled,led, and and while while cell cell wall wall ridges were were visible visible in in transmitted transmitted light, light, they hadthey not had not becomebecome lignified. lignified. Microtubules Microtubules ran ran parallel parallel to to the the wall wall ridges asas singlesingle bundles. bundles. (b )(b Once) Once cell cell wall wall ridges ridges had developedhad developed lignification,lignification, the themicrotubule microtubule bundles bundles split split toto runrun on on either either side side of theof ridgesthe ridges (arrows). (arrows). Bar in Bar (a) = in 50 (µam;) = bar50 inµm; (b )bar = 20 inµ m.(b) = 20 µm. 3. Discussion 3. DiscussionThe velamen is a specialized tissue in the outer layer of the orchid root, having cellThe walls velamen with patterned, is a specialized secondary tissue cell wallin the thickenings. outer layer This of cellthe layer,orchid which root, is having most cell wallsprominent with patterned, in epiphytic secondary orchids, cell is an wall adaptation thickenings. that optimizes This cell waterlayer, retentionwhich is [most23,26 ]prom- inentand in water epiphytic and nutrient orchids, intake is an fromadaptation the environment that optimizes [25], both water of retention which are [23,26] important and wa- factors for epiphytic orchids whose roots are not typically under moist soil. Although such ter and nutrient intake from the environment [25], both of which are important factors for wall thickenings are almost unique to orchid roots, similar patterned cell walls have also epiphyticbeen observed orchids covering whose theroots roots are of not several typica otherlly familiesunder moist of plants, soil. including Although the such fern wall thickeningsAsplenium [are30] andalmost related unique species to [orchid31]; the roots, Cyperaceae similar and patterned Velloziaceae, cell where walls thickenings have also been observedare multilayered covering [ 24the]; androots other of several monocots othe [32r]. families of plants, including the fern Asple- nium [30] and related species [31]; the Cyperaceae and Velloziaceae, where thickenings are3.1. multilayered Microtubules [24]; and Velamenand other Development monocots [32]. The secondary thickenings of the orchid velamen cell wall consist of cellulose mi- 3.1.crofibrils Microtubules orientated and Velamen in specific Development patterns, with the organization of these patterns being an important characteristic in taxonomic identifications [27,33]. Our observations of four differentThe secondary orchid species thickenings showed patternsof the orchid consistent vela withmen thesecell wall previous consist characterizations. of cellulose micro- fibrilsThe orientated velamen develops in specific from patterns, living postmeristematic with the organization cells that undergo of these secondary patterns cellbeing wall an im- portantdeposition characteristic and subsequent in taxonomic programmed identifications cell death to[27,33]. leave theOur shell observations of the cell wall. of four The differ- entcoalignment orchid species of cellulose showed deposition patterns consistent and microtubules with these is well previous known characterizations. [3], and cellulose The velamensynthase develops complexes from that living synthesize postmeristematic cellulose microfibrils cells that coalign undergo with and secondary travel along cell wall depositionmicrotubules and insubsequent both primary programmed [9,33] and secondary cell death cell to wallsleave [ 11the,12 shell], so itof is the no cell surprise wall. The coalignmentthat microtubules of cellulose ran parallel deposition to the depositionand microtubules of the cellulose is well ridges known during [3], velamenand cellulose development in orchid roots. synthaseA single,complexes previous that study synthesize of velamen cellulose cell wall microfibrils development coalign in Ansellia withused and transmis- travel along microtubulession electron in microscopy both primary to report [9,33] the and association secondary of microtubulescell walls [11,12], with the so laterit is no stages surprise thatof microtubules wall thickening ran development, parallel to butthe nodeposition evidence of was the observed cellulose for ridges “cytoplasmic during pre-velamen developmentpatterning” orin alignmentorchid roots. of microtubules with the early stages of wall thickening develop- mentA single, [33]. However, previous our study observations, of velamen reported cell wall in a development preliminary form in Ansellia in Miltoniopsis used transmis-[21] sion electron microscopy to report the association of microtubules with the later stages of wall thickening development, but no evidence was observed for “cytoplasmic pre-pat- terning” or alignment of microtubules with the early stages of wall thickening develop- ment [33]. However, our observations, reported in a preliminary form in Miltoniopsis [21] and repeated more extensively in this study for a wider range of species, have confirmed extensive microtubule alignment with wall thickenings, with apparently strongly
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and repeated more extensively in this study for a wider range of species, have confirmed extensive microtubule alignment with wall thickenings, with apparently strongly bundled microtubules aligning to the ridges formed due to the secondary deposition of cellulose microfibrils. Thus, cytoskeletal organization in velamen cells is consistent with other sec- ondary cell wall thickenings where microtubules coalign with wall deposition, including tracheary elements in various plants [12–14,34–36] and cortical phi thickenings [19].
3.2. The Unusual Pattern of Microtubules That Flank the Cell Wall Ridges The observation that microtubules initially align with the developing secondary wall ridges and then develop into a pattern where parallel microtubules flank the ridges as the velamen becomes more mature is intriguing. We observed this pattern previously in Miltoniopsis [21], and it was observed in all four orchid species used in this study. Moreover, orthogonal sectioning demonstrated that these microtubules truly flanked the cell wall ridges and were not a single band of microtubules that was partially out of the focal plane. This flanking microtubule pattern might best be explained by microtubules being involved in widening the cell wall ridges once they have formed. This flanking pattern has been reported before during secondary wall development of tracheary cells by immunofluorescence in Zinnia tissue culture cells that develop into vessel elements [13,34], with GFP-based probes in Arabidopsis tissue cultures also differentiating into xylem [14] and during xylem differentiation in Arabidopsis roots [12]. Similar flanking microtubules have also been observed around the cell wall ridges (ingrowths) deposited during the development of xylem transfer cells in wheat [37]. Thus, the secondary wall formation of velamen cells is similar to that of the xylem and other secondary cell walls, with aligned microtubules directly overlaying secondary wall deposition during the initial stages of secondary thickening formation and subsequently reorganizing to flanking positions at later stages.
3.3. Developing Orchid Roots as a Model System for Secondary Cell Wall Investigations? Orchid roots, and the velamen in particular, provide an interesting system in which to study various developmental paths. Recently, for example, velamen development was used to investigate the control of programmed cell death [38], while the velamen has also been suggested as a model system for investigating the uptake and flow of water through porous material [26]. In this study, we used immunofluorescence microscopy to answer the question as to whether velamen formation in orchid roots might be developed as a model system for live cell imaging of secondary cell wall formation. Such a model system might, ideally, have the following characteristics: (i) secondary cell wall development occurring in a developmentally recognizable se- quence at specific sites; (ii) secondary cell wall development showing similar characteristics to ‘important’ sec- ondary cell walls, such as xylem development; (iii) secondary cell wall development in surface layers that are readily accessible for microscopy; (iv) a system readily adapted to tissue culture; (v) the ability to stably transform tissues so that they express fluorescent protein constructs; (vi) the availability of genomic information and access to mutants; (vii) and limited autofluorescence that would confound live cell imaging experiments. No currently used experimental system displays all these characteristics, with lignin autofluorescence associated with secondary wall development being problematic across a broad range of wavelengths [39], although the recent development of transgenic Arabidopsis lines in which epidermal cells can be reprogrammed to develop secondary cell walls with the use of inducible promoter systems [16–18] comes close. However, the orchid velamen system, and in particular Laelia anceps, would provide some advantages as a model system for secondary cell wall studies, especially if longer wavelength fluorescent proteins, such as mCherry or the recently developed near infrared proteins [40], were used. Not only are plants commercially available and readily grown in tissue culture, but the velamen Plants 2021, 10, 1358 12 of 16
also has highly visible secondary thickenings in a cell layer readily accessible at the root surface. The development of these cell wall ridges is controlled by the organization of the microtubules within the cells and follows patterns consistent with xylem development, and as with developing tracheary cells, the velamen cells then undergo programmed cell death. While the experiments conducted to date have used immunofluorescent labelling of fixed tissues to investigate microtubule development and were thus limited in scope because of the need to either use sectioned material or cell wall digests—as was the case for observing the outer layers of the velamen—the best understanding of the roles for microtubules in cell wall formation would require live cell imaging. Fusions of fluo- rescent proteins to tubulin and cellulose synthase have redefined our understanding of cell wall formation in both primary and secondary cell walls [9,11,12]. For the orchid root system to be useful for such live cell imaging experiments, transformation would be required. Fortunately, extensive protocols for growing and splitting orchid plants in culture exist, and a range of orchid species has now been stably transformed using a combination of gene gun- and Agrobacterium-based approaches, including Cymbidium [41], Dendrobium [42], Oncidium [43] and Phalaenopsis [44]. Additionally, while neither of the orchid species (Phalaenopsis equestris and Dendrobium catenatum) for which genomes are publicly available [45,46] proved suitable for imaging when tested, several further genomes have been published more recently (Dendrobium officinale and Apostaceae shengen)[47], with the likelihood of more being published in the near future.
4. Materials and Methods 4.1. Plant Material Various orchid species (Table1) were purchased from commercial growers and local suppliers and grown in orchid mix (Osmocote, Scotts Australia, Bella Vista NSW, Australia) in a greenhouse at 24 ◦C/18 ◦C (day/night) under partial shade.
Table 1. Orchid species investigated.
Velamen Thickening Class (as Defined by Porembski and Barthlott 1988) Species [24] Dendrobium sp. dendrobium Laelia anceps epidendrum Miltoniopsis sp. cymbidium Phalaenopsis sp. vanda
4.2. Fixing Root Tissue
Roots were fixed in PME solution (50 mM Pipes pH 7.2, 2 mM EGTA, 2 mM MgSO4) supplemented by 0.1% (v/v) Triton X-100, 3.7% (v/v) formaldehyde and 0.1% (v/v) dimethyl sulfoxide for a minimum of 1–2 h [48]. Excised roots were vacuum infiltrated with fixative for at least the first 15 min to aid penetration of the fixative through the velamen and into the root. Prior to analysis, fixed roots were washed in phosphate buffered saline (PBS; 131 mM NaCl, 5.1 mM Na2HPO4, 1.56 mM KH2PO4, pH 7.2).
4.3. Cell Wall Labelling Hand sections of fixed root material were rinsed in distilled water and stained for lignin with either berberine hemisulfate (0.1% (w/v) in water, 5 min, Sigma, St. Louis, MO, USA) [49] or basic fuchsin (0.001% (w/v) in water, 5 min; Sigma) [50]. Samples were also labelled for cellulose with pontamine fast scarlet 4B (0.1% (w/v) in 150 mM NaCl, 5 min; Sigma) [51–53]. Sections were mounted in glycerol on slides.
4.4. Immunolabelling of the Cytoskeleton Immunolabelling of root sections followed published procedures, with two different approaches used to allow antibodies access to fixed cytoplasm [19,21,50,54] in regions of the root tip near where the velamen was developing (Figure1). For immunolabelling, whole Plants 2021, 10, 1358 13 of 16
roots were extracted in PME supplemented with 1% (v/v) Triton X-100 (1 h), permeabilized in methanol (−20 ◦C, 15 min) and rehydrated in phosphate-buffered saline. In the first approach, longitudinal sections about 1 mm thick were cut from the surface of roots by hand with a double-sided razor blade, and the sections treated for ~15 min with cell wall digest enzymes (1% (w/v) pectoylase YC, 0.2% (w/v) macerozyme, 1.0% (w/v) BSA and 0.1% (v/v) Tween-20 dissolved in PBS buffered to pH 6.0). This approach allowed for antibody labelling of the cells in the outermost layer of the velamen. Alternatively, ~100 µm thick sections were cut after whole roots were embedded in polymerized acrylamide gel (21% (v/v), Biorad, Hercules, CA USA) [19], an approach that was suitable for labelling cells in the inner part of the velamen. After washing in PBS, sections were blocked in incubation buffer (PBS containing 1% (w/v) bovine serum albumin (BSA) and 0.5% (v/v) Tween-20, 15 min) before being incubated in mouse monoclonal anti-α-tubulin (clone B512, Sigma, diluted 1/1000 in incubation buffer, 1 h). After multiple washes in PBS, sections were incubated in a mixture of secondary antibodies (goat antimouse coupled to fluorescein and goat antimouse coupled to Cy5; Jackson ImmunoResearch, West Grove, PA, USA, diluted 1/200 in incubation buffer, 1 h). The use of the two secondary antibodies allowed for visual screening for microtubule labelling, looking for fluorescein fluorescence, but allowed for confocal imaging using Cy5 at a wavelength where background autofluorescence was minimized. After several washes in PBS, sections were mounted in antifade agent (type AF1, Citiflour, London, UK).
4.5. Confocal Microscopy Root sections were observed by confocal microscopy with an Olympus FV1000 micro- scope using 10× NA 0.30 dry, 20× NA 0.60 dry and 30× NA 1.05 silicone oil immersion lenses. Fluorescence from fluorescein and berberine resulting from excitation at 473 nm was collected from 500–550 nm and basic fuchsin and pontamine were excited at 559 nm with emission collected from 570 to 620 nm, while Cy5 excited at 635 nm was imaged from 650–760 nm. Lignin autofluorescence was excited at 405 nm and imaged from 420 to 460 nm. Transmitted light images were collected concurrently in either bright-field, DIC or polarization modes. Z-stacks through multiple cell layers were collected with step sizes ranging from 0.5 to 2.0 µm and, for berberine and basic fuchsin imaging of lignin fluorescence, were collected with the pinhole minimized to improve resolution in the Z-direction. All images were processed through ImageJ (FIJI installation version 1.51j, NIH, Bethesda, MD, USA) to select appropriate cell layers and to generate three dimensional rotations, and with standard tools in Adobe Photoshop version CS4 (version 11.0.1, Adobe Systems, San Jose, CA, USA). For some experiments, a Leica SP5 confocal microscope was used with 20× NA 0.7 and 63× NA 1.3 glycerol immersion lenses. Excitation wavelengths were 488, 561 and 633 nm, and similar emission windows were used.
5. Conclusions We have shown that the velamina of four different orchid species that are readily available show different patterns of secondary cell wall development and that in the case of Laelia and Dendrobium, these secondary cell walls are highly structured in the cell layer immediately at the surface of the root. The formation of these secondary cell walls is controlled by microtubules, and as with other secondary cell walls, the microtubules initially align parallel to the cell wall ridges but subsequently develop a flanking pattern running along either side of the maturing wall ridge. In conclusion, we suggest that our data demonstrate that the orchid velamen, and in particular the velamen layers of Laelia anceps and Dendrobium, may prove to be worth developing as live cell imaging systems for studying the formation of the secondary cell wall. We note, however, that the orchid velamen shows extensive patterning beyond the structures seen in this study, with 11 different velamen patterns having been defined [24]. Further surveys of orchid taxa may identify a species where not only are transformation protocols and sequence information Plants 2021, 10, 1358 14 of 16
available, but novel secondary cell walls are also present whose study might be informative concerning secondary cell walls in general.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/plants10071358/s1. Supplementary Movie S1.avi: Miltoniopsis: animation of confocal imag- ing of lignin in a cross section through the velamen. Supplementary Movie S2.avi: Miltoniopsis: animation of confocal imaging of lignin in longitudinal section through the outer velamen. Supple- mentary Movie S3.avi: Laelia: animation of confocal imaging of lignin in a cross section through the velamen. Supplementary Movie S4.avi: Laelia: animation of confocal imaging of lignin in longitudinal section through the outer velamen. Supplementary Movie S5.avi: Dendrobium: animation of confocal imaging of lignin in a cross section through the velamen. Supplementary Movie S6.avi: Dendrobium: animation of confocal imaging of lignin in longitudinal section through the outer velamen. Sup- plementary Movie S7.avi: Phalaenopsis: animation of confocal imaging of lignin in a cross section through the velamen. Supplementary Movie S8.avi: Phalaenopsis: animation of confocal imaging of lignin in longitudinal section through the outer velamen. Author Contributions: N.A.I., D.W.M. and D.A.C. designed the experiments; N.A.I., M.A. and D.A.C. performed the experiments; N.A.I., D.W.M. and D.A.C. drafted the manuscript. All authors have read and agreed to the published version of the manuscript. Funding: This research was supported by a Malaysian Ministry for Education PhD scholarship (SLAB) to N.A.I., by grant 316.17 from the Australian Orchid Foundation to D.A.C. and by funding from the School of Biological Sciences at the University of Canterbury. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are available on request from the corresponding author. Acknowledgments: At the University of Canterbury, we acknowledge the assistance of Dave Conder in the maintenance of the orchids, while at the University of Newcastle, the help of Joseph Enright and Agnes Kovacs in growing orchids is also gratefully acknowledged. We thank Jaimie Baker (University of Newcastle) for help with the immunolabelling of samples and also acknowledge the assistance of Murray Baker (School of Molecular Sciences, The University of Western Australia) for providing orchid samples from his collection. Conflicts of Interest: The author declares no conflict of interest.
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Nurul A. Idris 1,2, Maketelana Aleamotu ʻ a 3, David. W McCurdy 3 and David A. Collings 2,3,4,5,*
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