Bordered pit-pair membranes of green sapwood Douglas-fir after alteration by pectinase enzymes followed by critical point drying were examined with the scanning electron microscope to confirm and expand results of earlier reported observations with other microscopic equipment. Micrographs of treated bordered pit-pair membranes with various degrees of pectin removal clearly showed the spatial relationship of torus structure. The technique used permits easy cleavage of the torus that, in turn, reveals in great detail the inner organization of microfibrils in the torus sandwich. Indications are that the initial pectinase dissolution of the torus is initiated in regions of plasmodesmata. Elasticity of the microfibrils in water or ethanol is vividly displayed

Sachs, I. B.

Tschernitz, J. L.

English

Wood and Fiber Science (E-Journal)

OBSERVATIONS O N  MICROFIBRIL ORGANIZATION O F  DOUGLAS-FIR BORDERED PIT-PAIR MEMBRANES BY SCANNING ELECTRON MICROSCOPY J. L. Tschernitz and I .  B. Sachs' C:lrr.lnical Engineer and Technologist, respectively, Forest Products Laboratory' Forest Setvice, USDA, Madison, WI 53705 (Reccaivc~cl 2 October 1973) ARSTHACT Borderc:d pit-pair rnen~l)~.ancs of green sapwood Douglas-fir after alteration by pectinase r.nzy1rrc.s followed 1)y critical point drying were examined with the scanning electron ~t~icroscope to confirll~ and expand results of earlier reported observations with other ~~licroscopic cquipl~lent. hlicrograplrs of treated bordered pit-pair membranes with various degretls of pcxctin ren~oval clearly showcd the spatial relationsllip of torus structrrre. The tectiniclue used permits easy clcavage of the torus that, in turn, reveals in great detail the ir~ner organization of tnicrofibrils in the torus sandwich. Indications are that the initial pcctinase dissolution of the tortls is initiated in regions of plas~nodesmata. Elasticity of tlrc, ~lricrofibrils in water or ethanol is vividly displayed. Atltlitioncrl keyr~ortls: Pscrr(/ots~rgc~ rtlcrlzicsii Val-. g/u~rct~, wood s t r ~ ~ c t ~ ~ r y ,  y ~ ~ z y l l l a t i ~  degradation. INTRODUCTION The structure of the bordered pit-pair n~cmbrane in conifer tracheids still draws deliberate scrutiny because of its cardinal importaiicc~ in wood pernleability. Because of thr nc.ed to understand movernent of liquids and gases in or through wood protlucts and of the variability sometimes observed, it is important that we increase our u~~derstanding of wood anatomy and the lnechanics that control the flow of fluids through wood. The objective here was to present, as recorded by scanning electron microscopy, obsc.rvations of the organization of thc: Inargo and the torus of Douglas-fir pit ~ncrnbra~lc. This work reprc>sents an ex- te~lsion of an earlier investigation (Tscher- rlitz 1973) in which enzyme action within the bordered pit-pair membrane was used to cllhallcc creosote treatability of po1t.s. --- ' 'l'lrr a~~t l rors  tlra~rk Richard E. Kinncy and 'Thvodorr Miallowski of this Laboratory for their technical assistance in this study. ' Maintained in cooperation wi t l~  tllc Univcsrsity of Wisconsin. Thc effects of mild dissolution of proto- pectin ( the native pectin substance that gives rise to soluble pectin and pectinic acids or to both on hydrolysis [Wood 1960 1 ) in the bordered pit-pair membrane are investigated. The selective degradation of the torus by the enzyme allows broad aspects of the structural detail of the mcm- brane to become evident. PROCEDURE AN11 OBSERVATIONS Enxy ~ n e  treatment Specimens, longitudinal cores %-inch in diameter by %-inch long, of Douglas-fir green sapwood ( Pseudotsuga menziesii var. gluucn (Beissn.) Franco) were treatcd by infusion with one solution composed of 2.0% (volume) Rohm and Haas Pectin01 59L, 1.0% (weight) ammonium oxalate at pH 5.5, and 0.15% sodium benzoate, and held submerged at 30 C (Tschernitz 1973) for varying times. After a period of solvent exchange in ethanol (Sachs and Kinney 1972), the spcciincns were split radially under ethanol. Splitting the specimens under c.thanol after pectin reinoval with M'OOI) AN> ICIHER 332 WINTER 1975, V. 6(4) OBSERVATlONS ON hiICROFIl3RILS IN BORDERED PITS 333 FIG. 1. Mature sapwood bordered pit-pair membrane of Douglas-fir after critical point ( C o t )  drying. The close spacing of microfibrils indicates the margo has indeed been dried without the presence of a retreating interfacial surface. (No enzyme treatnient.) 20,000X. NOTE: In all fig- ures, thc arrow is one n~icronleter long. pclctinase may be thc critical part of the treatment. Splitting in the dry condi- tion, even with the weakened torus struc- ture, does not permit as good a cleavage and exposure of the organizational ele- ments of the torus. A modification of the critical point drying technique was used whereby the final solvent, ethanol, was removed by exchange with liquid carbon dioxide followed by an expansive loss of the carbon dioxide as gas (described by \Vcatherwax and Caulfield [I9711 ) . Pit-pair structure 1,igllt and electron microscope studies havc established that the torus is the thickened portion of the Pinaceae pit membrane and that bordered pits arc normally complementary pits in the walls of cclls contiguously united in a pair. The scanning electron microscope reveals that the bordered pit-pair membrane of Douglas-fir before pectinase treatment dis- plays a margo of radiating and interwoven microfibrils extending from the annulus to what appears to be a centrally thickened torus ( Fig. 1 ) . Interwoven microfibrils and circularlv oriented microfibrils from the torus lend credence to the torus defi- nition of Bauch et al. (1972), that the torus is the central part of a pit membrane that call be distinguishcd from the margo by the lack of capillary interspaces due to removal of an~orphous substances during 334 J. 1,. TSCIIEHUITZ AND I. U. SACHS FIG. 2. Enzyme-degraded bordered pit-pair FIG. 4. With one torus removed in its en- lnernbrane shows a few of the circnlarly arranged tirety frorn pit-pair membrane during specimen microfibrils intermixed with some supporting mi- preparation, orientation of microfibrils between crofihrils jnst under torus. (Enzyrne treatment pit-pair tori can be seen. (Enzyme treatment time: 16 11.) 1 1 , 5 0 0 ~ .  tinie: 16 h . )  5 , 0 0 0 ~ .  Frc. 3. The torus raised by specimen preparation opens to show microfibrils, margo, and primary \vall or jn$t n~icrofibrils and margo sandwiched between pit-pair tori. (Enzyme treatment time: 16 11.) 7,700 x . OBSERVATIONS OX h1ICROFIBnILS IN HOHDERED PITS 335 Flc. 5. Raised torlls discloses that some spokelike n~icrofibrils of margo extend into and are a part of ~l~icrofibrils sandu-iched between pit-pair tori. (Enzyme treatnlent time: 16 h.) 11 ,250~ .  cell ~naturation. Frcy-Wysling et al. ( 1956) showed thnt the circularly directed l~licrofibrils of the torus are laid down tluring differcntiation of the trachcid wall of Pinus syluestris. In thc altered mature torus of Douglas-fir, the circularly arranged 1)unclles of microfibrils become evident in a band form approximately 1 to 2 p n ~  in width after dissolution of torus matrix ma- terials (Fig. 2) .  It is obvious that the circularly oriented ~nicrofibrils are not merely supported by the underlying micro- fibrils, but additionally there is some inter- weaving of the ring microfibrils with the \upporting microfibrils (Fig. 2) .  Studics have shown that the chemical composition of the bordered pit-pair mem- brane consists of cellulose, hemicellulose, pcctin, and lignin. Trying to allocate the cl~c~mical components to inorphological units of t11c I~orderrd pit-pair membrane, Nicholas and Thomas (1968), Bauch et al. ( 1968), Jutte and Levy ( 1971), Tschernitz (1973), and Meyer (1974) used various cnzymes and observed that in the sapwood of some conifers pectinase degrades the torus, cellulase ruptures the microfibrils of the margo, and the margo is not affected by hemicellulasc. However, this selective breakdown of the pit membranes was not possible ill dried Douglas-fir because thc sapwood contained phenolic compounds (Bauch et al. 1970; Bauch and Berndt 1973). In the work reportcd here using grcen sapwood, phenolic compounds if present did not interfere with the treating solution a\ they normally do in heartwood. 336 J. I,. TSCHERXITZ AND I. B. SACHS FIG. 6. Spokelike microfibrils of margo appear to extend from torus to annulus. (Enzyme treat- 111t~nt timc.: 3 11. ) 13,000 X .  Snnrltcich con~truction of torus After 16 h of cnzyrnc (pcctinase) trcat- nlcmt, the lamellate structure of the torus b c ~ a m c  distinct, four larnellae were evi- dent. Nicholas and Thomas (1967) dem- onstratcd that steaming altered torus struc- ture in a manncr so that the swelling torces induced by methacrylate embedding cauwd a delamination of the pit membrane into three distinct layers. In this study, four distinct layers of the torus were ob- served. TWO layers of randomly arranged microfibrils, indistinguishable from primary walls or remnants thereof, are sandwiched betwclell the tori-pair (Figs. 3 and 4) .  111 Fig. 5, the raised torus after enzyme preparatory treatment, reveals that some of the spokelike microfibrils of the margo arc of the nctwork of microfibrils sand- wiched between the tori. Sachs and Kinney (1972) suggested that the spokelike ap- pearance of the margo may result from a rapidly drying interface. Before drying occurs, the rnargo is a dense network of microfibrils similar in density to thosc found between the tori (Fig. 1). That some microfibrils do not break and form a network of radiating strands may be due to the overlay and interweaving of the circularly arranged microfibrils, whose presence may prevent or slow the disloca- tion of the microfibrils during drying. In addition, as the tori become separated by pcctinasc treatment, linking microfibrils appear to be extending from the circularly oriented microfibrils of the torus to the I'I(:. 7. Wi th  partial pc~ctin rrmoval, plas~>rodesinata (arl.o\v) she\\ t.~~largc,inc.nt as  primary poilrt of c2rizy~lrc> ;~ttac,k. (1Cnzy111c ~ I . ( * ~ I ~ I I I O I I ~  tiliie: 2 11.) 1 2 , 0 0 0 ~ .  iu~nulus ( Fig. 6 ) .  The sandwich construc- ti011 of t h ~  torus plus the) intcrwovcn ar- rangc>lncant of niicrofi1)rils tcmds further to support thv contc~ntion that thc torus may ;I colllpositc. structure, (Saclls 1963). Intc~rspc~rsccl ill  the tori arc: :L nun1l)cr of olx~ni~igs npprosi~ilatcly 80 nm in diameter (Fig. 7 ) .  Thcsc, may 1)c thc location of l ' o r ~ ~ l c ~  plusmod(wmata that pc~ni,tmtc the lxi111ary 1r7all i l l  its clarly dcvclop~ncnt and arc, 1atc.r still visiblc in thc pit mcm1)ranc. Plasmodcsma c~c~uals cytoplasm plus cis- tcr11a (Esau 1965). Aftcr 2 h in thc pcctinasc solution with partial pectin removal, some of thc open- ings appcar empty (Fig. 7 ) .  The openings il~crc~asc. in diillnctcar with timck ill the cn- zymc: solutioll so that nftc'r 16 h, tlicy appvar 1argc:r (Fig. S ) .  Thc presc,ncc and thr. proximity of thc~sc opcliings suggest that thc plasmodcsmata integrity or archi- tc,cture is destroyed by thc action of thc cmzymc trc.:~t~nc,nt. Pc.1-haps as thcs proto- pc,ctin is Ilytlrolyzc~d by enzyme action, thc plaslnodcsmata drop away 1)c~causc. the support for thcni is clin~inatctl. This \\~oul(l indicate that thc plasmod(~srl1ata may 1)e sl~rroundcd or c~ml~cdclcd in protopc,ctin. Eftrsticit!y of nlicrofibri1.v Thc elasticity of thc microfibrils in 1vatc.r or ethanol bccomcs fairly cvidc~it by 01,- sc,r\ring a calcium osalatcx crystal that was dcri\,cd f ro~n  thc cnzylnc, treating solution that hail l)c,comc, c~ltrappc,d in tllc, nlargo ( Fig. $1).  Calciulll oxalatc crystals that 938 T. L. TSCIIEUNITZ AND I. I3. SACUS 1'1(;. 8. \'aids in t o r ~ ~ s  fro111 1.enlova1 of pl.otopcc.ti~l s~lggrst that plasn~odes~nata (Fig. 7 )  are lost. j I<~lz!.~r~c. trcsatlrlc~~t titl~(s: l(i 1 1 . )  11,250X. ocet~rrc~l a\ an impurity in the trcating rc1agc>nt were iclcntitied by X-ray diffrac- tio11 (13. I;. Caultic~ld, pcrsonal communica- tion, 1972). Many microfibrils in the figure -inclliding cxcccdingly tinc microtibrils- ~ c ~ ~ ~ , i i n  intact and appear strctchcd ovc,r th(1 \)ro'id surface‘ ot thc crystal. Thc cry\t,tl \upport i~pp,u-c~ntly prc.\7cwtcxtl tlis- location ot thc tinc microfi1)rils of the In'ugo whil(1 they wcrv sii1)joctc~tl to intcr- t,ic.ial dryiiiq str(~ss('4. ICnzymc prctrcatnic~nt of grccn sapwood ot Douglas-fir in corn1)ination with tech- niclue5 ot scan~iing clcctron microscopy has rc,sultc~l in the‘ following: 1. (:lc>:ir tlc,linc.ation of thc spatial rcla- tionship of the microanatomy of the bor- dcrvd pit-pair mcmbranc. 2. Indication that splitting undcr water or c~thanol providcs easy clcavage of the torus in conjnnction with c~nzynlcx trcbat- mc5nt. 3. A dct,~il(~el visual dcmonstrntion of microfil~ril oric.~~tation within t h ~  torus structurc3 that confirms the prcscnce and the mamicxr of distri1)ution ot pc,ctiil in the torus. 4. Reconfirmation ancl cla1)oration of the sandwich structure ot thc pit-pair mcinbrane. 5. Although l)y 110 nichans pro\~c,tl, thc pllotoniicrographs stroilgly imply that thc initial perforation ot thc torus is in and ,il1011t thc~ l~lnsmodcsmata. OBSERVATIONS ON blICROFIHRILS I N  HOHDEREII PITS 339 I .  $1. Calcilmi oxalate crystal (similar crystals identified by X-ray diffraction) entrapped in inargo of l)o~.tlrred pit-pair ntcinbranc illustrates elasticity of fine microfibrils. (Enzyme treatment time: 1 11. ) 18.250 x . 6. I3y 11biilg tllc, critical point tcch~licluc., r~~icrofibrils of t h ~  i ~ ~ a r g o  appc;lr elastic ill \vatu or ethanol. hlc.ution of trade or propric.t;cry nallrcs is for i~~forlr~ation purposes only ailcl docs not imply ( ~ I I C ~ O ~ S ~ ~ I I I C D ~  l ~ y  the Forest Scrvicc. of the U.S. I>c.pal.t~~~crit of Ag~.icllltl~rc. REFERENCES I~Av( : I I ,  ., A \ I )  11. HERSI)T. 1073. Variability of tlir chcmical compositio~i of pit mem- 1)1.anc.s i l l  bordered pits of gyntnosperins. Wootl Sci. Tecl~nol. 7:6-19. , W. LIESE, ANI)  F. BERNU.~. 1970. Bio- logical investigations for the improvement of the pcr~nf~ability of soft\\,oods. Holzforscliung 24 ( 6 ) : 199-205. , W. LIESE, ANI) F. SCHOLZ. 1068. WLer die fintwicklr~ng und stofffiche Zl~sammrnset- z1117g der Iloftiipfclmcnibrancn \Ion Liing- stracl~ritlcn i l l  Col~ifcrn. Ilolzforsch~tng 22 ( 5 )  :144-153. , W. LIESE, AS]) 11. SCF~UI,TZE. 1072. The 111orphological variability of the bordered pit rnenlbl.a~~es in gymnosperlils. Wood Sci. Technol. 6 :  165-184. Esav, K. 1965. Plant anatotl~y. 2nd ed., John Wiley and Sons, Inc., N. Y. P. 44.  HEY-WYSSLING, A., H. H. BOSSIIAIII), ANI) K. MUHLETHALEH. 1956. The sl~hmicroscopic forinatioit of bordered pits. Planta 47:115- 126. JUTYE, S. hf . ,  AKI)  J .  F. LEVY. 1971. Thc struc- turc and degradation of bordered pits in gymnosperm tracheids cs revealed by the scanning reflection electron microscope. Acta Bot. Neerl. 20(5  ) 3453-466. XIEYER, R. W. 1974. Effect of enzymc treat- mcnt oil bordered-pit ultrastructure, permea- bility, arid toughness of the sapwood of three \vesterr1 conifers. Wood Sci. 6(3):220-230. NICIIOLAS, L). D., AND R. J. TITOMAS. 1967. In- 340 J. L,. TSCIIIRR'ITZ AN]) I. H.  SACIIS f l i ~ ( ' ~ ~ c , c ,  ok slcxat~ling ~ I I  tl~c. r~ltrastrllcture of Irordr.rc.tl pit 111c.11111ranc in lol~lolly pine. For. 1'1.od. J .  18( 1 ) :57-59. -- , A Y I )  R. J .  T I I ~ A I ~ ~ S .  1868. Tl~c. ilrf111- (.nee, of cllzylncys on  st~-rlct~~rc. and permea- I~ility of 1ol)lolly pine. Proc. ,4WPA 64:70-76. S,\~.IIS, I. R. 1063. 'Tori~s of thr I~ordered pit ~rrc~~~rl) ra~lc  in conifers. Natr~rc. 198:!)06,007. , A N I )  R. E. KISNEY. 1072. An "i~ltra- filtc.1.'' in tlic nrargo of 11ratiu.e. 1)orclertd pits? 1'1.o~. Electron hlicrosc. Soc. Ai.1. 13:206,207. 'Tsc:ir@l~u~.~.z, J  L. 197:3. Enzynlo ~ n i u t ~ ~ r e  im-proves ct-eosotct trcatnient of kiln-dried Rocky hlonntain Douglas-fir. For. Prod. J. 2 3 ( 3 ) :  30-38. WEATIIEHWAX, R. C., ASI) 11. F. CAULFIELI). 1971. Celli~lost~ aerogels: An  improved 111rtIiod for preparing a highly expanded form of dry ct~llillose. Tappi 54(6):985,086.  woo^,, R. K. S. 1960. Pectic and ccllnlolytic enzymes in plant disease. Ann. Rev. Plant Phys. 2 :299--322. 

Observations On Microfibril Organization of Douglas-Fir Bordered Pit-Pair Membranes By Scanning Electron Microscopy

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Observations On Microfibril Organization of Douglas-Fir Bordered Pit-Pair Membranes By Scanning Electron Microscopy

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