lignin formation in wheat coleoptile cell walls · the dry tissue was extracted with 6% kohat room...

Plant Physiol. (1971) 48, 596-602

Lignin Formation in Wheat Coleoptile Cell Walls

A POSSIBLE LIMITATION OF CELL GROWTH'

Received for publication May 3, 1971

F. W. WHITMOREDepartment of Forestry, Ohio Agricultural Research and Development Center, Wooster, Ohio 44691

ABSTRACT

Four growth-influencing compounds-hydroxyproline, 2,2'-dipyridyl, 2-chloroethylphosphonic acid, and indoleaceticacid-were used to examine the relationship between ligninformation and growth of wheat coleoptile sections. Hydroxy-proline and 2-chloroethylphosphonic acid, at low concentra-tions, inhibited growth and increased lignin content. Dipyridyl,which promoted coleoptile elongation, decreased lignin con-tent. Indoleacetic acid caused a 300% increase in growth at0.1 mM but resulted in lignin content no different from con-trols with no auxin. Chemical and anatomical evidence isgiven which indicates that lignin is present in the epidermalcell walls of the wheat coleoptile. It is thus possible that bond-ing between lignin and hemicellulose may have some influenceon coleoptile growth.

Wardrop (20) has hypothesized that lignification may limitenlargement of plant cells by immobilizing the hemicellulosematrix, thereby preventing surface growth. His suggestionpertained to woody cells which undergo secondary wallthickening, and in which lignin forms a major component ofthe cell wall. Coleoptile cells, however, also have a secondarytype of wall layer which is formed at the end of the extensionphase (1). Experiments described in this report show thatlignin may be synthesized in the epidermal cells of the wheatcoleoptile. As a result of bonding between lignin and hemi-cellulose (5, 7), factors which control lignification may therebyinfluence the termination of elongation of coleoptile cells. Therelationship between lignin biosynthesis and coleoptile elonga-tion was investigated.

MATERIALS AND METHODS

Incubation and Nitrobenzene Oxidation. Wheat seeds (Tri-ticunz vulgare cultivar Knox or Redcoat) were grown for 72hr in the dark in vermiculite. Coleoptile sections 9 mm longwere cut 3 mm from the tips, and the leaves were removed.The sections were floated in water for 1 to 2 hr before thetreatment incubation. Twenty-five to 50 sections were in-cubated in 50-ml Erlenmeyer flasks in 2- to 4-ml solutions. Thebasic medium for incubation was 50 mm sucrose; 2.5 mmpotassium maleate buffer, pH 4.8; the lignin precursor U-"C-L-phenylalanine, 15 mc/mmole, 1 to 5 1ic per flask: and hydroxy-

'Ohio Agricultural Research and Development Center JournalPaper 41-71.

proline, 2, 2'-dipyridyl, 2-chloroethylphosphonic acid (Am-chem Ethrel 68-240), and IAA as specified in the "Results"section. Incubation temperature was 25 C. Flasks were gentlyshaken for 20 hr in a darkroom.At the end of the incubation period, length of the sections

was measured. Sections were ground in a mortar, washed twicewith 80% (v/v) ethanol, centrifuged, then washed once withethanol-ether (1:1, v/v). To isolate vanillin, a characteristicoxidation product of lignin, the dried tissue was placed in aglass or Teflon centrifuge tube with 0.3 ml of nitrobenzene and2 ml of 2 N NaOH and heated in an iron bomb for 2 hr at160 C. The cooled solution was filtered and extracted 3 timeswith ether. The aqueous solution was acidified to pH 3 withHCl and extracted 3 times with ether. The ether extract wasevaporated to dryness and dissolved in ethanol. Portions ofthe ethanol solution were spotted on 500-, silica gel thinlayer plates and developed in benzene-glacial acetic acid, 9:1,v/v. The plates were sprayed with 0.2% 2,4-dinitrophenyl-hydrazine to locate marker and extracted vanillin. Vanillinspots were scraped off and eluted with ethanol, portions ofthe eluates were dried on planchets, and radioactivity wasmeasured. Vanillin from the tissue was identified by co-chromatography of authentic samples in two other solvent sys-tems: ethyl acetate-2-propanol-water, 65:24:11, v/ v; and 1-butanol-acetic acid-water, 63:10:27, v/v. The benzene-aceticacid system was used exclusively in most experiments.Acid Hydrolysis of Whole Sections. Coleoptile sections in-

cubated in "C-phenylalanine were placed in a sealed glass tubecontaining 6 N HCl and heated to 110 C for 18 hr. A fewpieces of the tissue residue were washed, applied to microscopeslides with gelatin, then coated with autoradiographic strippingfilm for exposure. The remainder of the residue was countedafter drying on a planchet. The residue was removed fromthe planchet and hydrolyzed in 72% H2S04 by weight, for 1hr, at 23 C, to check for any cell wall carbohydrates whichmight have incorporated "C from phenylalanine.The acid was diluted to 3% and the sample was autoclaved

at 15 psi for 2 hr. The hydrolysate was neutralized withBa(OH)2, then separated by paper chromatography with ethylacetate-pyridine-water, 8:2: 1, v/v, and a running time of 24hr. Spots were detected with 3% o-aminodiphenyl hydro-chloride in 95% ethanol (18). Authentic glucose, galactose,mannose, arabinose, and xylose were included on thechromatograms for identification of the labeled cell wallsugars.

Differential Extraction of Vascular and Epidermal Tissue.Fifty coleoptile sections were incubated with 20 /tc of "C-phenylalanine. With the aid of a dissecting microscope. thetwo vascular bundles were removed from each section andkept separate from the remaining tissue. The two fractions,designated as vascular and epidermal, were ground in a mortarwith 80% ethanol. The tissues were washed in a 0.5% solution

596

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LIGNIN FORMATION IN COLEOPTILE CELL WALLS

of Tween 20, water, 80% and 100% ethanol, with centrifuga-tion between washings. Finally suspended in 3 ml of ethanol,each tissue fraction was divided into 3 approximately equalparts by pipetting. The portions were dried on planchets andtheir radioactivity was counted. One portion of each fractionwas then hydrolyzed in 6 N HCI for 5 hr at 15 psi in anautoclave, and one portion was hydrolyzed in 72% H,SO, for1 hr at room temperature, then diluted to 3% acid and auto-claved at 15 psi for 2 hr. The third portion of each fractionwas extracted with 16 ml of water, 0.15 g of sodium chloride,and 1 drop of glacial acetic acid at 100 C for 4 hr, with freshreagents added every hour (11). Extracts from the epidermalfraction by all 3 hydrolyses were tested for the presence ofradioactive phenylalanine and tyrosine by separation on silicagel thin layer chromatographic plates, using 1-propanol-ammonia, 70:30, v/v, as the solvent. The dried plates wereplaced in contact with x-ray film for exposure. Radioactivespots were compared with known phenylalanine and tyrosinespots run concurrently and detected with ninhydrin spray.

Isolation of Epidermal Cell Constituents. One hundred cole-optile sections were incubated 48 hr in 20 pc of "4C-phenyl-alanine, after which the vascular bundles were excised anddiscarded. The remaining epidermal and other tissues wereground in a mortar with water, centrifuged, and washed 4times with water. The water extract and washings werecombined and heated to boiling to precipitate protein. Afterthe protein was washed with hot water, it was suspended in 10ml of water with 0.05 ml of Tween 20, from which 0.1 mlwas removed for counting.The tissue was then washed in 80% ethanol, dried at 65 C,

and weighed. The dry tissue was refluxed 1 hr in 50 ml of amixture of equal parts benzene and ethanol. Next the tissuewas refluxed with water for 18 hr to extract pectic materials.The extract was filtered, and a portion was counted. Afterdrying, 5 mg of the 23 mg of tissue remaining after hotwater extraction was subjected to nitrobenzene oxidation andisolation of vanillin as described previously. The remainder ofthe dry tissue was extracted with 6% KOH at room tempera-ture for 64 hr. The extract was neutralized with ion exchangeresin, H+ form, and a portion was counted. The extract wasthen partitioned with 3 equal volumes of ethyl ether. Theether fraction was evaporated, and portions were examined bythin layer chromatography with two solvent systems: benzene-acetic acid, 9:1, v/v; and ethyl acetate-isopropanol-water,65:24: 11, v/v. Radioactive compounds were located byautoradiography and reaction with 2, 4-dinitrophenylhydra-zine.

After the initial KOH extraction, the tissue was extractedfor 5 days with 24% KOH and 4% H3BO3 under nitrogenand at room temperature (13). The extract was neutralizedwith resin and separated into ether and aqueous phases.The tissue remaining after alkali extraction was subjected

to 72% H2SO extraction to remove cellulose, as described inan earlier section. The residue was first extracted with 6 NHCl to remove protein, followed by nitrobenzene oxidationand isolation of vanillin.

Microautoradiography. Coleoptiles which had been in-cubated in the basic medium, including 1 ,uc of "4C-phenyl-alanine per ml, were dehydrated, embedded in paraffin, andsectioned transversely at 5 ,u thickness. Microautoradiographswere prepared with Kodak AR-10 stripping film (10). Ex-posure was 1 to 6 weeks.

Determination of Radioactivity. Cell wall residues remain-ing after hydrolyses were filtered onto cellulose acetate filtersand repeatedly washed with water followed by ethanol. Thefilters were cemented to planchets and counted in a gas flow

detector with an efficiency of about 25%. Liquid extracts weredried on planchets and counted in the same way.

RESULTSLignin Formation in Epidermal Cells. Microautoradio-

graphs made from transverse sections of coleoptiles incubatedin labeled phenylalanine showed that radioactive compounds,insoluble in water and organic solvents used in preparationof the sections, were most concentrated in the vascularbundles and the inner and outer epidermal cell walls (Fig. 1).The radioactive compounds resulting from phenylalaninepresumably were both protein and lignin. In order to removeprotein, several whole sections were hydrolyzed in 6 N HCl.Hydrolysis destroyed the structure of the coleoptile cylinder,leaving particles which appeared upon microscopic examina-tion to be epidermal wall fragments. The fragments stainedvery lightly in safranin. Microautoradiographs showed uni-formly distributed radioactivity (Fig. 2).To check the possibility that the residual radioactivity in

the cell wall particles might have been contained in celluloseor other HCI-resistant carbohydrate, a sample was hydro-lyzed in H2SO, resulting in further destruction of the frag-ments. Glucose was the only sugar detected in the hydrolysateafter chromatographic separation, and it contained less than0.5% of the radioactivity originally in the HCl-hydrolyzedfragments. The lack of radioactivity in carbohydrate was notsurprising since the coleoptiles were furnished with adequatenon-labeled sucrose during incubation. The radioactivityremaining in the fragments after protein hydrolysis was there-fore presumed to be lignin. This conclusion was supportedby isolation of radioactive vanillin from epidermal peels.The stability of radioactive compounds in epidermal and

vascular tissue formed from labeled phenylalanine was investi-gated further. Vascular bundles were excised from thecoleoptile sections and the two resulting tissue fractions,vascular and epidermal, were subjected to three treatmentswhich differ in their attack on protein, carbohydrate, andlignin. The vascular fraction contained some epidermal tissue,but the epidermal fraction was entirely free of vascular tissue.Autoclaving the tissue in 6 N HCI, which hydrolyzes protein aswell as noncellulosic carbohydrate, removed about 40% ofthe radioactivity from the epidermal fraction and about 25%from the vascular fraction (Table I). Primary hydrolysis with72% H2SO0, followed by autoclaving in 3% acid, removesnearly all carbohydrate and some cell wall protein. This treat-ment decreased the radioactivity of the epidermal tissue by65%, and the vascular tissue by about 50%. The delignifyingagent sodium chlorite reduced the radioactivity of both frac-tions to less than 1%. Most of the radioactivity removed byHCl and H2SO0 was in protein, as evidenced by identificationof nearly all the radioactivity in the hydrolysates as phenyl-alanine and tyrosine. Protein also was hydrolyzed by sodiumchloride, but the large reduction in residual radioactivity bythis treatment is presumed to result from the removal of lignin.The mineral acids are poor lignin solvents.

Further evidence for the presence of lignin in epidermalcell walls was obtained by the isolation of lignin degradationproducts in several fractions of the tissue after extraction bythe procedures of Lamport (13). Table II summarizes theradioactivity found in eight major fractions of tissue obtainedafter sections had been incubated in "4C-phenylalanine. Beforeextraction, all the vascular bundles were removed and dis-carded, so that no xylem elements were included.

After the initial grinding of the tissue in water to extractcytoplasmic contents, samples of the residue were counted.The total water-insoluble radioactivity was 1,004,000 cpm. Aportion of the tissue remaining after hot water extraction was

Plant Physiol. Vol. 48, 1971 597



Plant Physiol. Vol. 48, 1971

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FIG. 1. Microautoradiograph of part of a transverse section of wheat coleoptile. The section was cut from a 72-hr-old coleoptile and incubatedwith '4C-phenylalanine for 20 hr. The most intense radioactivity was located in the xylem elements of the vascular bundle (v), and in the innerand outer epidermal-cells (e).

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599Plant Physiol. Vol. 48, 1971

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Table I. Removal of Radioactivity Formned in Coleoptile Sectionisfrom '4C-phenylalanine by Tlhree Solvenits of Cell

Wall ConistituentsSee "Materials and Methods" for experimental conditions of

hydrolysis. The cpm values were determined on tissue maceratedand washed immediately after a 20-hr incubation in labeledphenylalanine, and again on the same tissue after hydrolysis.

Epidermal Tissue i ascular Tissue

TreatmentBefore After Before After

treatment treatment treatment treatment

cpm cpan

6 N HCI 300,790 185,620 305,040 228,86072c-% H2SO4 296,450 115,660 297,270 143,680NaClO2 308,990 1,590 304,230 1, 170

Table II. Radioactivity ini Frcactionis of Coleoptile Tissiue aifterInicuibationt in 1 4C- Pheniylalaninle

One hundred coleoptile sections were incubated with 20,uc ofthe labeled amino acid for 48 hr. Vascular bundles were removedbefore extraction. Tissue was ground in a mortar with water. Thetotal incorporated radioactivity not removed by the initial waterextraction was 1,004,000 cpm. See "Materials and Methods" fordetails of extraction.

Fraction

I. Water-soluble, heat-precipitated

II. Benzene-ethanolIII. Hot waterIV. 6%" KOH

A. Ether-soluble

B. Water-solubleV. 24'% KOH, 4%''

H3BO3A. Ether-solubleB. Water-soluble

VI. 72%-/C H2SO4VII. 6 N HCIVIII. Residue

Main Components

Cytoplasmic protein

Fats, waxes

Pectic material, protein

Phenolic aldehydes. acidsFerulic acidCarbohydrate, protein

Phenolic aldehydes, acidsCarbohydrates, proteinCellulose, proteinProteinLignin

Total, major fractions

subjected to nitrobenzene oxidation. Vanillin isolated fromthis degradation contained the equivalent of 80,730 cpm forthe entire sample; hence about 8% of the radioactive carbonin the cell walls was found in vanillin.The largest amount of radioactivity, more than half of the

total contained in the tissue, was removed by 6% KOH. Thisfraction contained hemicellulose and some protein. More thanhalf the radioactivity in the 6% KOH fraction was soluble inether when neutralized and therefore was not protein. Ferulicacid, commonly released by lignin in alkali extraction (5), wasthe major radioactive product in the ether fraction.The second hemicellulose fraction, extracted with 24%

KOH and 4% H3B03, yielded less than 1% of the ether-solubleradioactivity obtained by extraction with 6% KOH. Most ofthe coleoptile lignin thus is soluble in 6% KOH.The cellulose fraction, extracted with 72% H2SO0, con-

tained very little radioactivity. The residue from this extractioncontained both lignin and protein. In order to remove protein,

the residue was hydrolyzed in 6 N HCl, which removed 2,600cpm, leaving a solid residue which contained 12,250 cpm,presumed to be lignin. The residue was subjected to nitro-benzene oxidation, yielding only 60 cpm in vanillin. In studieson wood lignin, it has been found that yields of vanillin fromsulfuric acid lignins are very low, apparently because ofchanges in the phenylpropane structures during acid extraction(5).The results given in Table II strongly indicate the presence

of lignin in epidermal cells, especially because of the rela-tively high yield of radioactive ferulic acid in the 6% KOHextract. This ferulic acid could not have existed in thetissue as a free compound, because it would have been re-moved earlier by 80% ethanol, benzene-ethanol, or hot water.

Relationship of Lignin Formation and Coleoptile Elonga-tion. In the following experiments, differences in radioactivityof vanillin produced by nitrobenzene oxidation were con-sidered as indicating differences in lignin synthesized from thelabeled phenylalanine substrate. Nitrobenzene oxidation alsoproduced p-hydroxybenzaldehyde, but its radioactivity wasnot used in estimating lignin because it can be formed fromradioactive tyrosine in protein, as well as from lignin (6).Radioactive syringaldehyde was not detected.

Free hydroxyproline reduces cell elongation, especiallyauxin-induced cell elongation (8, 9). Furthermore, hydroxy-proline has some influence on the formation of cytoplasmicand wall-bound peroxidase isozymes in growing coleoptile sec-tions (22). Peroxidase is the terminal enzyme in lignin syn-thesis from phenylpropane derivatives (14). Because of therelationships between hydroxyproline and peroxidase, theeffect of the imino acid on lignin formation was investigated.

Hydroxyproline at 0.5 and 1 mm caused an increase inlignin content over that formed in the controls (Fig. 3A). At2 mm, lignin content was slightly less than in control tissue.Inhibition of the growth of the sections was approximatelythe same at all levels of hydroxyproline.

Dipyridyl, which blocks hydroxylation of proline and hasbeen found to increase the growth of soybean hypocotyl sec-tions (4), resulted in increased growth of coleoptile sections atlow levels and strong inhibition of lignin synthesis at all levelstested (Fig. 3B).

Ethylene can increase peroxidase activity in plant tissue(17). An experiment, similar to the foregoing, was designedwith the use of 2-chloroethylphosphonic acid, which releasesethylene after absorption into plant cells (21). As Figure 3Cshows, the lowest level of 2-chloroethylphosphonic acid used,1 ,ul/liter of Amchem Ethrel 68-240, resulted in increasedlignin synthesis while inhibiting growth of the coleoptile sec-tions. At 5 and 10 ,ul/liter, both lignin synthesis and growthwere decreased.

In none of the three experiments is it known how the treat-ment compounds influenced lignin biosynthesis. Hydroxypro-line and ethylene under some conditions can affect peroxidaseactivity, and experiments in this laboratory showed thatdipyridyl at concentrations of 0.1 mM and higher depressactivity of crude cytoplasmic peroxidase. Dipyridyl, however,is a general respiratory inhibitor and probably interferes withall iron-activated enzymes.

Since dipyridyl may inhibit respiration, thus causing a

general decrease in all cell wall biosynthesis, the relationshipbetween lignin and growth was examined in another growthpromoter, IAA. Figure 3D shows a slightly different relation-ship from that of dipyridyl. Auxin increased the growth ofcoleoptile sections to a maximum of 300% of the controls,but lignin synthesis remained about the same as controls uipto 0.1 mm, above which it decreased along with growth. At 1mM. auxin still caused growth of 150% of controls, but lignin

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Table III. Uptake of PhenzylalanizieSamples of 20 coleoptile sections were incubated in 2.5 mM

potassium maleate buffer, pH 4.8, 50 mm sucrose, 0.33 ,uc 14C-phenylalanine, and the treatment compound, for 4 hr at 20 C.After washing and flushing out the leaf cavities with water, thesections were ground in 80% ethanol. Counts were made on the80%,'c ethanol extracts and the insoluble residue which includedcell walls. Each value is the mean of 2 samples of 10 sections.

TreatmeDt Soluble in Insoluble inTreatment ~~80% Ethanol 80%o Ethanol

cprn/1O scclsons

Control 4,590 11,325IAA, 0.1 mm 7,340 13,515Dipyridyl, 0.1mM; 3,470 11,6852-Chloroethylphosphonic acid, 0.1 4,520 11,960

,ul/literHydroxyproline, 1 mm 4,310 10,825

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FIG. 3. Relationships between lignin content and growth ofcoleoptile sections after incubation in four growth-influencingcompounds. Included in all media were 2 ,uc of '4C-phenylalanine,50 mM sucrose, and 2.5 mm potassium maleate buffer, pH 4.8.Sample units contained 25 coleoptile sections each and were incu-bated at 25 C for 20 hr. Growth was expressed as the differencebetween the length of the sections after 20 hr and the initial lengthof 9 mm. Lignin was estimated by determining the cpm in vanillinisolated after nitrobenzene oxidation of the sample of 25 sections.The points located off the curves and connected by vertical linesrepresent individual samples of 25 sections; thus, each point forgrowth is the mean of 25 sections, and each point for lignin is thetotal cpm in vanillin for 25 sections. Controls are the zero levelsof each compound, and their values are set at 100%. The actualmean values for controls in all the experiments were 8.4 mm persection for growth and 1240 cpm in vanillin per section.

content was reduced to less than half that of the controls. Thecurves of Figure 3D are similar to those of Figure 3B(dipyridyl) in that lignin content relative to the control isalways lower than relative growth. IAA, like dipyridyl, de-creases the activity of peroxidase (22), but certainly not by ageneral decrease in respiration.The significance of the results in Figure 3 is that the growth

inhibitors hydroxyproline and 2-chloroethylphosphonic acidresult in lignin contents, expressed as a percentage of controls,which are higher than growth values relative to controls, atall treatment levels. Conversely, the growth promotersdipyridyl and IAA give relative lignin contents which are

consistently lower than relative growth values.The effect of the growth-influencing substances on uptake

of phenylalanine are shown in Table III. Except for IAA, noneof the compounds exerted much effect on total uptake in the4-hr period. The increase in uptake of phenylalanine causedby IAA is similar to that reported for glucose in auxin-inducedgrowth (3). No parallel increase in lignin content resulted (Fig.3D). The soluble fraction of the dipyridyl treatment was lowerin radioactivity than the control; however, the insoluble frac-tion was about the same as the control. With the possibleexception of dipyridyl, then, it seems unlikely that the syn-thesis of lignin could be controlled by uptake of the precursorfrom the medium.

In none of the experiments of Figure 3 were the vascularbundles removed. It is extremely difficult to excise vascularbundles in such a way as to leave uniform amounts ofepidermal tissue in each section. Lignin estimates based onvanillin therefore include xylem elements. Because of this,the possibility cannot be eliminated that some of the influenceof the treatments in terms of lignin formation was exerted onthe xylem elements.

DISCUSSION

Several points must be raised concerning the relationshipof the experiments to the hypothesis that lignification limitscell expansion. First, there is the question of whether theisolation of vanillin arising from labeled phenylalanine is a

good indication of lignin formation. The widely used ligninestimation based on weighing Klason lignin after acid hydrol-ysis (5) is not suitable for living cells which have a fairly highprotein content, because the sulfuric acid digestion does notremove all the cell wall protein (13). Lignin cannot be isolatedin a pure form with present methods (14). The isolation of

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Plant Physiol. Vol. 48, 1971 601

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602 WHITMORE

vanillin after nitrobenzene oxidation is generally accepted asa reliable indicator of the presence of lignin (18).

There is no doubt that L-phenylalanine is a good precursorfor lignin (14); however, its introduction into tissue may in-crease the pool of the free amino acid to the extent that morelignin is formed than would be under normal physiologicallevels of the precursor. The facts that lignin is formed fromthe precursor and the amount varies with treatment indicatethat a biosynthetic pathway exists in epidermal cells and thatit is sensitive to control, whether or not the normal conditionis one of limited phenylalanine.

Finally, there is the question of whether the lignified part ofthe epidermal wall is in a structural position to limitextension. The outer epidermal wall in wheat and oat cole-optiles is very thick and is covered with a thin cuticle (16).Wall fragments isolated after HCl hydrolysis (Fig. 2) may becuticle, for their areas were much too great to be walls of in-dividual epidermal cells. The fact that the fragments containglucan and lignin, however, suggest that they might be piecesof the thick outer wall noted by O'Brien (15). If this is thecase, the outer wall fragments would have to be common toseveral epidermal cells, because of their large areas. An elec-tron microscopic study of developing coleoptiles, perhaps withautoradiography of lignin deposition, may help clarify thispoint.A central question is whether lignification begins before

the cell has reached its final size. If it does not, then ligninformation cannot limit the size of the cell, but only make thecell wall more rigid. If lignin formation does limit the sizeof the cell, it could do so by controlling the rate of wall expan-sion over the entire extension phase or by causing the termina-tion of cell growth without influencing the earlier rate ofexpansion. Studies by Adamson et al. (1) suggest that thelatter situation may be the more likely, since the appearanceof secondary wall layers coincides closely with the end of cellgrowth. A process controlling only the termination of cellgrowth such as lignification might, however, influence the rateof elongation of the entire coleoptile. As Avery and Burk-holder (2) have shown, regions of the coleoptile elongate atdifferent rates. This could be caused by the termination ofextension of increasing numbers of cells in the coleoptileregions having decreasing rates of elongation.

If lignin indeed is formed in epidermal cells of monocotcoleoptiles, it should be considered a possible factor in thesystem controlling elongation of this organ. Hemicellulosesseem to be the carbohydrate wall constituents most directlyinvolved in controlling cell expansion (3). It is with hemicellu-lose that lignin is believed to form covalent bonds, probablyether linkages, at least in woody cells (7). Lignin may thusform stabilizing cross-linkages, similar to those proposed forextension (13), within the hemicellulose matrix of coleoptileepidermal cells.

In an analysis of differential growth in coleoptiles, Thimannand Schneider (19) found that the epidermal cells were undertension, while the inner cells were under compression duringelongation. This means that a growth-limiting phenomenon wasmore advanced in the epidermal cells than in the inner cells,and it is the outer cells which apparently contain lignin. Ofcourse, the xylem elements also contain lignin, but their


secondary walls are in the form of spiral thickenings allowingfor extension.The results of these experiments do not confirm suffi-

ciently the hypothesis of limitation of coleoptile growth bylignin formation. No causal relationship between lignificationand growth limitation was established. The results, however,are consistent with those predicted by the hypothesis. Ligninformation is increased by treatments which increase peroxidaseactivity and which reduce elongation of coleoptiles, but bothcould be the result of some other process limiting cell growth,such as the metabolism of hydroxyproline-rich protein(13).

Ackitowledfsne0t-Ithank lMrs.D. R. Abernatlhy for tecliical assistance.

LITERATURE CITED

1. ADAaMsoN, D.. V. 1i. K. Low, AND H. ADAMSON. 1968. Transitions betwveendlifferent plhases of growth in cells from etiolated pea stems, Jertisalemnaiticlioke tubers, and wheat coleoptiles. In: F. Wightman and G. Setter-field, eds., Biochemistry and Physiology of Planit Growth Substances.Rtunge Press, Ottawa. pp. 505-520.

2. AV-ERY, G. S., JlR. AND P. R. BURKHOLDER. 1936. Polarized growtls an(d cellstuIlies on the Arcsia coleoptile, phytolhormone test object. Bull. TorreyBot. Club 63: 1-15.

3. BAKER, D. B. AND P. M. RAY. 1965. Direct and indirect effects of atuxin oncell wall syntlhesis in oat coleoptile tissue. Plant Physiol. 40: 345-352.

4. BARNE-TT. N. M. 1970. Dipyridyl-induced cell elongation and inlsibition ofcell wall hydroxyproline biosynthesis. Plant Physiol. 45: 188-191.

5. BRAUNS, F. E. AN-D D. A. BRAUNS. 1960. The Chemiiistr-y of Lignin. Aca-(lemiic Press, Ncew York.

6. BROWN, S. A., K. G. TAN-NER, AND J. E. STON\E. 1953. Studies of ligiinbiosyntliesis using isotopic carb,on. II. Slhort-term experiments wsith14CO2. Can. J. Cliem. 31: 755-760.

7. 11owxN-iL:L, H. 1I. 1971. Stability of the lignin-carbolhvdrate comnplex. Tappi50: 473-477.

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lignin formation in wheat coleoptile cell walls · the dry tissue was extracted with 6% kohat room...

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