dluring - plant physiology · pentoses and ascorbic acid do not react and there is negligible...

COMIPOSITION OF DEVELOPING PRIMARY WALL IN ONION ROOT TIP CELLS.1. QUANTITATIVE ANALYSES 1, 2

WILLIAM A. JENSEN AND MARY ASHTONDEPARTMENT OF BOTANY, UNIVERSITY CF CALIFORNIA, BERKELEY

An understanding of the factors influencing anddirecting cell development must rest on knowledge ofthe changes occurring during the course of cell de-velopment on both a morphological and biochemicallevel. Previous work on onion root tips has outlinedsome of these changes that occur early in the develop-ment of the cell (7, 8, 9, 11). The wall is one of themost conspicuous parts of the cell and undergoes greatchanges during the life of the cell. Very little isknown, however, of the chemical composition of thewall dluring the early stages of formation. Theanalyses reported here attempt to enlarge the pictureof the composition of the developing primary cell wall.

The root tip is an excellent material in which tostudy cell development, as the various stages are ar-ranged in approximately linear order. Thus, theanalysis of consecutive sections yields information onchanges accompanying cell development. The cell(levelops rapidly, particularly during the early stages,and to follow the development, sufficiently thin sec-tions must be used. The first 20 consecutive 100 #sections of the onion root tip are analyzed in the pres-ent work.

The usual methods of cell wall analysis based ongravinietric measurements or on paper chroma-tography could not be used on this level. Lignin isnot present in this region of the root and as all theother wall components can be analyzed in terms ofcarbohydrates (hexoses and pentoses) or simple car-bohydrate derivatives (hexuronic acids), colorometricprocedures were used. Microchemical techniqueswere developed to handle the material and measurethe sugars. A standard extraction procedure of thexvall components is used and the various extractsanalyzed for the amount of hexuronic acids, hexoses,and pentoses present. The results are expressed asper cell and per unit surface area per cell.

MATERIALS AND METHODS

Sets of Alliurn cepa var. white globe were placedon vials of tap wvater and kept in the dark at 250 C.Roots between 1 and 2 cm in length developed in 48hours. The first 5 to 10( mm of carefully selected uni-form roots were excised and quickly frozen in iso-

1 Received July 16, 1959.2 Supported in part by a research grant from the Na-

tional Science Foundation (G-4286).

pentane cooled with liquid nitrogen. The root tipswere then placed in methyl alcohol at -30° C. Themethyl alcohol was changed once during the first 24hour interval and three times during the second 24hours, finally being replaced with toluene at the sametemperature. A fter 2 hours the roots were brought toroom temperature and the toluene changed. Theyvere then paraffin infiltrated and imbedded. Theroots were carefully aligned in groups of three duringthe imbedding.

The roots were sectioned transversely on a reg-ular Spencer microtome at either 10 or 25 it. As theanalyses required 100 o segments of the root, ten 10 itsections or four 25 o sections were collected andplaced in a 1 ml centrifuge tube. Actually, when 10A sections were used only nine were placed in the tube,while the tenth was mounted on a microscope slideand stained. These sections acted as controls on thealignment of the roots and the condition of the tissue.The 10 A sections were used in preliminary analyseswhile the 25 A sections were used in the later replica-tions. As the analyses required nine 100 A segments,nine roots were cut and the identical sections placedin the same tube. This was facilitated during im-bedding by aligning roots in groups of three in theparaffin.

Once the tissue was placed in the centrifuge tubesit was never removed. Changing the various liquidsin the tubes without loss of tissue was a very criticalstep. Pipettes draxvn from glass tubing were drawn asecon(l time so that the opening was narrower indiameter than the smallest section. A holder rigidlysupported the pipette which was connected to a flaskand ultimately to a vacuum line. A stopcock in thetop of the flask controlled the pressure in the flask andhence the rate of removal of the liquid. The centri-fuge tube containing the tissue and solution was raisedto the pipette by means of a small movable platform.A magnifying lens mounted in front of the tube, anda mirror mounted at one side aided in observing thetissue and in aligning the tube with the pipette. Carewas taken to keep the tip of the pipette just below thesurface of the liquid and to remove the liquid slowly.'Where the liquid removed was to be saved, a rubberbulb with a small hole in the top replaced the vacuumline. By closing the hole with a finger, the bulb couldbe used to draw off the liquid. Opening the holeequalized the pressure in the bulb and the liquid re-mained in the pipette and was not carried up into thebulb. The same stands for pipette and tube wereused as in the case of the vacuum line. The tubes ineither case were usually centrifuged in a small micr-

313

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PLANT PHYSIOLOGY

centrifuge to force the tissue to the bottom before re-moval of the liquid.

The tubes were covered with aluminum foil wher-ever possible to reduce the amount of dust and lintfalling into the tubes. Aluminum blocks 6in X 6in X2ill in which 100 holes YSin in diameter and 78in indepth lhadl been drilled, were used for heating or cool-inig the tubes. These gave uniform heat conductionon a hot plate and could be coole(d in a deep freeze.

The tissue was deparaffinized with toluene, rinsedlwvith 100 % ethyl alcohol, and air dried. Analysesfor total carbohydrate, lhexoses, pentoses, and hex-uronic acids were made directly on the tissue at thispoint., otherwise the sections wvere extracted accor(ingto the following sclhedlule:

Tissue

H,O 25° C.16 hrs then txxo 3 hr periods

Soluble Residue

0.5 NH,3 Oxalate 90)° C.Three 1 hr periods

Soluble

Soluble

Residue

4 % NaOH 250 C.6 lhrs

Residue

17.5 c NaOH 250 C.6 hrs

Soluble Residue

The extracts contaiining the soluble fraction were inieaclh case combinedl and brought to dryness in an ovenat 500 C. In the case of the 4 % NaOH extract, anequal volume of 4 % acetic acid was added to neu-tralize the base an(d then the solution evaporated. Atall the unlderlined points in the above schedule, theextracts or residlues were measured for amount ofliexoses. pentoses, and hexuronic acids present. Ofall the cell wall components fractionated in thisschemiie, only the 4 % NaOH and the 17.5 % NaOHlsoluble fraction had to be determined by difference inthe residues before and after extraction, rather thandirectly on the extract. This was necessary when itNvas found that after treatment with NaOH neitherthe hexoses nor pentoses would react in the color re-actions necessary for their determination.

This extractioil schedule was worked out withgreat care. Longer and more numerous extractionperiods were tested in most cases but the schedulegiven above proved to give the most satisfactoryseparation of w-all conmponents. Wherever possiblealternate procedures wNere used to verify particular

fractions. The ammonium oxalate extraction was re-placed by extraction wNith 1N HC1 and similar resultswere obtained. Balance sheets for the various car-bohydrates were also run combining various extrac-tion procedures.

The extraction procedure usedl here is similar tobut not identical with that of Boroughs and Bonnei(4) anlcl differs from that of Jermvn and Isherwoodl(10) principally in not first extracting with 70 1alcohol and obtaining a cold water and anmmoniumoxalate fraction instead of a single hot water fraction.It also (liffers from that of Bishop, Bayley, and Set-terfield (2) where a 10 % NaOH and a 17.5 CNaOH extraction are combined so that only oneNaOH fraction was measure(l. Burstr6m (5), whogenerally folloxved Jermyn and Isherwood also com-bined the alkali soluble fractions. A search of theliterature revealedl no two identical extraction sclhed-ules. This indicates that every material must be han-(Iled as a separate case.

The procedures listed below were used for the car-bohvdrate anialAyses. They are based on miacro orsemi-micro procedures (1,6), but in all cases havebeen further reduce(d in volume and modified.

I. TO'IAL PENTOSE AND HEXOSE-ORCINAL REAC-T10N. To the samnple wNas added 50 ul H10, 50 Alfreshly prepared 2 ' orcinal in 20 11cH2S04 and500 Ml 60 % H,SO. The mixture was heated for30 minutes at 800 C in an aluminum block and thencooled in a second, cold aluminum block. A "flea"of glass coated ferrum reductuml was adlde(d anid thesolution stirred by nmoving the flea wvith a magnet.The absorption was miieasuredl at 425 mMi in a Beck-man spectrophotonmeter mo(lel DU. The range ofthis procedure is 0.5 to 10 Ag + 0.1 Ag. Glucose, fruc-tose, galactose, mannose, ribose. and arabinose giveequal absorption per Ag. while xvlose gives twice thisabsorption. Glucuronic an(d galacturonic acidls givevalues of less than a tenth of the hexoses and pentoseswhile ascorbic acid gives no color at all. The orcinalreaction was used to measure the total combined hex-oses an(l pentoses in the untreatedl tissue.

II. HEXURONIc ACIDS-CARBAZOLE REACTION.To the samples was addedl 50 Ml H.O an(d 300 Al con-centrated H1SS04. The tubes were heated for 20minutes at 1000 C in an aluminum block and thencooledl in a cold aluminum block for 5 minutes. Then10 Al of a freshly prepared solution of 0.1 %,o carba-zole in 95 % ethyl alcohol was added. A flea wasused to stir the solution andl the absorption measuredat 535 mny in a Beckman spectrophotometer. Theprocedure has a range of 0.5 to 5 Mg + 0.1 Mg. Glu-curonic and galacturonic acids give identical values.Pentoses and ascorbic acid do not react and there isnegligible interference fronm hlexoses. The carbazolereaction was used to mleasure hexuronic acid in theuntreated tissue and in the various extracts.

III. PENTOSES-CYSTEINE REACTION. The tubescontaining the samples were coole(d in a coldl alumi-nunm block andl 50 Ml cold H. 0 an( 200 Ml coldl concen-

314


315JENSEN AND ASHTON-COMPOSITION OF PRIMARY WALL

tratecl H.2SO4 were added. After shaking, the tubeswere removed from the block, fleas added, and thetubes allowed to come to room temperature. Thesolutions were kept at room temperature for 2 hours-with regular stirring. Then 5 of freshly prepared3 % aqueous cysteine was added and the mixturestirred. After 15 minutes, the absorption at 390 mILand at 425 my was read in a Beckman spectrophoto-meter. The difference of absorption at 390 mA to425 myA is proportional to the amount of pentosepresent. The range is 0.5 to 5 Mg 0.1 Ag. Therewvas no reaction to hexoses, hexuronic acids, or ascor-bic acidl. The greatest difficulty with this procedureis that xylose gives 4.5 times the color per Ag as doesarabinose or ribose. In all cases the pentose dataare expressed as arabinose. This cysteine reactionAas used to measure pentoses in the untreated tissue,-tlhe extracts, and the tissue residues.

IV. HEXOSES-CYSTEINE REACTION. To thesample was added 50 Al H,O. The tubes were thenplaced in an ice water bath and 500 Al of a six partH.,SO4: one part H..O mixture was added. After afew mlinutes the tubes were removed from the icewater, allowed to warm to room temperature, and-then heated in boiling water for 3 minutes. Theywere then cooled in a water bath at room temperatureanid 100 Ml 3 % aqueous cysteine solution added. Thesolutions were stirred with a flea and the absorptionat 415 mM and 380 mM read in a Beckman spectrophoto-meter. The difference in absorption at 415 mM and380 mIML equals the amount of hexose present. Therange of the procedure is 0.5 to 5.0 Mg + 0.1 Mg.Glucose, fructose, galactose, and mannose give almostidentical absorption values. There is no interferencefronm pentoses, hexuronic acids, or ascorbic acid.This cysteine reaction was used to measure hexoses inthe untreated tissue, the extracts, and the tissueresidue.

V. TOTAL HEXOSES AND PENTOSES-INDOLE RE-ACTION. To the sample was added 50 Ml H,O, 500 Ml75 % H..SO4 and 20 Ml 1 % alcoholic (95 % ethylalcohol) indole solution. This mixture was heatedfor 10 miniutes at 1000 C and cooled. The absorptionw-as measured at 470 mM in a Beckman spectrophoto-meter. The range of the procedure is 0.5 to 5.0 Mg+ 0.1 Ag for hexoses and pentoses. Glucose, fructose,mannose. galactose, arabinose, and ribose have equalabsorption wllile xylose is 11 times higher. Galac-turonic and glucuronic acid react intensively whileascorbic acid gives less intense, but still appreciable,color. Consequently, the indole test was used onlyon the 4 % and 17.5 % NaOH tissue residue whereneither the hexuronic acids nor ascorbic acid ispresent.

In all of the carbohydrate analyses two kinds ofstandards were used. In one the standards weretreated the same as the tissue or extract and carriedthrough the entire procedure. This standard actedas a control of the effect various chemicals and heat-ing periods might have on the carbohydrates. The

second standard was run only through the color reac-tion. In most cases there was little difference be-tween the two standards.

The carbohydrate procedures given above wereworked out in great detail. All hydrolysis times andtemperatures were tested and complete absorptioncurves run in every case. Each procedure was testedfor the possibility of interference by higher concen-trations of sugars not normally reactive in that pro-cedure. Even at ranges of more than five times theamount of the carbohydrate normally present in thetissue, no interference in any of the tests was found.

Attempts were made to remove the protoplasmfrom the cells before analysis of the wall by treatmentwith alcoholic KOH. These attempts were aban-doned after determinations showed large losses ofhexuronic acidls, hexoses, and pentoses. As there islittle hexuronic acid in the cell other than that asso-ciated with the cell wall and the total amount of pen-toses found in all the nucleic acids in the cell give aninsignificant color under the conditions used here, itwas concluded that not only the protoplasm but muchof the cell wall material was being removed. Onionroot tips do not contain starch and it would appearsafe to assume that the significant amount of the hex-uronic acids, hexoses, and pentoses, is either asso-ciated witlh the cell wall or is soluble.

The roots were analyzedc morphologically in thesame way as reported earlier (9). A group of bulbsdifferent from that used in previous work was usedin this study. The roots from the new group ofbulbs had slightly dlifferent cell numbers and cell di-mensions. For these reasons the cell numiiber. meancell cross sectional area, mean cell length, and meancell surface area are presented here. The numberof mistosis also were counte(l.

RESULTS

MORPHOLOGY. The first 2 mm of the root tip arerepresented diagrammatically in figure 1. The graphimmediately below the diagram presents the cell num-ber per 100 A section. The upper curve is the totalcell number while the lower represents the numberof cells per section minus the number of rootcap cells.Figure 1 presents the average cross sectional area.length, and surface area of the cells in each 100 A sec-tion. The percent of nuclei in division is also pre-sented in figure 1. A detailed morphological analysisof the onion root has been published (9) and completediscussion of the morphology of the root will not beundertaken here.

The general pattern of cell development can easilvbe seen from the graphs of figure 1. The cell, basalto the apical initials, first enters a period of marke-lradlial enlargement. This period lasts from 400 to1,400 A from the tip. Elongation begins some 800to 1,000 M from the tip and extends beyond the 2 mlof the tip analyzed here. From 800 to 1,400 A thereis a period in which the cell is both enlarging radiallyand elongating. The amount of cell elongation, how-ever, is small until after approximately 1,400 A from


PLANT PHYSIOLOGY

the tip. The period where elongation and radial en-largement overlap will be termed the first period orphase of elongation, while elongation after 1,400 uwill be referred to as the second phase. This patternis in general agreement with that of Scott et al (11).

The distribution of cell divisions must also beconsidered in relation to the various stages of celldevelopment. The apical initials are very low innumber of cells in division. This feature of the roothas been discussed elsewhere (9). During the periodof radial enlargement, the number of cells in divisionincreases and the maximum number of divisions oc-curs at the beginning of elongation. During the firstphase of elongation, cell divisions are as numerous asduring the first phase of radial enlargement. As thesecond phase of elongation is approached, cell divi-sions decrease and by 1,600 A have ceased. Thispattern of divisions is similar to that reported earlierby Jensen and Kavaljian (9). In the roots used bythem, however, the rootcap was more extensive andthe first 2 mm of the root analyzed, essentially didnot contain the second period of elongation. More-over, they used fluctuating temperatures while aconstant, slightly elevated (25° C) temperature wasused in the present work.

The cells of the rootcap in front of and surround-ing the apical initials undergo rapid development re-sulting in large, heavy walled cells. The presence ofthe rootcap cells surrounding the apical initials mustnot be forgotten when considering the data from theearly sections which contain large numbers of root-cap cells as well as the apical initials and other non-

rootcap cells. The amount of carbohydrate present inthe apical initials can be calculated if it is assumedthat the amount of carbohydrate per rootcap cell im-mediately in front of the apical initials, is the same asin the rootcap cells surrounding the apical initials.Since the amount of carbohydrate in the rootcap cellsin front of the apical initials is known from sectionII, and the number of rootcap cells present in sectionIII is known from the morphological analysis, thensimple multiplication of these two figures will resultin the amount of carbohydrate in section III that willbe accounted for by rootcap cells. If this figure isthen subtracted from the total amount of carbohydratein section III, the remainder will be the carbohydratepresent in the apical initials. Dividing this figureby the number of apical initials, results in an estimateof the amount of carbohydrate present per cell of theapical initials. This figure is really only an approxi-mation, but it is nonetheless interesting and useful.

CARBOHYDRATE DETERMINATIONS. The results ofthe carbohydrate determinations on the whole tissueand after the various extraction procedures, are pre-sented graphically per cell and per L-2 surface area percell, in figures 2 and 3. All data have been expressedper uM or per iLIM so that the data on the varioussugars can be compared directly.

Before describing the results a word must be saidregarding terminology. Two general systems of cellwall terminology are apparently in existence. One isbased on solubility criteria so that all hot water orammonium oxalate soluble materials become pectin

7 LLL DIVWISN I NITIALS I

12 13 1415 1 718 19 20DISTANCE FROM TIP IN HUNDREDS OF MICRONS

z

zw-JS

FiG. 1 (left graph). Number of cells per 100 ,u section. (. = total number of cells per section, + = non-rootcap cells per section) and percent cells in division. The diagram of the root at the top is on the same scale asthe graph.

(Right graph) Average cell cross section area, length, and surface area expressed as a function of distance fromtip of section analyzed.

316


JENSEN AND ASHTON-COMPOSITION OF PRIMARY WALL

TOTAL HEXOSE, PENTOSE, ANDHEXURONIC ACID

41

3!

21

2'

HE XO SE

~5

~5

0

5-

1 2 3 4 5 6 7 8 9 10 I 12 13 14 15 16 17 20AMMONIUM OXALATE SOLUBLE 45

( 900) HEXOSE0 40

0 H. ACID0 35

30'

0 &° ZPENTOSE 25

20

10.

5

2 3 4 5 6 7 8 9 10 11 12 13 14 5 16 17 18 19 20

17.5 % No OH SOLUBLE(25°)

PENTOSE

/~~~

,/'EXOSE

o~~~OeO0-0-0-0-- 0-00

9C

sC

70

50

40

30

20

10

WATER SOLUBLE( 250)

H. ACID

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19204% NaOH SOLUBLE

(250)

.4ENTOSE,/

. 02 3 4 6 7 8 9 U II 12 131415 16 17 18I9/

175%,NaOIH INSOLUBLE(250) /

0'

HEXOSE

0

//

,0,0-0~~

ON

0

f r .-I I A I . I I I1 23 456 7891011121314151617181920 1 23456 78910I 1 12131415161718992ODISTANCE OF SECTION ANALYZED FROM THE TIP IN HUNDREDS OF MICRONS

FIG. 2. Results of carbohydrate analyses expressed per cell. Open circle is hexose; closed circle is pentose; half-open circle is hexuronic acid. The squares indicate calculated values for the apical initials (see text for explanation).

25

22

20

17

15

317

0

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1I25

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T 750

50

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45

40

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PLANT PHYSIOLOGY

AMMONIUM OXALATE SOLUBLEo (900)

°\W _°-O0- HEXOSE

a 01.0~~~~~~

PENTOSEa

17.5% NaOH SOLUBLE(25)

a

PENTOSE

0 0

0 00

0-0-

* * * *

0 ~~ ~ ~~ o0,-0O-0o0ooEXOSE°8° -°-D-o-°-°--

WATER SOLUBLE(25-)

0

a

4% No OH SOLUBLE(250)

PENTOSE_*_ *-

,-

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0-0 * 0 0°- ° H.ACID0p00

(

a

17.5% NoOH INSOLUBLE(25)

'9.5

17 18 19 20 I 2 3 4 5 6 7 8

DISTANCE FROM TIP IN HUNDREDS OF MICRONS

FIG. 3. Results of carbohydrate analyses expressed per surface area per cell. Open circle is hexose; closedcircle is pentose; half-open circle is hexuronic acid. Squares indicate calculated values for apical initials.

318

oI

I


JENSEN AND ASHTON-COMPOSITION OF PRIMARY WN'ALL

an(l alkali soluble materials become hemicelluloses(10). The other is based on chemical compositioncriteria so that only pure polygalacturonic acid com-pounds are pectic substances, while hemicellulosescontain hexuronic acid and pentose, and noncellulosicpolysaccharides composed of hexoses and pentosesmay be found either in the hot water-ammonium oxa-late soluble or alkali soluble fraction (3). This pic-ture is furtlher complicated by dividing the pectic sub-stances (in a chemical sense as polygalacturonic acid)into pectin and protopectin on solubility character-istics. Only cellulose and lignin are clearly uncon-troversial terms.

The following definitions will be used: pectin-water soluble hexuronic acids; protopectin-water in-soluble, ammonium oxalate soluble hexuronic acids;pectic substances-the sunm of the water and ammoni-um oxalate soluble hexuronic acids; soluble non-cellu-losic polysaccharides-water insoluble, ammoniumoxalate soluble hexoses and pentoses; hemi-cellulose-ammonium oxalate insoluble, 4 % NaOH solublehexuronic acids and pentoses; insoluble non-cellulosicpol)ysaccharides-4 % NaOH insoluble, 17.5 % NaOHsoluble hexoses and pentoses; cellulose- 17.5 %NaOH insoluble hexose.

Quantitative comparison of the present data withprevious work is almost impossible. This is becausethe present data is expressed per cell and almost nodata on a similar basis exist in the literature. More-over, it is not possible to obtain the dry weight ofthe sections mlaking it impossible to express the pres-ent (lata in terms of percent dry weight as is commonin tlle literature.

To understan(d the implications of the data ex-presse(l per unit surface area per cell it is necessaryto realize that there need be no connection betweenthe surface area of the cell and the area of the wall.WVhen the data are expressed per unit area they meanthat the amiiount of wall material per cell is being com-pared with the surface area of the cell. If the amountof wall material and the area of the cell both increaseuniformly from one section to another the amount ofwall material per unit area will remain constant; if thewall miiaterials increase more than the surface area,the amiiount of wall material per unit area will in-crease. There is no asumption that the wall is con-tinuous or entire. From figure 4, it is clear. that ex-cept for the cell in rapid elongation, the wall is not asimlple. entire sheet surrounding the cell. When theunit area (lata are viewed in this wa) an(d the factorsinvolved in changes in surface area are examined, apicture of cell w,all development can be outlined.

The imlportant points of the determiiinations canbe sulmmiiiarized thus:

A. TOTAL HEXURONIc ACID, HEXOSE, AND PEN-TOSE. The three major groups of carbohydratespresent in the root tip show, in the analysis of thetotal tissue. a unity and relationship that belies thediversity of their distribution within the cell wall.The cells of the root cap are high in both pentosesandl hexoses w-hile relativelv low%v in hexuronic acids.

All three groups are relatively low in the cells under-going radial enlargement and increase proportionallyto the increase in surface area of the cell. Correlateddirectly with the beginning of elongation there is anincrease in amount of all three groups of carbohy-drates. This increase in amount is greater than theincrease in surface area of the cell. As the cellsenter the second phase of elongation, the amount ofhexuronic acids, pentoses, and hexoses continues toincrease, but only the hexoses continue to increaseat a rate greater than the increase in surface area.

These determinations of total hexuronic acid,hexose, and pentose were also used as a check on themeasurement of the various fractions. When thehexuronic acid, hexose, and pentose content of thevarious fractions are added together they should equalthe total content of these carbohydrates measured di-rectly. The direct and indirect total amounts werefound to differ by less than 3 % for the hexoses andlless than 8 % for the hexuronic acids. The pentosedifference was almost 30 %, however, with the in-direct total being higher than the direct. The pen-toses are the most difficult to measure of the threecarbohydrates, requiring a procedure that does notallow the tissue to be heated. This fact makes ithighly probable that the direct total amount is lowerthan the indirect total because of difficulties in obtain-ing complete hydrolysis of the wall.

B. WATER SOLUBLE FRACTION. All three groupsof carbohydrates were found in the water soluble frac-tion in relatively large amounts. As the tissue wasin contact only with toluene or absolute methyl orethyl alcohol from the time of initial freezing untilthe water extraction, we believe that no measurableamount of carbohydrate was lost.

The hexuronic acids may compose part or all ofthe water soluble pectins. The cells of the root cap

FIG. 4. Microscopic structure of cell wall. A. Wallof cells in radial enlargement. B. Wall of cells in transi-tion zone. C. Wall of rapidly elongating cells.

319


PLANT PHYSIOLOGY

L,.M Pi jN T W .

..~~~~~

L4i WATER SOLUBLEA PECTIC SUBSTANCES

B -- HEMICEL JLOSENON CELLULOSIL

POLYSACCH ARIDE SCCE UI CSE

JEHEXURONIC A Ctf[.

AM3NL___. r

0, W/C - W Y- '~ ETA N t - N

PE rh .,@

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DA20p- N-4 OTlAPICAL RADI A... R ANS1-r'.N ITIAL. ENLARGEMENT

STAG ES OF CELL -DEVELOPMENT

320

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11



are relatively high in hexuronic acid. The cells inradial enlargement contain a relatively low amountwhich appears to be directly proportional to the sur-face area of the cell. As soon as elongation begins,howexer, the amount of hexuronic acid increases at arate greater than the increase in surface area. As thecell reaches the second phase of elongation, the rateof increase of the hexuronic acid falls below the rateof increase of surface area and the amount of hexur-onic acid per unit area progressively declines.

Hexose content of the root cap cells is also high,particularly in the extreme cells. In the cells inra(lial enlargement the amount of hexose is lowerthan in the rootcap. As cell elongation begins. hex-ose content per cell increases. This increase, how-ever, is directly proportional to the increase in cellsurface area so that the amount per unit area remainsconstant.

The pentose content per cell is similar to the hex-ose pattern except that at the beginning of elongationthere is a small but consistent drop in pentose amountper unit area.

C. 0.5 % AMMONIUM OXALATE SOLUBLE.Again, all three groups of carbohydrates were foundand in relatively large amounts.

The hexuronic acids, which may be consideredthe protopectins and the pectates, are present inamounts directly proportional to the cell surface areain all stages of development represented in the first2 mnm of the root including the rootcap. The proto-pectins make up 6 % of the total carbohydrate contentof the first section and increase to 10 % by the eighthsection and then remain relatively constant. The sumof the pectin and protopectin follows a similar pattern,starting at 13 %, increasing to 18 % and then remain-ing constant.

Hexoses, probably in the form of a non-cellulosicpolysaccharide, are present in large amounts in cellsthat have undergone or are undergoing cell elongation.The cells of the rootcap appear very high in hexosesand undoubtably influence the amount per unit areabetween 500 to 800 A from the tip. During radialenlargement the amount of hexose is relatively lowand directly proportional to cell surface area. Thisproportionality is maintained during early elongationbut as the cells enter the second phase of elongationthere is a sharp rise and a new proportionality at ahigher level is established.

Pentoses, probably in the form of a non-cellulosicpolysaccharide, are present in large amounts in thecells of the rootcap. Cells in radial enlargement andearly elongation contain relatively less pentoses butthe amount is directly proportional to the increase insurface area. As the rate of elongation increases,

the increase in pentose content does not remain pro-portional but decreases so that the amount of pentoseper unit area of the wall decreases.

D. 4 % NaOH SOLUBLE FRACTION. This frac-tion contained only hexuronic acid and pentoses andthus fits one commonly accepted definition of thepolyuronide hemicelluloses (3).

The hexuronic acid content per cell is low in thecell in radial enlargement and increases as the cellsenter elongation. In all phases of cell developmentthe amount per cell is (lirectly proportional to surfacearea.

Pentose content per cell is directly proportionalto surface area only during the very early stages ofcell development. During this periodl the ratio ofpentose to hexuronic acid is 1: 1. While the cellsstill are undergoing radial enlargement, the amountof pentose per cell begins to increase at a rate greaterthan the increase in cell surface area. This conditionpersists until the second phase of elongation whenthe amount of pentose decreases slightly in relationto increase in surface area of the cell.

This pattern of hexuronic acid and pentose changesuggests that the hemicellulose present consists ofchains of hexuronic acid-pentose units in a ratio of1: 1. The extra pentose molecules may form crosslinkages between the hexuronic acid-pentose chains.

E. 17.5 % NaOH SOLUBLE. This fraction con-tained only hexose with an occasional trace of pentoseand represents almost pure cellulose.

The hexose content of the rootcap is high whilethe cells in radial enlargement are relatively low. Ascell elongation begins, the hexose content of the wallbegins to increase at a rate greater than the increasein surface area. When cell elongation enters the sec-ond phase, however, the rate of increase of hexose percell becomes constant in relation to the increase insurface area.

DiscUSSION

The composition of the primary cell wall in theroot tip presents a surprisingly complicated picture.The problem is to relate the changes in the chemicalcomposition of the wall with the morphological de-velopment of the cell. The region of the root analyzedcontains cells of the rootcap. the apical initials, andcells in the following stages of morphological develop-ment: radial enlargement, a transition stage whereradial enlargement and elongation are both going on.and elongation. The data for the apical initials andthe various stages of cell development are summarizedin figure 5.

FIG. 5. Composition of cell wall expressed as a function of stages of development of cell. The noncellulosicpolysaccharides referred to in the legend are the insoluble noncellulosic polysaccharides of the text. The hexuronicacid content of the water soluble and pectic substances fraction are shown because of importance of the hexuronicacids in definitionis of pectin and protopectin. The black bars in both cases represent the amount of hexuronic acidin each fraction.

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PLANT PHYSIOLOGY

The cells of the rootcap actually are present inall stages of development but the mature, fully (liffer-entiated cells predominate. These cells are large andpossess very thick walls. From the chemical analysesit is clear that the walls contain large amiiounts of cellu-lose an(d soluble non-cellulosic polysaccharides. Thecells also contain fairly large amiiounts of water solublehexoses, pentoses, and( hexuronic aci(ls. Protopectin,hemicellulose, andl the insoluble non-cellulosic polv-sacclharides ar-e present in mluclh lower amounts andare (lirectly proportional to cell surface area.

The apical initials represent a relatively smalliroulp of cells that can only le treate(l indlirectly onthe blasis of calculations previously (lescribed. Thestriking feature of the apical initials is their low inci-(lence of division. They appear to be metabolicallyinactive insofar as they have been examined. A1or-phologically. the apical initials are slniall cells \iththin. (lelicate appearing cell walls that in no senseforml a continiuous slheet of mlaterial. On a per cellbasis, the apical initials are low in all cell wall com-p)onents, except the insoluble pentose non-cellulosicpolysaccharides, when comparedl with the other cellsin the root (fig 5). On a per unit surface area basis,lio\\-ex-ei- all the pentose containing fractions, exceptthe pentose in hemicellulose. are either equal to orgreater than the same fractiolns in the cells in radialenlargement. Pectin ai(l lprotolpectin are low oneither basis but the wall is proportionally richer inproto;)ectin than is the w\ all in radlial enlargement.Cellulose is also low hut the proportion of celluloseto pectic substances is the samiie in the ap)ical initialsall(l the cells in radial enlargement. It is clear thatthe cells of the apical initials (lo not lack cellulose.nor0 are very high in pectic subistances or composedonly of pectic substances and(l cellulose.

The cells in radial enlargemiielnt are increasing)rimarily in (liamleter with little increase in averagelengtlh. Cell (divisions al-e numlerotus (Iiring this stageand incr-ease in numiiber. The wvall is morphologicallya loose open structure writh large gaps (the primarypit fields) running at right angles to the long axis ofthe cell (fig 4a). The Nall is low! in all carbohydratesaltlhoughl all components are present in rouglhly equalamiiounts. If all the water awl amminionium oxalatesolubile carbohydrates are takeni togetlher and treatedas pectic substances they amiiounit to somiie three timesthe anmoulnt of cellulose piresenit. Tf, howev\7-er, onlythe hexul-onic acids of these fractions are consideredas pectic substances they are theln e(qual to the celluloseplresenit. During this periodl of radial enlargementall the \vall components increase uniformly in relationtoi one alnotlher and in direct proportion to the increasein cell surface area. The cells in ra(lial enlargemilentare often considered as the meristem and treated asbeing simple meristematic cells. Tn this connectionit slhioul(d be emphasized again thlat the wall does notlack cellulose nor is it compose(d primarily of pecticsubstances.

Radial enlargement continues until approximately1.400 A from the tip. w\hile elongation begins some800 to 900 A from the tip. Thts, there is a region

lhere the cell is still increasing in (liamleter wvhileat the same timle beginning to increase in length.Cell division in the root reaches a peak at the begin-ning of this stage ancl decreases as the stage progressesan(l elongation becomes more pronounce(l. Durl-ingthis stage the mlorplhology of the wrall chalnges to aiore continuous structure with the larger gaps beingfille(d in, although still visible, and the primary pitfieldls hecoming smaller (fig 4b). This change inthe morphology of the wall is reflectedl in the amiioutntand composition of the various wall components. Nllthe carholhydrate colmiponents of the wall increase (Ilir-inlg this lerio(l hut three (pectin, soluble pentose noln-cellulosic polysaccharide. and cellulose) increasemore than the s-urface area of the cell.

As radial enlargement ends andl several hlunld(redmicrons later cell (livisions cease, the cell enters thestage of rapidl elongation. The morphology of the-xvall changes againi, becoming continuous with all bhutthe final traces of the primary pit fields nearly in-visible (fig 4c). The change to straight elolngationhas somiie very interestilng effects on the carhohydratesof the cell wall. All components continue to increaseper cell hut it is in relation to surface area that surprising changes occur. From 1.600 to 2.000 A frolmithe tip a steady state seems to exist between tlle in-creases in cell xvall components and(lthe increase incell surface area (all cell w-all components increase(lirectlv propol-tiolnal to surface area). This steadystate has heen arrived at (lifferentlv,lhoNevel-, fol- thedifferent comlpolnelnts. In the case of protopectiln aln(dhemicellulosic lhexuronic acid no change Nas involvedas they remiiained directly proportional to surface areathrough all the stages of development. For cellulosean(l pectin w-hichl wsere from the beginning of eloniga-tion increasing faster than the surface area, the finalstate is a restilt of synthesis now becoming (lirectlvproportional to grow-th in area. For soluble llexosenoncellulosic polysaccharides and the insoluble n1on1-cellulosic polysaccharides, the encd of radlial enlarge-ment resulte(d in a periocl of increasedl synthesis thatterminate(l in the establishment of a new propol-tioln-ality to surface area but at a higher level. Tn all casesthe en(d result is a condition where eachl componentis being synthesized in (lirect proportion to the in-crease in surface area of the cell. The comipositionof the wall is (lifferent. hovever, thaln it \\was before.Per unit area, the wall is higlher in cellulose. pectilnhemicellulose, soluble hexose noncellulosic polysac-charide, and( the insoluble noncellulosic polysacclha-rides wvhile remaining constant w\ith regard to proto-pectin an(I water soluble hexose. Only xvater solublepentose (lecreased in amount.

What essentially appears to be happeninig (lurinigthis stage of (levelopment is that the gaps in the cellwall associatedl with radial enlargemiient are beingfilled in with cellulose, pectin, hemicellulose, soltublehexose noncellulosic polysaccharide, and the insolublenoncellulosic polysaccharides. This accounts for theincrease of these comliopnients per unit areal immiile(liate-lv after the cessation of ra(lial enlargemlenit a(In for the

322



fact that they then become directly proportional tosurface area at a higher level while the wall appearsno thicker, if not thinner, morphologically. Whenthis stage is compared with the preceding one whereboth radial enlargement and elongation are occurringsimultaneously, it seems possible that the amount ofelongation in the earlier stage may be limited by thefact that the wall is still too fragmentary for rapidelongation to occur. Only after the wall is an ap-proximately continuous unit, can elongation occur toany great extent. This point of conversion wouldappear to be a logical place to study the effect ofgrowth substances on roots.

The data presented here are difficult to comparewith other cell wall analyses due to differences in thetechniques and the means of expression employed.Insofar as comparison is possible the dlata reportedhere are in general agreement with other analyses ofprimary walls using quantitative bioclhemical tech-niques (3, 4, 5, 10). The notable exception is thewvork of Bishop et al (2), who reported small amountsof pectic substances in the primary wall of Avenacoleoptiles. The striking difference in the materialsmay, however, very well account for the differencesin the results. We agree with Bishop et al in the con-clusion that the hemicelluloses and noncellulosic poly-saccharides may be more important in cell wall (le-velopment than generally recognized.

SUMMARYThe first 20 consecutive 100 As sections of the onion

root tip were analyzed for water soluble, 0.5 % am-monium oxalate soluble, 4 % NaOH soluble, 17.5 %NaOH soluble, and 17.5 % NaOH insoluble hexose.pentose, and hexuronic acids. The data were ex-pressed per cell and per A2 surface area per cell.

Besides the rootcap and apical initials, this regionof the root contains the following stages of cell de-velopment: radial enlargement, a transition stagewhere both radial enlargement and elongation occur.and elongation. On the basis of the present analysisthe cell wall in these stages can be characterized.The rootcap cell is high in cellulose and soluble non-cellulosic polysaccharides, fairly high in water solublehexoses and pentoses and pectin, and low in proto-pectin, hemicellulose, and insoluble noncellulosic poly-saccharides. The apical initials were low in all cellwall components although all were present, the pecticsubstances and cellulose being present in eqjualamounts. In radial enlargement the wall was stilllow in all components but the amounts of the major

components were approximately equal. During thetransition stage all components increased per cellwhile on a per unit area basis the only component thatshowed an increase that could be correlated with thebeginning of elongation was pectin. When radial en-largement ceased there was a marked increase in theamount of cellulose, pectin, soluble hexose noncellu-losic polysaccharide, and insoluble noncellulosic poly-saccharides per unit area. This increase can be cor-related to morphological changes in the wall. Duringthe stage of elongation all components increased indirect proportion to the increase in surface area ofthe cell.

LTTERATURE CITED1. ASHWELL, G. 1957. Colorimetric analysis of

sugars. In: Methods in Enzymology, S. P. Colo-wick and N. 0. Kaplan, eds. Academic Press,New York.

2. BISHOP, C. T., S. T. BAYLEY, and G. SETTERFIELD1958. Chemical constitution of the primary cellwalls Avena coleoptiles. Plant Physiol. 33: 283-289.

3. BONNER, J. 1950. Plant Biochemistry. AcademicPress, New York.

4. BOROUGHS, H. and J. BONNER 1953. Effects of in-doleacetic acid on metabolic pathways. Arch. Bio-chem. Biophys. 46: 279-290.

5. BURSTR6M, H. 1958. The influence of growth reg-ulators on the composition of the cell wall.Fysiogr. Sallskapet. Forhandl. I. Lund 28: 53-64.

6. DISCHE, Z. 1955. New color reactions for the de-termination of sugars in polysaccharides. In:Methods of Biochemical Analysis, D. Glick, ed.Vol. 2. Interscience Publishers, New York.

7. JENSEN, W. A. 1958. The nucleic acid and proteincontent of root tip cells of Vicia faba and A iliu1tcepa. Exp. Cell Research 14: 575-583.

8. JENSEN, W. A. 1958. Carbohydrate content of theroot tip cells of Alliun cepa. Plant Physiol. 33:64-65.

9. JENSEN, W. A. and L. G. KAVALJIAN 1958. Ananalysis of cell morphology and the periodicity ofdivision in the root tip of Alliumni cepa. Amer. Jour.Bot. 45: 365-372.

10. JERMYN, M. A. and F. A. ISHFRWOOD 1956.Changes in the cell wall of the pear during ripen-ing. Biochem. Jour. 64: 123-132.

11. ScoTT, F. M., K. HAMrNER, E. BAKER, and E. BOWLER1956. Electron microscope studies of cell wallgrowth in onion root. Amer. Jour. Bot. 43: 313-324.

12. WHALEY, WA. G., L. W. MERICLE, and C. HEILMSCH1952. The wall of the meristematic cell. Amer.Jour. Bot. 39: 20-26.

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dluring - plant physiology · pentoses and ascorbic acid do not react and there is negligible...

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