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Patterns of cellulose synthesis in maize root-tips
0
196i by _kademic Press Inc.
Experimental
Cell Research 46, 495-510 (1967)
PATTERNS
495
OF CELLULOSE
SYNTHESIS
IN MAIZE ROOT-TIPS A
CHEMICAL
R.
AND
M.
_XUTORADIOGRAPHIC
ROBERTS’
and V.
S.
STUDY
BUTT
Botany School, South Parks Road, Oxford, .England Received
September
16, 1966
THE plant cell wall consists mainly of carbohydrates derivatives (the hexuronic acids) and in mature tissues known to occur in the composition
and carbohydrate lignin. Changes are
of the wall during the development
of cells
120, 211. Such changes can be suitably studied in root-tips because the primary meristem is confined to a small region near the tip. Segments have been analysed at recessive distances from this point along the root axis to obtain values for the carbohydrate composition in cells of increasing developmental age [9, 131. The first part of this paper is similarly concerned withthe composition of the cell wall of the primary root of maize. However, analyses of this kind, even when very thin segments are used, of necessity overlook differences that might have occurred between tissues, and the biochemical origin of such divergence
cannot
techniques
on fixed sections
However, fractions
be traced.
there
It is only by use of essentially that distinctions
are few histochemical
and individual
sugar
components
histochemical
of this kind might be made. tests whereby
different
might be identified
cell
wall
on a tissue
section. Jensen [la] attempted to overcome these problems by staining the cell walls in transverse and longitudinal sections of Mium root-tips to a crimson-red
colour
by the periodic
acid-Schiff
stain
(PAS).
The
relative
intensity of colour gave an arbitrary measure of the amount of polysaccharide. He extracted adjacent sections from the same root for pectin and hemicellulose,
with ammonium
the residues
and
oxalate
a non-extracted
and strong alkali, section
respectively,
so that he was able
and stained to estimate
roughly the relative contributions of the fractions to ,the cell malls of diRerent regions. The information, however, was too imprecise to allow detailed measurements
of biochemical
1 Present address: Department 14214, U.S.A.
change
occurring
of Biology, State University
during
development,
but it
of P\;ewYork at Buffalo, New Uork
Experimental
Cell Research 46
496
R. &I. Roberts
and
1,‘. S. Butt
did indicate that considerable differences in c.ell wall composition were found between tissues quite close to the apex. A more precise location of changes occurring in root-tips has been used in studying patterns of protein synthesis. Labelled amino acids were supplied to growing seedlings and the radioactivity incorporated into protein, trac.ed by means of autoradiography [3]. The degree of blackening of the autoradiograph reflected arbitrarily the amount of isotope incorporated below, so that by counting silver grains above different regions of the section a nearquantitative picture of the variations in protein synthesis within the tip could be inferred. In this work, the same method was employed in order to follow patterns of cellulose synthesis after root-tips of maize seedlings had been incubated in radioac.tive glucose solution. Materials other than cellulose, which is insoluble in concentrated alkali, were removed from sections of the root with 24 per cent (w/v) KOH and autoradiographs prepared of the residues. Incorporated radioactivity revealed the location of cellulose synthesis in the root-tip.
METHODS Materials Radioactive
Plant
was obtained as a freeze dried powder of from the Radio-chemical Centre, Amersham, Bucks,
chemicals.-1*C-6-D-glucose
specific activity England.
4.1 &/Limole
material.-After
sodium hypochlorite
maize grain (Orla 266) had been surface treated in dilute (1 per cent available chlorine) for 10 min and then soaked in tap
water for 8 hr, they were allowed to germinate in moist sphagnum moss for 64 hr at 25” in darkness. Seedlings with primary roots 3-6 cm in length were selected, and the terminal 1 cm root-tip excised. All root-tips were used within 1 hr after excision. Smaller segments from the terminal root-tip were cut with a guillotine of razor blades, separated by 1 mm copper spacers. Incubations Incubations were performed in standard Warburg flasks containing 3 ml of fluid at 25”. The medium was maintained sterile by the inclusion of penicillin (10 /cg/ml). During 3 hr, excised tips increased in length approximately 10 per cent. When intact seedlings were employed, the young plants were supported upon muslin above beakers containing the aerated medium into which the terminal 2 cm of the roots were dipped. The grain was kept moist by wet cotton wool and the whole system enclosed to keep out light; incubations were performed at room temperature (19”). Histological methods Preparation of sections.-Roots drated and embedded in paraffin Ezperimentul
Cell Reseureh 46
were fixed in acetic acid-ethanol (I : 2 v/v), dehywax (m.p. 56”). Longitudinal sections 6 p and trans-
Patterns
of cellulose synthesis
497
verse sections 8 p thick were mounted on gelatin coated slides as recommended by Messrs Kodak Ltd. Approximately median longitudinal sections could be recognised on the paraffin ribbon. These were mounted on separate slides and examined under the microscope. The most median section and its immediate neighbours were selected for further treatment. They were dried at 40”, the paraffin removed with xylene, and transferred to water through a series of ethanol dilutions. E&action of sections.-One of the longitudinal sections from each root was no-t extracted to provide the pattern of total isotope incorporation into ethanol-xylenewater insoluble compounds. The remainder were extracted with 21 per cent (w/v) KOH, a manner modified from that described by Jensen [12]. This treatment with alkali completely removed the geIatin adhesive from the slides so that material requires extreme care in handling. The slides were laid flat and facing upwards while alkali (0.3-0.5 ml) was pipetted onto the section so that it was completely immersed. The sections were left for 24 hr under a dish to prevent evaporation, after which the remaining solution was removed using a filter paper wick, and finally allowed to dry out in an oven at 40”. During this, the section came to adhere strongly to the glass. The remaining crystals of potassium hydroxide were removed by means of 50 per cent (v/v) ethanol and the slides finally dried in the oven. As many as 75 per cent of the sections may be lost or severely displaced during the overall procedure. Those which survived were used to provide the pattern of I% incorporation into the cellulose fraction. Preparation of autoradiographs.-Autoradiographs were prepared by application of stripping film (Kodak ARlO). The preparations were stored in contact with the film at - 15’ for a period to obtain clear definition of the silver grain deposits. Estimation of silver grain deposition.-The number of silver grains were counted in an area 24 p x 24 ,Uof the field of view under an eyepiece fitted with a graticule square divided into a number of smaller squares of known dimension. Attempts were made to count along serial lines from the meristem in the epidermis, inner and outer cortex and the central pith cells of the stele. Here, the epidermis was the outermost tissue of the root and easily traced. The outer cortex was defined as that tissue 36-60 ,u from the inner surface of the epidermal wall and the inner cortex as that adjacent to the endodermis. The pith cells lie in the centre of the stele, but only exceptionally were sections sufficiently median that pith cells in all stages of development could be traced from their origin in the meristem to a position 5 mm behind it. Confusion can easily arise by mistaking some of the juvenile vascular elements for pith cells; these, of course, have a different synthetic activity. Non-specific production of silver grains was shown to be unlikely since non-radioactive sections processed in the usual way showed no increase over backgrouud count. The likely absorption or binding of traces to cell wall of cytoplasm was shown to be unlikely since roots killed with alcohol sh.owed no ability to incorporate tracer into insoluble materials after incubation for 6 b.r in isotope solution. By counting silver grains, figures for isotope incorporation into the cell wall of different tissues were obtained. The relative incorporation of IX per unit cell wall area can be calculated if the dimensions of cells are known and they are assumed to be cylinders. If the number of silver grains per unit volume of tissue equals n, then the number of silver grains per cell will be nr2h x n (where r is the radius and h is the length of the cells in that particular region of the rootj. The surface area of t.he cell equals Erperitnenfal
Cell Research 46
498
R. M. Roberts
and ?‘. S. Butt
2nrh + 2nr2 so that the silver grains per unit cell surface will then be n/2(t/r + I/h) or rnj2 when h >r. Staining of sections.-All sections were stained prior to application of the film by
the PAS (periodic acid-Schiff) method as described for plant tissue by Jensen [13], but modified here to reduce the intensity of staining by oxidising with 0.5 per cent (w/v) periodic acid for only 7 min. The Schiff reagent (Feulgen) was prepared according to Darlington and La Cour [4]. Cell walls and cytoplasm are stained a brilliant crimson red, whilst cytoplasm and nucleus remain clear. The stain had no effect on the autoradiograph. Fractionation
of roots
At the end of incubations, the solutions were decanted and the roots washed on a sintered disc with distilled water. They were then homogenised in a motor-driven glass (Potter) homogeniser with cold 80 per cent (v/r) ethanol. This was centrifuged at 750-1000 x g for 5 min at room temperature and the precipitate washed repeatedly with 80 per cent ethanol. The insoluble fraction, which was assumed to be essentially similar to the material present on sections prior to extraction, and the soluble fraction plus washings, which was assumed to be similar to that removed during fixation, embedding and subsequent deparaffinisation of roots and sections, were analysed separately. Methods of cell wall fractionation were determinedby those applied successfully in this and later work to the extraction of sections, and developed here to provide more detailed chemical analysis. (1) The pectic fraction was extracted with a 0.5 per cent (w/v) solution of ammonium oxalate at 90” for 6 hr. The soluble polysaccharide was precipitated by addition of 10 volumes of 95 per cent (v/v) ethanol. (2) Hemicellulose was recovered from the residue insoluble in ammonium oxalate by treatment with two successive portions of 24 per cent (w/v) potassium hydroxide at room temperature for 24 hr. When the alkali had been neutralised with glacial acetic acid and 10 volumes of ethanol added, the solution was left to stand for about 1 hr, at the end of which the precipitate was centrifuged at 1000 g for IO min to form a glutinuous residue. (3) The washed residue from alkali extraction was the u-cellulose fraction. Hydrolysis
of polysaccharide
fractions
Each fraction was hydrolysed by heating with 3.5 per cent (v/v) H&O, for 12 hr in sealed glass tubes in an oven at 100”. It was necessary to first dissolve the a-cellulose fraction in 72 per cent (v/v) H,SO, and then to dilute twenty fold. After 12 hr, solutions were neutralised by addition of BaCO, and centrifuged to remove insoluble salts. The supernatant was evaporated to dryness over P,Os in uacuo. Chromatography
The sugars released by hydrolysis were dissolved in water and portions applied to Whatman No. 1 papers. After chromatography, sugars were detected with aniline hydrogen phthalate spray, and developed by heating at 105” [22]. Two descending solvent systems were used. Experimenfal Cell Research 46
Patterns
of cellulose synthesis
499
Solvent il.-Benzene:n-butanol:pyridine: water (I : 5: 5:3j [lo]. At IS”, a clear separation of the neutral sugars present in maize root hyd.rolyses was achieved after 40 hr using the upper phase of this solvent system. Solvent B.-Ethyl acetate:pyridine: acetic acid:water (5: 5: I :3j [S]. At the end of 16 hr, neutral sugars were separated less satisfactorily than in Solvent A but amino acids separated well for a one dimensional system. These mere detected by spraying with 0.1 per cent ninhydrin in n-butanol and heating it in an oven at 105” for 5 min. For quantitative estimation of sugars and uranic acids on chromatograms, the areas corresponding with known marker spots running in parallel on the same paper were eluted with water, and the reducing sugar content of the eluate was estimated. Carbohydrate
estimations
Total carbohydrafe by sulphonated a-naphthol [5].-The purple colour developed when a solution of a-naphthol in sulphuric acid is heated with a sample of carbohydrate material on a boiling water bath for IO min was measured at 555 mp in a Unicam S.P. 500 spectrophotometer. The method gives a linear relationship between colour and carbohydrate content for a number of sugars up to 50 ,ug glucose or its molar equivalent for other sugars. Small amounts of ammonium oxalate or potassium hydroxide in extracts of pectin and hemicellulose respectively did not affect the determinations at the dilutions necessarily employed. The a-cellulose was made soluble in 72 per cent (v/v) H,SO, and aliquots of this solution taken for estimation. Reducing sugar using alkaline ferricyanide.-Reducing sugar in eluates was measured by a modification [S] of Folin’s original method [7]. The method is sensitive to 1 pg of reducing sugar and a linear relationship existed between optical density and molar sugar concentration for glucose, galactose, xylose, arabinose, and glucuronic and galacturonic acids over the range 5 to 20 pg. Detection of radioactive areas on chromatograms.-Chromatograms were placed in contact with Ilford “Ilfex” X-ray film for 2 to 4 weeks.
RESULTS Pectin hemicellulose
and cellulose content of roots
The fractions derived from the alcohol insoluble material were isolated in the soluble state and their carbohydrate content determined in terms of equivalents of glucose by taking small aliquots and estimating these by means of sulphonated a-naphthol. In three separate determinations on groups of 20 root-tips, the pectins comprised only about 13 per cent of the total carbohydrate, whilst the cc-cellulose constituted about 30 per cent, and the predominant hemicellulose about 50 per cent (Table I>. In the 1 cm root-tip, is high, this therefore, the pectin content is low, and, while the hemicellulose may be over-estimated if some of this fraction passes into ammonium oxalate. Experimental Cell Research 46
R. &I. Roberts and V. S. Buti
500
The a-cellulose may also be an overestimate if the hemicellulose has not been completely removed. The changes which take place over the first 10 mm of the root were determined in 1 mm segments cut from groups of 50 root-tips (Figs. 1 and 2). In terms of glucose equivalents, all fractions increase over the first 3 segments TABLE I. Composition
of the cell wall of maize root-tip. Hemicellulose 96
Pectin 96 Total carbohydrate Sugar residue: Uranic acid Galactose Glucose Arabinose Xylose Total
13.5kO.5
(3)
55.s+1.6
11
G
22
16 26 23
19
33 15 100
u-Cellulose Pb (3)
30.7k1.3 (3)
Mainly glucose Traces of others
29 100
to give a peak. At this stage cells are fully extended radially, but extended only a little longitudinally, so that the number of cells and the total amount of cell wall material in each segment is very high. As far as segment tive, there is a marked fall for each fraction as cells elongate, but subsequently, the cellulose content increases slightly. Only after segment 8 is there a rise in hemicellulose. Looked at from a percentage basis, the pectin content is highest in youngest segments, but falls markedly through the extending regions into the nonelongating zone. A similar pattern is evident for the first four segments for hemicellulose, but from that point there is a slight positive increase. By contrast, although there is little change over the first two segments, there is a marked increase in cellulose through the extending region which becomes constant only when cells no longer increase in size. Sugar components of the cell wall fractions Chromatography of the hydrolysed fractions revealed the presence of the c.omponent sugars. The uranic acid content of both pectin and hemicellulose is very low, and it was not possible to distinguish between galacturonic, glucuronic or possibly methyl substituted uranic. acids. Galactose, glucose, arabinose and xylose are all present in both pectin and hemicellulose, but the proportions in which they are founcl are quite different. Quantitatively, the Erperimental
Cell Research 46
Patterns of cellulose synthesis
501
hemicellulose fraction is characterised by higher levels of glucose and xylose, while the pectin fraction is richer in uranic acid, galactose and arabinose (Table I). The relatively large amounts of glucose found in hemicellulose may have originated in part from starch. However, sections cut from roots at this stage and stained by PAS revealed that starch was not present in large IOC I-
O-
5c
O-
5-
s
O- 1 2 3 4
1 I 5 ! z”;bzr9
I
I
10 /
segment
1 I
o-
I
Fig. 1. Fig. l.-Insoluble carbohydrate ceklose (0 ); cc-cellulose (A).
1
8
I
I
I
,
1 2 3 4 5 6 7 8 9 IO segment number
Fig. 2. content
of mm segments of maize root-tip.
Fig. a--Relative percentage contributions of pectin, hemicellulose of segments of maize root-tip. For key, see Fig. 1.
Pectin
and a-cellulose
( l ); hemi-
to the cell walls
amounts and was unlikely to cause significant errors in the determination of cell mall carbohydrates. In the cc-cellulose fraction, glucose predominated to the virtual exlusion of all other sugars suggesting that most of the noncellulosic polysaccharides had been removed by alkali treatment. Cell dimensions.-In order to interpret the data obtained from autoradiographs on isotope incorporation per unit tissue volume in terms of the inc,orporation per unit cell surface, it was nec.essary to know the dimensions of cells on tissue sections. The average changes in length and diameter of c.ells, from four of the major tissues are shown in Figs. 3 and 4. The measurements were made from a series transverse and longi.tudinal sections from a number of roots of similar diameters, and each point represents an average 33 - 671816
Experimental
Cell Research 46
502
R. hf. Roberts and V. S. Butt
for several values. No allomanc.es were made for shrinkages occurring during fixation and embedding procedures. Although different cells do not expand laterally at the same rate, they all achieve their full diameters by 2 mm. Growth in length does not become rapid until the later stages of radial expansion. Cells of different lineages extend at different rates and this is presumed to be
300 :3
,’
M =
_/--____---I’ ,/’ ,’1’
t2 200 5 5 =” 0 100
1
Fig. 3.-Length epidermis ( n ).
2
3 distance
4 from
of cells along the maize root-tip.
5 6 cap junction
Pith (A);
7 (mm)
8
inner cortex
9
10
(0);
outer cortex
(0 );
related to the frequenc.y of transverse cell divisions in the meristematic region. Growth is largely completed by 7mm, so that in tissues beyond this point, cells can be considered to be fully extended. The length of pith cells beyond 5 mm was approximated by assuming that the growth in these cells must keep pace with that in other tissues. This was made necessary because it was difficult to follow single c.ells for their whole length when they exceeded about 200 p. The incorporation of label from 14C-6-D-glucose into cellulose.-h initial unpublished experiments it was shown that in short incubation periods isotope from l”C-U-n-glucose (1 ,I&; 1 pmole/3 ml) only incorporated into cell wall material and not into protein as well. Moreover, in addition to 14C in hexose residues, substantial labelling of pentoses in polpsaccharides also occurred. However, when 1”C-6-D-gluCOse (1 ,xC; 0.25 pmole/3 ml water) was supplied to exc.ised root-tips for 3 hr, no tracer passed into arabinose and xylose units of the mall, to agree with the observation that this carbon is lost Experimental
Cell Research 46
Patterns of celldose synthesis
503
by decarboxylation in pentose synthesis. Some protein had become labelled because various amino acids contained W. There was faint radioactivity associated with galactose residues after hydrolysis of pectin, but, in hemicellulose, most of the tracer was in glucose with a fainter darkening of the autoradiograph above the area on the chromatogram corresponding with
01
Fig. k-Diameter
' 1 distance
2 from
3 4 cap junction
of cells along the maize rod-tip.
/ 5 (mm)
For key, see Fig. 3.
galactose. The 14C in c.ellulose was entirelv Y in glucose, and there was no evidence for contamination by any other radioactive materials in the insoluble residue, after alkali extraction. Sections of maize root extracted by strong alkali, therefore, showed an isotope distribution likely to indicate an incorporation of label into cellulose during the 3 hr incubation. This allowed patterns of cellulose synthesis to be ascertained by autoradiography. Investigation of sections from several root-tips revealed that although individual. roots varied greatly in the total amount of isotope incorporated, the same general patterns of silver grain production were observed along the rootaxis. From the data presented in Fig. 5 a for the numbers of silver grains associated with a standard area of the field of view at increasing distances from the cap junction in the pith, inner and outer cortical tissues, and epidermis of a single root-tip. The relative incorporation of 14C per unit cell wall area was c.alculated (Fig. 5 b), because this was considered to provide a more suitable basis for studying developmental changes occurring in the wall. However, because of the great variation in mall thic.kness observed in the epidermis, the ealculation could not be considered valid for this tissue, and has not been included. Experimental
Cell Research 46
R. M. Roberts and V. S. Butt
504
In the epidermis there is a rapid build up of a 15 to 20 ,u thick outer wall within 0.3 mm of the initial cells. This wall becomes markedly thinner in cells beyond 2 mm which are actively elongating. However, even in this tissue, in cells which are much longer than they are broad, the number of silver grains per unit tissue volume is roughly proportional to the incorporation per unit
1
2 distance
3 from
4 5 cap junction (mm)
6
7
Fig. 5.-Relative rates of incorporation of n-glucose into cellulose in the first cm of a root apex of maize determined from an autoradiograph of a median section extracted with alkali. a, per unit volume; b, per unit cell surface. For key, see Fig. 3.
cell surface, and isotope incorporation reached a broad maximum at about 3 mm and then slowly declined. It can also be seen that a substantial amount of isotope was found in cells beyond 6.5 mm, although these cells had ceased to elongate. In the early formation of the young outer wall, although silver grains were deposited throughout its depth, they were concentrated mostly towards its inner surface (i.e. adjoining the protoplast). Experimental
Cell Research 46
Patterns of cellulose synthesis
50.5
In the inner cortex and pith, the incorporation was very low in cell walls which were growing relatively slowly in the first mm, but subsequently there was a rapid increase to reach a maximum at about 3 mm in both tissues. The number of silver grains per unit cell wall area was then about ten times as high as at 0.5 mm. This was followed by a decline in incorposation even though c.ell extension continued at a constant rate, i.e. the rate of incorporation on a cell wall area basis was falling off steeply. Within cell walls of the outer cortex a similar broad maximum was found between 2.5 and -1.5 mm, followed by a steady decline. Again, a low density of silver grains was found above young cells in the first mm. Incorporation into root cap cells was low except in certain cells on the margins where a fairly high silver grain density was observed. However, by comparing the extracted section with the unextracted control, it was observed that most of the original isotope incorporated into these cells had been removed by treatment with strong alkali [14]. In cortical and epidermal tissues, at least, there was no indication, as revealed by the autoradiographs, of a sudden change in cellulose synthesis when cells ceased to elongate. This could partly have been disguised, however, by the changes in c.ell position which occurred during the incubation so that cells approaching the end of their elongation stage at excision would have become displaced slightly after a further 6 hr and stopped elongating altogether in an older part of the root. Neither was there evidence that in growing cells synthesis of cellulose was confined mainly to the ends of these cells (tip growth). Cellulose deposition, exc.ept in certain appreciably thickened cell corners of some cortical cells, occurred evenly along the n-all. However, the rate of incorporation was generally very low in young cells which were extending slowly, but reached a maximum during the early stages of elongation long before cell wall extension was complete. Experiments with intact seecllings.-There was the possibility that the pattern of incorporation of l% into cellulose in excised root-tips was a consequence of physiological changes occurring after excision and that the data obtained was not applicable to root-tips developing normally on intact seedlings. Consequently, experiments were devised similar to those described, but employing whole, three day old maize plants. No significant differeuces wese observed between these and excised roots in the changes in cellulose synthesis occurring along the root axis, so that all results on excised tips are assumecl to be applicable to the roots of intact seedlings. Excised tips were preferred in these and subsequent experiments, however, because it was possible to obtain greater control over their metabolism after excision and to make determinations of their metabolic activity in a closed system. Experimental
Cell Research 46
R. 111.Roberts and I’. S. Butt
506
DISCUSSION
In this paper, two methods of investigating developmental changes in the carbohydrate composition of the cell malls of maize root-tips cells have been used. The analysis of segments of roots to yield values for a series of cell preparations of increasing developmental age showed that the pec,tins, which constitute only a small percentage of the cell wall, characteristically contribute most extensively in younger regions. This agreed with the results of Fuller [9] with Tricia, who used thicker slices than in the work reported here. Jensen and Ashton [13] with Mium employed very thin slices, analysing the roots at successive distances of 100 p. They also showed that pectins were highest in meristematic regions and that their percentage contribution fell with increasing age of the cells. However, even in immature regions of maize root-tips, the hemicellulose constitutes the bulk of the cell wall material and there is no evidence that very young walls are comprised mainly of pectin. The fall observed in the content of polysaccharides, particularly of the pectin and hemicellulose, between mm segments 3 and 6 seems to be too great to be explained merely by the increasing separation of the end walls of the cells during growth. In the third mm from the tip of the root, cells were in the early stages of elongation; on average, epidermal and cortic.al cells were over 20 p in length and some of the older pith cells in the segment were as long as 70 ,u. If all cells are assumed to increase tenfold in length from 20 to 200 ,u between 3 and 7 mm and remain relatively unchanged in diameter, then the expected fall in the polysaccharide content of the segments between these regions is about 30 per cent. The actual fall is considerably greater than this. B similar “thinning” of c.ell walls of hvena coleoptile during growth promoted by ausin, has been noticed [19]. This lack of stoichiometry between cell wall synthesis and gTowth would suggest that the deposition of non-cellulosic polysaccharides in particular, is not related directly to the extension of the wall. The low values for uranic acids in the isolated pectins are similar to those obtained on oat coleoptile [2]. Such low levels of uranic acid in pectin make unlikely the possibility that polyuronides are directly involved in the control of cell wall extensibility [17]. However, it is possible that the uranic acids have been extensively broken down during isolation, because extraction with ammonium oxalate may lead to pectin degrading by transelimination [l] and some breakdown of uranic acid almost certainly occurs during hydrolysis. Nevertheless, we have shown (in unpublished experiments) that when Experimental
Cell Research 46
Patterns
of cellulose synthesis
507
labelled glucuronic a&l is supplied .to maize root-tips, uronos$ units in pectin become labelled at only about one-fifth the rate of pentosyl units. Similar results have been obtained using 1”C-2-myo-inositol as a precursor of pectin and hemicellulose in maize roots (J. Deshusses, R. M. Roberts and I?. Loewus, unpublished results). This does indicate that the uranic acid content of these roots is indeed low. The residue remaining after the extraction of the cell wall with alkali is cellulose. The proportion of cellulose in the cell wall was markedly lower in the two youngest segments of the root-tip than in older segments. The percentage increased with the onset of rapid extension and suggested that cellulose synthesis occurred at a low rate in young meristematic tissues and is laid down only extensively when cell walls begin to elongate more rapidly. Ray also observed increases in cellulose proportionately greater than other components of the wall, in the extending regions of oat coleoptile segments during auxin-induced growth, and suggested that there map be some association between cellulose synthesis and the growth process [16]. Clearly, however, the analysis of root segments is imprecise because it relies on gross analysis of all the cells in a partic.ular region of the root. Trends of development are risualised which are characteristic of all cells even though different tissues may varp considerably in their cell wall composition. In addition unless the segments are very thin, short term changes in c.ell wall composition must of necessity be overlooked. Thus, the first mm of the roottip, as well as containing the bulk of the meristem, contains the whole of the root-cap, within which a complete developmental sequence of cells is found, progressing from the thin walled initials to the thickened cells on the cap margins. From the autoradiographs on the other hand, it was possible to follow precisely the changes in c.ellulose synthesis occurring in different tissues. The autoradiographs supported the conclusion that cellulose synthesis was 10~ in the cell walls of meristematic cells of all tissues, including the initials of the root-cap. Maximum incorporation in tortes, pith and epidermis was achieved between 3 and 4 mm from zero, after which a marked decline set in, even though cell extension was proceeding linearly. Cellulose was deposited evenly along the wall, except in certain thickened areas, particularly those adjoining some of the cortical air spaces. There was no evidence for tip or other localised features of growth in any tissues. This observation agrees with the evidence of other workers [ll, lS] who have shown that during extension growth of parenchyma and epidermal cells of oat c.oleoptiles, cellulose synthesis is not localised in any way. It is unlikely, however, that the deposition of new microExperimental
Cell Research 46
R. AI. Roberts and V. S. Butt
50s
fibrils is a direct factor controlling elongation because cellulose synthesis was not observed to keep pace with the rate of cell extension beyond 4 mm from the tip. Although this type of autoradiographic investigation allows the changes in incorporation of isotope occurring in different tissues to be followed with some precision, a number of objections have to be borne in mind when assessing the results. It may be argued that the distributions observed in different tissues primarily reflect differences in the availability of the labelled compound within the organ. For instance, the high incorporation of 14C into the epidermis from the radioactive glucoses might be due to the ready access to outer cells. Nevertheless, this is a thick-walled tissue which would demand a high rate of autoradiographs of root-tips have polysaccharide synthesis. Furthermore been prepared from actively photosynthesising maize plants grown in an atmosphere of 14C0, [15]. It was shown that the epidermis incorporated high levels of 14C, even though in this case, isotope had been supplied endogenously from the shoot. These observations suggest that the heavy inc.orporation in the epidermis stems from exceptionally active synthesis, and does not arise from the position of the tissue. Similarly, the high incorporation of isotope into cell walls adjoining the cortical air spaces of the inner cortex might indicate no more than the penetration of labelled compound at these points. Again however, these regions were noticeably thick-walled, and since it is unlikely that compounds can be incorporated into the wall without first entering the cell, even if cell wall deposition may be organised at the surface of the cytoplasm, it is likely that the unequal deposition of isotope arises from synthetic differences. However, because the incubation times employed in these experiments were relatively short and the possibility that the penetration of glucose into the deeper lying tissues might be an important factor in determining the pattern of silver grains on the autoradiographs, quantitative comparisons on the amount of incorporation occurring between tissues were avoided because these might not necessarily represent precise differences in the respective rates of their polysaccharide synthesis. It was assumed, however, that comparisons between cells of different age within the same tissue, whether cortex, pith or epidermis, were valid. It is significant that no visible change in the pattern of isotope incorporation was observed near the cut ends of the root where isotopically labelled glucose was readily available throughout the experiment. There are, therefore, several objections which might be seriously levelled against this type of investigation. It does provide, however, a useful procedure for localising the deposition of cellulose within a tissue where no satisfactory, Experimental
Cell Research 46
Patterns of cellulose synthesis quantitative method previously existed, and could investigations on other developing cell systems.
509 be usefully
employed
in
SUMMARY
By analysis of mm segments of maize root-tip, it has been shown that pectic substances, which are relatively poor in uranic acid but rich in arabinose and galactose, provide only 13 per cent of the cell wall polysaccharides. Their contribution is highest in young cells. The hemicelluloses constituted over 50 per cent of all polysaccharides throughout the root. cc-Cellulose increases during elongation from about 20 per cent of all polysaccharide in the younger region to about 35 per cent in cell walls of growing ~11s. Patterns of cellulose synthesis in excised and intact roots were also investigated by autoradiography. During short incubation periods with 14C-6-oglucose, most of the label incorporated into the ethanol-insoluble fsaction was recovered, after hydrolysis, in hesose. When this residue was extracted with alkali, the insoluble cc-cellulose contained 4C in glucose units only. This incorporation was presumed to be related to the amount of cellulose synthesised during the incubation. Autoradiographs of sections extracted with alkali revealed, therefore, the incorporation pattern into cellulose within the roe-t. Cellulose was synthesised only slowly in cell walls of the cap and meristematic region. As cells began to extend more rapidly, there was a sharp rise in incorporation, increasing to a maximum in the pith, inner and outer cortex and epidermis between the third and fourth mm behind the cap-junction. Cells at this stage had not completed their growth, and no direct relationship could be inferred to exist between deposition of cellulose and extension of the wall. REFERENCES 1. BARRETT, A. J. and NORTHCOTE, D. H., Biochem. J. 94, 617 (1965). 2. BISHOP, C. T., BAYLEY, S. T. and SETTERFIELD, G., PIanf Physiol. 33, 283 (1958). 3. CLOWES, F. A. L., J. Expfl Bof. 11, 81 (1960). 4. DARLINGTON, C. D. and LACOUR, L. F., The Handling of Chromosomes. 3rd ed. Allen and Unwin Ltd, London, 1960. R. L. and \VOLFROM, M. L. (eds.), Methods in Carbohydrate 5. DISCHE, Z., in WHISTLER, Chemistry, Vol. 1, p. 478. Academic Press, New York, 1962. 6. FISCHER, F. G. and D~RFEL, H., Hoppe-Seyler’s 2. Physiol. Chem. 302, 186 (1955). 7. FOLIN, O., J. Biol. Chem. 77, 421 (1928). S. FULLER, Ii. W., in Automation in der analytischen Chemie, p. 319. Technicon GmbH, Frankfurt, 1965. 9. __ Biochem. J. 701 18 p. (1959). 10. GAILLARD, D. E. G., Nature 171, 1160 (1953). 11. GORHAM, P. R. and COLVIN, J. R., Expfl Cell Res. 13, 187 (1957). 12. JENSEN, W. A., Am. J. Botany 47, 287 (1960). Experimental
Cell Research 46
R. M. Roberts and V. S. Butt
510
13. JENSEN, W. A. and ASHTON, M., Plant Physiol. 35, 313 (1960). 14. JUNIPER, B. E., and ROBERTS,R. III., J. Roy Microscop. Sot. 85, 63 (1966). 332 (1953). 15. R~BIDE&, G. s. and MERICLE, L. W., Plun~PhysioZ.2k, 16. Ray, P. M., Biochem. J. 89, 141 (1963). 17. SET&F&D, G. and B.%YL&Y,S.‘T., &I. Reu. Plant Physiol. 12, 35 (1961). 1s. __
Can. J. Botany 35, 135 (1957).
19. __ Ann. Botany (London) 21, 633 (1957). 20. THORNBER, P. J. and NORTHCOTE,D. H., Biochem. J. 81, 455 (1961). 21. __ ibid. 81, 449 (1961). 22. WILSON, C. iVI., Snul. Chem. 31, 1199 (1959).
Experimental
Cell Research 46
196i by _kademic Press Inc.
Experimental
Cell Research 46, 495-510 (1967)
PATTERNS
495
OF CELLULOSE
SYNTHESIS
IN MAIZE ROOT-TIPS A
CHEMICAL
R.
AND
M.
_XUTORADIOGRAPHIC
ROBERTS’
and V.
S.
STUDY
BUTT
Botany School, South Parks Road, Oxford, .England Received
September
16, 1966
THE plant cell wall consists mainly of carbohydrates derivatives (the hexuronic acids) and in mature tissues known to occur in the composition
and carbohydrate lignin. Changes are
of the wall during the development
of cells
120, 211. Such changes can be suitably studied in root-tips because the primary meristem is confined to a small region near the tip. Segments have been analysed at recessive distances from this point along the root axis to obtain values for the carbohydrate composition in cells of increasing developmental age [9, 131. The first part of this paper is similarly concerned withthe composition of the cell wall of the primary root of maize. However, analyses of this kind, even when very thin segments are used, of necessity overlook differences that might have occurred between tissues, and the biochemical origin of such divergence
cannot
techniques
on fixed sections
However, fractions
be traced.
there
It is only by use of essentially that distinctions
are few histochemical
and individual
sugar
components
histochemical
of this kind might be made. tests whereby
different
might be identified
cell
wall
on a tissue
section. Jensen [la] attempted to overcome these problems by staining the cell walls in transverse and longitudinal sections of Mium root-tips to a crimson-red
colour
by the periodic
acid-Schiff
stain
(PAS).
The
relative
intensity of colour gave an arbitrary measure of the amount of polysaccharide. He extracted adjacent sections from the same root for pectin and hemicellulose,
with ammonium
the residues
and
oxalate
a non-extracted
and strong alkali, section
respectively,
so that he was able
and stained to estimate
roughly the relative contributions of the fractions to ,the cell malls of diRerent regions. The information, however, was too imprecise to allow detailed measurements
of biochemical
1 Present address: Department 14214, U.S.A.
change
occurring
of Biology, State University
during
development,
but it
of P\;ewYork at Buffalo, New Uork
Experimental
Cell Research 46
496
R. &I. Roberts
and
1,‘. S. Butt
did indicate that considerable differences in c.ell wall composition were found between tissues quite close to the apex. A more precise location of changes occurring in root-tips has been used in studying patterns of protein synthesis. Labelled amino acids were supplied to growing seedlings and the radioactivity incorporated into protein, trac.ed by means of autoradiography [3]. The degree of blackening of the autoradiograph reflected arbitrarily the amount of isotope incorporated below, so that by counting silver grains above different regions of the section a nearquantitative picture of the variations in protein synthesis within the tip could be inferred. In this work, the same method was employed in order to follow patterns of cellulose synthesis after root-tips of maize seedlings had been incubated in radioac.tive glucose solution. Materials other than cellulose, which is insoluble in concentrated alkali, were removed from sections of the root with 24 per cent (w/v) KOH and autoradiographs prepared of the residues. Incorporated radioactivity revealed the location of cellulose synthesis in the root-tip.
METHODS Materials Radioactive
Plant
was obtained as a freeze dried powder of from the Radio-chemical Centre, Amersham, Bucks,
chemicals.-1*C-6-D-glucose
specific activity England.
4.1 &/Limole
material.-After
sodium hypochlorite
maize grain (Orla 266) had been surface treated in dilute (1 per cent available chlorine) for 10 min and then soaked in tap
water for 8 hr, they were allowed to germinate in moist sphagnum moss for 64 hr at 25” in darkness. Seedlings with primary roots 3-6 cm in length were selected, and the terminal 1 cm root-tip excised. All root-tips were used within 1 hr after excision. Smaller segments from the terminal root-tip were cut with a guillotine of razor blades, separated by 1 mm copper spacers. Incubations Incubations were performed in standard Warburg flasks containing 3 ml of fluid at 25”. The medium was maintained sterile by the inclusion of penicillin (10 /cg/ml). During 3 hr, excised tips increased in length approximately 10 per cent. When intact seedlings were employed, the young plants were supported upon muslin above beakers containing the aerated medium into which the terminal 2 cm of the roots were dipped. The grain was kept moist by wet cotton wool and the whole system enclosed to keep out light; incubations were performed at room temperature (19”). Histological methods Preparation of sections.-Roots drated and embedded in paraffin Ezperimentul
Cell Reseureh 46
were fixed in acetic acid-ethanol (I : 2 v/v), dehywax (m.p. 56”). Longitudinal sections 6 p and trans-
Patterns
of cellulose synthesis
497
verse sections 8 p thick were mounted on gelatin coated slides as recommended by Messrs Kodak Ltd. Approximately median longitudinal sections could be recognised on the paraffin ribbon. These were mounted on separate slides and examined under the microscope. The most median section and its immediate neighbours were selected for further treatment. They were dried at 40”, the paraffin removed with xylene, and transferred to water through a series of ethanol dilutions. E&action of sections.-One of the longitudinal sections from each root was no-t extracted to provide the pattern of total isotope incorporation into ethanol-xylenewater insoluble compounds. The remainder were extracted with 21 per cent (w/v) KOH, a manner modified from that described by Jensen [12]. This treatment with alkali completely removed the geIatin adhesive from the slides so that material requires extreme care in handling. The slides were laid flat and facing upwards while alkali (0.3-0.5 ml) was pipetted onto the section so that it was completely immersed. The sections were left for 24 hr under a dish to prevent evaporation, after which the remaining solution was removed using a filter paper wick, and finally allowed to dry out in an oven at 40”. During this, the section came to adhere strongly to the glass. The remaining crystals of potassium hydroxide were removed by means of 50 per cent (v/v) ethanol and the slides finally dried in the oven. As many as 75 per cent of the sections may be lost or severely displaced during the overall procedure. Those which survived were used to provide the pattern of I% incorporation into the cellulose fraction. Preparation of autoradiographs.-Autoradiographs were prepared by application of stripping film (Kodak ARlO). The preparations were stored in contact with the film at - 15’ for a period to obtain clear definition of the silver grain deposits. Estimation of silver grain deposition.-The number of silver grains were counted in an area 24 p x 24 ,Uof the field of view under an eyepiece fitted with a graticule square divided into a number of smaller squares of known dimension. Attempts were made to count along serial lines from the meristem in the epidermis, inner and outer cortex and the central pith cells of the stele. Here, the epidermis was the outermost tissue of the root and easily traced. The outer cortex was defined as that tissue 36-60 ,u from the inner surface of the epidermal wall and the inner cortex as that adjacent to the endodermis. The pith cells lie in the centre of the stele, but only exceptionally were sections sufficiently median that pith cells in all stages of development could be traced from their origin in the meristem to a position 5 mm behind it. Confusion can easily arise by mistaking some of the juvenile vascular elements for pith cells; these, of course, have a different synthetic activity. Non-specific production of silver grains was shown to be unlikely since non-radioactive sections processed in the usual way showed no increase over backgrouud count. The likely absorption or binding of traces to cell wall of cytoplasm was shown to be unlikely since roots killed with alcohol sh.owed no ability to incorporate tracer into insoluble materials after incubation for 6 b.r in isotope solution. By counting silver grains, figures for isotope incorporation into the cell wall of different tissues were obtained. The relative incorporation of IX per unit cell wall area can be calculated if the dimensions of cells are known and they are assumed to be cylinders. If the number of silver grains per unit volume of tissue equals n, then the number of silver grains per cell will be nr2h x n (where r is the radius and h is the length of the cells in that particular region of the rootj. The surface area of t.he cell equals Erperitnenfal
Cell Research 46
498
R. M. Roberts
and ?‘. S. Butt
2nrh + 2nr2 so that the silver grains per unit cell surface will then be n/2(t/r + I/h) or rnj2 when h >r. Staining of sections.-All sections were stained prior to application of the film by
the PAS (periodic acid-Schiff) method as described for plant tissue by Jensen [13], but modified here to reduce the intensity of staining by oxidising with 0.5 per cent (w/v) periodic acid for only 7 min. The Schiff reagent (Feulgen) was prepared according to Darlington and La Cour [4]. Cell walls and cytoplasm are stained a brilliant crimson red, whilst cytoplasm and nucleus remain clear. The stain had no effect on the autoradiograph. Fractionation
of roots
At the end of incubations, the solutions were decanted and the roots washed on a sintered disc with distilled water. They were then homogenised in a motor-driven glass (Potter) homogeniser with cold 80 per cent (v/r) ethanol. This was centrifuged at 750-1000 x g for 5 min at room temperature and the precipitate washed repeatedly with 80 per cent ethanol. The insoluble fraction, which was assumed to be essentially similar to the material present on sections prior to extraction, and the soluble fraction plus washings, which was assumed to be similar to that removed during fixation, embedding and subsequent deparaffinisation of roots and sections, were analysed separately. Methods of cell wall fractionation were determinedby those applied successfully in this and later work to the extraction of sections, and developed here to provide more detailed chemical analysis. (1) The pectic fraction was extracted with a 0.5 per cent (w/v) solution of ammonium oxalate at 90” for 6 hr. The soluble polysaccharide was precipitated by addition of 10 volumes of 95 per cent (v/v) ethanol. (2) Hemicellulose was recovered from the residue insoluble in ammonium oxalate by treatment with two successive portions of 24 per cent (w/v) potassium hydroxide at room temperature for 24 hr. When the alkali had been neutralised with glacial acetic acid and 10 volumes of ethanol added, the solution was left to stand for about 1 hr, at the end of which the precipitate was centrifuged at 1000 g for IO min to form a glutinuous residue. (3) The washed residue from alkali extraction was the u-cellulose fraction. Hydrolysis
of polysaccharide
fractions
Each fraction was hydrolysed by heating with 3.5 per cent (v/v) H&O, for 12 hr in sealed glass tubes in an oven at 100”. It was necessary to first dissolve the a-cellulose fraction in 72 per cent (v/v) H,SO, and then to dilute twenty fold. After 12 hr, solutions were neutralised by addition of BaCO, and centrifuged to remove insoluble salts. The supernatant was evaporated to dryness over P,Os in uacuo. Chromatography
The sugars released by hydrolysis were dissolved in water and portions applied to Whatman No. 1 papers. After chromatography, sugars were detected with aniline hydrogen phthalate spray, and developed by heating at 105” [22]. Two descending solvent systems were used. Experimenfal Cell Research 46
Patterns
of cellulose synthesis
499
Solvent il.-Benzene:n-butanol:pyridine: water (I : 5: 5:3j [lo]. At IS”, a clear separation of the neutral sugars present in maize root hyd.rolyses was achieved after 40 hr using the upper phase of this solvent system. Solvent B.-Ethyl acetate:pyridine: acetic acid:water (5: 5: I :3j [S]. At the end of 16 hr, neutral sugars were separated less satisfactorily than in Solvent A but amino acids separated well for a one dimensional system. These mere detected by spraying with 0.1 per cent ninhydrin in n-butanol and heating it in an oven at 105” for 5 min. For quantitative estimation of sugars and uranic acids on chromatograms, the areas corresponding with known marker spots running in parallel on the same paper were eluted with water, and the reducing sugar content of the eluate was estimated. Carbohydrate
estimations
Total carbohydrafe by sulphonated a-naphthol [5].-The purple colour developed when a solution of a-naphthol in sulphuric acid is heated with a sample of carbohydrate material on a boiling water bath for IO min was measured at 555 mp in a Unicam S.P. 500 spectrophotometer. The method gives a linear relationship between colour and carbohydrate content for a number of sugars up to 50 ,ug glucose or its molar equivalent for other sugars. Small amounts of ammonium oxalate or potassium hydroxide in extracts of pectin and hemicellulose respectively did not affect the determinations at the dilutions necessarily employed. The a-cellulose was made soluble in 72 per cent (v/v) H,SO, and aliquots of this solution taken for estimation. Reducing sugar using alkaline ferricyanide.-Reducing sugar in eluates was measured by a modification [S] of Folin’s original method [7]. The method is sensitive to 1 pg of reducing sugar and a linear relationship existed between optical density and molar sugar concentration for glucose, galactose, xylose, arabinose, and glucuronic and galacturonic acids over the range 5 to 20 pg. Detection of radioactive areas on chromatograms.-Chromatograms were placed in contact with Ilford “Ilfex” X-ray film for 2 to 4 weeks.
RESULTS Pectin hemicellulose
and cellulose content of roots
The fractions derived from the alcohol insoluble material were isolated in the soluble state and their carbohydrate content determined in terms of equivalents of glucose by taking small aliquots and estimating these by means of sulphonated a-naphthol. In three separate determinations on groups of 20 root-tips, the pectins comprised only about 13 per cent of the total carbohydrate, whilst the cc-cellulose constituted about 30 per cent, and the predominant hemicellulose about 50 per cent (Table I>. In the 1 cm root-tip, is high, this therefore, the pectin content is low, and, while the hemicellulose may be over-estimated if some of this fraction passes into ammonium oxalate. Experimental Cell Research 46
R. &I. Roberts and V. S. Buti
500
The a-cellulose may also be an overestimate if the hemicellulose has not been completely removed. The changes which take place over the first 10 mm of the root were determined in 1 mm segments cut from groups of 50 root-tips (Figs. 1 and 2). In terms of glucose equivalents, all fractions increase over the first 3 segments TABLE I. Composition
of the cell wall of maize root-tip. Hemicellulose 96
Pectin 96 Total carbohydrate Sugar residue: Uranic acid Galactose Glucose Arabinose Xylose Total
13.5kO.5
(3)
55.s+1.6
11
G
22
16 26 23
19
33 15 100
u-Cellulose Pb (3)
30.7k1.3 (3)
Mainly glucose Traces of others
29 100
to give a peak. At this stage cells are fully extended radially, but extended only a little longitudinally, so that the number of cells and the total amount of cell wall material in each segment is very high. As far as segment tive, there is a marked fall for each fraction as cells elongate, but subsequently, the cellulose content increases slightly. Only after segment 8 is there a rise in hemicellulose. Looked at from a percentage basis, the pectin content is highest in youngest segments, but falls markedly through the extending regions into the nonelongating zone. A similar pattern is evident for the first four segments for hemicellulose, but from that point there is a slight positive increase. By contrast, although there is little change over the first two segments, there is a marked increase in cellulose through the extending region which becomes constant only when cells no longer increase in size. Sugar components of the cell wall fractions Chromatography of the hydrolysed fractions revealed the presence of the c.omponent sugars. The uranic acid content of both pectin and hemicellulose is very low, and it was not possible to distinguish between galacturonic, glucuronic or possibly methyl substituted uranic. acids. Galactose, glucose, arabinose and xylose are all present in both pectin and hemicellulose, but the proportions in which they are founcl are quite different. Quantitatively, the Erperimental
Cell Research 46
Patterns of cellulose synthesis
501
hemicellulose fraction is characterised by higher levels of glucose and xylose, while the pectin fraction is richer in uranic acid, galactose and arabinose (Table I). The relatively large amounts of glucose found in hemicellulose may have originated in part from starch. However, sections cut from roots at this stage and stained by PAS revealed that starch was not present in large IOC I-
O-
5c
O-
5-
s
O- 1 2 3 4
1 I 5 ! z”;bzr9
I
I
10 /
segment
1 I
o-
I
Fig. 1. Fig. l.-Insoluble carbohydrate ceklose (0 ); cc-cellulose (A).
1
8
I
I
I
,
1 2 3 4 5 6 7 8 9 IO segment number
Fig. 2. content
of mm segments of maize root-tip.
Fig. a--Relative percentage contributions of pectin, hemicellulose of segments of maize root-tip. For key, see Fig. 1.
Pectin
and a-cellulose
( l ); hemi-
to the cell walls
amounts and was unlikely to cause significant errors in the determination of cell mall carbohydrates. In the cc-cellulose fraction, glucose predominated to the virtual exlusion of all other sugars suggesting that most of the noncellulosic polysaccharides had been removed by alkali treatment. Cell dimensions.-In order to interpret the data obtained from autoradiographs on isotope incorporation per unit tissue volume in terms of the inc,orporation per unit cell surface, it was nec.essary to know the dimensions of cells on tissue sections. The average changes in length and diameter of c.ells, from four of the major tissues are shown in Figs. 3 and 4. The measurements were made from a series transverse and longi.tudinal sections from a number of roots of similar diameters, and each point represents an average 33 - 671816
Experimental
Cell Research 46
502
R. hf. Roberts and V. S. Butt
for several values. No allomanc.es were made for shrinkages occurring during fixation and embedding procedures. Although different cells do not expand laterally at the same rate, they all achieve their full diameters by 2 mm. Growth in length does not become rapid until the later stages of radial expansion. Cells of different lineages extend at different rates and this is presumed to be
300 :3
,’
M =
_/--____---I’ ,/’ ,’1’
t2 200 5 5 =” 0 100
1
Fig. 3.-Length epidermis ( n ).
2
3 distance
4 from
of cells along the maize root-tip.
5 6 cap junction
Pith (A);
7 (mm)
8
inner cortex
9
10
(0);
outer cortex
(0 );
related to the frequenc.y of transverse cell divisions in the meristematic region. Growth is largely completed by 7mm, so that in tissues beyond this point, cells can be considered to be fully extended. The length of pith cells beyond 5 mm was approximated by assuming that the growth in these cells must keep pace with that in other tissues. This was made necessary because it was difficult to follow single c.ells for their whole length when they exceeded about 200 p. The incorporation of label from 14C-6-D-glucose into cellulose.-h initial unpublished experiments it was shown that in short incubation periods isotope from l”C-U-n-glucose (1 ,I&; 1 pmole/3 ml) only incorporated into cell wall material and not into protein as well. Moreover, in addition to 14C in hexose residues, substantial labelling of pentoses in polpsaccharides also occurred. However, when 1”C-6-D-gluCOse (1 ,xC; 0.25 pmole/3 ml water) was supplied to exc.ised root-tips for 3 hr, no tracer passed into arabinose and xylose units of the mall, to agree with the observation that this carbon is lost Experimental
Cell Research 46
Patterns of celldose synthesis
503
by decarboxylation in pentose synthesis. Some protein had become labelled because various amino acids contained W. There was faint radioactivity associated with galactose residues after hydrolysis of pectin, but, in hemicellulose, most of the tracer was in glucose with a fainter darkening of the autoradiograph above the area on the chromatogram corresponding with
01
Fig. k-Diameter
' 1 distance
2 from
3 4 cap junction
of cells along the maize rod-tip.
/ 5 (mm)
For key, see Fig. 3.
galactose. The 14C in c.ellulose was entirelv Y in glucose, and there was no evidence for contamination by any other radioactive materials in the insoluble residue, after alkali extraction. Sections of maize root extracted by strong alkali, therefore, showed an isotope distribution likely to indicate an incorporation of label into cellulose during the 3 hr incubation. This allowed patterns of cellulose synthesis to be ascertained by autoradiography. Investigation of sections from several root-tips revealed that although individual. roots varied greatly in the total amount of isotope incorporated, the same general patterns of silver grain production were observed along the rootaxis. From the data presented in Fig. 5 a for the numbers of silver grains associated with a standard area of the field of view at increasing distances from the cap junction in the pith, inner and outer cortical tissues, and epidermis of a single root-tip. The relative incorporation of 14C per unit cell wall area was c.alculated (Fig. 5 b), because this was considered to provide a more suitable basis for studying developmental changes occurring in the wall. However, because of the great variation in mall thic.kness observed in the epidermis, the ealculation could not be considered valid for this tissue, and has not been included. Experimental
Cell Research 46
R. M. Roberts and V. S. Butt
504
In the epidermis there is a rapid build up of a 15 to 20 ,u thick outer wall within 0.3 mm of the initial cells. This wall becomes markedly thinner in cells beyond 2 mm which are actively elongating. However, even in this tissue, in cells which are much longer than they are broad, the number of silver grains per unit tissue volume is roughly proportional to the incorporation per unit
1
2 distance
3 from
4 5 cap junction (mm)
6
7
Fig. 5.-Relative rates of incorporation of n-glucose into cellulose in the first cm of a root apex of maize determined from an autoradiograph of a median section extracted with alkali. a, per unit volume; b, per unit cell surface. For key, see Fig. 3.
cell surface, and isotope incorporation reached a broad maximum at about 3 mm and then slowly declined. It can also be seen that a substantial amount of isotope was found in cells beyond 6.5 mm, although these cells had ceased to elongate. In the early formation of the young outer wall, although silver grains were deposited throughout its depth, they were concentrated mostly towards its inner surface (i.e. adjoining the protoplast). Experimental
Cell Research 46
Patterns of cellulose synthesis
50.5
In the inner cortex and pith, the incorporation was very low in cell walls which were growing relatively slowly in the first mm, but subsequently there was a rapid increase to reach a maximum at about 3 mm in both tissues. The number of silver grains per unit cell wall area was then about ten times as high as at 0.5 mm. This was followed by a decline in incorposation even though c.ell extension continued at a constant rate, i.e. the rate of incorporation on a cell wall area basis was falling off steeply. Within cell walls of the outer cortex a similar broad maximum was found between 2.5 and -1.5 mm, followed by a steady decline. Again, a low density of silver grains was found above young cells in the first mm. Incorporation into root cap cells was low except in certain cells on the margins where a fairly high silver grain density was observed. However, by comparing the extracted section with the unextracted control, it was observed that most of the original isotope incorporated into these cells had been removed by treatment with strong alkali [14]. In cortical and epidermal tissues, at least, there was no indication, as revealed by the autoradiographs, of a sudden change in cellulose synthesis when cells ceased to elongate. This could partly have been disguised, however, by the changes in c.ell position which occurred during the incubation so that cells approaching the end of their elongation stage at excision would have become displaced slightly after a further 6 hr and stopped elongating altogether in an older part of the root. Neither was there evidence that in growing cells synthesis of cellulose was confined mainly to the ends of these cells (tip growth). Cellulose deposition, exc.ept in certain appreciably thickened cell corners of some cortical cells, occurred evenly along the n-all. However, the rate of incorporation was generally very low in young cells which were extending slowly, but reached a maximum during the early stages of elongation long before cell wall extension was complete. Experiments with intact seecllings.-There was the possibility that the pattern of incorporation of l% into cellulose in excised root-tips was a consequence of physiological changes occurring after excision and that the data obtained was not applicable to root-tips developing normally on intact seedlings. Consequently, experiments were devised similar to those described, but employing whole, three day old maize plants. No significant differeuces wese observed between these and excised roots in the changes in cellulose synthesis occurring along the root axis, so that all results on excised tips are assumecl to be applicable to the roots of intact seedlings. Excised tips were preferred in these and subsequent experiments, however, because it was possible to obtain greater control over their metabolism after excision and to make determinations of their metabolic activity in a closed system. Experimental
Cell Research 46
R. 111.Roberts and I’. S. Butt
506
DISCUSSION
In this paper, two methods of investigating developmental changes in the carbohydrate composition of the cell malls of maize root-tips cells have been used. The analysis of segments of roots to yield values for a series of cell preparations of increasing developmental age showed that the pec,tins, which constitute only a small percentage of the cell wall, characteristically contribute most extensively in younger regions. This agreed with the results of Fuller [9] with Tricia, who used thicker slices than in the work reported here. Jensen and Ashton [13] with Mium employed very thin slices, analysing the roots at successive distances of 100 p. They also showed that pectins were highest in meristematic regions and that their percentage contribution fell with increasing age of the cells. However, even in immature regions of maize root-tips, the hemicellulose constitutes the bulk of the cell wall material and there is no evidence that very young walls are comprised mainly of pectin. The fall observed in the content of polysaccharides, particularly of the pectin and hemicellulose, between mm segments 3 and 6 seems to be too great to be explained merely by the increasing separation of the end walls of the cells during growth. In the third mm from the tip of the root, cells were in the early stages of elongation; on average, epidermal and cortic.al cells were over 20 p in length and some of the older pith cells in the segment were as long as 70 ,u. If all cells are assumed to increase tenfold in length from 20 to 200 ,u between 3 and 7 mm and remain relatively unchanged in diameter, then the expected fall in the polysaccharide content of the segments between these regions is about 30 per cent. The actual fall is considerably greater than this. B similar “thinning” of c.ell walls of hvena coleoptile during growth promoted by ausin, has been noticed [19]. This lack of stoichiometry between cell wall synthesis and gTowth would suggest that the deposition of non-cellulosic polysaccharides in particular, is not related directly to the extension of the wall. The low values for uranic acids in the isolated pectins are similar to those obtained on oat coleoptile [2]. Such low levels of uranic acid in pectin make unlikely the possibility that polyuronides are directly involved in the control of cell wall extensibility [17]. However, it is possible that the uranic acids have been extensively broken down during isolation, because extraction with ammonium oxalate may lead to pectin degrading by transelimination [l] and some breakdown of uranic acid almost certainly occurs during hydrolysis. Nevertheless, we have shown (in unpublished experiments) that when Experimental
Cell Research 46
Patterns
of cellulose synthesis
507
labelled glucuronic a&l is supplied .to maize root-tips, uronos$ units in pectin become labelled at only about one-fifth the rate of pentosyl units. Similar results have been obtained using 1”C-2-myo-inositol as a precursor of pectin and hemicellulose in maize roots (J. Deshusses, R. M. Roberts and I?. Loewus, unpublished results). This does indicate that the uranic acid content of these roots is indeed low. The residue remaining after the extraction of the cell wall with alkali is cellulose. The proportion of cellulose in the cell wall was markedly lower in the two youngest segments of the root-tip than in older segments. The percentage increased with the onset of rapid extension and suggested that cellulose synthesis occurred at a low rate in young meristematic tissues and is laid down only extensively when cell walls begin to elongate more rapidly. Ray also observed increases in cellulose proportionately greater than other components of the wall, in the extending regions of oat coleoptile segments during auxin-induced growth, and suggested that there map be some association between cellulose synthesis and the growth process [16]. Clearly, however, the analysis of root segments is imprecise because it relies on gross analysis of all the cells in a partic.ular region of the root. Trends of development are risualised which are characteristic of all cells even though different tissues may varp considerably in their cell wall composition. In addition unless the segments are very thin, short term changes in c.ell wall composition must of necessity be overlooked. Thus, the first mm of the roottip, as well as containing the bulk of the meristem, contains the whole of the root-cap, within which a complete developmental sequence of cells is found, progressing from the thin walled initials to the thickened cells on the cap margins. From the autoradiographs on the other hand, it was possible to follow precisely the changes in c.ellulose synthesis occurring in different tissues. The autoradiographs supported the conclusion that cellulose synthesis was 10~ in the cell walls of meristematic cells of all tissues, including the initials of the root-cap. Maximum incorporation in tortes, pith and epidermis was achieved between 3 and 4 mm from zero, after which a marked decline set in, even though cell extension was proceeding linearly. Cellulose was deposited evenly along the wall, except in certain thickened areas, particularly those adjoining some of the cortical air spaces. There was no evidence for tip or other localised features of growth in any tissues. This observation agrees with the evidence of other workers [ll, lS] who have shown that during extension growth of parenchyma and epidermal cells of oat c.oleoptiles, cellulose synthesis is not localised in any way. It is unlikely, however, that the deposition of new microExperimental
Cell Research 46
R. AI. Roberts and V. S. Butt
50s
fibrils is a direct factor controlling elongation because cellulose synthesis was not observed to keep pace with the rate of cell extension beyond 4 mm from the tip. Although this type of autoradiographic investigation allows the changes in incorporation of isotope occurring in different tissues to be followed with some precision, a number of objections have to be borne in mind when assessing the results. It may be argued that the distributions observed in different tissues primarily reflect differences in the availability of the labelled compound within the organ. For instance, the high incorporation of 14C into the epidermis from the radioactive glucoses might be due to the ready access to outer cells. Nevertheless, this is a thick-walled tissue which would demand a high rate of autoradiographs of root-tips have polysaccharide synthesis. Furthermore been prepared from actively photosynthesising maize plants grown in an atmosphere of 14C0, [15]. It was shown that the epidermis incorporated high levels of 14C, even though in this case, isotope had been supplied endogenously from the shoot. These observations suggest that the heavy inc.orporation in the epidermis stems from exceptionally active synthesis, and does not arise from the position of the tissue. Similarly, the high incorporation of isotope into cell walls adjoining the cortical air spaces of the inner cortex might indicate no more than the penetration of labelled compound at these points. Again however, these regions were noticeably thick-walled, and since it is unlikely that compounds can be incorporated into the wall without first entering the cell, even if cell wall deposition may be organised at the surface of the cytoplasm, it is likely that the unequal deposition of isotope arises from synthetic differences. However, because the incubation times employed in these experiments were relatively short and the possibility that the penetration of glucose into the deeper lying tissues might be an important factor in determining the pattern of silver grains on the autoradiographs, quantitative comparisons on the amount of incorporation occurring between tissues were avoided because these might not necessarily represent precise differences in the respective rates of their polysaccharide synthesis. It was assumed, however, that comparisons between cells of different age within the same tissue, whether cortex, pith or epidermis, were valid. It is significant that no visible change in the pattern of isotope incorporation was observed near the cut ends of the root where isotopically labelled glucose was readily available throughout the experiment. There are, therefore, several objections which might be seriously levelled against this type of investigation. It does provide, however, a useful procedure for localising the deposition of cellulose within a tissue where no satisfactory, Experimental
Cell Research 46
Patterns of cellulose synthesis quantitative method previously existed, and could investigations on other developing cell systems.
509 be usefully
employed
in
SUMMARY
By analysis of mm segments of maize root-tip, it has been shown that pectic substances, which are relatively poor in uranic acid but rich in arabinose and galactose, provide only 13 per cent of the cell wall polysaccharides. Their contribution is highest in young cells. The hemicelluloses constituted over 50 per cent of all polysaccharides throughout the root. cc-Cellulose increases during elongation from about 20 per cent of all polysaccharide in the younger region to about 35 per cent in cell walls of growing ~11s. Patterns of cellulose synthesis in excised and intact roots were also investigated by autoradiography. During short incubation periods with 14C-6-oglucose, most of the label incorporated into the ethanol-insoluble fsaction was recovered, after hydrolysis, in hesose. When this residue was extracted with alkali, the insoluble cc-cellulose contained 4C in glucose units only. This incorporation was presumed to be related to the amount of cellulose synthesised during the incubation. Autoradiographs of sections extracted with alkali revealed, therefore, the incorporation pattern into cellulose within the roe-t. Cellulose was synthesised only slowly in cell walls of the cap and meristematic region. As cells began to extend more rapidly, there was a sharp rise in incorporation, increasing to a maximum in the pith, inner and outer cortex and epidermis between the third and fourth mm behind the cap-junction. Cells at this stage had not completed their growth, and no direct relationship could be inferred to exist between deposition of cellulose and extension of the wall. REFERENCES 1. BARRETT, A. J. and NORTHCOTE, D. H., Biochem. J. 94, 617 (1965). 2. BISHOP, C. T., BAYLEY, S. T. and SETTERFIELD, G., PIanf Physiol. 33, 283 (1958). 3. CLOWES, F. A. L., J. Expfl Bof. 11, 81 (1960). 4. DARLINGTON, C. D. and LACOUR, L. F., The Handling of Chromosomes. 3rd ed. Allen and Unwin Ltd, London, 1960. R. L. and \VOLFROM, M. L. (eds.), Methods in Carbohydrate 5. DISCHE, Z., in WHISTLER, Chemistry, Vol. 1, p. 478. Academic Press, New York, 1962. 6. FISCHER, F. G. and D~RFEL, H., Hoppe-Seyler’s 2. Physiol. Chem. 302, 186 (1955). 7. FOLIN, O., J. Biol. Chem. 77, 421 (1928). S. FULLER, Ii. W., in Automation in der analytischen Chemie, p. 319. Technicon GmbH, Frankfurt, 1965. 9. __ Biochem. J. 701 18 p. (1959). 10. GAILLARD, D. E. G., Nature 171, 1160 (1953). 11. GORHAM, P. R. and COLVIN, J. R., Expfl Cell Res. 13, 187 (1957). 12. JENSEN, W. A., Am. J. Botany 47, 287 (1960). Experimental
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13. JENSEN, W. A. and ASHTON, M., Plant Physiol. 35, 313 (1960). 14. JUNIPER, B. E., and ROBERTS,R. III., J. Roy Microscop. Sot. 85, 63 (1966). 332 (1953). 15. R~BIDE&, G. s. and MERICLE, L. W., Plun~PhysioZ.2k, 16. Ray, P. M., Biochem. J. 89, 141 (1963). 17. SET&F&D, G. and B.%YL&Y,S.‘T., &I. Reu. Plant Physiol. 12, 35 (1961). 1s. __
Can. J. Botany 35, 135 (1957).
19. __ Ann. Botany (London) 21, 633 (1957). 20. THORNBER, P. J. and NORTHCOTE,D. H., Biochem. J. 81, 455 (1961). 21. __ ibid. 81, 449 (1961). 22. WILSON, C. iVI., Snul. Chem. 31, 1199 (1959).
Experimental
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