Low-pH effects were studied on the extension of isolated fresh, methanol-killed and frozen-thawed collocytes. Fresh and frozen-thawed samples responded to low pH. This response decreased with increasing differentiation. A yield stress was found for frozen-thawed samples. The significance of the response during growth and differentiation is discusse

Jaccard, Michel

Pilet, Paul-Emile

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Plant & CellPkysial. 18: 88S-891 (1977)Extensibility and rheology of collenchyma cells :II. Lovv-pH effect on the extension of collocytes isolatedfrom high- and low-growing materialMichel Jaccard and Paul-Emile PiletInstitute of Plant Biology and Physiology, University of Lausanne,1005 Lausanne, PI. de la Riponne, Switzerland(Received February 21, 1977)Low-pH effects were studied on the extension of isolated fresh, methanol-killed andfrozen-thawed collocytes. Fresh and frozen-thawed samples responded to low pH.This response decreased with increasing differentiation. A yield stress was found forfrozen-thawed samples. The significance of the response during growth and differentia-tion is discussed.Creep relaxation parameters have been reported for young and senescent col-locytes from the petiole of Apium graveolms (5, 13). For better understanding ofcollocyte wall extension, both growth and differentiation patterns as related to thephyllotaxy of the petioles and their age should be analyzed. At least three para-meters can be discussed: longitudinal growth and length of collocytes, and the weightof their wall fraction. Collocyte length was reported to increase with cell wall thick-ness during differentiation; while growth decreased at the end of differentiation(21). Collocytes and epidermal cells were also suggested to be characterized bypassive growth (19). Consequently, in elongation analysis, an external force can beapplied to the collocytes in order to mimic the tension due to the other tissues.Recently, others have observed, when using isolated epidermis of Aoena coleoptile andPisum stem that frozen-thawed and fresh tissues responded to low pH by a timedependent extension very similar to the response of intact coleoptiles while methanol-killed segments did not (2, 3, 23).The pH effect on extension may be characterized by a similar short time re-sponse in both in vitro (frozen-thawed tissue, external force applied) and in vivoassays (fresh tissue, turgor pressure as the extending force) (17). For peeled Aoenacoleoptile, maximal stimulation in the elongation has been found at pH 5 in both invivo and in vitro expermients (18), while the optimal growth response of peeled seg-ments of Pisum was at pH 3.5 for both in vivo and in vitro assays (2, 25). We notehere that the xyloglucan metabolism can be changed by auxin and low pH; thexyloglucan level increased in a cold water extract of the wall fraction (6, 8, 9).The experiments described below were carried out to understand better theaction of low pH on the wall extension of collocytes in relation to their growth anddifferentiation.883884 M. Jaccard and P. E. PUctMaterials and methodsPlants of Apium graveoUns were cultivated in a greenhouse with controlled tem-perature and humidity. When the free part (no leaf lamina) of the external petioles(Fig. 1) measured 22±2 cm (it was designated as the "basal part" of the petiole),plant was used for the experiments.For growth analysis, India ink marks were made at 5-mm intervals on the basalpart. After 10 days, the elongation of each zone was measured and expressed in %of the initial length.Collocyte bundles were prepared as previously reported (5). In the presentstudy, fibers (2 to 3 cm long) were removed from the top of the basal part Sevento eight fibers were obtained from each petiole. They were immediately placed in20 ml of buffered solution (citric acid 0.01 M-disodiumhydrogenophosphate 0.02 M)and separated according to position of the basal part.To measure length, the collocytes were incubated for 10 hr in a pectinasemixture 0.4% pecinol (Rohm, Germany) 16% saccharose. The solution was thenvigorously shaken and the length was determined microscopically. Similar observa-tions were done using 12% chromic oxide.For extension experiments, fresh, frozen-thwaed and methanol-killed collocyteswere used. Frozen- thawed collocytes were prepared as follows: the samples wereFig. 1. Diagram showing a leaf of Apium graveolcns andthe material used in the present assays. Primary and secondary(1,2) ncrvurca; free part of the petiole (3); lamina (4);the 3-cm segment(s) employed for the experiments.Rheology of collocytes: low pH effects 885KOH H 2 040%Fig. 2. Diagrammatic presentation of the instrument used for testing coUocyte extension in relation to low-pHeffects (see text).quickly frozen and kept at — 20°C; before use, they were thawed at room temperatureand washed with buffer solution. The methanol-killed collocytes were prepared byincubation for 5 min in boiling methanol. The extension of the isolated collocyteswas traced with a creep apparatus slightly modified (Fig. 2) from that described pre-viously (5). The sample was inserted between a lower (LC) and an upper clamp(UP) attached to a pivoting beam. The collocytes were stretched by a force (F)applied on the other side of the beam by hanging a weight or using a dynamometer.Extension of the fiber caused rotation of the beam which was detected by a TESAtransducer (PA), amplified (AP) and transmitted to a Metrohm recorder (E 1).For some measurements, a differential function of extension was required; a differ-entiator (S/St) was used after the amplifier and another (E 2) was used for only theextension rate. To analyze the fresh material, an air pump (P), followed by twowashing bottles (40% KOH and distilled water) was connected to a porous stone (C)which passed air through the liquid in fine bubbles. Solution changing was con-trolled by an automatic clock system (H) which switched on filler pumps (A or B)and a draining pump (D). The regularity of the liquid level was controlled bycontact electrodes (M).Creep determinations were performed as previously reported (5). Externalforce F=10 g was applied to frozen-thawed specimens (T=l min).The weight of the wall fraction (expressed as length unit g/cm) was estimatedusing a standard method (1, 12). The tension applied was calculated by dividingthe force applied (F) by the weight of the wall fraction, assuming that the cell walldensity was about 1 (1, 11); the tension was expressed in dynes/cm2.In the extension experiments, the force applied (F) was 8 g for fresh collocytesand it was changed for frozen-thawed and methanol-killed specimens. Eachextension result represents the measurement of at least 10 collocyte bundles repeatedtwice. For growth and differentiation data, the values were obtained using at leastfour plants.886 M. Jaccard and P. E. PilctResults and discussionYoung petioles are small and situated in the inner part of the plant. Differ-entiated petioles are long and located on the exterior. The petioles arranged on aspiral may represent a growth and differentiation gradient for collenchyma. Growthof the basal part of the petiole and the weight of the wall fractions of the collocytes(Fig. 3), length of collocytes and total strain etot1 (Fig. 4) are given as functionsof the relative length Lr of the basal part of the petiole:Lr=Lp/Lmax ( I )Lp: length of the basal part of the petioleLmu: length of the basal part of the tallest petiole of the plantThe relative length of the basal part is a more usuable value than the range oflocation. The basal parts with a small Lr are located on the top of the celery shootand are arranged higher on the spiral than parts with a greater Lr. The significantgrowth of this basal part seems to occur in the 3 cm just below the first nervure. Nodramatic gradient of growth is apparent in this zone. As indicated in Fig. 3,growth decreased when Lr increased, while the weight of the wall fractions increasedwhen the growth was low. Fig. 4 shows that collocyte length increased with in-creasing Lr, but total strain «tot decreased when Lr was enhanced. Similar resultswere previously reported for fresh tissues (5).TOO80ogj «>uTI 20a.o o500 £c5I400 S-tlI300 F1.00.0200 I1000.00.1 0 2 0 4 0.6 0.8 1.0RELATIVE LENGTH L rFig. 3.0 2 0 4 0.6 OS 1.0RELATIVE LENGTH L rFig. 4.Fig. 3. Relationships between the growth of the 3-an basal part {just below the first nervure) of the petiole, theweight of the wall fraction ofcollocytes preparedfrom the same region of the petiole and the relative length Lr of thebasal part. Measurements were taken after 10 dayi of growth. Linear regression of the experimentaldata is reported. Points and vertical lines represent the mean value for a sample ±the confidentinterval (P=0.05).Fig. 4. Relationships between the length of the collocytes of the 3-cm basal part, the total strain tut of frozen-thawed collocytes (F= 10 g) and the relative length Lr. Points and vertical lines represent the mean value± the standard error." For critical discussion of this parameters, see Ref. 5.Rheology of collocytes: low pH effects 887Fig. 5. The pH effect with time on theexUnsion (absolute and relative values) qfisolaltdfresh collocates prepared from high-growingmaterial (F=8g).Our data clearly show that the basal part with an Lr of 0.1 to 0.2 is high-growing material while the basal part with an Lr of 0.8 to 1.0 is low-growing material.Fig. 5 shows that low pH induced extension of isolated fresh collocytes of high-growing material with a lag period of 1-2 min. The response of fresh tissues in-creased with decreasing pH. When pH was changed from an acidic to a neutralvalue, the extension stopped rapidly; this extension was reversible up to a secondextension with an acidic pH (Fig. 6). Other buffers (acetate, glycine) have alsobeen tested and similar results were obtained.To express the extension rate of collocytes for pHn, the following equation canbe proposed:ERn=dL/dt-l/Lo (expressed in %XlO-4/min)dL/dt: elongation rate for a collocyte bundle.LQ : initial length of the collocyte bundle.(II)Thus, ERj, ER4 and ER3 are the extension rates of collocytes in buffered solu-tions of pH 5, pH 4 and pH 3, respectively. The extension rate was measured10-20 min after the change of solution or when the extension was at a steady state.The pH effect on the extension rate can be easily expressed:at pH 5:at pH 3: JER8=ER3—ERfl(III)(IV)When calculating the J ER values, the precision limit of the instruments usedwas 1%X 10~4/min. When a logarithmic scale was used, the zero value of id ERwould be transformed in 1% X 10~4/min. As already reported, the pH 3 responsescould include a different behavior of extension from that obtained for pH 5 (24).Fig. 7 shows the effects of pH 5 and 3 on Lr. The decrease of the pH responseswas correlated with the growth of basal part (see Fig. 3). Note that the same dropwas observed for the relative value for both pH 3 and 5 in relation to Lr. However,for L r = 1.0, the effect of pH 3 was tenfold that of pH 5, which was minimal. Sucha difference could be related to the decrease of turgor pressure of the collocytes fortreatment at low pH.888 M. Jaccard and P. E. Pilct07asOS01| 0 3i a:K 01aor•pHS PH* /71^jpHti/ 1pHt1000too2Xo 10crUJ120 60 70TIHEtMIN)0.8 O.e 1.0RELATIVE LENOIH L hFig. 6. Fig. 7.Fig. 6. Effect of pH changes on the extension (absolute values) of isolated fresh colloyctes prepared from kigh-growing petioles (F=8g).Fig. 7. 7fa pH effect on the extension rate ( ± standard error) of isolated fresh collocytes in relation to relativelength Lr{F=8g).The data of Table 1 support this explanation. Frozen-thawed collocytesbehaved like fresh collocytes and the extension rate did not significantly differ exceptfor pH 3 for low-growing collocytes. In such case, they exhibited a lower responsethan fresh collocytes. It has to be noticed that they lost turgor pressure andthat biochemical activity, i.e., the ability to synthesize new cell wall material,was reduced (17). However, some enzymes (glucosidases and galactosidases)seem protected against this kind of treatment (23).Methanol-killed collocytes exhibited a very small response to low pH; this wasalready observed for epidermic cells (24). But the decrease of their extensionFig. 8. The pH effect on the extension rat* ( ± standarderror) of frozen-thazotd collocyUs prepared from high-growing (HG) and low-growing (LG) material.10 20 30FORCE F (9)Rheology of collocytcs: low pH effects 889Table 1 pH Effects on fresh (I), methanol-kUUd (II) and frozen-thawed (III) coUenchjma bundles preparedfrom Hgh-(HG) and low-growing (LG) petioles at afore* of 10 gIIIIIIRelative lengt)HG0.20.20.2LG1.01.01.0pHSEffgin^HG2.40±0.10'0.88±0.152.24±0.23FU)LG0.000.000.00nin pH 3 Effect iilog(<HG2.95±0.10'1.58±0.302.83±0.114ER3)LG1.20±0.20'0.000.00* Standard deviation given for each value.ability was (when reported in relative values) identical for both pH 3 and 5. Infact, the methanol-killed samples lost most of their protein content in boilingmethanol (24). Thus, the cell wall materials may have been modified by suchdrastic treatment.Most tissues (17, 23) show a yield stress for the pH effect on extension whenfrozen-thawed. Frozen-thawed collocytes of high-growing material displayedsimilar characteristics (Fig. 8). For low F, only small values were found for A ER$.In contrast, collocytes of low-growing material showed no significant response.This could be explained by the fact that the cell wall is thicker than in high-growingcollocytes. The pH effect on collocytes from low- and high-growing materials to thesame force or stress is given in Table 2. Stress large enough to induce a pH effecton collocytes of high-growing material had no effect on collocytes of low-growingmaterial. This could be correlated to a change of the cell wall which takes placeat the end of the differentiation and which must be in connection with a decrease ofpermanent strain ep.2 Some observations also indicate that rheological parametershave been related to an increase of cellulose content in the cell wall (7). Otherstudies on collenchyma cell wall have shown that the increase of differentiation wasfollowed by a large accumulation of cellulose (20). Such data could very wellexplain, at least partially, the present results.Table 2 pH Effects on force and stress in frozen-thawed collocytes prepared from high-(HG) and low-growing(LG) petiolesForce or stress appliedForce-10 gStress =13.4x10" dynes/cm«HGLGHGLGTotal strain(tot3. 50±0. 301.15±0.303. 60 ±0.302.40±0.45Standard deviation given for each value.Strain ratio«ol/«tot0.45±0.040. 72±0.040.45±0.040. 72±0.04pH Effect in «log (JER,)2.42±0.100.02.42±0.100.0% x 10-</minlog (JER,)2.94±0.110.02.94±0.110.0" This parameter hai been critically discussed for a similar material in Ref. 5.890 M. Jaccard and P. E. PilletConclusionGrowing collocytes exhibit a similar low-pH response to that of epidermal cells.Such responses to pH 3 and 5 do not differ, except in the intensity of the extension.The response to low pH decreased when the growth decreased at the end of differ-entiation. It has been previously reported that NAA induces increase in both thelength of collocytes and their extensibility (14) and that NAA acts on the cell wall bydecreasing the pH as IAA does (10,16). At the end of growth, the wall changes froma low-pH sensitive into a low-pH nonsensitive one (22). In addition, experimentson low-growing material have shown that the isolated collocytes are not sensitiveto NAA (unpublished results). However, it has been noticed that pH-sensitivetissues do not necessarily respond to auxin (4). In contrast, fusicoccin inducesgrowth by decreasing the pH of the wall (10) and by acting also on the balancebetween endogenous IAA and ABA (14, 15). Such pH and growth interactionscould probably be better understood by analyzing the comparative effects of NAAand fusicoccin on collocyte extension (work in preparation).References( 1) Cleland, R.: Extensibility of isolated cell walls: measurement and change during cell elonga-tion. Planta 74: 197-209 (1967).( 2) Cleland, R. and D. L. Rayle: Hydrogen ion entry as a controlling factor in the acid-growthresponse of green pea stem section. Plant Physiol. 55: 547-549 (1975).( 3) Durand, H. and D. L. Rayle: Physiological evidence for auxin-induced hydrogen ion secretionand the epidermal paradox. Planta 114: 185-193 (1973).( 4) Evans, M.: Rapid responses to plant hormones. Aim. Rev. Plant Physiology 25:195-223 (1974).( 5 ) Jaccard, M. and P. E. PUet: Extensibility and rheology of coUenchyma: I. Creep relaxationand viscoelasticity of young and seneicent cells. Plant & Ctli Physiol. 16: 113-120 (1975).(6) Jacobs, M. and P. M. Ray: Promotion of xyloglucan metabolism by acid pH. Plant Physiol.56: 373-376 (1974).( 7 ) Kawamura, H., S. Kamisaka and Y. Masuda: Regulation of lettuce hypocotyl elongation bygibberellic acid. Correlation between cell elongation, stress relaxation properties of the cellwall and wall polysaccharide content. Plant & Cell Physiology 17: 23-34 (1976).( 8) Labavitch, J. M. and P. M. Ray: Relationship between promotion of xyloglucan metabolismand induction by indoleacetic acid. Plant Physiol. 54: 499-502 (1974).( 9 ) Labavitch, J. M. and P. M. Ray: Turnover of cell wall polysaccharides in elongating pea stemsegments, ibid. 53: 669-673 (1974).(10) Marri, E., P. Lado, F. Rasi-Caldognon and R. Colombo: Correlation between proton ex-trusion and stimulation of cell enlargement Effects of fusicoccin and of cytolcinins on leaffragment and isolated cotyledons. Plant Sc. Letters 2: 139-150 (1975).( / / ) Monro, J. A., D. Penny and R. W. Bailey: The organisation and growth of primary cell wallsof lupin hypocotyl. Photochemistry 15: 1193-1198 (1976).(12) Olson, C. A., J. Bonner and D. J. Morre: Force extension analysis of Avena coleoptile cell walls.Planta66: 127-133 (1965).(13) Pilct, P. E., M. Jaccard and R. Magliocco: Mesure de rextensibilite des cellules de collen-chyme. C. R. Acad. Sc. 277: 1637-1640 (1973).(14) Pilet, P. E. and J. Cl. Roland: Growth and extensibility of collenchyme cells. Plant Sri. Letters2:203-207(1974).(15) Pilet, P. E.: Action of fusicoccin and abtcuic acid on root growth, ibid. 5: 137-140 (1975).(16) Pilet, P. E.: Fusicoccin and auxin effects on root growth, ibid. 7: 81-84 (1976).Rheology of collocytes: low pH effects 891(17) Rayle, D. L. and R. Qeland: The in vitro acid growth response: Relation to in vivo growthresponse and auxin action. Planta 104: 282-296 (1972).(18) Rayle, D. L.: Auxin-induced hydrogen ion secretion in Avena coleoptiles and its implication.ibid 114:63-73 (1973).(19) Roelofsen, P. A.: Ultraitructure of the wall in growing cells and its relation to the directionof growth. Adv. Bot. Rtstarthl: 69-149 (1965).(20) Roland, J. Cl.: Organisation de la membrane paraplasmique du collenchymc. J. de Micro-scopie 5: 323-348 (1966).(21) Roland, J. Cl: Recherches en microscopic photonique et en microscopic e'lectronique surl'origine de la differentiation des cellules du collenchymc. Ann, dts Sc. Nat., Bot. (Paris) 8: 141-214 (1967).(22) Roland, J. Cl. and P. E. Pilet: Implications du plasmalemme et de la paroi dans la croissancedcs cellules vege" tales. Exptricnlia 30: 441 -451 (1974).(23) Tanimoto, E. and M. Igari: Correlation between ^-galactosidase and auxin-induced elonga-tion growth in etiolated pea stem. Plant Gt Cell Physiol. 17: 673-682 (1976).(24) Yamagata, Y., R. Yamamoto and Y. Masuda: Auxin and hydrogen ion action on light-grownpea epicotyl segment II. Effect of hydrogen ions on extension of isolated epidermis, ibid 15:873-341 (1974).(25) Yamamoto, R., K. Maki, Y. Yamagata and Y. Masuda: Auxin and hydrogen ion action onlight-grown pea epicotyl segment. I. Tissue specificity of auxin and hydrogen ion action, ibid.15: 823-831 (1974).

Extensibility and rheology of collenchyma cells: II. Low-pH effect on the extension of collocytes isolated from high- and low-growing material

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