Planta
Planta 0988)176:60-67
9 Springer-Verlag1988
Calcium and the mechanical properties of soybean hypocotyl cell walls: Possible role of calcium and protons in cell-wall loosening Sarbjit S. Virk and Robert E. Cleland Department of Botany, KB-15, University of Washington, Seattle, WA 98195, USA
Abstract. The role of calcium in the mechanical strength of isolated cell walls of soybean (Glycine max (L.) Merr. cv. Wayne) hypocotyls has been investigated, using the Instron technique to measure the plastic extensibility (PEx) of methanolboiled, bisected hypocotyl sections and epidermal strips, and atomic absorption spectroscopy to measure wall calcium. Plastic extensibility was closely correlated with the growth rate of intact soybean hypocotyls. Removal of calcium from isolated cell walls by ethylene glycol-bis(2-aminoethyl ether)N,N,N',N'-tetraacetic acid (EGTA) or low pH increased PEx, while addition of calcium decreased PEx; both effects were reversible. The amount of calcium removed and the increase in PEx at pH 4.5 were strongly dependent upon the chelating ability of the buffer anion. There was a direct correlation between the amount of calcium removed from the wall by EGTA or acid and the increase in PEx. Removal of up to 60% of the calcium increased PEx of half-sections up to two fold, but further loss of calcium caused a much greater increase in PEx. With epidermal strips, PEx increased only when calcium was reduced below a threshold. At pH 3.5, there was an additional increase in PEx after a lag of about 2 h; this additional increase may be the result of acid-induced cleavage of a different set of load-bearing bonds. We conclude that calcium bridges are part of the load-bearing bonds in soybean hypocotyl cell walls, and that breakage of these crosslinks by apoplastic acid participates in wall loosening. Acid-induced solubilization of wall calcium may be one mechanism involved in wall loosening of dicotyledonous stems. EGTA = ethylene glycol-bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid; PEx = Instron plastic extensibility Abbreviations:
Key words: Calcium and cell walls - Cell enlargement - Glycine (cell enlargement) - Hypocotyl Plastic extensibility
Introduction The ability of plant cells to enlarge is determined, to a large extent, by the extensibility of their cell walls (for reviews see Cleland 1971; Taiz 1984), which in turn is a function of the spectrum of loadbearing bonds that exist in the walls. BennettClark (1956) suggested that calcium crosslinks between pectic carboxyl groups (calcium bridges) constitute the principal load-bearing bonds in growing cell walls, and that breakage of the calcium bridges might be the mechanism of auxin-induced wall loosening and thus cell enlargement. While addition of calcium to live Avena coleoptile sections inhibited growth (Cooil and Bonner 1956) and decreased tissue deformability (Tagawa and Bonner 1957), studies with isolated Avena coleoptile cell walls (Cldand and Rayle 1977) showed that the plastic extensibility (PEx), as assayed by the Instron technique, was unaffected by conditions that cause the addition or removal of calcium from the walls. It was concluded that calcium bridges are not load-bearing bonds in Avena coleoptile cell walls. Since then, several authors have suggested that calcium crosslinks might be important in the mechanical properties of the cell walls of dicotyledonous plants. For example, the extension of Helianthus hypocotyl walls was increased by ethylene glycol-bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) (Soll and B6ttger 1982), while that of pea epicotyl walls was increased by Mg z+ and
S.S. Virk and R.E. CMand : Calcium and the mechanical properties of soybean hypocotyl cell walls
decreased by C a 2+ (Nakajima etal. 1981). No measurements of wall calcium were made on the extending tissue, however, and thus the relationship between wall calcium and wall extensibility could not be determined. Our research was undertaken to determine the relationship between calcium content and the mechanical strength of soybean hypocotyl cell walls. We used boiled cell walls in order to reduce complications caused by possible enzymatic acid-induced changes in wall extensibility. Since the cuticle is a barrier to the entry and exit of calcium ions and chelators (Prat et al. 1984) even in boiled tissues, bisected sections were employed. The Instron technique was used to evaluate the mechanical strength of the walls. The plastic extensibility, PEx, is a measure of the ability of walls to undergo viscoelastic extension (Cleland 1967; Fujihara etal. 1978), and is closely correlated with but not identical to the cell-elongation parameter, wall extensibility (Cleland 1967, 1984). It will be shown here that load-bearing calcium crosslinks exist in soybean hypocotyl walls, and it will be proposed that apoplastic acidification may promote wall loosening in these walls by solubilization of this calcium. Material and methods Plant material. Seeds of soybean (Glycine max (L.) Merr. cv. Wayne, Champaign County Seed Co, Champaign, Ill., USA) were surface-sterilized in 10% NaOC1 for 20 min, then soaked for 1 h in water, planted in wet vermiculite, and the seedlings were allowed to grow at 25 + 1~ C in continuous dim red light (5 gmol.m- 2.s- 1; red fluorescent tubes) for 4 d. Hypocotyls of about 3 cm length were used for all experiments. Sections, 20 mm long, and cut starting 2~4 mm below the hook, were preincubated for I h in water and were then bisected longitudinally into equal halves with a home-made bisector. In some experiments, epidermal strips were removed from 15-ram halfsections with fine forceps. The bisected sections and the epidermal strips were boiled in absolute methanol for 7-8 min to inactivate enzymes and release the protoplasmic contents. Ca 2+ in the cytoplasm became absorbed onto the walls during the boiling, as no Ca 2+ was released to the outside solution (data not shown). Groups of 20 half-sections or 25-30 epidermal strips were then incubated in 20 ml of experimental solutions (unless otherwise stated). The experimental solutions used are described in table headings and figure legends. After incubation, half-sections or strips were washed in water, and stored in fresh methanol until used for wall-extensibility measurements. The pH of each incubation solution was measured at both the start and end of the incubation; if they did not agree within 0.1 pH unit, the treatment was discarded. Plastic tubes were used for all incubations, because it was found that some calcium could leach out from acid-washed Pyrex glass tubes in the presence of EGTA or low pH. Measurement of plastic extensibility. Wall extensibility was measured by the Instron technique, as described by Cleland (1967). Half-sections were placed between the clamps and extended
61
twice to 60 g load at the rate of 3 cm.min-1. This force was selected because it gave a longitudinal stress on the walls comparable to that of turgor (calculations not given). Epidermal strips were extended twice to 20 g. From the slopes of the first and second extensions at 50 g (half-sections) or 16 g (epidermal strips), total, elastic and plastic extensibility values were determined. The values were expressed as tissue plastic extensibility (PEx), with units of percent extension/100 g load, rather than as plastic compliance values. This means that differences in average hypocotyl diameter (such as occurred in the experiments of Figs. 3 and 4) will result in different PEx values, even when the actual plastic wall compliances are the same. The Instron clamps were covered with Parafilm to avoid calcium contamination of the sections. Calcium measurement. After extension in the Instron assay, the half-sections were oven-dried (70~ for 48 h), weighed, then extracted with concentrated nitric acid for 4 h (1 ml/20 halfsections or 30 epidermal strips), and calcium was measured with an atomic absorption spectrophotometer (Model 303 ; Perkin-Elmer, Norwalk, Conn., USA). Calcium is expressed as gg- (g DW)- x of the walls. Growth measurements. Groups of ten 10-mm sections were weighed, then incubated in 3 ml of 10 mM K-phosphate buffer, pH 7, + 1 mM KC1, + 50 mM CaC12, +_10 gM indole-3-acetic acid or fusicoccin. After 3 h, the sections were reweighed, boiled in methanol, and their PEx was determined. Growth as increase in fresh weight was calculated from the initial and final weights. Replication. Each datum point is the average for ten half-sections or fifteen epidermal strips. Each experiment was repeated at least three times. Standard errors were calculated for each datum point and were less than 10% unless otherwise indicated. Both PEx and the calcium content of the walls were found to vary, depending upon the exact location along the hypocotyl where the section was cut, and the diameter of the sections, which varied from experiment to experiment. As a result, absolute values from one experiment could not always be compared to those from a different experiment (e.g. Figs. 3, 4). On the whole, however, the experiments were reproducible from one day to another.
Results T h e m e c h a n i c a l s t r e n g t h o f s o y b e a n h y p o c o t y l cell walls was assayed by m e a s u r i n g the plastic extensibility, P E x , b y the I n s t r o n t e c h n i q u e . A l t h o u g h P E x is n o t i d e n t i c a l to w a l l e x t e n s i b i l i t y (the cellul a r g r o w t h p a r a m e t e r ) , t h e i r r e l a t i o n s h i p is s h o w n b y the close c o r r e l a t i o n b e t w e e n P E x a n d t h e growth of s o y b e a n hypocotyl sections u n d e r a variety o f g r o w t h c o n d i t i o n s (Fig. 1). T h e p l a s t i c e x t e n s i b i l i t y o f b o i l e d , b i s e c t e d soyb e a n h y p o c o t y l sections was altered b o t h by the a d d i t i o n a n d the r e m o v a l o f c a l c i u m (Fig. 2). W h e n the h a l f - s e c t i o n s w e r e i n c u b a t e d i n 50 m M CaC12 + 5 m M N a - a c e t a t e b u f f e r , p H 6.0, for 3 h, tissue c a l c i u m i n c r e a s e d b y o v e r s e v e n f o l d a n d P E x d e c r e a s e d , r e l a t i v e to tissues t r e a t e d w i t h b u f f e r a l o n e . W h e n t h e s e c t i o n s were i n c u b a t e d i n b u f f e r w i t h 0.1 m M E G T A f o r 3 h, w a l l c a l c i u m w a s re-
62
S.S. Virk and R.E. Cleland: Calcium and the mechanical properties of soybean hypocotyl cell walls
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of soybean hypocotyl cell walls. Bisected, methanol-boiled sections were incubated for 3 h in 50 mM 2-(N-morpholino)ethane sulfonic acid-2-amino-2-(hydroxymethyl)-l,3-propanediol (Mes-Tris) buffer, pH 6.0, containing 50 m M of the chloride salt of the indicated cation. Plastic extensibility (PEx) and elastic extensibility (EEx) were then measured with the Instron technique. Initial value was 12.1 +0.5 for EEx and 12.0_+0.5 for PEx
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Fig. 3. Effect of acidic pHs on PEx of methanol-boiled soybean hypocotyls. Boiled, bisected sections were incubated in 50 mM K-phosphate, pH 3 7, for 3 h, then PEx was measured with an Instron. Bars= +SE, n = 2 0
T i m e (Hours) Fig. 2. Reversible effect of addition or removal of calcium on PEx of soybean hypocotyl cell walls. Bisected, methanol-boiled sections were incubated in 5 mM Na-acetate, pH 6.0, with addition of 0.1 mM E G T A or 50 m M calcium chloride. After 3 h, PEx and calcium content of one set of sections was measured, while other sets were either returned to the same solution (pH 6.0) or transferred to the opposite conditions (CaC12 or EGTA) for 4 h before analysis. Numbers are the calcium content of the walls in gg-(g DW) 1. Bars=SE, n = 14
duced by about 50% and PEx was increased. The effects on PEx of both calcium addition and removal were reversed when the sections were subsequently treated for an additional 4 h under the opposite conditions. Calcium was the only divalent cation to cause a decrease in PEx. Neither M g 2+ n o r M n 2+ at the same concentration (50 mM) had any effect
(Table 1). This contrasts with the report of Nakajima et al. (1981) that M g 2+ could increase the extensibility of isolated epidermal cell walls of pea. The lack of effect of Mn 2 + is interesting in view of the report of Tepfer and Taylor (1981) that this ion is as effective as Ca 2 + in binding to bean-stem cell walls and in inhibiting acid-extension. Neither Na + nor K + had a consistent promotive effect on PEx. Wall loosening appeared to be promoted by H + ; as the pH of a 50 m M K-phosphate buffer was reduced from 7 to 3, PEx increased in proportion to the decrease in pH (Fig. 3). However, this effect was apparently not only in response to the protons, but in part a result of the ability of the anion to extract calcium from the walls (Table 2). Those buffers at pH 4.5 which caused the greatest
S.S. Virk and R.E. Cleland: Calcium and the mechanical properties of soybean hypocotyl cell walls
63
Table 2, Effect of the anion in an acidic buffer on calcium con-
tent and PEx of soybean hypocotyl cell walls. Bisected, methanol-boiled sections were incubated for 3 h in the indicated buffer (50 mM), PEx was measured and then the calcium content of the same sections was determined by atomic absorption spectroscopy. Initial value (no incubation) for PEx was 17.7-t1.0 and for calcium content was 715 • 15 Buffer
Mes-Tris, pH 7.0 Mes-Tris, pH 4.5 K-maleate, pH 4.5 Na-phthalate, pH 4.5 Na-acetate, pH 4.5 K-citrate, pH 4.5 K-phosphate, pH 4.5 K-phosphate-citrate, pH 4.5
PEx Wall calcium (% extension/100 gload) pg.(gDW) i 19.1 • 1.7 20.8 _1.1 21.3-t- 1.1 22.4 • 2.4 25.2• 28.2• 29.8 • 1.9 33.2•
698 • 6 683-t-50 638-1- 1 649 • 58 554• 505• 490_+ 31 364• 8
removal of wall calcium (citrate, phosphate, and phosphate-citrate) also caused the largest increases in PEx. The ability of citrate to extract wall calcium is well known (e.g. Jarvis et al. 1984), but the almost equal ability of phosphate to extract wall calcium has apparently not been recognized previously. 2-(N-Morpholino)ethanesulfonic acid-2amino-2-(hydroxymethyl)-l,3-propanediol (MesTris) buffer at pH 4.5 caused neither calcium loss from the wall nor increased PEx relative to the pH 7.0 control. The reason for this lack of effect is not known. Na-acetate, whose anion would not be expected to bind calcium effectively and thus extract wall calcium directly, did remove calcium from the wall at pH 4.5 and increased PEx. In this case the calcium very probably was extracted by the H +. We elected to use Na-acetate as our standard buffer in these experiments. The effects on PEx of short-term incubation with buffers at different pHs and with E G T A were determined, and PEx was plotted as a function of the calcium content remaining in the walls at the end of the incubation. A strong correlation between wall Ca 2 § and PEx was obtained (Fig. 4). Regardless of the mechanism of removal of wall calcium (by H § anion or EGTA), the increase in PEx was proportional to the amount of calcium removed, as long as the calcium content of the walls did not fall below about 200 pg Ca 2+. (g DW) -1. However, if longer incubations in E G T A (up to 10 h) were used, more calcium was removed and PEx increased at a much greater rate (Fig. 5). This indicates that the calcium removal at first consisted primarily of calcium which was not involved in calcium crosslink.s, and only secondarily of load-bearing calcium bridges. It is sug-
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Fig. 4. Relationship between PEx of methanol-boiled soybean sections and their calcium content. Different levels of calcium in the walls were created by incubation of methanol-boiled halfsections before PEx was measured with an Instron. K-phosphate (50 mM), pH 3, 3 h 9 K-phosphate (50 mM), pH 7_+ EGTA, 3 h 9 Na-acetate (50 mM), pH 6 • E G T A for varying times. A Na-acetate (50 mM), pH 3, 3 h 9 Na-acetate (50 mM), pH 6, 3 h 9 different buffers (50 mM), pH 4.5, 3 h | no incubation, or Mes-Tris (50 mM), pH 7, 3 h
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Fig. S. Effect of extensive removal of wall calcium on PEx of soybean hypocotyls. Boiled, bisected sections were incubated in 50 mM Na-acetate, pH 6.0+ 1 mM E G T A for up to i0 h. PEx and calcium content of the walls were then determined. The points can be fitted to two straight lines with greatly different slopes
gested that when this non-load-bearing calcium was gone, then the further loss of calcium was the more difficult to remove crosslinked calcium. Its removal led to large increases in PEx. The two phases of calcium-mediated wall loosening can also be seen from the time course of the effect of low p H on PEx and calcium removal (Fig. 6). During the first 2 h in low p H almost 2/3 of the calcium was removed, with only a limited
64
S.S. Virk and R.E. Cleland: Calcium and the mechanical properties of soybean hypocotyl cell walls Wall Calcium ~ug/g DW 200 400 600
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Fig. 6. Time course of acid-induced increase in PEx in soybean hypocotyls. Boiled sections were incubated in 50 m M Na-acetate at pH 3.5 (e) or pH 6.0 (o) for varying periods of time. Solutions were changed once after 3 h. Numbers represent wall calcium ( g g - ( g D W ) - 1 ; means of three experiments). The SE for Ca was less than 8%. Insert: relationship between PEx and wall calcium
increase in PEx. During the next 4 h, calcium continued to be lost at a much lower rate, and PEx increased sharply. A plot of calcium versus PEx (insert, Fig. 6), shows the two phases of the calcium/PEx relationship more clearly. Although all longitudinal walls in the hypocotyl contribute to the total wall strength, it is the increase in extensibility in the epidermal-subepidermal walls that is the cause of the auxin-induced cell enlargement (Masuda and Yamamoto 1972; Taiz 1984). In order to assess the contribution of calcium crosslinks in these cells to the mechanical properties of the whole tissue, methanol-boiled epidermal strips were incubated for up to 9 h in 50 m M Na-acetate, p H 6.0, with or without I m M EGTA, and PEx and wall calcium was measured. A plot of calcium versus PEx (Fig. 7) again shows two phases in the calcium/PEx relationship. In this case, removal of calcium had almost no effect on extensibility until a threshold of calcium was reached. When calcium was reduced below this threshold (about 200 gg. (g D W ) - l), PEx increased sharply in proportion to the amount of calcium removed. In general, then, the properties of half-sections are similar to those of the epidermal-subepidermal walls. The increase in PEx during the first couple of hours after addition of acidic buffers appeared to be largely the result of H § and anion-mediated extraction of the calcium. Over longer time periods, however, acid itself must have had a second effect on PEx in addition to its extraction of calcium. As shown in Fig. 8, when sections which had
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Wall Calcium (,ug/g DW) Fig. 7. Effect of removal o f wall calcium on PEx of isolated epidermal walls of soybean hypocotyls. Groups of 25-30 methanol-boiled epidermal strips were incubated in 50 m M Na-acetate, pH 6 . 0 _ 1 m M EGTA. After 1~9 h, sections were washed, PEx and wall calcium was determined. Some sections were incubated 1 h in E G T A and then returned to solutions-EGTA for up to 8 h
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Time (Hours) Fig. 8. Evidence for an acid-induced increase in PEx in boiled soybean hypocotyl walls unrelated to calcium removal. Boiled, bisected sections incubated for 2 h in 50 m M Na-acetate at pH 3.5, or at 6.0 with or without 1 m M EGTA, then subjected to analysis or transferred to fresh solution of the same kind or from pH 6 + E G T A to solutions without E G T A at pH 6 or 3. After 2 h, PEx and calcium content were determined. Numbers are wall calcium (gg. (g D W ) - 2). Note that sections transferred from pH 6 + E G T A to pH 3.5 had a much higher PEx, but the same calcium level as sections retained in pH 6 + EGTA
been incubated for 2 h at pH 6.0 with E G T A were incubated for an additional 2 h at p H 6.0 with E G T A or at p H 3.5, the removal of calcium was almost the same. However, PEx increased much more in the presence of the low-pH solution. Thus it would appear that two factors are influencing wall extensibility of soybean hypocotyl cell walls; the amounts of calcium crosslinks and of at least one additional acid-labile link in the walls.
S.S. Virk and R.E. Cleland: Calcium and the mechanical properties of soybean hypocotyl cell walls Discussion The role of calcium as part of the load-bearing bonds in cell walls has been a matter of some controversy. H o w does one assess this role? There are two main types of assay. The first is to measure the mechanical properties of cell walls after metabolic activity has been stopped by boiling the tissue in methanol or by freezing-thawing. The properties can be measured by the Instron, creep or stress relaxation assays (Cleland 1971; Taiz 1984); in theory, all give the same qualitative information about the viscoelasticity of the walls (Fujihara et al. 1978). The relationship between these measurements and wall extensibility, which is one of the parameters which control the rate of cell elongation, is not completely settled (Taiz 1984), but the Instron parameter PEx appears to be closely correlated with wall extensibility (Cleland 1967, 1984; Fig. 1). A second assay measures the facilitated creep of isolated cell walls when under applied tension (Cleland 1983; Cleland et al. 1987). Facilitated creep occurs in response to acid-induced cleavage of load-bearing bonds in many tissues (Cleland 1986). This assay evaluates the capacity of walls to undergo facilitated creep rather than measuring the wall extensibility that existed in live tissues at the time the walls were isolated. In this study we have used the Instron technique and have determined the effect of calcium removal and addition to the wall on the plastic extensibility, PEx. The data presented here are for methanol-boiled half-sections and epidermal strips. We have determined the calcium concentration for each set of sections after measurement of PEx by the Instron technique, so that the relationship between calcium content and the mechanical strength of the walls could be determined precisely. We show here that for soybean hypocotyl cell walls, added calcium reduced PEx, and removal of calcium with acidic buffers or E G T A increased PEx. The quantitative relationship between calcium removal and PEx is not simple. Removal of up to 2/3 of the calcium associated with the walls of half-sections resulted in some increase in PEx, with the amount of calcium removed being proportional to the increase in PEx. However, when more calcium was removed, the proportionality changed, and the increase in PEx was far greater. For epidermal strips, on the other hand, extensibility increased only after wall calcium had been lowered below a threshold. This indicates that the effect of calcium on PEx at calcium levels above this threshold is the consequence of calcium crosslinks in the inner tissues (cortex and vascular bun-
65
dles), while the effect of calcium removal at levels below the threshold is the consequence of the breakage of calcium crosslinks in the epidermal walls only or in the inner tissues as well. In assessing these data, several points should be kept in mind. First of all, PEx is not necessarily proportional to total wall calcium. The calcium content of the walls when first isolated was far from saturation. According to Yamaoka and Chiba (1983), a maximum of 12% of the uronic acids of soybean cell walls are bound to calciurn ions. Thus when the walls were incubated in CaClz, the calcium content increased as much as sevenfold with only a small decrease in PEx. Added calcium was absorbed onto the wall but produced few additional load-bearing crosslinks. Treatment of walls with E G T A increased PEx to the same extent, whether or not the walls were pretreated with calcium. Wall calcium, however, was three times higher in calcium pretreated walls (Fig. 2). It is important to recognize that the majority of wall calcium was not involved in calcium crosslinks, and could be removed without any change in PEx. Presumedly this was calcium associated with carboxyl groups in non-loadbearing layers of the walls (Taiz 1984) or calcium associated with a single carboxyl group, which failed to form a calcium crosslink between two carboxyl groups. It must also be remembered that the extractions may remove more wall components than just calcium ions. Jarvis (1982) found that the calcium chelator cyclohexanediaminetetraacetic acid (CDTA) extracted wall polysaccharides in addition to calcium. Buffers such as Tris-maleate have been found to decrease the calcium-binding capacity of isolated pea epicotyl cell walls, which indicates that they must have removed some wall polysaccharides (Baydoun and Brett 1984). The large increase in PEx after prolonged treatment with EGTA (10 h) or p H 3.5 (5 h) (Figs. 5, 6) could be the result of extraction of wall polysaccharides in addition to wall calcium. These data may seem to be in conflict with previous results with cell walls of other dicotyledons. For example, Courtney and Morr6 (1980) could find no effect on PEx of calcium added in vitro to boiled pea stem sections; however, since the cuticle was intact, it is likely that the calcium never reached the epidermal cell walls. Jarvis et al. (1984) concluded that they could not find a correlation between the amount of calcium removed by citrate from celery petiole collenchyma, and wall extensibility, but in fact they also found that extensibility increased greatly when the wall calcium fell below some threshold. Nakajima etat. (1981)
66
S.S. Virk and R.E, Cleland: Calcium and the mechanical properties of soybean hypocotyl cell walls
found that added calcium ions decreased the creep of pea epidermal cell walls, while Mg 2+ increased it. Actual wall calcium measurements were not made on the tissues subjected to mechanical analysis, however. In Arena coleoptile cell walls, an increase in wall extensibility by acid appears to occur in response to an activation of wall-loosening enzymes by low pH, as indicated by the fact that inactivation of proteins by boiling or by removal by proteolytic enzymes renders the walls incapable of being loosened by acid (Rayle and Cleland 1977). In soybean hypocotyl walls, PEx can still be increased by acid, even after the walls have been boiled. This effect is, in part, the consequence of removal of calcium by the acid as well as by the accompanying anion. However, over longer periods of time, protons seem to be able to induce pronounced wall loosening by a second, calciumindependent mechanism. Whether this involves a breakage of hydrogen bonds in the walls, is the result of extraction of some polysaccharide component of the wall, the result of acid-activation of wall loosening enzymes that are resistant to boiling methanol, or is caused by some other mechanism, remains to be determined. Our data indicate that calcium crosslinks are important as load-bearing bonds in soybean hypocotyl cell walls. The crosslinks are probably not the classic "egg box" calcium-pectate interactions (Rees 1969), but more complex interactions involving wall polyuronides (Tepfer and Taylor 1981). Wall-loosening during auxin-induced growth could involve a breakage of these crosslinks. It has been shown that auxin causes proton excretion in soybean hypocotyls (Rayle and Cleland 1977). The protons may cause solubilization of the calcium involved in crosslinks, and thus may contribute to an increase in wall extensibility. However, it would appear that wall extensibility is also dependent upon a second set of load-bearing bonds that are cleaved under acidic conditions and that do not involve calcium. Both sets of bonds may have to be broken in soybean hypocotyl walls in order for maximum wail extension to occur. If so, the mechanism of wall-loosening in these walls in response to auxin would be fundamentally different from that in Arena coleoptiles. It is of interest that Moll and Jones (1981) suggested that the gibberellin-induced increase in wall extensibility which occurs in lettuce hypocotyl walls involves an uptake of calcium into the cells from the walls. However, the calcium solubilized by the protons does not need to be taken up into the cells, but can simply remain in the apoplastic
solution as long as the solution remains acidic. Alternatively, the calcium can be displaced from the load-bearing inner cell wall layers to the mechanically-unimportant outer regions of the wall. This research was supported by contract DE-AM0676ER73019 from the U.S. Department of Energy.
References Baydoun, E.A.-H., Brett, C.T. (1984) The effect of pH on the binding of calcium to pea epicotyl cell walls and its implications for the control ofcel! extension. J. Exp. Bot. 35, 18201831
Bennett-Clark, T.A. (1956) Salt accumulation and mode of action of auxin. A preliminary hypothesis. In: The chemistry and mode of action of plant growth substances, pp. 284~ 291, Wain, R.L., Wightman, F., eds. Butterworths, London Cleland, R.E. (1967) Extensibility of isolated cell wails: measurement and changes during cell elongation. Planta 74, 197-209 Cleland, R.E. (1971) Cell wall extension. Annu. Rev. Plant Physiol. 22, 19~222 CMand, R.E. (1983) The capacity for acid-induced wall loosening as a factor in the control of Arena coleoptile cell elongation. J. Exp. Bot. 34, 676-680 Cleland, R.E. (1984) The Instron technique as a measure of immediate-past wall extensibility. Planta 160, 514-520 Cleland, R.E. (1986) The role of hormones in wall loosening and plant growth. Aust. J. Plant Physiol. 13, 93 103 Cleland, R.E., Rayle, D.L. (1977) Reevaluation of the effect of calcium ions on auxin-induced elongation. Plant Physiol. 60, 709-712 Cleland, R.E., Cosgrove, D., Tepfer, M. (1987) Long-term acidinduced wall extension in an in-vitro system. Planta 170, 379 385 Cooil, B., Bonner, J. (1957) Effects of calcium and potassium ions on the auxin-induced growth of Arena coleoptile sections. Planta 48, 696-723 Courtney, J.S., Morr6, D.J. (1980) Studies on the role of wall extensibility in the control of cell expansion. Bot. Gaz. 141, 56-62 Fujihara, S., Yamamoto, R., Masuda, Y. (1978) Viscoelastic properties of plant cell walls. III. Hysteresis loop in the stress-strain curve at constant strain rate. Biorheology 15, 87-97 Jarvis, M.C. (1982) The proportion of calcium-bound pectin in plant cell walls. Planta 154, 344-346 Jarvis, M.C., Logan, A.S., Duncan, H.J. (1984) Tensile characteristics of collenchyma cell walls at different calcium contents. Physiol. Plant. 61, 81-86 Masuda, Y., Yamamoto, R. (1972) Control of auxin-induced stem elongation by the epidermis. Physiol. Plant. 27, 109 115 Moll, C., Jones, R.L. (1981) Calcium and GA-induced elongation of lettuce hypocotyl sections. Planta 152, 450-456 Nakajima, N., Morikawa, H., Igasashi, S., Senda, M. (1981) Differential effects of calcium and magnesium on mechanical properties of pea stem cell walls. Plant Cell Physiol. 22, 1305-1315 Prat, R., Goeissaz, M.-B., Goldberg, R. (1984) Effects of Ca 2+ and Mg 2+ on elongation and H + secretion of Vigna radiata hypocotyl sections. Plant Cell Physiol. 25, 1459 1467 Rayle, D.L., Cleland, R.E. (1977) Control of plant cell enlargement by hydrogen ions. Curr. Top. Devel. Biol. 11, 187-214
S.S. Virk and R.E. Cleland: Calcium and the mechanical properties of soybean hypocotyl cell walls Rees, D.A. (1969) Structure, conformation, and mechanism in the formation of polysaccharide gels and networks. Adv. Carbohydr. Chem. Biochem. 24, 267 332 Soll, H., B6ttger, M. (1982) The mechanism of proton-induced increase in wall extensibility. Plant Sci. Lett. 24, 163-171 Tagawa, T , Bonner, J. (1957) Mechanical properties of the Arena coleoptile as related to auxin and to ionic interactions. Plant Physiol. 32, 207 212 Taiz, L. (1984) Plant cell expansion; regulation of cell wall mechanical properties. Annu. Rev. Plant Physiol. 35, 585657
67
Tepfer, M., Taylor, I.E.P. (1981) The interaction of divalent cations with pectic substances and their influence in acidinduced cell wall loosening. Can. J. Bot. 59, 1522 15125 Yamaoka, T., Chiba, N. (1983) Changes in the coagulating ability of pectin during the growth of soybean hypocotyls. Plant Cell Physiol. 24, 1281 1290
Received 17 January; accepted 31 May 1988
Planta 0988)176:60-67
9 Springer-Verlag1988
Calcium and the mechanical properties of soybean hypocotyl cell walls: Possible role of calcium and protons in cell-wall loosening Sarbjit S. Virk and Robert E. Cleland Department of Botany, KB-15, University of Washington, Seattle, WA 98195, USA
Abstract. The role of calcium in the mechanical strength of isolated cell walls of soybean (Glycine max (L.) Merr. cv. Wayne) hypocotyls has been investigated, using the Instron technique to measure the plastic extensibility (PEx) of methanolboiled, bisected hypocotyl sections and epidermal strips, and atomic absorption spectroscopy to measure wall calcium. Plastic extensibility was closely correlated with the growth rate of intact soybean hypocotyls. Removal of calcium from isolated cell walls by ethylene glycol-bis(2-aminoethyl ether)N,N,N',N'-tetraacetic acid (EGTA) or low pH increased PEx, while addition of calcium decreased PEx; both effects were reversible. The amount of calcium removed and the increase in PEx at pH 4.5 were strongly dependent upon the chelating ability of the buffer anion. There was a direct correlation between the amount of calcium removed from the wall by EGTA or acid and the increase in PEx. Removal of up to 60% of the calcium increased PEx of half-sections up to two fold, but further loss of calcium caused a much greater increase in PEx. With epidermal strips, PEx increased only when calcium was reduced below a threshold. At pH 3.5, there was an additional increase in PEx after a lag of about 2 h; this additional increase may be the result of acid-induced cleavage of a different set of load-bearing bonds. We conclude that calcium bridges are part of the load-bearing bonds in soybean hypocotyl cell walls, and that breakage of these crosslinks by apoplastic acid participates in wall loosening. Acid-induced solubilization of wall calcium may be one mechanism involved in wall loosening of dicotyledonous stems. EGTA = ethylene glycol-bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid; PEx = Instron plastic extensibility Abbreviations:
Key words: Calcium and cell walls - Cell enlargement - Glycine (cell enlargement) - Hypocotyl Plastic extensibility
Introduction The ability of plant cells to enlarge is determined, to a large extent, by the extensibility of their cell walls (for reviews see Cleland 1971; Taiz 1984), which in turn is a function of the spectrum of loadbearing bonds that exist in the walls. BennettClark (1956) suggested that calcium crosslinks between pectic carboxyl groups (calcium bridges) constitute the principal load-bearing bonds in growing cell walls, and that breakage of the calcium bridges might be the mechanism of auxin-induced wall loosening and thus cell enlargement. While addition of calcium to live Avena coleoptile sections inhibited growth (Cooil and Bonner 1956) and decreased tissue deformability (Tagawa and Bonner 1957), studies with isolated Avena coleoptile cell walls (Cldand and Rayle 1977) showed that the plastic extensibility (PEx), as assayed by the Instron technique, was unaffected by conditions that cause the addition or removal of calcium from the walls. It was concluded that calcium bridges are not load-bearing bonds in Avena coleoptile cell walls. Since then, several authors have suggested that calcium crosslinks might be important in the mechanical properties of the cell walls of dicotyledonous plants. For example, the extension of Helianthus hypocotyl walls was increased by ethylene glycol-bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) (Soll and B6ttger 1982), while that of pea epicotyl walls was increased by Mg z+ and
S.S. Virk and R.E. CMand : Calcium and the mechanical properties of soybean hypocotyl cell walls
decreased by C a 2+ (Nakajima etal. 1981). No measurements of wall calcium were made on the extending tissue, however, and thus the relationship between wall calcium and wall extensibility could not be determined. Our research was undertaken to determine the relationship between calcium content and the mechanical strength of soybean hypocotyl cell walls. We used boiled cell walls in order to reduce complications caused by possible enzymatic acid-induced changes in wall extensibility. Since the cuticle is a barrier to the entry and exit of calcium ions and chelators (Prat et al. 1984) even in boiled tissues, bisected sections were employed. The Instron technique was used to evaluate the mechanical strength of the walls. The plastic extensibility, PEx, is a measure of the ability of walls to undergo viscoelastic extension (Cleland 1967; Fujihara etal. 1978), and is closely correlated with but not identical to the cell-elongation parameter, wall extensibility (Cleland 1967, 1984). It will be shown here that load-bearing calcium crosslinks exist in soybean hypocotyl walls, and it will be proposed that apoplastic acidification may promote wall loosening in these walls by solubilization of this calcium. Material and methods Plant material. Seeds of soybean (Glycine max (L.) Merr. cv. Wayne, Champaign County Seed Co, Champaign, Ill., USA) were surface-sterilized in 10% NaOC1 for 20 min, then soaked for 1 h in water, planted in wet vermiculite, and the seedlings were allowed to grow at 25 + 1~ C in continuous dim red light (5 gmol.m- 2.s- 1; red fluorescent tubes) for 4 d. Hypocotyls of about 3 cm length were used for all experiments. Sections, 20 mm long, and cut starting 2~4 mm below the hook, were preincubated for I h in water and were then bisected longitudinally into equal halves with a home-made bisector. In some experiments, epidermal strips were removed from 15-ram halfsections with fine forceps. The bisected sections and the epidermal strips were boiled in absolute methanol for 7-8 min to inactivate enzymes and release the protoplasmic contents. Ca 2+ in the cytoplasm became absorbed onto the walls during the boiling, as no Ca 2+ was released to the outside solution (data not shown). Groups of 20 half-sections or 25-30 epidermal strips were then incubated in 20 ml of experimental solutions (unless otherwise stated). The experimental solutions used are described in table headings and figure legends. After incubation, half-sections or strips were washed in water, and stored in fresh methanol until used for wall-extensibility measurements. The pH of each incubation solution was measured at both the start and end of the incubation; if they did not agree within 0.1 pH unit, the treatment was discarded. Plastic tubes were used for all incubations, because it was found that some calcium could leach out from acid-washed Pyrex glass tubes in the presence of EGTA or low pH. Measurement of plastic extensibility. Wall extensibility was measured by the Instron technique, as described by Cleland (1967). Half-sections were placed between the clamps and extended
61
twice to 60 g load at the rate of 3 cm.min-1. This force was selected because it gave a longitudinal stress on the walls comparable to that of turgor (calculations not given). Epidermal strips were extended twice to 20 g. From the slopes of the first and second extensions at 50 g (half-sections) or 16 g (epidermal strips), total, elastic and plastic extensibility values were determined. The values were expressed as tissue plastic extensibility (PEx), with units of percent extension/100 g load, rather than as plastic compliance values. This means that differences in average hypocotyl diameter (such as occurred in the experiments of Figs. 3 and 4) will result in different PEx values, even when the actual plastic wall compliances are the same. The Instron clamps were covered with Parafilm to avoid calcium contamination of the sections. Calcium measurement. After extension in the Instron assay, the half-sections were oven-dried (70~ for 48 h), weighed, then extracted with concentrated nitric acid for 4 h (1 ml/20 halfsections or 30 epidermal strips), and calcium was measured with an atomic absorption spectrophotometer (Model 303 ; Perkin-Elmer, Norwalk, Conn., USA). Calcium is expressed as gg- (g DW)- x of the walls. Growth measurements. Groups of ten 10-mm sections were weighed, then incubated in 3 ml of 10 mM K-phosphate buffer, pH 7, + 1 mM KC1, + 50 mM CaC12, +_10 gM indole-3-acetic acid or fusicoccin. After 3 h, the sections were reweighed, boiled in methanol, and their PEx was determined. Growth as increase in fresh weight was calculated from the initial and final weights. Replication. Each datum point is the average for ten half-sections or fifteen epidermal strips. Each experiment was repeated at least three times. Standard errors were calculated for each datum point and were less than 10% unless otherwise indicated. Both PEx and the calcium content of the walls were found to vary, depending upon the exact location along the hypocotyl where the section was cut, and the diameter of the sections, which varied from experiment to experiment. As a result, absolute values from one experiment could not always be compared to those from a different experiment (e.g. Figs. 3, 4). On the whole, however, the experiments were reproducible from one day to another.
Results T h e m e c h a n i c a l s t r e n g t h o f s o y b e a n h y p o c o t y l cell walls was assayed by m e a s u r i n g the plastic extensibility, P E x , b y the I n s t r o n t e c h n i q u e . A l t h o u g h P E x is n o t i d e n t i c a l to w a l l e x t e n s i b i l i t y (the cellul a r g r o w t h p a r a m e t e r ) , t h e i r r e l a t i o n s h i p is s h o w n b y the close c o r r e l a t i o n b e t w e e n P E x a n d t h e growth of s o y b e a n hypocotyl sections u n d e r a variety o f g r o w t h c o n d i t i o n s (Fig. 1). T h e p l a s t i c e x t e n s i b i l i t y o f b o i l e d , b i s e c t e d soyb e a n h y p o c o t y l sections was altered b o t h by the a d d i t i o n a n d the r e m o v a l o f c a l c i u m (Fig. 2). W h e n the h a l f - s e c t i o n s w e r e i n c u b a t e d i n 50 m M CaC12 + 5 m M N a - a c e t a t e b u f f e r , p H 6.0, for 3 h, tissue c a l c i u m i n c r e a s e d b y o v e r s e v e n f o l d a n d P E x d e c r e a s e d , r e l a t i v e to tissues t r e a t e d w i t h b u f f e r a l o n e . W h e n t h e s e c t i o n s were i n c u b a t e d i n b u f f e r w i t h 0.1 m M E G T A f o r 3 h, w a l l c a l c i u m w a s re-
62
S.S. Virk and R.E. Cleland: Calcium and the mechanical properties of soybean hypocotyl cell walls
A "0
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Table 1. Effect of calcium and other cations on wall extensibility
I
o
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of soybean hypocotyl cell walls. Bisected, methanol-boiled sections were incubated for 3 h in 50 mM 2-(N-morpholino)ethane sulfonic acid-2-amino-2-(hydroxymethyl)-l,3-propanediol (Mes-Tris) buffer, pH 6.0, containing 50 m M of the chloride salt of the indicated cation. Plastic extensibility (PEx) and elastic extensibility (EEx) were then measured with the Instron technique. Initial value was 12.1 +0.5 for EEx and 12.0_+0.5 for PEx
20
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Cation
Extensibility (% extension/100 g load)
X UJ
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l
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% Increase in F W Fig. 1. Correlation between the Instron extensibility (PEx) and the growth of soybean hypocotyl sections as influenced by various growth conditions. Groups of 10 sections were incubated for 3 h in 10 mM K-phosphate buffer, pH 7, +1 m M KC1, _+50 m M CaC12 and _+10 ~tM indole-3-acetic acid (IAA) or fusicoccin (FC). Numbers in the figure refer to the following conditions: 1) Buffer control 2) +CaC12 3) + IAA
4) IAA + CaC12 5) FC 6) FC + CaC12
No cation Na + K+ Mg 2+ Mn 2+ Ca 2+
03 ~
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12.2 + 0.4 /3.1 +0.5 12.7_+0.3 12.3_+0.2 11.9+0.5 11.5-+0.4
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Fig. 3. Effect of acidic pHs on PEx of methanol-boiled soybean hypocotyls. Boiled, bisected sections were incubated in 50 mM K-phosphate, pH 3 7, for 3 h, then PEx was measured with an Instron. Bars= +SE, n = 2 0
T i m e (Hours) Fig. 2. Reversible effect of addition or removal of calcium on PEx of soybean hypocotyl cell walls. Bisected, methanol-boiled sections were incubated in 5 mM Na-acetate, pH 6.0, with addition of 0.1 mM E G T A or 50 m M calcium chloride. After 3 h, PEx and calcium content of one set of sections was measured, while other sets were either returned to the same solution (pH 6.0) or transferred to the opposite conditions (CaC12 or EGTA) for 4 h before analysis. Numbers are the calcium content of the walls in gg-(g DW) 1. Bars=SE, n = 14
duced by about 50% and PEx was increased. The effects on PEx of both calcium addition and removal were reversed when the sections were subsequently treated for an additional 4 h under the opposite conditions. Calcium was the only divalent cation to cause a decrease in PEx. Neither M g 2+ n o r M n 2+ at the same concentration (50 mM) had any effect
(Table 1). This contrasts with the report of Nakajima et al. (1981) that M g 2+ could increase the extensibility of isolated epidermal cell walls of pea. The lack of effect of Mn 2 + is interesting in view of the report of Tepfer and Taylor (1981) that this ion is as effective as Ca 2 + in binding to bean-stem cell walls and in inhibiting acid-extension. Neither Na + nor K + had a consistent promotive effect on PEx. Wall loosening appeared to be promoted by H + ; as the pH of a 50 m M K-phosphate buffer was reduced from 7 to 3, PEx increased in proportion to the decrease in pH (Fig. 3). However, this effect was apparently not only in response to the protons, but in part a result of the ability of the anion to extract calcium from the walls (Table 2). Those buffers at pH 4.5 which caused the greatest
S.S. Virk and R.E. Cleland: Calcium and the mechanical properties of soybean hypocotyl cell walls
63
Table 2, Effect of the anion in an acidic buffer on calcium con-
tent and PEx of soybean hypocotyl cell walls. Bisected, methanol-boiled sections were incubated for 3 h in the indicated buffer (50 mM), PEx was measured and then the calcium content of the same sections was determined by atomic absorption spectroscopy. Initial value (no incubation) for PEx was 17.7-t1.0 and for calcium content was 715 • 15 Buffer
Mes-Tris, pH 7.0 Mes-Tris, pH 4.5 K-maleate, pH 4.5 Na-phthalate, pH 4.5 Na-acetate, pH 4.5 K-citrate, pH 4.5 K-phosphate, pH 4.5 K-phosphate-citrate, pH 4.5
PEx Wall calcium (% extension/100 gload) pg.(gDW) i 19.1 • 1.7 20.8 _1.1 21.3-t- 1.1 22.4 • 2.4 25.2• 28.2• 29.8 • 1.9 33.2•
698 • 6 683-t-50 638-1- 1 649 • 58 554• 505• 490_+ 31 364• 8
removal of wall calcium (citrate, phosphate, and phosphate-citrate) also caused the largest increases in PEx. The ability of citrate to extract wall calcium is well known (e.g. Jarvis et al. 1984), but the almost equal ability of phosphate to extract wall calcium has apparently not been recognized previously. 2-(N-Morpholino)ethanesulfonic acid-2amino-2-(hydroxymethyl)-l,3-propanediol (MesTris) buffer at pH 4.5 caused neither calcium loss from the wall nor increased PEx relative to the pH 7.0 control. The reason for this lack of effect is not known. Na-acetate, whose anion would not be expected to bind calcium effectively and thus extract wall calcium directly, did remove calcium from the wall at pH 4.5 and increased PEx. In this case the calcium very probably was extracted by the H +. We elected to use Na-acetate as our standard buffer in these experiments. The effects on PEx of short-term incubation with buffers at different pHs and with E G T A were determined, and PEx was plotted as a function of the calcium content remaining in the walls at the end of the incubation. A strong correlation between wall Ca 2 § and PEx was obtained (Fig. 4). Regardless of the mechanism of removal of wall calcium (by H § anion or EGTA), the increase in PEx was proportional to the amount of calcium removed, as long as the calcium content of the walls did not fall below about 200 pg Ca 2+. (g DW) -1. However, if longer incubations in E G T A (up to 10 h) were used, more calcium was removed and PEx increased at a much greater rate (Fig. 5). This indicates that the calcium removal at first consisted primarily of calcium which was not involved in calcium crosslink.s, and only secondarily of load-bearing calcium bridges. It is sug-
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Fig. 4. Relationship between PEx of methanol-boiled soybean sections and their calcium content. Different levels of calcium in the walls were created by incubation of methanol-boiled halfsections before PEx was measured with an Instron. K-phosphate (50 mM), pH 3, 3 h 9 K-phosphate (50 mM), pH 7_+ EGTA, 3 h 9 Na-acetate (50 mM), pH 6 • E G T A for varying times. A Na-acetate (50 mM), pH 3, 3 h 9 Na-acetate (50 mM), pH 6, 3 h 9 different buffers (50 mM), pH 4.5, 3 h | no incubation, or Mes-Tris (50 mM), pH 7, 3 h
^
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-~ 3 0
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400 600 800 Calcium ,ug/g DW
Fig. S. Effect of extensive removal of wall calcium on PEx of soybean hypocotyls. Boiled, bisected sections were incubated in 50 mM Na-acetate, pH 6.0+ 1 mM E G T A for up to i0 h. PEx and calcium content of the walls were then determined. The points can be fitted to two straight lines with greatly different slopes
gested that when this non-load-bearing calcium was gone, then the further loss of calcium was the more difficult to remove crosslinked calcium. Its removal led to large increases in PEx. The two phases of calcium-mediated wall loosening can also be seen from the time course of the effect of low p H on PEx and calcium removal (Fig. 6). During the first 2 h in low p H almost 2/3 of the calcium was removed, with only a limited
64
S.S. Virk and R.E. Cleland: Calcium and the mechanical properties of soybean hypocotyl cell walls Wall Calcium ~ug/g DW 200 400 600
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Fig. 6. Time course of acid-induced increase in PEx in soybean hypocotyls. Boiled sections were incubated in 50 m M Na-acetate at pH 3.5 (e) or pH 6.0 (o) for varying periods of time. Solutions were changed once after 3 h. Numbers represent wall calcium ( g g - ( g D W ) - 1 ; means of three experiments). The SE for Ca was less than 8%. Insert: relationship between PEx and wall calcium
increase in PEx. During the next 4 h, calcium continued to be lost at a much lower rate, and PEx increased sharply. A plot of calcium versus PEx (insert, Fig. 6), shows the two phases of the calcium/PEx relationship more clearly. Although all longitudinal walls in the hypocotyl contribute to the total wall strength, it is the increase in extensibility in the epidermal-subepidermal walls that is the cause of the auxin-induced cell enlargement (Masuda and Yamamoto 1972; Taiz 1984). In order to assess the contribution of calcium crosslinks in these cells to the mechanical properties of the whole tissue, methanol-boiled epidermal strips were incubated for up to 9 h in 50 m M Na-acetate, p H 6.0, with or without I m M EGTA, and PEx and wall calcium was measured. A plot of calcium versus PEx (Fig. 7) again shows two phases in the calcium/PEx relationship. In this case, removal of calcium had almost no effect on extensibility until a threshold of calcium was reached. When calcium was reduced below this threshold (about 200 gg. (g D W ) - l), PEx increased sharply in proportion to the amount of calcium removed. In general, then, the properties of half-sections are similar to those of the epidermal-subepidermal walls. The increase in PEx during the first couple of hours after addition of acidic buffers appeared to be largely the result of H § and anion-mediated extraction of the calcium. Over longer time periods, however, acid itself must have had a second effect on PEx in addition to its extraction of calcium. As shown in Fig. 8, when sections which had
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Wall Calcium (,ug/g DW) Fig. 7. Effect of removal o f wall calcium on PEx of isolated epidermal walls of soybean hypocotyls. Groups of 25-30 methanol-boiled epidermal strips were incubated in 50 m M Na-acetate, pH 6 . 0 _ 1 m M EGTA. After 1~9 h, sections were washed, PEx and wall calcium was determined. Some sections were incubated 1 h in E G T A and then returned to solutions-EGTA for up to 8 h
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Time (Hours) Fig. 8. Evidence for an acid-induced increase in PEx in boiled soybean hypocotyl walls unrelated to calcium removal. Boiled, bisected sections incubated for 2 h in 50 m M Na-acetate at pH 3.5, or at 6.0 with or without 1 m M EGTA, then subjected to analysis or transferred to fresh solution of the same kind or from pH 6 + E G T A to solutions without E G T A at pH 6 or 3. After 2 h, PEx and calcium content were determined. Numbers are wall calcium (gg. (g D W ) - 2). Note that sections transferred from pH 6 + E G T A to pH 3.5 had a much higher PEx, but the same calcium level as sections retained in pH 6 + EGTA
been incubated for 2 h at pH 6.0 with E G T A were incubated for an additional 2 h at p H 6.0 with E G T A or at p H 3.5, the removal of calcium was almost the same. However, PEx increased much more in the presence of the low-pH solution. Thus it would appear that two factors are influencing wall extensibility of soybean hypocotyl cell walls; the amounts of calcium crosslinks and of at least one additional acid-labile link in the walls.
S.S. Virk and R.E. Cleland: Calcium and the mechanical properties of soybean hypocotyl cell walls Discussion The role of calcium as part of the load-bearing bonds in cell walls has been a matter of some controversy. H o w does one assess this role? There are two main types of assay. The first is to measure the mechanical properties of cell walls after metabolic activity has been stopped by boiling the tissue in methanol or by freezing-thawing. The properties can be measured by the Instron, creep or stress relaxation assays (Cleland 1971; Taiz 1984); in theory, all give the same qualitative information about the viscoelasticity of the walls (Fujihara et al. 1978). The relationship between these measurements and wall extensibility, which is one of the parameters which control the rate of cell elongation, is not completely settled (Taiz 1984), but the Instron parameter PEx appears to be closely correlated with wall extensibility (Cleland 1967, 1984; Fig. 1). A second assay measures the facilitated creep of isolated cell walls when under applied tension (Cleland 1983; Cleland et al. 1987). Facilitated creep occurs in response to acid-induced cleavage of load-bearing bonds in many tissues (Cleland 1986). This assay evaluates the capacity of walls to undergo facilitated creep rather than measuring the wall extensibility that existed in live tissues at the time the walls were isolated. In this study we have used the Instron technique and have determined the effect of calcium removal and addition to the wall on the plastic extensibility, PEx. The data presented here are for methanol-boiled half-sections and epidermal strips. We have determined the calcium concentration for each set of sections after measurement of PEx by the Instron technique, so that the relationship between calcium content and the mechanical strength of the walls could be determined precisely. We show here that for soybean hypocotyl cell walls, added calcium reduced PEx, and removal of calcium with acidic buffers or E G T A increased PEx. The quantitative relationship between calcium removal and PEx is not simple. Removal of up to 2/3 of the calcium associated with the walls of half-sections resulted in some increase in PEx, with the amount of calcium removed being proportional to the increase in PEx. However, when more calcium was removed, the proportionality changed, and the increase in PEx was far greater. For epidermal strips, on the other hand, extensibility increased only after wall calcium had been lowered below a threshold. This indicates that the effect of calcium on PEx at calcium levels above this threshold is the consequence of calcium crosslinks in the inner tissues (cortex and vascular bun-
65
dles), while the effect of calcium removal at levels below the threshold is the consequence of the breakage of calcium crosslinks in the epidermal walls only or in the inner tissues as well. In assessing these data, several points should be kept in mind. First of all, PEx is not necessarily proportional to total wall calcium. The calcium content of the walls when first isolated was far from saturation. According to Yamaoka and Chiba (1983), a maximum of 12% of the uronic acids of soybean cell walls are bound to calciurn ions. Thus when the walls were incubated in CaClz, the calcium content increased as much as sevenfold with only a small decrease in PEx. Added calcium was absorbed onto the wall but produced few additional load-bearing crosslinks. Treatment of walls with E G T A increased PEx to the same extent, whether or not the walls were pretreated with calcium. Wall calcium, however, was three times higher in calcium pretreated walls (Fig. 2). It is important to recognize that the majority of wall calcium was not involved in calcium crosslinks, and could be removed without any change in PEx. Presumedly this was calcium associated with carboxyl groups in non-loadbearing layers of the walls (Taiz 1984) or calcium associated with a single carboxyl group, which failed to form a calcium crosslink between two carboxyl groups. It must also be remembered that the extractions may remove more wall components than just calcium ions. Jarvis (1982) found that the calcium chelator cyclohexanediaminetetraacetic acid (CDTA) extracted wall polysaccharides in addition to calcium. Buffers such as Tris-maleate have been found to decrease the calcium-binding capacity of isolated pea epicotyl cell walls, which indicates that they must have removed some wall polysaccharides (Baydoun and Brett 1984). The large increase in PEx after prolonged treatment with EGTA (10 h) or p H 3.5 (5 h) (Figs. 5, 6) could be the result of extraction of wall polysaccharides in addition to wall calcium. These data may seem to be in conflict with previous results with cell walls of other dicotyledons. For example, Courtney and Morr6 (1980) could find no effect on PEx of calcium added in vitro to boiled pea stem sections; however, since the cuticle was intact, it is likely that the calcium never reached the epidermal cell walls. Jarvis et al. (1984) concluded that they could not find a correlation between the amount of calcium removed by citrate from celery petiole collenchyma, and wall extensibility, but in fact they also found that extensibility increased greatly when the wall calcium fell below some threshold. Nakajima etat. (1981)
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S.S. Virk and R.E, Cleland: Calcium and the mechanical properties of soybean hypocotyl cell walls
found that added calcium ions decreased the creep of pea epidermal cell walls, while Mg 2+ increased it. Actual wall calcium measurements were not made on the tissues subjected to mechanical analysis, however. In Arena coleoptile cell walls, an increase in wall extensibility by acid appears to occur in response to an activation of wall-loosening enzymes by low pH, as indicated by the fact that inactivation of proteins by boiling or by removal by proteolytic enzymes renders the walls incapable of being loosened by acid (Rayle and Cleland 1977). In soybean hypocotyl walls, PEx can still be increased by acid, even after the walls have been boiled. This effect is, in part, the consequence of removal of calcium by the acid as well as by the accompanying anion. However, over longer periods of time, protons seem to be able to induce pronounced wall loosening by a second, calciumindependent mechanism. Whether this involves a breakage of hydrogen bonds in the walls, is the result of extraction of some polysaccharide component of the wall, the result of acid-activation of wall loosening enzymes that are resistant to boiling methanol, or is caused by some other mechanism, remains to be determined. Our data indicate that calcium crosslinks are important as load-bearing bonds in soybean hypocotyl cell walls. The crosslinks are probably not the classic "egg box" calcium-pectate interactions (Rees 1969), but more complex interactions involving wall polyuronides (Tepfer and Taylor 1981). Wall-loosening during auxin-induced growth could involve a breakage of these crosslinks. It has been shown that auxin causes proton excretion in soybean hypocotyls (Rayle and Cleland 1977). The protons may cause solubilization of the calcium involved in crosslinks, and thus may contribute to an increase in wall extensibility. However, it would appear that wall extensibility is also dependent upon a second set of load-bearing bonds that are cleaved under acidic conditions and that do not involve calcium. Both sets of bonds may have to be broken in soybean hypocotyl walls in order for maximum wail extension to occur. If so, the mechanism of wall-loosening in these walls in response to auxin would be fundamentally different from that in Arena coleoptiles. It is of interest that Moll and Jones (1981) suggested that the gibberellin-induced increase in wall extensibility which occurs in lettuce hypocotyl walls involves an uptake of calcium into the cells from the walls. However, the calcium solubilized by the protons does not need to be taken up into the cells, but can simply remain in the apoplastic
solution as long as the solution remains acidic. Alternatively, the calcium can be displaced from the load-bearing inner cell wall layers to the mechanically-unimportant outer regions of the wall. This research was supported by contract DE-AM0676ER73019 from the U.S. Department of Energy.
References Baydoun, E.A.-H., Brett, C.T. (1984) The effect of pH on the binding of calcium to pea epicotyl cell walls and its implications for the control ofcel! extension. J. Exp. Bot. 35, 18201831
Bennett-Clark, T.A. (1956) Salt accumulation and mode of action of auxin. A preliminary hypothesis. In: The chemistry and mode of action of plant growth substances, pp. 284~ 291, Wain, R.L., Wightman, F., eds. Butterworths, London Cleland, R.E. (1967) Extensibility of isolated cell wails: measurement and changes during cell elongation. Planta 74, 197-209 Cleland, R.E. (1971) Cell wall extension. Annu. Rev. Plant Physiol. 22, 19~222 CMand, R.E. (1983) The capacity for acid-induced wall loosening as a factor in the control of Arena coleoptile cell elongation. J. Exp. Bot. 34, 676-680 Cleland, R.E. (1984) The Instron technique as a measure of immediate-past wall extensibility. Planta 160, 514-520 Cleland, R.E. (1986) The role of hormones in wall loosening and plant growth. Aust. J. Plant Physiol. 13, 93 103 Cleland, R.E., Rayle, D.L. (1977) Reevaluation of the effect of calcium ions on auxin-induced elongation. Plant Physiol. 60, 709-712 Cleland, R.E., Cosgrove, D., Tepfer, M. (1987) Long-term acidinduced wall extension in an in-vitro system. Planta 170, 379 385 Cooil, B., Bonner, J. (1957) Effects of calcium and potassium ions on the auxin-induced growth of Arena coleoptile sections. Planta 48, 696-723 Courtney, J.S., Morr6, D.J. (1980) Studies on the role of wall extensibility in the control of cell expansion. Bot. Gaz. 141, 56-62 Fujihara, S., Yamamoto, R., Masuda, Y. (1978) Viscoelastic properties of plant cell walls. III. Hysteresis loop in the stress-strain curve at constant strain rate. Biorheology 15, 87-97 Jarvis, M.C. (1982) The proportion of calcium-bound pectin in plant cell walls. Planta 154, 344-346 Jarvis, M.C., Logan, A.S., Duncan, H.J. (1984) Tensile characteristics of collenchyma cell walls at different calcium contents. Physiol. Plant. 61, 81-86 Masuda, Y., Yamamoto, R. (1972) Control of auxin-induced stem elongation by the epidermis. Physiol. Plant. 27, 109 115 Moll, C., Jones, R.L. (1981) Calcium and GA-induced elongation of lettuce hypocotyl sections. Planta 152, 450-456 Nakajima, N., Morikawa, H., Igasashi, S., Senda, M. (1981) Differential effects of calcium and magnesium on mechanical properties of pea stem cell walls. Plant Cell Physiol. 22, 1305-1315 Prat, R., Goeissaz, M.-B., Goldberg, R. (1984) Effects of Ca 2+ and Mg 2+ on elongation and H + secretion of Vigna radiata hypocotyl sections. Plant Cell Physiol. 25, 1459 1467 Rayle, D.L., Cleland, R.E. (1977) Control of plant cell enlargement by hydrogen ions. Curr. Top. Devel. Biol. 11, 187-214
S.S. Virk and R.E. Cleland: Calcium and the mechanical properties of soybean hypocotyl cell walls Rees, D.A. (1969) Structure, conformation, and mechanism in the formation of polysaccharide gels and networks. Adv. Carbohydr. Chem. Biochem. 24, 267 332 Soll, H., B6ttger, M. (1982) The mechanism of proton-induced increase in wall extensibility. Plant Sci. Lett. 24, 163-171 Tagawa, T , Bonner, J. (1957) Mechanical properties of the Arena coleoptile as related to auxin and to ionic interactions. Plant Physiol. 32, 207 212 Taiz, L. (1984) Plant cell expansion; regulation of cell wall mechanical properties. Annu. Rev. Plant Physiol. 35, 585657
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Tepfer, M., Taylor, I.E.P. (1981) The interaction of divalent cations with pectic substances and their influence in acidinduced cell wall loosening. Can. J. Bot. 59, 1522 15125 Yamaoka, T., Chiba, N. (1983) Changes in the coagulating ability of pectin during the growth of soybean hypocotyls. Plant Cell Physiol. 24, 1281 1290
Received 17 January; accepted 31 May 1988
