JXB Advance Access originally published online on November 16, 2006
Journal of Experimental Botany 2006 57(15):3989-4002; doi:10.1093/jxb/erl166
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
RESEARCH PAPER |
Calcium pectate chemistry controls growth rate of Chara corallina
College of Marine Studies and College of Agriculture and Natural Resources, 700 Pilottown Road, University of Delaware, Lewes, DE 19958, USA
* To whom correspondence should be addressed. E-mail: boyer{at}cms.udel.edu
Received 16 February 2006; Accepted 21 August 2006
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionConclusionsAppendixReferences |
|---|
Pectin, a normal constituent of cell walls, caused growth rates to accelerate to the rates in living cells when supplied externally to isolated cell walls of Chara corallina. Because this activity was not reported previously, the activity was investigated. Turgor pressure (P) was maintained in isolated walls or living cells using a pressure probe in culture medium. Pectin from various sources was supplied to the medium. Ca and Mg were the dominant inorganic elements in the wall. EGTA or pectin in the culture medium extracted moderate amounts of wall Ca and essentially all the wall Mg, and wall growth accelerated. Removing the external EGTA or pectin and replacing with fresh medium returned growth to the original rate. A high concentration of Ca2+ quenched the accelerating activity of EGTA or pectin and caused gelling of the pectin, physically inhibiting wall growth. Low pH had little effect. After the Mg had been removed, Ca-pectate in the wall bore the longitudinal load imposed by P. Removal of this Ca caused the wall to burst. Live cells and isolated walls reacted similarly. It was concluded that Ca cross-links between neighbouring pectin molecules were strong wall bonds that controlled wall growth rates. The central role of Ca-pectate chemistry was illustrated by removing Ca cross-links with new pectin (wall ‘loosening’), replacing vacated cross-links with new Ca2+ (‘Ca2+-tightening’), or adding new cross-links with new Ca-pectate that gelled (‘gel tightening’). These findings establish a molecular model for growth that includes wall deposition and assembly for sustained growth activity.
Key words: Calcium, cell wall, Chara corallina, magnesium, pectin, turgor pressure, wall growth
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionsAppendixReferences |
|---|
This work was undertaken to investigate the growth-accelerating activity of pectin, a normal cell wall constituent of plants. The activity was first reported by Proseus and Boyer (2006b) and was detected as a reversal of declining growth in isolated cell walls, returning wall growth to the rapid rates characteristic of living cells. Because the pectin is a wall constituent, this finding suggests a link between growth and the deposition of new cell wall.
When plant cells grow, their size is determined by their walls that typically extend 10–100-fold (Roberts, 1994). This large extension would cause the walls to become much thinner if new wall was not deposited simultaneously, and several studies indicate that deposition maintains the thickness to within a factor of about two (Taiz, 1984; Kutschera, 1990, and references therein; Bret-Harte et al., 1991). Under the electron microscope, the new primary wall appears to integrate seamlessly with the old one, and new polymers line the inside of the old wall and extend into the old one (Northcote and Pickett-Heaps, 1966; Ray, 1967; Morrison et al., 1993).
The wall extension is driven by turgor pressure (P) that stretches the walls irreversibly as load-bearing bonds naturally break and re-form between wall polymers, causing the polymers to slip past each other under the tension from P (Passioura and Fry, 1992; Passioura, 1994). Recent evidence indicates that these P also are necessary for much of the deposition of new cell wall (Proseus and Boyer, 2006a). Polysaccharides presented to the wall are often too large to enter the wall spontaneously, but are concentrated in the periplasm and moved into the old wall by P (Proseus and Boyer, 2005). The high concentrations cause pectins to cross-link spontaneously (Proseus and Boyer, 2006a). Importantly, the rejuvenating activity of pectin reported by Proseus and Boyer (2006b) acted on walls isolated from the cytoplasm and thus unable to synthesize new wall.
The chemical mechanisms controlling the rate of wall extension are uncertain for plants and, consequently, the growth activity of pectin is of considerable interest. In the following work, the activity was investigated by maintaining P at growth-promoting levels while pectins were supplied to isolated walls or living cells in an otherwise normal culture environment.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionsAppendixReferences |
|---|
Plant material
The experiments were conducted with single internode cells of the alga Chara corallina (Klein ex. Willd., em. R.D.W.) grown at 22–23 °C as described by Proseus and Boyer (2006b). Young, growing internodes were obtained from the apex of the thallus to ensure that the cells had primary walls. Mature internodes were obtained from the base of the thallus. For live cells, P was measured, changed, and controlled by using a pressure probe (Steudle and Zimmermann, 1974) to remove or add cytoplasmic solution without altering the external medium, as described by Zhu and Boyer (1992). For isolated walls, isolation and attachment to the pressure probe were carried out according to Proseus and Boyer (2005, 2006a, b). P was controlled as in the live cells, except the lumen, previously occupied by the cytoplasm, was filled with mineral oil and the oil was removed or added to the lumen with the probe. P were comparable for the live cells and isolated walls. Wall extension was measured simultaneously in the same cells or walls using a position transducer by the methods described by Proseus et al. (1999). All experimental manipulations were conducted at 22–23 °C in the culture medium in which the plants were grown except as noted.
PGA and EGTA
Polygalacturonic acid (poly-
-(1–4)-D-galacturonate, 80% potassium salt, denoted as PGA) was used for most of the experiments. Obtained from citrus albedo (Sigma, St Louis, MO, USA), its molecular weight was 170 kDa (about 945 saccharide residues) measured by size exclusion chromatography at the Complex Carbohydrate Research Center, Athens, GA, USA. The chromatography was conducted by eluting the PGA in ammonium formate (pH 4.8) on a Superose 75 column (Amersham Biosciences, San Francisco, CA, USA). For experiments, this PGA was added to culture medium taken directly from the Chara cultures and filtered to 0.2 µm to remove cells and debris. The final PGA concentration was 35 µM (6 mg ml–1). The pH of the solution was measured, and if necessary, adjusted to 7 before use. For a few trials the PGA solution was dialysed for 72 h at 4 °C against 4.0 l of water changed twice using dialysis cassettes having a molecular weight cut-off of 3500 (Pierce, Rockford, IL, USA).
In one experiment, citrus albedo PGA of small molecular weight (1–3.5 kDa, i.e. 5–19 saccharide residues) was obtained from a second source (Complex Carbohydrate Research Center, Athens, GA, USA) (PGA-CCRC). Its molecular weight was determined at the Complex Carbohydrate Research Center by anion exchange chromatography on a Dionex Carbo-Pak PA1 column (Dionex Corp, Sunnyvale, CA, USA) with NaOH as eluant (1 ml min–1). Solutions of PGA-CCRC were prepared at 6 mg ml–1, pH 7, in culture medium filtered as above.
In other experiments, 2.5 mM EGTA (ethylene glycol-bis(ß-aminoethyl ether) N,N,N',N'-tetra-acetic acid) (Sigma, St Louis, MO, USA) was prepared with filtered culture medium and adjusted to pH 7 with NaOH before use.
All solutions were stored at 4 °C and used within 3 d or frozen at –20 °C and used within 2 weeks.
PGA-pectinase digest
For some experiments, a solution containing 170 kDa PGA (6 mg ml–1, Sigma) was prepared with endopectinase from Aspergillus niger (1.25 mg ml–1, approximately 4–11 units ml–1) (MP Biomedicals, Aurora, OH, USA, Lot R2162, EC 3.2.1.15
[EC]
) in water at pH 5. The PGA/pectinase mixture was incubated on a rotary shaker (40 rpm) at 40 °C for 90 min to decrease the molecular weight of the PGA. The resulting PGA-digest was transferred to a new vial without transferring any insoluble flocculent remaining in the bottom of the reaction vial. The solution was boiled gently for 10 min to stop the reaction, cooled to room temperature and adjusted to pH 7 with NaOH.
In order to detect the decrease in molecular weight during the digestion, the gelling ability of the PGA-digest was assessed periodically by adding Ca2+ to a sample obtained from the digest. At each sampling, 0.1 ml of 1 M CaCl2 was added to the 1 ml sample of the digest, in a preweighed test tube. The solution was vortexed briefly and centrifuged at 700 g for 10 min (Damon/IEC HN-SII centrifuge). The supernatant was poured into a separate preweighed tube leaving the gelled PGA in the first tube. Both tubes were dried to a constant mass at 80 °C and the dry weights were recorded.
Analysis of cell wall cations
After removing both ends of the cell, the walls were isolated according to Proseus and Boyer (2005, 2006a) and rinsed twice in water for 1 min. The walls from several cells were pooled until a total length of 70 mm of wall was present. The pooled sample was placed in 5 ml of water or PGA solution (35 µM, 170 kDa) or EGTA solution (2.5 mM) held in acid-washed (10% HCl for 24 h) test tubes that were capped. All equipment contacting these preparations was acid-washed prior to use. After incubating the walls for 1.5 h at 23 °C in the water (untreated walls) or PGA or EGTA solutions (referred to as PGA or EGTA extracts), the walls were removed, blotted on tissue paper, and transferred to separate dry tubes. The walls in the dry tubes were digested in 0.25 ml of purified HNO3 (Fisher Optima brand, Lot 127052) for at least 1 h at 23 °C and heated to 120 °C (uncapped) over an electric plate for 5–8 min until the walls were visibly dissolved. The tubes were cooled, brought to 5 ml with water, recapped and stored at 4 °C prior to analysis. HNO3 without cell walls served as a blank. Other PGA and EGTA tubes did not receive cell walls and served as additional blanks. Another tube received 5 ml of culture medium filtered to 0.2 µm. This procedure was replicated four times, and all cells were obtained from the same culture vessel.
The digested cell walls, culture medium, extracts, PGA and EGTA blanks, and the HNO3 blanks were analysed for Ca and Mg content by inductively-coupled plasma (ICP) spectrometry on a Jobin-Yvon model JY70Plus spectrometer (Jobin-Yvon, Edison, NJ, USA). In addition to the Ca and Mg analyses, the content of Zn, Cu, B, Fe, K, and Na were determined in the cell walls, culture medium, and HNO3 blank. Linear calibration for each element was performed prior to each analysis session. The ICP-reported values for Ca and Mg in the HNO3 blank were used to correct the values reported from tubes containing cell wall material to account for any ions introduced with the acid. Likewise, ICP-reported values from the PGA and EGTA blanks were subtracted from the values reported from the PGA and EGTA extracts.
Volume and dry weight of cell walls
A mean cell radius was measured in 10 intact, fully turgid cells using a digital caliper. Transverse slices of cell wall were prepared with a razor blade from each of the 10 cells, stained for 30 min in 0.1 mg ml–1 Congo red, and viewed under a light microscope to measure wall thickness. The isolated walls were dried to constant mass at 80 °C and dry mass was measured by weighing.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionsAppendixReferences |
|---|
Growth activity of PGA and EGTA
Recent work from this laboratory established that irreversible growth occurred in isolated Chara walls and was driven by P in every respect like live cells except that the rate decelerated while the live cells continued to grow rapidly (Proseus and Boyer, 2006b). Supplying PGA to the isolated walls restored rapid growth that lasted for several hours. Therefore, it seemed possible that the growth of live cells might also respond to PGA, implicating pectin in the living system. Placing PGA in the culture medium around live cells (Fig. 1A, B) or isolated cell walls (Fig. 1C, D) caused a similar rapid increase in elongation rates in both systems. Replacing the PGA medium with fresh culture medium returned elongation to the rates seen prior to the PGA exposure. Mature cells did not grow in normal medium and exhibited minimal response to the PGA (Fig. 1E, F). Identical trials with dialysed PGA solution gave results identical to those in Fig. 1 (data not shown), eliminating the possibility that traces of small molecular weight compounds influenced the results. Elongation rates did not respond to replacing the normal medium with water (pH 7) (Fig. 1G, H). Although the cell grew particularly vigorously in Fig. 1G, H, the same results were obtained with replicate cells that grew more slowly.
|
The culture medium normally developed a pH around 8 during the growth of the plants, but there was little effect of lowering the pH to 4.5 (Fig. 2A) or 3.5 (Fig. 2B). Growth in the live cells proceeded at the same rate as at pH 8.1 (Fig. 2A) or increased slightly (Fig. 2B). PGA remained active and growth accelerated whenever PGA was added to the medium, regardless of pH as low as 3.5 (Fig. 2A, B) or as high as 11 (data not shown). Measurements immediately before and after each experiment indicated that the pH of the medium was always stable within 0.3 of a unit except at a pH of 3.5 where the pH rose as much as 1.3 units. It should be noted that these experiments were conducted in the culture medium without additional buffering because anionic buffers such as citrate or succinate can act as chelators of ions.
|
The synthetic chelator EGTA mimicked the effect of PGA on elongation when the EGTA was supplied at a low concentration of 2.5 mM. There was a rapid acceleration when the EGTA was added to the medium around the live cells (Fig. 3A, B) or isolated walls (Fig. 3C, D). There was a deceleration to the original rates when the EGTA was replaced by normal culture medium.
|
Elemental concentration in culture medium and cell walls
Table 1 shows that inorganic elements in the wall were Ca and to a lesser extent Mg. Other elements were below the detection limits for the measurement. Likewise in the culture medium, Ca2+ and Mg2+ were the dominant inorganic ions with K+ and B detected in small amounts. By expressing these concentrations as mmol kg–1, a simple comparison of medium and wall concentrations indicates that the wall accumulated large amounts of Ca and Mg from the dilute culture medium.
|
Extraction of walls with EGTA or PGA
ICP analysis revealed that 2.5 mM EGTA or 35 µM PGA used in the above experiments removed Ca and Mg from the walls (Fig. 4). In Fig. 4A, untreated walls had a mean Ca concentration of 270 mmol kg–1 dry wall, while exposure to the EGTA or PGA solution for 1.5 h reduced the concentration to 14 and 88 mmol kg–1 dry wall, respectively (Fig. 4A). The Ca removed from the walls was recovered in the solution (white bars, Fig. 4A). The recovery confirmed that 95% and 67% of the Ca had been removed by the EGTA and PGA, respectively. Similarly for Mg, untreated walls had a mean concentration of 70 mmol kg–1 dry wall (Fig. 4B), and EGTA removed all of the Mg from the walls after 1.5 h. PGA removed nearly all of the Mg (Fig. 4B). The Mg removed from the walls was fully recovered in the EGTA or PGA solutions (white bars, Fig. 4B).
|
Quenching the growth activity of PGA
Because Ca was the most abundant inorganic element in the walls and was partially removed by treatment with EGTA or PGA, it seemed possible that excess Ca2+ might quench the activity of PGA. Excess CaCl2 (50 mM) added to normal culture medium moderately inhibited elongation in isolated walls (Fig. 5A) while P was held at 0.5 MPa (P trace not shown). Increasing Ca2+ concentrations to 100 mM did not cause additional inhibition (not shown). If PGA was added to the culture medium, growth in the isolated walls accelerated but, when followed by 50 mM CaCl2, the acceleration was completely quenched (Fig. 5B). The Ca2+ caused the PGA to gel around the cell, enveloping the wall, probe tip, and transducer wire, and elongation became near zero. Mg2+ did not cause a gel to form and instead formed a loose precipitate with PGA (data not shown). Likewise, CaCl2 quenched EGTA-stimulated elongation in the isolated walls (Fig. 5C), but no gelling occurred. The wall resumed elongation at a rate close to the original after the CaCl2 was added.
|
In order to test whether the action of PGA depended on its gelling action, the PGA was digested with pectinase. Aliquots from the PGA/pectinase digestion showed a progressive loss of gelling ability in the presence of Ca2+ (Fig. 6A) until, after 30 min of pectinase treatment, no gelling was detected. Prior to digestion, adding the PGA solution to a live cell evoked the anticipated acceleration response, and replacing the PGA with fresh culture medium returned growth to the original rate (Fig. 6B, C). The small molecular weight PGA-digest after 90 min of pectinase digestion retained these stimulatory effects (Fig. 6D, E). As with the large molecular weight PGA (Fig. 5B), the rapid elongation in PGA-digest could be quenched by the addition of 50 mM CaCl2, shown for an isolated wall in Fig. 7A, B. However, in contrast to Fig. 5B, growth did not cease in the digest because gelling did not occur.
|
|
All of the PGA tests described above used PGA from a single source (Sigma). Consequently, citrus PGA from a second source was tested (designated PGA-CCRC, approximate molecular weight of 1.1–3.6 kDa). PGA-CCRC applied to live cells (Fig. 8B) caused an elongation response indistinguishable from the action of 170 kDa PGA supplied by Sigma (Fig. 1A).
|
Response to extreme Ca removal
In the above experiments, the removal of 95% of the wall Ca and the entire wall Mg with EGTA accelerated growth in the isolated wall. If the EGTA concentration was increased to 25 mM around isolated walls to remove further wall Ca content, i.e., to decrease wall Ca below the 5% remaining when treated with 2.5 mM EGTA (Fig. 4A), wall acceleration was followed by an abrupt loss of P (Fig. 9A, B). The oil in the lumen leaked out and could be seen as an oil droplet bulging from the wall, indicating that the wall had burst. Depending on the cell, the droplet appeared at various positions along the length of the wall. The wall became flaccid when the droplet appeared but the wall otherwise remained intact.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionsAppendixReferences |
|---|
The experiments indicate that PGA extracted Ca and Mg from the cell wall and thus accelerated the growth rate of the wall and living cells. Ca appeared to be the critical element because the wall retained its integrity with only 5% of the normal Ca complement and no Mg. The wall burst if more of the Ca was removed. Ca2+ was able to form a gel with large PGA, but Mg2+ did not. Also, Metraux and Taiz (1977) found that Ca2+ inhibited wall extension in Nitella while Mg2+ had the opposite effect. It thus appears that wall Mg played little part in the growth response.
Nevertheless, charophyte cell walls possess a large capacity for multivalent cation binding and cross-linking because the walls are rich in PGA (Morrison et al., 1993; Popper and Fry, 2003). The carboxyl groups are largely unmethylated and free to dissociate (Anderson and King, 1961; Morikawa and Senda, 1974; Gillet et al., 1992, 1998; Morrison et al., 1993; Gillet and Liners, 1996; Popper and Fry, 2003). Provided there are regions of at least 10–20 consecutive unmethylated GalA residues in each PGA molecule, clusters of two or possibly four adjacent PGA molecules will form non-covalent interpolymer associations with divalent cations (Grant et al., 1973; Morris et al., 1982; Powell et al., 1982; Jarvis, 1984; Jarvis and Apperley, 1995; Ralet et al., 2001; Willats et al., 2001). These associations form junction zones cross-linking the PGA, and a gel can result if Ca2+ is the cross-linking cation (Jarvis, 1984; Jarvis and Apperley, 1995; Willats et al., 2001). The large PGA used in this study contained approximately 750 galacturonic acid residues (80% of 945 residues in 170 kDa polymer), which provided ample opportunity for Ca cross-links and accounted for the gelling with Ca2+. If the PGA was digested to a smaller molecular weight, gelling did not occur but the digested PGA remained active in growth acceleration, probably because Ca-complexing activity required a minimum of only two GalA binding sites per Ca.
It is worth noting that the amount of 170 kDa PGA causing growth to accelerate was 17.9 times more than necessary to bind all of the cations in the medium+wall+reagents (see W in Table 2, Appendix). The PGA was thus in excess, but had a low concentration (6 mg g–1). The PGA remained soluble because the amount of calcium in the medium+wall+reagents was too small to form sufficient cross-links for gelling, and adding more Ca2+ caused a gel to form. By contrast, PGA in the periplasm of the living cells is likely to have a higher concentration than in the medium. For example, at growth-promoting P of 0.5 MPa, 170 kDa PGA would have a concentration of about 140 mg g–1 or 24 times higher than in the medium according to Proseus and Boyer (2006a). At this concentration, gelling occurs whether Ca2+ is present or not (Proseus and Boyer, 2006a). Consequently, gelling is likely to be a central feature of wall assembly in charophytes.
Load-bearing by Ca-PGA
The Ca and Mg accumulated by Chara walls from the culture medium occupied about 39% of the available charged groups in the walls (estimated, see U+V in the table in the Appendix). Because about 95% of this Ca and essentially all of the Mg could be removed without losing wall integrity, the tensions generated by P could be borne by only a few Ca-PGA cross-links. That these were critical load-bearing bonds was evident when some of the remaining Ca was removed with high concentrations of EGTA (25 mM) and the wall burst at normal P of 0.46 MPa. Confirmation of this load-bearing ability of Ca-PGA occurred when a Ca-PGA gel formed around an isolated wall. The new Ca-PGA cross-links were strong enough to inhibit wall growth fully.
The wall matrix of Chara also contained xylose suggesting the presence of xyloglucan (Preston, 1974), but Popper and Fry (2003) were unable to detect xyloglucans characteristic of terrestrial species. In pea stems, exogenous xyloglucan bound to the cell walls and decreased growth rates, which suggested a load-bearing function (Takeda et al., 2002). Xyloglucans are thought to cross-bridge between pectins and cellulose microfibrils with intertwining segments of the molecules or with hydrogen bonds (Passioura and Fry, 1992; Passioura, 1994; Fry, 2004) or covalent bonds (Thompson and Fry, 2000). Being uncharged, they would not participate in cation binding in Chara, but if present they might help the matrix and microfibrils retain some coherency after Ca was removed by the EGTA.
Gillet et al. (1992) reported a direct relationship between PGA content and cation binding capacity of Nitella cell walls. When Gillet and Liners (1996) exposed Nitella walls to concentrated solutions of the Ca chelator CDTA (20 mM), the walls eventually developed a delaminated appearance when viewed in an electron microscope, suggesting disassembly. Less Ca-bound PGA was detected in the walls. These results align with the high-EGTA experiment in which 25 mM EGTA removed most of the Ca and Mg from the wall and the wall burst. By contrast, the growth experiments of the present work used much lower concentrations of PGA and EGTA than in the Gillet and Liners (1996) experiments. Some Ca remained bound, wall strength returned when the PGA or EGTA was washed away with culture medium, and growth resumed at the original rate. This recovery suggests re-binding of Ca to previously vacated PGA sites rather than wall dis-assembly as in Gillet and Liners (1996).
Chelation by PGA
The affinity of the walls for Ca2+ is reflected in a pK of 3.5 for the dissociation of Ca-PGA (Van Cutsem and Gillet, 1983; Jarvis, 1984). EGTA, which is a chelator with particularly strong affinity for Ca2+ (pK of 11 according to Martell and Smith, 1974), displayed cation extraction and growth accelerating activity similar to PGA. Therefore, the growth accelerating action of PGA lies in its chelation chemistry.
When culture medium containing PGA or a moderate concentration of EGTA was replaced with fresh culture medium, the external chelating activity was removed and new Ca2+ and Mg2+ were supplied as a normal part of the fresh medium (2.92 µmol, M+O in the table in the Appendix). The new supply was in excess of the amount needed to replace the Ca and Mg in the wall completely (0.025 µmol, H+J in the table in the Appendix). The growth returned to the rate prior to exposure to the PGA or EGTA, as expected if there is a dynamic equilibrium between free Ca2+ and bound Ca in the Ca-PGA complex. Operationally, the equilibrium allowed wall cations to be removed by adding the chelator to the culture medium or replaced in the wall by washing away the chelator with fresh Ca2+-containing culture medium.
Metraux and Taiz (1977) found that exposing Nitella walls to monovalent ions increased extension rates while multivalent ions (except Mg2+) tended to decrease the rates. These ions probably displaced wall Ca, and increased or decreased extension might then reflect the differing ability to form cross-links with pectin. Van Cutsem and Gillet (1983) found that H+ could displace wall Ca. A source of H+ is available at the inner face of the cell wall where acid is produced by the action of plasma membrane H+-ATPases in live Nitella and Chara cells (Hope and Walker, 1975; Metraux et al., 1980). In this periplasmic space, the pH is maintained between 4.5 and 5.0, and with the medium at pH 7 or 8, a pH gradient was probably present across the wall in the live cells. However, decreasing the pH of the medium had little effect on their growth perhaps because substantial Ca2+ was always present in the culture medium, and wall Ca may not have been displaced. It thus seems unlikely that the pH gradient played a role in cell growth under the culture conditions.
Growth acceleration by PGA
After maturation, charophytes develop a rigid secondary wall with thick helical bands of cellulose-rich wall laid down along the inner face of the primary wall (Metraux, 1982; Morrison et al., 1993; Toole et al., 2002). In the present work, new PGA supplied to such mature cells lacked activity. PGA was active only with the primary wall, whose properties were distinguished by a large temperature response of growth in the isolated wall and a critical P that had to be exceeded before growth occurred, shown by Proseus and Boyer (2006b). These properties indicated that the load-bearing bonds in the wall are strong and require large activation energy before growth occurs. Only a few bonds would have sufficient energy.
From the present experiments, Ca-PGA cross-links under load from P are identified as the strong bonds. Based on bursting of the walls when most Ca was removed from the wall, the Ca-PGA cross-links were clearly strong enough to bear the longitudinal load. All of the measurements were made at a P around 0.5 MPa, i.e. typical of growing Chara cells (Proseus et al., 1999, 2000) and above the 0.35 MPa threshold needed to begin breaking the strong bonds (Proseus and Boyer, 2006b). Because the Ca cross-links are in dynamic equilibrium with free Ca2+ in the wall and thus break and re-form spontaneously, the cross-links would tend to slip irreversibly under tension from P, accounting for irreversible deformation of the wall at growth-promoting P (Proseus and Boyer, 2006b). The amount of Ca-PGA cross-linkage in the wall was thus the key to the changes in wall deformability with P and temperature, and thus the rate of growth.
According to this hypothesis, isolated walls would display a deceleration in growth if they were not supplied with new PGA. Isolated walls undergo this type of deceleration (Metraux et al., 1980; Taiz et al., 1981; Taiz and Richmond, 1984; Proseus and Boyer, 2006b). In the absence of new PGA, the spontaneous slippage of the tethering Ca-PGA in the wall would extend the wall and cause more Ca-PGA cross-links to become taut tethers (Passioura and Fry, 1992; Passioura, 1994). The accumulation of taut Ca-PGA would increase the population of polymers sharing the load of P and causing growth to decelerate. On the other hand, if new PGA was supplied, some of the wall Ca would be removed. The accumulation of taut tethers might then be prevented and allow growth to continue without deceleration, as shown by Proseus and Boyer (2006b).
Together, these concepts identify three types of PGA action that affect growth rates. They are most readily seen in isolated walls, and a close comparison of Figs 5B and 7 shows their action in Fig. 10. Figure 10A indicates that P was identical for the comparison and exceeded the critical P for growth. In Fig. 10B, isolated walls exposed to these P experienced accelerated growth when new 170 kDa PGA or digested 170 kDa PGA was supplied. The PGA chemistry would remove moderate amounts of Ca from the wall, probably decreasing the number of cross-links and increasing wall deformability (arrow 1, Fig. 10B; wall ‘Loosening’ in Fig. 10C). Replacing the PGA with fresh medium allowed new Ca2+ to enter from the external medium and replenish the vacated cross-links in the wall. For PGA that did not gel, wall growth returned nearly to its rate before PGA addition (upper arrow 2, Fig. 10B; ‘Ca2+ Tightening’, in Fig. 10C). If enough new Ca2+ entered from the external medium to cause gelling, new cross-links formed (lower arrow 2, Fig. 10B; ‘Gel Tightening’, in Fig. 10C). The wall markedly tightened with these new cross-links, and wall growth ceased. This suggests that the new cross-links were strong, and assuming they would form in newly assembled wall, tightening actions would follow loosening actions in the same PGA molecules.
|
Molecular model for growth
Evidence from this study indicates that these three types of PGA action may apply not only to isolated walls, but also to living cells. A tentative model for growth in living cells is offered in Fig. 11 for a single molecular unit of growing wall structure. The unit consists of two cellulose microfibrils embedded in and bonded to xyloglucans and pectins. The high tensile strength of the transversely-oriented cellulose prevents the wall from expanding laterally (Probine and Preston, 1962; Preston, 1974; Jarvis, 1984; Ridley et al., 2001; Baskin, 2005) while Ca-PGA controls the expansion longitudinally. This pattern of wall polymers causes wall extension to be anisotropic, and the microfibrils to spread apart as the cells elongate.
|
With P present, some of the Ca-PGA in the wall is taut and load-bearing (Fig. 11A). The tension straightens the PGA, constraining its motion and increasing its order. If the tension diminishes, the PGA spontaneously returns to a more random, mobile configuration, as expected when the wall deforms elastically as described by Proseus et al. (1999). The Ca-PGA equilibrates with free Ca2+ in the wall and exchanges Ca with it. At growth-promoting P above 0.35 MPa, the cross-links in the Ca-PGA come under sufficient load to slip gradually during the exchange with free Ca2+, as suggested in a model by Proseus and Boyer (2006b). With slippage, however, the stretching causes additional Ca-PGA to come under load, and growth decelerates unless new PGA is released to the periplasmic space by the cytoplasm. With new PGA (Fig. 11B), new anionic charge is present and extracts some of the Ca previously cross-linked to PGA in the wall. The wall loosens and growth accelerates. The high turgor causes some of the new PGA to enter the wall, as shown by Proseus and Boyer (2005) for large polysaccharides. The transfer of Ca to the new PGA depletes Ca in the existing wall and favours the entry of new Ca2+ from outside (Fig. 11C). The new Ca2+ binds to the vacated PGA sites and re-strengthens the wall. Growth returns toward its original rate. However, the extracted Ca now bound to the new PGA can begin cross-linking to the existing wall and to other new Ca-PGA (Fig. 11D). If the molecular weight of the new PGA is large, a gel-like matrix forms at the inner wall face and inside the existing wall where new PGA has penetrated, as shown for polysaccharides by Proseus and Boyer (2005, 2006a). Moreover, if P is above the critical level, gelling can occur without new Ca2+ (Proseus and Boyer, 2006a). The wall thickens and is markedly strengthened by this new deposition and assembly of wall material.
Accordingly for living cells, the rate of cell growth appears to be maintained by the rate of release of new PGA to the periplasm. Loosening, Ca2+-tightening, and gel-tightening take place but in a continuum (Figs 10, 11). Because Proseus and Boyer (2005) found that large polysaccharides accumulate with high concentration in the periplasm and can enter small interstices in the existing wall when P is high, the new wall interpenetrates the old wall as shown in Fig. 11B–D, and integrates seamlessly with the existing wall. The new PGA deposited together with other wall constituents not only control growth rate, but also prevent structural failure as part of the growth process. The model thus suggests that the rate of deposition is roughly in proportion to the rate of growth in turn linked to the rate of supply of new PGA and other wall constituents.
The model is consistent with the finding that P required for growth are also required for rapid wall deposition in the live cells, as reported by Proseus and Boyer (2006a). The model does not require wall enzymes, which is consistent with the continued growth activity of isolated Chara cell walls after boiling (Proseus and Boyer, 2006b). The orientation of Ca cross-links is in agreement with previous studies of the longitudinal orientation of some of the pectins and transverse orientation of the Ca bridges in the charophyte wall under stress (Morikawa and Senda, 1974; Morikawa et al., 1974; Richmond et al., 1980). The model may add to the seminal findings of Baker and Ray (1965) that the growth-accelerating action of auxin in Avena is linked to an increased deposition of non-cellulosic constituents in the wall (Ray and Baker, 1965). Auxin's action was partially prevented by 10 mM Ca2+ supplied externally (Baker and Ray, 1965), resembling the present results with Ca2+.
Proseus et al. (1999, 2000) gave a prominent role to xyloglucans and enzymes in P-driven growth in Chara, with an uncertain role for wall deposition. However, Proseus and Boyer (2006b) could not demonstrate a role for wall enzymes and instead attributed P-driven growth to intrinsic polymer properties in the wall. Because the present work does not implicate xyloglucans in longitudinal load-bearing and identifies a central role for Ca-PGA in the deformation of the wall, the original xyloglucan and enzyme-based mechanism visualized by Proseus et al. (1999, 2000) should be modified. A newer visualization would centre on wall deposition and the chemical properties of pectins themselves, as suggested here.
|
Conclusions |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionsAppendixReferences |
|---|
Several chemical properties of pectin appear important for Chara growth. (i) A strong affinity of PGA for Ca2+ created a cross-linked Ca-PGA complex in the wall sufficiently strong to bear the load of P parallel to the long axis of the cell. By bearing this load, Ca-PGA under tension controlled cell length while cellulose microfibrils appeared to prevent cell widening. (ii) In the wall, free Ca2+ rapidly interchanged with Ca-PGA. During the interchange under high tension from P, load-bearing Ca-PGA appeared to slip and cause irreversible wall deformation. The deformation extended the wall but put more Ca-PGA under tension. The rate of deformation would decelerate probably because of the increased amount of taut Ca-PGA. (iii) If new PGA was supplied, its chelating ability removed Ca from the wall, probably including the taut Ca-PGA. Wall structure was loosened and irreversible deformation accelerated. New Ca2+ prevented this loosening. (iv) If the new PGA molecules were large, new Ca cross-links formed a gel structure in and around the existing wall that could retard wall deformation. Together with the tendency of P to concentrate the PGA and cause spontaneous gelling, the cross-links would balance the accelerating action of the same PGA.
It is proposed that these chemical properties control the growth rate of Chara cells. The growth acceleration by new PGA and growth retardation by Ca binding/gelling of the same PGA may be the molecular mechanism for this control. Accordingly, the rate at which metabolism synthesizes and delivers new pectin to the periplasm would control wall loosening for irreversible deformation by P. At the same time, Ca2+ binding and the action of P to concentrate the new PGA would enhance gelling. With gelling, wall assembly and strengthening would keep pace with wall deformation. New wall would be deposited at about the rate of cell enlargement.
|
Appendix |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionsAppendixReferences |
|---|
Table 2 Measured and calculated values for various parameters in the primary cell walls of Chara internodes and in the culture medium in the experimental apparatus used in this study
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Calculations are for a typical growing cell.
a Fresh cells had a radius of 0.365 mm and a wall thickness of 0.005 mm to give a volume of 1.14x10–8 L mm–1 cell length. Dry mass was 0.49x10–8 kg mm–1 cell length.
b From Morrison et al. (1993) and Popper and Fry (2003).
c Primary stoichiometry assumed to be 2:1 for PGA/divalent cations, 1:1 for EGTA/divalent cations from Martell and Smith (1974).
|
Acknowledgements |
|---|
We thank Lincoln Taiz for sharing ideas from his important early work on charophyte growth. Thanks also are given to Joe Scudlark for assistance with the elemental analyses, and are extended to the Complex Carbohydrate Research Center for the gift of PGA having small molecular weight. Support for TEP from an Okie Fellowship from the College of Marine Studies is gratefully acknowledged.
|
Abbreviations |
|---|
EGTA, ethylene glycol-bis(ß-aminoethyl ether) N,N,N',N'-tetra-acetic acid, a chelator;
L, change in length; P, turgor pressure; PGA, polygalacturonate; PGA-CCRC, PGA from Complex Carbohydrate Research Center. |
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionsAppendixReferences |
|---|
Anderson DMW and King NJ. (1961) Polysaccharides of the Characeae. III. The carbohydrate content of Chara australis. Biochimica et Biophysica Acta 52 449–454.[Medline]
Baker DB and Ray PM. (1965) Relation between effects of auxin on cell wall synthesis and cell elongation. Plant Physiology 40 360–368.
Baskin TI. (2005) Anisotropic expansion of the plant cell wall. Annual Review of Cell and Developmental Biology 21 203–222.[CrossRef][ISI][Medline]
Bret-Harte MS, Baskin TI, Green PB. (1991) Auxin stimulates both deposition and breakdown of material in the pea outer epidermal cell wall, as measured interferometrically. Planta 185 462–471.
Fry SC. (2004) Primary cell wall metabolism: tracking the careers of wall polymers in living plant cells. New Phytologist 161 641–675.[CrossRef][ISI]
Gillet C, Cambier P, Liners F. (1992) Release of small polyuronides from Nitella cell walls during ionic exchange. Plant Physiology 100 846–852.
Gillet C and Liners F. (1996) Changes in distribution of short pectic polysaccharides induced by monovalent ions in the Nitella cell wall. Canadian Journal of Botany 74 26–30.
Gillet C, Voué M, Cambier P. (1998) Site-specific counterion binding and pectic chains conformational transitions in the Nitella cell wall. Journal of Experimental Botany 49 797–805.
Grant GT, Morris ER, Rees DA, Smith PJC, Thom D. (1973) Biological interactions between polysaccharides and divalent cations: the egg box model. FEBS Letters 32 195–198.
Hope AB and Walker NA. (1975) The physiology of giant algal cellsNew York, NY Cambridge University Press.
Jarvis MC. (1984) Structure and properties of pectin gels in plant cell walls. Plant, Cell and Environment 7 153–164.
Jarvis MC and Apperley DC. (1995) Chain conformation in concentrated pectic gels: evidence from 13C NMR. Carbohydrate Research 275 131–145.
Kutschera U. (1990) Cell-wall synthesis and elongation growth in hypocotyls of Helianthus annuus L. Planta 181 316–323.[ISI]
Martell AE and Smith RM. (1974) Amino acids. Critical stability constantsNew York, NY Plenum Press Vol. 1.
Metraux J-P. (1982) Changes in cell-wall polysaccharide composition of developing Nitella internodes. Planta 155 459–466.[CrossRef]
Metraux J-P, Richmond P, Taiz L. (1980) Control of cell elongation in Nitella by endogenous cell wall pH gradients. Multiaxial extensibility and growth studies. Plant Physiology 65 204–210.
Metraux J-P and Taiz L. (1977) Cell wall extension in Nitella as influenced by acids and ions. Proceedings of the National Academy of Sciences, USA 74 1565–1569.
Morikawa H and Senda M. (1974) Oriented structure of matrix polysaccharides in and extension growth of Nitella cell wall. Plant and Cell Physiology 15 1139–1142.
Morikawa H, Tanizawa K, Senda M. (1974) Infrared spectra of Nitella cell walls and orientation of carboxylate ions in the walls. Agricultural and Biological Chemistry 38 343–348.
Morris ER, Powell DA, Gidley MJ, Rees DA. (1982) Conformation and interactions of pectins. I. Polymorphism between gel and solid states of calcium polygalacturonate. Journal of Molecular Biology 155 507–516.[CrossRef][ISI][Medline]
Morrison JC, Greve LC, Richmond PA. (1993) Cell wall synthesis during growth and maturation of Nitella internodal cells. Planta 189 321–328.[CrossRef]
Northcote DH and Pickett-Heaps JD. (1966) A function of the Golgi apparatus in polysaccharide synthesis and transport in root-cap cells of wheat. Biochemical Journal 8 159–167.
Passioura JB. (1994) The physical chemistry of the primary cell wall: implications for the control of expansion rate. Journal of Experimental Botany 45 1675–1682.
Passioura JB and Fry SC. (1992) Turgor and cell expansion: beyond the Lockhart equation. Australian Journal of Plant Physiology 19 565–576.
Popper ZA and Fry SC. (2003) Primary cell wall composition of bryophytes and charophytes. Annals of Botany 91 1–12.
Powell ER, Morris ER, Gidley MJ, Rees DA. (1982) Conformations and interactions of pectins. II. Influence of residue sequence on chain association in calcium pectate gels. Journal of Molecular Biology 155 517–531.[CrossRef][ISI][Medline]
Preston RD. (1974) The physical biology of plant cell wallsLondon Chapman and Hall.
Probine MC and Preston RD. (1962) Cell growth and the structure and mechanical properties of the wall in internode cells of Nitella opaca. II. Mechanical properties of the walls. Journal of Experimental Botany 13 111–127.
Proseus TE and Boyer JS. (2005) Turgor pressure moves polysaccharides into growing cell walls of Chara corallina. Annals of Botany 95 967–979.
Proseus TE and Boyer JS. (2006a) Periplasm turgor pressure controls wall deposition and assembly in growing Chara corallina cells. Annals of Botany 98 93–105.
Proseus TE and Boyer JS. (2006b) Identifying cytoplasmic input to the cell wall of growing Chara corallina. Journal of Experimental Botany 57 3231–3242.
Proseus TE, Ortega JKE, Boyer JS. (1999) Separating growth from elastic deformation during cell enlargement. Plant Physiology 119 775–784.
Proseus TE, Zhu G-L, Boyer JS. (2000) Turgor, temperature, and the growth of plant cells: using Chara corallina as a model system. Journal of Experimental Botany 51 1481–1494.
Ralet M-C, Dronnet V, Buchholt HC, Thibault J-F. (2001) Enzymatically and chemically de-esterified lime pectins: characterization, polyelectrolyte behavior and calcium binding properties. Carbohydrate Research 336 117–125.[CrossRef][ISI][Medline]
Ray PM. (1967) Radioautographic study of cell wall deposition in growing plant cells. Journal of Cell Biology 35 659–674.
Ray PM and Baker DB. (1965) The effect of auxin on synthesis of oat coleoptile cell wall constituents. Plant Physiology 40 353–360.
Richmond PA, Metraux J-P, Taiz L. (1980) Cell expansion patterns and directionality of wall mechanical properties in Nitella. Plant Physiology 65 211–217.
Ridley BL, O'Neill MA, Mohnen D. (2001) Pectins: structure, biosynthesis, and oligogalacturonide-related signaling. Phytochemistry 57 929–967.[CrossRef]