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Plant Cell Expansion: Unzipped by expansins
STEPHEN C. FRY
PLANT CELL EXPANSION
Unzipped by expansins Newly discovered plant proteins, known as expansins, break hydrogen bonds in the cell wall and may play an important role in plant growth. A striking feature of plant development is the dramatic increase in volume of many cells that accompanies plant growth. Most cell divisions in plants take place in localized pockets of tissue called meristems, such as the one at the shoot tip. Meristem cells are small by plant standards, typically about 0.5 picolitres in volume. Some of them stop dividing - they 'drop out' behind the advancing meristem and form the stem of the plant. If that were the end of the story, the stem would be a very short organ. A tree would have a trunk about the height of a daffodil. However, the non-dividing cells that emerge from the base of the meristem start expanding and routinely reach about 1000 picolitres, 2000 times their original volume. The amount and direction of cell expansion ultimately define the size and shape of the plant. Not surprisingly, therefore, cell expansion is tightly regulated by plant hormones, light, pH and other factors. Allow a bean to germinate in the dark and its shoot grows tall and spindly; in the light it grows short and stocky. Our long-standing inability (see Mark 4:27, for example) to understand the control of plant growth has prompted a considerable research effort aimed at elucidating the underlying processes. Plant cell expansion is driven by the tendency of the cell to take up water by osmosis, which creates hydrostatic pressure in the cell, stretching the cell wall. However, the magnitude of this pressure is not usually the factor that controls the rate of cell expansion. That privilege goes to the cell wall itself, which either expands readily - as in dark-grown bean shoots - or resists expansion in the face of a given hydrostatic pressure. The wall's susceptibility to extension is not simply a physical attribute of its structural polymers, but depends on the action of proteins in the wall. Isolated cell walls, when stretched with a constant force, exhibit a gradual, irreversible extension ('creep'), typically achieving a 30-40% extension over several hours before breaking. This creep is stopped by treatments likely to inactivate proteins. The wall can well be regarded as a living part of the cell; it is not an inert excrescence (the 'ergastic substance' of the older plant physiology textbooks). Which structural molecules of the cell wall do the creeping, and which molecules catalyze the process? Current ideas focus on a hemicellulose-cellulose 'trellis'. Girder-like microfibrils, made of cellulose, are thought to be tethered by long strings of hemicellulose that adhere to the microfibrils by hydrogen bonds and may
cross-link them. The growth (in area) of a cell wall requires the microfibrils to move apart and/or to slip relative to each other, and this requires breakage or lengthening of the tethers. Chemically speaking, the major hemicellulose likely to serve a tethering role in the growing walls of dicotyledonous plants is xyloglucan; in the grass family, mixed-linkage 3-(1--3),(1-44)-glucan outweighs xyloglucan. The breakage or lengthening of tethers is likely to be catalyzed by proteins, of which growing plant cell walls contain a diverse range, including rigid extensins [1], mucilaginous arabinogalactan-proteins [1,2], numerous enzymes [3] and hundreds of proteins with no known function [4]. The enzymes include hydrolases - cellulase, pectinase, 3-galactosidase and so on - that might attack structural polysaccharides. Correlations have been reported between the activities of various such enzymes and plant growth rate. For example, cellulase is induced when high doses of the hormone auxin promote the lateral swelling of pea stems. Cellulase is interesting in this respect, because its favoured substrates are xyloglucan and mixed-linkage 3-(1-43),(1--44)-glucan - the postulated tethers [5]. A novel, non-hydrolytic, wall enzyme, xyloglucan endotransglycosylase (XET) - recently discussed in these pages [6] - may also play a part in the catalysis of creep. In brief, XET cuts a xyloglucan chain and re-anneals the cut end to another xyloglucan molecule. This 'cutting and pasting' of a plant cell wall polymer resembles what happens to the peptidoglycan of a bacterial wall during growth. Correlations have frequently been found between XET activity and plant growth rate [7,8], suggesting that XET has a role in the mechanism of plant cell expansion. Recently [9-13], novel proteins known as expansins have been added to the list of wall-located catalysts. Discovered in Dan Cosgrove's laboratory at Pennsylvania State University, expansins were identified directly by their effect on the susceptibility of cucumber cell walls to extension. Let us first consider the assay system used [9]. Segments of elongating hypocotyl - the stem-like organ between the cotyledons and the root - excised from cucumber seedlings were killed by freezing and thawing (to eliminate the cells' hydrostatic pressure and to prevent further wall synthesis), clamped and stretched longitudinally. The segments 'crept' - they underwent a slow but irreversible increase in length. Segments that had been pre-treated in hot water or with proteinase, to inactivate
© Current Biology 1994, Vol 4 No 9
815
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Current Biology 1994, Vol 4 No 9
wall proteins, failed to creep. Curiously, segments that had been boiled in methanol and then re-hydrated still crept. Creep had a slightly acidic pH optimum and was inhibited by A13 + ions and several other toxins, whereas fluoride, cyanide and ascorbate all stimulated creep. A very informative finding [10] was that the ability of the hot-water-treated segments to creep could be restored by the addition of novel proteins - the expansins - isolated from fresh cell walls. This elegant reconstitution system unambiguously demonstrated the ability of expansins to loosen cell walls. When XET was tested in the same way it did not promote creep [11], indicating either that XET activity was not a limiting factor for wall expansion in the cucumber hypocotyls tested or that exogenous XET failed to permeate the cell wall to its required site of action. Expansins occur in the cell walls of both dicots and grasses [12]. Two expansins purified from cucumber walls have molecular weights of 29 000 and 30 000; they are hydrophobic, non-glycosylated proteins. The ability of these purified expansins to 'loosen' heat-inactivated cell walls was found to be sensitive to A13+, and their loosening activity has an acidic pH optimum. They are inactivated by heating in water but not in methanol. These properties, especially the last, strongly suggest that expansins played an essential role in the creep that had been observed in unheated cell walls. How do expansins work? An intelligent guess might be that they are enzymes that, like XET, catalyze the cleavage of a polysaccharide. This guess, however, seems to be
wrong: expansins exhibit no enzyme activity in any assay yet tried. The breakthrough in understanding how expansins may work came from a beautifully low-tech experiment: the discovery that expansins will weaken Whatman No. 3 filter paper [13]. Filter paper is almost pure cellulose, the fibres of which are held together - not very strongly, as most of us will have demonstrated to our cost with a clumsily held glass rod - by hydrogen bonds. Expansin, at a concentration of only 1.7 x 10-7M, gradually broke these hydrogen bonds, promoting the ability of a taut strip of paper to extend until, after about an hour, the paper tore. Cellulase also weakened the filter paper, but by partially hydrolysing it, something expansin is incapable of. Concentrated (8M) urea, a hydrogenbond-breaking agent, had an effect on filter paper somewhat similar to that of expansins; however, urea weakened the paper maximally within about two seconds, suggesting that it acted on all the accessible hydrogen bonds simultaneously, in contrast to expansin's gradual action. This difference is not surprising. given that the urea had to be applied at 50 million times the concentration of expansin to evoke a comparable effect. The gradualness of expansin's effect on cellulose suggests that each expansin molecule acts once, then moves, and then repeats the cycle over and over again. Precisely what expansin does to the plant cell wall remains unclear; the following scenario - illustrated in Figure 1 - is necessarily speculative. In loosening cell walls, expansin seems unlikely to break cellulose-cellulose
Fig. 1. A model of how a hemicellulose chain may tether adjacent microfibrils, showing also how expansin molecules may act as wedges that prise the hemicellulose off the microfibrils by breaking hydrogen bonds (red lines). The white arrows represent the outward force exerted (as turgor pressure) by the swelling cell on its wall. The work done by this force, as adjacent microfibrils are gradually separated, should ensure that broken hydrogen bonds do not readily reform after an expansin molecule has passed along the hemicellulose chain.
DISPATCH bonds, as microfibrils remain intact during growth. Thus, the breakage of hydrogen bonds in filter paper may be a side issue. Perhaps the real trick of expansin in vivo is to break hemicellulose-cellulose hydrogen bonds. Expansin could lengthen inter-microfibrillar tethers if it caused hemicellulose chains to detach from microfibrils. Cosgrove's group believes that expansins have a higher affinity for hemicellulose-coated cellulose than for pure cellulose [13]. Therefore, given free range in the cell wall, an expansin molecule ought to latch on to a hemicellulose-cellulose complex (a 'junction zone'). The expansin would then break a group of hydrogen bonds, locally prising the hemicellulose off the microfibril. The tautness of a tethering hemicellulose chain would make it difficult for these hydrogen bonds to reform. The expansin would then no longer find itself located at a hemicellulose-cellulose junction zone; its decreased affinity would let it move along to the nearest adjacent junction zone and repeat the process. In this way, a single expansin molecule could gradually unzip a long stretch of cellulose-bound hemicellulose, lengthening a tether and thus permitting the cell to expand. The story critically depends on expansin being able to break hemicellulose-cellulose as well as cellulose-cellulose hydrogen bonds; experimental verification is eagerly awaited.
'non-covalent reaction' that they catalyse, while not the last word, is an important step towards understanding how plants grow. References 1. 2. 3. 4. 5. 6. 7. 8.
9. 10. 11.
If cellulase, XET and expansin all act on xyloglucan causing hydrolysis, transglycosylation and hydrogenbond-breakage, respectively - we next need to sort out the distinctive contribution of each. While it would be premature to build precise models, it is attractive to think that expansin could, by unzipping xylpglucan from the microfibrils, make the hemicellulose more accessible as a substrate to cellulase and/or to XET. Growth is far too important for plants to rely on any single control point; interactions between the diverse catalysts and substrates are to be expected. The discovery of expansins and the
12.
13.
Showalter AM: Structure and function of plant cell wall proteins. PlantCell 1993, 5:9-23. Komalavilas P, Zhu J-K, Nothnagel EA: Arabinogalactan-proteins from the suspension culture medium and plasma membrane of rose cells. J Biol Chem 1991, 266:15956-15965. Fry SC: Primary cell wall metabolism. In Oxford Surveys of Plant Molecular and Cell Biology 2. Edited by Miflin BJ: Oxford: Oxford University Press, 1985:1-42 Corke FMK, Roberts K, Smith E: Cell wall proteins and cell growth. J Exp Bot 1994, 45(suppl):29. Fry SC: Cellulases, hemicelluloses and auxin-stimulated growth: a possible relationship. Physiol Plant 1989, 75:532-536. Fry SC: Plant cell expansion: loosening the ties. Curr Biol 1993, 3:355-357. Potter I, Fry SC: Xyloglucan endotransglycosylase activity in pea internodes: effects of applied gibberellic acid. Plant Physiol 1993, 103:235-241. Pritchard J, Hetherington PR, Fry SC, Tomos AD: Xyloglucan endotransglycosylase activity, microfibril orientation and the profiles of cell wall properties along growing regions of maize roots. J Exp Bot 1993, 44:1281-1289. Cosgrove DJ: Characterization of long-term extension of isolated cell walls from growing cucumber hypocotyls. Planta 1989, 177:121-130. McQueen-Mason SJ,Durachko DM, Cosgrove DJ: Two endogenous proteins that induce cell wall extension in plants. Plant Cell 1992, 4:1425-1433. McQueen-Mason SJ,Fry SC, Durachko DM, Cosgrove DJ: The relationship between xyloglucan endotransglycosylase and in-vitro cell wall extension in cucumber hypocotyls. Planta 1993, 190:327-331. Li ZC, Durachko DM, Cosgrove DJ: An oat coleoptile wall protein that induces wall extension in vitro and that is antigenically related to a similar protein from cucumber hypocotyls. Planta 1993, 191:349-356. McQueen-Mason SJ,Cosgrove DJ: Disruption of hydrogen bonding between wall polymers by proteins that induce plant wall extension. Proc Natl Acad Sci USA 1994, 91:6574-6578.
Stephen C. Fry, Division of Biological Sciences, The University of Edinburgh, Daniel Rutherford Building, The King's Buildings, Edinburgh EH9 3JH, UK.
817
PLANT CELL EXPANSION
Unzipped by expansins Newly discovered plant proteins, known as expansins, break hydrogen bonds in the cell wall and may play an important role in plant growth. A striking feature of plant development is the dramatic increase in volume of many cells that accompanies plant growth. Most cell divisions in plants take place in localized pockets of tissue called meristems, such as the one at the shoot tip. Meristem cells are small by plant standards, typically about 0.5 picolitres in volume. Some of them stop dividing - they 'drop out' behind the advancing meristem and form the stem of the plant. If that were the end of the story, the stem would be a very short organ. A tree would have a trunk about the height of a daffodil. However, the non-dividing cells that emerge from the base of the meristem start expanding and routinely reach about 1000 picolitres, 2000 times their original volume. The amount and direction of cell expansion ultimately define the size and shape of the plant. Not surprisingly, therefore, cell expansion is tightly regulated by plant hormones, light, pH and other factors. Allow a bean to germinate in the dark and its shoot grows tall and spindly; in the light it grows short and stocky. Our long-standing inability (see Mark 4:27, for example) to understand the control of plant growth has prompted a considerable research effort aimed at elucidating the underlying processes. Plant cell expansion is driven by the tendency of the cell to take up water by osmosis, which creates hydrostatic pressure in the cell, stretching the cell wall. However, the magnitude of this pressure is not usually the factor that controls the rate of cell expansion. That privilege goes to the cell wall itself, which either expands readily - as in dark-grown bean shoots - or resists expansion in the face of a given hydrostatic pressure. The wall's susceptibility to extension is not simply a physical attribute of its structural polymers, but depends on the action of proteins in the wall. Isolated cell walls, when stretched with a constant force, exhibit a gradual, irreversible extension ('creep'), typically achieving a 30-40% extension over several hours before breaking. This creep is stopped by treatments likely to inactivate proteins. The wall can well be regarded as a living part of the cell; it is not an inert excrescence (the 'ergastic substance' of the older plant physiology textbooks). Which structural molecules of the cell wall do the creeping, and which molecules catalyze the process? Current ideas focus on a hemicellulose-cellulose 'trellis'. Girder-like microfibrils, made of cellulose, are thought to be tethered by long strings of hemicellulose that adhere to the microfibrils by hydrogen bonds and may
cross-link them. The growth (in area) of a cell wall requires the microfibrils to move apart and/or to slip relative to each other, and this requires breakage or lengthening of the tethers. Chemically speaking, the major hemicellulose likely to serve a tethering role in the growing walls of dicotyledonous plants is xyloglucan; in the grass family, mixed-linkage 3-(1--3),(1-44)-glucan outweighs xyloglucan. The breakage or lengthening of tethers is likely to be catalyzed by proteins, of which growing plant cell walls contain a diverse range, including rigid extensins [1], mucilaginous arabinogalactan-proteins [1,2], numerous enzymes [3] and hundreds of proteins with no known function [4]. The enzymes include hydrolases - cellulase, pectinase, 3-galactosidase and so on - that might attack structural polysaccharides. Correlations have been reported between the activities of various such enzymes and plant growth rate. For example, cellulase is induced when high doses of the hormone auxin promote the lateral swelling of pea stems. Cellulase is interesting in this respect, because its favoured substrates are xyloglucan and mixed-linkage 3-(1-43),(1--44)-glucan - the postulated tethers [5]. A novel, non-hydrolytic, wall enzyme, xyloglucan endotransglycosylase (XET) - recently discussed in these pages [6] - may also play a part in the catalysis of creep. In brief, XET cuts a xyloglucan chain and re-anneals the cut end to another xyloglucan molecule. This 'cutting and pasting' of a plant cell wall polymer resembles what happens to the peptidoglycan of a bacterial wall during growth. Correlations have frequently been found between XET activity and plant growth rate [7,8], suggesting that XET has a role in the mechanism of plant cell expansion. Recently [9-13], novel proteins known as expansins have been added to the list of wall-located catalysts. Discovered in Dan Cosgrove's laboratory at Pennsylvania State University, expansins were identified directly by their effect on the susceptibility of cucumber cell walls to extension. Let us first consider the assay system used [9]. Segments of elongating hypocotyl - the stem-like organ between the cotyledons and the root - excised from cucumber seedlings were killed by freezing and thawing (to eliminate the cells' hydrostatic pressure and to prevent further wall synthesis), clamped and stretched longitudinally. The segments 'crept' - they underwent a slow but irreversible increase in length. Segments that had been pre-treated in hot water or with proteinase, to inactivate
© Current Biology 1994, Vol 4 No 9
815
816
Current Biology 1994, Vol 4 No 9
wall proteins, failed to creep. Curiously, segments that had been boiled in methanol and then re-hydrated still crept. Creep had a slightly acidic pH optimum and was inhibited by A13 + ions and several other toxins, whereas fluoride, cyanide and ascorbate all stimulated creep. A very informative finding [10] was that the ability of the hot-water-treated segments to creep could be restored by the addition of novel proteins - the expansins - isolated from fresh cell walls. This elegant reconstitution system unambiguously demonstrated the ability of expansins to loosen cell walls. When XET was tested in the same way it did not promote creep [11], indicating either that XET activity was not a limiting factor for wall expansion in the cucumber hypocotyls tested or that exogenous XET failed to permeate the cell wall to its required site of action. Expansins occur in the cell walls of both dicots and grasses [12]. Two expansins purified from cucumber walls have molecular weights of 29 000 and 30 000; they are hydrophobic, non-glycosylated proteins. The ability of these purified expansins to 'loosen' heat-inactivated cell walls was found to be sensitive to A13+, and their loosening activity has an acidic pH optimum. They are inactivated by heating in water but not in methanol. These properties, especially the last, strongly suggest that expansins played an essential role in the creep that had been observed in unheated cell walls. How do expansins work? An intelligent guess might be that they are enzymes that, like XET, catalyze the cleavage of a polysaccharide. This guess, however, seems to be
wrong: expansins exhibit no enzyme activity in any assay yet tried. The breakthrough in understanding how expansins may work came from a beautifully low-tech experiment: the discovery that expansins will weaken Whatman No. 3 filter paper [13]. Filter paper is almost pure cellulose, the fibres of which are held together - not very strongly, as most of us will have demonstrated to our cost with a clumsily held glass rod - by hydrogen bonds. Expansin, at a concentration of only 1.7 x 10-7M, gradually broke these hydrogen bonds, promoting the ability of a taut strip of paper to extend until, after about an hour, the paper tore. Cellulase also weakened the filter paper, but by partially hydrolysing it, something expansin is incapable of. Concentrated (8M) urea, a hydrogenbond-breaking agent, had an effect on filter paper somewhat similar to that of expansins; however, urea weakened the paper maximally within about two seconds, suggesting that it acted on all the accessible hydrogen bonds simultaneously, in contrast to expansin's gradual action. This difference is not surprising. given that the urea had to be applied at 50 million times the concentration of expansin to evoke a comparable effect. The gradualness of expansin's effect on cellulose suggests that each expansin molecule acts once, then moves, and then repeats the cycle over and over again. Precisely what expansin does to the plant cell wall remains unclear; the following scenario - illustrated in Figure 1 - is necessarily speculative. In loosening cell walls, expansin seems unlikely to break cellulose-cellulose
Fig. 1. A model of how a hemicellulose chain may tether adjacent microfibrils, showing also how expansin molecules may act as wedges that prise the hemicellulose off the microfibrils by breaking hydrogen bonds (red lines). The white arrows represent the outward force exerted (as turgor pressure) by the swelling cell on its wall. The work done by this force, as adjacent microfibrils are gradually separated, should ensure that broken hydrogen bonds do not readily reform after an expansin molecule has passed along the hemicellulose chain.
DISPATCH bonds, as microfibrils remain intact during growth. Thus, the breakage of hydrogen bonds in filter paper may be a side issue. Perhaps the real trick of expansin in vivo is to break hemicellulose-cellulose hydrogen bonds. Expansin could lengthen inter-microfibrillar tethers if it caused hemicellulose chains to detach from microfibrils. Cosgrove's group believes that expansins have a higher affinity for hemicellulose-coated cellulose than for pure cellulose [13]. Therefore, given free range in the cell wall, an expansin molecule ought to latch on to a hemicellulose-cellulose complex (a 'junction zone'). The expansin would then break a group of hydrogen bonds, locally prising the hemicellulose off the microfibril. The tautness of a tethering hemicellulose chain would make it difficult for these hydrogen bonds to reform. The expansin would then no longer find itself located at a hemicellulose-cellulose junction zone; its decreased affinity would let it move along to the nearest adjacent junction zone and repeat the process. In this way, a single expansin molecule could gradually unzip a long stretch of cellulose-bound hemicellulose, lengthening a tether and thus permitting the cell to expand. The story critically depends on expansin being able to break hemicellulose-cellulose as well as cellulose-cellulose hydrogen bonds; experimental verification is eagerly awaited.
'non-covalent reaction' that they catalyse, while not the last word, is an important step towards understanding how plants grow. References 1. 2. 3. 4. 5. 6. 7. 8.
9. 10. 11.
If cellulase, XET and expansin all act on xyloglucan causing hydrolysis, transglycosylation and hydrogenbond-breakage, respectively - we next need to sort out the distinctive contribution of each. While it would be premature to build precise models, it is attractive to think that expansin could, by unzipping xylpglucan from the microfibrils, make the hemicellulose more accessible as a substrate to cellulase and/or to XET. Growth is far too important for plants to rely on any single control point; interactions between the diverse catalysts and substrates are to be expected. The discovery of expansins and the
12.
13.
Showalter AM: Structure and function of plant cell wall proteins. PlantCell 1993, 5:9-23. Komalavilas P, Zhu J-K, Nothnagel EA: Arabinogalactan-proteins from the suspension culture medium and plasma membrane of rose cells. J Biol Chem 1991, 266:15956-15965. Fry SC: Primary cell wall metabolism. In Oxford Surveys of Plant Molecular and Cell Biology 2. Edited by Miflin BJ: Oxford: Oxford University Press, 1985:1-42 Corke FMK, Roberts K, Smith E: Cell wall proteins and cell growth. J Exp Bot 1994, 45(suppl):29. Fry SC: Cellulases, hemicelluloses and auxin-stimulated growth: a possible relationship. Physiol Plant 1989, 75:532-536. Fry SC: Plant cell expansion: loosening the ties. Curr Biol 1993, 3:355-357. Potter I, Fry SC: Xyloglucan endotransglycosylase activity in pea internodes: effects of applied gibberellic acid. Plant Physiol 1993, 103:235-241. Pritchard J, Hetherington PR, Fry SC, Tomos AD: Xyloglucan endotransglycosylase activity, microfibril orientation and the profiles of cell wall properties along growing regions of maize roots. J Exp Bot 1993, 44:1281-1289. Cosgrove DJ: Characterization of long-term extension of isolated cell walls from growing cucumber hypocotyls. Planta 1989, 177:121-130. McQueen-Mason SJ,Durachko DM, Cosgrove DJ: Two endogenous proteins that induce cell wall extension in plants. Plant Cell 1992, 4:1425-1433. McQueen-Mason SJ,Fry SC, Durachko DM, Cosgrove DJ: The relationship between xyloglucan endotransglycosylase and in-vitro cell wall extension in cucumber hypocotyls. Planta 1993, 190:327-331. Li ZC, Durachko DM, Cosgrove DJ: An oat coleoptile wall protein that induces wall extension in vitro and that is antigenically related to a similar protein from cucumber hypocotyls. Planta 1993, 191:349-356. McQueen-Mason SJ,Cosgrove DJ: Disruption of hydrogen bonding between wall polymers by proteins that induce plant wall extension. Proc Natl Acad Sci USA 1994, 91:6574-6578.
Stephen C. Fry, Division of Biological Sciences, The University of Edinburgh, Daniel Rutherford Building, The King's Buildings, Edinburgh EH9 3JH, UK.
817