Proc. Nadl. Acad. Sci. USA Vol. 91, pp. 7387-7389, August 1994 Commentary Expansins: Proteins that promote cell wall loosening in plants Lincoln Taiz Biology Departent, University of California, Santa Cruz, CA 95064

It was July 7, 1912, and Harry Houdini, iri against the cell wall, which exerts aa This domain is embedded in a second the company of a bevy of dutiful report- counter force on the protoplast, discour network, pectic polysaccharides. The ers, was going to perform one of hissaging further water uptake. Ifthe osmotic pectic polysaccharides, rich in uronic greatest escapes, from a barge floating inigradient is sufficient, however, water willI acid residues, can form cross-links based the middle of the East River in Newvcontinue to enter the cell for a time, andI on calcium bridges and other ionic inter- York. First he was shackled in leg irons enormous hydrostatic turgor pressures; actions. Structural proteins form a third two pairs of handcuffs, and elbow irons. can build up, distending the wall to its; interlocking network. The latter may in- Then he was crammed into a sturdy elastic limits. But the expanding proto- terweave through the other two domains, wooden crate, 40 inches x 22 inches x 24Iplast does not merely out-muscle the forming a "warp and weft" structure (6). inches, and the lid was nailed shut and wall, like Houdini kicking out the sides ofr Such models, while useful for wall bio- reinforced with steel bands. For addedIhis box. Rather, the protoplast releases chemists, tell us little about the mecha- effect, the box was given a further wrap-*unidentified "wall-loosening factors" nism of wall extension. Which of the ping of stout ropes and fitted with 200Iwhich alter the wall's mechanical prop- myriad interactions in the cell wall con- pounds of lead weights. Following the erties and allow it to undergo a process trols the rate of stress relaxation? obligatory hype and fanfare, the com- called "stress relaxation." During stress The inspiration for this commentary pletely sealed crate, with the manacled relaxation, reversible wall extension is article is that a major breakthrough ap- Houdini inside, was lowered over the converted to a permanent, irreversible pears to have been achieved. Daniel Cos- side of the barge. Even the skeptics on extension, dramatically reducing both grove and his colleagues at Pennsylvania board must have been silenced as the box the wall stress and the turgor-pressure. State University have described a new slowly sank out of sight into the murky It's a bit like breaking in a new pair of class of cell wall proteins, "expansins," depths below. Seconds passed. Predict- shoes: the shoe size hardly changes at all, which may represent the key players in ably, just when the frantic onlookers but your feet can sure tell the difference! the elusive wall-loosening mechanism were about to demand that the box be Except that in the case of plant cells, no (7). At the very least they account for one raised, the smiling magician broke the sooner do the walls "relax" than more type of wall extension. Expansins have surface, leaving his gaping admirers to water enters the protoplast and the wall is been shown to increase the extensibility wonder how on earth he did it (1). again elastically distended. Plant cell ex- of isolated cell walls without causing A similar, but even more baffling, co- pansion is thus a continuous cycle of time-dependent weakening (8). Expan- nundrum on a microscopic scale has been biochemical stress relaxation followed by sins are activated at acid pH and are thus vexing plant physiologists for over half a turgor-driven elastic extension. (If shoes prime candidates for the wall-loosening century. Plant protoplasts are born swad- could do this, one pair would last a life- agents that operate during growth medi- dled inside tiny boxes called cell walls. time!) The important point to remember ated by proton extrusion (8). How do These walls are quite rigid by any stan- is that cell expansion is driven by passive expansins work? In a recent issue of dard, able to resist internal hydrostatic water uptake, whereas stress relaxation these Proceedings, McQueen-Mason and pressures of up to 10 atmospheres. The requires biochemical input (unlike break- Cosgrove (9) provide an exciting clue: strength of cell walls arises from their ing in your shoes). Any interference with expansins disrupt the hydrogen bonds in peculiar composition: tough, slender cellular metabolism immediately brings paper. To appreciate the significance of rods of crystalline cellulose microfibrils stress relaxation, and therefore wall ex- this finding, let us briefly review what is embedded in a glue-like matrix of pec- tension, to a grinding halt. known about the physiology ofplant cell tins, hemicelluloses, and proteins. In- When I last reviewed this topic 10 expansion. deed, the cell walls of plants can be years ago (4), thejury was still out on the thought of as the prototype for modern identity ofthe chemical bonds that had to Auxin and the Acid Growth Hypothesis advanced composites such as fiberglass be broken for stress relaxation to occur. and graphite fiber-reinforced resins It is generally agreed that the matrix The vast majority of studies carried out which are widely used as lightweight sub- controls the rate ofwall extension, while on plant cell expansion have concerned stitutes for steel. Yet despite this formid- the orientation of the cellulose mi- the action of the growth hormone auxin ible barrier, plant cells manage to expand crofibrils determines the direction of cell (indole-3-acetic acid). Auxin promotes 10 to 100 times their original size during expansion. Unfortunately, the matrix of the rapid elongation of excised sections normal development. For decades, plant primary walls is bewilderingly complex, of elongating tissues such as dicot stems physiologists have wondered how on a veritable jungle of molecular lianas in- or the coleoptiles of grass seedlings. earth they do it. tertwined and coiled about each other. In Since the 1970s, the "acid growth hy- a recent review, Carpita and Gibeaut (5) pothesis," first propounded by Hager et Relaxing the Wall proposed a structure for the typical an- al. (10) and by Rayle and Cleland (11), has giosperm primary wall based on three been the dominant paradigm for studies The biophysical basis ofplant cell expan- intermolecular networks. The first is of auxin action. According to the classi- sion is fairly well understood (2, 3). The made up ofthe cellulose microfibrils with cal model, auxin-induced proton extru- driving force is the osmotic uptake of xyloglucan chains hydrogen bonded to sion lowers the pH of the cell wall, water. As water enters the cell the pro- their surface and forming a continuous thereby activating wall-loosening en- toplast expands, but it quickly bumps lattice connecting adjacent microfibrils. zymes. The activated wall-loosening en- 7387 Downloaded by guest on September 23, 2021 7388 Commentary: Taiz Proc. Natl. Acad. Sci. USA 91 (1994) zymes cleave load-bearing bonds, allow- But the properties of wall extension are experiments between the Cosgrove and ing wall extension to proceed. While not inconsistent with hydrolytic cleavage. As Fry labs, neither purified XET nor the universally embraced by all workers in irreversible enzymes, hydrolases would xyloglucan nonasaccharide induced any the field (12), the acid growth hypothesis be expected to cause progressive weak- cell wall loosening as measured by the has the advantage over all other compet- ening of the wall, and the rate of acid- same in vitro assay (21). Suddenly, the ing models that it is supported by an induced wall extension should accelerate wall-loosening mechanism had a face, impressive body of evidence (13). The with time. Yet when frozen-thawed stem and it was soon given a name: "expan- five basic pillars upon which the model is or coleoptile sections are placed under sins." Dan Cosgrove would have pre- built are as follows: (i) stem and coleop- tension at acid pH, they can extend at a ferred the term "extensins," but this tile sections, which have been rendered constant rate for many hours. Thus, wall name had already been claimed for an- permeable to protons by abrading the strength is maintained during acid-in- other cell wall protein, one that is rich in waxy cuticle, exhibit a growth response duced extension, even in the absence of hydroxyproline. It now appears that "ex- to mildly acidic buffers; (ii) isolated cell wall synthesis. The irreversibility of hy- tensin" probably functions as a wound- walls obtained by freeze-thawing the tis- drolytic enzyme reactions is also incon- inducible protein that slows pathogen at- sue exhibit acid-induced extension in in sistent with the rapid inhibition ofgrowth tack, and has little or nothing to do with vitro extension experiments; (iii) stem or by metabolic inhibitors or neutral buff- regulating wall loosening during cell coleoptile sections pump protons extra- ers. Finally, Cosgrove and Durachko (16) growth (4, 22). Unfortunately, inappro- cellularly in response to auxin with the found a lack of correlation between au- priate monikers in plant physiology die same kinetics as auxin-induced growth; tolysis and extension of walls isolated hard (witness the reverse order of pho- (iv) auxin-induced growth can be blocked from cucumber hypocotyls. Ofparticular tosystems I and II in the Z scheme), and by neutral buffers and by in- significance is the observation that max- we are probably stuck with "extensin" hibitors of the plasma membrane H+- imal autolytic release of sugars occurred for the foreseeable future. ATPase (14); and (v) other agents that at pH 6.5, which is above the threshold This brings us at last to the latest in- elicit proton extrusion, such as the fungal for acid-induced extension. stallment in the expansin saga, which ap- phytotoxin fusicoccin, also promote cell If wall strength is to be maintained peared recently in these pages (9). Once elongation. during extension, bond cleavage must be expansins had been shown to promote Even if the acid growth hypothesis for accompanied by the formation of new acid-induced wall loosening in vitro, the auxin action is incorrect, no one ques- bonds. Albersheim (17) and Fry (18) have burning questionbecame, How do they do tions that fusicoccin works this way, or proposed that the wall-loosening enzyme it? Is expansin an enzyme? And ifso, what that acidic buffers can trigger transient is most likely a transglycosylase, which type? As noted earlier, the number of cell expansion. Therefore, elucidating transfers the glycosidic linkage from one possible targets for expansin action in the the mechanism ofacid-induced extension sugar residue to another. Recently, a native wall is legion, making detection is an iitiportant first step toward a more xyloglucan endotransglycosylase (XET) nearly impossible. To avoid this difficulty, global understanding of wall loosening. has been identified in the cell walls of McQueen-Mason and Cosgrove tested Acid-iriluced extension is also more ac- many plant species (19, 20). This enzyme the ability of expansin to mechanically cessible experimentally than most other has the ability to cleave xyloglucan weaken pure cellulosic paper, with posi- aspects, of wall loosening since it can be chains and to reform the glycosidic link- tive results! In both creep and stress re- readilydemonstrated in vitro. In the first age with a new partner. When xyloglucan laxation studies, expansin weakened the of an important series of papers from the fragments are added to native walls they mechanical strength of paper, and the Cosgroye lab, the acid-induced "creep" can become incorporated into existing effect was promoted by acid pH. More- (long-term wall extension) of frozen- xyloglucan chains through the action of over, expansin induced extension without thawed cucumber hypocotyls was char- this enzyme (20). But does XET mediate any detectable cellulolytic activity. In the acterized (8). Acid-induced creep was acid-induced wall loosening? authors' words, "Because paper derives abolished by pretreating the wall with its mechanical strength from hydrogen boiling water, proteases, or Cu2+. In con- Enter Expansins bonding between cellulose microfibrils, trast, sulfhydryl-reducing agents were we conclude that expansins can disrupt stimulatory. In addition, acid-induced Having convinced themselves of the ex- hydrogen bonding between cellulose fi- extension exhibited a Q1o of 3.8 between istence of a wall-loosening enzyme, the bers.... In the growing cell wall, expan- 20'C and 300C, whereas the Q1o ofgel-sol Cosgrove team set about to purify it. sin action is likely to catalyze slippage transitions for pectin solutions was only They used a simple and elegant ap- between cellulose microfibrils and the 1.35 over the same temperature range. proach. Isolated cell cucumber hypo- polysaccharide matrix... " (9). The Taken together, the results strongly im- cotyl walls were incubated in a buffer ready reversibility of hydrogen bonds plicate the participation of a wall- containing 1 M NaCl, and the resulting nicely explains why the wall is not pro- loosening enzyme. extract was tested for the ability to pro- gressively weakened during the wall- mote wall extension in vitro. The results loosening process. As the matrix polysac- Hydrolases vs. Transglycosylases were spectacular. In the second paper of charides shift their positions relative to the series, McQueen-Mason et al. (7) the cellulose microfibrils, new hydrogen Cell wall hydrolytic enzymes, such as showed that they could restore the ability bonds are formed as rapidly as the old cellulases, hemicellulases, and pecti- ofwater-boiled cucumber hypocotyl sec- ones are broken. nases, had long been considered poten- tions to respond to acid by incubating tial candidates for wall loosening en- them in proteins extracted from non- Enzymes, Chaperonins, or Andtifez? zymes. Such enzymes are present in cell boiled hypocotyl walls. The reconsti- walls and account for the autolytic activ- tuted walls exhibited the classical acid- Expansins are able to promote wall ex- ity of frozen-thawed cell wall prepara- induced extension between pH 5.0 and tension at a dry weight ratio of only tions (5). Do hydrolases regulate cell wall 3.0, and they mimicked the response of 1:10,000 (expansin to cell wall), suggest- loosening during acid-induced exten- native walls in all respects. Moreover, ing that they act as catalysts rather than sion? Qne of the early effects of auxin in when the cell wall extracts were fraction- structural components of the wall. The dicots Appears to be the turnover of cell ated, two polypeptides, 29 and 30 kDa, binding of expansins to cellulose is en- wall xyloglucan, consistent with a role copurified with the creep-inducing activ- hanced by the presence of bound hemi- for xyl.glucanases in wall extension (15). ity. In a separate set of collaborative cellulose (D. J. Cosgrove, personal com- Downloaded by guest on September 23, 2021 Commentary: Taiz Proc. Natl. Acad. Sci. USA 91 (1994) 7389 munication), indicating that expansinis proton extrusion is sufficient to cause wal 5. Carpita, N. C. & Gibeaut, D. M. (1993) may recognize the cellulose-hemicellu- loosening. But accurately determining the Plant J. 3, 1-30. lose interface. Could expansins represent pH of the cell wall is no trivial undertak 6. Lamport, D. T. A. (1986) in Cellulose: a new type of enzyme that catalyzes thee ing. In the first and most elegant of suck Structure, Modification and Hydrolysis, eds. Young, R. A. & Rowell, R. M. (Wi- breakage of hydrogen bonds betweer studies, Jacobs and Ray (27) used a mi ley, New York), pp. 77-90. polysaccharide chains? A possible anal croelectrode to measure the pH of the 7. McQueen-Mason, S., Durachko, D. M. ogy is the DnaB protein, which breaksssolution in the cavity left by a punctured & Cosgrove, D. J. (1992) Plant Cell 4, hydrogen bonds between nucleic acidi cell in corn coleoptiles. They found thatt 1425-1433. base pairs and serves as a helicase during auxin caused the pH of the solution, pre- 8. Cosgrove, D. J. (1989) Planta 177, 121- DNA replication in Escherichia coli (23). sumed to be in equilibrium with the cell 130. Alternatively, expansins may act more wall, to drop from 6.0 to as low as 4.5. In 9. McQueen-Mason, S. & Cosgrove, D. J. like chaperoning, promoting polysaccha- contrast, Schopfer, measuring the effect (1994) Proc. NatI. Acad. Sci. USA 91, ride conformations that discourage hy-*of auxin on the pH at which acid growth 6574-6578. drogen bond formation. begins, deduced a much smaller auxin- 10. Hager, A., Menzle, H. & Krauss, A. My favorite analogy for expansin ac- induced reduction in wall pH, from 5.25 to (1971) Planta 100, 47-75. tion is the antifreeze polypeptides (AFPs) pH 5.0 (12). By the same "null titration" 11. Rayle, D. & Cleland, R. E. (1970) Plant of fish. These proteins bind to the sur- method Schopfer found that fusicoccin Physiol. 46, 250-253. wall 12. Schopfer, P. (1993) Plant Physiol. 103, faces of ice crystals, preventing growth lowered the pH to 3.9 (12). Compar- 351-357. of the lattice (24). The structure and ing Schopfer's values for auxin-induced 13. Rayle, D. & Cleland, R. E. (1992) Plant molecular mechanism of the AFP from acidification with McQueen-Mason et Physiol. 99, 1271-1274. winter flounder are known (25). The pro- al.'s curve for the dependence of recon- 14. Jacobs, M. & Taiz, L. (1980) Proc. Natl. tein consists ofa single amphiphilic a-he- stituted extension on pH (7), we see that Acad. Sci. USA 77, 7242-7246. lix, one side ofwhich is hydrophilic while a pH drop from 5.25 to 5.0 is sufficient to 15. Labavitch, J. M. & Ray, P. M. (1974) the other side is hydrophobic. The hy- nearly double expansin activity. Thus, Plant Physiol. 53, 669-673. drophilic side of the a-helix binds to the even using the more conservative esti- 16. Cosgrove, D. J. & Durachko, D. M. surface of the ice crystal, exposing the mates of Schopfer, the evidence is com- (1994) J. Exp. Bot., in press. hydrophobic face to the liquid phase. pelling that expansins play an important 17. Albersheim, P. (1976) in Plant Biochem- Perhaps the a-helices of expansin are role in auxin-induced cell enlargement. istry, eds, Bonner, J. & Varner, J. E. arranged similarly, with the polar sides Other cell wall-loosening mechanisms (Academic, New York), pp. 226-277. are bound to be discovered. For exam- 18. Fry, S. C. (1989) Physiol. Plant. 75, 532- binding to cellulose. Ifthere were enough 536. expansin molecules scattered about in ple, in oat coleoptiles, expansin does not 19. Smith, R. C. & Fry, S. C. (1991) Bio- critical locations in the wall it might pre- fully restore acid-induced extension in chem. J. 279, 529-535. vent "polysaccharide freezing" in this boiled walls (26). There are also exam- 20. Fry, S. C., Smith, R. C., Renwick, way. The stimulatory effect ofacid could ples, as in the case ofgibberellin-induced K. F., Martin, D. J., Hodge, S. K. & be explained if the a-helices were more growth, in which rapid wall extension Matthews, K. J. (1992) Biochem. J. 282, stable at acid pH. Fortunately, we may occurs without any detectable increase in 821-828. not have to wait long for an answer, since proton extrusion (28). Indeed, the very 21. McQueen-Mason, S. J., Fry, S. C., Du- an expansin cDNA clone has now been complexity of plant cell walls implies rachko, D. M. & Cosgrove, D. J. (1993) isolated from which a secondary struc- multifactorial regulation. The signifi- Planta 90, 327-331. ture can be derived (D. J. Cosgrove, per- cance ofthe discovery ofexpansins is not 22. Showalter, A. M., Bell, J. N., Cramer, sonal communication). that the problem has been solved, but C. L., Bailey, J. A., Varner, J. E. & that it has given us our first glimpse into Lamb, C. J. (1985) Proc. Natl. Acad. wall Sci. USA 82, 6551-6555. Perspective the black box of cell loosening, 23. Lehninger, A. L., Nelson, D. L. & Cox, which is ample cause for celebration. The M. M. (1993) Principles ofBiochemistry Do expansins play an essential role in many years it has taken us to reach this (Worth, New York). normal plant growth? It depends on one's point is eloquent testimony that Nature 24. Feeney, R. E., Burcham, T. S., & Yeh, view of the acid growth hypothesis. It guards her secrets even more jealously Y. (1986) Annu. Rev. Biophys. Biophys. may, after all, be a coincidence that the than magicians. Chem. 15, 59-78. spatial distribution of growth in both cu- 25. Yang, D. S. C., Sax, M., Chakrabartty, cumber hypocotyls (8) and oat coleop- 1. Amazing Randi (1976) Houdini, His Life A. & Hew, C. L. (1988) Nature (London) andArt (Grosset & Dunlap, New York). 333, 232-237. tiles (26) correlates with the ability ofthe 2. Cosgrove, D. J. (1986) Annu. Rev. Plant 26. Cosgrove, D. J. & Li, Z.-C. (1993) Plant walls to respond to acidic buffers. And it Physiol. 37, 377-405. Physiol. 103, 1321-1328. may also be a coincidence that expansins 3. Cosgrove, D. J. (1993) New Phytol. 124, 27. Jacobs, M. & Ray, P. M. (1976) Plant just happen to promote wall loosening at 1-23. Physiol. 58, 203-209. acidic pH values. The controversy boils 4. Taiz, L. (1984) Annu. Rev. Plant Physiol. 28. Stuart, D. A. & Jones, R. L. (1978) down to whether or not auxin-induced 35, 585-657. Planta 42, 135-142. Downloaded by guest on September 23, 2021