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Cell wall extensibility controls the rate of plant cell growth. It is determined by intrinsic mechanical properties of wall polymers and by wall proteins modifying these polymers and their interactions. Heat-inactivation of endogenous cell wall proteins inhibited acid-induced extension of onion epidermis peels transverse to the net cellulose alignment in the cell wall but not parallel to it. In the former case the acid-induced extension could be controlled by expansins and in the latter case by pectins restricting shear between microfibrils. Heat-inactivated cell walls stretched transversely to the net cellulose orientation extended faster at pH 5.7 and slower at pH 4.5 compared to native walls. Expansins seem to be inactive at pH 5.7, so that faster extension may result from heat-induced viscous flow of pectins and conformational changes in the cuticle of the epidermis. This stimulation of wall extension is not seen at pH 4.5 as it is outweighed by the inhibitory effect of expansin heat-inactivation. Thus, cell wall extension in higher plants might be controlled by a complex interplay between protein-dependent and protein-independent mechanisms, the result of which depends on pH and preferential orientation of main wall polymers.
It is usually considered that the rate of plant cell growth is controlled by cell wall extensibility.1 Extensibility is determined by intrinsic mechanical properties of wall polymers and by wall proteins modifying these polymers and their interactions. 2 Current research is mostly focused on the protein-dependent regulation of wall extensibility and pays less attention to protein-independent mechanisms. Heat-inactivation has traditionally been used to eliminate activity of endogenous wall proteins.3,4 After this treatment wall extension is entirely determined by the mechanics of its polymers allowing, for instance, the effect of an exogenous protein to be studied. Our recent data on cell wall heat-inactivation demonstrate a complex interplay between protein-dependent and protein-independent mechanisms of control of wall extension in higher plants, the output of which is highly dependent on pH and polymer orientation in the cell wall.5,6
We studied the effects of exogenously applied xyloglucan endotransglucosylase/hydrolase (XTH) enzymes on the extension of heat-inactivated onion epidermis walls to estimate a possible involvement of these enzymes in the control of the wall extensibility.5,6 In addition to specific effects of XTHs, we found that boiling in water for 15 seconds had a different effect on the acid-induced wall extension parallel and transverse to the net cellulose orientation in onion epidermis.5 In the former case the stimulatory effect of a pH 4.5 buffer on the wall extension was not significantly inhibited by the heat-inactivation, while in the latter case it was abolished (Fig. 1). This shows that the acid-induced wall extension transverse to the net cellulose orientation is for the greater part protein-mediated. It could be controlled by expansins3 and might involve separation of parallel microfibrils.7 The acid-induced extension parallel to the net cellulose alignment is for the greater part protein-independent and could involve shear between microfibrils.8
To our knowledge, the protein-independent acid-induced extension has not been demonstrated in higher plants, but it was described in charophycean algae.9,10 In Nitella short-term boiling in water had no effect on the extension of internodal walls at pH 4.5.9 The matrix of charophycean cell walls is composed mainly of polygalacturonic acid (PGA)11,12 while no xyloglucans are present.11 This pectin cross-linked by Ca2+ seems to be a load-bearing component of the charophycean walls.13 As PGA is unable to directly cross-link cellulose microfibrils,14 PGA-PGA interactions could be responsible for the protein-independent acid-induced extension in charophytes. Interestingly onion cell walls contain more pectin and less xyloglucan (about 50% and 10% of the wall dry weight, respectively)15 than typical angiosperm walls.16 Apparently the ancient protein-independent mechanism of acid-induced extension of charophytes still exists in the walls of higher plants rich in pectin and poor in xyloglucan where it interacts with the expansin-mediated mechanism. The fact that the former mechanism seems to prevail over the latter in the direction parallel to the net cellulose alignment (Fig. 1) can be explained by a preferential orientation of pectin in the onion epidermis.17 The PGA backbone was shown to be co-aligned with cellulose microfibrils, whereas the carbonyl side groups of PGA are orientated approximately perpendicular to them.17 Apparently such preferential orientation of pectin restricts shear but not separation of microfibrils, explaining the observed orientation-dependent differences in the mechanism of the acid-induced wall extension (Fig. 1).
Subsequently we focused on the wall extension transverse to the net orientation of cellulose in the onion epidermis.6 Boiling in water for 15 seconds not only decreased the subsequent wall extension at pH 4.5 as mentioned above (Fig. 1, right bars),5 but, surprisingly, also increased it at pH 5.7 (reviewed in ref. 6). A similar finding was reported time ago for heat-inactivated cell walls of Nitella.9 We have explained the opposite effect of pH on the basis of different pH optima for cell wall loosening and tightening proteins.6 Nevertheless an alternative or additional mechanism, by analogy with Nitella,9 could involve a limited heat-induced degradation of some labile bonds within the wall, which renders it more extensible. This weak stimulatory effect on wall extension is not seen at pH 4.5 as it is outweighed by the strong inhibitory effect of the heat-inactivation of expansins, cell-wall loosening proteins active at acidic pH values.3 However, expansins seem to be inactive at pH ≥ 5.7,3 in native cell walls, and the weak effect due to limited wall degradation by heat-inactivation is only revealed in the absence of their background activity.
The nature of the labile bonds broken in the wall during boiling is unknown. Obviously, the covalent bonds within/between wall polymers are too strong to be broken by boiling for 15 s. The likely candidates are ionic interactions between pectins, the wall polymers demonstrating higher gel-sol transitions and viscous flow at elevated temperatures.18 Other candidates are non-covalent interactions between cutin and waxes constituting plant cuticles.19 It was shown that cutin changes conformation20 and waxes melt21,22 at temperatures well below 100°C. Plant cuticles affect the mechanics of the underlying cell walls23 and become more extensible at higher temperatures.24 We might indeed observe this effect in the isolated onion epidermis as it is covered by a hydrophobic cuticle.
To sum up, our data show that wall extension in higher plants seems to be controlled by a complex interplay between the ancient protein-independent and the more recent protein-mediated mechanisms. The result of this interplay depends on pH, relative proportions of main wall polymers as well as on their preferential orientation.
This work was supported by research grants of the Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT, Vlaanderen), the Interuniversity Attraction Poles Programme—Belgian State—Belgian Science Policy [IUAP VI/33], the Research Foundation—Flanders (FWO) [Grant WO038.04 N and G.0101.04] and the University of Antwerp (BOF-NOI).
Addendum to: Van Sandt V, Suslov D, Verbelen J-P, Vissenberg K. Xyloglucan endotransglucosylase activity loosens a plant cell wallAnn Bot200710014671473 doi: 10.1093/aob/mcm248. and Maris A, Suslov D, Fry SC, Verbelen J-P, Vissenberg K. Enzymic characterization of two recombinant xyloglucan endotransglucosylase/hydrolase (XTH) proteins of Arabidopsis and their effect on root growth and cell wall extensionJ Exp Bot20096039593972 doi: 10.1093/jxb/erp229.
Previously published online: www.landesbioscience.com/journals/psb/article/11771