4.3.6  Biochemical processes in walls of growing cells

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Rapid physical changes in cell walls (Section 4.3.5) have long been ascribed to wall loosening and rigidifying factors. Evidence that these rheological changes occur almost instantaneously, coupled with the abundance of cell wall proteins, have prompted a search for enzymes as wall-modifying factors. In principle, relatively few enzymes could account for quick changes in wall plasticity, such as that observed in Nitella, particularly if tension within the wall could be relieved simply by breakage and re-formation of H-bonds between wall polymers. However, it is more likely that many enzymes are required to loosen, rigidify and stabilise the range of wall polymers found in higher plant cell walls. The interplay be-tween f and Pth in wall modifications, depending on environ-mental conditions, cell type and ontogeny, also points to a high level of molecular complexity underlying wall rheology.

Early experiments on acid-induced wall growth (Section 4.1.3(b)) raised the possibility that protons contribute to rapid wall loosening. Specifically, H+ could activate cell wall enzymes with an acid pH optimum by lowering wall pH below 5. This view of cell wall loosening is still largely hypothetical because so few components of the wall loosening process, especially enzymes, have been identified. Indications that H+ concen-trations in cell walls influence growth rates come from the blue light inhibition of cucumber seedling growth — depolarisation of plasma membranes (reduced H+ extrusion) precedes wall stiffening (Section 4.3.5), raising the possibility that wall alkalinisation inhibits growth. A second line of evidence comes from the expansins (growth-inducing proteins) which are highly active in walls of living cells at pH 4.5 but not at pH 6.8 (Section 4.3.6(b)).

(a)  Extension of the cell wall matrix

Ordered assembly of cell walls as they are exocytosed from plasma membranes is critical for processes such as growth and morphogenesis. The three-dimensional structure of walls arises through interaction of several families of molecules: inextensible cellulose microfibrils lie embedded in a carbohydrate-rich matrix of hemicelluloses (e.g. xyloglucans and glucuronoarabinoxylans), pectins, proteins and sometimes lignin (Table 4.2). Further biochemical changes to some of these molecules occur within walls, including cross-linkage reactions leading to polymerisation. Whether the response of walls to abrupt changes in P is being considered (as in Section 4.3.5), or steady growth of an elongating tissue, molecular events in cell walls are important.

The first clue to regulation of cell wall relaxation is that growth of most plant cells is not isodiametric (equivalent stretching in all directions). Ethylene application can induce cells to balloon around their girth but most cells elongate according to a strict polarity set down throughout the organ. Cellulose microfibrils in cell walls prior to initiation of growth tend to be transversely oriented (at 90°) to the axis of growth (Figure 4.19). The inextensible nature of cellulose microfibrils is good physical evidence that elongating cells are constrained from increasing in girth and must grow by separation of adjacent microfibrils. This places the focus on the non-cellulosic polymers of the cell wall (the so-called gel matrix) as participants in growth.

(b)  Enzymes and wall tension

Species variation in the carbohydrate composition of cell walls suggests that different enzymes are required to act on these substrates in different species. Xyloglucan, a xylose–glucose polymer, is a prime candidate for binding cellulose microfibrils together in dicotyledonous plants where it is abundant. Xyloglucan adheres strongly to cellulose in a strong stoichiometric relationship. An enzyme capable of cleaving and rejoining xyloglucan chains has also been isolated, providing the elements theoretically required for a biochemical model of wall extensibility. The activity of this enzyme (xyloglucan endotransglycosylase or XET) in young barley seedlings does not peak exactly in the region of maximum leaf growth but a role for XET in growth is suggested by the stimulatory effect of gibberellic acid on both XET activity and growth (Smith et al. 1996). XET might have more functional significance in dicotyledons in which xyloglucan is often a very abundant wall polymer. Glucanases isolated from maize cell walls enhance auxin-stimulated growth of maize coleoptiles, antibodies to glucanase blocking the effect, but no further indication of the role of these enzymes in growth has emerged.

Other experiments have demonstrated that a class of cell wall proteins, expansins, are potential agents for catalysing cell wall yielding in vivo (McQueen-Mason 1995). Figure 4.24a shows that sharp gradients in growth along the hook of a cucumber hypocotyl are paralleled by a gradient in extension of these tissues when stretched under acid conditions (Figure 4.24b) but not at neutral pH (Figure 4.24c). When tissues were killed by boiling, extension was blocked (Figure 4.24d). Hypocotyl extension requires acid pH and non-denatured proteins.

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Figure 4.24 Distribution of growth and wall extension at four positions along a cucumber hypocotyl. (a) Growth rate is most rapid near the hook. (b) Hypocotyl segments were frozen, thawed, abraded and stretched under a 20 g load in an acidic buffer (pH 4.5), revealing most rapid extension in the fast—growing hook. (c) When measured at pH 6.8, segments extended very little. (d) Segments in which enzymes were denatured by boiling did not extend under the load (From McQueen—Mason 1995; reproduced with permission of Journal of Experimental Botany)

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Figure 4.25    Extension of apical 1 cm segments of abraded cucumber hypocotyls which were either frozen and thawed or boiled (proteins denatured) prior to application of a load. After boiling alone there was negligible extension but addition of a crude preparation of ‘apical wall proteins’ extracted from rapidly growing cell walls induced hypocotyls to stretch. Hypocotyl segments which were frozen and thawed began to stretch only after transfer from neutral pH (6.8) to acid pH (4.5), demonstrating the dependence of wall extension on acid conditions (From McQueen—Mason et al. 1992; reproduced with permission of Plant Cell)

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Figure 4.26 (a) Extension activity assayed in crude preparations containing wall proteins extracted from tissues at four positions along the axis of a cucumber hypocotyl (see Figure 4.25). Protein extracted from these four positions was assayed by adding it to apical hypocotyl segments that had been frozen, thawed, abraded and clamped into an extensometer prior to stretching. Note that even non-growing tissues (position 4) had wall proteins capable of inducing extension. (b) Segments which were boiled to denature wall proteins stretched more on addition of partially purified expansin if they were taken from the growth zone of the hypocotyl, indicating a loss of expansin sensitivity as cells exited the growth zone (From McQueen-Mason et al. 1992; reproduced with permission of Plant Cell)

Two lines of evidence point to expansins having a direct role in cell extension. First, preparations of proteins from growing cell walls caused boiled cell walls to stretch sig--
n-ificantly at a pH of 4.5 but not at a pH of 6.8 (Figure 4.25). Purification of this showed that a 25 kD expansin was capable of catalysing wall extension. Second, while expansin activity does not decline in tissues as they exit the growth zone (Figure 4.26a), sensitivity of these tissues to added expansin does decrease (Figure 4.26b). This might be interpreted as a decreasing availability of substrates for expansin to act on in expanded walls. In the case of expansins, it is proposed that the ‘substrates’ are hydrogen bonds between hemicelluloses and cellulose microfibrils. Expression of expansin-encoding genes, release of the enzyme into cell walls, wall acidification and accessibility to H-bonds might all play a role in determining wall rheology. Expansin applied to meristem surfaces alters the development of leaf initials, suggesting multiple roles for expansin as a wall-loosening factors. Undoubtedly, further enzymes which play a role in wall loosening in other species and tissues await discovery.

What is clear is that the biophysical consequences of instantaneous changes in tension in cell walls (e.g. induced by changes in P) will be followed by a phalanx of biochemical events including wall polymer synthesis and altered gene expression. Sustained expansion of plant cell walls cannot be explained simply by inexorable wall hydrolysis; if it were, cell walls would weaken to breaking point during growth. The ‘setting’ of long-term cell expansion rates is likely to hinge on biochemical events underlying wall relaxation and reinforcement.

(c)  Cessation of cell wall expansion

Molecular events leading to cessation of wall expansion are even less well understood than those which initiate growth. A common view is that sufficient tension develops over time in the molecules cross-linking cellulose microfibrils (e.g. xyloglucans) to prevent further wall expansion. Essentially, when a cell has reached its final dimensions its wall is ‘locked’ into a final, hardened conformation. Molecules with a specific role in growth cessation are thought to be exocytosed into cell walls, providing either substrates for cross-linkage reactions or enzymes catalysing cross-linkage of pre-existing wall polymers. Identification of cross-linkage reactions between
moieties found in the cell wall have led to a search for their presence in vivo. For example, ferulic acid residues in grass cell walls can cross-link to produce di-ferulic acid and potentially stiffen walls through formation of a polysaccharide-lignin network. Unfortunately, in rice coleoptiles the abundance of the di-ferulic form bore no relation to growth cessation.

Secondary cell walls generally form after primary walls have ceased to grow but the familiar rigidity of secondary cell walls (e.g. wood) is mostly viewed as distinct from stiffening of primary walls. Lignification of primary walls commences earlier than once thought and is a possible factor in growth cessation (Müsel et al. 1997). Such a response might be controlled through release of peroxide into walls in much the same way as seen in walls subject to fungal attack. Peroxidases are targeted as candidates for the catalysis of these reactions.

The number of non-cellulosic polymers coming under tension as a cell wall expands will rise if there is not a continual release of that tension, probably through enzyme action. Therefore the degradation of enzymes responsible for polymer cleavage might also play a role in cessation of growth. However, experiments with expansins are a good reminder that the sensitivity of the wall (making substrate for enzymes) is also an important factor in growth cessation. Understanding rigidification of this complex matrix of polymers demands input from the disciplines of biology, chemistry and physics. Combining established techniques with novel approaches to the study of individual cells (e.g. Fourier-Transform Infra-red microspectroscopy and the cell pressure probe) will bring new insights to the molecular basis of wall expansion.

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