Summary: Numerous examples have been presented of enzyme activities, assayed in vitro, that appear relevant to the synthesis of structural polysaccharides, and to their assembly and subsequent degradation in the primary cell walls (PCWs) of higher plants. The accumulation of the corresponding mRNAs, and of the (immunologically recognized) proteins, has often also (or instead) been reported. However, the presence of these mRNAs, antigens and enzymic activities has rarely been shown to correspond to enzyme action in the living plant cell. In some cases, apparent enzymic action is observed in vivo for which no enzyme activity can be detected in in‐vitro assays; the converse also occurs. Methods are reviewed by which reactions involving structural wall polysaccharides can be tracked in vivo. Special attention is given to xyloglucan endotransglucosylase (XET), one of the two enzymic activities exhibited in vitro by xyloglucan endotransglucosylase/hydrolase (XTH) proteins, because of its probable importance in the construction and restructuring of the PCW's major hemicellulose. Attention is also given to the possibility that some reactions observed in the PCW in vivo are not directly enzymic, possibly involving the action of hydroxyl radicals. It is concluded that some proposed wall enzymes, for example XTHs, do act in vivo, but that for other enzymes this is not proven. Contents I. Primary cell walls: composition, deposition and roles 642 II. Reactions that have been proposed to occur in primary cell walls 645 III. Tracking the careers of wall components in vivo: evidence for action of enzymes in the walls of living plant cells 656 IV. Evidence for the occurrence of nonenzymic polymer scission in vivo? 666 VI. Conclusion 667 References 667

Major differences in primary cell wall (PCW) components between non‐vascular plant taxa are reported. (1) Xyloglucan: driselase digestion yielded isoprimeverose (the diagnostic repeat unit of xyloglucan) from PCW‐rich material of Anthoceros (a hornwort), mosses and both leafy and thalloid liverworts, as well as numerous vascular plants, showing xyloglucan to be a PCW component in all land plants tested. In contrast, charophycean green algae (Klebsormidiumflaccidium, Coleochaetescutata and Chara corallina), thought to be closely related to land plants, did not contain xyloglucan. They did not yield isoprimeverose; additionally, charophyte material was not digestible with xyloglucan‐specific endoglucanase or cellulase to give xyloglucan‐derived oligosaccharides. (2) Uronic acids: acid hydrolysis of PCW‐rich material from the charophytes, the hornwort, thalloid and leafy liverworts and a basal moss yielded higher concentrations of glucuronic acid than that from the remaining land plants including the less basal mosses and all vascular plants tested. Polysaccharides of the hornwort Anthoceros contained an unusual repeat‐unit, glucuronic acid‐α(1→3)‐galactose, not found in appreciable amounts in any other plants tested. Galacturonic acid was consistently the most abundant PCW uronic acid, but was present in higher concentrations in acid hydrolysates of bryophytes and charophytes than in those of any of the vascular plants. Mannuronic acid was not detected in any of the species surveyed. (3) Mannose: acid hydrolysis of charophyte and bryophyte PCW‐rich material also yielded appreciably higher concentrations of mannose than are found in vascular plant PCWs. (4) Mixed‐linkage glucan (MLG) was absent from all algae and bryophytes tested; however, upon digestion with licheninase, PCW‐rich material from the alga Ulva lactuca and the leafy liverwort Lophocolea bidentata yielded penta‐ to decasaccharides, indicating the presence of MLG‐related polysaccharides. Our results show that major evolutionary events are often associated with changes in PCW composition. In particular, the acquisition of xyloglucan may have been a pre‐adaptive advantage that allowed colonization of land.

The plant cell-wall matrix is equipped with more than 20 glycosylhydrolase activities, including both glycosidases and glycanases (exo- and endo-hydrolases, respectively), which between them are in principle capable of hydrolysing most of the major glycosidic bonds in wall polysaccharides. Some of these enzymes also participate in the ‘cutting and pasting’ (transglycosylation) of sugar residues—enzyme activities known as transglycosidases and transglycanases. Their action and biological functions differ from those of the UDP-dependent glycosyltransferases (polysaccharide synthases) that catalyse irreversible glycosyl transfer. Based on the nature of the substrates, two types of reaction can be distinguished: homo-transglycosylation (occurring between chemically similar polymers) and hetero-transglycosylation (between chemically different polymers). This review focuses on plant cell-wall-localized glycosylhydrolases and the transglycosylase activities exhibited by some of these enzymes and considers the physiological need for wall polysaccharide modification in vivo. It describes the mechanism of transglycosylase action and the classification and phylogenetic variation of the enzymes. It discusses the modulation of their expression in plants at the transcriptional and translational levels, and methods for their detection. It also critically evaluates the evidence that the enzyme proteins under consideration exhibit their predicted activity in vitro and their predicted action in vivo. Finally, this review suggests that wall-localized glycosylhydrolases with transglycosidase and transglycanase abilities are widespread in plants and play important roles in the mechanism and control of plant cell expansion, differentiation, maturation, and wall repair.

We have developed a method for the colocalization of xyloglucan endotransglycosylase (XET) activity and the donor substrates to which it has access in situ and in vivo. Sulforhodamine conjugates of xyloglucan oligosaccharides (XGO–SRs), infiltrated into the tissue, act as acceptor substrate for the enzyme; endogenous xyloglucan acts as donor substrate. Incorporation of the XGO–SRs into polymeric products in the cell wall yields an orange fluorescence indicative of the simultaneous colocalization, in the same compartment, of active XET and donor xyloglucan chains. The method is specific for XET, as shown by competition experiments with nonfluorescent acceptor oligosaccharides, by negligible reaction with cello-oligosaccharide–SR conjugates that are not XET acceptor substrates, by heat lability, and by pH optimum. Thin-layer chromatographic analysis of remaining unincorporated XGO–SRs showed that these substrates are not extensively hydrolyzed during the assays. A characteristic distribution pattern was found in Arabidopsis and tobacco roots: in both species, fluorescence was most prominent in the cell elongation zone of the root. Proposed roles of XET that include cell wall loosening and integration of newly synthesized xyloglucans could thus be supported.