A distinguishing feature of plant cells is the presence of cortical microtubules adjacent to the plasma membrane (37). It has been noted since the discovery of cortical microtubules that the orientation of cortical microtubules in expanding cells is similar to that of cellulose microfibrils (38). This led to the hypothesis that the deposition of cellulose is oriented by an interaction between cellulose synthase and the microtubules, an idea that was reinforced by many observations of correlations between microtubule and microfibril organization which have been comprehensively and critically reviewed by Baskin (39). In the model of Giddings and Staehelin (40), as recast in an influential textbook (41), the movement of cellulose synthase is constrained by a close association between cortical microtubules and the plasma membrane, much like a bumper car bouncing along between rails of cortical tubulin. It is generally assumed that the energy of polymerization provides the motive force that moves the cellulose synthase complex through the membrane.

However, as noted in a recent critique of the model, there is no direct evidence for involvement of microtubules in microfibril orientation and many inconsistencies mediate against the idea (42). For instance, short treatment of Arabidopsis with the microtubule destabilizing drug oryzalin or the microtubule stabilizing drug taxol caused no apparent change to the orientation of cellulose microfibrils in cells that expanded during the treatment, as visualized by field emission scanning electron microscopy (43, 44). Long treatments caused changes in cellulose orientation but these may have been due to effects on the orientation of cell division. Similarly, when microtubule polymerization was impaired by shifting the temperature-sensitive mor1-1 mutant to non-permissive temperature, cellulose microfibrils exhibited a similar pattern of deposition as in controls (45, 46).

Recently, Paredez and coworkers (47) produced a functional N-terminal YFP fusion to CESA6 that complemented the corresponding mutant in Arabidopsis. When expressed under the native promoter, a substantial amount of the fusion protein accumulates in the Golgi apparatus where it assembles into distinct particles that can be seen to move to the plasma membrane. This is compatible with previous evidence from electron microscopy indicating that cellulose synthase rosettes assemble in the Golgi (48). Within less than a minute of arriving in the plasma membrane, the cellulose synthase particles begin moving in linear paths at a constant rate of about 300 nm min-1, somewhat slower than the rate observed by Hirai and coworkers (49) on tobacco membrane sheets. This is reminiscent of yeast chitin synthase III, in which activity is regulated by a specialized mechanism of vesicle sorting coupled with endocytic recycling (50). In this model, chitin synthase is maintained inside specialized vesicles called chitosomes (TGN/early endosome vesicles) and is trans­ported to the specific sites of function where it becomes activated. Inactivation occurs via endocytosis. Because plant Golgi do not synthesize cellulose, it is apparent that the cellulose synthase complexes observed there are not active but that they become activated upon arrival at the plasma membrane. Rosettes have also been estimated to have only a 20 minutes lifetime in moss (51), which may suggest that they are also dissociated or endocytosed.

When viewed in cells in which the microtubules are labeled with CFP, the YFP-labeled cel­lulose synthase particles can be seen to move along the microtubules. Importantly, inhibition of tubulin polymerization with oryzalin rapidly leads to strong disruptions of the normal patterns of movement of the cellulose synthase particles that aggregate in patterns resem­bling meandering streams. Similarly, treatment of seedlings with Morlin, a novel inhibitor of microtubule treadmilling and membrane attachment, caused stalling of the cellulose syn­thase complexes (52). Thus, from live cell imaging it is readily apparent that microtubules exert a strong effect on the orientation of cellulose synthase movement (which presum­ably reflects cellulose synthesis) (47). However, Paredez and coworkers (47) observed that after relatively long periods of oryzalin treatment, when most or all of the cortical micro­tubules have depolymerized, the cellulose synthase particles resume movement in relatively straight parallel paths. The rigidity of cellulose probably explains why no guidance is nec­essary to ensure that cellulose synthase moves in relatively straight lines. It is not clear what orients the pattern of deposition in these cells but models for the formation of oriented patterns of cellulose based on geometric considerations have been proposed (53) and may be testable in these experimental materials. These observations suggest that both sides of the microtubule-microfibril alignment debate are correct and that the discrepancies and inconsistencies between experiments reflect the limitations of using static imaging methods and different treatment times and conditions. The availability of the new imaging tools outlined here should facilitate a resolution of the matter.

Alignment of GFP-labeled cellulose synthase with microtubules was previously reported by Gardiner and coworkers (54), who used an N-terminal fusion of GFP to the xylem-specific CESA7 (irx3) protein. Because of difficulties viewing the vascular tissues by confocal mi­croscopy, the images of this GFP:CESA7 construct are difficult to discern. However, it appears that the distribution of fluorescence is not uniform and there are bands of fluorescence that are perpendicular to the long axis of the cells. Attempts to colocalize tubulin with CESA7 using immunofluorescence methods (54) indicate a similar pattern. However, the resolu­tion of the images was not high enough to provide a critical analysis. Treatment with the microtubule assembly inhibitor, oryzalin, rapidly reduced the banding pattern. Given the technical limitations of working with xylem-localized markers, the observations of Gardiner and coworkers (54) appear to be entirely consistent with the more recent work of Paredez and coworkers (47).

A surprising twist to the microtubule-cellulose synthase story was the observation that in tobacco protoplasts, inhibition of cellulose synthase activity prevented the development of oriented microtubule arrays (55). These data are consistent with the hypothesis that cellulose microfibrils or cellulose synthase, directly or indirectly, provide spatial cues for cortical microtubule organization. Similarly, microtubule organization in spruce pollen tubes was altered by isoxaben (56), and the orientation of microtubules in Arabidopsis root epidermal cells was disrupted by DCB (46).

In Arabidopsis, two genes (At5g17310 and At3g03250) encode proteins that share high aa sequence identity to each other (93%) and to the well-characterized potato and barley UDP-Glc PPase (>80%). Recombinant At5g17310 (UGlcPP1) expressed in E. coli utilizes only Glc-1-P and UTP to form UDP-Glc. UGlcPP1 is specific for both UTP and Glc1P since TTP, GTP, ATP or other sugar-1-phosphates are not substrates for this enzyme (414). A crystal structure of UGlcPP2 and At3g03250 has been submitted (Wesenberg, G. E., Phillips, G. N., Jr., Bitto, E., Bingman, C. A., Allard, S. T.M.). Early biochemical work established that UDP-Glc PPase is inhibited by UDP-Xyl. If this inhibition occurs in vivo, it would imply that UDP-Xyl, in addition to gene expression, regulates the UDP-glucose pool, and thus, the NDP-sugar pool available for wall synthesis. Recent analysis of rice plants where one of the two rice UDP-Glc PPase genes, ugpl, was suppressed, suggests that the production of UDP-Glc during pollen development is critical for callose deposition (417).

The above considerations leave little doubt that the primary aggregates of cellulose emerging from individual rosettes are likely to have a long-period helical character. Depending on the cooperative association of rosettes during biogenesis and their relative mobility within the plasma membrane, these primary aggregates will come together to form a secondary aggregate that may vary in relative organization and have a longer period. At this and higher levels of assembly of native celluloses, tissue and species specificities are expected to arise. One of the key determinants will be the degree to which the synthase rosettes act cooperatively; we anticipate that this is one key point of entry of distinctive genomic information. The variability of higher levels of aggregation is illustrated in Figure 6.2, where it is obvious that
individual fibrils often occur in pairs or triads, and these in turn can be intimately integrated in higher level associations into aggregates of multiple fibrils.

In developing a foundation for experimental studies of native celluloses that explore the relationship between structure and genomic information, it is helpful to consider alternative patterns of aggregation and assess whether they might be altered during isolation from higher plant tissues, whether for experimental studies or in industrial processes that use different celluloses as feedstocks. To accomplish this, a number of models have been represented in the same manner as was done in Figure 6.3, in order to explore how the different nanofibrils with helical structures might come together in the aggregation to form the next higher or secondary level of nanostructure.

To facilitate visualization of factors that enter into aggregation of nanofibrils, Figure 6.11 was developed. In panel A, a 6 by 6 nm nanofibril is represented both as a single nanofibril

A

with the specified helical period and then as an assembly of nine 2 by 2 helical nanofibrils of the same period. The 2 by 2 nm fibrils were considered representative of the most elementary nanofibrils formed. We recognize at the outset that while the square cross section may be stable at the 20 by 20 nm level, it is not likely to be stable at the 2 by 2 or 6 by 6 levels. Surface phenomena would lead to their transformation to polygonal cross sections of higher order. However, we believe panel A to be a helpful intermediate representation because the helical pattern is more clearly visualized.

To improve the approximation to reality, the aggregates in panel B were constructed. First, the corners of the 2 by 2 fibrils in panel A were removed so that the cross section becomes octagonal and can more closely approximate the circular or ellipsoidal polygonal cross sections usually observed for higher plants. The fibrils were then assembled in three different modes. In A, the fibrils were twisted individually and then the assembly also twisted. In B, the individual fibrils were each subjected to a twist of 90° over the 300-nm period, and then packed as closely as possible without their surfaces intersecting. Finally in C, the fibrils were collectively subjected to the twist. These of course represent the most simply visualized members of secondary patterns of aggregation, and other patterns are expected to occur also as a result of variability in patterns of cooperative associations of synthase rosettes. A number of circumstances can be envisioned for the further aggregation of the nanofibrils. In the pattern represented by C at the left of panel B, nanofibrils retain a coherence of order relative to each other that might allow them to come together to form a fibril similar to the single fibril in panel A, but with the corners rounded off. This case is illustrated in detail in Figure 6.12.

The type of aggregation shown in Figure 6.12 may in fact be responsible for formation of the types of fibrils that occur when cellulose is deposited alone in the cell wall, as in nanofibrils of cotton or ramie. The same would apply to aggregation of elementary nanofibrils in algae, though their assembly processes differ from those of higher plants. Pattern B of Figure 6.12 would not be mechanically stable, so we believe this pattern is unlikely to occur in load-bearing tissues. It may occur in other contexts where its distinctive character fits selected functions in the cell wall. The pattern A shown on the right side of panel B in Figure 6.11 is the most likely pattern of aggregation when cellulose is deposited in the presence of other cell wall constituents that might influence the progress of the aggregation.

When one considers flexibility of the fibrils and the possible nearest neighbor, lateral interactions between them, it is not possible to anticipate which pattern of aggregation occurs in any particular plant tissue. And it is at this level of aggregation that we believe the balance between the Ia and the Ip forms is established. When the most elementary nanofibrils co-aggregate with adjacent ones, one can expect the patterns of hydrogen bonding to be modified. It seems very likely that the inherent self-assembly characteristic of the cellulose molecule in its native conformation is more a function of its skeletal organization. The hydrogen bonding patterns appear to be secondary determinants of the aggregation. This, of course, is also consistent with findings from the molecular modeling program.

From a mechanical point of view, we believe the most likely pattern in load-bearing tissues of higher plants is pattern A on the right in panel B of Figure 6.11. This view is influenced, in part, by the pattern being the closest approximation to patterns used at the macroscopic level in the design and construction of cables and ropes. This pattern is likely the most efficient load-bearing structure. These visualizations are derived from construction of mathematical models and are not artistic depictions. However, we recognize that there are other patterns of secondary aggregation.

A cad-4 cad-5 (cad-c cad-d) double mutant of Arabidopsis (ecotype Wassilewskja) was also recently successfully generated (57) and subsequently comprehensively analyzed (71). Figure 7.13E shows the prostrate phenotype that results from the double mutation relative to that of wild type (71); as anticipated, the dynamic modulus properties were also substan­tially reduced, this being a further indication of a structurally weakened vascular apparatus (71). Several other important features were identified following the comprehensive study of different stages of growth/development and polyphenolic deposition into the cell walls of the double mutant, using both chemical degradation and 13C NMR spectroscopic analyses. Specifically, the double mutant contained very small amounts of monolignols (circa 10% of the polyphenolics present), as well as polymeric p-hydroxycinnamaldehyde moieties. At plant maturity, these constituted together about 11.3% of the cell wall residue, in contrast to lignin in the wild-type line which was almost double this amount (~22.5%). Chemical degradation (thioacidolysis) analyses though established that the total amount of monomer — cleavable 8-0-4′ interunit linkages, i. e., monolignol 1,3, and 5 andstyryl — O-aryl ether (sub­structure XII, Figure 7.11B), etc. derived substructures closely mirrored that for monolignol — derived lignin deposition (Figure 7.14D) at the same stages of growth/development. [This was not previously recognized by other researchers (57) since their analyses of the styryl — O-aryl ether-derived substructures (XII, Figure 7.11B) were lower by almost an order of magnitude, due to a lack of either authentic standards and/or correct response factors.]

That is, the 8-0-4′ interunit linkage frequency was again apparently directly propor­tional to polyphenolic content in a manner somewhat analogous to that of lignification proper in the wild-type line; however, the p-hydroxycinnamaldehyde deposition was ter — minated/aborted prematurely (see arrowhead, Figure 7.14D). Perhaps significantly, this metabolic “checkpoint” — arresting polyphenolic deposition — also appears to be coincident to that of termination of H-lignin deposition in the pC3H line, suggesting a common mech­anism is in place for terminating/aborting both processes. Interestingly, 8-5′ linkages were also very evident in the p-hydroxycinnamaldehyde isolates, but in this case with substruc­ture IX (Figure 7.11B); others [e. g., substructures X and XI (239)] were not detected (71).

Taken together, it is proposed that the double mutant has attempted, in a futile manner, to produce a poly-p-hydroxycinnamaldehyde facsimile (of inferior structural properties) to that of monolignol 1-, 3-, and 5-derived lignins. This can thus be provisionally envisaged to occur through very limited substrate degeneracy during template polymerization, that would normally be operative for constitutive macromolecular lignin assembly. On the other hand, the poor biophysical/structural integrity properties of the phenotype so obtained presumably provide useful insight as to why poly-p-hydroxycinnamaldehydes did not evolve as a substitute for the monolignols 1, 3, and 5 in lignification proper.

While using our definition for a classical model above allows us to evaluate the energy of a given arrangement of atoms, and potentially to optimize the structure to a local minimum energy conformation when connected to an optimization algorithm, this approach has only limited scientific value. This is especially true when one wants to study a processive enzyme such as the cellulose hydrolysis enzyme CBH I. In such a situation, it is the dynamics of the system that are of interest to researchers trying to uncover its mode of action with the aim of ultimately improving its efficiency. Thus, to obtain such dynamical properties it is necessary to use the potential energy equation discussed above, and the corresponding gradients (or forces) to propagate the system through time. This is achieved by using dynamics methods that are collectively termed molecular dynamics (MD).

8.4.1 Dynamics methods

The workhorse of the MD methodologies and programs is the dynamics engine that treats the system as a classical mechanical system and integrates Newton’s equations of motion based on the force field that is applied. This amounts to initiating some velocities for the atoms, determining forces (the negative gradient of the potential), and then propagating the velocities and adjusting them for the forces one small step at a time. An analytical solution to Newton’s equations of motion for even a four-atom molecule using a typical all-atom force field does not exist and thus it is necessary to employ numerical techniques. Numerous numerical algorithms exist for solving the differential equations that arise from Newton’s equations of motion, two popular formulations being the predictor-corrector methods (47) and finite difference methods. The most commonly used are the finite difference methods and so the following discussion centers on these methods.

Until recently, pretreatments have usually been designed for extensive removal of hemi — cellulose in order to improve the enzymatic hydrolysis of cellulose. Thus, the impact of the enzymatic hydrolysis of fiber-bound hemicellulose on cellulose hydrolysis has not been considered important, as evaluated by the number of publications. The amount of hemi — cellulose in the solid substrate resulting from pretreatment varies from about 1% to up to 25%, depending upon the pretreatment. Acidic and high temperature pretreatments tend to hydrolyze and remove more hemicellulose, while alkaline and low temperature processes generally leave higher residual hemicellulose in the solids and greater amounts of oligomers in the hydrolyzates. Ammonium fiber expansion/explosion (AFEX) is essentially a dry pro­cess and results in virtually no change in the solids composition, as there is no liquid phase to partition components.

The removal of xylans during the pretreatment has been shown to correlate with the hydrolyzability of the raw material (87). There was a clear correlation with the residual hemicellulose content of pretreated spruce and the degree of hydrolysis. It is however, difficult to conclude whether this correlation is affected also by other factors, such as further chemical modifications caused by the severity of the pretreatment. In another study, various raw materials with different levels of residual hemicellulose contents did not seem to follow this hypothesis. Neither residual xylan nor glucomannan seemed to correlate with the degree of cellulose hydrolysis when additional p-glucosidase was supplemented. On the other hand, xylanase activity in the preparation was shown to increase the hydrolysis conversion and rate (88).

Hemicellulolytic activity in commercial cellulase preparations has been expected to pro­vide the necessary hydrolysis of the residual hemicellulose in the solid matrix. The activities of accessory enzymes in the commercial preparations vary, and in most reports, are not even measured. Their role in the solubilization of matrix bound hemicelluloses can only be speculated. Even less is known about the effect of these unquantified activities on the soluble oligomer fractions resulting from pretreatment. Although extensive studies on the activities required for enzymatic hydrolysis of these compounds have not been carried out, interest and research into this area are rapidly expanding. From the known structures and known enzyme activities, one can begin to evaluate enzyme mixes for their efficacy on these complex substrates.

A large number of pretreatment approaches have been investigated across a variety of biomass feedstock types. Published studies are widely available and there are several re­view articles available that provide a general overview of the field (10-14). Unfortunately, standard experimental and analytical methodologies have not been utilized across much of the published pretreatment literature, making it difficult to conduct comparative eval­uations based on published findings. Recently, several pretreatment research teams across North America have undertaken the first broad-ranging coordinated effort to develop com­parative process performance and economic evaluation data for several leading pretreatment options. While this collaboration, known as the Biomass Refining Consortium for Applied

Fundamentals and Innovation (CAFI), does not fully encompass all possible pretreatment technologies or potential biomass feedstocks, it does serve as a model for how comparative data can be developed and made available for various stakeholders and potential commer­cializes of biomass conversion technologies. A series of papers that cover the comparative findings from a recently completed CAFI project on a common corn stover feedstock have been published (15-22).

14.5.1 Physical pretreatments

14.5.1.1 Comminution

Most pretreatment approaches require that collected biomass undergo some degree of me­chanical size reduction prior to introduction into a pretreatment reactor. Woody biomass can be chipped in a manner similar to that commonly practiced in the pulp and paper industry. Depending on the pretreatment process and associated heat and mass transfer considerations, woody biomass is commonly comminuted to particle sizes smaller than typical wood pulping chips, as pretreatment processes are often practiced at much shorter residence times than wood pulping processes. Other biomass feedstocks, such as agricul­tural residues and herbaceous energy crops, can be coarsely chopped during or after the feedstock harvesting operation. Again, further comminution may be employed on these feedstock types as well, depending on the pretreatment process and associated heat and mass transfer considerations.

Intensive comminution of various biomass types has been practiced as an actual pretreat­ment process, without any further pretreatment prior to enzymatic hydrolysis. Methods include various types of ball milling (dry, wet, and vibratory processes), other types of attrition milling, compression milling, and wet or dry disk refining (23-27). While these methods can increase the enzymatic digestibility of the comminuted biomass by increasing the available surface area and by decrystallizing cellulose, most studies have concluded that the high mechanical power requirements cause comminution to be cost-prohibitive for use as a stand-alone pretreatment in a biomass to ethanol conversion process.

14.5.1.2 Irradiation

The use of high-energy electron beam and microwave energy sources as a biomass pretreat­ment approach has been investigated. These methods are believed to mechanically disrupt plant cell wall structure and decrease the crystallinity of cellulose, resulting in an increased enzymatic digestibility of cellulose. Issues with cost, energy intensity, and the practical­ity of applying such approaches in commercial processes have limited the development of irradiation as a viable pretreatment approach (12, 28).

The recombinant cellulolytic strategy involves engineering non-cellulolytic organisms that exhibit high product yields by producing a heterologous cellulase system enabling cellulose utilization. Early research advances have been reviewed previously (15). The yeast, S. cere — visiae, is a promising host organism for this strategy because of its high ethanol productivity at high yields, high osmo — and ethanol-tolerance, natural robustness in industrial processes, ease of genetic manipulation, and generally regarded as safe status due to its long association with the food and beverage industries. Cellulases from bacterial and fungal sources have been transferred to S. cerevisiae, enabling the hydrolysis of cellulosic derivatives (15), or growth on cellobiose (63, 64).

Three recombinant enzymes — Trichoderma reesei endoglucanase II, T. reesei cellobiohy — drolase II, as well as Aspergillus aculeatus p — glucosidase cellulase — have been co-expressed in S. cerevisiae via individual fusion proteins with the C-terminal-half region of a-agglutinin (65). However, this recombinant strain cannot grow on cellulose using these recombinant cellulases, possibly because of poor recombinant cellulase expression or low enzyme activity or both.

van Zyl and coworkers (66) were the first to produce recombinant S. cerevisiae that can grow on pure insoluble cellulose by expressing two recombinant cellulases — the T. reesei endoglucanase (EG I) and the S. fibuligera p-glucosidase (BGL 1). The resulting strain was able to grow on phosphoric acid swollen cellulose (PASC) through simultaneous production of sufficient extracellular endoglucanase and p-glucosidase activity. Anaerobic growth was observed on the medium containing 10 g/L PASC as sole carbohydrate source with concomi­tant ethanol production of up to 1.0 g/L. Since crystalline cellulose hydrolysis requires three types of cellulases (endoglucanase, cellobiohydrolase, and p-glucosidase) to work together (24, 25), it is still a challenge to develop recombinant cellulolytic microorganisms that can express high levels of these cellulases to support cell growth on crystalline cellulose.

To achieve the self-supporting growth based on recombinant cellulases, it is appropriate to estimate the feasibility of cellulase expression levels. On the basis of the sufficiency of ex­pression of growth-enabling heterologous enzymes, the level of enzyme expression required to achieve a specified growth rate maybe calculated as a function of enzyme-specific activity (63). For growth enabled by cellulases with specific activities in the range available, required expression levels are well within the range reported in the literature (1-10% of cellular pro­tein) (67, 68). Protein expression at this level has been reported in both S. cerevisiae (67) and E. coli (69), although not to date for active cellulases. On the other hand, nature has created a diversity of cellulolytic microorganisms. With time, we anticipate that recombi­nant cellulolytic microorganisms with activity on crystalline cellulose will be created in the laboratory.

Some plant cell wall polysaccharides contain small amounts of ester-linked hydroxycin — namic acid derivatives such as p-courmaric and ferulic acid (291). These ester-linked acid derivatives can undergo oxidative coupling to form dehydrodimers that may lead to cell wall polysaccharide cross-linking (291, 292) and may also be involved in the formation of polysaccharide-lignin complexes (293, 294). The ester-linked hydroxycinnamic acids are more abundant in the walls of the monocotyledonous group known as the commelinids, including the grass family (295) and such walls are particularly rich in hydroxycinnamic acids linked to the hemicellulose arabinoxylan (291), and to a lesser extent the hemicellu — lose xyloglucan. However, ester-linked hydroxycinnamic acids have also been shown to be linked to pectins in plants such as spinach and sugar beet (291,296-298). The types of link­ages associated with pectin include the following feruloylated arabinan and (1^4)-linked D-galactosyl oligosaccharides that presumably originate from side chains from RG-I: O — (6-O-frans-feruloyl)-p-D-galactopyranosyl-(1^4)-D-galactose, O-(2-O-trans-feruloyl)- a-L-arabinofuranosyl-(1^5)-L-arabinose, and O-a-L-arabinofuranosyl-(1^3)-O-(2- O-trans-feruloyl-a-L-arabinofuranosyl)-(1^5)-L-arabinose [see (291) and references therein] and O-[5-O-(feruloyl)-Ara]-(1^5)-[2-O-(feruloyl)-Ara]-(1^5)-Ara (299).

The mode ofsynthesis offeruloylated hemicellulose and pectin has received some, albeit, limited study. There is evidence that hemicellulosic arabinoxylan can be feruloylated by both feruloyl-glucose and feruloyl-CoA precursors/substrates, although the precise role of these substrates in the synthesis of feruloylated arabinoxylan within the cell (likely in the Golgi) or in the cell wall remains unclear (300, 301). A Golgi or sub-Golgi fraction from parsley suspension-cultured cells was able to transfer ferulic acid from feruloyl-CoA onto endogenous polysaccharide acceptors. However, since the identity of the polysaccharide(s) that was feruloylated was not determined (302), it is not clear whether the enzyme activity identified was involved in pectin or hemicellulose feruloylation.

Recently, Mitchell and coworkers (303), using a bioinformatics approach to identify genes highly expressed in cereals during the late stages of arabinoxylan synthesis, identified cereal Pfam family PF02458 genes, members of the CoA-acyl transferase superfamily, as candidate feruloyltransferases. However, enzymatic confirmation that these genes are actually involved in feruloylation has not been presented.

What controls the supply and flux of UDP-GlcA in plants is still debatable and it is very likely that different plant species adopt different mechanisms to control the supply of UDP — GlcA. In maize, mutants lacking the activity of one UDPGDH isoform have a reduction in the content of Ara, and Xyl in hemicellulose. This suggests that in maize, UDPGDH-A is a major supplier of the UDP-pentoses and that the myo-inositol oxidation pathway (not

“bifunctional ADH”) cannot compensate the flux of “sugar” to the formation of UDP-GlcA. In Arabidopsis mutants lacking MIOX1 and 2 isoform activities, no significant differences in monosaccharide amount or composition were observed in wall polysaccharides when compared to wildtype. Thus, it is likely that the major contributor for flux of NDP-sugars in plants is UDP-Glc. As mentioned above, it is possible that the myo-inositol pathway and the salvage pathway operate in a tissue-specific manner, for example, during pollen tube growth. During pollen tube germination and growth, large amounts of pectin are degraded. It is likely that the free sugars are recycled back by kinases and the activity of Sloppy, to readily form an available pool of NDP-sugars. This pool will provide NDP-sugars for growth of the pollen tube. Pollen tubes are one of the fastest growing cells known (1 cm h-1) and mutants lacking Sloppy have a pollen phenotype (415). A similar regulation scenario likely occurs during seed germination with seeds that store a large amount of phytic acid, as discussed above.