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.