Two types of cellulose synthesis complex knit the plant cell wall together

Plant secondary cell walls (SCWs) compose most of Earth’s renewable fibers and biomass (1), and they have irreplaceable roles in the plant lifestyle, ecosystem cycles, carbon sequestration, and human industry. Nonetheless, much remains unknown about how these strong, cellulose-rich polymer networks are synthesized and assembled. Watanabe et al. (2) generate key insights into the dynamic regulation of the cellulose synthase (CESA) enzyme family during the transition between the synthesis of the primary cell wall (PCW), which surrounds all expanding plant cells, and the SCW, which confers specialized functions to some cells. The PCW and SCW become unified despite being successively synthesized and having different matrix polymers and cellulose content. This structural coherency is related to the little-explored “transition period” of cell wall synthesis (3–6), the focus of Watanabe et al.’s research (2). Their results provide clues about how differences in SCW properties of diverse plants may arise and point to the isomer-specific regulation of intracellular membrane protein trafficking, leading to important future research directions.

Based on in silico structural comparison with a bacterial enzyme (7), each CESA synthesizes one β-1,4-glucan chain while simultaneously exporting the polymer into the cell wall space. The individual CESAs are organized within a cellulose synthesis complex (CSC), which has six lobes and an average 21.4-nm hexagonal diameter as measured where the transmembrane helices cross the plasma membrane (8) (Fig. 1). Eighteen CESAs are predicted to exist within one CSC (8), which facilitates the coalescence of multiple glucan chains into a microfibril. Each CSC moves within the plasma membrane as it spins a strong, partially crystalline, cellulose microfibril in its wake (9). The locations of CSCs in the membrane and their direction of movement are affected by, but not entirely dependent on, cortical microtubules (6, 9, 10). For example, cortical microtubule banding precedes the deposition of the banded SCW thickenings of primary xylem elements. This banding pattern focuses the cellular events required for SCW cellulose synthesis, including the CSCs that Watanabe et al. (2) analyzed in terms of their composition and dynamic behavior during the transition between PCW and SCW synthesis.

Fig. 1.

Abundant CSCs viewed by freeze-fracture transmission electron microscopy in the transition period of a differentiating primary xylem element of Zinnia elegans. The transmembrane helices of the CSCs are visualized on the interior face of the fractured plasma membrane bilayer. The transition period was diagnosed by the relatively dense banded CSCs in a plasma membrane region that did not yet curve inward, as it would have once SCW synthesis progressed further. The CSCs are arbitrarily encircled in green or magenta to symbolize Watanabe et al.’s (2) discovery that both PCW and SCW CSCs cofunction in the plasma membrane during the transition between PCW and SCW synthesis. Samples for the main image were prepared by conventional unidirectional shadowing, whereas the Inset shows a CSC (in moss) after optimized rotary shadowing as in a study by Nixon et al. (8). The white scale bar in the main image represents 100 nm, and the black scale bar in the Inset represents 20 nm.

After the relevant plant genes were discovered in the mid-1990s, scientists surprisingly found that a minimum of six CESA isomers is required for normal seed plant development to proceed: three isomers for PCW cellulose synthesis and three others for SCW cellulose synthesis. The resulting difference between PCW and SCW CSCs correlates with the existence of six main clades (sequence groups) of CESAs in seed plants (11). This pattern is conserved despite variation in CESA gene number between species. For example, weedy Arabidopsis thaliana has 10 CESA genes within these six clades, whereas woody Populus trichocarpa has 17 unique CESA genes (12) and cotton fiber-producing Gossypium hirsutum has 32 CESA genes (13). Before the research of Watanabe et al. (2), we had little understanding of how PCW CSCs and SCW CSCs might cofunction or function differently (5, 6, 14).

The Experimental System

Watanabe et al. (2) crossed multiple independent transgenic lines of A. thaliana to generate their powerful experimental line. Young living seedlings coexpressing three fluorescently tagged proteins (a PCW CESA, a SCW CESA, and tubulin) were crossed with a background line in which SCW formation can be chemically induced via the up-regulation of an SCW-specific transcription factor (15). After induction, the surface epidermal cells transdifferentiated to xylem cells, which allowed high-quality imaging of dynamic CESA behavior over a time course with and without pharmacological antagonists. The progression of transdifferentiation in each cell was internally and dynamically marked by the presence of the different CESAs, as well as whether microtubules and CESAs adopted the banded pattern typical of primary xylem elements. Both the PCW and SCW CSCs colocated with banded microtubules, indicating that each type of CSC can interact in a similar way with the microtubules involved in SCW formation. Further experiments led to a temporal and robust picture of CESA/CSC behavior over four time points within 18 h, representing the early-, mid-, late-, and posttransition periods relative to pure PCW or SCW synthesis.

Dynamic Behavior of Different Types of CSCs During the Transition Period

The colocalization of distinct fluorophores tagging the PCW and SCW CESAs showed that their export to the plasma membrane could occur within the same Golgi vesicles. However, the PCW and SCW CSCs assembled separately, as shown by different average rates of movement of the tagged PCW or SCW CESAs once they were in the plasma membrane. The three PCW CESAs and three SCW CESAs were abundant in the plants (as detected with isoform-specific antibodies in protein blots), and the PCW CSCs and the SCW CSCs cofunctioned at the plasma membrane (as detected by live cell imaging) for about 1 h midtransition. Photobleaching experiments showed that the delivery of PCW CSCs to the membrane via Golgi vesicles slowed after SCW CSCs were present.

Both the PCW and SCW CESAs were also detected in poorly defined endomembrane compartments (16, 17), sometimes called small CESA-containing compartments, with a role in CESA endocytosis and/or recycling. In the late-transition period, the PCW CESAs appeared by themselves in another unidentified small endomembrane compartment, and then finally in the lytic vacuole. Protein blotting confirmed that the PCW CESAs were degraded at this time. Further experiments as proposed by Watanabe et al. (2) will reveal how the degradation of particular CESA isoforms is regulated while others are present in the cell, as well as the nature of the unique compartment that hosts the CESAs in route to the lytic vacuole.

Isomer-Specific Differences in CESA Functionality

Watanabe et al. (2) show that the CSCs traditionally related to PCW or SCW cellulose synthesis moved at different average rates, which is a proxy for the rate of CESA catalysis in vivo, given the continuous formation of a linear cellulose microfibril. The SCW CSCs moved about 1.7-fold faster, on average, than the PCW CSCs during early- to midtransition, even when the two types of CSCs were functioning in the same membrane. Therefore, variances in experimental conditions could not have caused the difference. The faster movement of the SCW CSCs occurred even though they were near the same cortical microtubule bundles as the PCW CSCs, which supports a likely difference in catalysis rate that can be investigated further. In addition to the typically observed higher density of SCW CSCs (5, 14), a faster rate of catalysis correlates logically with the higher cellulose content of thicker SCWs. Others have shown that variation in the speed of PCW CSCs can affect cellulose crystallinity and its saccharification efficiency (18), which suggests that the biophysical properties of cellulose produced by PCW and SCW CSCs may differ. Interestingly, the speed of the SCW CSCs was substantially lower in late transition, indicating the possibility of multilayered regulation of the catalysis rate. In another indication of differences between CESA isoforms, the PCW CSCs were uniquely affected by isoxaben, a cellulose synthesis inhibitor.

Broader Implications for Biomaterials

Future experiments can investigate the sequence and/or regulatory differences that dictate the unique assembly of PCW versus SCW CSCs, the potential for faster catalysis of the SCW CESAs, and the basis of the differential sensitivity of the PCW and SCW CESAs to isoxaben. In addition, the cofunction of two distinct types of CSCs during the transition period may have broader implications for the structure and strength of biomaterials. For example, cotton fibers have a multiday transition period during which the genes for numerous PCW and SCW CESAs are coexpressed (3). At this time, a thin transitional cell wall layer with intermediate cellulose content is laid down that contributes strongly to the strength of the mature fibers (19, 20). In the woody tree, Populus x euramericana, both PCW-type CSCs and SCW-type CSCs were involved in secondary xylem formation with different effects on wood properties in gene suppression experiments (21). In wood- and fiber-producing species with proliferated CESA protein families, future research will likely reveal many ways in which CSCs are employed in noncanonical ways. Watanabe et al. (2) have opened the door to many research avenues that will allow us to fully explain and beneficially manipulate the synthesis of abundant renewable biomaterials.