In Vitro Versus in VivoCellulose Microfibrils from Plant Primary Wall Synthases: Structural Differences

Abstract

Detergent extracts of microsomal fractions from suspension cultured cells of Rubus fruticosus (blackberry)were tested for their ability to synthesize in vitro sizable quantities of cellulose from UDP-glucose. Both Brij 58 and taurocholate were effective and yielded a substantial percentage of cellulose microfibrils together with (133)-␤-D-glucan (callose). The taurocholate extracts, which did not require the addition of Mg 2؉ , were the most efficient, yielding roughly 20% of cellulose. This cellulose was characterized after callose removal by methylation analysis, electron microscopy, and electron and x-ray synchrotron diffractions; its resistance toward the acid Updegraff reagent was also evaluated. The cellulose microfibrils synthesized in vitro had the same diameter as the endogenous microfibrils isolated from primary cell walls. Both polymers diffracted as cellulose IV I , a disorganized form of cellulose I. Besides these similarities, the in vitro microfibrils had a higher perfection and crystallinity as well as a better resistance toward the Updegraff reagent. These differences can be attributed to the mode of synthesis of the in vitro microfibrils that are able to grow independently in a neighbor-free environment, as opposed to the cellulose in the parent cell walls where new microfibrils have to interweave with the already laid polymers, with the result of a number of structural defects.

Indaziflam Herbicidal Action: A Potent Cellulose Biosynthesis Inhibitor

“…Traditional biochemical methodologies used to illustrate drug molecular mechanisms are not yet applicable to CBIs. To date, purification of functionally active celluloseproducing CSCs or CESAs has been challenging (Lai- Kee-Him et al, 2002) and insufficiently robust to enable in vitro drug affinity-binding assays. Furthermore, despite a crystallized bacterial CESA homolog (Morgan et al, 2013), both CESAs and CSCs have sufficiently diverged over time so that CBIs do not exhibit activity on bacteria (Tsekos, 1999;Morgan et al, 2013;Sethaphong et al, 2013).…”

A single heterologously expressed plant cellulose synthase isoform is sufficient for cellulose microfibril formation in vitro

“…In these studies, the cellulose synthase was BcsA, which requires a membrane-anchored, periplasmic subunit (BcsB) for catalytic activity. Plant cellulose biosynthetic activity has been demonstrated in membrane extracts from several plant tissues (15)(16)(17), but none of the catalytically active synthases has been purified to date. Because of these difficulties, it was speculated that the plant enzymes also might require an additional, weakly associated factor for catalytic activity, similar to the BcsA-B complex in bacteria.…”

“…Assuming a stacking distance of about 5 Å between glucan chains within a cellulose microfibril, the estimated width of the PttCesA8-produced fiber is sufficient to accommodate at least 18-24 glucan chains. Indeed, comparable cellulose fibers have been synthesized from solubilized Physcomitrella patens CesA5 (17), with a similar estimated diameter of 20-30 Å, as well as from blackberry membrane extracts (16).…”

“…In particular, treatment of cellulosic tissues with Updegraff reagent [80% acetic acid and concentrated nitric acid at a 10:1 (vol:vol) ratio] (25) has been shown to hydrolyze noncellulosic cell wall materials efficiently as well as isolated glucan chains not organized into fibrils (16,26). Hence, resistance to acid hydrolysis correlates with a higher-order organization of cellulose.…”

Plant cell walls: the skeleton of the plant world

“…comm.). While the in vitro cellulose synthase assay conditions have been significantly improved (Lai-Kee-Him et al 2002;Pelosi et al 2003;Colombani et al 2004), synthesis rates are still quite low compared with those observed in vivo (~10%) and assay conditions need to be empirically defined and optimised for each plant system. Several studies have implicated other proteins in the CSC.…”