Carbohydrate-binding modules promote the enzymatic deconstruction of intact plant cell walls by targeting and proximity effects

Abstract

Cell wall degrading enzymes have a complex molecular architecture consisting of catalytic modules and noncatalytic carbohydrate-binding modules (CBMs). The function of CBMs in cell wall degrading processes is poorly understood. Here, we have evaluated the potential enzyme-targeting function of CBMs in the context of intact primary and secondary cell wall deconstruction. The capacity of a pectate lyase to degrade pectic homogalacturonan in primary cell walls was potentiated by cellulose-directed CBMs but not by xylan-directed CBMs. Conversely, the arabinofuranosidase-mediated removal of side chains from arabinoxylan in xylan-rich and cellulose-poor wheat grain endosperm cell walls was enhanced by a xylan-binding CBM but less so by a crystalline cellulose-specific module. The capacity of xylanases to degrade xylan in secondary cell walls was potentiated by both xylan- and cellulose-directed CBMs. These studies demonstrate that CBMs can potentiate the action of a cognate catalytic module toward polysaccharides in intact cell walls through the recognition of nonsubstrate polysaccharides. The targeting actions of CBMs therefore have strong proximity effects within cell wall structures, explaining why cellulose-directed CBMs are appended to many noncellulase cell wall hydrolases.

Fig. 1.

Effect of pectate lyase (Pel10A) treatments on the detection of the JIM5 pectic HG epitope in primary cell walls of transverse sections of tobacco stem pith parenchyma. (A) Indirect immunofluorescence microscopy of JIM5 binding after Pel10A (no treatment, Pel10A alone, CBM2a·Pel10A, CBM15·Pel10A). JIM5-tagged FITC fluorescence is shown, as observed on the left half of each micrograph. The right half of each micrograph shows the same micrograph with overlaid false colors reflecting fluorescence intensities. (Insets) Images of JIM5 fluorescence combined with Calcofluor White fluorescence (blue) show higher magnification of cell walls in the region of intercellular spaces. (Scale bar: 200 μm.) (B) Histogram showing relative fluorescence intensities reflecting Pel10A treatments on JIM5 binding to the sections. The cellulose-binding modules CBM2a and CBM3a show a positive impact on the appended pectate lyase, whereas the xylan-binding modules CBM15 and CBM2b-1-2 show no impact. The results are expressed as percentages of the remaining fluorescence compared with a control without enzymatic treatment. The histogram shows mean ± SEM.

Fig. 2.

Indirect immunofluorescence microscopy of CBMs and monoclonal antibodies binding to transverse sections of wheat grain endosperm cell walls and effect of arabinofuranosidase treatments on the detection of the LM10 xylan epitope. (A) Lack of binding of LM10 indicated that the endosperm (e) cell walls are heavily arabinosylated. The xylan-binding module CBM2b-1-2 binds effectively and cellulose-directed CBM2a does not bind effectively to equivalent cell walls. (Scale bar: 500 μm.) (B) Indirect immunofluorescence detection of the LM10 epitope in wheat grain sections after treatments with the arabinofuranosidase Abf51A alone or appended with CBM2a or CBM2b-1-2. (Scale bar: 500 μm.) (C) Histogram showing relative LM10-associated fluorescence in wheat grain endosperm cell walls after no treatment and treatments with Abf51A constructs. The results are expressed as percentages of fluorescence relative to corresponding micrographs showing maximum fluorescence (i.e., LM10 epitope detection after CBM2b-1-2·Abf51A treatment). LM10 epitopes are revealed by the loss of arabinosyl residues. The histogram shows mean ± SEM.

Fig. 3.

Impact of appended single CBMs, an enzyme mixture, and an appended tandem CBM on the action of Xyl10B (A) and Xyl11A (B) catalytic domains (CDs) on CBM2b-1-2 recognition of xylan in secondary cell walls of transverse sections of tobacco stem. The results are expressed as percentages of the remaining CBM2b-1-2:GFP fluorescence compared with the control without enzymatic treatment. In all cases, the same final molarities of CDs were used for the Xyl10B derivatives (50 nM) (A) and the Xyl11A derivatives (250 nM) (B). Histograms show mean ± SEM.

Fig. 4.

Binding properties of CBM3a, CBM2b-1-2, and the tandem CBM3a·CBM2b-1-2 module to isolated polymers and intact cell walls. (A) Effective binding of CBM3a, CBM2b-1-2, and CBM3a·CBM2b-1-2 to ligands in vitro using microtiter plate assays. (B) Indirect immunofluorescence analysis of CBM3a, CBM2b-1-2, and CBM3a·CBM2b-1-2 binding to transverse sections of tobacco stems (Upper) and pea stems (Lower). All probes bind effectively to the secondary cell walls of phloem fibers (pf) and xylem cells (x). In addition, CBM3a binds to cortical primary cell walls (*) in both stems. (Scale bar: 200 μm.)