Insights into the xylan degradation system of Cellulomonas sp. B6: biochemical characterization of rCsXyn10A and rCsAbf62A.

Valorization of the hemicellulose fraction of plant biomass is crucial for the sustainability of lignocellulosic biorefineries. The Cellulomonas genus comprises Gram-positive Actinobacteria that degrade cellulose and other polysaccharides by secreting a complex array of enzymes. In this work, we studied the specificity and synergy of two enzymes, CsXyn10A and CsAbf62A, which were identified as highly abundant in the extracellular proteome of Cellulomonas sp. B6 when grown on wheat bran. To explore their potential for bioprocessing, the recombinant enzymes were expressed and their activities were thoroughly characterized. rCsXyn10A is a GH10 endo-xylanase (EC 3.2.1.8), active across a broad pH range (5 to 9), at temperatures up to 55 °C. rCsAbf62A is an α-L-arabinofuranosidase (ABF) (EC 3.2.1.55) that specifically removes α-1,2 and α-1,3-L-arabinosyl substituents from arabino-xylo-oligosaccharides (AXOS), xylan, and arabinan backbones, but it cannot act on double-substituted residues. It also has activity on pNPA. No differences were observed regarding activity when CsAbf62A was expressed with its appended CBM13 module or only the catalytic domain. The amount of xylobiose released from either wheat arabinoxylan or arabino-xylo-oligosaccharides increased significantly when rCsXyn10A was supplemented with rCsAbf62A, indicating that the removal of arabinosyl residues by rCsAbf62A improved rCsXyn10A accessibility to ß-1,4-xylose linkages, but no synergism was observed in the deconstruction of wheat bran. These results contribute to designing tailor-made, substrate-specific, enzymatic cocktails for xylan valorization. KEY POINTS: • rCsAbf62A removes α-1,2 and α-1,3-L-arabinosyl substituents from arabino-xylo-oligosaccharides, xylan, and arabinan backbones. • The appended CBM13 of rCsAbf62A did not affect the specific activity of the enzyme. • Supplementation of rCsXyn10A with rCsAbf62A improves the degradation of AXOS and xylan.

Identification of an algal xylan synthase indicates that there is functional orthology between algal and plant cell wall biosynthesis.

Insights into the evolution of plant cell walls have important implications for comprehending these diverse and abundant biological structures. In order to understand the evolving structure-function relationships of the plant cell wall, it is imperative to trace the origin of its different components. The present study is focused on plant 1,4-ß-xylan, tracing its evolutionary origin by genome and transcriptome mining followed by phylogenetic analysis, utilizing a large selection of plants and algae. It substantiates the findings by heterologous expression and biochemical characterization of a charophyte alga xylan synthase. Of the 12 known gene classes involved in 1,4-ß-xylan formation, XYS1/IRX10 in plants, IRX7, IRX8, IRX9, IRX14 and GUX occurred for the first time in charophyte algae. An XYS1/IRX10 ortholog from Klebsormidium flaccidum, designated K. flaccidumXYLAN SYNTHASE-1 (KfXYS1), possesses 1,4-ß-xylan synthase activity, and 1,4-ß-xylan occurs in the K. flaccidum cell wall. These data suggest that plant 1,4-ß-xylan originated in charophytes and shed light on the origin of one of the key cell wall innovations to occur in charophyte algae, facilitating terrestrialization and emergence of polysaccharide-based plant cell walls.

Overexpression of INFLORESCENCE DEFICIENT IN ABSCISSION activates cell separation in vestigial abscission zones in Arabidopsis.

Plants may shed organs when they have been injured or served their purpose. The differential pattern of organ abscission in different species is most likely the result of evolutionary adaptation to a variety of life styles and environments. The final step of abscission-related cell separation in floral organs of wild-type Arabidopsis thaliana, which only abscises sepals, petals, and stamens, is controlled by INFLORESCENCE DEFICIENT IN ABSCISSION (IDA). Here, we demonstrate that Arabidopsis 35S:IDA lines constitutively overexpressing IDA exhibit earlier abscission of floral organs, showing that the abscission zones are responsive to IDA soon after the opening of the flowers. In addition, ectopic abscission was observed at the bases of the pedicel, branches of the inflorescence, and cauline leaves. The silique valves also dehisced prematurely. Scanning electron microscopy indicated a spread of middle lamella degradation from preformed abscission zone cells to neighboring cells. A transcript encoding an arabinogalactan protein (AGP) was upregulated in the 35S:IDA lines, and large amounts of AGP were secreted at the sites of abscission. AGP was shown to be a constituent of wild-type floral abscission zones during and soon after cell separation had been completed. We suggest that the restricted expression pattern of IDA precludes abscission of nonfloral organs in Arabidopsis.