Abstract
Hemicelluloses, such as xyloglucan, xylan and mannans, consist of a heterogeneous array of plant-derived polysaccharides that form the plant cell wall. These polysaccharides differ from each other in their structure and physiochemical properties, but they share a β-(1,4)-linked sugar backbone. Hemicelluloses can be hydrolyzed by plant-cell-wall-degrading enzymes (PCWDEs), which are widely distributed in phytopathogenic microbes. Recently, it has become apparent that phytophagous beetles also produce their own PCWDEs. Our previous work identified genes encoding putative mannanases belonging to the subfamily 10 of glycoside hydrolase (GH) family 5 (GH5_10) in the genomes of the leaf beetle, Gastrophysa viridula (Chrysomelidae, Chrysomelinae; one gene), and of the bean beetle, Callosobruchus maculatus (Chrysomelidae, Bruchinae; four genes). In contrast to proteins from other GH5 subfamilies, GH5_10 proteins are patchily distributed within the tree of life and have so far hardly been investigated. We addressed the following questions: Are beetle-derived GH5_10s active PCWDEs? How did they evolve? What is their physiological function? Using heterologous protein expression and enzymatic assays, we show that the G. viridula GH5_10 protein is an endo-β-1,4-mannanase. We also demonstrate that only one out of four C. maculatus GH5_10 proteins is an endo-β-1,4-mannanase, which has additional activity on carboxymethyl cellulose. Unexpectedly, another C. maculatus GH5_10 protein has evolved to use xylan instead of mannans as a substrate. RNAi experiments in G. viridula indicate (i) that the sole GH5_10 protein is responsible for breaking down mannans in the gut and (ii) that this breakdown may rather be accessory and may facilitate access to plant cell content, which is rich in nitrogen and simple sugars. Phylogenetic analyses indicate that coleopteran-derived GH5_10 proteins cluster together with Chelicerata-derived ones. Interestingly, other insect-derived GH5_10 proteins cluster elsewhere, suggesting insects have several independent evolutionary origins.
Highlights
The plant’s primary cell wall is a complex structure consisting of polysaccharides and proteins that encase and protect growing plant cells
GalM is a polysaccharide consisting of a pure mannose backbone decorated with evenly distributed galactose moieties, whereas GluM is a straight-chain polysaccharide consisting of a backbone that alternates unevenly between mannose and glucose moieties with occasional branching
We previously reported that the transcriptomes of two phytophagous beetles, G. viridula and C. maculatus, harbor transcripts encoding GH5_10 putative mannanases [21]
Summary
The plant’s primary cell wall is a complex structure consisting of polysaccharides and proteins that encase and protect growing plant cells. To cellulose and pectins, the hemicellulose network—made of polysaccharides such as xyloglucan, xylan and mannans—is one of the main constituents of the plant’s primary cell wall [1,2]. Mannans may function as seed storage and/or structural components [5,6]. The backbone of the polymer is composed of mannose residues linked together by β-1,4-glycosidic bonds. In galactomannan, this backbone is substituted with α-1,6-linked galactose residues. In contrast to mannan and galactomannan, the backbone of glucomannan is made up of randomly alternating mannose and glucose residues linked together by β-1,4-glycosidic bonds [8]
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