A mixed-linkage (1,3;1,4)-β-D-glucan specific hydrolase mediates dark-triggered degradation of this plant cell wall polysaccharide

Abstract

The presence of mixed-linkage (1,3;1,4)-β-D-glucan (MLG) in plant cell walls is a key feature of grass species such as cereals, the main source of calorie intake for humans and cattle. Accumulation of this polysaccharide involves the coordinated regulation of biosynthetic and metabolic machineries. While several components of the MLG biosynthesis machinery have been identified in diverse plant species, degradation of MLG is poorly understood. In this study, we performed a large-scale forward genetic screen for maize (Zea mays) mutants with altered cell wall polysaccharide structural properties. As a result, we identified a maize mutant with increased MLG content in several tissues, including adult leaves and senesced organs, where only trace amounts of MLG are usually detected. The causative mutation was found in the GRMZM2G137535 gene, encoding a GH17 licheninase as demonstrated by an in vitro activity assay of the heterologously expressed protein. In addition, maize plants overexpressing GRMZM2G137535 exhibit a 90% reduction in MLG content, indicating that the protein is not only required, but its expression is sufficient to degrade MLG. Accordingly, the mutant was named MLG hydrolase 1 (mlgh1). mlgh1 plants show increased saccharification yields upon enzymatic digestion. Stacking mlgh1 with lignin-deficient mutations results in synergistic increases in saccharification. Time profiling experiments indicate that wall MLG content is modulated during day/night cycles, inversely associated with MLGH1 transcript accumulation. This cycling is absent in the mlgh1 mutant, suggesting that the mechanism involved requires MLG degradation, which may in turn regulate MLGH1 gene expression.

Disruption ofBrachypodiumlichenase alters metabolism of mixed‐linkage glucan and starch

“…Barley isoenzyme EI was induced in dark-incubated barley seedlings and MLG content decreased significantly (Roulin et al, 2002). A similar result was found in maize that the expression of MLGH1, a gene encoding lichenase, was increased during the night (Kraemer et al, 2021). To determine whether genes encoding lichenases in B. distachyon are induced by darkness and to examine further the response of lch1 mutants to darkness, 3-week-old wildtype and lch1 plants were incubated under a 16-h light/8-h dark cycle (0 h), or in complete darkness for 24, 48, and 72 h (Figure S8).…”

“…MLG content in leaves of dark-incubated plants decreased, which is consistent with the induction of EI by darkness. Recently, Kraemer et al (2021) identified a lichenase in maize named MLGH1, and this enzyme is involved in MLG degradation during the night. The mutant mlgh1 contained more MLG and showed no significant growth defects.…”

Forgotten Actors: Glycoside Hydrolases During Elongation Growth of Maize Primary Root

“…Barley HvEI-HvEII enzymes are 1,3;1,4-β- D -glucan endohydrolases ( Varghese et al, 1994 ). The Zm00001d038049 gene matches the maize GRMZM2G137535 gene, which was demonstrated to be an MLG-degrading enzyme in maize leaves ( Kraemer et al, 2021 ). The protein content of Zm00001d038049 in roots was negligible ( Supplementary Figure 3 ).…”

“…At present, GHs are thought to participate in cell wall polysaccharide turnover; although neither cause nor effect of this intensive metabolism is understood yet ( Barnes and Anderson, 2018 ). For instance, MLG turnover is coupled to the day and night cycle in maize leaves via the expression of an MLG-specific hydrolase ( Kraemer et al, 2021 ). GHs take part in cellulose microfibril formation ( Nicol et al, 1998 ; Lane et al, 2001 ; Szyjanowicz et al, 2004 ) and control the angle of cellulose microfibrils ( Derba-Maceluch et al, 2015 ).…”

Chromosome-scale genome assembly of the diploid oat Avena longiglumis reveals the landscape of repetitive sequences, genes and chromosome evolution in grasses

“…Intragenic complementation has not been observed among alleles with mutations in different CSLD1 domains of Lotus japonicas [96], suggesting that the CSLD1 has not yet undergone the complete functional differentiation. CslF6 and CslH have a functional role in the synthesis of mixed-linkage (1,3;1,4)-β-glucan (MLG [97]), and the sequence divergence of the Csl genes we found is likely a reflection of their functional divergence although MLG synthesis is tightly regulated and thus maximizing the yield of end-product cellulose might be difficult. For example, isoforms may utilize the same donor but a different acceptor molecule in the synthesis of the same polysaccharide, and thus, having multiple genes may be a requirement for synthesis of some types of plant polysaccharides [58].…”