Evidence that endo‐1,4‐β‐glucanases act on cellulose in suspension‐cultured poplar cells

Abstract

SummarySuspension-cultured poplar (Populus alba) cells produce two distinct endo-1,4-b-glucanases, one of which is released in the extracellular culture medium and the other localized in their walls. Two cDNA clones, PopCel1 and PopCel2, isolated from a poplar cDNA library, encode the extracellular and the wallbound endo-1,4-b-glucanases, respectively, based upon deduced amino acid sequences. The products of these two genes contained domains conserved in endo-1,4-b-glucanase (family 9) and showed 91.5% amino acid identity. The levels of both PopCel1 and PopCel2 mRNAs increased during the lag phase of growth and decreased rapidly during the linear phase. After the levels had decreased, they were again increased by addition of sucrose to the culture medium and further enhanced by the addition of 2,4-dichlorophenoxyacetic acid (2,4-D) in the presence of sucrose. The accumulation of the mRNAs was correlated with the solubilization of cello-oligosaccharides. Cello-oligosaccharides and xyloglucan were also solubilized from the wall preparations of poplar cells incubated with enzyme preparations from the extracellular culture medium and walls. An antibody against both PopCel proteins reduced the production of cello-oligosaccharides by the extracellular enzyme by 90% and that by the wall-bound enzyme by 55%, and also prevented xyloglucan solubilization. The results show that the accumulation of poplar endo-1,4-b-glucanases is regulated indirectly by auxin in the presence of sucrose and can act on cellulose in suspension-cultured poplar cells.

Arabidopsis endo‐1,4‐β‐glucanases are involved in the formation of root syncytia induced by Heterodera schachtii

“…Upregulation of AtCel2 triggered by high sucrose concentration in the medium indicates its involvement in cellulose biosynthesis as previously suggested by Ohmiya et al (2000) and Shani et al (2006) for other endo-1,4-b-glucanases. Putative cell wall substrates of secreted endo-1,4-bglucanases are non-crystalline cellulose and xyloglucans.…”

“…Other studies revealed that expression of endo-1,4-bglucanases depends on addition of sucrose as shown for PopCel1 and PopCel2 (Ohmiya et al, 2000) and for AtCel1 . Based on these results the authors suggest that the EGases are involved in cellulose biosynthesis.…”

“…Based on in silico analysis of promoter regions of AtCel2 and KOR3 that revealed the presence of several sugar-responsive motifs in promoters of both genes we performed an experiment with plants growing in media supplemented with additional sucrose. Its basis was the observation that syncytia are enriched with sucrose and some reports that indicated a sucrose-dependent regulation of endo-1,4-b-glucanase genes (Ohmiya et al, 2000;Shani et al, 2006). Quantitative RT-PCR with RNA isolated from 7-day-old root tissue growing on media with 6% sucrose shows that AtCel2 is upregulated, while expression of KOR3 does not change ( Figure 5).…”

“…Their potential substrates are cellulose and xyloglucan. Ohmiya et al (1995Ohmiya et al ( , 2000 analysed two endo-1,4-b-glucanases from poplar (Populus alba), PopCel1 and PopCel2, and suggested that these enzymes hydrolyse non-crystalline regions of cellulose and loosen cell walls by releasing xyloglucans from cellulose microfibrils. This irreversible modification is thought to promote cell wall expansion.…”

Poplar Carbohydrate-Active Enzymes. Gene Identification and Expression Analyses

“…Other cellulases in GH9, lacking the membrane-spanning domain, had ESTs that were broadly distributed among poplar tissues (Table III). These cellulases may act on amorphous cellulose in the cell wall and, by doing so, increase cell wall plasticity (Ohmiya et al, 2000(Ohmiya et al, , 2003Park et al, 2003).…”