Recombinant Expression and Characterization of the Cytoplasmic Rice β-Glucosidase Os1BGlu4

Abstract

The Os1BGlu4 β-glucosidase is the only glycoside hydrolase family 1 member in rice that is predicted to be localized in the cytoplasm. To characterize the biochemical function of rice Os1BGlu4, the Os1bglu4 cDNA was cloned and used to express a thioredoxin fusion protein in Escherichia coli. After removal of the tag, the purified recombinant Os1BGlu4 (rOs1BGlu4) exhibited an optimum pH of 6.5, which is consistent with Os1BGlu4's cytoplasmic localization. Fluorescence microscopy of maize protoplasts and tobacco leaf cells expressing green fluorescent protein-tagged Os1BGlu4 confirmed the cytoplasmic localization. Purified rOs1BGlu4 can hydrolyze p-nitrophenyl (pNP)-β-d-glucoside (pNPGlc) efficiently (k cat/K m = 17.9 mM−1·s−1), and hydrolyzes pNP-β-d-fucopyranoside with about 50% the efficiency of the pNPGlc. Among natural substrates tested, rOs1BGlu4 efficiently hydrolyzed β-(1,3)-linked oligosaccharides of degree of polymerization (DP) 2–3, and β-(1,4)-linked oligosaccharide of DP 3–4, and hydrolysis of salicin, esculin and p-coumaryl alcohol was also detected. Analysis of the hydrolysis of pNP-β-cellobioside showed that the initial hydrolysis was between the two glucose molecules, and suggested rOs1BGlu4 transglucosylates this substrate. At 10 mM pNPGlc concentration, rOs1BGlu4 can transfer the glucosyl group of pNPGlc to ethanol and pNPGlc. This transglycosylation activity suggests the potential use of Os1BGlu4 for pNP-oligosaccharide and alkyl glycosides synthesis.

Integrated Genome-Scale Analysis Identifies Novel Genes and Networks Underlying Senescence in Maize

“…A highly significant SNP (no. 53) on chromosome 8 lies in gene ZmBLGU42 (Zm00001d012159; Figure 7A) which putatively encodes for a b-glucosidase orthologous to rice Os1bglu4 (Rouyi et al, 2014). We found that, compared with PHG35, transcript levels of ZmBLGU42 were higher in B73 at 33, 39, and 42 DAA but identical in both inbred lines at 36 DAA ( Figure 7B).…”

“…Key insights into the role of ZmBLGU42, a b-glucosidase orthologous to Arabidopsis BGLU42 (Zamioudis et al, 2014) and rice Os1bglu4 (Rouyi et al, 2014), in senescence comes from the early onset of glucosidase activity in the B73 compared with PHG35. The b-glucosidase activity also increases during Arabidopsis leaf senescence (Patro et al, 2014), although the exact role of this process remains to be determined.…”

Rice Homeodomain Protein WOX11 Recruits a Histone Acetyltransferase Complex to Establish Programs of Cell Proliferation of Crown Root Meristem

“…From those that contain the WOX binding site and were downregulated in both GCN5 RNAi and wox11 mutant, we selected eight genes from representative categories (including metabolism, cell component, and hormone) for experimental validation. These genes included auxin transport-related OsPIN9, cell wall biosynthesis-related CESA9 (encoding cellulose synthase), CSLF6 (cellulose synthase-like; Jin et al, 2015), and OsEXPA23 (expansin precursor), and energy metabolism-related OsGLU5 (1,4 b-endoglucanase; Zhang et al, 2012), Os1bglu5 (b-glucosidase), Os9bglu33 (b-glucosidase; Rouyi et al, 2014), and OsALDO (fructose-bisphosphate aldolase, a key enzyme of glycolysis). qRT-PCR analysis indicated that all of these genes were indeed downregulated in crown roots of GCN5 and ADA2 RNAi and wox11 mutant plants compared with the respective wild type (Figures 6A to 6C).…”

Functional Characterization of CsBGlu12, a β-Glucosidase from Crocus sativus, Provides Insights into Its Role in Abiotic Stress through Accumulation of Antioxidant Flavonols

“…This process of glycosylation and deglycosylation offers an efficient mechanism that regulates the homeostasis of secondary metabolites and is not limited to plants only but extends to other domains of life like archaea, eubacteria, and animals. Furthermore, the subcellular localization, substrate specificity, and conditions under which ␤-glucosidases come into contact with their physiological substrates determine their biological role (10) and remain an area of intensive study. It is, therefore, important to deepen our knowledge on how plants regulate the individual members of ␤-glucosidases in response to developmental and environmental cues and how the released metabolites modulate the plant's physiological response.…”