Fig 1.
Structure of β-GGM and substrate specificity of mannanases.
(A) β-GGM structure, the single-letter codes, and CAZy families of Arabidopsis biosynthesis enzymes [4]. (B) The GH26 mannanase (CjMan26) and GH5 mannanase (AnGH5) have different substrate specificity. Both require Man residue at subsite -1 and accommodate either Glc or Man at subsite +1. Only AnGH5 allows the α-Gal side chain at -1 position.
Table 1.
List of plant samples studied in this work.
Fig 2.
Diverse mannan structures in angiosperms.
(A) Comparison of CjMan26 digestion products. Tomato fruits have a digestion pattern containing both β-GGM and galactoglucomannan. GAGM, GBGM, GAGAGM, and GBGAGM arise from β-GGM. GMM, MM arise from galactoglucomannan. Some monocots and ANA-grade have unannotated oligos (U1-4, red asterisks). The samples shown here are of leaf origin except for tomato (fruits) and Nymphaea thermarum (petiole). M: marker lane (standard oligo saccharides). (B) Sequential digestion of CjMan26 digestion products from Austrobaileya scandens (leaf). U2 and U4 migrated to U1 and U3, respectively after α-galactosidase treatment. U1 and U3 were partially digested after β-glucosidase treatment and completely degraded by the following β-mannosidase treatment. The number of “n” is estimated to be three, based on the migration. M: marker lane (standard oligo saccharides).
Fig 3.
β-galactosidase-specific band shifts were not observed in monocots and basal angiosperms samples.
(A) Tomato fruits produced GBGM oligo after AnGH5 digestion, which migrated to GAGM after the following β-galactosidase treatment. M: marker lane (standard oligo saccharides). (B-D) AnGH5 digestion products from monocots (B) and ANA-grade (C and D) showed neither GBGM nor another β-galactosidase sensitive oligosaccharide. The samples in (B-D) are of leaf origin. M: marker lane (standard oligo saccharides).
Fig 4.
AtMBGT1 orthologues in rice and Amborella.
(A) Phylogenetic tree of GT47A-VI and VII. The tree was adapted from our previous publication [4]. OsGT47A-VII and AtrGT47A-VII were distantly located from eudicot genes in GT47A-VII subclade. (B) Expression analysis of rice genes. The expression level was presented as the median of biological triplicates. The source data were obtained in [37]. We obtained the curated form of data via the public database (bar.utoronto.ca/eplant) [46]. SAM: shoot apical meristem. Minimal data set is shown in S3 Table in S1 File.
Fig 5.
Complementation analysis of rice and Amborella GT47A-VII in Arabidopsis mbgt1.
Analysis of CjMan26 digestion products. GBGM is diagnostic for MBGT activity. While WT and p35S::AtMBGT1/mbgt1 have the GBGM band, mbgt1 and two GT47A-VII introduced lines (p35S::OsGT4We 7A-VII/mbgt1 and p35S::AtrGT47A-VII/mbgt1) did not produce the band. Three independent overexpression lines have been examined for the mannan structure analysis and one representative image is shown. M: marker lane (standard oligo saccharides).
Fig 6.
OsGT47A-VII is a galactosyltransferase on xyloglucan.
(A) Shoot growth phenotype in rice mutant (osgt47a-vii). Statistical test: t-test (n = 5~6. N.S. not significant. *P<0.05). (B) Root growth phenotype in rice mutant (osgt47a-vii). Statistical test: t-test (n = 5~6. *P<0.05). Minimal data set is shown in S4 Table in S1 File. (C) Xyloglucan structure analysis after XG5 xyloglucanase digestion (red asterisk: XLXG band, blue asterisk: XXLG band). Three independent overexpression lines have been examined and one representative image is shown. M: marker lane (standard oligo saccharides).