The DUF579 domain containing proteins IRX15 and IRX15-L affect xylan synthesis in Arabidopsis
Version of Record online: 2 FEB 2011
© 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd
The Plant Journal
Volume 66, Issue 3, pages 387–400, May 2011
How to Cite
Jensen, J. K., Kim, H., Cocuron, J.-C., Orler, R., Ralph, J. and Wilkerson, C. G. (2011), The DUF579 domain containing proteins IRX15 and IRX15-L affect xylan synthesis in Arabidopsis. The Plant Journal, 66: 387–400. doi: 10.1111/j.1365-313X.2010.04475.x
- Issue online: 26 APR 2011
- Version of Record online: 2 FEB 2011
- Accepted manuscript online: 28 DEC 2010 01:23AM EST
- Received 24 August 2010; revised 20 November 2010; accepted 21 December 2010.
Figure S1. Psyllium and analysis of the mucilaginous layer from the seed. (a) The mature psyllium plant, 3 months old. (b) Developing psyllium seeds. (c) Transverse section of psyllium seed late in development showing endosperm (E) and mucilagous layer (ML). (d) mucilaginous layer isolated form endosperm. Bar = 5 cm, A; 5 mm, B; 0.5 mm, C and D. (e) Non-cellulosic neutral monosaccharide composition of mucilaginous layers and endosperm at 10, 13 and 16 days post anthesis (DPA). Arabinose (Ara), rhamnose (Rha), fucose (Fuc), xylose (Xyl), mannose (Man), galactose (Gal) and glucose (Glc) are expressed as percentage of total recoverable neutral monosaccharide. Standard deviation bars are shown (n = 2). (f) Partial 13C–1H correlation (HSQC) spectrum showing the anomeric region of isolated mucilaginous layers from seeds 10 to 12 DPA. No signals were detected for the indicated reducing end structure residues. Galacturonic acid (GalA), rhamnose (Rha), xylose (Xyl).
Figure S2. Multiple sequence alignment of the Arabidopsis DUF579 protein family. Multiple sequence alignment was preformed using ClustalX 2.0.9 (Larkin et al., 2007) using the Gonnet protein weight matrix.
Figure S3. Transmembrane prediction of the Arabidopsis DUF579 protein family. Predictions were preformed using the TMHMM Server v. 2.0 [http://www.cbs.dtu.dk/services/TMHMM/; Krogh et al. (2001)].
Figure S4. Expression and phylogenetic tree of the Arabidopsis DUF579 protein family. The phylogenetic tree was preformed with TreeView X. Expression profiles were derived from the mircoarray data of Schmid et al. (2005). Y-axis maximum of the expression graphs is indicate in the upper left corner of each graph. The expression graph of Arabidopsis IRX10 is shown below for comparison. The number of ESTs found in psyllium mucilaginous layer is indicated for the respective genes.
Figure S5. The single knockout lines irx15 and irx15-L. (a) Diagram of Arabidopsis irx15 (At3g50220) and irx15-L (At5g67210) showing position of exons (solid boxes), 5′ and 3′ untranslated regions (open boxes), T-DNA insertions sites, and binding sites of primers used for genotyping insertion lines (small arrows). (b) RT-PCR of IRX15, IRX15-L, and UBQ10 (At4g05320) transcripts using Col-0, irx15, Ws, and irx15-L stem RNA. (c, d) Non-cellulosic neutral monosaccharide composition of alcohol-insoluble residue (AIR) of stem and silique from Col-0, irx15, Ws, and irx15-L. Arabinose (Ara), rhamnose, (Rha), fucose (Fuc), xylose (Xyl), mannose (Man), galactose (Gal) and glucose (Glc) are expressed as percentage of total recoverable neutral monosaccharide. Standard deviation bars are shown (n = 2).
Figure S6. Immunohistochemical analysis of stem sections using LM11 and LM15. Resin embedded stem sections 1 cm above the rosette from wild type cross (Col-0 x Ws) (a, c) and irx15 irx15-L (b, d) were labeled using LM11 (xylan; a, b), and LM15 (xylan; c, d). Bar = 60 μm.
Figure S7. Morphology of secondary cell walls of the lower stem. Toluidine Blue staining of vascular bundle and adjacent interfascicular fibers (a, b), and transmission electron microscopy of xylem cells (c, d) from wild type cross (Col-0 x Ws) (a, c) and irx15 irx15-L (b, d). Bar = 60 μm, a, b; 10 μm c, d.
Figure S8. Partial HSQC spectra showing the xylan non-anomeric region. The spectra are from the same spectra as in Figure 6. These spectra show how the xylan nonreducing ends are seen and can be quantified by their unique C/H4 and C/H5 correlations, as well as some of the xylitol end-groups resulting from NaBH4 reduction of the reducing endgroups, as well as some of the other correlations that can be reasonably assigned.
Table S1. Characterization of stems.
Table S2. Expression level of selected genes involved in secondary cell wall formation in Arabidopsis.
Table S3. Characterization by NMR and carbohydrate dot blot assay of glucuronoxylan from Arabidopsis stems extracted with 1 m KOH.
Appendix S1. Experimental procedures: cDNA library construction, Quantitative dot blot analysis using carbohydrate specific antibodies, Crystalline cellulose analysis, Lignin analysis, qRT-PCR analysis performed on Arabidopsis stems.
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