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. 2020 Jan;18(1):5-7.
doi: 10.1111/pbi.13198. Epub 2019 Jul 20.

A dominant negative approach to reduce xylan in plants

Andrew G Brandon  1   2 , Devon S Birdseye  2 , Henrik V Scheller  1   2
Affiliations

A dominant negative approach to reduce xylan in plants

Andrew G Brandon et al. Plant Biotechnol J. 2020 Jan.
No abstract available

Keywords: antimorphic mutation; cell walls; dominant negative mutation; glycosyltransferase; lignocellulosic biofuels; protein complex; xylan.

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Conflict of interest statement

AGB and HVS are inventors on a patent application related to the work described here.

Figures

Figure 1
Figure 1
(a) Sequence alignment of IRX10 (At1g27440) homologs: A. thaliana (IRX10‐L, AT5G61840), P. trichocarpa (XP_002297880.1, POPTR_0001s12940g), O. sativa (Os01g0926700), B. distachyon (BRADI_2g59410), P. ovata (IRX10_6, APW77260.1), P. patens (XP_001753186) and H. sapiens (EXT1, AAB62718.1, 35% id.). (b) Representative plants from the T1 generation. EVC, empty vector control; IRX10, plant transformed with unmutated IRX10. (c) Immunoblot using anti‐FLAG antibody showing transgene expression from T2 lines. (d) Alcohol‐insoluble residue (AIR) prepared from 6 cm of basal inflorescence stem of fully senesced plants as previously described. AIR samples (1–3 mg) were hydrolysed with 2M trifluoroacetic acid for 1 h at 120°C, lyophilized, resuspended in water and centrifuged at 20 000  g for 10 min. The supernatant was analysed by high‐performance anion exchange chromatography on an ICS 5000 instrument (Thermo Fisher) using a CarboPac™ PA20 column (3 × 150 mm, Thermo Fisher). Bars show average ± SD (n = 3–5). P‐values indicate significance of difference in xylose content compared to EVC. (e) Immunofluorescence labelling of stem cross sections (left panels, scale bars 100 μm) with the xylan‐specific LM10 antibody (PlantProbes, Leeds, UK), and toluidine blue O‐stained cross sections (right panels, scale bars 25 μm). The basal regions of inflorescence stems (3 cm above the rosette) were collected from 10‐week‐old plants and fixed overnight at 4°C in 4% paraformaldehyde, 50 mm PIPES, 5 mm EGTA, 5 mm MgSO 4, pH 6.9. The stems were embedded in 7% agarose, and 100‐μm sections were made using a Leica VT1000S vibratome. For immunolabelling, sections were washed three times with phosphate‐buffered saline (PBS) pH 7 and incubated overnight with a 10‐fold dilution of LM10 antibody in PBS. The sections were then incubated with fluorescein isothiocyanate‐conjugated goat anti‐rat secondary antibodies for 1 h and imaged with a Zeiss LSM710 confocal microscope with ZEN2010 software (Zeiss). Phloem (ph) and xylem (xy) are labelled with arrows and arrowheads indicating irregular and normal vessels, respectively. (f) Xylan synthase activity in transgenic plants. All reactions were performed in a total of 25 μL containing 10 mm MnCl2, 1% (v/v) Triton X‐100 and 50 mm MES, pH 6.5. The assays included 2 μg ANTS‐labelled xylohexaose, 200 μm UDP‐Xyl and microsomal membranes (50 μg total protein). Reactions were incubated for 2 h at 30°C, terminated by heating (100°C, 3 min) and centrifuged at 10 000  g for 10 min. Supernatants (15 μL) were mixed with 15 μL 3M urea, and the samples (5 μL) were analysed by separation on large format Tris‐borate acrylamide gel prepared as described elsewhere and electrophoresed at 200 V for 30 min followed by 1000 V for 1.5 h. The gels were visualized using a G‐box (Syngene, www.syngene.com) with UV detection filter and UV tubes (365 nm emission). Band intensities for Xyl7 and Xyl8 were detected and quantified automatically with GeneTools (Syngene). The sum of the two bands was used as a measurement for XylT activity. Significant differences from EVC are indicated (P < 0.01, ANOVA and Dunnett's multiple comparison test).
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