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Secondary cell wall characterization in a BY-2 inductive system

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Abstract

Plant secondary cell walls (SCW) are likely to serve as major sources of sugars for biofuel production. Yet, current knowledge of SCW biosynthesis and modifications is far from complete. Cell culture has been suggested as an ideal system for identifying and characterizing cell wall biosynthetic genes. This knowledge will underpin strategies to improve bioenergy feedstocks, and their subsequent fermentation. We previously established a glucocorticoid receptor (GR)-mediated post-translational inducible system of VASCULAR-RELATED NAC DOMAIN7 (VND7) fused to the activation domain of the herpes virus VP16 in tobacco Bright Yellow-2 (BY-2) cells. VND7 is a master transcription factor that controls xylem vessel element differentiation. Its activation by dexamethasone (DEX) resulted in the induction of vessel element formation including cell wall thickening and cell death at high frequency. Here we investigate the kinetics of SCW-related genes after DEX induction in tobacco. Homologues of Arabidopsis cellulose and xylan biosynthetic genes were cloned, and used to conduct kinetic RT-qPCR. Results show an increase in SCW gene expression in BY-2 cells 10 h after DEX induction, with a maximum observed at 24 h. Biochemical analyses of the DEX-induced lines reveal increased cellulose to non-cellulosic polysaccharide ratio, as well as time-dependent increases in characteristic SCW polymers, such as xylan and mannan, indicating the deposition of true SCW. Therefore, we conclude that the BY2::VND7-VP16-GR cell suspension culture is a convenient in vitro system that can be used as an efficient tool to elucidate the regulation of SCW formation through RNA transcript and biochemical analysis.

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Acknowledgments

We are grateful to H. Igarashi for setting up the BY-2::VND7-VP16-GR lines and providing invaluable advices on cell culture systems. We would like to thank M. Araki, A. Ihara, S. Oyama and A. Takebayashi for their technical assistance. This work was supported in part by the RIKEN Plant Science Center. The work conducted by JCM and ZZ was supported by a grant from the BBSRC: BB/G016240/1 BBSRC Sustainable Energy Centre Cell Wall Sugars Programme (BSBEC) to PD.

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Authors and Affiliations

  1. RIKEN, Plant Science Center, Yokohama, 230-0045, Japan

    Nadia Goué, Morgane Josserand & Taku Demura

  2. Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QW, UK

    Jenny C. Mortimer, Zhinong Zhang & Paul Dupree

  3. Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, 630-0192, Japan

    Yoshimi Nakano & Taku Demura

  4. Biomass Engineering Program Cooperation Division, RIKEN Center for Sustainable Resource Science, Yokohama, 230-0045, Japan

    Misato Ohtani & Taku Demura

  5. Forestry and Forest Products Research Institute, Tsukuba, 305-8687, Japan

    Koichi Kakegawa

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  1. Nadia Goué
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  2. Jenny C. Mortimer
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  3. Yoshimi Nakano
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Corresponding authors

Correspondence to Nadia Goué or Misato Ohtani.

Electronic supplementary material

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11240_2013_354_MOESM1_ESM.pdf

Supplementary Table 1 Summary of the candidate genes and primer sets used in this study. Degenerate primers were designed using IUPAC’s code for nucleotide degeneracy (PDF 282 kb)

Supplementary Table 2 Primer sets used for quantitative RT-PCR analysis (PDF 13 kb)

11240_2013_354_MOESM3_ESM.pdf

Supplementary Figure 1 Phylogenetic relationship of NtCESA4 (S1A), NtCESA7 (S1B), NtCESA8 (S1C), NtIRX8 (S1D), NtIRX9 (S1E) and NtIRX10 (S1F) with related sequences. Sequences are from the following species: At, Arabidopsis thaliana, Al, Arabidopsis lyrata, Bd, Brachypodium distachyon, Bl, Betula luminifera, Bn, Brassica napus, Bo, Bambusa oldhamii, Bp, Betula platyphylla, Cm, Cucumis melo, Ec, Eucalyptus camaldulensis, Egl, Eucalyptus globulus, Eg, Eucalyptus grandis, Gb, Gossypium barbadense, Gh, Gossypium hirsutum, Ghe, Gossypium herbaceum, Gk, Gossypioides kirkii, Gm, Glycine max, Gr, Gossypium raimondii, Hv, Hordeum vulgare, Jc, Jatropha curcas, Lj, Lotus japonicus, Ll, Leucaena leucocephala, Mt, Medicago truncatula, Np, Nicotiana plumbaginifolia, Nt, Nicotiana tabacum, Os, Oriza sativa, Pb, Populus balsamifera, Pe, Phyllostachys edulis, Pg, Picea glauca, Pm, Pseudotsuga menziesii, Pp, Physcomitrella patens, Pr, Pinus radiata, Ps, Picea sitchensis, Pt, Pinus taeda, Ptd, Populus tremuloides, Pti, Populus trichocarpa, Pto, Populus tomentosa, Ptxa, Populus tremula x Populus alba, Ptxt, Populus tremula x Populus tremuloides, Pu, Populus ussuriensis, Rc, Ricinus communis, Sb, Sorghum bicolor, Sl, Solanum lycopersicum, Sm, Selaginella moellendorffii, So, Saccharum officinarum, Sp, Shorea parvifolia, Ss, Salix sachalinensis, St, Solanum tuberosum, Ta, Triticum aestivum, Th, Thellungiella halophila,Vv, Vitis vinifera, Ze, Zea elegans, Zm, Zea mays, Zv, Zinnia violacea (PDF 2795 kb)

11240_2013_354_MOESM4_ESM.pdf

Supplementary Figure 2 (a) Time-course of BY-2 cell transdifferentiation after DEX induction. Count, amount of BY-2 cell transdifferentiation between 0 and 3 days after induction. Error bars indicating standard deviation for 3 counts of 200 BY-2 cells are shown. (b) BY-2:VND7-VP16-GR suspension cell culture 2 days after DEX induction showing one xylem vessel element transdifferentiation and two undifferentiated BY-2 cells (PDF 42 kb)

11240_2013_354_MOESM5_ESM.pdf

Supplementary Figure 3 Quantitative RT-PCR analysis from 0 to 3 Day After DEX Induction (DAI) of (a) AtVND7 in the BY-2::VND7-VP16-GR background, (b - g) genes of interest in the BY-2 wild type (WT) background. Bars show relative expression levels for each gene normalized with UBQ2 expression levels in BY-2 cell suspension culture with DEX induction (+DEX) and uninduced BY-2 cell suspension culture (-DEX). Error bars indicate standard deviation of 3 independent biological replicates (PDF 632 kb)

11240_2013_354_MOESM6_ESM.pdf

Supplementary Figure 4 Quantity of xylan in differentiating tobacco BY2 cells, calculated by PACE analysis. Error bars indicate standard deviation of 2 independent biological replicates (PDF 17 kb)

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Goué, N., Mortimer, J.C., Nakano, Y. et al. Secondary cell wall characterization in a BY-2 inductive system. Plant Cell Tiss Organ Cult 115, 223–232 (2013). https://doi.org/10.1007/s11240-013-0354-7

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