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. 2010 Jul;153(3):906-14.
doi: 10.1104/pp.110.154013. Epub 2010 May 20.

VASCULAR-RELATED NAC-DOMAIN6 and VASCULAR-RELATED NAC-DOMAIN7 effectively induce transdifferentiation into xylem vessel elements under control of an induction system

Masatoshi Yamaguchi  1 , Nadia Goué, Hisako Igarashi, Misato Ohtani, Yoshimi Nakano, Jennifer C Mortimer, Nobuyuki Nishikubo, Minoru Kubo, Yoshihiro Katayama, Koichi Kakegawa, Paul Dupree, Taku Demura
Affiliations

VASCULAR-RELATED NAC-DOMAIN6 and VASCULAR-RELATED NAC-DOMAIN7 effectively induce transdifferentiation into xylem vessel elements under control of an induction system

Masatoshi Yamaguchi et al. Plant Physiol. 2010 Jul.

Abstract

We previously showed that the VASCULAR-RELATED NAC-DOMAIN6 (VND6) and VND7 genes, which encode NAM/ATAF/CUC domain protein transcription factors, act as key regulators of xylem vessel differentiation. Here, we report a glucocorticoid-mediated posttranslational induction system of VND6 and VND7. In this system, VND6 or VND7 is expressed as a fused protein with the activation domain of the herpes virus VP16 protein and hormone-binding domain of the animal glucocorticoid receptor, and the protein's activity is induced by treatment with dexamethasone (DEX), a glucocorticoid derivative. Upon DEX treatment, transgenic Arabidopsis (Arabidopsis thaliana) plants carrying the chimeric gene exhibited transdifferentiation of various types of cells into xylem vessel elements, and the plants died. Many genes involved in xylem vessel differentiation, such as secondary wall biosynthesis and programmed cell death, were up-regulated in these plants after DEX treatment. Chemical analysis showed that xylan, a major hemicellulose component of the dicot secondary cell wall, was increased in the transgenic plants after DEX treatment. This induction system worked in poplar (Populus tremula x tremuloides) trees and in suspension cultures of cells from Arabidopsis and tobacco (Nicotiana tabacum); more than 90% of the tobacco BY-2 cells expressing VND7-VP16-GR transdifferentiated into xylem vessel elements after DEX treatment. These data demonstrate that the induction systems controlling VND6 and VND7 activities can be used as powerful tools for understanding xylem cell differentiation.

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Figures

Figure 1.
Figure 1.
Transgenic Arabidopsis plants expressing VND6-VP16-GR and VND7-VP16-GR chimera proteins. A, Schematic of the VND6-VP16-GR and VND7-VP16-GR constructs. 35S, CaMV 35S promoter; VP16, activation domain of herpes virus VP16 protein; GR, hormone-binding domain of rat GR; NOS, terminator of nopaline synthase. B, RT-PCR analysis of VND6, VND7, VP16, and UBQ10 expression in the wild-type (Columbia [Col]) and transgenic plants. C, Seven-day-old seedlings were soaked in water with (+DEX) or without (−DEX) for 4 d. Scale bar = 1 cm.
Figure 2.
Figure 2.
Transdifferentiation of various types of cells into xylem vessel elements in Arabidopsis. Seven-day-old VP16-GR-2-2 (A–C), VND7-VP16-GR-8-2 (D–H, J), and VND6-VP16-GR-6-1 (I) transgenic plants were treated with DEX for 4 d. A to F, Induction of transdifferentiation by activation of VND7-VP16-GR in the mature region of the roots (A and D), leaf blades (B and E), and petioles (C and F). Scale bar = 50 μm. G to J, Secondary wall thickening was observed in guard cells (G), columella cells (H), root hair cells (I), and trichome cells (J). Scale bar = 20 μm in G and H; scale bar = 50 μm in I and J.
Figure 3.
Figure 3.
Secondary wall patterns in transdifferentiated cells induced by the activation of VND6-VP16-GR and VND7-VP16-GR in Arabidopsis. Seven-day-old VP16-GR-2-2 plants (A and D), VND6-VP16-GR-1-1 plants (B and E), and VND7-VP16-GR-10-2 plants (C and F) were treated with DEX for 4 d. Epidermal cells in the leaves (A–C) and roots (D–F) were stained with safranin and observed under a confocal laser-scanning microscope. Scale bar = 50 μm.
Figure 4.
Figure 4.
Expression profiles of genes involved in the xylem vessel element differentiation in Arabidopsis. A, RT-PCR analysis of genes encoding enzymes involved in xylem vessel element differentiation. B, RT-PCR analysis of MYB transcription factors regulating secondary wall synthesis. Fourteen-day-old VND6-VP16-GR-6-1 and VND7-VP16-GR-8-2 plants were treated with (+) or without (−) DEX for 1 d, and then total RNA was extracted.
Figure 5.
Figure 5.
Analysis of cell wall composition in Arabidopsis. A, Monosaccharides from the acid-hydrolyzed alcohol-insoluble residue were analyzed by HPAEC-PAD. Data are the means ± sd, n = 2. *P > 0.1: Student's t test relative to the wild type (Columbia [Col]). B, PACE fingerprint of xylan in the cell walls of seedlings. Oligosaccharides released by NpXyn 11A treatment of alcohol-insoluble residues were derivatized with 2-aminonaphthalene trisulfonic acid and separated by PAGE.
Figure 6.
Figure 6.
The tobacco BY-2 in vitro system of xylem vessel element formation. A, Frequency of xylem vessel element formation over 7 d of culture. Data are the means of three lines ± sd. B, Xylem vessel elements induced in suspension culture of BY-2 cells 3 d after DEX addition. The secondary cell wall was stained using calcofluor (blue). Scale bar = 30 μm.
Figure 7.
Figure 7.
Induction of xylem vessel element transdifferentiation in transgenic poplar plants. The leaves of wild-type (A and D) transgenic poplar carrying VND6-VP16-GR (B and E) or VND7-VP16-GR (C and F) were incubated in water with (D–F) or without (A–C) DEX for 7 d. Scale bar = 50 μm.
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