Transcription switches for protoxylem and metaxylem vessel formation

Abstract

Land plants evolved xylem vessels to conduct water and nutrients, and to support the plant. Microarray analysis with a newly established Arabidopsis in vitro xylem vessel element formation system and promoter analysis revealed the possible involvement of some plant-specific NAC-domain transcription factors in xylem formation. VASCULAR-RELATED NAC-DOMAIN6 (VND6) and VND7 can induce transdifferentiation of various cells into metaxylem- and protoxylem-like vessel elements, respectively, in Arabidopsis and poplar. A dominant repression of VND6 and VND7 specifically inhibits metaxylem and protoxylem vessel formation in roots, respectively. These findings suggest that these genes are transcription switches for plant metaxylem and protoxylem vessel formation.

Figure 1.

The Arabidopsis in vitro xylem vessel element formation system. (A) Xylem vessel elements induced in Arabidopsis suspension cells cultured for 7 d. Bar, 50 μm. (B) Frequency of xylem vessel element formation. Results are means ± SD (n = 3). (C) Hierarchical clustering of developmentally regulated genes during in vitro xylem vessel element formation. Colors indicate expression levels at 0 d after induction as follows: (magenta) high; (yellow) medium; (cyan) low.

Figure 2.

Characterization of the VND genes. (A) Neighbor-joining tree of selected NAC-domain proteins of zinnia and Arabidopsis. Amino acid sequences were aligned by using the MAFFT program. (B) Expression patterns of VND1 to VND7 during in vitro xylem vessel element formation revealed by microarray analysis. (C) Expression of VNDp::YFP–NLS in Arabidopsis roots. Images of differential interference contrast (DIC) and YFP fluorescence were merged. Bars, 100 μm.

Figure 3.

Overexpression phenotypes of VND6 and VND7. (A) Longitudinal DIC images of Arabidopsis hypocotyls. (B) Longitudinal confocal laser scanning microscope (CLSM) images of Arabidopsis roots. (C) CLSM image of epidermis of Arabidopsis rosette leaf. (D) CLSM images of epidermis in the poplar leaves. (PX) Protoxylem vessel; (MX) metaxylem vessel. Bars: A,B, 100 μm; insets in A, 20 μm; C, insets in B, 10 μm; D, 50 μm.

Figure 4.

Dominant repression phenotypes of VND6 and VND7 in Arabidopsis. (A–C) Fourteen-day-old seedlings. (D–F) Longitudinal DIC images of vascular bundles in a middle region of the 9-d-old roots. White arrows indicate protoxylem vessels. Black and red arrowheads indicate primary and central metaxylem vessels, respectively. (A,D) Wild type. (B,E) VND6–SRDX. (C,F) VND7–SRDX. Bars: A–C, 10 mm; D–F, 20 μm.

Figure 5.

Effects of phytohormones on expression levels of VND6 and VND7 in cultured hypocotyls. (F) Hormone free; (K) 50 ng/mL kinetin; (D) 500 ng/mL 2,4-dichlorphenoxyacetic acid; (BL) 1 μM brassinolide. Relative expression levels between VND6 and UBQ, and VND7 and UBQ are shown as compared with the hormone-free condition. Results are means ± SD (n = 3).