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Kubo, 1 Supplementary Materials Supplementary Figure Legends Supplementary Figure S1. Schematic representation of the vasculature in an Arabidopsis root. (A) Image of transverse section. (B) Image of longitudinal section. Supplementary Figure S2. Seven-d-old roots of immature xylem vessel-specific promoter::reporter lines. (A,D) ATXYN3p::CFP-NLS. (B,E) IRX3p::YFP-NLS. (C,F) LACCASEp::YFP-NLS. (A-C) Wild type (Col-0) background. (D-F) 35S::VND7 background. Fluorescent and DIC images were merged. Note that nucleus with fluorescence in immature xylem vessels is cylinder-shaped, while that in other cells is globular- or egg-shaped. Arrowheads indicate ectopic expression of the reporter genes in 35S::VND7. Bars: 50μm. Supplementary Figure S3. Amino acid alignments among Z567 and selected Arabidopsis NAC-domain proteins (AtNACs). Amino acid sequences of AtNACs were obtained from the AGRIS database (Davuluri et al 2003). Alignments were calculated by MAFFT (http://bioinformatics.uams.edu/mafft/) and drown by MacVector 7.2.2. Conserved NAC sub-domains are indicated by black lines (NAC I to NAC V). Red lines indicate conserved C-terminal sub-domains of Z567 and the VND family. Supplementary Figure S4. Expression of VNDp::GUS. (A-C) Five-d-old root of VND6p::GUS. (D) Five-d-old root of VND7p::GUS. Arrows indicated immature protoxylem vessels. (E-H) Aerial parts of 7-d-old VND2p (E), VND3p (F), VND5p (G), and VND7p::GUS (H) plants. Bars: A,D, and inset in D, 100 μm; B,C, 10 μm; E-H, 500 μm. Supplementary Figure S5. Seven-d-old roots of the VND-YFP lines driven by 35S. (A) 35S::VND6-YFP. (B) 35S::VND7-YFP. Images of DIC and YFP were merged. Inset in B indicates a CLSM image of the 35S::VND7-YFP line. Red signals by propidium iodide-staining show cell shapes in the root. Arrows indicate transdifferentiated xylem vessel elements with fluorescent signal of VND7-YFP in nucleus. Bars: 100μm. Supplementary Figure S6. Nine-d-old roots and hypocotyls of the VND-SRDX lines. (Left)
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Kubo, 1

Supplementary Materials

Supplementary Figure Legends

Supplementary Figure S1. Schematic representation of the vasculature in an

Arabidopsis root. (A) Image of transverse section. (B) Image of longitudinal section.

Supplementary Figure S2. Seven-d-old roots of immature xylem vessel-specific

promoter::reporter lines. (A,D) ATXYN3p::CFP-NLS. (B,E) IRX3p::YFP-NLS. (C,F)

LACCASEp::YFP-NLS. (A-C) Wild type (Col-0) background. (D-F) 35S::VND7

background. Fluorescent and DIC images were merged. Note that nucleus with fluorescence

in immature xylem vessels is cylinder-shaped, while that in other cells is globular- or

egg-shaped. Arrowheads indicate ectopic expression of the reporter genes in 35S::VND7.

Bars: 50µm.

Supplementary Figure S3. Amino acid alignments among Z567 and selected Arabidopsis

NAC-domain proteins (AtNACs). Amino acid sequences of AtNACs were obtained from

the AGRIS database (Davuluri et al 2003). Alignments were calculated by MAFFT

(http://bioinformatics.uams.edu/mafft/) and drown by MacVector 7.2.2. Conserved NAC

sub-domains are indicated by black lines (NAC I to NAC V). Red lines indicate conserved

C-terminal sub-domains of Z567 and the VND family.

Supplementary Figure S4. Expression of VNDp::GUS. (A-C) Five-d-old root of

VND6p::GUS. (D) Five-d-old root of VND7p::GUS. Arrows indicated immature

protoxylem vessels. (E-H) Aerial parts of 7-d-old VND2p (E), VND3p (F), VND5p (G), and

VND7p::GUS (H) plants. Bars: A,D, and inset in D, 100 µm; B,C, 10 µm; E-H, 500 µm.

Supplementary Figure S5. Seven-d-old roots of the VND-YFP lines driven by 35S. (A)

35S::VND6-YFP. (B) 35S::VND7-YFP. Images of DIC and YFP were merged. Inset in B

indicates a CLSM image of the 35S::VND7-YFP line. Red signals by propidium

iodide-staining show cell shapes in the root. Arrows indicate transdifferentiated xylem

vessel elements with fluorescent signal of VND7-YFP in nucleus. Bars: 100µm.

Supplementary Figure S6. Nine-d-old roots and hypocotyls of the VND-SRDX lines. (Left)

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Wild type. (Center) VND6-SRDX. (Right) VND7-SRDX. White arrows indicate points where

protoxylem vessels begin to differentiate. Black and red arrowheads indicate points where

primary and central metaxylem vessels begin to differentiate, respectively. In wild type

roots, the first protoxylem vessels differentiate around where root hair formation begins.

Soon after the protoxylem vessel formation, primary metaxylem vessels differentiate on the

inner side of protoxylem vessels. Central metaxylem vessel usually differentiates after roots

fairly develop. Interestingly, in VND6-SRDX roots, central metaxylem vessel is hardly

observed. In VND7-SRDX roots, protoxylem vessel formation is delayed or repressed

significantly. Bar: 5 mm.

Supplementary Figure S7. Tissue-dependent expression of VND6 and VND7 in the cultured

hypocotyls. (F) Hormone free. (K+D) 50 ng/ml Kinetin and 500 ng/ml

2,4-Dichlorphenoxyacetic acid. (K+D+BL) Kinetin, 2,4-D , and 1 µM brassinolide.

Expression of VND6p and VND7p::GUS was examined in the hypocotyls cultured for 5 d in

the presence of indicated phytohormons. Bars: 500µm and 20µm (insets).

Supplementary method for microarray (MIAME format)

Experimental design: materials for the microarray experiments. Fifteen ml of

Arabidopsis Col-0 suspension cells was transferred to 35 ml of a fresh modified Murashige

and Skoog (MS) medium supplemented with 1 µg/ml 2,4-dichlorphenoxyacetic acid and 3%

sucrose every 7 d, and subcultured on a rotary shaker at 120 rpm in the dark at 22 °C. For xylem vessel element induction, a 7.5 ml aliquot of 7-d-old subcultured cells was transferred

into 42.5 ml of fresh medium that included 1 µM brassinolide and 10 mM H3BO3, and

cultured as described above. Sample without medium were collected, frozen in liquid nitrogen

and stored at -80°C.

Samples used. Two different samples were used from time-course of Arabidopsis culture

system. Total RNA was prepared by using phenol/SDS method (Nishitani et al. 2001) and

purified by using RNeasy Mini Kit (Qiagen). Two independent experiments were performed.

Extract labeling. Microarray analysis was performed using ATH1 GeneChips™ (Affymetrix).

Ten µg of total RNA was reverse transcribed using the SuperScript Choice system for cDNA

synthesis (Invitrogen) according to the protocol recommended by Affymetrix. The

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oligonucleotide used for priming was 5’-ggccagtgaattgtaatacgactcactatagggaggcgg-(t)24-3’

(Amarsham). Double-stranded cDNA was cleaned by phenol:chloroform extraction and the

aqueous phase removed by centrifugation through Phase-lock Gel (Eppendorf). In vitro

transcription was performed on 1 µg of cDNA using the Enzo BioArray High Yield RNA

transcript labelling kit (Enzo Diagnostics). The cRNA was cleaned using RNAeasy clean-up

columns (Qiagen). The cRNA was fragmented by heating in 40 mM Tris-acetate pH 8.1, 100

mM KOAc, 30 mM MgOAc. The Affymetrix eukaryotic hybridization controls were added to

the sample prior to hybridization as per manufacturer’s instructions.

Hybridization conditions. Ten µg of fragmented cRNA were hybridised (45°C, 16 h).

Hybridization was controlled by use of the GeneChip™ Eukaryotic Hybridization Control Kit

(Affymetrix). Washing and staining was performed in a Fluidics Station 400 (Affymetrix)

using the protocol EukGE-WS2v4 and scanned in an Affymetrix GeneChip scanner.

Data analysis. Data analysis was performed using Microarray Suite ver. 5 (Affymetrix) and

GeneSpring 7.1 (Silicon Genetics). The 50th percentiles of all averaged values obtained from

the duplicated experiments were used as the first synthetic positive controls for each gene.

The value for each gene was divided by these synthetic positive controls. Subsequently, the

50th percentiles of the normalized values for each gene were used as their own synthetic

positive controls, and the normalized value for each gene was divided by its own synthetic

positive control. After this double normalization, genes with a greater than eight-fold change

in expression over the culture time course (i.e., the maximum normalized value divided by the

minimum normalized value was > 8) were chosen. Among them, any gene whose flag was

absent at all time points was discarded as being unreliable data. Finally, selected genes were

clustered using the QT clustering function with a minimum cluster size = 10, a minimum

similarity = 0.9, and a similarity measure standard correlation.

Array design. Affymetrix ATH1 GeneChip

Supplementary References

Davuluri, R.V., Sun, H., Palaniswamy, S.K., Matthews, N., Molina, C., Kurtz, M., and

Grotewold, E. 2003. AGRIS: Arabidopsis gene regulatory information server, an

information resource of Arabidopsis cis-regulatory elements and transcription factors.

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BMC Bioinformatics 4: 25.

Nishitani, C., Demura, T., and Fukuda, H. 2001.Primary phloem-specific expression of a

Zinnia elegans homeobox gene. Plant Cell Physiol. 42: 1210-1218.

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