Article Figures & Data
Figures
- Figure 1.
Three tissue samples can be isolated from the root-hypocotyl. A, Extensive secondary growth is evident in the root-hypocotyl of an 8-week-old Arabidopsis plant. Lignified vessels and fibers of secondary xylem are stained blue with TBO. Nonlignified primary cell walls of cells in secondary xylem, secondary phloem, and nonvascular tissues are stained pink with ruthenium red. B, Nonvascular tissue of the outer bark can be separated from secondary phloem, yielding the nonvascular sample for expression profiling. C, Secondary phloem can be separated from secondary xylem, yielding the phloem-cambium and xylem samples for expression profiling. Free-hand transverse sections were prepared from fresh tissue just prior to staining. Tissues were dissected after staining. nv, Nonvascular; pc, phloem-cambium; sp, secondary phloem; sx, secondary xylem; x, xylem. Bars = 50 μm.
- Figure 2.
Ratio-intensity plots of between-tissue comparisons with statistically biased genes (≥3.29 sds, P ≤ 0.001) highlighted. The vertical axis plots the log2 of the ratio of the normalized MSIs for the same gene between two tissues: X versus PC (A), X versus NV (B), and PC versus NV (C). The horizontal scale specifies the log10 of the product of the two MSIs for the same gene between two tissues. Genes with between-tissue means below 50 (0.1 times the among-genes mean) were considered insignificant regardless of ratio due to the noise in that region. Correlations (R2 values) are shown in the lower right corner for each between-tissue comparison.
- Figure 3.
“Triangle plot” of the relative MSIs among three tissues. Each gene is represented by a point whose proximity to each of the tissue-labeled corners reflects the relative expression in that tissue (see “Materials and Methods” for the numerical formula). Only genes with MSI values ≥200 are shown. Genes expressed in a single tissue lie in an extreme corner, while those expressed equally in all three lie in the center. Genes with signals significantly (P ≤ 0.001) higher in one tissue relative to both other tissues are highlighted in red (so-called “one-tissue genes”). Genes with signals not significantly different for two tissues but higher in both of those tissues relative to the third are highlighted in green (so-called “two-tissue genes”). Genes with signals significantly biased in only one pair-wise comparison or those not significantly biased in any pair-wise comparison are shown as large and small black points, respectively.
- Figure 4.
Promoters for CLV1, MYR1, ZFWD1, XND1/ANAC104, and At1g20160 direct expression of reporters in vascular tissues as predicted from genome-wide transcript profiles of isolated vascular tissues. A, GFP expression driven by the promoter for CLV1 localized to the cambial zone and secondary phloem. A, Inset, The secondary phloem of wild-type control plants exhibited no detectable green fluorescence. B and C, GUS activity driven by the promoter for the phloem-cambium-biased, G2-like transcription factor MYR1 was detected in the secondary phloem of the root (B) and inflorescence stem (C). D and E, GUS activity due to the MYR1 promoter was also detected throughout the vascular tissue of the leaf (D), where it localized predominantly to the abaxial (phloem) side of the vascular bundle, as shown in a transverse section through a midvein (E). F, GUS activity driven by the promoter for the xylem-biased gene ZFWD1 was limited to vascular tissues. G, Higher magnification of the area within the black box (F) revealed that ZFWD1p::GUS expression was associated with xylem cells (arrow). H, XND1/ANAC104 promoter-driven GUS activity visible beneath the bark of roots on 8-week-old plants (H, inset) was localized to xylem vessels, shown here following isolation of two adjacent vessels (one mature GUS-negative and one immature GUS-positive) from secondary xylem. I, XND1p::GUS expression in the shoot was limited to senescing leaves, where it was evident in xylem cells surrounding mature vessels (arrows) and immature vessels (arrowhead) in the midvein. J, XND1p::GUS expression in the primary tissues of seedling roots was limited to TEs, as shown for metaxylem cells (arrow) adjacent to mature protoxylem cells. K, GUS activity driven by the promoter for a xylem-biased subtilisin-like Ser protease At1g20160 was localized predominantly in xylem cells surrounding mature vessels (arrows) and additional cells on the adaxial (xylem) side of the midvein. L, Vascular tissue-localized expression for At1g20160p::GUS is shown in a representative leaf. A, The free-hand transverse section was prepared from a 6-week-old (or 5-week-old for inset) hypocotyl, and cell walls were counterstained with propidium iodide prior to detection of GFP by confocal microscopy. B, C, E, I, and K, Free-hand transverse sections of root-hypocotyl, stem, or petiole were prepared following histochemical staining for GUS activity. D, Whole-mount leaf from a 5-week-old plant. F and G, Whole-mount cotyledon from a 3-d-old seedling. H, Xylem vessels were isolated from a root similar to that shown in the inset following histochemical staining for GUS activity. J, Whole-mount root from a 3-d-old seedling. L, Whole-mount leaf from a 4-week-old plant. cz, Cambial zone; nv, nonvascular tissue; p, phloem; x, xylem. Bars = 100 μm (A, inset in A, and B); 50 μm (C); 25 μm (E, G, H, I, J, and K); 500 μm (inset in H). B, Broken lines indicate the limit of phloem or nonvascular tissues.
- Figure 5.
RT-PCR results for selected tissue-biased genes are consistent with predictions from genome-wide transcript profiles of isolated vascular tissues. Ethidium bromide-stained gels show products of RT-PCR for tissue-biased genes selected from xylem (X; At1g02250 and At1g32770), phloem-cambium (PC; At2g03500 and At3g04030), and nonvascular (NV; At3g26450) gene sets. Numbers of PCR cycles used were 27 for At3g26450 and actin (ACT7) and 30 for At1g02250, At1g32770, At2g03500, and At3g04030. Different PCR cycle numbers and annealing temperatures were evaluated before these representative experiments were selected for presentation.
Tables
- Table I.
Relative signal intensities and tissue biases for known Arabidopsis marker genes for xylem, phloem, or nonvascular peripheral cells
Locus Gene Symbol Description Log2 (X versus PC)a Log2 (X versus NV) Log2 (PC versus NV) Tissue Bias, This Report Published Localization Reference At4g18780 IRX1 Cellulose synthase catalytic subunit 4.7* 3.8* −0.9 X Xylem TEs Gardiner et al. (2003) At5g17420 IRX3 Cellulose synthase catalytic subunit 4.1* 4.1* 0.0 X Xylem TEs Gardiner et al. (2003) At5g44030 IRX5 Cellulose synthase catalytic subunit 4.0* 4.1* 0.1 X Xylem TEs Gardiner et al. (2003) At4g35350 XCP1 Cys proteinase 6.8* 6.2* −0.6 X Xylem TEs Funk et al. (2002) At1g20850 XCP2 Cys proteinase 6.2* 6.5* 0.3 X Xylem TEs Funk et al. (2002) At5g57350 AHA3 Plasma membrane ATPase 3 (proton pump) −6.0* −1.2 4.8* PC Phloem CCs DeWitt and Sussman (1995) At1g79430 APL G2-like transcription factor −6.2* −1.4 4.8* PC Phloem SE/CCs Bonke et al. (2003) At1g05760 RTM1 Jacalin lectin family protein −4.2* 1.5 5.8* PC Phloem SEs Chisholm et al. (2001) At1g22710 SUC2 Suc transporter/Suc-proton symporter −3.4* 0.7 4.0* PC Phloem CCs Stadler and Sauer (1996) At1g12110 NRT1.1/CHL1 Nitrate/chlorate transporter −0.8 −3.5* −2.6* NV Epidermis (hypocotyl guard cells) Guo et al. (2003) At1g08090 NRT2.1 High-affinity nitrate transporter −1.3 −6.1* −4.9* NV Endodermis, cortex, epidermis Nazoa et al. (2003) ↵a Log2 of the signal ratio for the three pair-wise tissue comparisons (X versus PC, xylem versus phloem-cambium; X versus NV, xylem versus nonvascular, PC versus NV, phloem-cambium versus nonvascular), where a positive value indicates a higher signal for the first member of the pair-wise comparison and a negative value indicates a higher signal for the second member of the pair-wise comparison. Log2 values representing pair-wise comparisons between significantly different signals are indicated by a single asterisk. For reference: a log2 value of 4 is equivalent to a 16-fold difference between signals, while a log2 value of 6 is equivalent to a 64-fold difference between signals.
- Table II.
Relative signal intensities and tissue biases for genes known or proposed for this report to have roles in vascular tissue differentiation or function
Locus Gene Symbol Description Log2 (X versus PC)a Log2 (X versus NV) Log2 (PC versus NV) Tissue Bias, This Reportb Role in Vascular Tissue Differentiationc Reference At4g32880 ATHB-8 Homeobox-Leu zipper family 2.6* 4.4* 1.8* X Vascular differentiation Baima et al. (2001) At1g30490 ATHB-9/PHV Homeobox-Leu zipper family 2.0* 2.4* 0.4 X Vascular bundle organization McConnell et al. (2001) At2g34710 ATHB-14/PHB Homeobox-Leu zipper family 1.7* 2.9* 1.3 X Vascular bundle organization McConnell et al. (2001) At1g52150 ATHB-15 Homeobox-Leu zipper family 2.8* 3.6* 0.9 X ? At5g60690 REV/IFL1 Homeobox-Leu zipper family 2.0* 3.5* 1.5 X Vascular patterning Zhong and Ye (2004) At1g02250 ANAC005 NAC domain 2.3* 2.5* 0.3 X ? At1g32770 ANAC012 NAC domain 4.5* 3.6* −0.8 X ? At2g46770 ANAC043 NAC domain 3.4* 2.6* −0.8 X ? At4g28500 ANAC073 NAC domain 2.3* 5.1* 2.8 X ? At4g28530 ANAC074 NAC domain 1.9* 1.6* −0.3 X ? At5g64530 XND1/ANAC104 NAC domain 5.9* 6.4* 0.5 X ? At1g63910 MYB103 R2R3 MYB 2.0* 3.0* 1.0 X ? At4g33450 MYB69 R2R3 MYB 4.2* 3.0* −1.2 X ? At1g17950 MYB52 R2R3 MYB 5.6* 2.3* −3.2* X ? At3g46130 MYB48 R2R3 MYB 3.6* 3.8* 0.1 X ? At5g12870 MYB46 R2R3 MYB 3.6* 2.9* −0.8 X ? At1g66230 MYB20 R2R3 MYB 4.8* 5.7* 0.9 X ? At1g79430 APL G2-like transcription factor −6.2* −1.4 4.8* PC Vascular differentiation Bonke et al. (2003) At3g12730 G2-like transcription factor −3.6* −0.6 3.0* PC ? At5g18240 MYR1 G2-like transcription factor −4.8* −0.3 4.5* PC ? At4g37750 ANT AP2 domain transcription factor −2.9* −0.6 2.3* PC ? At1g75820 CLV1 CLAVATA1 receptor kinase −3.6* 1.0 4.5* PC ? At4g20270 CLV1-like receptor kinase −2.8* 0.8 3.6* PC ? At2g31085 CLE6 CLAVATA3/ESR-related 6 −3.6* −0.2 3.4* PC ? At1g69970 CLE26 CLAVATA3/ESR-related 26 −4.6* −0.8 3.9* PC ? At2g28810 Dof transcription factor −3.6* −1.1 2.5* PC ? At5g62940 Dof transcription factor −2.9* 0.3 3.2* PC ? At1g07640 OBP2 Dof transcription factor −4.9* −1.6 3.3* PC ? Kang and Singh (2000) At1g32240 KAN2 G2-like transcription factor −4.2* −3.9* 0.2 PC/NV Vascular bundle organization Emery et al. (2003) At4g17695 KAN3 G2-like transcription factor −3.6* −3.2* 0.4 PC/NV Vascular bundle organization Emery et al. (2003) At5g64080 XYP1 Lipid transfer protein (LTP) family protein −1.9* −1.8* 0.1 PC/NV Vascular patterning Motose et al. (2004) At2g01830 AHK4/WOL/CRE1 His kinase 0.9 2.3* 1.4 NS Vascular differentiation Inoue et al. (2001); Mahonen et al. (2000); Scheres et al. (1995) At1g20330 CVP1 S-Adenosyl-Met-sterol-C-methyltransferase −1.3 −0.8 0.5 NS Vascular patterning Carland et al. (2002) At3g52940 FK C-14 sterol reductase −0.7 −0.4 0.3 NS Vascular patterning Jang et al. (2000) At1g13980 GNOM/EMB30 Unclassified −0.1 −0.3 −0.2 NS Vascular patterning Steinmann et al. (1999) At1g19850 MP/IAA24 IAA protein 24 0.4 1.7 1.3 NS Vascular differentiation Hardtke and Berleth (1998) At1g73590 PIN1 Auxin efflux carrier protein 1.0 2.2* 1.3 NS Vascular patterning Galweiler et al. (1998) ↵a Log2 of the signal ratio for the three pair-wise tissue comparisons as described for Table I. Log2 values representing pair-wise comparisons between significantly different signals are indicated by a single asterisk.
↵b NS, Not significantly tissue biased in any pair-wise comparison or tissue biased in only one pair-wise comparison. Other abbreviations introduced in text.
↵c Terms used to describe vascular tissue roles for characterized genes are from Ye (2002). ?, Genes identified for this report that may play roles in vascular tissue differentiation or function.
- Table III.
Summary of the six root-hypocotyl gene sets organized by functional category
Within each tissue-biased gene set, the percentage of genes in each functional category is shown followed by the number (in parentheses) of genes in each category. The single highest-ranking category (for xylem/phloem-cambium and xylem/nonvascular) or two highest-ranking categories (for xylem, phloem-cambium, nonvascular, and phloem-cambium/nonvascular) for each gene set, from among the classified genes, are shown in bold.
Functional Category Tissue-Biased Gene Set Xylem Phloem-Cambium Nonvascular Xylem/Phloem-Cambium Xylem/Nonvascular Phloem-Cambium/Nonvascular Biogenesis of cell wall 11 (35) 9 (18) 10 (16) 0 5 (2) 12 (28) Cell rescue, defense, cell death, and aging 7 (24) 13 (28) 17 (26) 14 (4) 24 (9) 8 (20) Cellular communication/signal transduction 11 (34) 14 (30) 3 (4) 35 (10) 3 (1) 11 (26) Cytoskeleton <1 (1) 0 0 0 0 0 DNA/RNA binding 0 0 3 (4) 0 0 <1 (1) Energy 2 (5) 2 (4) 1 (2) 0 0 1 (3) Lignin biosynthesis 6 (21) 1 (2) 6 (10) 0 5 (2) 2 (5) Metabolism 9 (28) 10 (21) 20 (31) 10 (3) 11 (4) 19 (46) Protein destination 0 0 0 0 0 0 Proteolysis 6 (18) 5 (11) 1 (2) 3 (1) 11 (4) 6 (15) Secondary metabolism 4 (12) 5 (11) 5 (8) 0 8 (3) 3 (8) Transcription 10 (33) 9 (18) 8 (12) 7 (2) 5 (2) 10 (24) Transport facilitation 11 (34) 7 (15) 11 (17) 17 (5) 3 (1) 10 (25) Unclassified 23 (74) 25 (53) 14 (22) 14 (4) 24 (9) 17 (40) Set total (319) (211) (154) (29) (37) (241) - Table IV.
Relative signal intensities and tissue biases for selected genes involved in hormone metabolism or transport
Locus Gene Symbol Description Log2 (X versus PC)a Log2 (X versus NV) Log2 (PC versus NV) Tissue Bias, This Reportb Role in Hormone Metabolism/Transport Reference At2g38120 AUX1 Amino acid/auxin permease 1.5* 2.0* 0.6 X Auxin transport Bennett et al. (1996) At1g30040 GA2OX2 GA 2-oxidase 3.6* 4.6* 1.1 X Gibberellic acid inactivation At2g22330 CYP79B3 Cytochrome P450, converts Trp to IAOx −3.3* −0.9 2.4* PC Auxin biosynthesis Hull et al. (2000) At1g15550 GA4 GA 3-β-dioxygenase/GA 3-β-hydroxylase −3.5* 0.8 4.4* PC Gibberellic acid biosynthesis Williams et al. (1998) At5g19040 IPT5 Adenylate isopentenyltransferase 5/cytokinin synthase −6.0* −2.2 3.9* PC Cytokinin biosynthesis Takei et al. (2004) At3g63110 IPT3 Adenylate isopentenyltransferase 3/cytokinin synthase −1.2 2.0* 3.2* X/PC Cytokinin biosynthesis Takei et al. (2004)
Supplemental Data
Supplemental Figure and Table Data
Files in this Data Supplement:
- Supplemental Data - Table I Data
- Supplemental Data - Table II Data
- Supplemental Data - Table III Data
- Supplemental Data - Table IV Data
- Supplemental Data - Table V Data
- Supplemental Data - Table VI Data
- Supplemental Data - Table VII Data
- Supplemental Data - Table VIII Data
- Supplemental Data - Fig 1 Data
