Genetic Regulation of Vascular Tissue Patterning in Arabidopsis

Correlation of Phloem Patterns with Xylem Patterns.

(A), (C), and (E) Fluorescence microscopy of phloem sieve cells stained with aniline blue.

(B), (D), and (F) Corresponding dark-field images of xylem elements. Wild-type cells are shown in (A) and (B), cvp1 cells are shown in (C) and (D), and cvp2 cells are shown in (E) and (F). Magnification ×30.

Wild-Type Morphology and Root Anatomy of cvp Mutants.

(A) to (D) Detached cotyledons from 7-day-old seedlings show wild-type morphology in the wild type (A) and in the cvp1 (B), cvp2 (C), and cvp1 cvp2 (D) mutants.

(E) to (H) Seven-day-old seedlings show wild-type seedling growth and phenotype. (E) shows a wild-type seeding, (F) shows a cvp1 seedling, (G) shows a cvp2 seedling, and (H) shows a cvp1 cvp2 seedling.

(I) and (J) Longitudinal view of 7-day-old cleared roots of the wild type (I) and cvp1 (J). (J) shows axialization in the vascular cylinder of cvp1.

(K) and (L) Transverse sections of wild type (K) and cvp1 (L). In (L), 7-day-old roots illustrate wild-type internal anatomy of cvp1.

Bars in (A) to (D) = 250 μm; bars in (E) to (H) = 500 μm; bars in (I) and (J) = 80 μm; bars in (K) and (L) = 25 μm.

Map Positions of CVP1 and CVP2.

Numbers represent recombination frequencies ±se between CVP genes and linked molecular markers. The recombination frequencies of m235, CVP1, and m59 were determined from scoring 72 F₂ plants (144 chromosomes), and the frequencies of NCC1, CVP2, and PVV4 were determined from scoring 43 F₂ plants (86 chromosomes).

Procambium Patterns in Embryonic Cotyledons.

Procambial tissue is observed as strands of lightly stained cells in the embryonic cotyledons.

(A) Wild type.

(B) cvp1.

(C) cvp2.

(D) cvp1 cvp2.

Bar in (D) = 31 μm for (A) to (D).

Anatomy of Cotyledon Vascular Bundles in cvp Mutants Visualized by Paradermal Plastic Sectioning of 5-Day-Old Cotyledons.

(A) Wild-type vein shows longitudinal files of elongating provascular cells. Vascular bundles normally are oriented obliquely, resulting in sections with short stretches of veins.

(B) cvp1 wild-type-like vein.

(C) cvp1 mutant vein illustrates improper alignment of cell files and incorrect division planes of provascular cells (arrowheads).

(D) Abnormal cell expansion within cvp1 vascular bundle (arrowheads). Arrows indicate misshapen xylem cells (tracheary elements) that stain a turquoise blue with toluidine blue dye.

(E) cvp2-1 vein showing elongated provascular cells arrayed in longitudinal files.

(F) cvp1 cvp2 mutant vein that is isolated (discontinuous) from other veins.

Bar in (A) = 31 μm; bars in (B) to (E) = 40 μm; bar in (F) = 25 μm.

Transverse Sections of Cotyledons from 5-Day-Old Seedlings.

(A) Wild type.

(B) cvp1 showing normal midvein and abnormal lateral vein.

(C) cvp1 showing misaligned lateral vein in longitudinal orientation.

(D) cvp2 showing midvein and a lateral vein composed of 10 vascular elements.

(E) cvp2 with arrow designating same lateral vein as shown in (D) at a distal point and composed of a single vascular element.

Note the wild-type epidermal and mesophyll cells (particularly the palisade cells) in (B) to (E). le, lower epidermis; lv, lateral vein; mv, midvein; p, palisade cells (which elongate and are arranged parallel to each other within the adaxial subepidermal layer); ue, upper epidermis.

Bars in (A) to (D) = 31 μm; bar in (E) = 25 μm.

Vascular Bundle Patterns in cvp Leaves.

(A) to (D) Safranin-O-stained first rosette leaves from 14-day-old wild-type (A), cvp1 (B), cvp2 (C), and cvp1 cvp2 (D) plants. In (B) and (D), note the wild-type venation pattern in cvp1 and unattached vascular bundles (arrows) and premature termination of tertiary veins (only a few are indicated with asterisks) in the cvp2 and cvp1 cvp2 leaves.

(E) to (H) Cleared cauline leaves from wild-type ([E] and [F]) and cvp2 ([G] and [H]) plants show the increase in free vein endings (indicated with asterisks in [H]) in cvp2 mutants. Boxed regions in (E) and (G) are shown at a higher magnification in (F) and (H), respectively.

(I) to (P) Developmental stage analysis of the first rosette leaf of the wild type ([I], [K], [M], and [O]) and cvp2 ([J], [L], [N], and [P]) at 7 days ([I] and [J]), 9 days ([K] and [L]), 12 days ([M] and [N]), and 16 days ([O] and [P]) after sowing shows that there is no delay in vein initiation in cvp2 mutants. Arrows indicate the midvein ([I] and [J]), an open apical loop in a cvp2 mutant leaf (L), unconnected cvp2 veins (N), and open apical loops (P). Asterisks indicate some of the free tertiary vein endings (P). Transparent spiked structures are trichomes.

Bars in (A) to (D), (E), (G), (O), and (P) = 800 μm; bars in (F) and (H) = 300 μm; bars in (I) and (J) = 80 μm; bars in (K) and (L) = 250 μm; bars in (M) and (N) = 320 μm.

Inflorescence Stems in cvp1 Plants.

(A) Transverse section of inflorescence stem displaying wild-type internode lengths. Xylem (x) and lignified sclerenchyma (s) cell walls appear dark blue. Lightly staining phloem bundles (p) are associated with xylem.

(B) Transverse section of inflorescence stem displaying decreased internode lengths. Note increased production of xylem and lignified sclerenchyma.

(C) cvp1 plant displaying inflorescence stems with normal (a) and decreased (b) internode lengths.

Bars in (A) and (B) = 400 μm.

Sensitivity to Auxin and an Inhibitor of Polar Auxin Transport.

Plants were grown on synthetic media containing different concentrations of either IAA (A) or triiodobenzoic acid (TIBA) (B), an inhibitor of polar auxin transport. Root lengths were measured after growth on these media for 10 days. WT, wild type.