The polycotyledon mutant of tomato shows enhanced polar auxin transport

Abstract

The polycotyledon mutant of tomato (Lycopersicon esculentum L. cv Ailsa Craig) showed altered development during embryogenesis and during vegetative and reproductive phases. The phenotype was pleiotropic and included the formation of extra cotyledons, changes in leaf shape, increased number of flowers (indeterminacy) with abnormal floral organs, the formation of epiphyllous structures, and altered gravitropism. The earliest defects were observed at the transition from the globular to the heart stage of embryogenesis with the formation of multiple cotyledons. Epidermal cells in the mutant embryo were smaller and less expanded compared with wild type. Examination of polar auxin transport (PAT) showed a striking enhancement in the case of the mutant. Increase in PAT did not appear to be caused by a decrease in flavonoids because the mutant had normal flavonoid levels. Application of 2,3,5-triiodobenzoic acid, an inhibitor of polar transport of auxin, rescued postgermination phenotypes of young seedlings. Our analysis reveals a level of control that negatively regulates PAT in tomato and its contribution to plant development and organogenesis.

Figure 1.

Morphology of poc plants throughout life cycle. A, poc seedlings (clockwise from top right): wild-type seedlings (arrow); dicot [1+(2)], note two midveins; dicot with two halves of curled cotyledons; dicot [1+(2)] with a partially fused cotyledon; tetracot; and tricot. B, Fused poc cotyledons showing two midveins running parallel in the petiole. Wild type (left). C, poc dicot seedling showing smaller and more rounded cotyledons. Wild type (left). D, Phenotype of 3.5-month-old plants. Wild type (left), poc A (second from left), poc B (third from left), poc C (right). E, Adult leaves (sixth node onwards) of 3-month-old wild type and poc mutant. In poc mutant, the leaf abnormalities vary from the simple lanceolate leaf to leaf with variable number of leaflets, reduction in size, and loss of lobing in leaflets. Wild type (upper and lower left), poc B (upper row), poc A (middle row), poc C (lower row). Wild-type leaves (upper and lower left). F, Epiphyllous structures (arrows) on poc leaves appearing near the junction of petiolule to the rachis. G, poc inflorescence (right) showing multiple blooming flowers with abnormal phyllotactic arrangement. H, poc flower (right) showing the absence of anther cone and shorter petals. I, poc petal (right) with an appendage on stamen-facing side (arrow). J, poc sepal (right) with a petal-like sector. Note the absence of trichomes (arrow). K, Twisted and short stamens of poc mutant lacking fusion to form anther cone. Wild type (left). L, Fusion of stamens of poc to the carpel. M, Appearance of a new inflorescence from inside of a fully differentiated flower of poc mutant (arrow).

Figure 2.

Comparison of the development of embryo in poc mutant (A, C, E, G, and I) and wild-type plants (B, D, F, H, and J). A and B, Globular stage 10 DAP. C and D, Late globular stage (13 DAP). E and F, Triangular/early heart stage (15 DAP). G and H, Late heart/torpedo stage (18 DAP). I and J, Magnification of medial epidermal cells from the right-hand margin region of embryos shown in E and F, respectively. Arrowheads, Cell boundaries in an epidermal cell file. Scale bars: A to D, 10 μm; E to G, 20 μm; and I and J, 5 μm.

Figure 3.

Scanning electron micrograph of epidermal cells of light-grown seedlings. A and B, One-week-old cotyledons showing that the poc (B) has smaller cells. C and D, One-week-old hypocotyls showing that poc (D) have shorter and less broad cells. Scale bars in A to D: 50 μm.

Figure 4.

Auxin polar transport in the presence or absence of TIBA. A, Auxin polar transport was measured using the method of Okada et al. (1991) in stem sections of 5-week-old light-grown plants. The basal end of stem segments was submerged in a solution containing [¹⁴C] IAA in an Eppendorf tube (Eppendorf Scientific, Westbury, NY) and after 4 h, a 5-mm section from non-submerged end of segments was excised, and the amount of radioactivity was determined. Error bars = se of five replicates. B, Auxin polar transport was measured using the method of Daniel et al. (1989) in stem sections of 4-week-old light-grown plants. The stem segments were sandwiched on glass microscopic slides between the receiver and donor blocks of agar, and the setup was placed vertically in a humid chamber. After 4 h of incubation, the amount of radioactivity was counted in the receiver blocks. Error bars = se of five replicates. C, Auxin efflux rate was measured by the retention of the amount of [¹⁴C] IAA in the hypocotyls of 3-week-old light grown plants. The cut segments of hypocotyls were floated on a solution containing [¹⁴C] IAA either with or without TIBA. Then, segments were incubated for another 2 h in the same buffer without IAA/TIBA and counted for radioactivity. Error bars = se of five replicates. D, Absorption spectrum of flavonoids extracted from stems of 4-week-old light-grown poc and wild-type plants.

Figure 5.

Hypocotyl and root growth of seedlings in the presence of TIBA and IAA. A, Morphology of 9-d-old dark-grown wild-type (a and c) and poc (b and d) seedlings in the presence (c and d) or absence (a and b) of TIBA (0.5 μm). B, Morphology of 9-d-old light-grown wild-type (a and c) and poc (b and d) seedlings in the presence (c and d) or absence (a and b) of TIBA (0.5 μm). C, Effect of various concentrations of TIBA on elongation of hypocotyl length of 10-d-old dark-grown poc seedlings compared with the wild type. D, Effect of various concentrations of TIBA on elongation of root lengths of 9-d-old light-grown poc seedlings compared with wild type. E, Effect of various concentrations of IAA on root elongation of 9-d-old light-grown wild-type seedlings compared with poc. F, Effect of IAA in promoting elongation of hypocotyl segments of 5-d-old dark-grown poc seedlings compared with wild type.

Figure 6.

Transverse sections showing the vasculature in hypocotyls of 15-d-old light-grown poc seedlings in the presence or absence of TIBA (10 μm). Sections were taken from the middle portion of hypocotyls and stained with safranine. A, Wild type; B, wild type + TIBA; C, poc mutant; D, poc mutant + TIBA. Note the difference in placement of the vascular bundles between mutant and wild type. Scale bars in A to D: 50 μm.

Figure 7.

Analysis of gravitropic response in seedlings. A, Kinetics of gravitropic response in the hypocotyls of 7-d-old light-grown wild-type and poc seedlings. Seedlings grown in plastic cuvettes were reoriented 90° relative to the gravity vector. The curvature was determined by photographing the seedlings at the given time intervals using a PC camera. B, Gravitropic response in the roots of 1-d-old light-grown wild-type and poc seedlings. Seedlings grown on vertical agar plates were reoriented 90° relative to the gravity vector. The curvature was determined by photographing the roots at the given time intervals using a PC camera.