Induction of monozygotic twinning by ascorbic acid in tobacco

Abstract

Embryo development in plants initiates following the transverse division of a zygote into an apical, proembryo cell and a basal cell that gives rise to the suspensor. Although mutants affected in embryo development through changes in cell division have been described, little is known about the control of the first zygotic division that gives rise to the proembryo. Ascorbic acid (Asc) promotes cell division by inducing G(1) to S progression but its role in embryo development has not been examined. In this study, we show that the level of dehydroascorbate reductase (DHAR) expression, which recycles Asc and regulates Asc pool size, affects the rate of monozygotic twinning and polycotyly. DHAR-induced twinning resulted from altered cell polarity and longitudinal instead of transverse cell division that generated embryos of equal size. Direct injection of Asc into ovaries phenocopied DHAR-induced twinning. Twinning induced by Asc was developmentally limited to the first two days after pollination whereas polycotyly was induced when the level of Asc was elevated just prior to cotyledon initiation. This work describes the first example of gene-directed monozygotic twinning and shows that Asc regulates cell polarity during embryo development.

Figure 1. Ascorbate-induced twinning and polycotyly.

A–F, Twin progeny emerging from DHAR-overexpressing tobacco seed. G–H, Triplet progeny emerging from DHAR-overexpressing tobacco seed. I–J, Twin progeny emerging from WT seed that developed from ovaries injected with 10 mM Asc on the day of pollination. K–N, Tricotyledon progeny from DHAR-overexpressing tobacco seed in various states of fusion. O–P, Tetracotyledon progeny from DHAR-overexpressing tobacco seed. Q–T, Monocotyledon progeny from DHAR-overexpressing tobacco seed where (T) the fused cotyledon was cleared of chlorophyll to reveal two main veins and plane of fusion. U, Wild-type seedling cleared of chlorophyll.

Figure 2. Frequency of ascorbate-induced twinning and polycotyly.

Frequency of twinning and polycotyly resulting from crosses between WT and DHAR-overexpressing tobacco. Crosses between wild-type (WT) and tobacco transformed with wheat DHAR (D_Ta) or tobacco DHAR (D_Nt) were performed. The histograms represent the frequency of twinning (black bars, left scale) and cotyledon abnormalities (i.e., polycotyly or single fused cotyledon)(white bars, right scale) measured in the resulting seed.

Figure 3. DHAR activity in leaves and floral organs of D_Ta, D_Nt, and WT plants.

DHAR activity was measured in leaves or the indicated floral organs in DHAR-overexpressing (D_Ta), DHAR-overexpressing (D_Nt), and wild-type (WT) plants.

Figure 4. Analysis of the genetic relationship among twin and triplet progeny.

(A) Inheritance of the D_Ta or D_Nt transgenes when transmitted through pollen (m) or egg (f) homozygous for the transgene. Inheritance of the D_Nt transgene in twin (Tw) and triplet (Trp) progeny from D_Nt pollen (D_Nt-m) when used to fertilize (1) D_Ta or (2) WT flowers (WT-f). Inheritance of the D_Ta transgene in twin and triplet progeny from D_Ta pollen (D_Ta-m) when used to fertilize (3) D_Nt or (4) WT flowers. (B) Inheritance of the D_Ta or D_Nt transgenes when transmitted through pollen (m) or egg (f) hemizygous for each transgene. (1) Inheritance of the D_Nt and D_Ta transgenes in twin and triplet progeny from eggs hemizygous for the D_Ta and D_Nt transgenes (D_Ta/D_Nt-f) when fertilized by WT pollen (WT-m). (2) Inheritance of the D_Nt and D_Ta transgenes in twin and triplet progeny from pollen hemizygous for the D_Ta and D_Nt transgenes (D_Ta/D_Nt-m) when used to fertilize WT flowers. (3) Inheritance of the D_Nt transgene in twin and triplet progeny from pollen hemizygous for D_Nt transgene present in 2 copies in the genome (D_{Nt(2 copies)}-m) when used to fertilize D_Ta flowers (D_Ta-f). p: PCR of tobacco containing the appropriate DHAR transgene to serve as a positive control. n: PCR of wild type tobacco to serve as a negative control.

Figure 5. Development of twin and triplet embryos in polyembryonic seed.

A–D, Developing WT zygotes showing growth prior to the first zygotic division. E–H, Developing zygotes in D_Ta ovules showing (E) horizontal position of nucleoli prior to the first zygotic division, (F) presence of twin zygotes in adjacent proximity with vertical position of nucleoli in one of the zygotes, (G) presence of twin zygotes in adjacent proximity with vertical position of nucleoli in both zygotes, (H) presence of triplet zygotes. I–J, Developing embryos at 5 DAP from (I) WT and (J) D_Ta plants showing twin proembryos at the same developmental stage. K–L, Developing embryos at 6 DAP from (K) WT and (L) D_Ta plants. Cells of the protoderm are evident in the embryo proper of the WT embryo not present in the twin D_Ta embryos as the latter are delayed in their development. M–N, Developing embryos at 8 DAP from (M) WT and (N) D_Ta plants. O–Q, 10 DAP developing embryos from (O) WT and (P) D_Ta-double and (Q) D_Ta-triple embryo seed. R–T, 12 DAP developing embryos from (R) WT and (S) D_Ta-double and (T) D_Ta-triple embryo seed. ac, apical cell; b, basal cell; e, embryo; ep, embryo proper; p, protoderm; s, suspensor; zy, zygote. Scale bars represent 50 µm.

Figure 6. Twinning is induced by Asc or compounds involved in Asc biosynthesis or recycling.

Frequency of twinning and polycotyly in progeny of WT ovaries injected with (A) Asc, GL, dehydroascorbate (DHA), glutathione (GSH), H₂O₂, or malate at the concentrations indicated on the day of pollination. The histograms represent the frequency of twinning (black bars, left scale) and cotyledon abnormalities (i.e., polycotyly or single fused cotyledon)(white bars, right scale) observed following germination.

Figure 7. Hormonal control of twinning.

Frequency of twinning and polycotyly in progeny of WT ovaries injected with gibberellic acid (GA₃), indole acetic acid (IAA), aminoethoxyvinylglycine (AVG), 2-chloroethylphosphonic acid (CEPA) that produces ethylene, or the cytokinin, benzyladenine (BA) at the concentrations indicated on the day of pollination. The histograms represent the frequency of twinning (black bars, left scale) and cotyledon abnormalities (i.e., polycotyly or single fused cotyledon)(white bars, right scale) observed following germination.

Figure 8. Determination of the developmental stage receptive to Asc-induced twinning and polycotyly.

5 mM Asc (white bars) or GL (black bars) was injected into WT ovaries the day of pollination or on days after pollination as indicated. A, The frequency of twinning and (B) polycotyly observed in the resulting progeny. The horizontal line represents the spontaneous frequency of polyembryony or cotyledon abnormalities.