Fertilization-independent seed development in Arabidopsis thaliana

Abstract

We report mutants in Arabidopsis thaliana (fertilization-independent seed:fis) in which certain processes of seed development are uncoupled from the double fertilization event that occurs after pollination. These mutants were isolated as ethyl methanesulfonate-induced pseudo-revertants of the pistillata phenotype. Although the pistillata (pi) mutant has short siliques devoid of seed, the fis mutants in the pi background have long siliques containing developing seeds, even though the flowers remain free of pollen. The three fis mutations map to loci on three different chromosomes. In fis1 and fis2 seeds, the autonomous endosperm nuclei are diploid and the endosperm develops to the point of cellularization; the partially developed seeds then atrophy. In these two mutants, proembryos are formed in a low proportion of seeds and do not develop beyond the globular stage. When FIS/fis plants are pollinated by pollen from FIS/FIS plants, approximately 50% of the resulting seeds contain fully developed embryos; these seeds germinate and form viable seedlings (FIS/FIS). The other 50% of seeds shrivel and do not germinate; they contain embryos arrested at the torpedo stage (FIS/fis). In normal sexual reproduction, the products of the FIS genes are likely to play important regulatory roles in the development of seed after fertilization.

Figure 1

Pollination-independent silique and seed development in a FIS/fis mutant plant. (A) Autonomous silique and seed development of a pi/pi FIS3/fis3 plant; unpollinated (B) and pollinated (C) silique from a pi/pi FIS/FIS plant also are shown. Note the comparable development of fertilization-independent seed (A, arrow) and sexually developed seed (C, arrow). (Bar = 1 mm.)

Figure 2

Cryo-scanning electron microscopy micrographs of ovules and seeds of fis mutants and fertilized wild-type plants. Developing ovules [nucellar column (n) protruding from the inner integument (ii) and the outer integument (oi) as shown in B] of (A) wild-type, (B) fis1/fis1 homozygote, (C) fis2/fis2 homozygote, and (G) FIS3/fis3 heterozygote. (D) Sexually fertilized seeds(s) of pi/pi FIS/FIS plants 7 days after fertilization. Unfertilized ovules shrivel (arrow). Seeds developing without fertilization(s) of (E) fis1/fis1 homozygote, (F) fis2/fis2 homozygote, and (H) FIS3/fis3 heterozygote. Collumella (c) on the surface of (I) sexually fertilized seed of wild type and (J) autonomously developing fis2 homozygous seeds. (Bar = 20 μm for A–C, G, I, and J, 100 μm for D–F, and 200 μm for H.)

Figure 3

Endosperm and embryo development of wild-type and fis seeds. (A) Longitudinal section through an ovule of an unfertilized pistillata (pi/pi) mutant. No endosperm or embryo development is visible. (B) Longitudinal section through an ovule of a 0.6-mm fruit from a pi/pi homozygote 3 days after fertilization with FIS/FIS pollen showing embryo (arrow). (C and D) Sections through an autonomously developing fis1 seed from a FIS1/fis1 heterozygous plant (from a 0.8-mm fruit). Notice cellularized endosperm (CE) and the embryo-like structure (D, arrow). (E) Section through an autonomously developing fis2 seed from a FIS2/fis2 heterozygote showing cellularized endosperm (CE) and a zygote-like structure (arrow) (from a 0.8-mm fruit). (F) Section through an autonomously developing fis3 seed from a 0.9-mm fruit from a FIS3/fis3 heterozygote showing free nuclear endosperm (arrowheads). (G and H) Autonomous endosperm development in a fis1/fis1 homozygote showing free nuclear coenocytic endosperm (CnE) (from a 1-mm fruit). (I) Embryo arrested at the globular stage 5 days after pollination of a fis1/fis1 homozygous plant with FIS/FIS pollen (arrow) (from a 1.2-mm fruit). (Bar = 50 μm for A–F and I and 100 μm for G and H.)

Figure 4

Pollination-induced silique and embryo development in a FIS1/fis1 heterozygote and a fis1/fis1 homozygote. (A) Torpedo stage Gus-positive (blue) embryos resulting from the pollination of a pi/pi FIS/FIS homozygous plant with a plant homozygous for a ³⁵S-Gus construct. (B) Torpedo stage Gus-positive (blue) and Gus-negative (white, arrow) embryos from the pollination of a fis1/fis1 homozygote with pollen from plants carrying a ³⁵S-Gus construct. (C) Silique development after self-pollination of (1) fis1/fis1, (2) FIS1/fis1, and (3) FIS1/FIS1 plants.

Figure 5

Wild-type and fis seed development. Seed development of wild-type Arabidopsis and fis mutants are compared at developmental phases (14). Phase 1 shows ovules connected to the ovary wall by the funiculus; in the subsequent phases, only the developing seed is shown. The relative size of the ovule compared with the developing seed is shown by the Inset. The lengths of siliques at the different phases are: phase 1: 0.29 ± 0.04 mm (0 HAF); phase 2: 0.60 ± 0.08 mm (36 HAF); phase 3: 1.00 ± 0.07 mm (72 HAF); and phase 4 1.26 ± 0.07 mm (120 HAF). a, b, and c represent different developmental types seen in the fis mutants. X, Y, and Z represent postulated genes other than FIS1, FIS2, and FIS3. A detailed description of the model is given in Discussion.