Maternal control of integument cell elongation and zygotic control of endosperm growth are coordinated to determine seed size in Arabidopsis

Abstract

We use Arabidopsis thaliana as a model to investigate coordination of cell proliferation and cell elongation in the three components that develop side by side in the seed. Two of these, the embryo and its nurturing annex, the endosperm, are placed under zygotic control and develop within the seed integument placed under maternal control. We show that integument cell proliferation and endosperm growth are largely independent from each other. By contrast, prevention of cell elongation in the integument by the mutation transparent testa glabra2 (ttg2) restricts endosperm and seed growth. Conversely, endosperm growth controlled by the HAIKU (IKU) genetic pathway modulates integument cell elongation. Combinations of TTG2 defective seed integument with reduction of endosperm size by iku mutations identify integument cell elongation and endosperm growth as the primary regulators of seed size. Our results strongly suggest that a cross talk between maternal and zygotic controls represents the primary regulator of the coordinated control of seed size in Arabidopsis.

Figure 1.

Influence of ttg2 Mutation, iku Mutations, and p35S:KRP2 Construct and Their Combinations on Endosperm Development and Seed Size. In the wild-type Landsberg erecta (Ler), mature seed size is uniform (A). Confocal section showing the organization of the wild-type seed at the heart embryo stage with the integument (i) surrounding the endosperm (ed) and the embryo (eb) (E). We observe the effect on seed size ([B] to [D]) and on endosperm development ([F] to [H]) of the ttg2 mutation ([B] and [F]), of the iku2 mutation ([C] and [G]), and of the combination of ttg2 and iku2 ([D] and [H]). In all genetic backgrounds showed, embryogenesis is not affected up to the heart embryo stage, after which, a high proportion of ttg2/ttg2; iku2/iku2 seeds abort ([D], arrowheads). (I) and (K) show material obtained from a cross between p35S:KRP2/+ ovule and wild-type Ler pollen. (J) shows seeds resulting from the cross between p35S:KRP2/+; iku1/+ plants with iku1/iku1 pollen. Arrowheads show seeds with iku1/iku1 endosperm and embryo with a size close to that of iku2/iku2 seeds (C). (L) shows a confocal section of such seeds at the embryo heart stage. Bars = 500 μm for (A) to (D), (I), and (F) and 38 μm for (E) to (H), (K), and (L).

Figure 2.

Early Endosperm Phenotype of ttg2 and iku2 Mutations and Their Combination. Whole-mount cleared seeds observed with differential interference contrast (DIC) microscopy of seed at the triangular embryo stage in wild-type Ler containing uncellularized endosperm (A) and in ttg2/ttg2 containing precociously cellularized endosperm of reduced size (arrowhead shows endosperm cell wall) (B). Cleared seeds at the mid-globular embryo stage in wild-type Ler containing uncellularized endosperm (C), in iku2/iku2 containing uncellularized endosperm of reduced size (D), and in a iku2/iku2; ttg2/ttg2 double mutant seed containing very precociously cellularized endosperm with an increased reduction in size (arrowhead shows endosperm cell wall) (E). Bar = 50 μm for all photographs. Magnification of the inset is twice the magnification of the main micrograph.

Figure 3.

Seed Neomorphic Phenotype in the iku2/iku2; ttg2/ttg2 Double Mutant. Dry seeds of wild-type Ler (A), ttg2/ttg2 (B), iku2/iku2 (C), and double mutant iku2/iku2; ttg2/ttg2 showing precocious germination (D). Bar = 100 μm for (A) to (D).

Figure 4.

Evaluation of the Mitotic Activity in the Maternal Seed Integument by Expression of the pCycB1;2:GUS Construct. This experiment has been done on pistils pollinated 12 h after emasculation. (A) to (C) GUS staining of cleared developing seeds carrying pCycB1;2:GUS construct harvested at 0, 2, and 4 d after pollination (DAP), respectively. Bars = 20 μm for (A) and 40 μm for (B) and (C). (D) Variations of the number of integument cells expressing the pCycB1;2:GUS construct from 0 to 4 DAP. (0 DAP corresponds to the emasculation time 12 h before pollination.)

Figure 5.

Reduction of Cell Proliferation in the Integuments Is Compensated for by an Increased Integument Cell Elongation Irrespective of the Presence of the iku1 Mutation. (A) Comparison of the cell number in the integument and the integument cell length between wild-type Ler seeds (blue bar) and a cross between p35S:KRP2/+ ovule and wild-type Ler pollen (red bar). Reduction of cell proliferation in the integument by overexpression of KRP2 in the integument is counterbalanced by integument cell elongation (A), leading to endosperm and seed sizes close to the wild type (Figures 1I and 1K). (B) Comparison of the cell number in the integument between iku1/iku1 seeds (blue bar) and seeds containing iku1/iku1 endosperm selected from a cross between p35S:KRP2/+; iku1/+ ovule and iku1/iku1 pollen (blue bar; [iku] means of iku phenotype). Compensation of the decreased integument cell proliferation by an increased integument cell elongation is observed as well when endosperm growth is reduced by iku1 (B), leading to endosperm and seed sizes close to iku1 (Figures 1J and 1L). (A) and (B) Compare the number of cells in the innermost layer (endothelium) of the integument with their length measured using confocal sections similar to those presented in Figure 1.

Figure 6.

Model of Seed Size Control in Arabidopsis. Seed size determination involves the zygotic control of endosperm growth by IKU class genes in addition to the maternal modulation of integument cell elongation by TTG2. These two pathways are integrated by a cross talk (blue arrow) and determine the final potential size of the seed. The integument cell number is regulated during the early phase of integument cell proliferation. However, as integument cell elongation compensates the cell number (red arrow) to accommodate to the endosperm size, integument cell proliferation does not have a strong influence on seed size.