Diphtheria Toxin-Mediated Cell Ablation Reveals Interregional Communication during Arabidopsis Seed Development

Abstract

Fertilization of the female gametophyte in angiosperm plants initiates a process of coordinated development of embryo, endosperm, and seed coat that ensures the production of a viable seed. Mutant analysis has suggested that communication between the endosperm and the seed coat is an important determinant in this process. In addition, cell groups within the embryo, derived from the apical and from the basal cell, respectively, after zygote division, concertedly establish a functional root meristem, and cells in the apical region of the embryo are hypothesized to repress cell divisions in the basal cell-derived suspensor. The available evidence for these interregional communication events mostly relies on the analysis of mutant phenotypes in Arabidopsis. To provide independent and direct evidence for communication events, we used conditional domain-specific expression of the diphtheria toxin A chain (DTA) in developing Arabidopsis seeds. By using a collection of cell-or tissue-type-specific promoters, we show that the mGAL4:VP16/UAS two-component gene expression allows reliable spatiotemporal and conditional expression of the GFP:GUS reporter and the DTA gene in the developing embryo and endosperm. Expression of DTA in the protoderm of the embryo proper led to excessive proliferation of suspensor cells, sometimes resulting in the formation of secondary embryos. Endosperm-specific expression of DTA caused complete cessation of seed growth, followed by pattern defects in the embryo and embryo arrest. Taken together, the results presented here substantiate the evidence for and underline the importance of interregional communication in embryo and seed development and demonstrate the usefulness of conditional toxin expression as a method complementary to phenotypic analysis of developmental mutants.Seed development in higher plants is characterized by the coordinated development of distinct tissues. Seed tissues mainly arise from cells and tissues of the female gametophyte that are formed before fertilization. The multinucleate female gametophyte is enclosed by several sporophytic maternal cell layers that constitute the ovule. In angiosperms, double fertilization by fusion of the egg cell and the central cell of the female gametophyte with the two sperm cells delivered by the pollen tube generates the diploid embryo and triploid endosperm, respectively. The embryo and endosperm both develop within the confines of the maternal tissue, now referred to as the seed coat, but each follows a different developmental program (for review on embryo and endosperm development, see Jü rgens and Mayer, 1994; Berger, 2003, respectively). Within the embryo, a complex but precise pattern of organs and cell types is laid down from a single cell, the zygote (Jü rgens and Mayer, 1994). Genetic controls are required to establish the embryo pattern and to ensure the proper initiation and relative positioning of distinct cell groups and organs, such as the meristems and vasculature. The endosperm first undergoes a series of synchronized ...

Direct evidence that suspensor cells have embryogenic potential that is suppressed by the embryo proper during normal embryogenesis

“…In addition, some available data are contradictory. Expressing LTP1::DTA (LTP1, lipid transfer protein 1; DTA, diphtheria toxin A chain) in the protoderm domain of embryos (21) to injure the embryo protoderm can induce excessive divisions in the suspensor (22). However, expression of the LTP1-driven BARNASE (Bacillus amyloliquefaciens ribonuclease) gene in the embryo, which results in defects in the basal tier of the embryo and hypophysis, leads to no signs of deregulation of suspensor development (23).…”

The Female Gametophyte and the Endosperm Control Cell Proliferation and Differentiation of the Seed Coat inArabidopsis

“…No significant growth of the seed coat is observed in seeds in which only the egg cell is fertilized as a result of the production of a single sperm cell in A-type cyclin-dependent kinase1 mutant pollen (Nowack et al, 2006). Similarly, ablation of endosperm in developing seeds results in extremely reduced seed coat development (Weijers et al, 2003). Thus, we propose that fertilization of the egg cell and consequent embryo development may not play a major role in seed coat growth.…”

“…The major phase of seed growth parallels the initial endosperm growth characterized by rapid successive rounds of mitoses devoid of cytokinesis (syncytial development) (Garcia et al, 2005). Specific production of diphtheria toxin A in the developing endosperm caused a rapid arrest of seed growth (Weijers et al, 2003). Genetic analyses of the HAIKU pathway revealed that reduced syncytial endosperm growth prevents cell elongation in the seed coat, leading to smaller seeds (Garcia 1 To whom correspondence should be addressed.…”

Natural Variation in the Degree of Autonomous Endosperm Formation Reveals Independence and Constraints of Embryo Growth During Seed Development in Arabidopsis thaliana

“…Interestingly, such checkpoint behavior was previously not found in plants in which the endosperm was genetically ablated, and Weijers et al (2003) found that embryogenesis could continue with very little endosperm although these embryos would eventually arrest. The driver line used for these ablation experiments becomes expressed 2 DAP, and a possible explanation is that this expression is already later than a putative seed size checkpoint.…”