Genetic subtraction profiling identifies genes essential for Arabidopsis reproduction and reveals interaction between the female gametophyte and the maternal sporophyte

Open Access et al. Johnston 2007 Volume 8, Issue 10, Article R204 Research Genetic subtraction profiling identifies genes essential for Arabidopsis reproduction and reveals interaction between the female gametophyte and the maternal sporophyte Amal J Johnston¤*‡, Patrick Meier¤*, Jacqueline Gheyselinck*, Samuel EJ Wuest*, Michael Federer*, Edith Schlagenhauf*, Jörg D Becker† and Ueli Grossniklaus* Addresses: *Institute of Plant Biology and Zürich-Basel Plant Science Center, Zollikerstrasse, University of Zürich, CH-8008 Zürich, Switzerland. †Centro de Biologia do Desenvolvimento, Instituto Gulbenkian de Ciência, Rua da Quinta Grande, PT-2780-156 Oeiras, Portugal. ‡Current address: Institute of Plant Sciences and Zürich-Basel Plant Science Center, ETH Zürich, Universitätstrasse, CH-8092 Zürich, Switzerland. ¤ These authors contributed equally to this work. Correspondence: Ueli Grossniklaus. Email: Published: 3 October 2007 Genome Biology 2007, 8:R204 (doi:10.1186/gb-2007-8-10-r204) Received: 9 February 2007 Revised: 10 September 2007 Accepted: 3 October 2007 The electronic version of this article is the complete one and can be found online at http://genomebiology.com/2007/8/10/R204 © 2007 Johnston et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. tified Expression <p>Genetic embryo 1,260 sac.</p> profiling genes subtraction expressed genes andessential in expression the embryo for profiling plant sac; reproduction a of total wild-type of 527 genes <it>Arabidopsis were identified </it>and for their a sporophytic expressionmutant in ovules lacking of mutants an embryo lacking sacan iden- Abstract Background: The embryo sac contains the haploid maternal cell types necessary for double fertilization and subsequent seed development in plants. Large-scale identification of genes expressed in the embryo sac remains cumbersome because of its inherent microscopic and inaccessible nature. We used genetic subtraction and comparative profiling by microarray between the Arabidopsis thaliana wild-type and a sporophytic mutant lacking an embryo sac in order to identify embryo sac expressed genes in this model organism. The influences of the embryo sac on the surrounding sporophytic tissues were previously thought to be negligible or nonexistent; we investigated the extent of these interactions by transcriptome analysis. Results: We identified 1,260 genes as embryo sac expressed by analyzing both our dataset and a recently reported dataset, obtained by a similar approach, using three statistical procedures. Spatial expression of nine genes (for instance a central cell expressed trithorax-like gene, an egg cell expressed gene encoding a kinase, and a synergid expressed gene encoding a permease) validated our approach. We analyzed mutants in five of the newly identified genes that exhibited developmental anomalies during reproductive development. A total of 527 genes were identified for their expression in ovules of mutants lacking an embryo sac, at levels that were twofold higher than in the wild type. Conclusion: Identification of embryo sac expressed genes establishes a basis for the functional dissection of embryo sac development and function. Sporophytic gain of expression in mutants lacking an embryo sac suggests that a substantial portion of the sporophytic transcriptome involved in carpel and ovule development is, unexpectedly, under the indirect influence of the embryo sac. Genome Biology 2007, 8:R204 http://genomebiology.com/2007/8/10/R204 Genome Biology 2007, Background The life cycle of plants alternates between diploid (sporophyte) and haploid (male and female gametophytes) generations. The multicellular gametophytes represent the haploid phase of the life cycle between meiosis and fertilization, during which the gametes are produced through mitotic divisions. Double fertilization is unique to flowering plants; the female gametes, namely the haploid egg cell and the homodiploid central cell, are fertilized by one sperm cell each. Double fertilization produces a diploid embryo and a triploid endosperm, which are the two major constituents of the developing seed [1]. The egg, the central cell, and two accessory cell types (specifically, two synergid cells and three antipodal cells) are contained in the embryo sac, also known as the female gametophyte or megagametophyte, which is embedded within the maternal tissues of the ovule. As a carrier of maternal cell types required for fertilization, the embryo sac provides an interesting model in which to study a variety of developmental aspects relating to cell specification, cell polarity, signaling, cell differentiation, double fertilization, genomic imprinting, and apomixis [1-3]. Out of the 28,974 predicted open reading frames of Arabidopsis thaliana, a few thousand genes are predicted to be involved in embryo sac development [1,4]. These genes can be grouped into two major classes: genes that are necessary during female gametogenesis and genes that impose maternal effects through the female gametophyte, and thus play essential roles for seed development. To date, loss-of-function mutational analyses have identified just over 100 genes in Arabidopsis that belong to these two classes [5-14]. However, only a small number of genes have been characterized in depth. Cell cycle genes (for instance, PROLIFERA, APC2 [ANAPHASE PROMOTING COMPLEX 2], NOMEGA, and RBR1 [RETINOBLASTOMA RELATED 1]), transcription factors (for instance, MYB98 and AGL80 [AGAMOUS-LIKE80]), and others (including CKI1 [CYTOKININ INDEPENDENT 1], GFA2 [GAMETOPHYTIC FACTOR 2], SWA1 [SLOW WALKER 1] and LPAT2 [LYSOPHOSPHATIDYL ACYLTRANSFERASE 2]) are essential during embryo sac development [6,15-23]. Maternal effect genes include those of the FIS (FERTILIZATION INDEPENDENT SEED) class and many others that are less well characterized [9,13,24]. FIS genes are epigenetic regulators of the Polycomb group and control cell proliferation during endosperm development and embryogenesis [7,10,12,25,26]. Ultimately, the molecular components of cell specification and cell differentiation during megagametogenesis and double fertilization remain largely unknown, and alternate strategies are required for a highthroughput identification of candidate genes expressed during embryo sac development. Although transcriptome profiling of Arabidopsis floral organs [27,28], whole flowers and seed [29], and male gametophytes [30-33] have been reported in previous studies, large-scale identification of genes expressed during female Volume 8, Issue 10, Article R204 Johnston et al. R204.2 gametophyte development remains cumbersome because of the microscopic nature of the embryo sac. Given the dearth of transcriptome data, we (...truncated)