Functional Analysis of Sporophytic Transcripts Repressed by the Female Gametophyte in the Ovule of Arabidopsis thaliana

Vielle-Calzada J-P (2013) Functional Analysis of Sporophytic Transcripts Repressed by the Female Gametophyte in the Ovule of Arabidopsis thaliana. PLoS ONE 8(10): e76977. doi:10.1371/journal.pone.0076977 Functional Analysis of Sporophytic Transcripts Repressed by the Female Gametophyte in the Ovule of Arabidopsis thaliana Alma Armenta-Medina 0 Wilson Huanca-Mamani 0 Nidia Sanchez-Leo n 0 Isaac Rodrguez-Are valo 0 Jean-Philippe Vielle-Calzada 0 Hector Candela, Universidad Miguel Hernandez de Elche, Spain 0 Grupo de Desarrollo Reproductivo y Apomixis, Laboratorio Nacional de Geno mica para la Biodiversidad y Departamento de Ingeniera Genet ica de Plantas, CINVESTAV Irapuato , Irapuato , Mexico To investigate the genetic and molecular regulation that the female gametophyte could exert over neighboring sporophytic regions of the ovule, we performed a quantitative comparison of global expression in wild-type and nozzle/sporocyteless (spl) ovules of Arabidopsis thaliana (Arabidopsis), using Massively Parallel Signature Sequencing (MPSS). This comparison resulted in 1517 genes showing at least 3-fold increased expression in ovules lacking a female gametophyte, including those encoding 89 transcription factors, 50 kinases, 25 proteins containing a RNA-recognition motif (RRM), and 20 WD40 repeat proteins. We confirmed that eleven of these genes are either preferentially expressed or exclusive of spl ovules lacking a female gametophyte as compared to wild-type, and showed that six are also upregulated in determinant infertile1 (dif1), a meiotic mutant affected in a REC8-like cohesin that is also devoided of female gametophytes. The sporophytic misexpression of IOREMPTE, a WD40/transducin repeat gene that is preferentially expressed in the L1 layer of spl ovules, caused the arrest of female gametogenesis after differentiation of a functional megaspore. Our results show that in Arabidopsis, the sporophytic-gametophytic cross talk includes a negative regulation of the female gametophyte over specific genes that are detrimental for its growth and development, demonstrating its potential to exert a repressive control over neighboring regions in the ovule. Current address; Laboratorio de Biotecnologa Vegetal; Facultad de Ciencias Agrono micas; Universidad de Tarapaca; Arica; Chile - Funding: This work was financially supported by grants from Consejo Nacional de Ciencia y Tecnologa, Consejo Estatal de Ciencia y Tecnologa de Guanajuato, the Howard Hughes Medical Institute, and the UC-MEXUS initiative. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. The ovules of flowering plants are most often formed as elongated primordia emerging from the inner surface of the young carpel, with the integument mounds initiating from periclinal divisions of the epidermal layer [1,2,3,4]. A differentiated ovule is composed of a nucellus and two integuments, and is usually attached by a funiculus to the placental tissue of the gynoecia. The integuments grow to progressively envelop the nucellus; by converging at the apex of the differentiated ovule, they form the micropyle, an extracellular narrow canal through which a pollen tube reaches the female gametophyte to deliver the sperm cells during double fertilization [5]. Through the funiculus, a vascular bundle extends from the placenta to the chalazal region. During early nucellar formation, a single meiocyte undergoes meiosis before giving rise to four haploid products, initiating the gametophytic generation and the formation of the female gametophyte [6]. In Arabidopsis thaliana, the megaspore mother cell (MMC) differentiates sub-epidermally, and undergoes meiosis to differentiate a single functional megaspore that divides mitotically to form a female gametophyte composed of the egg cell, two synergids, three antipodals at the chalazal region, and a binucleated central cell whose nuclei fuse prior to fertilization. Following double fertilization, the egg cell and the central cell give rise to the embryo and the endosperm respectively; while the function of synergids is to attract the pollen tube, the function of the antipodals remains unknown. In Arabidopsis, the investigation of the genetic basis and molecular mechanisms that regulate gametophytic development has recently benefited from large-scale transcriptional analysis and cell-specific isolation methods that allow gene expression comparisons between wild-type and mutant ovules lacking a differentiated female gametophyte, such as coatlicue (coa), nozzle/sporocyteless (spl), and determinant infertile1 (dif1) [7,8,9,10,11], Whereas the molecular nature of the gene affected in coa remains unknown, SPL/NZZ encodes a MADS-like transcriptor factor [12,13], and DIF1/SYN1 encodes a meiotic homologue of the Schizosaccharomyces pombe REC8/RAD21 cohesin gene [14,15]. Because distinct mutant phenotypes prevail in the ovules used for each of these experiments, a widely diverse collection of differentially expressed transcripts has been identified, likely due to deregulation of nonequivalent gene collections [11]. Several transcriptomes of distinct cell types of the wild-type female gametophyte are also available [16,17,18], allowing direct comparison of gene expression in gametophytic cells and their precursors. Unlike mature pollen, the differentiated female gametophyte maintains a tight physical contact with the maternal sporophyte that contributes to its protection and nourishment throughout its development [4]. Numerous pleiotropic effects caused by recessive mutations acting at the sporophytic level suggest a cross talk involving genetic and molecular factors that link integumentary and nucellar development to early stages of female gametophyte formation. For instance, Arabidopsis individuals defective in BELL1, AINTEGUMENTA, INNER NO OUTER and ABERRANT TESTA SHAPE show a variety of sporophytic defects all detrimental to the formation of the female gametophyte, suggesting a tight control of the sporophyte over the gametophyte [1,19,20,21]. Additional studies in other species showed that the maternal control of sporophytic tissues over the gametic precursor cells initiates early during ovule formation, before MMC differentiation. In rice, the leucine-rich repeat receptor-like kinase MULTIPLE SPOROCYTE1 (MSP1) prevents somatic cells to enter gametogenesis [22], whereas Oryza sativa TAPETUM DETERMINANT1 (OsTDL1A) controls gametic cell specification [23]. On the contrary, no evidence suggests that the female gametophyte could have an influence on the development or physiology of sporophytic cells. Except for the recurrent presence of persistent nucellar cells that are normally reabsorbed by the growing female gametophyte within the inner integumentary region, most gametophytic mutants do not appear to affect the morphology of the differ (...truncated)