Molecular dissection of apomixis – an asexual reproductive mode – is anticipated to solve the enigma of loss of meiotic sex, and to help fixing elite agronomic traits. The Brassicaceae genus Boechera comprises of both sexual and apomictic species, permitting comparative analyses of meiotic circumvention (apomeiosis) and parthenogenesis. Whereas previous studies reported local transcriptome changes during these events, it remained unclear whether global changes associated with hybridization, polyploidy and environmental adaptation that arose during evolution of Boechera might serve as (epi)genetic regulators of early development prior apomictic initiation. To identify these signatures during vegetative stages, we compared seedling RNA-seq transcriptomes of an obligate triploid apomict and a diploid sexual, both isolated from a drought-prone habitat. Uncovered were several genes differentially expressed between sexual and apomictic seedlings, including homologs of meiotic genes ASYNAPTIC 1 (ASY1) and MULTIPOLAR SPINDLE 1 (MPS1) that were down-regulated in apomicts. An intriguing class of apomict-specific deregulated genes included several NAC transcription factors, homologs of which are known to be transcriptionally reprogrammed during abiotic stress in other plants. Deregulation of both meiotic and stress-response genes during seedling stages might possibly be important in preparation for meiotic circumvention, as similar transcriptional alteration was discernible in apomeiotic floral buds too. Furthermore, we noted that the apomict showed better tolerance to osmotic stress in vitro than the sexual, in conjunction with significant upregulation of a subset of NAC genes. In support of the current model that DNA methylation epigenetically regulates stress, ploidy, hybridization and apomixis, we noted that ASY1, MPS1 and NAC019 homologs were deregulated in Boechera seedlings upon DNA demethylation, and ASY1 in particular seems to be repressed by global DNA methylation exclusively in the apomicts. Variability in stress and transcriptional response in a diploid apomict, which is geographically distinct from the triploid apomict, pinpoints both common and independent features of apomixis evolution. Our study provides a molecular frame-work to investigate how the adaptive traits associated with the evolutionary history of apomicts co-adapted with meiotic gene deregulation at early developmental stage, in order to predate meiotic recombination, which otherwise is thought to be favorable in stress and low-fitness conditions.

35The multicellular embryo, and ultimately the entire organism, is a derivative of the fertilized 36 egg cell. Unlike in animals, transcription factor networks orchestrating faithful egg 37 development are still largely unknown in plants. We have identified that egg cell 38 differentiation in Arabidopsis require interplay between evolutionarily conserved onco-protein 39 homologs RETINOBLASTOMA-RELATED (RBR) and redundant MYB proteins 40 MYB64/MYB119. RBR physically interacts with the MYBs; and with plant-specific 41 transcription factors belonging to the RWP-RK-domain (RKD) family and LEAFY 42 COTYLEDON1 (LEC1), which participate in development of egg cells and inherent stress 43 response. RBR binds to most of these egg cell-expressed loci at the DNA level, partially 44 overlapping with sites of histone methylation H3K27me3. Since deregulation of RKDs 45 phenocopies mutants of RBR and the MYBs in terms of cell proliferation in the egg cell 46 spatial domain, all the corresponding proteins are likely required to restrict parthenogenetic 47 cell divisions of the egg cells. Cross-talk among these transcription factors, and direct 48 regulation by RBR, govern egg cell development and expression of egg-to-zygotic polarity 49 factors of the WUSCHEL RELATED HOMEOBOX family. Together, a network of RBR-50 centric transcription factors underlies egg cell development and stress response, possibly, in 51 combination with several other predicted nodes. 52 53 54 55 Key words 56 egg cell | transcription factor | RETINOBLASTOMA RELATED | MYB | RKD | stress | 57 parthenogenesis 58 59 4 Author summary 60 61The RETINOBLASTOMA protein is one of the core components of the Eukaryotic 62 cell cycle, and corresponding evolutionary homologs have been implicated not only 63 to repress cell division but also to control differentiation and development. How 64 RETINOBLASTOMA RELATED (RBR) associate with other higher order regulators 65 to control faithful egg cell development in sexual plants is pivotal for manipulation of 66 successful reproduction in general, and engineering of parthenogenesis when 67 asexual or apomictic seed progeny are desirable over sexual plants. Using a suite of 68 molecular methods, we show that a RBR-associated transcription factor network 69 operates to specify egg cells in Arabidopsis. Complex cross-regulation within these 70 transcription factors seems to be necessary for successful maternal egg cell to 71 zygotic transition and reproductive stress response. Detailed genetic analysis 72 implicate that RBR and its interactive partners belonging to MYB and RWP-RK 73 transcription factor families are possibly required to prevent parthenogenesis of the 74 sexual egg cells. Novel RBR networks and stress nodes explained in this study 75 might help to improve our understanding of sexual and asexual reproduction. 76 77 78 79 80 Proper differentiation of the egg cells is pivotal for sexual reproduction as well as 81 parthenogenesis. In flowering plants, the egg cells are terminally differentiated within 82 the miniature ...