**4. Genes involved in polarity establishment and organ initiation are expressed in the embryo**

Embryogenesis is an important aspect of seed development, which involves cellular division and establishment of the embryo body plan. The process of embryo development is classified into ten major stages in rice. The first stage is the zygotic stage formed at 0 days after fertilization (DAP). The initial stages of post-zygotic development are characterized by the repeated cellular division without morphogenesis. This leads to the formation of a globular embryo, which can be observed from 1 to 3 DAP. It is the stage at which embryo specifies its body axis. Subsequently, formation and proper positioning of the shoot apical meristem (SAM), coleoptile primordium and the radicle primordium occurs at 4 DAP. This is followed by the origin of leaf primordium, which initiates at 5 DAP and is completed by 8 DAP, by the formation of all the three leaf primordia. With the enlargement of various organs, embryo morphogenesis is completed in rice by 10 DAP. This is followed by a stage of maturation which lasts till about 20 DAP, and thereafter, it enters into a dormant state [4, 5]. Profiling the RNA expression levels has greatly broadened the understanding of rice embryo development. Unlike animal embryos, both maternal and paternal genomes contribute equally in the process of embryogenesis and the plant embryonic development is majorly under zygotic control. Maternal-tozygotic transition (MZT) initiates 50 hours after fertilization [24–26]. Thus, zygotic genome gets "switched on" almost immediately after fertilization.

Microarrays coupled with laser microdissection (LMD) have made available the expression profiles of developing female gametophyte from the pre-meiotic to the mature embryo-sac stages in rice [27]. Similarly, expression profiles of stigma as well as embryo sac are also available from high-throughput RNA-Seq technology [28]. These cell-type specific expression analyses have been compared with various developmental stages of embryo to identify genes that are differentially expressed during the post fertilization period. Identification of such genes expands our idea of embryogenesis as many of them can be candidates for maintaining various stages or aspects of embryo morphogenesis. Microarray-based comparison of gametic and zygotic tissues has identified a total of 325 genes up regulated in zygote in comparison with the egg cell. Majority of these up regulated genes are involved in DNA/chromatin organization and their assembly is probably involved in the induction of genes participating in zygotic development. Further, methyltransferase 1 (MET1) and different TFs belonging to the homeobox proteins are highly up regulated in the zygote, apparently affecting polarity or asymmetric division in zygote. Specific METs show higher expression during early rice seed development and are essential for cytosine methylation, regulating the genes involved in various developmental processes [29]. Also, microarray analysis shows a significant transcript accumulation of one such member, *OsMET1–2* during the early seed stages [23]. This is an indication of the role played by various DNA methyl transferases (MTases) in gene regulation during early seed development (**Figure 1**). Microarray analysis has also shown that a total of 94 genes are down regulated in the zygotic stage. Subsequent gene ontology (GO) and pathway analyses suggest the involvement of these genes in metabolic pathways possibly associated with suppression of the maternal genes [26, 29]. Thus, various transcript analyses indicate the existence of an active zygotic genome during early seed development.

A systematic expression profiling at three developmental stages of the embryo categorized as early (3–5 DAP), middle (7 DAP) and late (14 DAP) have shown the expression of about 20,856 common genes, suggesting their role in housekeeping functions. However, many genes show specific expression in each category suggesting their involvement in imparting functions unique to the stage. These genes belong to different functional categories as metabolic processes, binding and cell part and cellular processes. About 1131 genes show specific expression at 3–5 DAP, possibly involved in determining the embryo axis. Polarized expression of different TF and transcription regulator (TR) genes has been identified at the apical-basal and dorsal-ventral axis in the globular embryo. This spatiotemporal expression of specific TFs and TRs might be involved in the establishment of early embryo patterning in rice [30]. Different phytohormone-related genes including GA biosynthetic genes, auxin efflux *PIN* genes, cytokinin A-type response regulators, and brassinosteroid (BR)-perception genes also show an embryonic axis-dependent expression. The repressor of GA-signaling *OSSLR1* shows a preferential expression in the basal region of the embryo. On the other hand, a GA biosynthetic gene, *OsGA20ox1* is expressed in the apical-dorsal region. Apical to basal auxin transport is initiated at the early globular stage by the auxin transport proteins, thus, regulating various aspects of embryonic pattern formation. Transcript accumulation for cytokinin response-regulator occurs in the apical-ventral region whereas BR and ethylene biosynthesis occurs in the basal region [31]. Among TFs, homeobox gene family members show differential expression during different phases of embryo development suggesting their inevitable roles during the process [4, 30]. Different MADS-box transcripts show seed-preferential expression with about 12 of them showing a specific expression in the seed, including Arabidopsis ABCDE class gene orthologs, suggesting their involvement in early embryo development [30, 32].

**4. Genes involved in polarity establishment and organ initiation are** 

Embryogenesis is an important aspect of seed development, which involves cellular division and establishment of the embryo body plan. The process of embryo development is classified into ten major stages in rice. The first stage is the zygotic stage formed at 0 days after fertilization (DAP). The initial stages of post-zygotic development are characterized by the repeated cellular division without morphogenesis. This leads to the formation of a globular embryo, which can be observed from 1 to 3 DAP. It is the stage at which embryo specifies its body axis. Subsequently, formation and proper positioning of the shoot apical meristem (SAM), coleoptile primordium and the radicle primordium occurs at 4 DAP. This is followed by the origin of leaf primordium, which initiates at 5 DAP and is completed by 8 DAP, by the formation of all the three leaf primordia. With the enlargement of various organs, embryo morphogenesis is completed in rice by 10 DAP. This is followed by a stage of maturation which lasts till about 20 DAP, and thereafter, it enters into a dormant state [4, 5]. Profiling the RNA expression levels has greatly broadened the understanding of rice embryo development. Unlike animal embryos, both maternal and paternal genomes contribute equally in the process of embryogenesis and the plant embryonic development is majorly under zygotic control. Maternal-tozygotic transition (MZT) initiates 50 hours after fertilization [24–26]. Thus, zygotic genome

Microarrays coupled with laser microdissection (LMD) have made available the expression profiles of developing female gametophyte from the pre-meiotic to the mature embryo-sac stages in rice [27]. Similarly, expression profiles of stigma as well as embryo sac are also available from high-throughput RNA-Seq technology [28]. These cell-type specific expression analyses have been compared with various developmental stages of embryo to identify genes that are differentially expressed during the post fertilization period. Identification of such genes expands our idea of embryogenesis as many of them can be candidates for maintaining various stages or aspects of embryo morphogenesis. Microarray-based comparison of gametic and zygotic tissues has identified a total of 325 genes up regulated in zygote in comparison with the egg cell. Majority of these up regulated genes are involved in DNA/chromatin organization and their assembly is probably involved in the induction of genes participating in zygotic development. Further, methyltransferase 1 (MET1) and different TFs belonging to the homeobox proteins are highly up regulated in the zygote, apparently affecting polarity or asymmetric division in zygote. Specific METs show higher expression during early rice seed development and are essential for cytosine methylation, regulating the genes involved in various developmental processes [29]. Also, microarray analysis shows a significant transcript accumulation of one such member, *OsMET1–2* during the early seed stages [23]. This is an indication of the role played by various DNA methyl transferases (MTases) in gene regulation during early seed development (**Figure 1**). Microarray analysis has also shown that a total of 94 genes are down regulated in the zygotic stage. Subsequent gene ontology (GO) and pathway analyses suggest the involvement of these genes in metabolic pathways possibly associated with suppression of the maternal genes [26, 29]. Thus, various transcript analyses

indicate the existence of an active zygotic genome during early seed development.

**expressed in the embryo**

30 Advances in Seed Biology

gets "switched on" almost immediately after fertilization.

A gradual transition in the transcript profile from early to late stages of embryo has been observed. A large number of genes are shared between the early and middle stages of embryo development although, unique expression of different genes is also observed [30, 31]. The genes up regulated in the early and middle stages of embryo development are majorly involved in amino acid, lipid and energy metabolism, nucleic acid replication/processing, signal transduction and transcriptional regulation. The enrichment of these pathway genes provides the energy required for the early developing embryo. As the embryo progresses towards the middle stage, additional genes as ribosomal protein components, translational machinery components are up regulated. Thus, the protein biosynthesis genes show a greater expression during the middle phase. There occurs a significant enhancement in the expression of genes belonging to different categories as the embryo progresses towards the maturation phase. Many of these genes show differential expression between 7 and 14 DAP embryos. Pathway and gene ontology studies suggest significant differences in the physiological processes that occur during early and late stages of rice embryogenesis [30]. Further, the maturation phase is characterized by the accumulation of protein modification and starch biosynthesis genes. Auxin related *Aux/ IAA*, *OsIAA18,* shows a significant up regulation during the middle and late stages of embryo development. Auxin-biosynthetic genes have been shown to be induced during different stages of embryo development [6, 14]. Also, the biosynthesis of ethylene is down regulated during embryo development by the enhanced level of ABA. Thus, the two phytohormones ABA and ethylene function antagonistically during embryo development in rice [14]. Additionally, GA also functions in the seed development process during maturation. Seed maturation process is majorly determined by the GA/ABA ratio [22]. To add, profiling studies have also identified the accumulation of long-lived mRNAs between 10 and 20 DAF within the embryo. Longlived mRNAs present in the mature dry seeds are required for proper seed germination. These majorly code for proteins related to the signaling of ABA, calcium ions and phospholipids as well as a heat shock protein HSP DNA J, essential for rice seed germination [33].
