**3. Functional intricacies of seed development revealed by atlases explain the conundrum of seed development**

Functional annotations of the enormous data generated by atlases further elaborate on the predominant activities occurring during seed development including their mode of regulation. The enrichment of genes involved in embryonic development in the transcriptome of endosperm suggests a possible communication between the embryo and the endosperm [11]. This exchange of information between the embryo and the endosperm highlights that a certain level of cross-talk might be necessary for their growth. Induction of expression of seed-specific genes after 5 days of flowering, when the endosperm development is accelerated and starch accumulation is initiated, suggests that majority of the seed-specific genes are associated with later stages of seed development and are required for seed filling and maturation [10]. Information about the key processes, pathways and genes involved in seed development can also be identified by comparative transcript profiling of tissues from various stages of development. Seed-specific proteins, seed allergens, genes involved in starch biosynthesis/degradation and ubiquitin-mediated protein degradation pathway show specific expression throughout the seed stages in comparison to four vegetative stages [6]. Transcription factors (TFs) have been reported to be enriched in both early and later stages of seed development suggesting their involvement throughout the process. In a study involving 48 organs of a j*aponica* rice variety, it has been found that out of 41 tissue-specific TFs obtained, 29 are seed-specific. These include several members of MADS, NAC, AP2-EREBP and CCAAT families that are expressed in a seed-specific manner with predominant expression either in the endosperm or the embryo and this expression is also driven by the stage of the tissues (**Figure 1**). TFs such as *VP1* and *LEC1*, which have been reported to be active during seed maturation, have been found to be present in the seed-specific category whereas AP2-EREBP TFs are found to express mostly in the embryo through its entire development [10]. In our study on an *indica* rice variety, it has been seen that 27 TFs families have higher number of members expressing in the seed stages, which include MYB, NAM, HSF, MADS, POZ, and bZIP. About 47 TFs are found to be specific to seed stages, of which most have specificity to S2 stage [6]. Such preferential expression patterns observed in various studies would imply that these TFs are administrating seed development by regulating downstream genes and pathways required for individual growth and development of the embryo and the endosperm at specific time points and stages.

Hormonal regulation is another indispensable component of the multi-faceted regulation of seed development. Rhythmic fluctuations in levels of hormones such as auxins, gibberellic acid (GA) and abscisic acid (ABA) have been observed during the course of seed development suggesting a complex interplay between these [18]. Different hormones have been known to be controlling different modules of seed development such as organ patterning, cell enlargement, desiccation and dormancy [19–22]. Seed-specific differential regulation of various hormones has been encountered in several reports. Auxin biosynthesis genes have been found to be induced during early stages of seed development [23]. This emphasizes on the role of auxins in the initial seed development processes that are mainly associated with active cell division in the embryo and the endosperm and organ initiation [4]. Along with auxins, implications for the role of gibberellin and ethylene have also been proposed. Genes associated with entkaurene biosynthesis, a precursor of gibberellin biosynthesis, were found to be up regulated in the S1 stage of seed development indicating the role of gibberellin in early phases of seed development [23]. A negative regulator of gibberellin signaling, *SLRL2* and a putative ethylene receptor that negatively regulates ethylene signaling, *OsETR2;2*, have been seen to be showing seed-specific expression [10]. These findings enumerate the significance of differential

regulation. The enrichment of genes involved in embryonic development in the transcriptome of endosperm suggests a possible communication between the embryo and the endosperm [11]. This exchange of information between the embryo and the endosperm highlights that a certain level of cross-talk might be necessary for their growth. Induction of expression of seed-specific genes after 5 days of flowering, when the endosperm development is accelerated and starch accumulation is initiated, suggests that majority of the seed-specific genes are associated with later stages of seed development and are required for seed filling and maturation [10]. Information about the key processes, pathways and genes involved in seed development can also be identified by comparative transcript profiling of tissues from various stages of development. Seed-specific proteins, seed allergens, genes involved in starch biosynthesis/degradation and ubiquitin-mediated protein degradation pathway show specific expression throughout the seed stages in comparison to four vegetative stages [6]. Transcription factors (TFs) have been reported to be enriched in both early and later stages of seed development suggesting their involvement throughout the process. In a study involving 48 organs of a j*aponica* rice variety, it has been found that out of 41 tissue-specific TFs obtained, 29 are seed-specific. These include several members of MADS, NAC, AP2-EREBP and CCAAT families that are expressed in a seed-specific manner with predominant expression either in the endosperm or the embryo and this expression is also driven by the stage of the tissues (**Figure 1**). TFs such as *VP1* and *LEC1*, which have been reported to be active during seed maturation, have been found to be present in the seed-specific category whereas AP2-EREBP TFs are found to express mostly in the embryo through its entire development [10]. In our study on an *indica* rice variety, it has been seen that 27 TFs families have higher number of members expressing in the seed stages, which include MYB, NAM, HSF, MADS, POZ, and bZIP. About 47 TFs are found to be specific to seed stages, of which most have specificity to S2 stage [6]. Such preferential expression patterns observed in various studies would imply that these TFs are administrating seed development by regulating downstream genes and pathways required for individual growth and development of the embryo and the

Hormonal regulation is another indispensable component of the multi-faceted regulation of seed development. Rhythmic fluctuations in levels of hormones such as auxins, gibberellic acid (GA) and abscisic acid (ABA) have been observed during the course of seed development suggesting a complex interplay between these [18]. Different hormones have been known to be controlling different modules of seed development such as organ patterning, cell enlargement, desiccation and dormancy [19–22]. Seed-specific differential regulation of various hormones has been encountered in several reports. Auxin biosynthesis genes have been found to be induced during early stages of seed development [23]. This emphasizes on the role of auxins in the initial seed development processes that are mainly associated with active cell division in the embryo and the endosperm and organ initiation [4]. Along with auxins, implications for the role of gibberellin and ethylene have also been proposed. Genes associated with entkaurene biosynthesis, a precursor of gibberellin biosynthesis, were found to be up regulated in the S1 stage of seed development indicating the role of gibberellin in early phases of seed development [23]. A negative regulator of gibberellin signaling, *SLRL2* and a putative ethylene receptor that negatively regulates ethylene signaling, *OsETR2;2*, have been seen to be showing seed-specific expression [10]. These findings enumerate the significance of differential

endosperm at specific time points and stages.

28 Advances in Seed Biology

**Figure 1.** Schematic representation of TFs involved in rice seed development. TF families whose members express during the five stages of seed development in the endosperm and embryo have been mentioned above and below the solid line, respectively.

regulation of these hormones in materializing seed development. The localized expression of different phytohormone-related genes during embryo development as well as their critical role in endosperm development and grain filling has been discussed in further sections.
