**5. Rice endosperm shows structural and developmental complexities**

The endosperm of rice occupies a major portion of the seed and defines the shape of the grain. It is the storehouse of nutrients including carbohydrates, lipids and storage proteins and serves as an important source of nutrition for the developing embryo. The formation of the endosperm starts with triple fusion, wherein the male nucleus fertilizes the bi-nucleate central cell to produce a triploid cell. Thereafter, it sequentially undergoes events of cell division, cell fate determination, tissue differentiation and programmed cell death (PCD) to produce the mature endosperm. Structurally, the endosperm features four major types of cells, the starchy endosperm, the aleurone cells, the transfer cells and the cells in the vicinity of the embryo [34]. The cells in the peripheral region, except those near the vascular tissue, form the aleurone layer which varies in thickness from one to five cell layers [35]. Cells immediately above the vascular bundles form the transfer cells. Cells enclosed within these two cell layers form the starchy endosperm. The embryo surrounding cells create the cavity in which the embryo is housed. These differentiated cell layers perform specific functions which are required by the embryo during its growth and afterwards for seed germination. Photosynthate (sucrose) produced in the leaves (source) is transported into the endosperm (sink) via the transfer cells [36, 37]. Endosperm surrounding region separates the embryo and the endosperm and might also be involved in providing nutrition to the embryo by apoplastic transport [38, 39]. Starchy endosperm stores starch and proteins that start accumulating soon after cellularization is complete [39]. The aleurone layer is composed of terminally differentiated cells that produce proteolytic, hydrolytic and cell wall degrading enzymes that digest the starch and proteins stored in the endosperm into sugars and amino acids for utilization by the growing embryo during seed germination [40, 41]. Hence, the development of endosperm is complex and singular owing to the modifications in its structure occurring through a short span of time and the accumulation of reserve materials and cell cycle activities that are switched on and off at precise time points.

The functional uniqueness of the endosperm is reflected in its transcriptome which has been found to be quite distinct from several other tissue types including reproductive stages and embryo [10]. Transcriptome analysis of three developmental stages of endosperm, spanning from during this time, has shown an overall decline in gene expression during this time. Even more down regulation has been observed in the later stages [12]. Studies also indicate that in the young endosperm stages (0–4 DAP), the number of specific genes increases with age suggesting that the complexity of molecular changes rapidly increases with progression of endosperm development [9]. In another study involving 7, 14 and 21 DAP endosperm tissue, it has been observed that the expression of specific genes can be clustered into distinct patterns. About 79 genes are expressed in all the three stages suggesting that they are constitutively required throughout endosperm development. A set of 32 genes express highly in 14 and 21 DAP indicating their role in nutrient accumulation and PCD. About 22 genes and 15 genes show higher expression in 7 DAP alone and in both 7 and 14 DAP, respectively. These genes can be presumed to be regulating cell proliferation and cellularization during initial development and synthesis and accumulation of storage compounds [42]. Thus, amalgamation of such information from transcriptome data with knowledge from previous developmental studies can be useful in generating knowledge about the functions performed by various genes in different stages.

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].

32 Advances in Seed Biology

**5. Rice endosperm shows structural and developmental complexities**

The endosperm of rice occupies a major portion of the seed and defines the shape of the grain. It is the storehouse of nutrients including carbohydrates, lipids and storage proteins and serves as an important source of nutrition for the developing embryo. The formation of the endosperm starts with triple fusion, wherein the male nucleus fertilizes the bi-nucleate central cell to produce a triploid cell. Thereafter, it sequentially undergoes events of cell division, cell fate determination, tissue differentiation and programmed cell death (PCD) to produce the mature endosperm. Structurally, the endosperm features four major types of cells, the starchy endosperm, the aleurone cells, the transfer cells and the cells in the vicinity of the embryo [34]. The cells in the peripheral region, except those near the vascular tissue, form the aleurone layer which varies in thickness from one to five cell layers [35]. Cells immediately above the vascular bundles form the transfer cells. Cells enclosed within these two cell layers form the starchy endosperm. The embryo surrounding cells create the cavity in which the embryo is housed. These differentiated cell layers perform specific functions which are required by the embryo during its growth and afterwards for seed germination. Photosynthate (sucrose) produced in the leaves (source) is transported into the endosperm (sink) via the transfer cells [36, 37]. Endosperm surrounding region separates the embryo and the endosperm and might also be involved in providing nutrition to the embryo by apoplastic transport [38, 39]. Starchy endosperm stores starch and proteins that start accumulating soon after cellularization is complete [39]. The aleurone layer is composed of terminally differentiated cells that produce proteolytic, hydrolytic and cell wall degrading enzymes that digest the starch and proteins stored in the endosperm into sugars and amino acids for utilization by the growing embryo during seed germination [40, 41]. Hence, the development of endosperm is complex and singular owing to the modifications in its structure occurring through a short span of time and the accumulation of reserve materials and cell cycle activities that are switched on and off at precise time points. The functional uniqueness of the endosperm is reflected in its transcriptome which has been found to be quite distinct from several other tissue types including reproductive stages and embryo [10]. Transcriptome analysis of three developmental stages of endosperm, spanning from during this time, has shown an overall decline in gene expression during this time. Even more down regulation has been observed in the later stages [12]. Studies also indicate that in the young endosperm stages (0–4 DAP), the number of specific genes increases with age suggesting that the complexity of molecular changes rapidly increases with progression of endosperm development [9]. In another study involving 7, 14 and 21 DAP endosperm tissue, it has been observed that the expression of specific genes can be clustered into distinct patterns. About 79 genes are expressed in all the three stages suggesting that they are constitutively required Endosperm involves the precise operation of several transcription factors throughout its course of development. About 1118 transcription factors belonging to 55 families have been reported to be expressing in early stages of endosperm development [12]. TFs have emerged as a major functional category in later stages (7–21 DAP) of endosperm development [42]. Expression pattern of TFs has also been indicated to be subjected to temporal regulation. Members of the transcription factor families such as, MADS, NAC, AP2-EREBP, MYB and CCAAT, have been observed to show higher expression in the endosperm (**Figure 1**). Out of these, *MADS* genes are expressed through the early stages (1–14 DAP), *AP2-EREBP* and *MYB* are expressed during early through middle stages (7–21 DAP), whereas, *NAC* and *CCAAT* are expressed through all stages (2–42 DAP) of endosperm development [10, 42]. MADS TFs have been shown to regulate endosperm development by a mechanism affecting the cytokinin level. Overexpression of *MADS29* activates the genes involved in starch biosynthesis and promotes the differentiation of proplastids to starch-containing amyloplasts [43]. In our study encompassing five different rice varieties, three NAC TFs exhibit seed-specific/preferential expression with significantly higher expression in S3-S5 stages suggesting their role in accumulation of storage reserves. They also show significant association with seed traits emphasizing their role in regulation of seed development [44]. Similarly, genome-wide analysis of 14 vegetative and reproductive tissues has indicated the expression of 21 C2 H2 proteins in seeds of which 12 are specific to seed tissue. The expression of these genes shows variable pattern among the five stages. Some of them are expressed from S1–S5, while most of them show higher expression in the later stages of seed development implying their function in both initial seed development and seed maturation [3]. In another report including three endosperm stages covering 3–10 DAP, different types of expression patterns of the transcription factors have been observed. Six TFs families including Dof are up regulated through 3–10 DAP. Three transcription factor families including GRAS are down regulated from 3 to 6 DAP then up regulated till 10 DAP. NF-YA family members on the other hand are up regulated from 3 to 6 DAP then down regulated until 10 DAP [12]. In summary, TFs are expressed throughout the development of the endosperm and their expression is highly preferential. The heterogeneity in the expression patterns of TFs is an indicator of the intricate molecular regulation of endosperm transcriptome probably required for proper completion of a stage and subsequent transition to another.

As mentioned previously, hormones are known to be regulators of embryo development and this raises the possibility of them being key ingredients in the regulatory network of endosperm development. In this context, it has been observed that several hormone response *cis*elements are present in the promoters of endosperm-specific genes that are expressed from 7 DAP to 21 DAP. The most abundant *cis*-elements belong to abscisic acid responses, including ABADESI1 and ABREMOTIFAOSOSEM. Since abscisic acid is a well-known hormone for desiccation and dormancy, which is associated with seed maturation, the aforesaid observation would imply that these processes are very eminent in endosperm and are initiated from the middle stages of development [45]. Along with ABA, *cis*-elements for gibberellic acid, such as GARE1OSREP1 and PYRIMIDINEBOXOSRAMY1A, auxins, ARFAT, and ethylene-responsive element, such as ERELEE4 have also been observed [42]. The simultaneous expression of the genes regulated by hormones unambiguously indicates the significance of hormonal interplay in the growth of endosperm. Although, the specific effects of these hormones can only be understood from detailed functional characterization studies.

One eccentric yet indispensable feature of endosperm development is the occurrence of two types of cell cycles at different stages of development. First is the free nuclear division without cellularization leading to syncytium formation in the initial stages of development (0–5 DAP), and second is endoreduplication that occurs in the later stages (8–10 DAP) and is associated with increasing cell size and endosperm volume [4]. In coherence with this information, two CDKs, *CDKB;1* and *CDKB;2*, have been found to be showing higher expression in early stages of endosperm (1–2 DAP). Also, one A-type cyclin and four B-type cyclins exhibit patterns of expression overlapping with these CDKs. It is noteworthy that cell cycle defects associated with the endosperm can influence the growth of the embryo. Knockdown of a rice cyclin gene, *CycB1;1*, results in the formation of a large embryo and abortive endosperm suggesting that normal mitotic activity of the endosperm is imperative for the development of the embryo as well [46].

One of the primary objectives of the endosperm is stocking of nutrients which will eventually be assigned to various metabolic pathways required for seed development process. Bulk of the endosperm is constituted by starch and prolamin storage proteins [47–49]. Transcriptome profiling studies advocate that genes associated with accumulation of starch and sugars are significantly up regulated in the endosperm. Genes related to starch metabolism and storage protein biosynthesis have been found to be among the highly up regulated genes as development progresses from 3 to 10 DAP. Also, 11 members of Dof TFs have been found to be up regulated in endosperm [12]. Dof TFs are known to be associated with synthesis of storage proteins in the endosperm [50]. Pathway studies have also indicated that in the endosperm, starch and sugar metabolism are highly up regulated followed by amino sugar and nucleotide sugar metabolism and carbon fixation by photosynthesis. It has also been observed that as endosperm moves from 3 DAP to 10 DAP most of the genes and pathways are down regulated except for those related to accumulation of storage materials [12]. Functional annotation of endosperm-specific genes from 7 DAP to 21 DAP have shown that seed storage protein, carbohydrate and energy metabolism, seed maturation, protein metabolism, lipid metabolism and transport emerge as the major categories. Seed storage proteins, including prolamins, glutelins and globulins have been reported to constitute the third largest category of endosperm-specific genes after transcription factors and stress responsive genes. Apart from this, overrepresentation of CATGCA motif or the RY element has been seen in the promoters of the endosperm-specific genes expressed from 7 DAP to 21 DAP. These genes show varied molecular function, including hydrolase activity, nutrient reservoir activity and transcription factor activity [42]. From these findings, it can be concluded that the endosperm starts gathering storage material quite early in its development which continues till maturation. This continuous process is controlled by the collaborative efforts of several genes, pathways and regulatory networks, which are primarily associated with synthesis and accumulation of starch and proteins.

desiccation and dormancy, which is associated with seed maturation, the aforesaid observation would imply that these processes are very eminent in endosperm and are initiated from the middle stages of development [45]. Along with ABA, *cis*-elements for gibberellic acid, such as GARE1OSREP1 and PYRIMIDINEBOXOSRAMY1A, auxins, ARFAT, and ethylene-responsive element, such as ERELEE4 have also been observed [42]. The simultaneous expression of the genes regulated by hormones unambiguously indicates the significance of hormonal interplay in the growth of endosperm. Although, the specific effects of these hor-

One eccentric yet indispensable feature of endosperm development is the occurrence of two types of cell cycles at different stages of development. First is the free nuclear division without cellularization leading to syncytium formation in the initial stages of development (0–5 DAP), and second is endoreduplication that occurs in the later stages (8–10 DAP) and is associated with increasing cell size and endosperm volume [4]. In coherence with this information, two CDKs, *CDKB;1* and *CDKB;2*, have been found to be showing higher expression in early stages of endosperm (1–2 DAP). Also, one A-type cyclin and four B-type cyclins exhibit patterns of expression overlapping with these CDKs. It is noteworthy that cell cycle defects associated with the endosperm can influence the growth of the embryo. Knockdown of a rice cyclin gene, *CycB1;1*, results in the formation of a large embryo and abortive endosperm suggesting that normal mitotic activity of the endosperm is imperative for the development of the embryo as

One of the primary objectives of the endosperm is stocking of nutrients which will eventually be assigned to various metabolic pathways required for seed development process. Bulk of the endosperm is constituted by starch and prolamin storage proteins [47–49]. Transcriptome profiling studies advocate that genes associated with accumulation of starch and sugars are significantly up regulated in the endosperm. Genes related to starch metabolism and storage protein biosynthesis have been found to be among the highly up regulated genes as development progresses from 3 to 10 DAP. Also, 11 members of Dof TFs have been found to be up regulated in endosperm [12]. Dof TFs are known to be associated with synthesis of storage proteins in the endosperm [50]. Pathway studies have also indicated that in the endosperm, starch and sugar metabolism are highly up regulated followed by amino sugar and nucleotide sugar metabolism and carbon fixation by photosynthesis. It has also been observed that as endosperm moves from 3 DAP to 10 DAP most of the genes and pathways are down regulated except for those related to accumulation of storage materials [12]. Functional annotation of endosperm-specific genes from 7 DAP to 21 DAP have shown that seed storage protein, carbohydrate and energy metabolism, seed maturation, protein metabolism, lipid metabolism and transport emerge as the major categories. Seed storage proteins, including prolamins, glutelins and globulins have been reported to constitute the third largest category of endosperm-specific genes after transcription factors and stress responsive genes. Apart from this, overrepresentation of CATGCA motif or the RY element has been seen in the promoters of the endosperm-specific genes expressed from 7 DAP to 21 DAP. These genes show varied molecular function, including hydrolase activity, nutrient reservoir activity and transcription factor activity [42]. From these findings, it can be concluded that the endosperm starts gathering storage material quite early in its development which continues till maturation.

mones can only be understood from detailed functional characterization studies.

well [46].

34 Advances in Seed Biology

Towards the end of its development, after the complete size has been attained and storage materials are being accumulated, the endosperm undergoes programmed cell death (PCD) which is initiated from 16 DAP in cereal seeds [4, 51, 52]. This results in degeneration of the storage cells of the endosperm surrounded by living cells of the aleurone layer. Although PCD has been less explored in rice endosperm, some reports of PCD genes from pollen tissues and rice protoplasts are available [11, 53, 54]. Transcriptome studies of endosperm have detected several PCD related genes. *AIP5*, a positive regulator of PCD in the tapetum, has been found to be up regulated, whereas, *hsp70,* a negative regulator of PCD in rice protoplasts, is down regulated in the endosperm. Apart from these, 11 PCD related genes have also been found to express in the endosperm [12]. PCD has also been implicated to be influenced by hormones. Ethylene and gibberellic acid have been suggested to be positive regulators and abscisic acid has been shown to be a negative regulator of cell death [51, 55]. Up regulation of six gibberellic acid pathway genes and down regulation of 20 abscisic acid pathway genes has been observed in the 6 and 10 DAP endosperm tissue [12]. These results provide additional support to the earlier reports and emphasize on the active involvement of these hormones in the regulation of PCD in the endosperm.
